Method for detecting immunoreactive compounds

FIELD: immunology.

SUBSTANCE: the present innovation deals with one-stage detection of a bound analyte with a conjugate consisted of an analyte-stereospecific compound (anti-analyte) being covalently conjugated with suspensoid particles of water-insoluble dyestuffs, as coumassi R-250, and/or acridine yellow, and/or acridine orange, and/or 2.4-nitrodiphenyl hydrazine, and/or fluorescein. Application of an anti-analyte with a colored suspensoid label in technology for obtaining conjugates for analysis of one-stage covalent binding leads to considerable simplification of synthesis procedure, increased economy, and reproducibility and higher sensitivity of detection systems. Thus, the innovation enables to improve sensitivity and reliability of stereospecific analysis and optimization of technology to obtain reagents applied in this process being necessary for detection (stereospecific conjugates).

EFFECT: higher accuracy of detection.

5 ex

The invention relates to the field of immunobiotechnology and can be used in the production of a highly sensitive test systems for (qualitative and quantitative) determination of antigens, antibodies and other immunoreactive compounds, as well as in technology asynchronically and reliable diagnostic tests based on stereospecific interactions, such as immunohistochemistry, genetic hybridization and ligand-receptor. The intended scope of the invention includes, in particular, the diagnosis of several diseases, including and especially dangerous: AIDS, hepatitis, chlamydia, botulism, tetanus, diabetes, etc. and the analysis of somatic States: early diagnosis of pregnancy, timing of ovulation and other

The proposed solution applies to objects, one of which is for carrying out another.

The analogue of the claimed objects to the technical essence and the achieved result is way stereospecific analysis using conjugates antianalytic with particles of colloidal gold and the way to obtain conjugates for this analysis (Roth j, Heitz P.U., Immunolabeling with the Protein A - Gold Technique: An Overview, Ultrastructural Pathology, 1989, 13:467-484).

In similar inquirey with immobilized on a solid phase carrier first antianemia defined ligand (an is lit) detects the second antianemia, labeled particles of colloidal gold. In the process of detection conjugate "Antianemic/colloidal gold provide direct visualization of specifically bound analyte. This eliminates an additional step of rendering the conjugate (as, for example, the substrate stage manifestations of enzyme conjugates in the dot-ELISA).

The main disadvantage of analog is the relatively low sensitivity associated with low chromophores (color in the visible range of the light spectrum) particles of colloidal gold, which is connected to both the optical characteristics due to the chemical nature of the colloids of gold, and the limited size of the particles of the colloid.

As for the method of obtaining conjugates antianalytic color suspensoids label, known for analogue use the non-covalent conjugation procedure, which essentially is a simple connection of freshly prepared metasta-stable colloid gold with antianemia in the area defined optimal concentrations of the latter, which for each specific antianalytic should be chosen in individual experiments. In connection to these two reagents are only non-chemical adsorption of communication that are much weaker than covalent bonds. The result is e obtained in the analog conjugates are not sufficiently stable, what complicates the possibility of their long-term storage and, in addition, imposes a number of significant restrictions on their use in the procedures of analysis - for example, does not allow using commonly used in the vast majority of solid-phase analysis in order to suppress nonspecific interactions detergents, which are able to disrupt non-covalent conjugate "Antianemic/colloidal gold. In General, the method-analogue has poor sensitivity and reliability.

The prototype is the patent of the Russian Federation No. 20899212 from 10.09.1997, in which the label was applied particles of colloidal carbon and dyes from a number of formisano, deformation and Sudan. The problem of improving system reliability analysis decided by covalent conjugation of the label with antianalytic.

These labels have a very high degree of optical density in the visible range of the spectrum, significantly higher than the optical density of all known color labels including colloidal gold, as well as chromophores products conversion precipitating substrates of enzyme labels in immunoassay version resynchronising analysis. The use of high optical density of the colloidal particles of carbon and dyes from a number of formisano, deformation and Sudan helped to resolve the problem is mu increase the sensitivity of systems resynchronising analysis.

The problem of increasing the reliability analysis and stability used in the analysis of the detecting reagents decided by covalent conjugation antianalytic with colored suspensoid particles. To do this, the particles were covered with a water-soluble polymer capable of relatively strongly absorb on the surface of the particles. Sorption of the polymer allows to obtain stable solutions suspensoid a color label. Processing of the received complex bifunctional reagent leads to covalent linking between the adsorbed molecules of the polymer and at the same time to implement on the surface of complex reactive groups of a bifunctional reagent. The subsequent connection is freed from excess conjugating reagent activated tag antianemia ends covalent proshivkoi molecules antianalytic to the surface color of the label.

The disadvantages of the prototype is that the production of carbon particles is a very tedious process, and formazane, deformatory and Sudan is not only limited produced by the domestic industry, but also quite expensive. In addition, in the production method of conjugate double-provided phase chromatography and the concentration process of the reagent, which not only leads to elongation, more complicated and expensive synthesis, but also to a significant, up to 25%, the sweat is Yam material.

The invention is aimed at solving problems: improving the sensitivity, reliability and versatility of the method of analysis, and optimization of the method of obtaining the conjugate.

The first task is solved by the use as the label of the second antianalytic particles of water-insoluble dyes: acridine orange, 2,4-nitrodiphenylamine, Kumasi R-250. The proposed labels have a very high degree of optical density in the visible range of the spectrum available, relatively cheap and do not require any preliminary preparation procedure for synthesis.

The optimization method of obtaining conjugate for detection and increase the sensitivity of detection systems solve the following way. Particles cover a water-soluble polymer, which is directly affine connection, i.e. antianemia, firmly adsorbed on the surface of the particles. Sorption of the polymer allows to obtain stable solutions suspensoid a color label. Adsorbed polymer exhibits in the aqueous phase is a functional group capable of covalently reacting with the Homo - and/or heterobifunctional conjugating compounds. Subsequent processing of the received complex bifunctional reagent, taken in a large molar excess relative to the functional groups of the polymer, about westlea in the presence in the reaction mixture antianalytic, leads to covalent linking between the adsorbed molecules of the polymer, the implementation on the surface of complex reactive groups of a bifunctional reagent and the covalent presence molecules antianalytic to the surface of the colored label. That is, the stabilization reaction, the activation and conjugation occur simultaneously. In the result, the inventive method allows to obtain the conjugates of strong covalent structures that are stable in solutions, including in the presence of high concentrations of detergents, and very stable during prolonged storage in a wide temperature range (which depend only on the natural characteristics antianalytic). Furthermore, covalently cross-linked polymer on the surface of particles is not the monolayer, as in the prototype, and multiclonal amplified structure, which leads to an increase in the number of reactive affinity groups antianalytic. One-step, unlike the prototype, the nature of the conjugation, excluding one-stage chromatographic purification stage and the concentration of the added benefit of making the inventive method is much easier in a procedural sense, much cheaper, more reliable, reproducible, regardless of the range of concentrations of those or other antianalytic. Created by way of a positive effect on the whole x is specified considerably more in comparison with similar reliability and versatility.

The following examples are provided only to demonstrate the capabilities of the present invention, but in no way limit both the range of possible technological methods of synthesis of specific detection reagents, and the area of possible application of the invention as a whole.

Cited examples of specific quantitative parameters, even in the area specified ranges are not fixed by the claims, because they aren't the principal terms of the proposed methods with respect to their significant differences, which in the broadest sense is the use of new non-ferrous suspensoids labels in the analysis and their one-step covalent conjugation with antianalytic.

The METHOD is AS follows

Obtaining conjugates color suspensoids labels with antianalytic (Protein A, Protein G, Streptavidin, Antibodies).

To 20 ml antianalytic 10-50 mg/ml in 0.01-0.2m phosphate buffer solution of pH 6-8,5 (FBI) add 0.1 to 2 grams of dry marks. The mixture is stirred on a magnetic stirrer or a rotary shaker type "Vortex" in a period of time sufficient to complete the preliminary peptization (wetting) of the particles. The resulting suspension is voiced in an ultrasonic disintegrator. Conditions of ultrasonic treatment suspension (intensity and powerfully is th the number and length of cycles of sound) picked so that we break the suspension while simultaneously (possible) cooling is not heated to temperatures above 40°and the total time of ultrasonic treatment on the suspension amounted to 120 minutes. The resulting effective ultrasonic disintegration of suspensoid centrifuged at 6000 g for 5-10 minutes to remove the remaining relatively large particles. Covered antianemia suspensoids particle labels with dimensions of 150 - 160 nm, contained in the supernatant, further treated with a solution of glutaraldehyde. The range of effective concentrations of the reagent - 1-25%, the processing time from 120 to 480 minutes. The resulting conjugates freed from excess antianalytic by centrifugation or gel filtration on a column of Separately (6B, 4B, 2B; CL-6B, CL-4B, CL-2B), Sephacryl S-300 or other suitable gels (Toyopearl, Ultragel and others), with the limit exceptions are not below one million daltons for globular proteins. Fraction released to idle the volume containing the conjugate are pooled and add BSA and glycerol to a final concentration of 1% and 20%.

According to the described scheme were synthesized conjugates: a G Protein - Acridine orange, a G Protein - Kumasi R-250, Streptavidin - 2,4-nitrodiphenylamine, Streptavidin - Kumasi R-250, the STI-HCG antibodies (AB 0421) - Kumasi R-250.

The working concentration of the conjugates used in the analyses is 0.03%, calculated on the dry weight of the label.

The method of determination of immunoreactive compounds is illustrated by the following examples.

Example 1. Immunochromatographic determination of human IgG using Protein conjugate G - Kumasi R-250 in comparison with Protein And labeled with colloidal carbon, and Protein And labeled with colloidal gold with a particle size of 40 nm.

In the analysis used strips of nitrocellulose (Bio-Rad) with a pore size of 5 μm with immobilized them in spots human IgG (applied amount of 0.002 ml) of multiples of 10 dilutions of 1 mg/ml, 100 μg/ml, 10 μg/ml, 1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, 100 PG/ml of the Edge strips, pre-blocked with 1% solution of casein in phosphate buffer solution containing 0.05% Tween-20 (FBI-TV), was placed in the solution of conjugates for 10-15 seconds (long enough to pass through capillary forces front reagents distance of about 10 mm), and then the FBI-TV. As the front of migrating diagnosticum zone immobilization of the ligand was observed manifestation in the form of colored spots analysis. The sensitivity of a detection system using a conjugate Protein G - Kumasi R-250 was 2 PG/spot, with the conjugate Protein a - Carbon - 20 PG/spot, with the conjugate Protein a - Gold - 200 PG/spot.

P is the iMER 2. Immunochromatographic determination of human IgG using Protein conjugate G - acridine orange in comparison with Protein And labeled with colloidal carbon, and Protein And labeled with colloidal gold with a particle size of 40 nm.

In the analysis used strips of nitrocellulose (Bio-Rad), similar to that described in example 1. The procedure of comparative analysis was carried out as in example 1, but instead of a conjugate Protein G - Kumasi R-250 as the detecting reagent used conjugate Protein G - acridine orange. The sensitivity of a detection system using a conjugate Protein G - acridine orange was 8 PG/spot, with the conjugate Protein a - Carbon - 20 PG/spot, with the conjugate Protein a - Gold - 200 PG/spot.

Example 3. Determination of anti-HIV antibodies on non-porous solid phase using a conjugate Protein G - Kumasi R-250.

As the solid phase used the tablets of white opaque polystyrene, on the inner surface of the hole which barbirolli in the form of spots (TFP) first ationality: "envI", which are synthesized in E. coli recombinant polypeptide containing antigenic determinants of glycoproteins gp120 and gp41 of HIV-1; "gagI", which are synthesized in E. coli recombinant polypeptide containing antigenic determinants of proteins P24 and R17 HIV-1; "polI", representing the th a synthesized in E. coli recombinant polypeptide, containing antigenic determinants polymerase R51 HIV-1; "envII", which are synthesized in E. coli recombinant polypeptide containing antigenic determinants of glycoprotein gp110 and gp38 HIV-2; control antigen specificity of the reaction (negative control), which is synthesized in E. coli recombinant polypeptide that does not contain antigenic determinants of HIV-1 and HIV-2, and the control antigen correctness of the reaction (internal positive control), which is a human IgG. In sensitized wells contributed 0.3 ml serum HIV-infected patients from dilutions that are multiples of two. After 30-minute incubation and washing FBI-TV in holes made conjugate Protein G - Kumasi R-250 for 15 minutes, then remove conjugate, watched the results of the analysis in clear contrast spots. Protein G in this case acts as a second antianalytic in relation to defined immunoreactive connection (HIV antibody). In parallel, the bound antibodies was determined by Protein And labeled with carbon, Protein And labeled with horseradish peroxidase, and Protein And labeled with colloidal gold with a particle size of 40 nanometers. The sensitivity of the assay was evaluated by final serum dilution giving distinct from background stain. In the result of comparative analysis of got the feeling italmost definition using the conjugate Protein G - Kumasi R-250 - 1/40960, carbon conjugate 1/20 .480, using enzyme conjugate - 1/1 .280 (standard procedure manifestations 4-chloronaphthalen) and are virtually indistinguishable results determine the conjugate Protein a - Gold.

Example 4. The dot-determination of anti-HIV antibodies on the porous solid phase using conjugate Streptavidin - 2,4-nitrodiphenylamine.

As the solid phase used strips (4 mm × 45 mm) nitrocellulose (Bio-Rad) with a pore size of 0.45 μm with immobilized them in the form of transverse lines first antianalytic, the same as in example 3.

Sensitized strips were placed in the grooves of the tablet, which was introduced in 1.0 ml of serum HIV-infected patients from dilutions that are multiples of two. After 30-minute incubation and washing FBI-TV in wells made a solution of biotinylated rabbit anti-human IgG antibodies. After 30 minutes, the antibody solution was removed, the strips were washed FBI-TV three times in grooves made conjugate Streptavidin - 2,4-nitrodiphenylamine for 15 minutes, then remove conjugate and wash strips FBI-TV, watched the results of the analysis in the form of colored lateral stripes in the areas of immobilization of the first antianalytic. The detection sensitivity according to the latest clearly visualized colored strip in a series of serial dilution of HIV-positive serum was 1/12800. Definition under similar conditions using as a detecting reagent enzyme conjugate Streptavidin-horseradish Peroxidase and 4-chloroptera as a precipitating substrate demonstrated sensitivity 1/16000.

Example 5. Dvuhsvetnoe immunofiltration (if) determination of human chorionic gonadotropin (HCG) using suspensoids conjugate anti-HCG antibodies (AB 0421) - Kumasi R-250.

Preparation connecting HCG filters.

In the 5-ám nitrocellulose filter with a diameter of 25 mm using a Hamiltonian syringe inflicted monoclonal anti-HCG antibodies (AB 0420) to the alpha subunit of HCG, 1 mg/ml in the FBI in the form of a vertical strip length 10 mm After drying of the filter after 30 minutes, put in the same place in the same strip, but perpendicular to it, the standard HCG from a solution of concentration 1 mg/ml in the FBI and after 30 minutes of drying, the filter was blocked by soaking in 1% casein in FBI-TV for 1 hour at room temperature, after which washed FBI-TV and again dried. The filter was placed in a fixture for immunofiltration (IFS), which is a plastic cylinder with a diameter of 27 mm and a height of 50 mm, with the upper part of the window in the form of holes with a diameter of 23 mm and filled with a highly porous paper sorbent with high absorption capacity.

Identify the bookmark standard HCG.

The standard HCG bred multiple of 2 FBI-TV and the urine of a healthy donor, added tween-20 0.1%. 120 μl of the standard dilution of HCG made in the wells for immunological reactions, was added 120 μl suspensoids conjugate anti-HCG antibodies (AB 0421) - Kumasi R-250 in your breeding FBI-TV. Immediately after this, the contents of the wells were transferred to the surface of the connecting filters in window fixtures to Interfax. After 1-2 minutes the results of the analysis was determined visually by the presence of (positive result: dark blue figure in the form of a "+" on white background) binding suspensoids conjugate in the area of immobilizing the first antibody. Sensitivity analysis in several series of tests designed system was 10 IU/L. the sensitivity meets the level diagnostic needs immunochemical determination of early pregnancy for a period of 1 week.

In case of an actual diagnosis in the absence of the sample HCG on the surface of the filter shows the picture as "-", which indicates a negative test for pregnancy.

TECHNICAL and ECONOMIC EFFICIENCY of the present invention is to increase the sensitivity and reliability analysis with direct visualization of analytes. Sensitivity analysis is determined by a high optical prototypicality labels and increasing the number of affine groups sewn to the surface of the particle labels. Reliability, efficiency and ease of analysis of determinate offer a completely new and ingenious way one-step synthesis of reagents for detection, which allows to obtain a very stable covalent conjugates antianalytic with colored suspensoids labels.

The technology of obtaining conjugates color suspensoids labels independent of unique or expensive equipment and objectively scarce or expensive reagents. Versatility and reliable reproducibility of the proposed method is proved in 32 series re-synthesis of conjugates with different antianalytic.

The proposed detection method due to its high sensitivity, reliability, simplicity and efficiency may become indispensable in the pockets of epidemiological risk in emergency situations involving the need for blood transfusion in the field field work health units, especially in countries with hot climate, as well as in other emergency situations. The invention can serve as a basis for creating effective asynchronically systems for simplified diagnostic testing and effectively used in a variety of "one patient-one test kits for outpatient, premolding and hospital analyses, is also in the mass "home" systems, suitable for individual use.

The method of determination of immunoreactive compounds, including the production of conjugate particles colored label with detecting antianemia followed by incubation of the conjugate with detektivami immunoreactive compound, characterized in that the detecting Antianemic kongugiruut with Kumasi R-250, or acridine orange, or 2,4-nitrodiphenylamine.