Covalently linked thermostable kinase for decontamination process validation

Abstract

A biological process indicator is provided for validating a treatment process in which the amount or activity of a contaminant in a sample is reduced. The indicator comprises a thermostable kinase covalently linked to a biological component, with the proviso that the biological component is not an antibody. Methods of preparing the indicator, and methods of using the indicator, are also provided.

Claims

1. A biological process indicator for validating a treatment process in which the amount or activity of a contaminant in a sample is reduced, wherein the indicator comprises a thermostable kinase covalently linked to a biological component, wherein the biological component consists of a molecule that mimics the sensitivity of the contaminant to the treatment process and wherein the molecule is selected from a group consisting of a blood protein, a fungal protein, a self-aggregating protein, a bacterial fimbrial protein, a bacterial toxin protein, a bacterial spore protein, a nucleic acid, a lipid, and a carbohydrate, with the proviso that the biological component is not an antibody, wherein: (a) the fungal protein is a hydrophobin, a fungal spore protein, a hyphal protein, a mycotoxin, or a fungal prion; and (b) the self-aggregating protein is a prion, a prion mimetic protein, an amyloid fibril, beta amyloid protein, tau protein, polyadenine binding protein, lung surfactant protein C, a hydrophobin, a chaplin, a rodlin, a gram positive spore coat protein, or a barnacle cement-like protein; wherein the thermostable kinase has a reference activity, as measured by a luminometer or luminescent assay, of at least 1,000,000 Relative Light Units (RLU) per mg kinase prior to the treatment process; wherein the thermostable kinase and the biological component are products of recombinant expression in bacteria; and wherein the biological process indicator is immobilized in or on a solid support.

2. The biological process indicator of claim 1, wherein said blood protein is a blood clotting protein, a serum protein, a platelet protein, a blood cell glycoprotein, or haemoglobin.

3. The biological process indicator of claim 2, wherein said blood clotting protein is fibrin, fibrinogen, or a transglutaminase substrate.

4. The biological process indicator of claim 1, wherein the nucleic acid is a DNA molecule or an RNA molecule.

5. The biological process indicator of claim 1, wherein the carbohydrate is a exopolysaccharide or a lipopolysaccharide, a peptidoglycan, a chitin, a glucan, a lignin, a mucin, a glycolipid, a glycoprotein, a spore extract, a polysaccharide from yeast capsules, or an invertebrate secretion.

6. The biological process indicator of claim 1, wherein the lipid is a glycolipid or a ganglioside.

7. The biological process indicator of claim 1, wherein the indicator is part of a biological matrix.

8. The biological process indicator of claim 7, wherein the biological matrix is a mimetic of the sample.

9. The biological process indicator of claim 7, wherein the biological matrix comprises one or more components consisting of blood, serum, albumin, mucus, egg, neurological tissue, food, culled animal material, or a commercially-available test soil.

10. The biological process indicator of claim 1, wherein the thermostable kinase is an adenylate kinase, an acetate kinase or a pyruvate kinase.

11. The biological process indicator of claim 1, wherein the thermostable kinase comprises an amino acid sequence comprising SEQ ID NOS:1-25, 31, 32, 34-36, 38, 40, 42, 48-50, 52, 54, 61, 67, 72, or 73.

12. The biological process indicator of claim 1, wherein the thermostable kinase is encoded by a DNA comprising a nucleotide sequence comprising SEQ ID NOS:26-30, 37, 39, 41, 42, 47, 49, 51, 53, or 60.

13. A biological process indicator of claim 1, wherein the indicator further comprises an agent to stabilize the kinase.

14. The biological process indicator of claim 13, wherein the stabilizing agent is a metal ion, a sugar, a sugar alcohol or a gel-forming agent.

15. The biological process indicator of claim 1, wherein the biological component and the kinase are linked together in the form of a fusion protein.

16. The biological process indicator of claim 1, wherein the biological process indicator is immobilized in or on the solid support by chemical cross-linking or adsorption.

17. The biological process indicator of claim 1, wherein the solid support is an indicator strip, a dip-stick or a bead.

18. A kit for use in validating a treatment process in which the amount or activity of a contaminant in a sample is reduced, comprising: (a) a biological process indicator comprising a thermostable kinase covalently linked to a biological component that mimics the sensitivity of the contaminant to the treatment process, consisting of a blood protein, a fungal protein, a self-aggregating protein, a bacterial fimbrial protein, a bacterial toxin protein, a bacterial spore protein, a nucleic acid, a lipid, and a carbohydrate, with the proviso that the biological component is not an antibody, wherein i. the fungal protein is a hydrophobin protein, a fungal spore protein, a hyphal protein, a mycotoxin, or a fungal prion; ii. the self-aggregating protein is a prion, a prion mimetic protein, a amyloid fibril, beta amyloid protein, tau protein, polyadenine binding protein, lung surfactant protein C, a hydrophobin, a chaplin, a rodlin, a gram positive spore coat protein, and a barnacle cement-like protein; and (b) a substrate for the thermostable kinase; wherein the thermostable kinase has a reference activity, as measured by a luminometer or luminescent assay, of at least 1,000.000 Relative Light Units (RLU) per mg kinase prior to the treatment process; wherein the thermostable kinase and the biological component are products of recombinant expression in bacteria; and wherein the biological process indicator is immobilized in or on a solid support.

19. The kit of claim 18, wherein the substrate for the thermostable kinase is ADP.

20. The kit of claim 18, further comprising luciferin/luciferase.

21. The kit of claim 18, further comprising a look-up table correlating the kinase activity of the indicator with the amount or activity of the contaminant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is now described in specific embodiments in the following examples and with reference to the accompanying drawings in which:

(2) FIGS. 1A, 1B, and 1C show activity of adenylate kinase (AK) enzymes after treatment at 70 C. (FIG. 1A), 80 C. (FIG. 1B) and 90 C. (FIG. 1C);

(3) FIGS. 2A and 2B show the thermal stability of a range of AK enzymes recombinantly expressed in E. coli. Genes encoding AK enzymes were cloned and expressed as described in Example 3. All genes were expressed from the vector pET28a except for Sacidocaldarius clone I which was expressed from pET3a as described previously. Expression levels were similar for each clone but a proportion of the Pyrococcus furiosus (P.fu) enzyme was in the insoluble fraction and this is likely to have reduced the amount of this enzyme being assayed. The thermal stability of the recombinant enzymes was measured following incubation at 80 C. for 30 minutes in a crude E. coli lysate at 10-fold serial dilutions from 1 mg/ml total cellular protein (such that column 12 is equivalent to 1 fg/ml total protein). Enzymes from Thermotoga maritima and Archaeoglobus fulgidus showed significantly greater stability than the other enzymes tested, although the remaining enzymes (Sulfolobus solfataricus (S. so P2), Aeropyrum pernix and P.fu) showed similar activity to the S. acidocaldarius enzyme used as the basis of previous assays (data labelled as Sac I);

(4) FIG. 3 shows gel electrophoretic analysis (SDS-PAGE) of the expression and purification of tAK-fibrin peptide fusions. Lane 1, Seeblue markers; Lane 2, whole cell homogenate; Lane 3, insoluble pellet (P1); Lane 4, supernatant (51); Lane 5, heat-treated S1insoluble pellet (P2); Lane 6, heat-treated S1soluble fraction (S2); Lanes 7-12, purified tAK-fibrin fractions eluted sequentially from Cibacron Blue affinity chromatography;

(5) FIGS. 4A and 4B show mass spectroscopy analysis of purified tAK (Sac)-Fibrin peptide fusions and wild type tAK (Sac). Purified Sac (FIG. 4A) and Sac-Fibrin (FIG. 4B) were applied to a silicon coated chip (NP), allowed to dry and matrix applied (Sinipinic acid) for SELDI mass spectroscopy analysis. Molecular weight is shown on the abscissa axis and the relative signal on the ordinate axis. The addition of the fibrin peptide results in an increase in molecular weight of Sac from 21170 Da (FIG. 4A) to 22188 Da (FIG. 4B) with no apparent degradation of said peptide. The respective dimer and trimer Sac species can also be observed at 42324 Da and 63448 Da for Sac, and 44357 Da and 66494 Da for Sac-fibrin;

(6) FIG. 5 shows gel electrophoretic analysis (SDS-PAGE) of solubilised and refolded Sup35-tAK (Sac) from clarified inclusion body preparations. Lane 1, SeeBlue Plus 2 marker; Lane 2; Sup t35AK, refolded from solubilised inclusion bodies (prepared in the presence of the reducing agent, DTT) in 20 mM Tris-HCl pH 8.5; Lane 3, as Lane 2 but using solubilised inclusion bodies prepared in the absence of DTT; Lane 4, as Lane 3, insoluble fraction; Lane 5, as Lane 2 but heat-treated at 75 C. for 10 min, soluble fraction; Lane 6, as Lane 3, heat-treated at 75 C. for 10 min, soluble fraction; Lane 7, as Lane 6, insoluble fraction; Lane 8, as Lane 7, washed pellet; Lane 9, as Lane 2 concentrated in dialysis tubing covered in PEG 10000; Lane 10, as Lane 1. E. coli RV308 expressing Sup35-tAK cultured at 30 C. in shake flasks to a final OD.sub.600 nm of 14. Centrifuged cell paste was resuspended in 20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100 at 0.05 culture volume. Cells were lysed by sonication. Inclusion bodies were isolated from the crude cell lysate by centrifugation and washed three times with 20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100. Final wet weight of washed pellet was 5 g/L culture. Inclusion bodies were resuspended at 15 mg/ml in 50 mM CAPS, pH 11 with 0.3% N-lauroylsarcosine and +/1 mM DTT, incubated for 1.75 hours at 20 C. and clarified by centrifugation at 12,000 rpm in Sorval centrifuge, SS34 rotor for 10 minutes. The supernatant containing the solubilised protein was then dialysed with 5 buffer changes (the first two with DTT and the remainder without) prior to use. Refolded Sup35-tAK could be prepared in soluble or insoluble forms by solubilising the inclusion bodies in the presence or absence of DTT as shown in lanes 2 and 4 respectively. Refolded, soluble, Sup35-tAK demonstrated stability against the above heat treatment;

(7) FIG. 6 shows electron microscopy analysis of tAK-Sup35 (Sac) fibril formation. Purified inclusion bodies were solubilised and refolded as described previously (FIG. 5). Fibril formation was induced by incubating at 4 C. for 24-72 hours at a protein concentration of 2 mg/ml. The samples were analysed by standard transmissive electron microscopy (TEM) techniques using uranyl acetate as the negative stain. Multiple polymeric species can be observed across the image (arrowed). No fibril species were observed at protein concentrations below 2 mg/ml;

(8) FIG. 7 shows cross-linking of tAK to purified porcine mucin using SPDP.

(9) Lane 1, molecular weight markers; Lane 2, tAK conjugated to mucin; Lane 3, tAK-mucin conjugate reduced using DTT. Following SPDP derivatisation of tAK and mucin as described previously, high molecular weight conjugate species are formed <200 kDa. No non-conjugated tAK is present in Lane 2 demonstrating highly efficient cross-linking between the two protein species. Reduction of the conjugate breaks the cross-linking bonds resulting in the appearance of free tAK as indicated in Lane 3;

(10) FIG. 8 shows processing of tAK mucin indicators in a washer-disinfector.

(11) tAK in mucin was prepared by adding unmodified tAK to porcine mucin and allowing tAK to dry on the indicator surface. tAK-mucin conjugate was prepared as described previously using SPDP to cross-link tAK and mucin. The indicators were processed in a validated washer-disinfector using 3E-zyme as the detergent;

(12) FIG. 9 shows SDS-PAGE analysis of the covalent attachment of tAK-fibrin fusion protein with fibrinogen to form a tAK-fibrin film. Lane 1, SeeBlue2 markers; Lane 2, 1:1000 ratio of tAK-fibrin to fibrinogen reactionnon-thrombin activated control; Lane 3, as Lane 2ratio of 1:500 tAK-fibrin: fibrinogen; Lane 4, as Lane 2, ratio of 1:250 tAK-fibrin: fibrinogen; Lane 5, as Lane 2, thrombin-activated; Lane 6, as Lane 3 thrombin-activated; Lane 7, as Lane 4 thrombin-activated. Reactions were incubated at 37 C. for 1 hour in the presence or absence of 5 U of thrombin, as required. Optimum covalent incorporation was achieved at a ratio tAK-fibrin: fibrinogen of 1:1000 (Lane 5) with higher molecular weight species (<198 kDa) formed as indicated, with a concomitant decrease of non-conjugate tAK-fibrin species (22.1 kDa).

SEQ ID NOS

(13) SEQ ID NO:1 Protein sequence of adenylate kinase from Sulfolobus solfataricus SEQ ID NO:2 Protein sequence of adenylate kinase from Sulfolobus acidocaldarius SEQ ID NO:3 Protein sequence of adenylate kinase from Sulfolobus tokodaii SEQ ID NO:4 Protein sequence of adenylate kinase from Pyrococcus furiosus SEQ ID NO:5 Protein sequence of adenylate kinase from Pyrococcus horikoshii SEQ ID NO:6 Protein sequence of adenylate kinase from Pyrococcus abyssi SEQ ID NO:7 Protein sequence of adenylate kinase from Methanococcus thermolithotrophicus SEQ ID NO:8 Protein sequence of adenylate kinase from Methanococcus voltae SEQ ID NO:9 Protein sequence of adenylate kinase from Methanococcus jannaschii SEQ ID NO:10 Protein sequence of adenylate kinase from Methanopyrus kandleri SEQ ID NO:11 Protein sequence of adenylate kinase from Methanotorris igneus SEQ ID NO:12 Protein sequence of adenylate kinase from Pyrobaculum aerophilum SEQ ID NO:13 Protein sequence of adenylate kinase from Thermotoga maritima SEQ ID NO:14 Protein sequence of adenylate kinase from Aeropyrum pernix SEQ ID NO:15 Protein sequence of adenylate kinase from Archaeoglobus fulgidus SEQ ID NO:16 Protein sequence of adenylate kinase from Pyrococcus abyssi (monomeric adenylate kinase (AdkE)) SEQ ID NO:17 Protein sequence of adenylate kinase from Pyrococcus furiosus genetically engineered to provide improved stability SEQ ID NO:18 Protein sequence of adenylate kinase from Pyrococcus horikoshii genetically engineered to provide improved stability SEQ ID NO:19 Protein sequence of adenylate kinase from Sulfolobus acidocaldarius genetically engineered to provide improved stability SEQ ID NO:20 Protein sequence of Acetate kinase from Thermatoga maritima SEQ ID NO:21 Protein sequence of Pyruvate kinase from Pyrococcus horikoshii SEQ ID NO:22 Protein sequence of Pyruvate kinase from Sulfolobus solfataricus SEQ ID NO:23 Protein sequence of Pyruvate kinase from Thermotoga maritima SEQ ID NO:24 Protein sequence of Pyruvate kinase from Pyrococcus furiosus SEQ ID NO:25 Protein sequence of Acetate kinase from Methanosarcina thermophila SEQ ID NO:26 DNA sequence encoding the adenylate kinase from Sulfolobus acidocaldarius SEQ ID NO:27 DNA sequence encoding the adenylate kinase from Sulfolobus acidocaldarius, wherein codon usage has been optimised for expression of the gene in E. coli. SEQ ID NO:28 DNA sequence encoding the adenylate kinase from Thermotoga maritima SEQ ID NO:29 DNA sequence encoding the adenylate kinase from, Thermotoga maritima, wherein codon usage has been optimised for expression of the gene in E. coli. SEQ ID NO:30 DNA sequence encoding the adenylate kinase from Archaeoglobus fulgidus, wherein codon usage has been optimised for expression of the gene in E. coli. SEQ ID NO:31 Protein sequence of adenylate kinase from Sulfolobus acidocaldarius, wherein codon usage has been optimised for expression of the gene in E. coli (SEQ ID NO:27). SEQ ID NO:32 Protein sequence of adenylate kinase from Thermotoga maritima, wherein codon usage has been optimised for expression of the gene in E. coli (SEQ ID NO:29). SEQ ID NO:33 Protein sequence of transglutaminase substrate SEQ ID NO:34 Protein sequence of thermostable adenylate kinase from Sulfolobus acidcaldarius fused at the N-terminus with a transglutaminase (Factor XIII) substrate sequence SEQ ID NO:35 Protein sequence of thermostable adenylate kinase from Sulfolobus acidcaldarius fused at the C-terminus with a transglutaminase (Factor XIII) substrate sequence SEQ ID NO:36 Protein sequence of thermostable adenylate kinase from Sulfolobus acidcaldarius fused at the N-terminus and C-terminus with a transglutaminase (Factor XIII) substrate sequence SEQ ID NO:37 DNA sequence of transglutaminase (Factor XIII) substrate sequence fused to the 5 end of adenylate kinase from Thermotoga maritima. SEQ ID NO:38 Protein sequence of adenylate kinase from Thermotoga maritima fused at the N-terminal with a transglutaminase (Factor XIII) substrate sequence. SEQ ID NO:39 DNA sequence of transglutaminase (Factor XIII) substrate sequence fused to the 3 end of adenylate kinase from Thermotoga maritima. SEQ ID NO:40 Protein sequence of adenylate kinase from Thermotoga maritime fused at the C-terminal with a transglutaminase (Factor XIII) substrate sequence. SEQ ID NO:41 DNA sequence of transglutaminase (Factor XIII) substrate sequence fused to both the 5 and 3 ends of adenylate kinase from Thermotoga maritima. SEQ ID NO:42 Protein sequence of adenylate kinase from Thermotoga maritime fused at the N- and C-terminal with a transglutaminase (Factor XIII) substrate sequence. SEQ ID NO:43 DNA sequence of complete Sup35 gene construct from Saccharomyces cerevisiae SEQ ID NO:44 Protein sequence of complete Sup35 from Saccharomyces cerevisiae SEQ ID NO:45 DNA sequence of sup35N (N-terminal domain) codon-biased for optimal expression in E. coli SEQ ID NO:46 Protein sequence of sup35N (N-terminal domain) SEQ ID NO:47 DNA sequence of E. coli codon biased adenylate kinase from Sulfolobus acidcaldarius fused at the N-terminus with Sup35 N-terminal domain from Saccharomyces cerevisiae SEQ ID NO:48 Protein sequence of adenylate kinase from Sulfolobus acidcaldarius fused at the N-terminus with Sup35 N-terminal domain from Saccharomyces cerevisiae SEQ ID NO:49 DNA sequence of E. coli codon biased adenylate kinase from Sulfolobus acidcaldarius fused at the C-terminus with Sup35 N-terminal domain from Saccharomyces cerevisiae SEQ ID NO:50 Protein sequence of adenylate kinase from Sulfolobus acidcaldarius fused at the C-terminus with Sup35 N-terminal domain from Saccharomyces cerevisiae SEQ ID NO:51 DNA sequence of Sup35N fused at the 5 end of adenylate kinase from Thermotoga maritima. SEQ ID NO:52 Protein sequence of adenylate kinase from Thermotoga maritima fused at the N-terminal with Sup35N. SEQ ID NO:53 DNA sequence of Sup35N fused at the 3 end of adenylate kinase from Thermotoga maritima. SEQ ID NO:54 Protein sequence of adenylate kinase from Thermotoga maritima fused at the C-terminal with Sup35N SEQ ID NO:55 DNA sequence encoding a short Sup35 peptide capable of aggregating to form amyloid fibrils; for use as a fusion peptide with tAK genes. SEQ ID NO:56 Sup35 derived amyloid peptide SEQ ID NO:57 DNA sequence encoding a Norovirus capsid protein (58 kDa) SEQ ID NO:58 Protein sequence of Norovirus capsid protein (58 kDa) SEQ ID NO:59 DNA sequence for a synthetic gene encoding a Norovirus capsid protein (58 kDa) optimised for expression in E. coli SEQ ID NO:60 DNA sequence for a synthetic gene encoding a Norovirus capsid protein (58 kDa) optimised for expression in E. coli fused at the 5 end of a gene encoding the tAK from Thermotoga maritima. SEQ ID NO:61 Protein sequence of a Norovirus capsid protein (58 kDa) fused at the N-terminus of the adenylate kinase from Thermotoga maritima. SEQ ID NO:62 Protein sequence of a bacteriophage MS2 coat protein SEQ ID NO:63 Protein sequence of a bacteriophage PP7 coat protein monomer SEQ ID NO:64 Protein sequence of a bacteriophage PP7 coat protein dimer SEQ ID NO:65 Protein sequence of E. coli CsgA SEQ ID NO:66 Protein sequence of Salmonella AgfA SEQ ID NO:67 Protein sequence of adenylate kinase from Thermotoga maritima fused to the N terminus of E. coli CsgA SEQ ID NO:68 Protein sequence of the hydrophobin 3 protein from Fusarium species SEQ ID NO:69 Protein sequence of the hydrophobin 5 protein from Fusarium species SEQ ID NO:70 Protein sequence of cement-like protein from Balanus albicostatus (19K) SEQ ID NO:71 Protein sequence of cement-like protein from Megabalanus rosa (20 k) SEQ ID NO:72 Protein sequence of fusion of the barnacle protein from Balanus albicostatus with the tAK from Thermotoga maritima; N-terminal fusion SEQ ID NO:73 Protein sequence of fusion of the barnacle protein from Balanus albicostatus with the tAK from Thermotoga maritima; C-terminal fusion SEQ ID NO:74 Protein sequence of Balanus albicostatus calcite-specific adsorbent SEQ ID NO:75 Protein sequence of a peptide derived from a barnacle cement protein SEQ ID NO:76 Protein sequence of a peptide derived from a barnacle cement protein SEQ ID NO:77 Protein sequence of a peptide derived from a barnacle cement protein

EXAMPLE 1

(14) Purification of Native Adenylate Kinase Enzymes

(15) Biomass was produced from twenty-four diverse thermophilic and hyperthermophilic microorganisms (Table 1).

(16) Eight members of the archaea were represented along with sixteen diverse aerobic and anaerobic bacteria. AKs from each of these organisms was purified by affinity chromatography using selective absorption and desorption from Cibacron Blue 3A (Blue Sepharose). All enzymes were further characterised and purified by gel filtration (Superdex G200). This enabled identification of the major AK fraction and estimation of molecular mass.

(17) TABLE-US-00002 TABLE 1 List of thermophilic organisms cultured to produce biomass for isolation of thermostable AKs. Organism Domain Growth T.sub.opt pH.sub.opt 1 Aeropyrum pernix Archaeon Aerobe 95 C. 7.0 2 Alicyclobacillus acidocaldarius Bacterium Aerobe 65 C. 3.5 3 Aquifex pyrophilus Bacterium Microaerophileeberophile 85 C. 6.5 4 Bacillus caldotenax BT1 Bacterium Aerobe 65 C. 7.0 5 Bacillus species PS3 Bacterium Aerobe 65 C. 7.0 6 Bacillus stearothermophilus 11057 Bacterium Aerobe 65 C. 7.0 7 Bacillus stearothermophilus 12001 Bacterium Aerobe 65 C. 7.0 8 Bacillus thermocatenulatus Bacterium Aerobe 65 C. 7.0 9 Clostridium stercocorarium Bacterium Anaerobe 55 C. 7.0 10 Meiothermus ruber Bacterium Aerobe 60 C. 6.5 11 Pyrococcus furiosus Archaeon Anaerobe 95 C. 7.5 12 Pyrococcus horikoshii Archaeon Anaerobe 95 C. 7.0 13 Pyrococcus woesei Archaeon Anaerobe 95 C. 7.0 14 Rhodothermus marinus Bacterium Aerobe 70 C. 6.5 15 Sulfolobus acidocaldarius 98-3 Archaeon Aerobe 75 C. 2.5 16 Sulfolobus shibatae B21 Archaeon Aerobe 75 C. 2.5 17 Sulfolobus solfataricus P2 Archaeon Aerobe 75 C. 2.5 18 Thermoanaerobacter ethanolicus Bacterium Anaerobe 65 C. 6.0 19 Thermoanaerobacter Bacterium Anaerobe 65 C. 6.5 thermosulfurogenes 20 Thermobrachium celere Bacterium Anaerobe 60 C. 7.0 21 Thermococcus litoralis Archaeon Anaerobe 85 C. 6.5 22 Thermus aquaticus YT1 Bacterium Aerobe 70 C. 8.0 23 Thermus caldophilus GK24 Bacterium Aerobe 70 C. 8.0 24 Thermus thermophilus HB8 Bacterium Aerobe 70 C. 8.0

EXAMPLE 2

(18) Analysis of Thermostability of Native Adenylate Kinases

(19) The thermostability at 70 C., 80 C., and 90 C. of adenylate kinases isolated from biomass from thermophilic organisms was assessed, and the results shown in FIG. 1A, 1B, 1C.

(20) The adenylate kinases were isolated from the biomass by affinity chromatography using selective absorption and desorption from Cibacron Blue 3A (Blue Sepharose). The samples eluted from the columns were diluted 1:10 000 and then 10 l of each added to a microtitre well. 2.5 l of apyrase was added to each well to destroy the ATP present from the elution buffer, and incubated at 37 C. for 30 minutes. The apyrase was inactivated by heat treatment at 65 C. for 20 minutes.

(21) ADP substrate was added and incubated at either 70 (panel A), 80 (panel B) or 90 C. (panel C) for 30 minutes and cooled to 25 C. before the addition of 10 l of D-luciferin-luciferase reagent. The ATP produced was measured as RLU on a plate luminometer.

EXAMPLE 3

(22) Expression and Purification of Recombinant Adenylate Kinases

(23) Clones expressing representative thermostable AKs were secured and recombinant thermostable AKs from the thermoacidophilic archaeon Sulfolobus acidocaldarius and the thermophilic bacterium, Bacillus stearothermophilus produced. The plasmids were transformed into E. coli and the cell extracts shown to contain protein bands on electrophoresis corresponding to the expected molecular masses of the AKs. Thermostable AK activity was measured after incubation at the appropriate temperature (80 C. for the Sulfolobus acidocaldarius AK and 60 C. for the Bacillus stearothermophilus AK).

(24) Purification methods for both thermostable AKs were established and included an initial heat treatment of incubation for 20 minutes at 80 C., to inactivate and aggregate proteins derived from E. coli, followed by affinity chromatography and gel filtration. The affinity chromatography involved adsorption of the enzyme to Blue Sepharose, followed by specific elution with a low concentration of AK co-factors (AMP+ATP and magnesium ions). The ATP and AMP (Sigma) in the elution buffer were degraded by incubation with mesophile apyrase, which is readily inactivated by subsequent heat treatment. Gel filtration chromatography was scaled up to utilise a preparation grade Superdex column to enable large quantities of both enzymes to be prepared.

(25) Primers were designed for PCR amplification of the AK genes from the thermophilic organisms identified during the screening of candidate native enzymes.

(26) The thermostable microorganisms were grown using individually defined growth conditions and genomic DNA isolated and used as templates for PCR amplification of the adenylate kinase genes from each organism. PCR amplified adenylate kinase genes from the thermophilic organisms, Thermotoga maritima, Aeropyrum pernix, Sulfolobus acidocaldarius and Sulfolobus solfataricus were sub-cloned into the vector, pET28a and transformed into a codon enhanced E. coli strain expressing rare tRNAs (Zdanovsky et al., 2000). This E. coli strain is suitable for enhancing expression levels of AT-rich genes.

(27) The success of the transformation was assessed by a mini-expression study, and the results analysed by SDS-PAGE of the culture supernatants before and after induction with IPTG. SDS-PAGE was also used to analyse the supernatants after inclusion of a heat treatment step, which consisted of heating the sample to 80 C. for 20 minutes prior to running on the SDS-PAGE gel to remove heat labile proteins present in the sample.

(28) Sequences:

(29) TABLE-US-00003 adenylatekinasefromSulfolobussolfataricus SEQIDNO:1 MKIGIVTGIPGVGKTTVLSFADKILTEKGISHKIVNYGDYMLNTALKEGY VKSRDEIRKLQIEKQRELQALAARRIVEDLSLLGDEGIGLIDTHAVIRTP AGYLPGLPRHVIEVLSPKVIFLLEADPKIILERQKRDSSRARTDYSDTAV INEVIQFARYSAMASAVLVGASVKVVVNQEGDPSIAASEIINSLM adenylatekinasefromSulfolobusacidocaldarius SEQIDNO:2 MKIGIVTGIPGVGKSTVLAKVKEILDNQGINNKIINYGDFMLATALKLGY AKDRDEMRKLSVEKQKKLQIDAAKGIAEEARAGGEGYLFIDTHAVIRTPS GYLPGLPSYVITEINPSVIFLLEADPKIILSRQKRDTTRNRNDYSDESVI LETINFARYAATASAVLAGSTVKVIVNVEGDPSIAANEIIRSMK adenylatekinasefromSulfolobustokodaii SEQIDNO:3 MSKMKIGIVTGIPGVGKTTVLSKVKEILEEKKINNKIVNYGDYMLMTAMK LGYVNNRDEMRKLPVEKQKQLQIEAARGIANEAKEGGDGLLFIDTHAVIR TPSGYLPGLPKYVIEEINPRVIFLLEADPKVILDRQKRDTSRSRSDYSDE RIISETINFARYAAMASAVLVGATVKIVINVEGDPAVAANEIINSML adenylatekinasefromPyrococcusfuriosus SEQIDNO:4 MPFVVIITGIPGVGKSTITRLALQRTKAKFRLINFGDLMFEEAVKAGLVK HRDEMRKLPLKIQRELQMKAAKKITEMAKEHPILVDTHATIKTPHGYMLG LPYEVVKTLNPNFIVIIEATPSEILGRRLRDLKRDRDVETEEQIQRHQDL NRAAAIAYAMHSNALIKIIENHEDKGLEEAVNELVKILDLAVNEYA adenylatekinasefromPyrococcushorikoshii SEQIDNO:5 MPFVVIITGIPGVGKSTITKLALQRTRAKFKLINFGDLMFEEALKLKLVK HRDEMRKLPLEVQRELQMNAAKKIAEMAKNYPILLDTHATIKTPHGYLLG LPYEVIKILNPNFIVIIEATPSEILGRRLRDLKRDRDVETEEQIQRHQDL NRAAAITYAMHSNALIKIIENHEDKGLEEAVNELVKILDLAVKEYA adenylatekinasefromPyrococcusabyssi SEQIDNO:6 MSFVVIITGIPGVGKSTITRLALQRTKAKFKLINFGDLMFEEAVKAGLVN HRDEMRKLPLEIQRDLQMKVAKKISEMARQQPILLDTHATIKTPHGYLLG LPYEVIKTLNPNFIVIIEATPSEILGRRLRDLKRDRDVETEEQIQRHQDL NRAAAIAYAMHSNALIKIIENHEDKGLEEAVNELVEILDLAVKEYA adenylatekinasefromMethanococcusthermolithotrophicus SEQIDNO:7 MKNKLVVVTGVPGVGGTTITQKAMEKLSEEGINYKMVNFGTVMFEVAQEE NLVEDRDQMRKLDPDTQKRIQKLAGRKIAEMVKESPVVVDTHSTIKTPKG YLPGLPVWVLNELNPDIIIVVETSGDEILIRRLNDETRNRDLETTAGIEE HQIMNRAAAMTYGVLTGATVKIIQNKNNLLDYAVEELISVLR adenylatekinasefromMethanococcusvoltae SEQIDNO:8 MKNKVVVVTGVPGVGSTTSSQLAMDNLRKEGVNYKMVSFGSVMFEVAKEE NLVSDRDQMRKMDPETQKRIQKMAGRKIAEMAKESPVAVDTHSTVSTPKG YLPGLPSWVLNELNPDLIIVVETTGDEILMRRMSDETRVRDLDTASTIEQ HQFMNRCAAMSYGVLTGATVKIVQNRNGLLDQAVEELTNVLR adenylatekinasefromMethanococcusjannaschii SEQIDNO:9 MMMMKNKVVVIVGVPGVGSTTVTNKAIEELKKEGIEYKIVNFGTVMFEIA KEEGLVEHRDQLRKLPPEEQKRIQKLAGKKIAEMAKEFNIVVDTHSTIKT PKGYLPGLPAWVLEELNPDIIVLVEAENDEILMRRLKDETRQRDFESTED IGEHIFMNRCAAMTYAVLTGATVKIIKNRDFLLDKAVQELIEVLK adenylatekinasefromMethanopyruskandleri SEQIDNO:10 MGYVIVATGVPGVGATTVTTEAVKELEGYEHVNYGDVMLEIAKEEGLVEH RDEIRKLPAEKQREIQRLAARRIAKMAEEKEGIIVDTHCTIKTPAGYLPG LPIWVLEELQPDVIVLIEADPDEIMMRRVKDSEERQRDYDRAHEIEEHQK MNRMAAMAYAALTGATVKIIENHDDRLEEAVREFVETVRSL adenylatekinasefromMethanotorrisigneus SEQIDNO:11 MKNKVVVVTGVPGVGGTTLTQKTIEKLKEEGIEYKMVNFGTVMFEVAKEE GLVEDRDQMRKLDPDTQKRIQKLAGRKIAEMAKESNVIVDTHSTVKTPKG YLAGLPIWVLEELNPDIIVIVETSSDEILMRRLGDATRNRDIELTSDIDE HQFMNRCAAMAYGVLTGATVKIIKNRDGLLDKAVEELISVLK adenylatekinasefromPyrobaculumaerophilum SEQIDNO:12 MKIVIVALPGSGKTTILNFVKQKLPDVKIVNYGDVMLEIAKKRFGIQHRD EMRKKIPVDEYRKVQEEAAEYIASLTGDVIIDTHASIKIGGGYYPGLPDR IISKLKPDVILLLEYDPKVILERRKKDPDRFRDLESEEEIEMHQQANRYY AFAAANAGESTVHVLNFRGKPESRPFEHAEVAAEYIVNLILRTRQKS adenylatekinasefromThermotogamaritima SEQIDNO:13 MMAYLVFLGPPGAGKGTYAKRIQEKTGIPHISTGDIFRDIVKKENDELGK KIKEIMEKGELVPDELVNEVVKRRLSEKDCEKGFILDGYPRTVAQAEFLD SFLESQNKQLTAAVLFDVPEDVVVQRLTSRRICPKCGRIYNMISLPPKED ELCDDCKVKLVQRDDDKEETVRHRYKVYLEKTQPVIDYYGKKGILKRVDG TIGIDNVVAEVLKIIGWSDK adenylatekinasefromAeropyrumpernix SEQIDNO:14 MKVRHPFKVVVVTGVPGVGKTTVIKELQGLAEKEGVKLHIVNFGSFMLDT AVKLGLVEDRDKIRTLPLRRQLELQREAAKRIVAEASKALGGDGVLIIDT HALVKTVAGYWPGLPKHVLDELKPDMIAVVEASPEEVAARQARDTTRYRV DIGGVEGVKRLMENARAASIASAIQYASTVAIVENREGEAAKAAEELLRLIKNL adenylatekinasefromArchaeglobusfulgidus SEQIDNO:15 MNLIFLGPPGAGKGTQAKRVSEKYGIPQISTGDMLREAVAKGTELGKKAK EYMDKGELVPDEVVIGIVKERLQQPDCEKGFILDGFPRTLAQAEALDEML KELNKKIDAVINVVVPEEEVVKRITYRRTCRNCGAVYHLIYAPPKEDNKC DKCGGELYQRDDKEETVRERYRVYKQNTEPLIDYYRKKGILYDVDGTKD IEGVWKEIEAILEKIKS Monomericadenylatekinase(AdkE)fromPyrococcusabyssi SEQIDNO:16 MNILIFGPPGSGKSTQARRITERYGLTYIASGDIIRAEIKARTPLGIEME RYLSRGDLIPDTIVNTLIISKLRRVRENFIMDGYPRTPEQVITLENYLYD HGIKLDVAIDIYITKEESVRRISGRRICSKCGAVYHVEFNPPKVPGKCDI CGGELIQRPDDRPEIVEKRYDIYSKNMEPIIKFYQKQGIYVRIDGHGSID EVWERIRPLLDYIYNQENRR

EXAMPLE 4

(30) Analysis of the Thermostability of Recombinant Adenylate Kinases

(31) The thermal stability of recombinant tAK enzymes was assessed in crude E. coli cell lysates.

(32) Cells were grown essentially as described in Example 3 and lysed by sonication. The AK activity of the crude extract was determined both before and after heat treatment at 80 C. for 30 minutes followed by 10-fold serial dilution

(33) The results (see FIGS. 2A and 2B) demonstrate that a wide variety of recombinant enzymes are suitable for the use in the method of the invention. In one embodiment, the AKs are those from T. maritima, A. fulgidus and S. solfataricus. Such enzymes are likely to provide a greater dynamic range for the bioluminescent assay, if required, to provide still further sensitivity.

EXAMPLE 5

(34) Genetic Modification of Adenylate Kinases to Improve Stability

(35) Site-directed mutants were constructed in the AK gene from P. furiosus, P. horikoshii and S. acidocaldarius as shown in Examples 6-8 and SEQ ID NOS:17-19 respectively, using standard methods known to those familiar with the art.

(36) In addition to specific changes identified in each gene, the regions underlined in the S. acidocaldarius sequence form the core packing region of the archaeal adenylate kinase trimer structure. Hence amino acid substitutions that disturb the packing of this region are likely to have a major effect in decreasing the thermal and physical stability of the enzyme. Conversely amino acid substitutions that improve the core packing, in particular hydrophobic residues with large side chains, may stabilise the enzyme to heat or other processes. Therefore in addition to the specific mutations already described a number of selective approaches were used with localised gene shuffling of related gene sequences in these regions (essentially as described in Stemmer (1994) Nature 370:389-391 and Crameri et al. (1996) Nature Biotech. 14:315-319) and random PCR-based mutagenesis using degenerate oligonucleotides or modified nucleotide mixes (e.g., Vartanian et al. (1996) Nucleic Acid Res. 24:2627-2633). A number of these modifications show altered stability when assessed by recombinant expression in E. coli and rapid assay of adenylate kinase activity in lysed cells at high temperature.

EXAMPLE 6

(37) Adenylate Kinases from Pyrococcus furiosus Genetically Engineered to Provide Improved Stability (SEQ ID NO. 17)

(38) TABLE-US-00004 MPFVVIITGIPGVGKSTITRLALQRTKAKFRLINFGDLMF EEAVKAGLVKHRDEMRKLPL(KTOE)IQRELQMKAAKKI (TTOA)EMAKEHPILVDTHATIKTPHGY(MTOL)LG LPYEVVKTLNPNFIVIIEATPSEILGRRLRDLKRDRDVET EEQIQRHQDLNRAAAIAYAMHSNALIKIIENHEDKGLEEA VNELVKILDLAVNEYA

(39) Mutations at one or more or all of the sites indicated modify the thermostability of the enzyme. In addition to the three defined changes highlighted, modification of the alanine at position 157 to another small hydrophobic residue (such as I, L) or larger hydrophobic residue (such as F) increases the thermostability of the recombinant protein. Hence, there are 35 variants possible through combination of modifications at these sites. Modification of amino acid 157 to a polar residue such as the T (as observed at the equivalent position in AdkA of P. horikoshii), S Y, D, E, K, R results in a decrease in stability.

EXAMPLE 7

(40) Adenylate Kinases from Pyrococcus horikoshii Genetically Engineered to Provide Improved Stability (SEQ ID NO. 18)

(41) The modification of either or both of the residues shown in bold and underlined increases the thermal stability of the enzyme (3 variants are possible).

(42) TABLE-US-00005 MPFVVIITGIPGVGKSTITKLALQRTRAKFKLINFGDLMF EEALKLGLVKHRDEMRKLPLEVQRELQMNAAKKIAEMAKN YPILLDTHATIKTPHGYLLGLPYEVIKILNPNFIVIIEAT PSEILGRRLRDLKRDRDVETEEQIQRHQDLNRAAAIAYAM HSNALIKIIENHEDKGLEEAVNELVKILDLAVKEYA

EXAMPLE 8

(43) Adenylate Kinase from Sulfolobus acidocaldarius Genetically Engineered to Provide Improved Stability (SEQ ID NO. 19).

(44) The modification of the underlined residues shown can increase the thermal stability of the enzyme.

(45) TABLE-US-00006 MKIGIVTGIPGVGKSTVLAKVKEILDNQGINNKIINYGDF MLATALKLGYAKDRDEMRKLSVEKQKKLQIDAAKGIAEEA RAGGEGYLFIDTHAVIRTPSGY(ATOM)PGLPSYV ITEINPSVIFLLEADPKIILSRQKRDTTRNRNDYSDESVI LETINFARYAATASAVLAGSTVKVIVNVEGDPSIAANEIIRSMK

EXAMPLE 9

(46) Expression of Acetate and Pyruvate Kinases

(47) Following the methods of Example 3, we expressed acetate and pyruvate kinases:

(48) TABLE-US-00007 AcetatekinasefromThermatogamaritima SEQIDNO:20 MRVLVINSGSSSIKYQLIEMEGEKVLCKGIAERIGIEGSRLVHRVGDEKH VIERELPDHEEALKLILNTLVDEKLGVIKDLKEIDAVGHRVVHGGERFKE SVLVDEEVLKAIEEVSPLAPLHNPANLMGIKAAMKLLPGVPNVAVFDTAF HQTIPQKAYLYAIPYEYYEKYKIRRYGFHGTSHRYVSKRAAEILGKKLEE LKIITCHIGNGASVAAVKYGKCVDTSMGFTPLEGLVMGTRSGDLDPAIPF FIMEKEGISPQEMYDILNKKSGVYGLSKGFSSDMRDIEEAALKGDEWCKL VLEIYDYRIAKYIGAYAAAMNGVDAIVFTAGVGENSPITREDVCSYLEFL GVKLDKQKNEETIRGKEGIISTPDSRVKVLVVPTNEELMIARDTKEIVEKIGR PyruvatekinasefromPyrococcushorikoshii SEQIDNO:21 MRRMKLPSHKTKIVATIGPATNSKKMIKKLIEAGMNVARINFSHGTFEEH AKIIEMVREQSQKLDRRVAILADLPGLKIRVGEIKGGYVELERGEKVTLT TKDIEGDETTIPVEYKDFPKLVSKGDVIYLSDGYIVLRVEDVKENEVEAV VISGGKLFSRKGINIPKAYLPVEAITPRDIEIMKFAIEHGVDAIGLSFVG NVYDVLKAKSFLERNGAGDTFVIAKIERPDAVRNFNEILNAADGIMIARG DLGVEMPIEQLPILQKRLIRKANMEGKPVITATQMLVSMTMEKVPTRAEV TDVANAILDGTDAVMLSEETAVGKFPIEAVEMMARIAKVTEEYRESFGIT RMREFLEGTKRGTIKEAITRSIIDAICTIGIKFILTPTKTGRTARLISRFKPKQWILAFS TREKVCNNLMFSYGVYPFCMEEGFNENDIVRLIKGLGLVGSDDIVLMTEG KPIEKTVGTNSIKIFQIA PyruvatekinasefromSulfolobussolfataricus SEQIDNO:22 MRKTKIVATLGPSSEEKVKELAEYVDVFRINFAHGDETSHRKYFDLIRTY APESSIIVDLPGPKLRLGELKEPIEVKKGDKIVFSQKDGIPVDDELFYSA VKENSDILIADGTIRVRVKSKAKDRVEGTVIEGGILLSRKGINIPNVNLK SGITDNDLKLLKRALDLGADYIGLSFVISENDVKKVKEFVGDEAWVIAKI EKSEALKNLTNIVNESDGIMVARGDLGVETGLENLPLIQRRIVRTSRVFG KPVILATQVLTSMINSPIPTRAEIIDISNSIMQGVDSIMLSDETAIGNYP VESVRTLHNIISNVEKSVKHRPIGPLNSESDAIALAAVNASKVSKADVIV VYSRSGNSILRVSRLRPERNIIGVSPDPRLAKKFKLCYGVIPISINKKMQ SIDEIIDVSAKLMQEKIKDLKFKKIVIVGGDPKQEAGKTNFVIVKTLEQQKK PyruvatekinasefromThermotogamaritima SEQIDNO:23 MRSTKIVCTVGPRTDSYEMIEKMIDLGVNVFRINTSHGDWNEQEQKILKI KDLREKKKKPVAILIDLAGPKIRTGYLEKEFVELKEGQIFTLTTKEILGN EHIVSVNLSSLPKDVKKGDTILLSDGEIVLEVIETTDTEVKTVVKVGGKI THRRGVNVPTADLSVESITDRDREFIKLGTLHDVEFFALSFVRKPEDVLK AKEEIRKHGKEIPVISKIETKKALERLEEIIKVSDGIMVARGDLGVEIPI EEVPIVQKEIIKLSKYYSKPVIVATQILESMIENPFPTRAEVTDIANAIF DGADALLLTAETAVGKHPLEAIKVLSKVAKEAEKKLEFFRTIEYDTSDIS EAISHACWQLSESLNAKLIITPTISGSTAVRVSKYNVSQPIVALTPEEKT YYRLSLVRKVIPVLAEKCSQELEFIEKGLKKVEEMGLAEKGDLVVLTSGV PGKVGTTNTIRVLKVD PyruvatekinasefromPyrococcusfuriosus SEQIDNO:24 MRRVKLPSHKTKIVATIGPATNSRKMIKQLIKAGMNVARINFSHGSFEEH ARVIEIIREEAQKLDRRVAILADLPGLKIRVGEIKGGYVELKRGEKVILT TKDVEGDETTIPVDYKGFPNLVSKGDIIYLNDGYIVLKVENVRENEVEAV VLSGGKLFSRKGVNIPKAYLPVEAITPKDFEIMKFAIEHGVDAIGLSFVG SVYDVLKAKSFLEKNNAEDVFVIAKIERPDAVRNFDEILNAADGIMIARG DLGVEMPIEQLPILQKKLIRKANMEGKPVITATQMLVSMTTEKVPTRAEV TDVANAILDGTDAVMLSEETAIGKFPIETVEMMGKIAKVTEEYRESFGLS RIREFMEIKKGTIKEAITRSIIDAICTIDIKFILTPTRTGRTARLISRFKPKQWILAFST NERVCNNLMFSYGVYPFCLEEGFDENDIVRLIKGLGLVESDDMVLMTEGK PIEKTVGTNSIKIFQIA AcetatekinasefromMethanosarcinathermophila SEQIDNO:25 MKVLVINAGSSSLKYQLIDMTNESALAVGLCERIGIDNSIITQKKFDGKK LEKLTDLPTHKDALEEVVKALTDDEFGVIKDMGEINAVGHRVVHGGEKFT TSALYDEGVEKAIKDCFELAPLHNPPNMMGISACAEIMPGTPMVIVFDTA FHQTMPPYAYMYALPYDLYEKHGVRKYGFHGTSHKYVAERAALMLGKPAE ETKIITCHLGNGSSITAVEGGKSVETSMGFTPLEGLAMGTRCGSIDPAIV PFLMEKEGLTTREIDTLMNKKSGVLGVSGLSNDFRDLDEAASKGNRKAEL ALEIFAYKVKKFIGEYSAVLNGADAVVFTAGIGENSASIRKRILTGLDGI GIKIDDEKNKIRGQEIDISTPDAKVRVFVIPTNEELAIARETKEIVETEVKLRSSIPV

EXAMPLE 10

(49) Preparation of a Fibrin-Based Indicator Device

(50) Preparation of tAK Fusions for Cross-Linking to Fibrin.

(51) A transglutaminase substrate sequence (MNQEQVSPLGGSEQ ID NO:33) is added on to the N-terminus, the C-terminus, or both N- and C-termini, of the thermostable adenylate kinase from S. acidocaldarius encoded by a codon optimised gene clone. (The transglutaminase substrate sequence is interchangeably referred to in these Examples as fibrin, the fibrin peptide or the transglutaminase substrate). This construct is transferred as an NdeI-SalI fragment into an in-house expression vector (pMTL 1015; as described in WO 2005/123764). The expression construct is confirmed by DNA sequencing and transferred into expressions hosts BL21 or RV308 for subsequent expression.

(52) Similarly, the resynthesised tAK gene from Thermatoga maritima (SEQ ID NO:29) is fused to the transglutaminase sequence in the three orientations identified above. The cloning and preparation of the expression system is also as described above.

(53) The fusion constructs can also be expressed in other expression vector-host combinations with the addition of affinity tags for subsequent purification. Particularly useful in this context are expression vectors which add 6-histidine tags on either the N- or C-terminus of the fusion proteins, modifications which aid purification and detection but do not interfere with the intrinsic properties of the fusion proteins. Vectors for this type of modification include pET series vectors (Novagen/Merck) and pQE series vectors (Qiagen).

(54) To generate material for the indicator devices the expression strains are grown initially in 8-liter fermenters essentially under static culture conditions. In brief, the strains are prepared as seed stocks and subsequently diluted into the 8-liters of growth media (modified terrific broth containing additional glucose). The cultures are grown under standard fermentation conditions until the cultures reached an optical density (OD at 600 nm) demonstrating that they are entering stationary conditions (typically at around an OD=5). The fermenters are then held under minimally aerated conditions for up to 12 hours prior to harvesting of material by continual centrifugation.

(55) Purification of tAK Fusions.

(56) The harvested material is then purified according to the following protocol.

(57) Buffer A: 20 mM Tris-HCl; 900 mM NaCl, pH 7.5

(58) Wash Buffer: 20 mM Tris-HCl; 200 mM NaCl, pH 7.5

(59) Buffer B: 20 mM Tris-HCl; 200 mM NaCl, pH 7.5; 10 mM ATP; 10 mM AMP; 10 mM MgCl.sub.2

(60) MgAc buffer: 15 mM MgAc (1M Fluka BioChemika), pH 6.8

(61) 1. Weigh frozen cell paste (10 g) and resuspend in 3 (30 ml) volume of Buffer A, pH 7.5. 2. Sonicate on ice (12,000 khz) using 25 cycles of 30 seconds on/30 seconds off Take 1 ml sample. 3. Sonicated cell solution is centrifuged at 6,000 rpm for 30 minutes at 4 C. Supernatant carefully poured off and 1 ml sample taken. 4. Supernatant is heat treated at 80 C. in a water bath for 20 minutes. 1 ml sample taken. (This step is an optional step depending on thermal stability of the fusion proteins). 5. Heat treated solution centrifuged at 6000 rpm for 30 minutes at 4 C. Pour off supernatant and take 1 ml sample. 6. Filter the sample with 0.2 m low binding filter before loading onto column. 7. Equilibrate Blue Sepharose Fast Flow column with 5 column volumes (CVs) of Buffer A. 8. Load the sample. Wash column with wash buffer at 0.2 ml/min overnight. 9. Elute protein with 100% buffer B at a flow rate of 1 ml/min collect product in 2.5 ml fractions. 10. Once all proteins have eluted wash column with 100% buffer B at 5 ml/min for 5 CV's. 11. Re-equilibrate column with 5 CV's buffer A. 12. Rinse column with 5 CV's 20% ethanol for storage at 4 C.

(62) Optionally, additional protein purification methods are applied to yield a higher purity product. Ion exchange chromatography on either SP-Sepharose Fast Flow or Q-Sepharose Fast Flow resins is particularly effective.

(63) The samples are then analysed using a standard assay format to identify fractions containing peak adenylate kinase activity. This is confirmed by SDS-PAGE analysis using standard techniques (see FIG. 3). In brief, the assay is carried out using the following protocol: 1. Dilute the purified tAK fusion 1:1000 and 1:10,000 in Mg Ac Buffer. Add 100 l per well. 2. Treat with Apyrase (50 l/well at 2.5 units per ml stock concentration; Sigma Grade VI Apyrase from potato) and incubate for 30 minutes at 30 C., with shaking, to remove ATP. 3. Incubate plate at 70 C. for 10 minutes to denature Apyrase. 4. Add 50 l/well of ADP (275 M ADP in MgAc buffer) and seal. Incubate at 70 C. for 20 minutes. 5. Remove plate and allow to cool to room temperature for 20 minutes, warm Luciferase/Luciferin (L/L) reagent to room temperature for 20 minutes. 6. Add 200 l ATP standard to 1 or 2 empty wells per plate. 7. Set up injectors on luminometer and prime them with L/L reagent (ATP reagent, Biotherma). Inject 30 l L/L reagent/well. 8. Read light generated immediately using luminometer.

(64) The fractions with peak kinase activity are then dialysed extensively against phosphate buffered saline (PBS pH 7.4) and stored until required. Confirmation of the presence of the added transglutaminase substrate sequence (i.e. the fibrin peptide) on the purified tAK is confirmed by SELDI mass spectroscopy analysis (see FIGS. 4A and 4B).

(65) Optionally a fusion can be prepared between tAK and the full length fibrinogen molecule to provide further means to incorporate the enzymatic activity within the fibrin film.

(66) Deposition of tAK Fusions onto a Solid Support.

(67) The tAK-fibrin fusion is diluted to around 200 g/ml in either PBS or bicarbonate buffer (pH 9.6) and applied to a solid support of 316L grade stainless steel, plastic, glass or textiles. The protein is allowed to adhere to the surface for up to 2 hours at room temperature or overnight at 4 C.

(68) Optionally, additional carrier molecules are added at this stage, e.g., sucrose at concentrations up to 1% w/v, albumin at up 1 mg/ml, pig mucin at up to 0.5% w/v. The addition of such carriers may be particularly important for certain types of indicator but the presence of the carrier should not interfere with subsequent interaction and cross-linking to the fibrin film applied in the next stage.

(69) Overlay of Fibrin-Containing Soil and Cross-Linking to Fibrin-tAK Fusion.

(70) A test soil (biological matrix) is then overlaid onto the tAK-fibrin fusion preparation adhered onto the surface as described above.

(71) A solution containing fibrinogen is added to effect the cross-linking of the indicator to the fibrin-containing test soil.

(72) A solution containing up to 3 mg/ml fibrinogen (containing Factor XIII), 2.5 mM CaCl.sub.2, and thrombin (up to 5 NIH units per ml) is mixed freshly and added to the coated surface of the solid support. The reaction is allowed to proceed at room temperature for up to 30 minutes, depending on the level of cross-linking required. Optionally, albumin (up to 80 mg/ml) and haemoglobin (up to 80 mg/ml) are added at this stage to provide a tougher and more realistic challenge for cleaning of a blood-like soil. After cross-linking, residual liquid is removed, and the indicator device is left to dry.

(73) FIG. 9 shows SDS-PAGE analysis of the covalent attachment of a tAK-fibrin fusion protein with fibrinogen to form a tAK-fibrin film.

(74) Optionally, the tAK-fibrin peptide fusion is added to the fibrin-containing test soil solution (biological matrix) prior its addition to the solid support surface. Cross-linking of the fibrin peptide to the matrix can be increased by adding more Factor XIII and/or extending the duration of the reaction. Cross-linking can also be enhanced by the use of the tAK fusion protein with fibrin peptides added to both ends of the molecule.

(75) Optionally a fibrinogen-tAK fusion could be added directly to this solution to provide further cross linkage of the indicator.

(76) Covalent Chemical Cross-Linking of tAK to Fibrin or Fibrinogen.

(77) The preparation of tAK-fibrin as a fusion protein has already been described above. However, tAK-fibrin may also be prepared by chemically joining tAK to fibrin, fibrin peptides or fibrinogen by a wide range of methods familiar to those working in the field. For example purified protein preparations for fibrinogen or fibrin are obtained from commercial sources (e.g., Sigma). The tAK from S. acidocaldarius is prepared as described above. The tAK is derivatised using the amide reactive reagent SPDP (SPDP (N-Succinimidyl 3-(2-pyridyldithio)-propionate; Pierce chemical company) according to the maufacturer's instructions. The fibrin or fibrinogen is also derivatised using the same protocol. The derivatised tAK is reduced by reaction with mercaptoethanol to yield a reactive sulfhydryl group. This is then mixed with the SPDP-derivatised fibrin causing the formation of covalent bonds between the two molecules. The concentrations of the reaction partners should be determined empirically following the guidelines within the manufacturer's instructions for SPDP.

(78) The chemically linked tAK-fibrin or fibrinogen can be used interchangeably or in addition to the fusion protein throughout the preceding description of this and subsequent examples.

EXAMPLE 11

(79) Uses of Fibrin-tAK Indicators

(80) Use in a Washer Disinfector.

(81) An indicator is prepared as described in Example 10. The solid support is a rectangular stainless steel strip 55 mm5 mm0.75 mm, which may be coated on one or both surfaces. One or several indicator strips are positioned within the chamber of the washer disinfector. Optimally, these may be positioned in sites which may be the most difficult to clean, providing the highest degree of certainty that the wash process has been effective. Alternatively, they may be positioned to monitor the function of multiple spray arms (i.e. where these may be independent of each other). The indicator strips are clipped to the shelves or other substructure of the washer-disinfector chamber to ensure that they do not move during the wash treatment. The orientation of the surrogate devices can be modified to provide further information about the efficacy of the wash process, for example by positioning them so that the coated surface are at right angles to the direction of water spray.

(82) The instrument load is added and the standard run cycle performed. At the end of the run the devices are removed from the chamber and the presence of residual tAK-fusion assessed, as outlined in Example 12, prior to the removal of the instruments and any subsequent processing. Optionally, devices can be removed during the wash process either by interrupting the process at carefully defined points or by using a machine that provides a method of withdrawing the indicator during the run.

(83) Use in Endoscope Test Procedure.

(84) The indicator device for monitoring an endoscope reprocessing system is essentially similar to that outlined above. A similar size indicator surface, representative of either the stainless steel components within an endoscope, the PTFE tubing or other relevant materials is placed within a tubular chamber. This is attached, via suitable screw, push or bayonet fittings to either the front end of the endoscope or the end which makes contact with patient tissues. This is placed within the endoscope reprocessing unit and the ends of the endoscope tubing and indicator device are coupled to the ports in the unit. The process is run as standard and the indicator device removed at the end of the run for analysis, prior to onward processing or the return of the endoscope to use.

EXAMPLE 12

(85) Means of Assessing Cleaning Performance

(86) The indicator device is removed at the end of the test process. The indicator is inserted into a hygiene monitor or luminometer reagent tube and processed according to the manufacturer's instructions, with the indicator device replacing the swab. The hand-held hygiene monitor provides a read-out in relative light units (RLUs) which is directly proportional to the concentration of ATP generated by the tAK-enzyme attached to the biological component. This in turn is directly proportional to the concentration of enzyme over the concentration range typically used for the indicator. The indicator devices are calibrated such that an RLU value below a pre-determined threshold value is indicative of at least a 3-log reduction (or potentially higher depending on the acceptance criteria) in the concentration of the tAK fusion which remains attached to the indicator surface. The batch release of processed instruments is based upon a reduction in the RLU value generated by the residual tAK fusion on the indicator device. This can be identified by either training operatives to return all batches of instruments above the threshold value or by calibrating the hygiene monitor to provide a simple pass or fail read-out based on the RLU value.

(87) The practical process for allowing onward processing of batches of surgical instruments or other processed products is as follows: 1. Insert indicator devices into pre-set positions within the chamber of the washer disinfector. Clip in place. 2. Add instrument load according to standard operating procedure. Close door and press run button 3. During the run, record any process parameters required by the standard operating procedure. 4. At end of the run record the time and any process parameters required by the standard operating procedure. 5. Switch on the hand-held hygiene monitor (SYSTEMSURE PLUS; Hygiena) and allow to calibrate. 6. Remove the indicator devices from the chamber and insert them into the reagent tube (ULTRASNAP; Hygiena). 7. Bend the reagent reservoir from side to side to expel all of the reagent down the sample tube (according to the manufacturer's instructions. 8. Shake the tube for 5 seconds. 9. Insert the tube into the hygiene monitor and record signal immediately. 10. Record the RLU value or Pass/Fail read out on the process run sheet. 11. Repeat steps 6-10 for any further indicator devices. 12. If any fails are observed, re-process the instruments starting at step 1. 13. At the end of each day download the results to a suitable data logger or computer terminal via the port attached. 14. Weekly and monthly check the Pass/Fail rate and record any trends in process fails (day of week, time of day, position within chamber, operator)

(88) This is an example of a suitable protocol, but a number of different reagent tubes or instruments (such as those prepared by BioTrace, Charm or other companies) would be suitable to enable such instrument release protocols.

EXAMPLE 13

(89) Preparation of tAK-Sup35 Fusion.

(90) Clones containing the N-terminal domain of Sup35 from Saccharomyces cerevisae fused to either the N- or C-terminus, or both termini, of adenylate kinases from either S. acidocaldarius or T. maraima are generated by standard DNA manipulation techniques. All clones are transferred as NdeI-SalI fragments into the pMTL1015 expression vector and their sequences verified. The expression constructs are used to transform BL21 or RV308 expression strains and the material grown in large scale fermentation conditions, but with minimal aeration.

(91) Expression and purification of a tAK-Sup35 fusion is essentially the same as for the fibrin-peptide fusions described in Example 10, except that the use of the thermal denaturation step (Step 4) is not part of the purification protocol. In brief, cell paste from the fermenter is resuspended in buffer A, and lysed by sonication. The cell debris is removed (no heat treatment is used standardly for these type of fusions) and the supernatant used for column purification as outlined in Example 10.

(92) Under certain growth conditions the fusion proteins may be insoluble, being apparent as inclusion bodies within the cells. In this case the cell pellets are prepared and lysed in the same way, but the resulting insoluble fraction, containing the inclusion bodies, is collected by centrifugation. This material is washed in a buffer (e.g., PBS) containing Triton X100 (up to concentrations of 5%). After each wash the pellet containing the fusion proteins is separated by centrifugation. After 5 washes the inclusion bodies are resolubilised in PBS containing 8M urea and agitated gently for up to 30 minutes. Any residual insoluble material is removed by centrifugation. The urea-solubilised material is dialysed against up to 510 volumes of PBS to remove the urea and allow the fusion proteins to refold. Optionally the urea may be removed more rapidly by spraying the urea-solubilised preparation through a fine gauge needle into 100 volumes of rapidly stirred PBS or buffer A as used for purification. The material is allowed to stand at room temperature with stirring for up to 30 minutes prior to subsequent processing. FIG. 5 shows a gel electrophoretic analysis (SDS-PAGE) of solubilised and refolded Sup35-tAK (Sac) from clarified inclusion body preparations.

(93) Subsequent purification of the fusions is carried out essentially as described in Example 10. The supernatant from either lysed cells or solubilised and refolded inclusion bodies is loaded onto a pre-equilibrated Blue Sepharose Fast Flow column. After extensive washing in buffer A and subsequently in wash buffer, the protein is eluted using buffer B. Peak fractions are determined by SDS-PAGE analysis and enzyme assay. Fractions are then pooled and dialysed into PBS.

(94) Conversion of tAK-Sup35 to an Amyloid Form.

(95) The Sup35-tAK fusions when assembled into fibrils are more representative of amyloid proteins such as prions which are key molecules against which to assess the efficacy of decontamination processes.

(96) The amyloid form of the Sup35-tAK fusions is generated by either refolding of the purified soluble protein or by modifying the conditions used for dialysis of the urea-resolubilised inclusion body preparations. In the first case, a conformational change is induced by exposure of the fusion proteins to conditions around pH 4 (e.g by dialysis into a suitably buffered solution at pH 7.4 optionally containing up to 1M NaCl). In the latter case, the resolubilised fusion proteins in 8M urea/PBS are dialysed for 6-12 hours at room temperature against 2M urea, 300 mM NaCl, in PBS (pH 7.4). Alternatively, the fibrilisation can be induced by dialysis against 20 mM Tris pH8.0 10 mM EDTA under similar incubation conditions. Electron microscopy is used to confirm the presence of fibrils (see FIG. 6).

(97) Optionally, the fusion proteins may be incorporated into fibrils containing normal Sup35. This is achieved by mixing the fusions with unfused Sup35 expressed in the same way, at ratios between 1:1 to 1:10 fusion:Sup35.

(98) Deposition of tAK-Sup35 Fusions onto Solid Support.

(99) Deposition of the fibrils onto a solid support is effected by simple protein adsorption in a suitable buffer (e.g., PBS pH 7.4 Bicarbonate buffer pH 9.6) in the presence of high levels of NaCl. The use of charged or precoated surfaces (e.g., plastics coated with Poly-L-lysine) is useful in providing surfaces which can more effectively bind the fusion proteins.

(100) Optionally, the fibrils may be deposited in a suitable carrier, such as sucrose (to 1%), pig mucin (up to 0.5%), or albumin (up to 1 mg/ml).

(101) Overlay of Test Soil

(102) A test soil (biological matrix) is then overlaid onto the amyloid preparation adhered onto the surface as described above.

(103) Suitable biological matrices in which the amyloid indicator is embedded include e.g., 0.5% mucin, with or without albumin, a commercial test soil (such as that manufactured by Browne's) or any one of the test soils identified in guidance documents issues by national and international standards committees (e.g., Edinburgh soil as detailed in HTM 01/01 (UK).

(104) Assembly of Amyloid Fibrils within the Test Soil

(105) Given the ability of amyloids to self-assemble in complex matrices it is possible for the amyloid-tAK fusion to be mixed with soil components prior to fibril formation and subsequent deposit onto surfaces. This provides further options for indicators in which the amyloid fibrils may be mixed and inter-chelated with other soil components providing a different type of matrix that may be harder to remove from surfaces.

EXAMPLE 14

(106) Uses of tAK-Sup35 Indicator

(107) Use of tAK-Sup35 Indicator for Assessing Prion Removal from Surfaces in a Washing Process.

(108) An indicator as described in Example 13 is prepared as fibrils and dried down onto a steel surface in the presence of 0.5% mucin. The indicator is placed within the chamber of a washer disinfector at pre-determined locations. The instrument load is added. The process is started as per the manufacturer's instructions and any process records completed. At the end of the process, and before any instruments are taken from the machine, the indicator devices are removed and assessed as described in Example 12.

(109) Use of tAK-Sup35 Indicator for Assessing Prion Inactivation in a Protease-Based Process.

(110) Indicators as described in Example 13 are prepared as fibrils with a high ratio of free Sup35:Sup35-tAK (in excess of 5:1) and deposited onto solid support strips in the presence of Edinburgh soil. The indicator devices are inserted into a pre-soak bath containing freshly made PRIONZYME (Genencor International) prion inactivation treatment (at 60 C., pH 12). The indicator strips are clipped to the side of the bath such that the ends of the indicators are within the bulk of the liquid. Instruments are added as required and processed for 30 minutes. The indicator devices are removed from the bath at the end of the process, prior to removal of the instruments and assessed as described in Example 12.

(111) Use of tAK-Sup35 Indicator for an Oxidative Process Aimed at Destroying Prions.

(112) An indicator as described in Example 13 is prepared as fibrils using only Sup35-tAK, and deposited onto a stainless steel surface (optionally in the presence of 0.1% w/v sucrose). The indicator is attached to the inside of the lid of a GENESIS container in which the instruments are prepared for processing and the lid closed. The container is inserted into the load chamber of a suitable processor for oxidative challenge (e.g., the 125 L ozone steriliser; TSO.sub.3 or a vapour phase hydrogen peroxide technology such as that described in published papers by Fichet et al. 2004; Lancet) and the process run according to manufacturers' instructions. At the end of the process, the GENESIS container is taken out of the chamber and the indicator devices are removed and processed as described in Example 12.

EXAMPLE 15

(113) Preparation of a Neurological Soil with tAK-Coupled Components

(114) Identification of Components Essential for a Neurological Test Soil.

(115) The critical target components encountered with neurosurgical processes that may remain attached to surgical instrument surfaces can be determined in experimental studies. Surgical instruments from neurosurgical wards may be treated in routine cleaning processes. Residual protein or other biological molecules can be solubilised from the surface of the instrument using partial acid hydrolysis, strong alkaline cleaners or the use of suitable lytic enzymes (e.g., proteases, nucleases, lipases). The principle molecules can then be determined using mass spectrometry techniques such as Surface-enhanced laser desorption ionisation (SELDI) or equivalents.

(116) The same analysis may be achieved by artificially soiling surgical instruments with human neurological material or equivalent samples from animal species (e.g., rodent brain homogenate).

(117) A representative neurological test soil will require a variety of components that include, inter alia, nerve derived proteins (e.g., neurofilament), lipids or glycolipids representative of neuronal environments (e.g., sialic-rich gangliosides) and carbohydrates. If the test soil is designed to address particular issues regarding specific diseases, such as diseases caused by protein aggregation (e.g., prion disease, Alzheimer's disease) then these components, or surrogates thereof, will also be valuable additions to the test soil.

(118) Cross-Linking of tAK-Fusions to Protein or Glycolipid.

(119) A recombinant tAK fusion can be made to neurofilament proteins or sub-domains thereof by using the methods essentially as described in Examples 10 and 13.

(120) In addition, cross-linking can be achieved without the need to generate recombinant expression clones. This may be particularly useful where heavily glycosylated proteins or glycolipids are linked to the tAK. In this case the protein or glycolipid is purified either from a suitable source or generated by expressing a suitable gene construct in a eukaryotic cell line (e.g., mammalian cell, baculovirus expression system). Purification may be via one of a variety of well known protein purification methods or by detergent solubilisation of membrane lipids. The purified material is then cross-linked to purified tAK using one of a wide range of coupling chemistries (e.g., SPDP (Pierce chemicals) used to link proteins via primary amines on proteins; treatment with meta-periodate used to open up carbohydrate groups allowing cross linking to glycolipids). Further suitable methods for effecting cross-linking of the tAK with carbohydrate or lipid-containing molecules are described in detail in Example 23

(121) Of particular use in this application is the covalent coupling of the tAK to biological components such as gangliosides, including those specific for neuronal cells (e.g., GT.sub.1b, GT.sub.1a) and those of general cell origin (e.g., GM.sub.1). Gangliosides are purified by standard methods involving detergent solubilisation, phase partitioning and differential centrifugation. Alternatively the indicator can be formulated using commercially available purified gangliosides.

(122) Conjugation of tAK to Nucleic Acid Components of Neurological Test Soil.

(123) The tAK is cross-linked to a suitable nucleic acid, either purified, generated synthetically or amplified from a template using PCR or similar techniques. The cross-linking can be achieved by incorporating a biotin label onto the nucleic acid, e.g., during synthesis and using a tAK-cross-linked to streptavidin.

(124) Deposition of Test Soil Components onto Solid Support.

(125) The deposition of one or more tAK indicators onto a solid support can be achieved as described in Examples 10 & 13. In brief, the tAK complexes are prepared in PBS or bicarbonate buffer (pH 9.6) and allowed to dry on a polycarbonate surface for 30 minutes at room temperature. Optionally, sucrose may be added up to concentrations of 1% w/v. The binding conditions are designed to favour attachment via the biological component rather than the tAK, for example by blocking remaining active groups on the surface of the tAK using a suitable surface modifying agent or by incorporation of high levels of NaCl.

(126) Optionally, the deposited neurological soil may be fixed by treatment with 70% ethanol or isopropanol. To achieve this, the indicator is incubated in 70% isopropanol for 30 minutes at room temperature. This mimics one of the commonly encountered processes which may increase the resistance of contaminating materials on surgical instruments, and therefore provides the indicator with an effective way of monitoring the removal of such materials.

EXAMPLE 16

(127) Preparation of Norovirus Capsid Protein (58 kDa)tAK Fusions

(128) A gene encoding a 58 kDa capsid protein from norovirus is generated as a synthetic construct. This clone is cloned onto the 5 end of the gene encoding the thermostable adenylate kinase from Thermotoga maritima to generate a single fusion protein. After sequence verification this clone is transferred into either a pMTL vector for expression in E. coli or into a baculovirus expression system (e.g., BacPAK expression system; Clontech) for expression in insect cell lines such as SF9 cells.

(129) Expression and Purification.

(130) Expression of the capsid protein-thermostable kinase fusions in E. coli is carried out essentially as described in the previous examples. The proteins are purified using a similar protocol on Blue Sepharose Fast Flow with no thermal denaturation step applied during the cell lysis protocol. Purified proteins are analysed by SDS-PAGE analysis and by enzymatic assay as described in the previous examples. The assembly of the fusion proteins into virus-like particles (VLPs) is promoted by altering the pH and salt concentration.

(131) Baculovirus expression and subsequent purification is carried out essentially as described in Jiang et al., (1992) Expression, self-assembly and antigenicity of the norwalk virus capsid protein, J Virology, 66, page 6527-6352.

(132) Deposition on Solid Supports.

(133) The purified VLP-tAKs are deposited onto a solid support suitable for validating clean-down and disinfection of surfaces used in food preparation or following outbreaks of norovirus in hospital settings.

(134) For validating the decontamination of food preparation surfaces, VLP-tAKs are prepared in a PBS buffer containing a crude food extract comprising egg albumin and sucrose. This matrix is coated onto a polyvinyl strip measuring 5 cm5 cm and allowed to dry for either 2 hours at room temperature or overnight at 4 C.

(135) For assessing the decontamination of a healthcare facility potentially contaminated following an outbreak of norovirus, the VLP-thermostable kinase indicators are made up in a preparation designed to mimic healthcare-related soiling. This included various blood-related proteins as described above, or one of the standard bed-pan soils enshrined in national and international decontamination standards. Particularly efficacious indicators are set up using textile solid supports representative of, for example, hospital curtains or gowns.

(136) Use of Norovirus VLP-tAK Fusions for Validation of Viral Removal Processes.

(137) The norovirus VLP-tAK fusions are particularly advantageous for validating the ability of processes to remove norovirus from water or food samples. They retain the size, charge and hydrophobicity properties of the parent virus and as such will mimic their behaviour in removal processes. This is particularly useful in this case as no culture method exists for norovirus and as such it is currently not possible to measure live virus clearance other than by RT-PCR, a potentially time consuming method.

(138) For example, the norovirus VLP-tAK fusions are put into a water source and filtered through a process designed to remove viral particles. Sufficient viruses are input to measure the required level of viral clearance. The numbers of VLPs post filtration are measured and assessed against the pre-determined pass/fail criteria.

EXAMPLE 17

(139) Generation of Bacteriophage MS2 Coat ProteintAK Fusions

(140) The generation of MS2 coat proteins and their spontaneous assembly into virus like particles has been described in a number of studies, for example in Peabody (2003), A viral platform for chemical modification and multivalent display, J Nanobiotechnology, vol 1, page 5.

(141) The protein sequence of MS2 coat protein capable of generating VLP when expressed in E. coli is given below (SEQ ID NO:62):

(142) TABLE-US-00008 MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRS QAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPV AAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIP SAIAANSGIY

(143) Constructs of MS2 coat proteins are generated with the tAK from Thermatoga maritima, fused at either the N- or C-terminus of the expressed protein. Depending on the location of the fusion this results in the incorporation of the tAK either within the lumen of the VLP or exposed on the surface. The two locations both have useful properties for their application as indicators. Optionally the MS2 coat protein may be modified by inclusion of a cysteine residue at position 15 in the native sequence (substitution threonine15 to cysteine). The VLP-tAK fusions are purified using a combination of the methods described for tAK fusions above (Examples 10 and 13) with additional ion exchange steps if required. The intact VLPs incorporating the tAK can also be purified on the basis of size exclusion using a Sepharose CL-4B column.

(144) Alternatively, purified tAK can be cross-linked to MS2 VLPs using chemical cross-linking reagents. In brief, tAK from S. acidocaldarius is derivatised with SPDP and reduced to yield a reactive sulfhydryl group. This is then mixed with the MS2 VLPs containing the T15C variant of the protein. This effects covalent disulfide bonds between the two partners. These types of covalently linked molecules can be used interchangeably with the genetic fusions throughout the remainder of these examples.

(145) Deposition of MS2 Coat ProteintAK Fusions on Solid Supports.

(146) The purified tAK-containing MS2-VLPs are deposited on surfaces in a similar way to the fusions described in the previous examples using standard protein adsorption techniques. Optionally, highly charged or hydrophobic surfaces may be used to provide an indication of viral removal from specific surfaces within process regimes.

(147) The VLP-tAK fusion may be deposited alone or may be contained within a suitable soil matrix designed to represent the relevant soiling encountered during the treatment process to be validated. For example, a bed-pan soil may be used for evaluation or validation of the removal of faecal material from either bed-pans, toilets or during diarrhoeal episodes.

(148) Use of MS2-tAK Fusions for Validating a Cleaning Regime.

(149) The MS2-VLP indicator is set up on a ceramic surface as described above. The ceramic indicator is exposed to the same cleaning chemistry as the bathroom surfaces to be cleaned, e.g., to diluted sodium hypochlorite at a dilution of approximately 2.5% (v:v), and under the same conditions (30 minutes at ambient temperature). At the end of the process the ceramic indicator is inserted into a hygiene monitor tube and the residual MS2-tAK measured using the method of Example 12. If cleaned below a pre-set threshold then the cleaning regime is deemed to have been successful. If not then repeat cleaning is required to minimise any risk of disease transmission.

(150) Use of MS2-tAK Fusions for Validating a Viral Removal Process.

(151) As the MS2-tAK VLPs mimic the size, surface charge and hydrophobicity of the parent virus and are capable of representing a wide variety of related viruses (e.g., polio virus), these indicators are extremely useful for validating viral removal processes in either a laboratory or field setting. The rapidity of the tAK assay provides significant advantages over traditional culture-based methods.

(152) For example, a water treatment system may be validated in situ using the MS2-tAK VLPs. Sufficient MS2-tAK VLPs are put into the input water in the treatment plant to provide a sufficient log clearance estimation for the efficacy of the process, as determined by national or local regulations. For example, 10.sup.9 VLP-tAKs per liter are put into the input water. The process is performed and approximately 1 ml samples of the post-process water is tested in a suitable hygiene monitor tube system (e.g., AQUA-TRACE, Biotrace UK). A value indicating less than 1 VLP-tAK per ml of water would be sufficient to demonstrate a 6-log clearance of viruses by the process employed. This could be done within 2 minutes of the process being completed rather than 16-24 hours that would be required for a standard culture-based method in E. coli.

(153) Such methods are relevant for validating a wide range of viral inactivation processes used widely in the healthcare, food, water or pharmaceutical industries. In the vast majority of cases it can replace the use of the parent MS2 bacteriophage, used widely to validate viral-removal processes, providing far more rapid and sensitive determination of removal. For example, water purification through ceramic microfilters (replacing the parent bacteriophage in Wegmann et al., 2007, Modification of ceramic microfilters with colloidal zirconia to promote the adsorption of viruses from water), treatment of water with gaseous chlorine (Clevenger et al., 2007, Comparison of the inactivation of Bacillus subtilis spores and MS2 bacteriophage by MIOX, ClorTec and hypochlorite, J Applied Microbiology, 103, p 2285-2290), validation of virucidal efficacy of hand washing (Jones et al., 1991, The use of bacteriophage MS2 as a model system to evaluate virucidal hand disinfectants, J Hospital Infection, 17, p 279-285). Other examples would be to validate, in process, the removal of virus particles from fractionated blood, cellular extract of human or animal origin, pharmaceutical products, food preparation (e.g., shell-fish extracts).

EXAMPLE 18

(154) Preparation of Further Kinase-VLP Fusions Suitable for Evaluating and Validating Viral Removal or Destruction

(155) The table below lists a series of VLP fusion proteins that are valuable in the development of indicators for assessing removal or inactivation of a range of viral pathogens. These represent either actual pathogens where the validation of removal may be essential, or model viruses capable of representing the removal of a range of related pathogens. The pathogens are from both the medical and veterinary field, and also encompass a range of known or possible zoonotic pathogens which may transmit from animals to humans.

(156) TABLE-US-00009 TABLE 2 Suitable biological components for preparing kinase-VLP fusions for evaluating and validating viral removal or destruction Recombinant Virus fragment Expression system Reference Bacteriophage, MS2 coat protein E. coli (pET3d Peabody et al., 2003, e.g., MS2, PP7 PP7 coat protein expression vector) Journal of Nanobiotechnology, 1, p5 Norwalk Capsid Baculovirus Reviewed in Grgacic and (norovirus) Anderson, 2006, Methods, 40, p60-65 Rotavirus VP2, VP6 and VP7 Baculovirus Reviewed in Grgacic and Anderson, 2006 SARS S, E and M Baculovirus Reviewed in Grgacic and (coronavirus) Anderson, 2006 Bluetongue VP2 Baculovirus Roy et al., 1994, Vaccine, 12, p805-811 Human viral major 293TT cells Buck et al., 2005, J papillomavirus structural protein, Virology, 79, p2839; L1 Buck et al., 2004, J. Virology, 78, p751 Hepatitis B Small envelope Yeast or mammalian Reviewed in Grgacic and protein (HBsAg) cells Anderson, 2006 Hepatitis C Core, E1 and E2 Baculovirus, yeast Reviewed in Grgacic and and mammalian Anderson, 2006 cells Influenza; both Haemagglutinin, Baculovirus, Reviewed in Grgacic and human strains neuraminidase and mammalian cells Anderson, 2006 (e.g., H5N1) matrix (M1 and avian optional) influenza strains. Poliovirus Capsid (VP0,1 and Baculovirus Reviewed in Grgacic and 3) Anderson, 2006 HIV Pr55gag, envelope Baculovirus, yeast Reviewed in Grgacic and and mammalian Anderson cells Dengue B Envelope (e) and Mammalian cells Purdy and Chang, 2005, premembrane/ Virology, 333, p239-250 membrane (prM/M)

(157) The protein sequence of bacteriophage PP7 coat protein monomer and dimer (Caldeira and Peabody, 2007, Journal of Nanobiotechnology, 5, p 10) is given below. Thermostable kinase genes may be fused to the C-terminus of either the monomer or dimer.

(158) TABLE-US-00010 PP7monomer (SEQIDNO:63) SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGA KTAYRVNLKLDQADVVDCSTSVCGELPKVRYTQVWSHDVTIVANSTEAS RKSLYDLTKSLVVQATSEDLVVNLVPLGR PP7dimer (SEQIDNO:64) MSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNG AKTAYRVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLY DLTKSLVATSQVEDLVVNLVPLGRYGSKTIVLSVGEATRTLTEIQSTAD RQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDSGLPKV RYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR

EXAMPLE 19

(159) Expression of KinaseBacterial Fimbriae Fusions for Use in Development of Indicators to Assess Biofilm Removal from Surfaces

(160) A fusion between the tAK from Thermotoga maritima and the CsgA protein from E. coli is generated by standard recombinant cloning familiar to those with knowledge of the art. The protein sequence generated is shown below.

(161) TABLE-US-00011 SequenceofE.coliCsgAprotein (SEQIDNO:65) MKLLKVAAIAAIVFSGSALAGVVPQYGGGGNHGGGGNNSGPNSELNIYQY GGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFGNS ATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATAHQ Y SequenceofadenylatekinasefromThermotoga maritimafusedtotheN-terminusoftheCsgA protein (SEQIDNO:67) MMAYLVFLGPPGAGKGTYAKRIQEKTGIPHISTGDIFRDIVKKENDELGK KIKEIMEKGELVPDELVNEVVKRRLSEKDCEKGFILDGYPRTVAQAEFLD SFLESQNKQLTAAVLFDVPEDVVVQRLTSRRICPKCGRIYNMISLPPKED ELCDDCKVKLVQRDDDKEETVRHRYKVYLEKTQPVIDYYGKKGILKRVDG TIGIDNVVAEVLKIIGWSDKGSGVVPQYGGGGNHGGGGNNSGPNSELNIY QYGGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFG NSATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATA HQY

(162) For expression the clone is transferred to a suitable expression vector such as pET 32a (Novagen) or pMAL-C2x (New England Biolabs) and the protein expressed in a suitable host strain (e.g., BL21) under normal growth conditions.

(163) Depending on the growth conditions the thermostable kinase-CsgA fusion may be expressed either solubly within the cytoplasm of the cells or as an insoluble inclusion body within the cell. In the former case purification is carried out as described in Examples 10 and 13. In the latter, inclusion bodies are isolated by centrifugation following cell lysis and washed extensively in buffer containing 1% Triton X100. Inclusion bodies are solubilised by suspension in 8M Urea or 6 M guanidine hydrochloride and then refolded by rapid dialysis in very low salt buffer (typically less than 20 mM NaCl).

(164) The generation of self assembled layers is mediated by incubating the purified and enzymatically active fusion protein with a hydrophobic surface (e.g., Teflon) in 10 mM Tris pH8. For hydrophilic surfaces such as stainless steel or glass the fusion is incubated in 50 mM Sodium acetate buffer pH4, optionally in the presence of 0.1& Tween 20. Elevated temperatures up to 80 C. may be used to enhance binding or to ensure uniform coverage of surfaces.

(165) Fusions with the equivalent protein sequences from other Gram-positive of Gram-negative organisms (e.g., AgfA from Salmonella species) can also be used.

(166) TABLE-US-00012 ProteinsequenceofSalmonellaAgfAprotein (SEQIDNO:66) MKLLKVAAFAAIVVSGSALAGVVPQWGGGGNHNGGGNSSGPDSTLSIYQY GSANAALALQSDARKSETTITQSGYGNGADVGQGADNSTIELTQNGFRNN ATIDQWNAKNSDITVGQYGGNNAALVNQTASDSSVMVRQVGFGNNATANQ Y

EXAMPLE 20

(167) Further Self-Assembling Peptides and Proteins for Generation of Biofilms

(168) The generation of further indicator devices containing tAK fusions with peptides capable of self-assembling into fibrils, or surface reactive biofilms is also provided. A list of suitable fusion partners is shown in the table below.

(169) TABLE-US-00013 TABLE 3 Suitable self-aggregating/assembling peptides and proteins for generation of biofilms Self aggregating proteins and Recombinant Expression peptides protein system Reference Sup 35 Sup 35 N-terminal E. coli, yeast Reviewed in Harrison domain or Sup35 et al., 2007, Rev peptide Physiol Biochem Pharmacol, 159, p1-77 Het S, Ure2, Rnq1, Native sequence or E. coli, yeast Reviewed in Harrison New 1 fragments thereof et al., 2007; Derkatch et al., 2007, Proc Natl Acad Sci USA, 101, p12934-12939 Beta amyloid A 1-32 E. coli, yeast; Reviewed in Harrison (Alzheimer's synthetic et al., 2007 disease) peptide Barnacle cement e.g 19 kDa protein E. coli, yeast Urushida et al., 2007, proteins from B. albicostatus; or baculovirus FEBS J., 274, p4336-4346; 20 kDa Nakano et al., protein from 2007, Megabalanus rosa; Biomacromolecules, 8, novel calcite p1830-1835; Kamino, dependent protein 2001, Biochem J., 356, from B. albicostatus p503-5077. Apolipoprotein A1 Residues 1-93 of E. coli, yeast, Andreola et al., 2003, J as an e.g., of ApoAI mammalian Biol Chem, 278, p2444 apolipoprotein cells disorders Tau (associated Proteins or peptides E. coli, yeast Reviewed in Harrison with alzheimer's containing residues or mammalian et al., 2007 disease) 306-311 cells (VQIVYK) Polyadenine Peptides containing E. coli, yeast Reviewed in Harrison binding protein 2 residues 2-11 or mammalian et al., 2007 (AAAAAAAAAA) cells Lung surfactant Peptides containing E. coli, yeast Reviewed in Harrison protein C residues 9-22 or mammalian et al., 2007 (VVVVVVVLVV cells VVIV) CgsA subunit Native sequence E. coli Gebbink et al., 2005, (adhesion to CsgA catalysed by Nat Rev Microbiol, 3, surfaces and CsGB sequence p333; Hammer et al., biofilm formation 2007 in E. coli) AgfA ((adhesion to Native sequence E. coli Reviewed in Harrison surfaces, cell-cell et al., 2007, Rev interactions and Physiol Biochem biofilm formation Pharmacol, 159, p1-77 in Salmonella spp) Amyloid forming Various native E. coli Described in Larsen et cell surface sequences al., 2008, Appl Env adhesins from floc Microbiol. On line forming and citation (Appl. Environ. filamentous Microbiol. bacteria in doi: 10.1128/AEM.0227 activated sludge 4-07v1) Herpes simplex Peptides containing E. coli or Cribbs et al., 2000, virus glycoprotein amino acids 22-42 mammalian Biochemistry, 39, B (gB) cells p5988-5994 Hydrophobins Native sequences or E. coli, yeast Gebbink et al., 2005; (from various derivative peptides or Pichia Sunde et al., 2007, fungal species e.g., containing the core pastoris Micron e-pub SC3 from 8-cysteine domain Schizophyllum of the hydrophobin. commune, RodA/B from Aspergillus fumigatus) Chaplins/Rodlins Chaplin proteins E. coli, yeast Gebbink et al., 2005 (Streptomyces spp) ChpA,B,C,D,E,F,G,H or pichia Rodlin proteins pastoris RdlA and RdlB and combinations thereof Gram positive P29a, P29b, GP85, E. coli, Walker et al., 2007, spore coat proteins and a SpoVM Clostridia Mol Micro., 63, p629-643 (e.g similar in analogue sequence or overall structure to those forming ribbon- appendages in Clostridium taeniosporum)

EXAMPLE 21

(170) Indicators for Monitoring Efficacy of Contact Lens Cleaning for Removal of Biofilms

(171) A range of bacteria and viruses pose a potential risk to contact lens wearers both in planktonic and biofilm forms. Indicator devices can be advantageously generated to monitor the effectiveness of cleaning methods for the removal of such organisms.

(172) The fimbriae fusions described above have can provide an indication of the removal of Gram-negative pathogens. Any member of the hydrophobin gene family is a suitable indicator fusion for the removal of fungal pathogens where these highly conserved molecules are the principle mediators of attachment. Hydrophobin genes, or equivalents from Fusarium species and Candida albicans, are suitable as these organisms represent one of the greatest threats for eye infection leading to ulceration and long term damage.

(173) Fusion proteins can be generated with any of these molecules and formulated within suitable films as described in the previous examples. These indicators can be incorporated as part of the wash chamber, in which the re-useable contact lens is to be cleaned. The process is performed for the appropriate length of time and the lens removed. The indicator is removed and the presence of active fusion protein assessed using a hygiene monitor in the usual way. If below the pre-set thresholds the contact lens is suitable for re-use. If above (failed then the contact lens must be re-processed or destroyed.

(174) TABLE-US-00014 Proteinsequenceofthehydrophobin3proteinfrom Fusariumspecies (SEQIDNO:68) MQFSTLTTVFALVAAAVAAPHGSSGGNNPVCSAQNNQVCCNGLLSCAVQV LGSNCNGNAYCCNTEAPTGTLINVALLNCVKLL Proteinsequenceofthehydrophobin5proteinfrom Fusariumspecies (SEQIDNO:69) MKFSLAAVALLGAVVSALPANEKRQAYIPCSGLYGTSQCCATDVLGVADL DCGNPPSSPTDADNFSAVCAEIGQRARCCVLPILDQGILCNTPTGVQD

EXAMPLE 22

(175) Generation of tAK Fusions to Cement-Like Proteins for Use in Determining Biofilm Removal

(176) tAK from Thermotoga maritima is fused to the 19 KDa protein from Balanus albicostatus and expressed as described above. Purification is effected from either the soluble or insoluble fraction. Refolding and subsequent deposition of the tAK-containing film onto a solid support is achieved as in Example 19. The thickness, rate of deposition and subsequent removal of the biofilm can be altered by modifying both the salt concentration and pH and by altering the concentration of the fusion protein.

(177) Protein sequences of barnacle cement-like proteins suitable for use in the invention are described below. The thermostable kinase may be fused to either the N-terminus or C-terminus of the cement proteins.

(178) TABLE-US-00015 Proteinsequenceofcement-likeproteinfromBalanusalbicostatus(19k) (SEQIDNO:70) VPPPCDLSIKSKLKQVGATAGNAAVTTTGTTSGSGVVKCVVRTPTSVEKKAAVGNT GLSAVSASAANGFFKNLGKATTEVKTTKDGTKVKTKTAGKGKTGGTATTIQIADAN GGVSEKSLKLDLLTDGLKFVKVTEKKQGTATSSSGHKASGVGHSVFKVLNEAETEL ELKGL Proteinsequenceofcement-likeproteinfromMegabalanusrosa(20k) (SEQIDNO:71) MKWFLFLLTTAVLAAVVSAHEEDGVCNSNAPCYHCDANGENCSCNCELFDCEAKK PDGSYAHPCRRCDANNICKCSCTAIPCNEDHPCHHCHEEDDGDTHCHCSCEHSHDH HDDDTHGECTKKAPCWRCEYNADLKHDVCGCECSKLPCNDEHPCYRKEGGVVSCD CKTITCNEDHPCYHSYEEDGVTKSDCDCEHSPGPSE ProteinsequenceoffusionofthebarnacleproteinfromBalanus albicostatuswiththetAKfromThermotogamaritima;N-terminalfusion (SEQIDNO:72) MRVLVINSGSSSIKYQLIEMEGEKVLCKGIAERIGIEGSRLVHRVGDEKHVIERELPDH EEALKLILNTLVDEKLGVIKDLKEIDAVGHRVVHGGERFKESVLVDEEVLKAIEEVSP LAPLHNPANLMGIKAAMKLLPGVPNVAVFDTAFHQTIPQKAYLYAIPYEYYEKYKIR RYGFHGTSHRYVSKRAAEILGKKLEELKIITCHIGNGASVAAVKYGKCVDTSMGFTP LEGLVMGTRSGDLDPAIPFFIMEKEGISPQEMYDILNKKSGVYGLSKGFSSDMRDIEE AALKGDEWCKLVLEIYDYRIAKYIGAYAAAMNGVDAIVFTAGVGENSPITREDVCS YLEFLGVKLDKQKNEETIRGKEGIISTPDSRVKVLVVPTNEELMIARDTKEIVEKIGRV PPPCDLSIKSKLKQVGATAGNAAVTTTGTTSGSGVVKCVVRTPTSVEKKAAVGNTG LSAVSASAANGFFKNLGKATTEVKTTKDGTKVKTKTAGKGKTGGTATTIQIADANG GVSEKSLKLDLLTDGLKFVKVTEKKQGTATSSSGHKASGVGHSVFKVLNEAETELEL KGL ProteinsequenceoffusionofthebarnacleproteinfromBalanus albicostatuswiththetAKfromThermotogamaritima;C-terminalfusion (SEQIDNO:73) VPPPCDLSIKSKLKQVGATAGNAAVTTTGTTSGSGVVKCVVRTPTSVEKKAAVGNT GLSVSASAANGFFKNLGKATTEVKTTKDGTKVKTKTAGKGKTGGTATTIQIADAN GGVSEKSLKLDLLTDGLKFVKVTEKKQGTATSSSGHKASGVGHSVFKVLEAETELEL KGLMRVLVINSGSSSIKYQLIEMEGEKVLCKGIAERIGIEGSRLVHRVGDEKHVIEREL PDHEEALKLILNTLVDEKLGVIKDLKEIDAVGHRVVHGGERFKESVLVDEEVLKAIEE VSPLAPLHNPANLMGIKAAMKLLPGVPNVAVFDTAFHQTIPQKAYLYAIPYEYYEKY KIRRYGFHGTSHRYVSKRAAEILGKKLEELKIITCHIGNGASVAAVKYGKCVDTSMG FTPLEGLVMGTRSGDLDPAIPFFIMEKEGISPQEMYDILNKKSGVYGLSKGFSSDMRD IEEAALKGDEWCKLVLEIYDYRIAKYIGAYAAAMNGVDAIVFTAGVGENSPITREDV CSYLEFLGVKLDKQKNEETIRGKEGIISTPDSRVKVLVVPTNEELMIARDTKEIVEKIGR

(179) Protein sequence of a novel barnacle cement proteins for use in the generation of thermostable kinase fusion proteins. The calcite dependent aggregation and adherence of this protein enable this type of indicator to monitor processes capable of removing mineral ions from aggregates in such a way as to destabilize and remove biofouling or biofilms. The thermostable kinase may optionally be fused to the N-terminus or C-terminus. Sequence from Mori et al., 2007, Calcite-specific coupling protein in barnacle underwater cement; FEBS J, 274, p 6436-6446. Protein sequence of

(180) TABLE-US-00016 Balanusalbicostatuscalcite-specificadsorbent (SEQIDNO:74) MKYTLALLFLTAIIATFVAAHKHHDHGKSCSKSHPCYHCHTDCECNHHHD DCNRSHRCWHKVHGVVSGNCNCNLLTPCNQKHPCWRRHGKKHGLHRKFHG NACNCDRLVCNAKHPCWHKHCDCFC

(181) Peptide sequence of a peptide derived from a barnacle cement protein for use in the formation of thermostable kinase-containing peptide biofilm preparations. Sequences derived from Nakano et al., 2007, Self assembling peptide inspired by a barnacle underwater adhesive protein; Biomacromolecules, vol 8, p 1830-1835.

(182) TABLE-US-00017 Peptide1 (SEQIDNO:75) SKLPCNDEHPCYRKEGGVVSCDCK Peptide2 (SEQIDNO:76) SKLPSNDEHPSYRKEGGVVSSDSK Peptide3 (SEQIDNO:77) KTITCNEDHPCYHSYEEDGVTKSDCDCE

(183) Use of the CementtAK Fusion for Monitoring Cleaning of Medical Devices

(184) The indicator described above is deposited onto stainless steel of a grade representative of surgical instruments using the deposition methods described in the previous examples. The device is inserted into a standard instrument load and the process performed as standard. The device is removed at the end of the process and the residual activity of the tAK fusion is correlated with removal of potentially infectious soil components.

(185) Use of the CementtAK Fusion for Monitoring Removal of Biofouling.

(186) The indicator described above can also be used to monitor the removal of biofouling in other contexts. For example the indicator may be attached to the bottom of a boat being subjected to cleaning for removal of barnacles and other marine biofilms. The indicator is subjected to the same process and assessed at the end of the procedure. Whilst visual removal of material is the key means of determining performance, the use of a more sensitive assay method allows an assessment of the removal of microscopic amounts of soiling which would provide a better primer for the re-establishment of the marine biofilm. Hence in this application the indicator provides both a demonstration of immediate efficacy and an indication of the longevity of the treatment.

EXAMPLE 23

(187) Cross Linking of tAK to E. coli or Staphylococcus aureus Exopolysaccaride

(188) The exopolysaccharide is generated by growing the relevant bacterial strain under standard growth conditions in either liquid, semi-liquid, biofilm or solid cultures familiar to those with knowledge of the art. Bacteria, typically towards the end of the logarithmic phase of growth are collected by resuspension (where required) and centrifugation. The cells are washed in low osmotic strength buffers (typically below 100 mM NaCl/NaPO.sub.4) usually at near neutral pH. The washing may be carried out by mixing vigorously for 1 hour at room temperature or overnight with gentle agitation at 4 C. Optionally an acidic preparation may be extracted using an acetate buffer at pH between 3 and 5. Limited cell perturbation may be achieved using very low energy sonication or by the addition of low levels of detergent. Preparations may be filter sterilised through a 0.2 m nitrocellulose or cellulose acetate filter prior to storage at 4 C. or 20 C.

(189) Cross-linking of the polysaccharides to tAKs can be achieved using a variety of coupling chemistries. In the first example the tAK from Sacidocaldarius is used. The coupling uses the heterobifunctional reagent ABH (p-Azidobenzoyl hydrazide; Pierce Chemical company product number 21510). The protocol is as follows. 1. Prepare a 20 mM periodate solution by dissolving 4.3 mg sodium metaperiodate in 1 ml 0.1M sodium acetate pH 5.5. Store on ice in the dark. 2. Add 1 ml of metaperiodate solution to 1 ml of the exopolysaccharide (EPS; or other glycoprotein, complex carbohydrate or lipid solution) at a concentration of at least 1 mg/ml carbohydrate. Incubate for 30 minutes at 4 C. 3. Dialyse overnight against phosphate buffered saline. 4. Prepare ABH by dissolving 1.8 mg in DMSO. 5. Add between 10 and 100 l of the ABH to the oxidised EPS solution generated in step 3 and incubate at room temperature for 2 hours. 6. Dialyse samples overnight to remove excess ABH. 7. Mix the ABH-derivatised EPS with purified tAK from S. acidolcaldarius prepared as described previously. The concentration of the tAK required to give the appropriate level of cross-linking may be determined empirically but will typically be in the range of 1-5 mg/ml. Incubate at room temperature for 30 minutes. 8. Expose the reaction mixture to UV light using a UV cross-linking apparatus or equivalent.

(190) In a second example of the chemistries available the heterobifunctional agent MPBH (4-[4-N-maleimidiophenyl]butyric acid hydrazide hydrochloride; Pierce Chemical company product 22305)) is used. The brief protocol is as follows: 1. tAK (e.g., from S. acidocladarius) with a reactive sulfhydryl is generated as described above by derivitisation with SPDP (Example 10) and subsequent reduction. Alternatively, tAK's with free cysteine residues (such as the tAK from Archaeoglobus fulgidus expressed in a recombinant form as described above) or with additional cysteine residues introduced by standard recombinant methods may be used. Protein is prepared at approximately 1-5 mg/ml 0.1M sodium phosphate 0.15M NaCl pH 7.2 or phosphate buffered saline. 2. Dissolve 3.5 mg MPBH in 1 ml of either dimethylformamide or dimethylsulfoxide to yield a 10 mM solution. 3. Add to the protein from step 1 to achieve a 5-10 fold molar excess of MPBH to protein and react for 2 hours at room temperature (or 4 hours at 4 C.). 4. Dialyse against 0.1M sodium phosphate 0.15M NaCl pH 7.2. 5. Prepare a 20 mM periodate solution by dissolving 4.3 mg sodium metaperiodate in 1 ml 0.1M sodium acetate pH5.5. Store on ice in the dark. 6. Add the 1 ml of metaperiodate solution to 1 ml of the exopolysaccharide (EPS; or other glycoprotein, complex carbohydrate or lipid solution) at a concentration of at least 1 mg/ml carbohydrate. Incubate for 30 minutes at 4 C. 7. Dialyse overnight against 0.1M sodium phosphate 0.15M NaCl pH 7.2. 8. Mix the derivatised sulfyhdryl-containing protein from section 4 with the oxidised EPS solution from step 7 and incubate for 2 hours at room temperature. Optionally separate the cross-linked from remaining free components using size exclusion chromatography.

(191) The methods described above are applicable to generating tAK conjugates with a wide range of complex carbohydrates, glycoprotein, glycolipids and other carbohydrate containing polymers including those from mammalian, bacterial, archaeal, plant or fungal origin.

(192) Use of Indicators Based on Exopolysaccharide-tAK Fusions.

(193) The EPS-tAK indicator is prepared in a suitable coating buffer such as phosphate buffered saline (pH 7.0-7.4), sodium bicarbonate (pH 9.0-9.6) or sodium acetate (pH4.0-5.5), optionally containing up to 500 mM NaCl at a relatively high concentration, e.g., 0.1-2.0 mg/ml. The solution is deposited onto a suitable solid support, such as surgical steel, plastics similar to catheters and lines, plastics used commonly in endoscopes. The interaction is allowed to proceed typically for 1-2 hours at room temperature and the coated surface allowed to dry at room temperature overnight prior to storage.

(194) Optionally other biological matrix components may be added either during the coating phase or subsequent to it.

(195) The indicator is included in the process to be monitored, e.g., within a washer disinfector cycle. The device is removed at the end of the process and inserted into hygiene monitor tubes to provide the read-out of the effective destruction and/or removal. The process is deemed effective if the value obtained is below the pre-determined thresholds of the hygiene monitor of luminometer. If successful the batch of instruments or material processed at the same time as the indicator may be used or passed on for subsequent processing. If deemed a fail the material must be reprocessed with a new indicator device.

EXAMPLE 24

(196) Further Examples of Complex Carbohydrates and Glycoconjugate Indicators

(197) A wide range of complex carbohydrate-containing molecules can be incorporated into indicator devices of the invention by covalent attachment to tAKs using methods such as those set out above (Example 23). Some further examples of these are provided in the table below.

(198) TABLE-US-00018 TABLE 4 Suitable carbohydrate-containing biological components Type of complex Organisms (various Type of process for indicator carbohydrate species and strains) applications EPS/LPS (sometimes Legionella, E. coli, Cleaning, decontamination, termed endotoxin) Staphylococcus species, sterilisation, (specific examples; Streptococcus species, biofilm removal, endotoxin removal Pseudomonas species, or destruction, surgical instrument Acinetobactor, Shigella, cleaning, medical product cleaning Campylobacter, Bacillus and decontamination) species, Lignin Filamentous fungi Biofilm removal and destruction. Removal Cell wall components Streptomyces Soil sterilisation Eap1p and equivalent Candida albicans and Biofilm removal, infection control cell surface related fungal organisms decontamination glycoproteins (Li et al., 2007, Eukaryotic cell, 6, p931-939) Spore extracts Bacillus species, Product sterilisation, room cleaning Clostridial species; other and decontamination spore formers Mucin preparations Mammalian species and Surgical instrument recombinant cell cultures decontamination, decontamination of surgical masks, outbreak control of respiratory virus outbreaks (e.g influenza, RSV) Brain-derived Mammalian species Evaluating/validating prion removal glycolipids technologies, decontamination of neurological instruments, samples etc. Invertebrate secretions Molluscan gels, Removal of biofouling Plant carbohydrates, Various plant species Removal or destruction of gums, resins, oils or contaminating materials on surfaces lipids or in products.

EXAMPLE 25

(199) Coupling of tAK to Mucus to Validate Processes Designed to Reduce Mucus Contamination of Medical Products

(200) Mucus is purified from a mucus-producing cell line such as normal human bronchial cells, cultured cells or is collected from sputum samples from patients. Washing in water or low salt solutions is sufficient to separate the mucin from most other components. Alternatively, purified mucin of animal origin e.g., porcine mucin, can also be used. The purified preparation is cross-linked to tAK using the methods described above, either to the protein component, through SPDP-coupling of the proteins as shown in FIG. 7, or to the carbohydrate component using the specific methods set out in Example 23. Deposition and subsequent use of the indicator is as described in Examples 23 and 24. FIG. 8 shows the results of the use of a tAK-mucin conjugate to monitor the removal of mucin in a washer-disinfector.