COMBINED COMPOSITE FOR STABILIZATION OF ACTIVE BIOLOGICAL MATERIALS, METHOD OF PRODUCTION AND USE THEREOF
20220162582 · 2022-05-26
Assignee
Inventors
Cpc classification
C12N11/089
CHEMISTRY; METALLURGY
G01N33/54313
PHYSICS
International classification
C12N11/04
CHEMISTRY; METALLURGY
C12N11/089
CHEMISTRY; METALLURGY
Abstract
A method for immobilizing in a sol-gel combined composite active or activable biological materials. The loss by leaching of the biological materials from the obtained combined composite is reduced while retaining the inherent biological activity. In addition, the composite obtained the method.
Claims
1-25. (canceled)
26. A method of production of a combined composite for stabilization of active or activable biological materials comprising the steps of: a) providing a simple composite in the form of a sol-gel immobilizing uniquely by encapsulation active or activable biological materials of one or more different natures, finely divided and with low water content; b) providing a second sol-gel immobilizing the simple composite of a), comprising: i) preparing a suspension of simple composite of a) in a phosphate-buffer solution 100 mM pH=6.8±0.2 at a mass/volume concentration between 0.83% and 3.34% in reference to the volume of the final reacting mixture; ii) effecting the hydrolysis of tetramethyl orthosilicate by acidic catalysis adding to tetramethyl orthosilicate an HCl solution at a concentration of 4.50 mM to 8.34 mM in the proportion of 28% of the final volume of this mixture; iii) adding the mixture from ii) to an equal volume of the suspension from i) at 25° C. to 30° C. allowing polymerization to occur until the obtained combined composite has a consistency that enables it to be fragmented; iv) fragmenting the combined composite obtained from iii) to granules of two classes of size: equal and smaller than approximately 2 mm.sup.3 and approximately between 2 mm.sup.3 and 4 mm.sup.3; v) optionally, incubating the fragmented combined composite of iv) during 24 hours in bovine serum albumin (BSA) 2.0% m/v solubilized in saline phosphate buffer 10 mM pH=7.3 in a proportion of three volumes of the incubating solution to one volume of combined composite; vi) maturating the fragmented combined composite by drying on a surface until a mass-decrease of about 70% and available water content (a.sub.w) of about 20 ppm; whereby a combined composite is obtained doubly immobilizing in sol-gel uniquely by encapsulation said active or activable biological materials, wherein the loss by leaching of said biological materials is reduced, being preserved its activity or activation capability.
27. The method according to claim 26, wherein in said combined composite the loss by leaching of said doubly immobilized biological materials is reduced and its activity or capability of activation is preserved for at least 20 cycles of use.
28. The method according to claim 26, wherein the simple composite of a) has an available water content (a.sub.w) of 10 to 15 ppm.
29. The method according to claim 26, wherein the simple composite of a) has a granulometry of from 100 to 110 μm.sup.3.
30. The method according to claim 26, wherein in step b) iii) the polymerization occurs in about 30 to 90 minutes.
31. The method according to claim 26, wherein the active or activable biological material is one or more from a biologically active protein such as an enzyme, a co-enzyme, and a immunologically active protein such as an antigen, an antibody, a hapten-protein, or any other member of a specific biological relationship.
32. The method according to claim 31, wherein the active or activable biological material is one or more from a immunologically active protein such as an antigen, an antibody, a hapten-protein and the method includes step v).
33. A combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material, obtainable by a method of claim 26.
34. The combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material according to claim 33, wherein the loss by leaching of said immobilized biological material is reduced and its activity is preserved for at least 20 cycles of use.
35. The combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material according to claim 33, wherein the immobilized active or activable biological material is one or more from a biologically active protein such as an enzyme, a co-enzyme, and an immunologically active protein such as an antigen, an antibody, a hapten-protein, or any other member of a specific biological relationship.
36. The combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material according to claim 33, wherein the immobilized simple composite, matured and finely divided has a granulometry of from 100 μm.sup.3 to 110 μm.sup.3.
37. The combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material according to claim 33, wherein said active or activable biological material is one or more of biologically active proteins such as an enzyme or a co-enzyme and the combined composite is to be used in biocatalysis.
38. The combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material according to claim 33, wherein said active or activable biological material is a specifically binding immunologically active protein and said combined composite is to be used for detection of its respective specific ligands.
39. The combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material according to claim 38, wherein said specifically binding immunologically active protein is an antigen, an antibody, a hapten-protein, and said combined composite is to be used for diagnosis of a pathology.
40. The combined composite that doubly immobilizes in sol-gel and uniquely by encapsulation active or activable biological material according to claim 33, for use in medical diagnosis.
41. A method of diagnosis of a pathology in a human or superior animal subject comprising the steps of: a) contacting a sample of biological fluid from the subject suspected to contain an analyte related to a pathology, in particular plasma/serum of blood, urine, supernatant of tissue maceration or any other fluid obtained from the subject, with the combined composite that doubly immobilize in sol-gel biological material that specifically binds to said analyte wherein the combined composite is a composite of claim 33; and b) detecting of specific binding between the analyte present in the sample and the biological material immobilized in the combined composite, the presence or absence of said specific binding indicating, respectively, an affirmative or negative diagnosis of the pathology in the subject.
42. The method of diagnosis according to claim 41, wherein the pathology is any disease that results from the presence of antigens and/or antibodies in the fluids and/or tissues of a human or superior animal subject.
43. The method of diagnosis according to claim 41, wherein the analyte in the sample is an antigen or an antibody and an immune-complex results from the specific binding.
44. The method of diagnosis according to claim 43, wherein the detection of specific binding comprises the steps of: a) contacting the (primary) immune-complex with a secondary antibody labelled with a detectable tracer, that binds specifically to the analyte of the biological sample; and b) detecting the tracer in the sample, directly or via its interaction with a particular reagent.
45. The method of diagnosis according to claim 44, wherein the detectable tracer in the secondary antibody is an enzyme and its detection is effected by revelation of colour after contact with its chromogenic substrate, or is a photo-sensible molecule and its detection is effected by fluorescence emission after luminous excitation with correspondent wavelength.
46. The method of diagnosis according to claim 41, additionally comprising a step c) of recycling the combined composite and the replication of steps a) to c) for at least 20 times.
47. An analytical chamber for detection of specific ligands of an immunologically active protein comprising as filling a combined composite immobilizing an immunologically active protein of claim 33, for use in medical diagnosis.
48. Device for detection of specific ligands of immunologically active proteins comprising a maximum of four groups of two chambers constructed in parallel, each of said groups being comprised of one chamber of analysis according to claim 47 and one chamber of negative control filled with combined composite immobilizing an immunologically non-active protein, for use in medical diagnosis.
49. The device according to claim 48, wherein each immunologically active protein immobilized in each combined composite filling each analytical chamber is associated to a different specific pathology.
50. The device according to claim 48, wherein it is reused for at least 20 times.
Description
4. BRIEF DESCRIPTION OF THE FIGURES
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5. DETAILED DESCRIPTION
Example 1—Composite Formulations
Example 1.1.—Simple Composite
[0075] In the preparation of an indicative volume of 20 ml of precursors of simple composite, it was followed the protocol described by Alstein and co-workers using as orthosilicate precursor 5.0 ml of tetramethyl orthosilicate (TMOS); 4.84 ml of HCl 2.5 mM; 1.0 ml of polyethyleneglycol 0.4 KDa. The mixture was agitated in a vortex until liquid medium presented transparent. Homogenization was exothermic and the mixture was then sonicated for 30 minutes. It was previously solubilized 180 to 500 mg of lyophilized protein, (e.g. enzyme, antigens/antibodies) in 10 m ml HEPES buffer 50 mM pH=7.5.
[0076] It was added 10 ml of the sonicated mixture to the same volume of HEPES buffer solubilizing protein. This mixture was homogenized while gelification occurred for about 6 minutes. Solidified gel was then finely fragmented and spread onto a glass surface forming a thin layer for room exposure drying. At maturation there was a loss of about 84% of volumic mass: from the initial liquid volume of 20.0 ml of precursor it was approximately obtained 3.0 g of matured composite.
[0077] The simple composite was then grinded for a granulometry lesser than 106 μm.sup.3. The water content of the matured simple composite (a.sub.w) was around 11 ppm. The matured simple composite had a protein concentration (mass of protein/mass of composite) among 0.5% and 1.8%.
[0078] For immune-essays it was also synthetized simple composite for negative control immobilizing bovine serum albumin with exactly the same mass of antigens/antibodies as described above.
Example 1.2.—Combined Composite
1.2.1. Failures of Immobilizing a Composite
[0079] In tempt of synthetizing a combined composite it was followed the conventional protocol of sol-gel preparation trying to immobilize one simple composite previously synthetized instead of a free protein. Having in knowledge the proton availability required for chemical attack of the orthosilicate structure it was experimented a fraction of 50 μl of HCl 100 mM at pH=7.0 buffered media with different volumes of water for hydrolysis of 1.5 ml TMOS in order to obtain a hydrophilic composite compatible with the afore describe simple composite.
[0080] It was found a water deficit in the reacting medium proportional to the amount of mass of simple composite despite having linearly followed the initial proportion mass-of-simple-composite versus volume-of-precursors. Consequently there was a complete failure in TMOS hydrolysis. Table 1 presents a compilation of the experiences which elucidate the difficulties of immobilizing a simple composite in a second sol-gel.
TABLE-US-00001 TABLE 1 experiences in tempting to immobilize a simple composite in sol-gel. Volume Volume Time (ml) of the (ml) of (min) Volume Volume mixture phosphate- of reaction Mass (mg) Volume (μl) (μl) (ml) H.sub.2O/HCl + buffer (PB) H.sub.2O/HCl <-> of simple HCl 100 mM H.sub.2O.sub.distilled TMOS TMOS 100 mM; pH = 7.0 TMOS composite RESULTS 50 not added 1.5 not not added not not added did not gelify controlled controlled 50 (added to PB) not added 1.5 2.0 1.0 not not added did not gelify controlled 50 (added to H.sub.2O) 100 to 600 1.5 1.7 to 2.2 1.0 not not added variable controlled 50 (+H.sub.2O) 540 1.5 1.0 1.0 not not added gelified controlled 50 (+H.sub.2O) 540 1.5 1.0 1.0 not 100 gelification few controlled consistent 50 (+H.sub.2O) 540 1.5 1.0 1.0 7 100 gelification but few robustness after drying 50 (+H.sub.2O) 540 1.5 1.5 1.0 a 2.0 in 2; in 100 variable 3 . . . in 7 50 (+H.sub.2O) 540 1.5 1.5 1.5 3 100 gelification in 14 sec. and robustness after drying 625 (+H.sub.2O) 6750 18.75 25.0 25.0 3 1660 did not gelify 63 (+H.sub.2O) 675 1.87 2.5 2.5 3 166 gelification in 1 min. Brittle composite after drying 50 (+H.sub.2O) 540 1.5 1.5 1.5 3 75 gelification in 14 sec. and robustness after drying
1.2.2. Practiced Formulation of the Combined Composite
[0081] The synthesis was effected with concentration of simple composite since 0.83% until 3.34% (m/v). In reference to a unit precursors volume of 3.0 ml it was dosed 25 mg to 100 mg of simple composite at granulometry lesser than 106 μm.sup.3 and with water content (a.sub.w) of circa 11 ppm. That simple composite was suspended in 1.5 ml of phosphate buffer (PB) that was prepared with 38 mM of Na.sub.2HPO.sub.4.2H.sub.2O; 62 mM of KH.sub.2PO.sub.4. PB was further adjusted pH=6.8±0.2 at 25° C. temperature (adding 1.0 M NaOH aliquots).
[0082] It was prepared an HCl solution whose concentration depended on higrophilia of the simple composite and conditioned by respective immobilized protein. For instance but not limited, for alkaline phosphatase [HCl]=4.5 mM; for mucin [HCl]=8.3 mM.
[0083] It was mixed 540 μl of distilled water with 50 μl of HCl solution and then added to 1.5 ml of TMOS. The mixture was homogenized during the initial 120 seconds and after 3 minutes 1.5 ml of that mixture was transferred to the same volume of PB suspension of simple composite. Gelification occurred in 10 to 15 seconds during which the media was homogenized in order to assure a homogeneous distribution of simple composite granules.
[0084] Polymerization went for 30 to 90 minutes after which the solidified gel was fragmented at granules of approximate volumetric dimensions less than 2 mm.sup.3 and between 2 mm.sup.3 and 4 mm.sup.3.
[0085] At immobilization of antigens/antibodies the solidified gel was incubated in 400 rpm orbital agitation during 24 hours in bovine serum albumin (BSA) at 2.0% (m/v). The solvent was phosphate buffer saline 10 mM pH=7.3±0.3 prepared with 7.6 mM Na.sub.2HPO.sub.4.2H.sub.2O; 2.4 mM KH.sub.2PO.sub.4; 137 mM NaCl; 2.7 mM KCl. The incubating volume was in a proportion of three volumes of the solution to one volume of solidified gel and after incubation it was transferred onto a clean-dry glass surface. From there on glass surfaces were changed for three times: at 24 hours, 48 h and 72 h of maturation.
[0086] The maturation of the combined composites either or not incubated in BSA, ran for seven days by exposure to room conditions after which there was a loss of volumetric mass of about 70% being the final water content a.sub.W≈19 ppm.
[0087] The protein concentration of the combined composite matured after BSA incubation was among 3.0 to 3.5 times the concentration found at the simple composite and without BSA incubation was in the range of 0.2 to 0.4.
[0088] After maturation the granule-size (G) separation was made and grouped in five classes:
[0089] G.sub.I(G)>1.0 mm.sup.3;
[0090] G.sub.II1.0 mm.sup.3>G>710 μm.sup.3
[0091] G.sub.III G>710 μm.sup.3>G>500 μm.sup.3
[0092] G.sub.IV G>500 μm.sup.3>G>300 μm.sup.3
[0093] G.sub.V G>300 μm.sup.3>G>106 μm.sup.3
Example 2—Time of Maturation
[0094] It was studied the loss of mass of the composites, simple and combined, along the exposure to room conditions. That monitoring was made from the moment of gelification until the assessed mass values variated less than 2.0%.
[0095] For both composites, samples of similar masses and with two orders-of-magnitude of mass were monitored in order to verify the maturation dependence from the volume of precursors (initial mass): they were studied samples of 1.5 and 3.0 ml.
[0096] From the obtained results it was inferred 72 and 50 hours the times of stabilization of mass-loss respectively for simple and combined composites. Consequently it was established 7 days as maturation interval for both formulation at the end of which the water contents were:
1. Simple composite a.sub.w=11±0.2 ppm;
2. Combined composite a.sub.w=19±0.6 ppm.
Example 3—Leaching Studies
3.1. Protein Quantification Method Used to Assess Protein Concentration of Composites and Supernatants
[0097] Determination of protein concentration was made by modified Lowry method. However the initial procedures did not guaranty a zero-mass-balance among initially existent protein in the composite samples and the protein transferred to the supernatant resulting from thermal-alkaline digestion (composite-sample submerged in 1.0 M NaOH during 10 min at 100° C. followed by ice incubation).
[0098] Methodology used herein was optimized in order to assure that all composite-samples were digested and inherent protein content was transferred to NaOH solution: mass of composite samples not bigger than 5.0 mg with granulometry lesser than 106 μm.sup.3. From each digestion medium 200 μl fractions were taken to be analysed.
[0099] Calibration of protein concentrations was made with BSA standard-solution (100% purity) at concentrations from 20 to 200 μg/ml: from each concentration 200 μl fractions were used to calibrate. Lowry-reagent was added (1.0 ml) to analysis-supernatant and BSA-standard solutions, and after 40 minutes 200 μl of Folin-Ciocalteau reagent (diluted 1:4) was added. After 10 min. of incubation liquid samples were absorbance read at 750 nm wavelength having samples been diluted when recorded values were higher than 1.0.
3.2. Protein Loss by Leaching
3.2.1. Simple Composite
[0100] It was firstly tested the leaching with samples of simple composite immobilizing three sorts of protein: bovine serum albumin (BSA), alkaline phosphatase (ALP) and generic antibodies (IgG).
[0101] The protein loads herein defined, were masses of lyophilized reagents added relatively to total volume of precursors still in sol-gel preparation. It was intended to obtain two groups of composites with different protein order-of-magnitudes of concentrations (1.0% and 10.0%) of BSA and ALP and one third typology with immobilized antibodies (IgG). So composite losses of protein were monitored in triple-essays in reference to different protein concentrations and using different proteins types.
[0102] The quantifications of protein contents retained in the composites were made before leaching (matured and dried composites) and after leaching essays at which the respective tested samples were 24 h dried at 40° C. followed by 10 days room exposure.
[0103] Having in mind protein leaching process would be proportional to area/volume ratio of grains two granulometries were tested: above 1 mm.sup.3 and under 750 μm.sup.3.
[0104] At first instance mass samples of 30 mg were used and experiences were performed by incubating matured fragmented composites in distilled water under orbital agitation of 200 rpm during 72 hours.
[0105] The incubation liquid volume was 1.0 ml in vials of 10 ml and at the end of each essay, the liquid media was decanted and centrifuged for 11 K rpm during 10 min. From the clarified supernatants three fraction of 200 μl were analysed in three separated quantification episodes to determine protein concentration in supernatants.
[0106] The procedures to determine after-leaching protein still retained in the composites followed the above described protocol of 5.0 ml sample-mass to ensure complete digestion of sol-gel and thus extensive release of immobilized protein to the analytical supernatant. Again three fraction of 200 μl were analysed in three separated protein concentration quantification episodes.
[0107] From the attained data mass-balance was made to quantify the losses of protein relative to the initial concentrations accordingly determined. On that view differentials were calculated among protein concentrations of the composites before and after leaching experiences. Additionally it was compared the amount of existent protein in the total mass of composite-samples (using previously mentioned data) before leaching and the total amount of protein in the volume of 1.0 ml incubating medium. Recorded data were averaged and respective numbers are presented at table 2.
TABLE-US-00002 TABLE 2 protein concentrations obtained at leaching tests of simple composite doped with different types of proteins and different protein concentrations. SIMPLE COMPOSITE BSA BSA ALP ALP IgG [protein].sub.in precursors (m/v) 1.0% 10.0% 1.0% 10.0% 0.5% [Protein].sub.immobilized in composite before leaching (μg/mg) 14.14 51.08 13.05 43.11 9.73 Δ [protein].sub.immobilized (μg/mg) Grain >1 mm.sup.3 0.85 9.63 0.69 5.37 1.14 BEFORE leaching − AFTER leaching Grain <750 μm.sup.3 4.25 11. 88 2. 47 9. 94 0. 93 Δ mass − protein (μg) Grain >1 mm.sup.3 182.77 783.37 26.95 584.48 43.93 Immobiliz..sub.before leaching − Grain <750 μm.sup.3 94.21 767.39 9.61 563.35 40.64 supernatant
[0108] Those results confirmed a somehow erosion of retained protein being much relevant as smaller the grain size: concentrations recorded at composites after leaching are as closer to the initial loads as bigger the essayed grain-size.
[0109] On the other hand with samples of lower granulometry there was a smaller difference between initial loads of composites and mass of protein solubilized in supernatant which is symptomatic of more transference of protein to the leaching media. The conjunct of results for the different protein types retained in simple composite and respective different loads confirm the leaching phenomena is proportional not only to the initial protein loads but to the grain-size as well.
[0110] The recorded data regarding IgG confirm results mentioned by Alstein and co-workers of an elevated retention of antibodies in this composite in which the difference before and after leaching is at the same order-of-magnitude for both granulometries.
[0111] Additionally it was tested the leaching of 5 mg and 60 mg mass-samples of simple composite doped with ALP at the maximum protein loads and minor granulometry at the same experimental conditions. The obtained numbers are presented at table 3 in which they are comparable with 30 mg mass-samples.
TABLE-US-00003 TABLE 3 protein concentrations obtained in leaching test of simple composite doped with alkaline phosphatase at the same concentration. but with samples of different mass. SIMPLE COMPOSITE: ALP-10% 5 mg 30 mg 60 mg [protein].sub.immobilized in composite before leaching. (μg/mg) 43.11 43.11 43.11 Δ[protein].sub.immobilized (μg/mg) Grain <750 μm.sup.3 11.70 9.94 4.23 BEFORE leaching - AFTER leaching Δ mass-protein (μg) Grain <750 μm.sup.3 28.60 563.35 1186.11 Immobilized.sub.before leaching - supernatant
[0112] The view of these figures permits to conclude the loss of retained protein was influenced by the spread-of-grain in the liquid media: for the same batch volume the differences of protein concentrations retained in the composite before and after leaching are as higher as the lower mass of the essayed samples.
[0113] On the other hand with greater-mass samples it is apparent a wider difference among initial protein loads of the composites and protein concentrations at leaching supernatants that means a lesser transference of protein into the incubation media comparing to the smaller-mass samples.
[0114] The effect of minor protein erosion with greater mass of simple composite for the same reaction-volume permits to deduce the more the simple composite will be used in the same batch-reactor volume the less protein total-loss will occur and consequently more stable will be the bioprocess activity of the doped simple composite.
3.2.2. Combined Composite
[0115] Having in mind the comparison with quantified protein loss recorded in simple composite the same experimental procedures were followed with combined composite immobilizing simple composite doped with the same protein which would further permit to monitor also the enzyme-activity response: alkaline-phosphatase.
[0116] It was synthetized combined composite immobilizing simple composite with the highest protein load (43.11 μg/mg) to obtain two groups of samples with different protein concentrations: 8.81 μg/mg and 23.14 μg/mg. Fragmented samples of 30 mg were essayed at small grain-size similar to previous experiments. Data treatment and experimental procedures were the same as those followed with simple composite leaching monitoring and table 4 presents the obtained results.
TABLE-US-00004 TABLE 4 protein concentrations obtained in leaching test of combined composite prepared from different masses of simple composite doped with alkaline phosphatase. COMBINED COMPOSITE [protein].sub.immobilized in composite before leaching (μg/mg) 8.81 23.14 Δ[protein].sub.immobilizaed (μg/mg) BEFORE leaching - AFTER leaching Grain < 750 μm.sup.3 0.55 0.78 Δ mass-protein (μg) Immobilized.sub.before leaching - supernatant Grain < 750 μm.sup.3 201.12 517.21
[0117] The combined composite with the initial highest protein concentration presented differential values before and after leaching of circa ten times lower than the simple composite (0.78 μg/mg versus 9.94 μg/mg) having a protein concentration about twice lower than the simple composite (23.14 μg/mg versus 43.11 μg/mg).
[0118] Likewise leaching essays using 5 mg and 60 mg mass-samples of combined composite were performed with low granulometry. Table 5 presents the obtained results.
TABLE-US-00005 TABLE 5 protein concentrations obtained in leaching test of combined composite doped with alkaline phosphatase at concentration of 23.14 μg/mg but with samples of different mass. COMBINED COMPOSITE 5 mg 30 mg 60 mg [protein].sub.immobilized in composite before leaching. (μg/mg) 23.14 23.14 23.14 Δ[protein].sub.immoloilizaed (μg/mg) BEFORE leaching - Grain <750 μm.sup.3 1.31 0.78 0.12 AFTER leaching Δ mass-protein (μg) Immobilized.sub.before leaching - Grain <750 μm.sup.3 43.33 517.21 1463.23 supernatant
[0119] The view of the figures confirm that trace-loss of protein is inversely proportional to the quantity of combined composite in the same liquid volume.
[0120] The last obtained results compared with simple composite results turn out to be evident the smaller loss of protein in combined composite: differences at protein retained in the composite before and after leaching. At the same time wider differences were recorded at combined composite comparing initial immobilized protein and protein content in supernatants which reflects greater retention grades.
3.2.3. Exposure to an Over-Concentrated Saline Medium
[0121] Simple and combined composite samples with the highest protein concentrations and smaller granulometries were exposed to high saline concentration solution 2.0 M NaCl in order to assess the loss of protein at a high ionic stress medium.
[0122] For both composites 60 mg mass-samples were tested by submerging 72 hours at 200 rpm orbital agitation, and protein quantification of leached composites and respective supernatants followed the same previous protocols. Table 6 presents the obtained data.
TABLE-US-00006 TABLE 6 protein concentrations obtained in leaching test of simple and combined composites doped with alkaline phosphatase and submerged in a solution of 2.0M NaCl. Exposition to NaCl 2.0M (samples of 60 mg) Simple Combined Composite Composite [protein].sub.immobilized in composite before leaching (μg/mg) 43.11 23.14 Δ [protein].sub.immobilized (μg/mg) Grain <750 μm.sup.3 4.90 0.61 BEFORE leaching - AFTER leaching Δ mass-protein (μg) Grain <750 μm.sup.3 784.66 1018.21 immobilized.sub.before leaching - supernatant
[0123] The recorded values regarding the difference between retained protein concentrations in composites before and after exposure to saline solution are in the same order-of-magnitude of those recorded by exposure to distilled water.
[0124] Comparing numbers of mass-protein within initial composites samples and mass-protein at supernatants at the end of the experiments a smaller difference is apparent at this case which could be symptomatic of a more intense transference of protein to the incubation media as reflex of the high ionic concentration.
[0125] These figures must however be seen as leaching solution was at an over-elevated saline concentration comparative to predicted usable physiological samples and time of exposure was considerable longer than it will be used at the preferable embodiment.
Example 4—Catalytic Essays
[0126] Having previously been verified that the invented formulation of combined composite has the capacity to immobilize/retain proteins, it was then intended to verify if one protein being an enzyme, is still active. In that context the activity of alkaline phosphatase (ALP; Sigma Aldrich Cat. No 10 567 752 001) was monitored firstly as free and then as immobilized enzyme within simple and combine composites.
[0127] Enzymatic essays recurred to spectrophotometric method to quantify the concentration of p-nitrophenol (pNP) as product of the standard-reaction of p-nitrophenylphosphate (pNPP) hydrolysis having in knowledge the respective 1:1 stoichiometry.
4.1. Enzyme Immobilized in the Simple Composite
[0128] After conventional free enzyme essays two samples of simple composite immobilizing ALP at precursors concentrations (m/v) of 8% and 10% were synthetized. At the end of 13 maturation days concentrations were respectively 4.8 and 6.2 mg.sub.protein/g.sub.composite.
[0129] Having in mind to reduce external access limitation of substrates molecules, the immobilizing matured composite samples were fragmented to grain-size at the range of 1 to 2 mm.sup.3. The essays were performed with simple composite samples of mass from 5.0 to 8.3 g and enzyme concentrations in batch-volumes were deduced from protein concentration of respective composites and correspondent mass of the samples.
[0130] Enzymatic essays were performed in 10 ml useful-volume reactors to which 3.6 ml of same buffer solution used in free-enzyme trials (phosphate buffer 100 mM pH=9.1), was added at room temperature. Kinetic studies started at the moment 0.4 ml of substrate solution (pNPP, solubilized at pH=9.1 buffer) was added and reactions ran for 28 minutes being collected 200 μl of liquid medium at 1:30 min. intervals. Initial substrate concentrations at the total reaction volumes were identical and around 3.0 mM. Data treatment had in account that along 28 min. time the reaction medium volume was progressively reduced by the collected samples but the mass of catalytic composite was the same.
[0131] The recorded values made possible to graph the kinetic profiles in function of enzyme loads as illustrated in
[0132] The obtained results turned evident that product concentrations were proportional to the mass of composite (with immobilized enzyme) for one same initial substrate concentration.
4.2. Combined Composite Immobilizing Simple Composite Doped with Enzyme
[0133] It was studied combined composite from the same formulation used at leaching tests (protein concentration of 23.14 μg/ml) and samples of mass from 14 to 20 mg were tested.
[0134] Grain-size of the samples, enzymatic essays experimental procedures and data treatment were exactly the same as those followed at simple composite kinetic studies. Similarly the quantification of existent enzyme in reaction media was made from the previously determined protein concentration of combined composite and the mass of samples used for each essay. Obtained resulted are presented in
[0135] Comparing maximum production of pNP, product of pNPP hydrolysis catalysed by the same enzyme immobilized in the two composite formulation it is evident that for identical masses of enzyme (11.65 mg.sub.simple-composite; 10.8 mg.sub.simple-composite versus 12 mg.sub.combined-composite; 11 mg.sub.combined-composite) and at a similar time reaction (15 min.) it was produced 8 to 9 times more of pNP with simple composite (40.57 mM; 26.76 mM) than with combined composite (5.03 mM; 3.03 mM).
[0136] Having in mind that combined composite of this invention is a coating of an enzyme-doped simple composite (encapsulated in a grain-size of compromise with robustness and external access of substrates) it is comprehensive that enzyme active-sites are less available and consequently, there is a lower production of enzymatic metabolite. However the lower catalytic efficiency is compensated by a more sustained retention of immobilized protein as early demonstrated by leaching studies.
Example 5—Drainage at Different Granulometry Profiles
[0137] It will now be presented the hydraulic tests performed with an aqueous dye solution and diluted blood permeating grained combined composite, in order to obtain residence-times compatible with utilization in a medical diagnostic device.
[0138] The combined composite herein tested was used as a filling-bed of a column also named analysis-chamber or reactor accordingly used at immune-essays or enzymatic tests. It was a rectangular box with dimensions of 4 cm.sub.length×3 cm.sub.width×1 cm.sub.depth made of transparent acrylic material that allowed to visualize the interior. The handling of its content was made by a drilled removable top-cap that once set at the box enabled elution of liquids through its interior. The surface of the opposite top was drilled as well to allow the exit of the liquids and the holes of both top-caps were 1.0 mm diameter.
[0139] Having in reference the useful mass value of 9 grams assessed by weighting the complete filling of water of the column (volumic-mass of 1 g/ml) it was planned to fill the column with grained composite. Such amount of mass was over-dimensioned relative to the application on diagnostic-device apparatus being estimated a final scale-down of 3:1. The aim of those studies was to test the drainage regime of liquid samples and thus grain-size was though as unique conditioning variable and accordingly it was used combined composite with same maturation grade.
[0140] Granulometry gradient profile was initially programmed to completely fill the useful volume and respective values were: [0141] Bottom layer: 15% (1.35 g)grain (G)>1.0 mm.sup.3; [0142] Bottom intermedium layer: 20% (1.80 g)
1.0 mm.sup.3>G>710 μm.sup.3; [0143] Central layer: 30% (2.70 g)
variable granulometry under 710 μm.sup.3; [0144] Top intermedium layer: 20% (1.80 g)
1.0 mm.sup.3>G>710 μm.sup.3; [0145] Top layer: 15% (1.35 g)
grain>1.0 mm.sup.3.
5.1. Monitoring Drainage of Dye Solution
[0146] After grain-size separation the two bottom layers were posed accordingly and the central layer was composed of grain-size ranged from 710 μm.sup.3 to 212 μm.sup.3. Immediate above layer was tried to be placed as programmed but only 1.6 g was able to be posed. The filling process was complied with washing with distilled water after posing each layer to improve grain compaction and reduce preferential run-off ways. Column was then left exposed 24 h to 40° C. dry-heat.
[0147] The essay started by measuring the volume of water saturating the column-content (adding water to the dry grained composite content until first drops came up at the bottom) useful liquid volume (ULV): 3.0 ml was measured. The propose was to assess the volume of retained water at such gradient profile. After saturation it was evident that at each added millilitre corresponded one millilitre drained at the bottom of the column, which proved that intending to incubate the whole content of the column, it should be added a liquid volume equal to ULV.
[0148] Additionally it was monitored the run-off time elapsed by each millilitre added until at least 900 μl were collected and such measuring was made for 25 trials at which 3 minutes was the average recorded times. Column was then left 24 h exposed to 40° C. dry-heat. Composite grain-gradient was again water saturated and one 1.0 ml fraction of Evans-blue (1.4 mg/100 ml) was eluted.
[0149] Absorbance calibration (λ=608 nm) of Evans-blue solution was made (since 1:1=1.426±6%; until 1:10=0.134±10%). Drainage flow of dye-solution was monitored by spectrophotometric readings of successive eluted/collected fractions of 1.0 ml distilled water and respective recorded values are presented in
[0150] After removing wet grained composite new dry grained composite was placed in the column following the same procedures except that central layer was composed by grain ranged from 710 μm.sup.3 to 300 μm.sup.3 and the upper layer was once again exclusively composed by grain 1.0 mm.sup.3>G>710 μm.sup.3 but this time took 1.16 g of grained composite. It was monitored the run-off time elapsed by each millilitre added until at least 900 μl was collected and such measuring was made for 30 trials at which 25 seconds was the average recorded time.
[0151] Column was then left 24 h exposed to 40° C. dry-heat. It was further assessed the volume of retained liquid (1.9 ml) and replicated the essay of dye-solution drainage. Collected data are presented at
[0152] After removing wet grained composite new dry grained composite was placed in the column following the same procedures except that central layer was composed by grain ranged from 710 μm.sup.3 to 500 μm.sup.3 and the upper layer was once again exclusively composed by grained composite 1.0 mm.sup.3>G>710 μm.sup.3 but this time took 0.98 g. It was monitored the run-off time elapsed by each millilitre added until at least 900 μl was collected and such monitoring was made for 25 trials at which 10 seconds was the average recorded time. Column was then left 24 h exposed to 40° C. dry-heat. It was further assessed the volume of retained liquid (1.0 ml) and replicated the essay of dye-solution drainage. Collected data are presented at
[0153] After removing wet grained composite new dry grained composite was placed in the column following the same procedures except that central layer was composed by grain ranged from 500 μm.sup.3 to 300 μm.sup.3 and the upper layer was once again exclusively composed by grained composite 1.0 mm.sup.3>G>710 μm.sup.3 but this time took 1.90 g. It was monitored the run-off time elapsed by each millilitre added until at least 900 μl was collected and such monitoring was made for 25 trials at which 1:30 minutes was the average recorded time.
[0154] Column was then left 24 h exposed to 40° C. dry-heat. It was further assessed the volume of retained liquid (2.0 ml) and replicated the essay of dye-solution drainage. Collected data are presented at
[0155] All of these results indicate that for one same volume occupied by this composite and permeated by water, the amount of retained liquid and the run-off times were conditioned by the smallest grain-size layer.
[0156] Table 7 illustrate these instances where values of granulometry and respective masses of bottom and bottom-intermedium layers were maintained. At same time variating granulometry of central layers but keeping its mass-content, resulted in variation of residence-time of permeating liquid.
TABLE-US-00007 TABLE 7 water drainage values with different grain-sizes of 2.7 g central-layer of combined composite. which also affected the useful volume of retained liquid and total mass held in the column. Intervals of Residence granulometry of the Total Volume of retained time central layer mass (g) liquid (ml) (seconds) 710 μm.sup.3 > G > 212 μm.sup.3 7.45 3.0 180 710 μm.sup.3 > G > 300 μm.sup.3 7.01 1.9 30 710 μm.sup.3 > G > 500 μm.sup.3 6.82 1.0 10 500 μm.sup.3 > G > 300 μm.sup.3 7.75 2.0 90
[0157] The drainage times of 1.0 ml of dye-solution for identical granulometry intervals at the central layer: [0158] 1. in the range of about 200 μm.sup.3: 90 seconds (500 μm.sup.3>G>300 μm.sup.3) that compares with 10 seconds (710 μm.sup.3>G>500 μm.sup.3); [0159] 2. for a wider range: 180 seconds (710 μm.sup.3>G>212 μm.sup.3) that compares with 30 seconds (710 μm.sup.3>G>300 μm.sup.3).
[0160] The drainage volumes for an identical granulometry intervals: [0161] a. in a range of about 200 μm.sup.3: 15 ml (500 μm.sup.3>G>300 μm.sup.3) that compares with 12 ml (710 μm.sup.3>G>500 μm.sup.3); [0162] b. for a wider range: 13 ml (710 μm.sup.3>G>212 μm.sup.3) that compares with 12 ml (710 μm.sup.3>G>300 μm.sup.3).
[0163] In summary in a compacted layer of combined composite of grain-size under 710 μm.sup.3 the permeation volume of an aqueous sample is proportional to the magnitude of the interval of grain-sizes and inversely proportional to the respective drainage times.
[0164] It was also evident that composite total mass within the column was inversely proportional to grain-size. Such finding would be expectable in face of compaction (as bigger as the smaller the grain) and consequently less inter-particles space that enables higher densities.
5.2. Monitoring the Drainage of Blood
[0165] Based on granulometry gradient found as better flowing regime of dye-solution (central layer 710 μm.sup.3>G>500 μm.sup.3) it was tested drainage of diluted blood. Samples collected from the inventor were anticoagulated with EDTA (50 mg/ml) and diluted with phosphate buffer saline 10 mM pH=7.3. Diluted samples of 1.0 ml were eluted followed by fractions of 1.0 ml of distilled water and run-offs were assessed by reading collected fraction at absorbance of 540 nm wavelength which targets haemoglobin (Hb) molecule. Calibration was made for Hb concentrations from 0.6 mg/ml (absorbance=0.314±7%) to 3.0 mg/ml (absorbance=1.635±4%). Sampled blood Hb concentration was 150 mg/ml (±1.0%). Results recorded from three trial are presented at
[0166] Before these results it is evident that diluted blood run-off occurred mainly after 4.0 ml of permeating water which agrees with previous data of dye-solution drainage. So it can be concluded that permeation of blood samples at dilutions herein referred was identical to run-offs recorded with Evans-blue solution concentrated at 1.4 mg/100 ml and those are two rheological identical liquid media.
[0167] Thus it seems reasonable to infer that drainage of 1:200 and 1:100 blood samples will have the same drainage regime at others gradient-grain of combined composite which enable to preview a scenario of good access to immobilized proteins (antigens/antibodies) by biological-sample fluids with viscosity and density similar to water.
5.2.1. Monitoring the Washing Drainage of Blood
[0168] According to the recorded run-off profiles either with Evans-blue or with blood solutions it was studied the washing drainage of the combined composite gradient-granulometries firstly permeated by blood and then using distilled water in a first fraction of 10.0 ml followed by five sequent elutions of 1.0 ml. At the series of three essays performed with 1:100 diluted blood it was collected 96±0.5% of added Hb right at the first fraction of 10.0 ml as illustrated in
[0169] Based on those results it was then studied the washing of the grained composite using lesser diluted samples of blood down to 1:30. The protocol in between every experimental episode, the grained content of the column was abundantly washed with distilled water and then 35° C. dried for 24 hours. Previous to addition of a new blood-sample the grained composite was saturated with 20.0 ml of distilled water.
[0170] It was monitored the clearance of the collected fractions at elution of three fractions of 10.0 ml of distilled water and one last of 1.0 ml and the obtained results are presented at
[0171] From the collected data it is evident that even for the most concentrated blood-samples the drainage of 1.0 ml analytical blood-unit-volume occurred mostly with elution of 10.0 ml water being that in third and fourth collected fractions, the Hb concentrations were lower than 1.0 mg/ml.
[0172] Having in mind these tests were performed with a total column-mass-filling of 6.82 g and the scale of these tests was also approximately 3:1 then at the final utilization scale the estimated mass of combined composite for the same gradient (relative proportions) will range among 1.5 g to 3.0 g. At this view and in a linear scaling of granulometry gradients herein studied, it is reasonable to estimate an effective washing volume of 10 ml.
Example 6—Combined Composite in Enzymatic Reactor
[0173] At this example it was studied the enzymatic activity for at least 20 cycles of activity of the combined composite filling afore described acrylic column and supplied by fractions of substrate-solution that permeated the grained-composite pushed by atmospheric pressure in a vertical plug-flow regime. The propose was to quantify the reaction-yield of conversion of p-nitrophenylphosphate (pNPP) into p-nitrophenol (pNP) catalysed by alkaline phosphatase immobilized in combined composite.
[0174] It was used the enzyme concentration threshold of correspondence among specific activity and load-of-enzyme: 0.4% (m/m). It was used the grain-size gradient previously found as the best run-off for enzymatic process: residence time of at least 1.5 minutes (central layers granulometry 500 μm.sup.3>G>300 μm.sup.3). Total amount of combined composite mass was 7.75 g and its compacting was effected to minimize preferential run-off ways. The volume of retained liquid was 2.0 ml.
[0175] After filling the column the experience started with abundant wash of the grained gradient composite at room temperature with the same eluent previously used in similar catalytic experience (phosphate buffer 100 mm pH=9.1) and herein used as eluent too. It was then added 4.0 ml of substrate solution (solubilized in pH=9.1) and after 15 minutes incubation the grained composite was permeated by three fractions of 2.0 ml of eluent followed by eight fractions of 1.0 ml. Quantification of metabolite concentration was direct from absorbance readings once the enzymatic reaction product was collected at one same volume added at respective elution fraction (contrarily to the batch process early described).
[0176] Data treatment started by calibration of concentrations of the product (pNP) for absorbance values (λ=405 nm) under 1.0: up to 56 μM (see the example
[0177] Abs.sub.405 nm values attained in each of the 11 collected fractions were converted to pNP concentration. Having in known the volume of each collected fraction it was computed the number of moles existent in each collected fraction. It was summed the number of collected moles of reaction product. Additionally knowing the substrate (pNPP) concentration it was computed the respective number of moles initially supplied based of the solution-volume fed to the reactor (see table 8).
TABLE-US-00008 TABLE 8 data treatment of the first experiment results at bioreactor of combined composite immobilizing alkaline phosphatase, on hydrolysis of p-nitrophenylphosphate. Mass of pNPP (mg) 5.9 [ALP] (mg/g) 4.31 Molar-weight pNPP (g/mol) 371.14 Solvent volume (mL) 50 [pNPP] (M) 0.0003179 Volume of added solution (mL) 4 Number of pNPP added moles 1.272E−06 Slope Origin ordinate [pNPP] (mM) 0.318 58.987 0.6876 Collections [pNP] Number of No Dilution Abs.sub.405 nm M moles pNP 1 (2 mL) 1 0.0748 5.10E−06 1.01997E−08 2 (2 mL) 1 0.0829 5.58E−06 1.11552E−08 3 (2 mL) 1 0.0756 5.15E−06 1.0294E−08 4 1 0.0723 4.95E−06 9.90472E−09 5 1 0.0708 4.86E−06 9.72776E−09 6 1 0.0631 4.41E−06 8.81936E−09 7 1 0.0575 4.08E−06 8.15871E−09 8 1 0.0498 3.63E−06 7.25031E−09 9 1 0.0495 3.61E−06 7.21491E−09 10 1 0.0389 2.98E−06 5.96439E−09 11 1 0.0314 2.54E−06 5.07958E−09 9.37687E−08 TOTAL
[0178] Knowing this reaction is 1:1 stoichiometry percentage conversion yields were calculated: product number of moles*100/substrate number of moles. At the first trial it was used a substrate concentration of 318 μM and the recorded yield was 7.4%. Next trial used a substrate solution one order-of-magnitude higher: 3.03 mM and the yield was 19.5%.
[0179] The third trial kept substrate concentration and prolonged incubation period for 20 minutes. The recorded yield was 25.7%. From that result it was inferred longer incubation time allowed a more extensive hydrolysis of the added substrate. Fourth trial was a replication of the third and the recorded yield was 24.1%.
[0180] At the fifth trial substrate concentration was reduced to ½ and the recorded yield was 23.1%, and at the next trial with the same conditions the value recorded was 29.7%.
[0181] At seventh trial substrate concentration was reduced to ¼ 750 μM. The recorded yield was 36.4%. That last trail was replicated for twice and recorded yields were: 38.9% and 35.6%.
[0182] At one next experimental episode three trial were performed replicating the last protocol and recorded yields were: 32.9%; 28.8% and 28.2%. At one new series of five trials the recorded yields were: 25.4%; 24.9%; 27.5%; 27.8% and 25.3%. At one other experimental episode of three trial the recorded yields were: 16.6%; 19.0% and 18.9%.
[0183] At the two following trails the recorded yields were: 21.7% and 21.1%. At one last experimental episode nine trial were performed and the recorded yields were: 18.2%; 16.0%; 15.5%; 16.7%; 16.3%; 14.6%; 13.9%; 14.0% and 19.0%.
[0184] Compilation of those mentioned figures is presented at
[0185] Before these results it is reasonable to conclude that combined composite is consistently applicable at immobilization of an enzyme, alkaline phosphatase, while retaining its activity. Having additionally in mind that experimental normalization was attained after seven essays of procedure adjustments it becomes reasonable to preview the utilization of this composite formulation for a wider number of cycles of more than those 20 initially pointed.
Example 7—Preferred Utilization of Combined Composite at Diagnostic Device
[0186] Now it will be described one preferential utilization of the invented combined composite applied to medical diagnosis onto a portable device constructed as a conjunct of vertical parallel operating units. It complies the maximum of 4 units and each unit includes two chambers, one analytical and one negative control chamber.
[0187] All the chamber are filled with grained combined composite in which the analytical one is filled with combined composite immobilizing an immunologically responsive protein, an antigen or an antibody associated with the diagnosis of a human being or superior animal pathology and the negative-control chamber is filled with combined composite immobilizing an immunologically non-responsive protein.
[0188] The functioning of the device is based on immune-diagnosis principles where a biological sample suspected to carry antibodies (or antigens) related to a pathology, by operating the device those molecules will ligate to respective specific ligands. The latter will be antigens (or antibodies) immobilized in the combined composites whose physical characteristics of pore-size enable the permeation of those liquid biological samples. After antigen-antibody ligation a primary immune-complex will be formed and the composite must be washed to remove the excess of debris and non-ligated proteins.
[0189] At the first instance of this example it was preliminary tested the response of the combined composite immobilizing a simple composite doped with mucin 1.8% (m/m). The combined composite was used to fulfil a test-column homologous to one device-unit. The test-column was filled with grained composite with granulometry gradient-profile analogous to blood drainage tests.
[0190] The immobilized antigen mucin is a molecule with a glycidic structure of sialic-acids homologous to surface-ligands of tumour cells (e.g. breast cancer). It was tested the formation of primary complex by ligation of an antibody-reagent kindly supplied by Glycoimmunology-Group of Science and Technology Faculty of Universidade Nova de Lisboa. Such antibodies had previously demonstrated a high ligation affinity to neoplasic tissues. The biologic samples at those tests were the supernatants from the culture of antibody-producer animal cells (hybridoma-cells).
[0191] After incubation of biological sample, the grained composite was washed to remove the excess of non-ligated antibodies. The detection of the formed immune-complexes was made by addition of a secondary antibody labelled with peroxidase. Such antibody had specific affinity to the referred primary antibody. The addition of the secondary antibody provided the formation of a secondary immune-complex: antigen.sub.immobilized-antibody.sub.biological-sample-antibody.sub.labelled. The grained composite was washed once again to remove the excess of non-ligated proteins.
[0192] It was then added the chromogenic substrate of peroxidase: 3,3′,5,5′-tetramethyl-benzidine. The substrate was degraded by the secondary antibody and consequently conferred blue-green colouring to the combined composite thus revealing the existence of the primary antibodies in the biological sample. The procedure was validated by the similar test performed with homologous grained combined composite doped with the non-immunogenic protein bovine serum albumin, at which the combined composite did not gain any colour as illustrated by
[0193] Recycling the grained combined composite enabled the respective re-utilization and that procedure was made by elution of a chaotropic solution that disrupted the ligations of primary and secondary antibodies. Grained combined composites were then washed to remove all the released proteins.
7.1. Preliminary Essay Protocol
[0194] At the preferential utilization of the invented combined composite in medical diagnosis preliminary essays must be performed in order to test Sensitivity and Specificity criteria.
[0195] Samples of combined composite immobilizing one same simple composite with a unique concentration of antigen/antibody/BSA will be tested. Protocol includes 8 essay-vials having each vial a sample of 100 mg of grained composite with granulometry range of 100 μm.sup.3 to 300 μm.sup.3: [0196] i) four essay-vials in which one of each vial will have a sample of grained combined composite immobilizing respectively 25; 50; 75; 100 mg of simple composite with BSA. Those masses of simple composite are relative to 3.0 ml of precursors when combined composite was synthetized. [0197] ii) four essay-vials in which one of each vial will have a sample of grained combined composite immobilizing respectively 25; 50; 75; 100 mg of simple composite with antigen/antibody. Those masses of simple composite are relative to 3.0 ml of precursors when combined composite will be synthetized.
7.1.1. Reaction Solutions
[0198] Phosphate Buffer Saline 10 mM pH=7.3±0.3 (25° C.)+0.05% (m/v) Tween-20 (PBS-T) used at: [0199] Dilution of the biological samples; [0200] Dilution of the secondary labelled antibody; [0201] Washing the grained combined composite.
[0202] Chromogenic peroxidase substrate: 3,3′,5,5′-tetramethy benzidine (TMB) used under dilution with distilled water.
[0203] Recycling solution Restore™ Western Blot Stripping Buffer; Thermo Scientific (Sb) used under dilution with distilled water.
7.1.2. Experimental Procedures by Vial
[0204] Step 1: Conditioning of the composite. [0205] Grained combined composite will be humidified with 2×2.5 ml PBS-T: Incubation time: 5 min. Liquid medium will then be decanted. [0206] Step 2: Elution of the biological sample. [0207] Biological liquid sample will initially be diluted (in PBS-T) at 1:10 for the respective volume of 2.5 ml. It is intended to obtain a result of unequivocal colouring of the composite doped with antigen (or antibody) and unequivocal clearance of the composite doped with BSA and so dilution grade of biological samples will be optimized since 1:5 to 1:13. Incubation time: 20 min. Liquid medium will then be decanted. [0208] Step 3: First wash with 2×2.5 ml PBS-T. Liquid medium will then be decanted. [0209] Step 4: Elution of the secondary antibody. [0210] Secondary antibody labelled with peroxidase will initially be diluted (in PBS-T) at 1:3333 for the respective volume of 2.5 ml. It is intended to obtain a result of unequivocal colouring of the composite doped with antigen (or antibody) and unequivocal clearance of the composite doped with BSA and so dilution grade will be optimized since 1:2000 to 1:5000. Incubation time: 5 min. Liquid medium will then be decanted. [0211] Step 5: Second wash with 4×2.5 ml PBS-T. Liquid medium will then be decanted. [0212] Step 6: Elution of the chromogenic substrate. [0213] Peroxidase chromogenic substrate, will initially be diluted with distilled water at 1:4 for the respective volume of 2.5 ml. It is intended to obtain a result of unequivocal colouring of the composite doped with antigen (or antibody) and unequivocal clearance of the composite doped with BSA and so TMB dilution grade will be optimized to 1:3. Incubation time: 10 to 20 min. Liquid medium will then be decanted.
[0214] Results: colouring of the combined composite is consequence of immune-complexes formation. Intensity of the attained colour in each of the essayed composites samples will give indication of analytical criteria of the method:
Sensitivity—the lesser mass of simple composite imprisoned at the combined composite (trendily 25 mg) that will enable to detect the existence of antibodies (or antigens) in biological samples;
Specificity—the greatest mass amount of simple composite imprisoned at the combined composite (trendily 100 mg) that will reveal no cross-reaction at: [0215] I. no colouring of the negative-control combined composite; [0216] II. no colouring of combined composite tested with biological samples containing different specificity antibodies (or antigens). [0217] Step 7: Recycling. [0218] Striping buffer, will initially be diluted with distilled water at 1:16 for the respective volume of 2.5 ml. According to the obtained clearance Sb dilution will be optimized since 1:10 to 1:32. Incubation time: 10 min. Liquid medium will then be decanted. [0219] Step 8: Third wash with 6×2.5 ml PBS-T. Liquid medium will then be decanted.
7.1.3. Filling of Chambers
[0220] This example refers the operation of the diagnosis medical device conceived for a maximum capacity of eight chamber as early mentioned. Each chamber had the useful internal volume of 9 cm.sup.3 and was filled as forwardly described.
[0221] After seven days maturation the grained combined composite was washed with distilled water and then dried for 3 hours at 37° C. Grain-size separation defined 5 granulometry classes:
[0222] G.sub.IG>1.0 mm.sup.3;
[0223] G.sub.II1.0 mm.sup.3>G>710 μm.sup.3
[0224] G.sub.III G>710 μm.sup.3>G>500 μm.sup.3
[0225] G.sub.IVG>500 μm.sup.3>G>300 μm.sup.3
[0226] G.sub.V G>300 μm.sup.3>G>106 μm.sup.3
[0227] The filling of each chamber was composed by 6 layers as referred below from top to bottom with respective mass of grain-size:
[0228] Layer 6: G.sub.I200 mg;
[0229] Layer 5: G.sub.III450 mg;
[0230] Layer 4: G.sub.IV450 mg;
[0231] Layer 3: G.sub.V 120 mg;
[0232] Layer 2: G.sub.II210 mg;
[0233] Layer 1: G.sub.I350 mg;
[0234] The compaction of each layer was optimized by eluting 3.0 to 5.0 ml of distilled water reducing so the formation of preferential run-off ways. Once deposited all the layers rest free-volume of the chamber was filled (with glass spheres of 0.8 to 1.2 mm diameter) up to 1.0 cm from the top. That upper space was left free in order to have a visible regurgitation window.
[0235] The whole piece of 8 chamber was left 48 hours at 37° C. and after that retained liquid volumes were quantified by eluting 10 ml of distilled water. By measuring after the collected water the differentials were 2.0±0.4 ml.
7.1.4. Operation of the Device
[0236] Biological samples placed at liquids-collector drained directly to analytical-chambers posed underneath as presented in
[0237] The minimal liquid volumes utilized on operation of the device were twice the values indicated below as each analysis-chamber was operated simultaneously with respective negative-control-chamber. [0238] Step 1: Conditioning of the composite. [0239] Grained combined composites were humidified with 10.0 ml PBS-T. [0240] Step 2: Elution of the biological sample. [0241] Biological liquid samples were diluted (in PBS-T) at 1:5 up to 1:13 for the respective volume of 4.0 ml. Incubation time: 20 min. [0242] Step 3: First wash with 10.0 ml PBS-T. [0243] Step 4: Elution of the secondary antibody. [0244] Mother solution of secondary antibody labelled with peroxidase was PBS-T diluted at 1:2000 up to 1:5000 for the respective volume of 4.0 ml. Incubation time: 5 min. [0245] Step 5: Second wash with 2 fractions of 10.0 ml PBS-T. [0246] Step 6: Elution of the chromogenic substrate. [0247] Peroxidase chromogenic substrate was diluted with distilled water at 1:4 up to 1:3 for the respective volume of 4.0 ml. Incubation time: 10 to 20 min.
[0248] Results: analytical-chambers filled with grained combined composites doped with mucin gained blue-green colour approximately proportional to mucin concentration and analytical-chambers filled with grained combined composites doped with BSA at mucin-concentration correspondence gained no colour. [0249] Step 7: Recycling.
[0250] Striping buffer was diluted with distilled water at 1:16 up to 1:10 for the respective volume of 7.0 ml. Incubation time: 10 min. [0251] Step 8: Third wash with 3 fractions of 10.0 ml PBS-T.
[0252] For the operation of the whole 8 chambers the liquid-volumes used were at the order of magnitude of linear correlation to the values referred to one chamber.