Competitive immunoassay test system for detecting a pyrogen

Abstract

The invention relates to a competitive immunoassay test system as well as a method to simply and rapidly detect pyrogens contained in a sample by means of the pyrogen binding domain of pattern recognition receptors. The test system comprises an assay carrier having at least one immobilized pyrogen binding domain of a pattern recognition receptor with a labeled, displaceable ligand, wherein the displacement of the ligand is indicated by a pyrogen contained in the sample by a color reaction of the labeling. The assay carrier preferably also has at least one immobilized ligand capture protein, which binds the displaced ligand and thereby initiates a color reaction of the labeling.

Claims

1. A competitive immunoassay test system for detecting at least one pyrogen contained in a liquid sample, the test system comprising: an assay carrier having: at least one immobilized pyrogen binding domain of at least one pattern recognition receptor (PRR) with at least one displaceable ligand comprising a first label, at least one immobilized ligand capture protein configured to bind the at least one displaceable ligand when it is displaced from the at least one immobilized pyrogen binding domain, and optionally a control compound comprising a second label, wherein the at least one immobilized pyrogen binding domain is selected from the group consisting of the pyrogen binding domains of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), a C-type lectin receptor (CLR), a cytosolic dsDNA-sensor (CDSS), a scavenger receptor, a mannose-binding lectin 2 (MBL-2) receptor, a glucan receptor and combinations thereof, and wherein the first label and the second label, when present, are the same or different, and are selected from the group consisting of fluorescent dyes, colored latex particles, soot particles, magnetic particles, quantum dots, colloidal selenium particles, gold nanoparticles, gold clusters, colloidal gold particles, enzymes, chemiluminescent compounds, streptavidin, avidin, and biotin, wherein the test system is configured so that binding of the at least one displaceable ligand to the at least one ligand capture protein, after displacement of the at least one displaceable ligand from the at least one pyrogen binding domain of the PRR by the at least one pyrogen contained in the liquid sample, is indicated by a color reaction of the first label.

2. The test system according to claim 1, wherein the assay carrier is a microtiter plate well, a reagent container, a test tube or a substrate capable of capillary flow.

3. The test system according to claim 2, wherein the substrate that is capable of capillary flow has the following areas in a direction of capillary flow each spaced apart from one another: a) a sample application area; b) a reaction area having the at least one immobilized pyrogen binding domain of at least one PRR with the at least one displaceable ligand; and c) an analysis area having the at least one immobilized ligand capture protein for binding the displaced labeled ligand.

4. The test system according to claim 3, further comprising: b) a control compound area having at least one adsorbed, labeled control compound that does not bind to the at least one pyrogen binding domain of the PRR or the at least one ligand capture protein; and d) a control area having at least one immobilized control compound capture protein for binding the at least one labeled control compound.

5. The test system according to claim 4, wherein the areas a), b), c) and d) are each spaced apart from one another and the areas b) and b) overlap each other or are spaced apart from each other.

6. The test system according to claim 1, wherein the first label and the second label, when present, are selected from the group consisting of fluorescein isothiocyanate (FITC), fluorescein, rhodamine, ATTO dyes, Texas Red, phycoerythrin derivatives, cyanine fluorescent dyes, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GOx), -D-galactosidase (-Gal), glucose 6-phosphate dehydrogenase (G6PD), acridinium ester, acridinium sulfonamide, and isoluminol.

7. The test system according claim 1, wherein the liquid sample is a clinical sample, a medical sample, a pharmaceutical sample, a food technology sample, or an environmental sample.

8. The test system according to claim 1, wherein the liquid sample is a clinical sample, and the clinical sample is of human or non-human animal origin.

9. The test system according claim 1, wherein the at least one immobilized pyrogen binding domain is the pyrogen binding domain of a toll-like receptor (TLR).

10. The test system according claim 1, wherein the at least one immobilized pyrogen binding domain is the pyrogen binding domain of one or more toll-like receptors (TLRs) selected from the group consisting of TLR 1, TLR 2, TLR 3, TLR 4, TLR 5, TLR 6, TLR 7, TLR 8, TLR 9 and heterodimers thereof.

11. The test system according to claim 1, wherein the at least one pyrogen to be detected is at least one selected from the group consisting of bacterial lipoproteins, bacterial lipopeptides, bacterial peptidoglycans, bacterial lipoteichoic acids, zymosan of yeast, double-stranded virus RNA, bacterial lipopolysaccharides (LPS), bacterial flagellin, unmethylated bacterial or viral DNA containing CpG, single-strand virus RNA and human heat shock protein 60.

12. The test system according claim 1, wherein the at least one immobilized pyrogen binding domain comprises the pyrogen binding domain of TLR4 and lipopolysaccharides (LPS) contained in the liquid sample are detected.

13. A method for detecting at least one pyrogen contained in a liquid sample, the method comprising: i) providing a test system according to claim 1, ii) bringing the liquid sample into contact with the assay carrier of the test system; and iii) analyzing a color reaction provided by the first label of the at least one displaceable ligand when the liquid sample comprises a pyrogen that displaces the at least one displaceable ligand from the at least one pyrogen binding domain, and the at least one displaceable ligand binds to the at least one ligand capture protein.

14. The method according to claim 13, wherein the assay carrier is a substrate capable of capillary flow and the liquid sample is applied to a sample application area in step ii) and conditions are provided and maintained for producing capillary flow from the sample application area to an analysis area of the substrate or to a control area of the substrate.

15. The method according to claim 14, wherein a pyrogen contained in the liquid sample is transported by capillary flow from the sample application area to a reaction area, where the pyrogen binds to the at least one immobilized pyrogen binding domain of the PRR and displaces the at least one displaceable ligand comprising the first label from the at least one immobilized pyrogen binding domain, and wherein the at least one displaceable ligand is transported to the analysis area where it binds to the at least one immobilized ligand capture protein and is detected by the color reaction of the first label.

16. The method according to claim 15, wherein the control compound is adsorbed in a control compound area and is transported by the capillary flow to the control area where the control compound binds to at least one immobilized control compound capture protein and is detected.

17. A competitive immunoassay test system for detecting at least one pyrogen contained in a liquid sample by analysis of a color reaction, the test system comprising: an assay carrier having: at least one immobilized pyrogen binding domain of at least one pattern recognition receptor (PRR) with at least one displaceable ligand comprising a first label, at least one immobilized ligand capture protein configured to bind the at least one displaceable ligand when it is displaced from the at least one immobilized pyrogen binding domain, and a control compound comprising a second label, wherein the first label and the second label, when present, are the same or different, and are selected from the group consisting of fluorescent dyes, colored latex particles, soot particles, magnetic particles, quantum dots, colloidal selenium particles, gold nanoparticles, gold clusters, colloidal gold particles, enzymes, chemiluminescent compounds, streptavidin, avidin, and biotin, wherein the test system is configured so that binding of the at least one displaceable ligand to the at least one ligand capture protein, after displacement of the at least one displaceable ligand from the at least one pyrogen binding domain of the PRR by the at least one pyrogen contained in the sample, is indicated by a color reaction of the label resulting from a contact of the liquid sample with the assay carrier.

18. A competitive immunoassay test system for detecting at least one pyrogen contained in a liquid sample, the test system comprising: an assay carrier having: at least one immobilized pyrogen binding domain of at least one pattern recognition receptor (PRR) with at least one displaceable ligand comprising a first label, at least one immobilized ligand capture protein configured to bind the at least one displaceable ligand when it is displaced from the at least one immobilized pyrogen binding domain, and optionally a control compound comprising a second label, wherein the first label and the second label, when present, are the same or different, and are selected from the group consisting of fluorescent dyes, colored latex particles, soot particles, magnetic particles, quantum dots, colloidal selenium particles, gold nanoparticles, gold clusters, colloidal gold particles, enzymes, chemiluminescent compounds, streptavidin, avidin, and biotin, wherein the test system is configured so that displacement of the at least one displaceable ligand from the at least one pyrogen binding domain by the at least one pyrogen contained in the liquid sample is indicated by a color reaction of the label.

19. The test system according claim 18, wherein the at least one immobilized pyrogen binding domain comprises a pyrogen binding domain of a toll-like receptor (TLR) and a pyrogen binding domain of a PRR selected from the group consisting of a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), a C-type lectin receptor (CLR), a cytosolic dsDNA sensor (CDSS), a scavenger receptor, a mannose-binding lectin 2 (MBL-2) receptor, a glucan receptor, and combinations thereof.

20. The test system according to claim 18, wherein the at least one immobilized pyrogen binding domain comprises at least one of toll-like receptor (TLR) 4 or TLR 9.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The figures show:

(2) FIG. 1 shows the immunoassay test system according to the invention, wherein the assay carrier is designed as a test tube and pyrogen binding domains of a pattern recognition receptor (PRR) are immobilized on the inner surface of the test tube.

(3) FIG. 2 shows the immunoassay test system according to the invention, wherein the assay carrier is configured as a microtiter plate well and pyrogen binding domains of a PRR and ligand capture proteins (antibodies) are immobilized on the base of the well.

(4) FIG. 3 shows the immunoassay test system according to the invention, wherein the assay carrier is configured as a substrate capable of capillary flow and pyrogen binding domains of a PRR are immobilized in the reaction area b) and a labeled control compound is absorbed and a sample containing a pyrogen is applied to the sample application area.

(5) FIG. 4 shows the displacement of the labeled ligand from the pyrogen binding domain by the pyrogen. At the same time, the adsorbed control compound is carried along by the capillary flow. In the results window, the labeled ligand is bound by the ligand capture proteins (antibodies) and the labeled control compound by the control compound capture proteins (antibodies) and thus detected.

(6) FIG. 5 shows another possible schematic structure of an assay carrier according to the invention.

(7) FIG. 6 shows the results of a PAMP assay, which was performed with the NIH 3T3 pNifty SEAP TLR4/CD14 reporter cell line. 25 ng/mL of LPS served as a positive control, and n.i. is the non-induced negative control. The substrate turnover (p-nitrophenyl phosphate) of the SEAP formed in the supernatant was analyzed photometrically after 60 min with a wavelength of 1=405 nm.

(8) FIG. 7 shows the flow rates of the membranes in the test strip structure with 300 L of sample solution. Structure of the test strip (width 2 cm): Standard 14 as the sample pad (1 cm length), various membranes (5 cm length) and CF3 as the adsorbent pad. It shows the flow rates of the following membranes: Fusion 5, Immunopore FP, HF as well as Fusion 5+Conjugate Pad GFCP10300 (1 cm length). 300 L of a Ponceau S solution was applied to the sample pad as the sample solution.

(9) FIG. 8 shows the investigation of various printing inks. To this end, cells from the NIH 3T3 pNifty SEAP TLR4/CD14 reporter cell line were printed in different printing inks on the substrate or pipetted manually and then a PAMP assay was conducted to test the functionality of the receptors. 25 ng/mL of LPS served as a positive control, and n.i. is the non-induced negative control. The substrate turnover (Quanti-Blue, InvivoGen) of the SEAP formed in the supernatant was [analyzed] after 30 min at a wavelength of 1=650 nm.

(10) FIG. 9 shows the fluorescence intensities of pyrogens bound to printed cells. NIH 3T3 pNifty SEAP TLR2/6 cells and corresponding reporter cells without receptors were printed on Immunopore membranes in a hyaluronic acid printing ink. The pyrogen binding of rhodamine-labeled Pam3CSK4 was measured with fluorescence photometry at l.sub.ex=549 nm and l.sub.em=566 nm. The cells were also pipetted manually on the membrane to check the printing process.

(11) FIGS. 10A-10C show the fluorescence intensities of pyrogens bound to printed and preserved cells. NIH 3T3 pNifty SEAP TLR2/6 cells and corresponding reporter cells without receptors were printed on Immunopore membranes in a hyaluronic acid printing ink and preserved in different ways. The pyrogen binding of rhodamine-labeled Pam3CSK4 was measured with fluorescence photometry at l.sub.ex=549 nm and l.sub.em=566 nm. The cells were also pipetted manually on the membrane to check the printing process. Preservation methods: FIG. 10A dried overnight at 37 C.; FIG. 10B fixed with 70% ethanol; FIG. 10C stored for 96 hours at 4 C.

(12) FIG. 11 shows the displacement of a displaceable ligand by LPS. To begin with, the TAMRA-labeled GLLHIFYIPRRDLKQLYFNL peptide was bound as the displaceable ligand to TLR4 (1.sup.st incubation) on Ni-NTA Magnetic Agarose Beads (Qiagen), on which TLR4 was immobilized via the his-tag. In the subsequent second incubation, bound peptide was displaced by Alexa Fluor 488-labeled LPS. The displacement was tracked using fluorescence microscopy and fluorescence photometric analysis (2.sup.nd incubation).

DETAILED DESCRIPTION

Example 1: Verification of LPS Through TLR4

(13) 1.1 Material Selection

(14) Pyrogens, bacteria, and the residues thereof are found all around us in our everyday lives and in production processes and can be inactivated only by dry heat greater than 180 C. As a result, the absolute freedom from pyrogens of all material used is essential (individual components of the immunoassay test system, solutions, buffers, substrates, consumable materials for laboratories, laboratory equipment). This is necessary, because pyrogens have a naturally high affinity for TLR4, i.e., the pyrogen binding domain, which is used here as a sensor element, bind thereto and therefore adversely affect or completely prevent the functionality of the immunoassay test system.

(15) All components of the immunoassay test system, such as substrates, blocking solutions, etc., were rigorously tested for their freedom from pyrogens using an established PAMP assay (DE 10 2006 031 483; EP 2 041 172). For this assay, the TLR4 receptor complex was stably transfected and expressed in a NIH 3T3 fibroblast cell line and a secreted alkaline phosphatase (SEAP) was stably integrated as the reporter gene. The activation of TLR4 by pyrogens leads to the activation of the transcription factor NF-B, which is turn induces the SEAP expression. Pyrogens, which are located in or on potential components of the immunoassay test system, can thus be detected by the expression of the reporter gene and then only pyrogen-free components can be selected for the structure of the immunoassay test system.

(16) FIG. 5 shows an example of the possible structure of an assay carrier according to the invention, which is configured as a substrate that is capable of capillary flow.

(17) The assay carrier of a test strip according to the invention, i.e., the substrate that is capable of capillary flow in the present case, is normally composed of a composite of several materials, each of which individually assumes a specific task. FIG. 5 schematically shows such a structure of a sample pad, conjugate pad, membrane and adsorbent pad.

(18) The sample to be analyzed is applied in the area of the sample pad, i.e., the sample application area. On the one hand, this guarantees a uniform and homogeneous sample transport and, on the other hand, can be used to retain particles. TLR4 pyrogen bind domains are immobilized on the overlapping conjugate pad, i.e., the reaction area. This can take place directly on the material or with the aid of particles such as agarose beads, which are kept here on site. In addition, the conjugate pad can contain buffer chemicals, blocking reactions or stabilizers. The separation between the control compound and the displaceable TLR4 ligand as the detection molecule takes place via the membrane. Both molecule types are captured at the end thereof via ligand capture proteins or control compound capture proteins in order to display the test result. The adsorbent pad (adsorbent material) at the end of the test strip absorbs excess liquid and guarantees the necessary capillary flow in the test strip.

(19) TABLE-US-00001 TABLE 1 Compilation of materials investigated for the substrate Function Designation Source of Supply Technical Data Sample pad CF3 GE Healthcare Sample and Adsorption pad, cotton linters, 322 m thickness Sample pad/ CFSP203000 Merck Millipore Cellulose, 83 m thickness, can be Adsorbent pad used as both a sample pad and an adsorbent pad Conjugate Standard 14 GE Healthcare Glass fiber, 355 m thickness release pad Conjugate GFCP103000 Merck Millipore Glass fiber, 48 m thickness release pad Membrane FF120HP GE Healthcare Nitrocellulose membrane (100 m), with plastic back (100 m), flow rate: 90-150 s/4 cm Membrane FF170HP GE Healthcare Nitrocellulose membrane (100 m), low viscosity, with plastic back (100 m), flow rate: 140-200 s/4 cm Membrane Immunopore GE Healthcare Nitrocellulose membrane (100 m), SP with plastic back (100 m), flow rate: 190-280 s/4 cm Membrane Immunopore GE Healthcare Nitrocellulose membrane (100 m), FP with plastic back (100 m), flow rate: 140-200 s/4 cm Membrane Millipore Merck Millipore Nitrocellulose High Flow Plus Membrane Protran BA85 Whatman Nitrocellulose, pore size: 0.45 m Adsorbent pad CF3 GE Healthcare Sample and adsorption pad, cotton linters, 322 m thickness Adsorbent pad CF5 GE Healthcare Absorption pad, cotton linters, 954 m thickness Special material Fusion 5 GE Healthcare Glass fiber with plastic back portion, material that simultaneously assumes all of the aforementioned functions

(20) The tested substrates (see Table 1) were ordered from GE Healthcare, Merck, Millipore and Whatman. However, all of them are not produced under sterile conditions, which could be shown in the material eluates tested in the PAMP assay (FIG. 6). The CF3, CF5 and CFSP203000 adsorbent pads in particular showed a very strong activation of the TLR4 receptors and thus a pyrogen contamination. Nevertheless, it is possible to use these materials for the structure of the test strip, because they do not come into direct contact with the reaction area on the opposite end of the test strip. Only the use of the CFSP203000 material as a sample pad and the use of the FF170 HP membrane were dispensed with. No activation of TLR4 could be detected for the materials of the most sensitive reaction area, the conjugate pad. Therefore, the tested materials are substantially suitable for the structure of the immunoassay test system according to the invention.

(21) An important parameter of the immunoassay test system is the flow rate of the liquid front in the test strip, because it determines the contact time with the pyrogen binding domain of the TLR4 receptor and thus the time that is available for displacement of the color-labeled, displaceable ligand via LPS. Therefore, to further characterize the substrates, a test strip with a width of 2 cm was structured as an example according to the diagram in FIG. 5. All test strips consisted of Standard 14 as a sample pad (1 cm length) and CF3 as an adsorbent pad (2 cm length). The flow rate of the Fusion 5, Immunopore FP, HF and Fusion 5+Conjugate Pad GFCP10300 (1 cm length) membranes, in each case with a length of 5 cm, were analyzed as follows: Different volumes (100/200/300/400/500 L) of a Ponceau S solution were applied to the sample pad. The uniformity of the migration, the absorbency of the materials and therefore the minimum required volume of the sample, as well as the time that the migration requires to reach a defined migration length of 3 cm were assessed. FIG. 7 presents the flow rates of the different membranes when applying 300 L of Ponceau solution.

(22) The Immunopore FP membrane clearly sets itself apart from the other 3 tested membranes, because it is hardly absorbent and has the highest flow rate of 40 sec/3 cm with a uniform migration. The Fusion 5 material in particular is a very absorbent material, which can easily absorb up to 500 L with greater sample volumes, however, it is not functional with sample volumes that are too low. By additionally combining the Fusion 5 with a conjugate pad (GFCP103000), absorbency is increased further and the flow rate drops to 10 sec/3 cm. Thus, various materials are available for different requirements with respect to sample volumes and test speed.

(23) In order to avoid non-specific bonds of the labeled control compounds and ligands on the tested membranes, the unoccupied membrane binding sites must be saturated, i.e., blocked. Roti Block (Carl Roth GmbH) showed itself to be the most suitable in terms of freedom from pyrogens, reproducibility and cytotoxic effect.

(24) 1.2 Supplying the Receptors

(25) Isolated TLR4 receptors or the pyrogen binding domains thereof were ordered for the immunoassay test system from the following sources: Recombinant TLR4 from NS0 cells (mouse myeloma cells), R&D Systems, 3146-TM-050/CF TLR4 protein fragment from wheat germ lysate, Abcam, ab112362 TLR4/CD284 from baculovirus insect cells, Hlzel Diagnostik GmbH, 10146-H08B-100

(26) TLR4/CD284 (Hlzel Diagnostik GmbH) in particular was used in the following.

(27) 1.3 Fluorescent Labeling of the Displaceable Ligand and the Control Compound

(28) To verify the displacement of the displaceable ligand and to verify the functionality of the test strip via the control compound, they must be detected after they are bound by corresponding capture proteins. Corresponding molecules are especially easy to detect by means of color labeling. The method of fluorescent labeling, the labeling with fluorophores, is advantageous here especially compared with the visual analysis of non-fluorescent dyes, because small quantities of the fluorophore can already be detected with corresponding detectors even beneath the visual detection limit. Another advantage of fluorescent labeling is the possibility of the parallel measurement of fluorophores of different colors.

(29) Especially suitable fluorophores are characterized by a strong absorption (a high extinction coefficient ), a high quantum yield (Q>0.7) and a large Stokes shift. In the present case, TAMRA was selected as the color label for the displaceable ligand and the control compound, because it has a high extinction coefficient of 95,000 L cm.sup.1 mol.sup.1 and is also very conspicuously visible.

(30) 1.4 Supplying the Displaceable Ligand

(31) Different small molecules and peptides were identified as displaceable ligands with the aid of the already described PAMP assay. The pyrogen E. coli LPS to be detected has a medium effective concentration (EC50) of 40 pg/mL in the PAMP assay with the NIH 3T3 pNifty SEAP TLR4/CD14 reporter cell line. So that LPS can displace a ligand bound to the receptor or the pyrogen binding domain thereof, it must have a lower binding affinity, which correlates with a lower EC50 value. Four peptides and two small molecules were identified as displaceable ligands with an agonistic effect with the aid of the PAMP assay: Small molecule T5635346 (2-hydroxy-4-methylphenyl)-morpholin-4-ylmethanone); IC50 of 7.7 mM Small molecule T6854457 (4-methyl-N-(2-oxoazepan-3-yl)benzenesulfonamide); IC50 of 2.6 mM

(32) TABLE-US-00002 KGETVNTTISFSFKGIKFSKpeptide(SEQIDNO:1); IC50> 200mM KMQYPISINVNPCIELKGSKpeptide(SEQIDNO:2); IC50> 200mM GLLHIFYIPRRDLKQLYFNLpeptide(SEQIDNO:3); IC50> 200mM KRKEVICRGSDDDYSFCRALpeptide(SEQIDNO:4); IC50> 200mM

(33) The affinity of a ligand to its receptor describes the tendency thereof to enter into a bond with the receptor. The greater affinity, the greater the association constant K.sub.a is. The strength of the receptor/ligand interaction can be determined experimentally with the aid of surface plasmon resonance spectroscopy (Biacore T200, GE Healthcare) and quantified by the so-called dissociation constant (K.sub.d). K.sub.d is thereby the reciprocal value of K.sub.a, a thermodynamic value, which indicates which portion of the ligand is bound on average to the receptor protein and has the dimension of a concentration. The Biacore chip system is based on a glass slide with a gold film in a flow cell. TLR4 was immobilized on the NTA sensor chip via its his-tag in accordance with the manufacturer's protocol and the to-be-tested ligands, control compounds and LPS were passed over it dissolved in HBS-P+ with 50 M EDTA at 20 C. with a flow rate of 10 L/min in respectively 5 concentrations. The bond with TLR4 is measured as a change in the refractive index in the flow cell as compared to a reference cell as a response difference in RU. 1 RU corresponds thereby to 1 pg of bound ligand. After ligand injection, buffer is used for rinsing in order to ideally recreate the initial state, complete disassociation of the ligand.

(34) Table 2 lists the binding properties, determined with the aid surface plasmon resonance spectroscopy, of the small molecule T5635346 with and without TAMRA labeling as well as of LPS of Salmonella minnesota labeled with Alexa Fluor 488. T5635346 has an affinity for TLR4 in the same order of magnitude as the LPS tested here. However, the binding affinity is reduced by 40 fold by the TAMRA labeling.

(35) TABLE-US-00003 TABLE 2 Binding properties of the small molecule T5635346 to TLR4 Mean Value Ligand in g/mol K.sub.d in M K.sub.a in 1/M T5635346 221 2.42E07 4,135,649 T5635346-TAMRA 723 8.50E06 117,633 LPS S. minnesota Alexa Fluor 2.83E07 4,203,447 488

(36) 1.5 Supplying the Control Compound

(37) Analogous to identifying the displaceable ligands, small molecules and peptides not binding to the receptor also were identified as control compounds: Small molecule T5874283 (1-(1,3-benzodioxol-5-yl)-3-[[4-(2-methylpropyl)morpholin-2-yl]methyl]urea) Small molecule T7054880 (1-[1-(3-methoxyphenyl)propan-2-yl]-3-(1H-pyrazol-4-yl)urea)

(38) TABLE-US-00004 FMMLGGLVRIpeptide(SEQIDNO:5) RMMWFGIMVpeptide(SEQIDNO:6) SGSPEEMLFCLEFVILHQPNSNpeptide(SEQIDNO:7)

(39) Surface plasmon resonance spectroscopy (see Section 1.4) was used to verify that the small molecules T7054880 and T5874283 selected as control compounds do not bind to TLR4 or bind to TLR4 with a similar affinity as LPS (see Table 3).

(40) TABLE-US-00005 TABLE 3 Binding properties of the control compounds to TLR4 Mean Value Ligand in g/mol K.sub.d in M K.sub.a in 1/M T5874283 335 1.02E07 9,803,922 T7054880 274

(41) 1.6 Immobilizing the Receptor on the Substrate

(42) In addition to immobilizing isolated receptors (see Section 1.2), cells from the reporter cell line were also printed on the Immunopore FP nitrocellulose membrane with the aid of a table-top robot mta TR300 printer (mta automation Inc.). First of all, suitable cell-compatible printing inks, such as e.g., inks based on hyaluronic acid (Lifecore Biomedicals), gelatin (Gelita AG) and carboxymethyl cellulose (Sigma-Aldrich GmbH) were tested for this. To this end, 100 l with an absolute cell count of 30,000 cells was respectively printed at a speed of 15 L/s per well of a 96-well microtiter plate or pipetted manually as a print control and the cells were incubated overnight at 37 C. On the second day, the cells were induced with 25 ng/mL of LPS and on the third day the secreted alkaline phosphatase in the supernatant was detected by the turnover of the Quanti-Blue substrate (InvivoGen) (FIG. 8).

(43) Based on the comparison of the cells printed in DMEM and the cells pipetted manually it was shown that the printing process had only a small impact on the cells as compared to pipetting manually. Of the printing media tested, a printing ink of 1% hyaluronic acid dissolved in DMEM medium was identified as suitable and used for the following experiments.

(44) To immobilize whole-cell reporter cells on membranes, NIH 3T3 pNifty SEAP TLR2/6 was printed on the Immunopore nitrocellulose membrane as an alternative to TLR4 expressed cells. A corresponding reporter cell line without TLR4 was also used to check a non-specific binding of the pyrogen. To this end, every four points was each printed with 40,000 cells for each test condition at a speed of 15 Ls on the Immunopore FP nitrocellulose membrane (1.5 cm1.5 cm). As a control, 40,000 cells in 100 L medium were pipetted manually in every four clone rings placed on the membrane in order to allow them to adhere in a stationary manner and to guarantee that the print pattern had similar test conditions for analysis. After incubation overnight at 37 C., on the following day, the Pam3CSK4 pyrogen with rhodamine labeling (6 ng/mL, Invivogen) that is specific for NIH 3T3 pNifty SEAP TLR2/6 was added and incubated at for 4 hours 37 C. to bind to the receptor. Prior to the fluorescence photometric measurement of the bound Pam3CSK4 with rhodamine labeling [at] l.sub.ex=549 nm and l.sub.em=566 nm, the individual samples were washed with PBS in order to remove non-bound pyrogen. FIG. 9 shows the results of the test described above for immobilizing the whole-cell reporter cells on membranes by means of printing methods. The vitality of the printed cells was of subordinate relevance for this test set-up. The main focus was that the membrane receptors were not damaged during the printing process and continued to have the ability for pyrogen binding. It was shown that by immobilizing by means of the printing methods, the membrane receptors are not damaged in direct comparison to the manually pipetted cells. In addition, it was shown that no undesired non-specific bonds occur between the pyrogen and membrane or the pyrogen and printing medium. Therefore, the printing offers the advantage that whole-cell sensor elements can be precisely positioned on a substrate in a spatially resolved manner.

(45) In addition to the structure of a test strip, the storage suitability thereof must also be considered. If whole-cell sensor elements, i.e., receptor cells, are applied to a surface without a protective layer, there is a risk that membrane receptors will get damaged when transferring the cells to a storable state and lose their functionality. Printing the cells may also be a solution for this problem, because the cells are deposited here in a gel-like printing ink, which protects the cells from external influences. For this purpose, whole-cell sensor elements were printed as described above and pipetted manually as a control. Appropriate samples were subjected to three different preservation and storage methods: Drying at 37 C. for 24 hours, fixing with 70% ethanol and storage at 4 C. for 96 hours. After preservation and storage, the bond of the specific pyrogen Pam3CSK4 with rhodamine labeling was tested as already described above.

(46) FIG. 10 shows the results of these investigations. As compared to the results in FIG. 9, higher values for the pyrogen binding to the control cell line and the membrane were always detected. It is a non-specifically bound pyrogen in this case. Roti Block can be used for blocking to reduce the non-specific pyrogen binding to the membrane. FIG. 10A shows the results after drying, FIG. 10B the results of the ethanol fixing and FIG. 10C the results after storage at 4 C. With a high background from non-specific pyrogen binding to the membrane and control cell line, a clear delta of the fluorescence intensity of more than 3000 a.u. (arbitrary units) was detected nevertheless in the case of all preservation and storage methods. The comparison of printed receptors cells to receptor cells pipetted manually, which are not embedded in a gel-like protective matric, showed that the surrounding protective layer prevents a loss of function of the TLRs with storage and preservation.

(47) 1.7 Antibodies as Specific Ligand Capture Proteins

(48) Specific capture proteins bind the displaceable ligand or the control compound in a spatially resolved manner. Antibodies in particular are suitable for this. They may be aimed either specifically at the displaceable ligand or the control compound or specifically against the fluorophore, which was used for the color labeling of the molecules. In Section 1.3, the fluorescence dyes, tetramethylrhodamine 5-carboxamido-(6-azidohexanyl) (TAMRA) and Alexa Fluor 488 were identified as suitable for labeling control compounds and displaceable ligands. Specific antibodies against these fluorophores are commercially available so that individual antibody production for selected displaceable ligands or control compounds may be dispensed with as the case may be, if the color properties of the fluorophore do not get lost when binding to the antigen.

(49) The effect of the antibody binding on the fluorophores, Alexa Fluor 488 and TAMRA, was investigated by means of a specific dot blot. For this purpose, two different concentrations of the dyes (100 and 500 ng for Alexa Fluor 488 as well as 7 and 35 ng for TAMRA) were dotted on a nitrocellulose membrane and incubated further with the specific antibodies. The binding of the antibodies to the respective fluorophore was immunodetected by the horseradish peroxidase conjugated on the antibodies. In the case of both fluorophores, it was shown with the aid of fluorescence microscopy that the antibodies bind specifically to their respective antigen and they thereby do not show any declines in the fluorescence intensity. As a result, the selected anti-TAMRA and anti-Alexa Fluor 488 antibodies are suitable as capture proteins for the structure of the immunoassay test system.

(50) 1.8 Displaceable Ligand Receptor Complex

(51) The central assay element of the immunoassay test system consists of the displaceable ligand receptor complex. It is cleaved by the presence of pyrogens, whereby the pyrogen displaces the displaceable ligand, binds to the receptor and the displaceable ligand is released. This was shown with the aid of Ni-NTA Magnetic Agarose Beads (Qiagen), bound on the TLR4 (Hlzel Diagnostik GmbH) by means of a his-tag (see FIG. 11).

(52) In a first step, magnetic agarose beads loaded with TLR4 were incubated with TAMRA-labeled GLLHIFYIPRRDLKQLYFNL peptide or with TAMRA-labeled small molecule T5635346 as the displaceable ligand for 1 hour at room temperature in binding buffer (50 mM NaH2PO2*2H2O; 300 mM NaCl; 20 mM Imidazol, pH 8.0). Non-bound ligands were removed in a wash step. In a second step, the magnetic agarose beads were incubated with the TLR4-displaceable ligand complex with Alexa Fluor 488-labeled Salmonella minnesota LPS in interaction buffer (binding buffer+0.05% Tween20). Non-bound ligands and any displaced ligands were removed in a subsequent wash step. Fluorescence images of the magnetic agarose beads were made after each incubation step. After the first incubation step, e.g., the binding of the peptide, i.e., of the displaceable ligand, is detectable as red dye on the bead surface via the TAMRA labeling. After the second incubation step, the displacement and release of the previously bound peptide is detectable as green dye on the bead surface through the binding of LPS via Alexa Fluor 488 labeling. FIG. 11 shows the associated fluorescence photometric analysis. The measured fluorescence of TAMRA drops from the first to the second incubation. This means that the TAMRA-labeled ligand was displaced. At the same time, however, the fluorescence of Alexa Fluor 488 increases from the first to the second incubation. This in turn means that the Alexa Fluor 488-labeled pyrogen LPS has occupied the previous binding sites of the TAMRA-labeled displaceable ligand.