Method for producing conjugate for performing stereospecific analysis

FIELD: medicine.

SUBSTANCE: method involves carrying out single-stage conjugate synthesis having stereospecific (affine) compound conjugated in covalent way to suspensoid carbon particles.

EFFECT: enabled direct analyte observation on solid phase surface as back spots; increased sensitivity of diagnosticum for performing immunochemical, gene-hybridizing and ligand-receptor studies and creating instrumentless rapid diagnosis test systems.

 

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 the technology of beinstantly and reliable diagnostics for genetic hybridization and ligand-receptor analysis. The intended scope of the invention includes the practice of analytical research in biology, medicine, agriculture, criminology, performed with fundamental or applied (diagnostic) purposes.

In immunoanalytical practice, the number of known ways, each of which can be divided into three stages: the formation of specific complex antigen-antibody, the introduction of the label and its visualization in one way or another. Such methods include radioimmunological assay (RIA) (Yalow RS, Berson SA - Nature, 1959, v.184, R-1649), enzyme-linked immunosorbent assay (ELISA) (Ngo TT & Lenhoff NM Enzyme-mediated immunoassay, 1985, Plenum Press, New York.), bio - and chemiluminescent immunoassays (J.S. Woodhead, Weeks I. - Pure & Appl. Chem. 1985, v.57, p.523-529). All these methods have a number of positive qualities: versatility, i.e. allow to determine any connection against which you can obtain specific antibodies; izberatelno the d and specificity; high sensitivity. However, alongside the advantages of the above (similar to the claimed method) analysis methods have significant drawbacks. The first is the health hazard posed by the introduction of isotope labels, preparation and implementation of RIA, and the use of toxic, carcinogenic Chromogens in IFA; dependence on complex and expensive recording equipment and limited available reagents used either in technology-based assays, either the test procedure.

Known systems analysis with direct tagging of specific analyte compounds (anti-analytes) optically dense particles or water-soluble colored polymers of different nature and sizes. Among these tags, latexes, including color (Tarcha et al., Abbot Labs., Eur. pat. appl. 89116594.6), erythrocytes (Guesdon J-L., AvrameasS., Ann. Immunol., 1980, v.131, R-396), non-metallic colloids sulfur, selenium, tellurium (Yost et al., Eur. pat. appl. 88110459.0), polymerised dyes Congo red, Tripney blue, Lissamine blue (Henry Robert P., US Pat. 4.452.886), colloidal gold (Roth j, Heitz P.U., Immunolabeling with the Protein A - Gold Technique: An Overview, Ultrastructural Pathology, 1989, 13:467-484). Use data labels allows you to visually determine the presence of analyte in homogeneous systems analysis (without separation of reactants) - such as agglutination to oline large particles (with sizes ranging from 0.2 to several micrometers) or the change in the spectral characteristics (color) specific agglutinating colloids, and in many heterogeneous systems Erythromelalgia and, more widely, Sedimentation-Adsorption (Immuno)Analysis; the dot-, Western-, southern-, Northern-blotting; immuno - and hybridelectric (Flow-Through tests), immunomagnetic (chromatography), and several others - staining zone specific binding of the analyte on non-porous or porous solid phase carrier. Despite the possibility of quantitative registration using a simple colorimeter, OTDR, densitometers, the main advantage of these methods is a direct visual determination of the analyte that conditionally releasing them to the group of so-called beinstantly methods.

Closest to the claimed object to the technical essence and the achieved result is a method of obtaining conjugates based on particles of colloidal carbon (Doublesin, Mbew, Achromatopsia. The way stereospecific analysis and the method for the conjugate for the stereospecific analysis. RF patent №20899212 from 10.09.1997).

In the prototype the problem of increasing the reliability analysis and stability used in the analysis of the detecting reagents solved by covalent conjugation of anti-analyte particles of carbon. For this purpose, the particles initially cover a water-soluble polymer which is inert ampipe the practical connection capable of relatively strongly absorb on the surface of the particles. Sorption of the polymer allows to obtain stable solutions suspensoid. Adsorbed polymer exhibits in the aqueous phase is a functional group capable of covalently reacting with the Homo-and/or heterobifunctional controlling compounds. Subsequent processing of the received complex bifunctional reagent, taken in a large molar excess relative to the functional groups of the polymer 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 next step is the release of the activated labels from excess conjugating reagent. A subsequent connection with the intact (unmodified) anti-analyte (affine connection) fails covalent proshivkoi molecules anti-analyte to the surface of a carbon label. In the described method allows to obtain the conjugates of strong covalent structures that are stable in solutions, including in the presence of high concentrations of detergents.

However, the multistep nature of congiaria creates a number of technological difficulties. First of all it concerns the necessity of a number of chromatographic process is ur cleanup: excess sorbed polymer in the first stage, and, as a consequence, the need for concentration; from an excess of activating a bifunctional reagent, which is again followed the procedure of concentration. This mnogodelnosti not only lengthens described in the prototype method, but also makes it time-consuming, a little tech, costly. In addition, the use of as sorbed on the surface of the particles of inert protein (BSA) leads to a decrease in the associated affine connection, which ultimately leads to reduced sensitivity stereospecific analysis conducted using such conjugates.

The additional advantage of making the inventive method is more repeatable regardless of the range of concentrations of those or other antianalytic. Created by way of a positive effect in General is characterized by significantly higher compared with the prototype reliability and versatility.

The aim of the invention is to develop a one-step method of obtaining stereospecific conjugates.

The goal is achieve through the use of the polymer, sorbed on the surface of carbon particles, molecules affine connection (antianalytic), which kongugiruut in one stage by adding a bifunctional reagent. The only phase chromatography is the removal of the C ready conjugate by conjugating compounds and molecules unbound antianalytic.

Incorporated in the inventive method the principle of one-step covalent conjugation is the opportunity for a very wide range of specific sorbed polymers and activating/conjugating reagents. This circumstance forces the applicant to submit the claims made in fundamental terms, try specifying which inevitably would have led to a narrowing of the scope of the claimed object.

The following specific examples describes a method for detecting conjugates made using glutaraldehyde as cross-linking (Homo)of a bifunctional reagent. The use of this substance is not fundamental to the method. Instead of glutaraldehyde can be used other known Homo - and heterobifunctional connection that the reactions of the second order is able to react preferably with the most versatile presented in possible antianalytic and intentionality primary amino groups (although for covalent conjugation with suspensoids labels such antianalytic as Fab′-fragments of antibodies, better can be, for example, maleimide reagents involved in the reaction of thiol-disulfide exchange).

The use of glutaraldehyde is p is impactfully, because additionally meets the requirement of universality of the proposed method is homobifunctional reagent, relative to the specific reactive with primary amino groups of proteins. In addition, an important factor is the availability and almost the lowest cost of glutaraldehyde, which also distinguishes it from other possible, the use of which in the framework of the proposed method from the economic point of view is obviously less profitable opportunity.

The method is as follows.

Example 1.

I. Receiving patinirovannaya reagents carbon labels.

To 20 ml of Protein solution And 1-50 mg/ml in 0.01-0.2 M Phosphate Buffer Solution of pH 6-8,5 (FBI) add 0.1 to 2 grams of carbon. 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) carbon particles. The resulting suspension is voiced in an ultrasonic disintegrator. Conditions of ultrasonic treatment suspension (intensity and power, 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°C. the resulting effective ultrasonic de the integration suspensoid centrifuged at 6000 g for 5-10 minutes to remove the remaining relatively large particles. Covered with Protein And suspensoids carbon particles with sizes 160-170 nm contained in the supernatant is ready for further conjugation.

Similarly prepared particles, peptizyme them in solutions of Protein G, streptavidin, antibodies under the same conditions.

II. Obtaining conjugates carbon labels with antianalytic (Protein A, Protein G, Streptavidin, Antibodies).

On a rotary shaker combine equal volumes patinirovannaya Protein And particles and solution of glutaraldehyde to a final concentration of the latter 1-25%. The time of conjugation from 30-120 minutes. Ready conjugate is released from the excess glutaraldehyde and unrelated antianalytic 3-4-fold 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%.

The conjugates of the carbon particles with Protein G, streptavidin, antibodies have a similar way.

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

The resulting conjugates were used in the us in nigeoplachivaemoy examples of analysis.

Example 1.

Immunochromatographic determination of human IgG using Protein a conjugate - Carbon in comparison with Protein And labeled with colloidal gold with a particle size of 40 nanometers.

In the analysis used strips of nitrocellulose (Millipore) with a pore size of 5 micrometers with immobilizerturning on them in spots human IgG (applied amount of 0.001 ml) from multiples of 10 a dilution of 1 mg/ml, 100 μg/ml, 10 μg/ml, 1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/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 a 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 black spots analysis. The sensitivity of a detection system using conjugase Protein a - Carbon was 10 PG/spot, with the conjugate Protein a - Gold - 100 PG/spot.

Example 2.

Serological determination of anti-HIV antibodies on non-porous solid phase using a conjugate Protein G - Carbon.

As the solid phase used the tablets for serial dilution of polystyrene, the inner surface of the hole which was covered by sum SinTe the practical peptides representing the determinants of the main structural proteins of HIV-1,2, which inflicted 0,001 ml rabbit serum against HIV antigens from dilutions are multiples of two. After 15-minute incubation and washing FBI-TV, wells contributed conjugate Protein G - Carbon for 15 minutes, then remove conjugate, watched the results of the analysis in clear contrast spots. In parallel, the bound antibodies was determined by Protein And labeled with colloidal gold with a particle size of 40 nanometers and Protein And labeled with horseradish peroxidase. The sensitivity of the assay was evaluated by final serum dilution giving distinct from background stain. In the comparative analysis of the received detection sensitivity using Carbon conjugate 1/40960, using enzyme conjugate - 1/1280 (standard procedure manifestations 4-chloronaphthalen) and are virtually indistinguishable results determine the conjugate Protein a - Gold.

Example 3.

The dot-hybridization analysis of DNA of phage lambda using conjugate Streptavidin-Carbon.

Multiples of 10 serial dilution of denatured DNA of phage lambda applicable in volumes of 1 µl on nitrocellulose membrane 0.45 µm. Mobilisierung micheneau DNA was hot for 2 hours at 80°With vacuum. Hybridization was performed using as a probe chemicalcompositions DNA of phage lambda. Washed and outreach to consumers was treated with blocking buffer with 0.5% colloidal casein and 1% gelatin in an hour and was carried out by determination of incubation with the working dilution of the conjugate of Streptavidin with Carbon for 30 minutes at room temperature and gentle shaking, followed 3 times washing FBI-TV before the final accounting results. Parallel detection was carried out by the standard conjugate Streptavidin-Horseradish Peroxidase with visualization of hybridized samples in a colorimetric reaction with o-dianisidine.

In the described comparative experiments in a number of reproducible series of sensitivity analyses using both systems definition using as suspensoids (4 different colors), and enzyme conjugates ranged from 1 to 0.1 picograms mistaway DNA to the point.

Example 4.

Dvuhsvetnoe immunochromatographic determination of human chorionic gonadotropin using suspensoids conjugate Carbon-Monoclonal Antibodies.

Preparation connecting HCG porous strips.

Transparent (PVC) thick (0.5 mm) rigid adhesive tape pasted a strip of 5-μm nitrocellulose filter with dimensions of 2*16 mm One of the narrow ends tightly, using the same backing, combined with the absorbing element (1*1 cm square tools the Oh highly porous chromatographic paper), considering in the subsequent the upper end. In the middle of the nitrocellulose strips with a thin line using the Hamiltonian syringe inflicted monoclonal anti-HCG antibodies (AB 0420), 1 mg/ml in the FBI. After drying the strips after 30 minutes after the application of binding antibodies strip was blocked by soaking in 1% casein in FBI-TV for 1 hour at room temperature, then washed FBI-TV and again dried.

The definition of a standard HCG.

The standard HCG bred multiple of 2 FBI-TV and the urine of a healthy donor, added tween-20 0.1%. To 50 µl of the standard dilution of HCG made in the wells of microtiter tablet, was added 50 μl of suspensoids conjugate anti-HCG antibodies (AB 0421) - Carbon in the working dilution of the FBI-TV. Immediately after that, the wells were lowered, the lower ends of the chromatographic strips with immobilizerturning they monoclonal anti-HCG antibodies. After 2-3 minutes, the results of the analysis was determined visually by the presence of (positive result: the black bars on a white background) or absence (negative) binding suspensoids conjugate in the area of immobilizing the first antibody. Sensitivity analysis in several series of tests designed system was 50 IU/L. the sensitivity meets the level diagnostic needs immunohistochemistry the distribution of early pregnancy for a period of 1 week.

The above examples illustrate the capabilities of the present invention and do not limit how 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 method.

Technical and economic efficiency of the proposed method lies in the substantial increase manufacturability synthesis stereospecific detection reagents (conjugates) by adnotatione procedures for conjugation, as well as to increase sensitivity and reliability stereospecific analysis with direct visualization of analytes. Sensitivity analysis is determined by the use of the new technological technique, which is to peptization carbon particles anti-analyte, which leads to an increase in the number of molecules of affine connections, conjugated with a carbon label.

The technology of obtaining conjugates carbon labels independent of unique or expensive equipment and objectively scarce or expensive reagents. Versatility and reliable reproducibility of the proposed JV is soba proved in 12 series of re-synthesis of conjugates with different anti-analytes.

The proposed conjugates can serve as the basis for the creation of effective beinstantly systems for simplified diagnostic testing and effectively used in a variety of "one patient - one test kits for outpatient, premolding and hospital analyses, including urgent clinical and epidemiological situation, as well as in the mass of "home" system, suitable for individual use.

A method of obtaining a conjugate for stereospecific analysis, including a connection antianalytic with suspensoid particles of colloidal carbon, wherein ationality in phosphate buffer add carbon, the mixture is stirred for peptization carbon particles and then treated in an ultrasonic disintegrator, received suspensoid centrifuged, then add a solution of glutaric aldehyde, the resulting conjugate is released from the excess glutaraldehyde and unbound antianalytic by centrifugation or gel filtration, and then to combined fractions containing the conjugate, add bovine serum albumin and glycerol.



 

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