Method for immunoferment detection of antigens

FIELD: biotechnology, analytical chemistry.

SUBSTANCE: claimed method includes providing of complexes between antigen molecules and specific antibodies on carrier surface, wherein said complexes are disclosed by addition of enzyme label thereto followed detection thereof based on formation of enzyme reaction product. Enzyme label addition is carried out by two protein interaction, namely bacterial ribonucleaze barnase and barstare, which is inhibitor thereof, wherein either of the two is added to immunoreagent, and the other one is added to enzyme label. Abovementioned complexes have high affinity, specificity, and binding constant of 1014 M-1.

EFFECT: new method for antigen detection.

6 dwg, 2 ex

 

The invention relates to biotechnology and analytical chemistry and can be used in the determination of biologically active substances in medical diagnosis, to characterize the quality of agricultural and food production, environmental monitoring, process control.

In modern practice, widely used immunoassay methods for the analysis of various substances (Gabaldon J.A., Maquieira, A., R. Puchades Current trends in immunoassay-based kits for pesticide analysis. Crit. Rev. Food Sci. Nutr. 1999; 39 (6): 519-538; Stefan R.L, van Staden J.F., Aboul-Enein H.Y. Immunosensors in clinical analysis. Fresenius J. Anal. Chem. 2000; 366 (6-7): 659-668; Lee N.A., Kennedy I.R. Environmental monitoring of pesticides by immunoanalytical techniques: validation, current status, and future perspectives. J. AOAC Int. 2001; 84 (5): 1393-1406; Luppa P.B., Sokoll L.J., Chan D.W. Immunosensors - principles and applications to clinical chemistry. Clin. Chim. Acta. 2001; 314 (1-2): 1-26), as the combination of antibodies and enzymes for the formation of detectable intermolecular complexes leads simultaneously to the specificity of the analysis, provide highly selective antibodies, and its sensitivity, provide a highly active enzyme labels. Methods enzyme-linked immunosorbent assay (ELISA) are divided into direct, in which the label is directly attached to immunoreagent (standard drug, antigen or specific antibody), and indirect, in which the introduction of the label in the immune complex is an additional stage of analysis (Egor is in the A.M., Osipov A.P., Dzantiev B.B., Gavrilova E.M. Theory and practice of immunoassay. // M: The High. SHK. 1991). Despite a long indirect immunoassay, in large part, its use is preferable. Indirect tagging prevents contact of the enzyme with the components of the sample, which potentially can cause inactivation. In addition, indirect immunoassay sidezym enzymatic conjugate is a universal reagent suitable for determining various antigens. Indirect labeling eliminates the need for each new task to synthesize specific conjugates and to characterize the properties obtained in the synthesis products.

When implementing indirect ELISA in modern practice, there are two main ways of introducing tags:

- the interaction of specific antibodies and enzyme labeled antivitamin antibodies;

- modification specific antibodies Biotin and their subsequent detection using conjugate avidin-enzyme or streptavidin-enzyme.

Although the first method allows you to work with native specific antibodies without any processing, interaction with antivirovym antibodies contributes to the analysis of substantial uncertainty, because depending on the properties antivitamin and specific is such antibodies efficiency of interaction between them can vary significantly. In this respect, the reaction of the Biotin-(strept)avidin, of course, more promising, since it is characterized by high binding constant is about 1015M-1(by 6-7 orders of magnitude surpassing reaction with antivirovym antibodies), the same for all interacting molecules and do not depend on individual properties of the drugs used (Hevey R.C., Malmros, M.K. Methods for the detection and determination of ligands. US Patent 4228237. October 14, 1980; Wilchek M, Bayer E.A. Applications of avidin-biotin technology: literature survey. Methods Enzymol. 1990; 184: 14-45; Diamandis E.P., Christopoulos BECAUSE The biotin-(strept)avidin system: principles and applications in biotechnology. Clin. Chem. 1991; 37 (5): 625-636; Lindqvist y, Schneider g Protein-biotin interactions. Curr. Opin. Struct. Biol. 1996; 6 (6): 798-803). Currently commercially available as a derivative of Biotin (activated esters, quickly and in a gentle manner to biotinylate antibodies or other proteins), and conjugates of streptavidin with a highly active enzyme label is horseradish peroxidase, alkaline phosphatase and others (Swanson P.E., M.R. Wick Commercial avidin-biotin-complex buffer kits. Am. J. Clin. Pathol. 1989; 91 (4, Suppl. 1): S43-44; Selvanayagam Z.E., Gopalakrishnakone P. Tests for detection of snake venoms, toxins and venom antibodies: a review on recent trends (1987-1997). Toxicon. 1999; 37 (4): 565-586; D.S. Wilbur, Pathare P.M., Hamlin D.K., P.S. Stayton, To, R., L.A. Klumb, Buhler K.R., Vessella R.L. Development of new biotin/streptavidin reagents for pretargeting. Biomol. Eng. 1999; 16 (1-4); 113-118; Sugiyama, K., Hoshino n, Tatsumi H., Fukuda S. Complexes containing crosslinked avidin, analytical method with the use of crosslinked avidin and analytical reagents and kits. US Patent 6787325. September 7, 2004).

Indirect them is uniformity analysis using Biotin-(streptavidin) in the most common embodiments includes the following steps (Avidin-Biotin Technology. Methods in Enzymology: Volume 184. John N. Abelson, Melvin I. Simon, Meir Wilchek, Edward A. Bayer, eds. San Diego, CA, USA. Academic Press, 1990):

A) before the analysis is carried out biotinylation specific antibodies with separation of the product of the synthesis of the conjugate containing one molecule antibodies and uncertain (varying for different molecules of the conjugate within the same synthesis) the number of molecules of Biotin - from unreacted activated Biotin and other low molecular weight components of the reaction medium;

B) as a result of specific immunochemical interactions (the list and the procedure which selects, based on the characteristics of the antigen and antibodies) on the surface of the tablet for ELISA is formed immobilized complex containing the antigen molecule (native or modified), biotinylated specific antibodies and, if necessary, additional reagents (second specific antibodies and other);

B) molecules that are not included in immobilized complexes are removed by washing tablet;

G) is added to the wells solution conjugate streptavidin-enzyme. After incubation is carried out washing tablet, which separates unreacted molecules of the conjugate from contacting the media. (If necessary acceleration analysis of conjugate streptavidin-enzyme may be introduced at the stage of formation of specifices the complexes.);

D) is added to the wells of the substrate, which is transformed by the molecules of the enzyme in the composition of the immobilized complexes with the formation of the products detected photometric, fluorometric, electrochemical or otherwise;

E) on the basis of the generated signal and the calibration is determined according to the content of the antigen in the sample.

One of the embodiments of the approach described in the article X.H. Song, Huhle g, Wang L.C., Harenberg J. "Quantitative Determination of PEG-Hirudin in Human Plasma Using a Competitive Enzyme-Linked Immunosorbent Assay." Thrombosis Research 2000; 99 (2): 195-202. Below is a summary of proposed by the authors of this article the methods of analysis discussed in this application as a prototype.

The antigen solution was introduced into the wells of microtiter plates to the ELISA plate and incubated over night at 4°C. After blocking of unoccupied plots of sorption tablets laundered from unreacted molecules. The test sample potentially containing the designated antigen, or their cultivation was made in the wells at the same time with specific antibodies against a defined antigen, and incubated. The number of immobilized antigen and cultivation of specific antibodies optimized on the basis of comparison results of the analysis of serial dilutions of both reagents (the so-called "shahmat the e titration"). After incubation, the wells were washed, made biotinylated individule antibodies, re-incubated and washed tablet. Next, the formed complexes were incubated with a solution of the conjugate streptavidin-peroxidase and again washed tablet. Incubated the wells with peroxidase substrate solution, the reaction was stopped by his transformation sulfuric acid and record the absorption of the product by using a photometer for microtiter plates. The concentration ofantigenfigured,usinga standard calibration curve obtained for samples made with known amounts of antigen.

Note that the introduction of the enzyme label via interaction of Biotin-(stept)avidin has significant drawbacks, not yet been known.

1. The chemical conjugation of antibodies with Biotin produces a mixture of products of different stoichiometry and therefore different properties. The ratio of products depends on controlled (initial concentration of reagents, duration of interaction, temperature, composition of the reaction medium)and uncontrollable factors (the presence of impurities, the degree of preservation of the modified Biotin reactivity, conformation state of the antibodies, and the degree of availability of reaction-CSP is one of the groups on their surface).

2. If necessary, contact conjugate (strept)avidin-enzyme to breakdown the results of the analysis are distorted in cases when the matrix sample contains Biotin, which is characteristic, in particular, the samples of meat (Kopinski J.S., J. Leibholz, W.L. Bryden on Biotin studies in pigs. 4. Biotin availability in feedstuffs for pigs and chickens. Br. J. Nutr. 1989; 62 (3): 773-780). Similar problems are also described for the analysis of urine, which, apparently, is connected with the audience in her Biotin metabolites (Raguseo R. And direct streptavidin-binding assay does not accurately quantitate biotin in human urine. J. Nutrition, 2001; 131 (8): 2208-2214), as well as in immunochemical characterization of helminths (Romaris F., Iglesias R., Garcia L.O., Leiro J., Santamarina M.T., E. Paniagua, Ubeira F.M. Free and bound biotin molecules in helminths: a source of artifacts for avidin biotin-based immunoassays. Parasitol. Res. 1996; 82 (7): 617-622). In the analysis of eggs present in the sample avidin (White NV 3rd. Vitamin-binding proteins in the nutrition of the avian embryo. J. Exp.Zool. Suppl. 1987; 1: 53-63) blocks Biotin on the surface of the modified antibodies, preventing the binding of the enzyme label, even if the analysis stage are carried out sequentially and the enzyme conjugate is not in contact with the sample.

The opportunity to work with molecular conjugates of standard composition (and thus, uniform properties) occurs at the transition from chemical conjugation to genetically engineered (Witkowski A., S. Daunert, M.S. Kindy, L.G. Bachas Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein. Anal. Chem. 1993; 65 (9): 1147-1151; V. Grigorenko, I. Andreeva, Borches So, Spener F., Egorov A. A genetically engineered fusion protein with horseradish buffer as a marker enzyme for use in competitive immunoassays. Anal. Chem. 2001; 73 (6): 1134-1139; Rau D., Kramer, K., Hock C. Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA. J. Immunoassay Immunochem. 2002; 23 (2): 129-143). However, for Biotin because of its non-protein nature, this approach is not applicable.

The objective of the invention is to develop a method enzyme immunoassay detection of antigens, in which the indirect introduction of the enzymatic label detected in immune complexes through protein-protein interactions with high affinity reaction, significantly exceeding the binding constant of the immunochemical interaction, and the possibility of genetically engineering obtain intermolecular conjugates standard composition.

Applicants are encouraged to introduce the enzyme label to use the reaction between bacterial enzyme ribonuclease (Barysau) and its inhibitor burstrom. This interaction is characterized by a high affinity (binding constant of the complex is 1014M-1) and specificity comes in a wide range of parameters of the reaction medium and is independent of the presence of any competing or inhibiting factors (R.W. Hartley Barnase-barstar interaction. Methods Enzymol. 2001; 341: 599-611). And barnase, and barstar are small proteins (mass 12 and 10 kDa, respectively) be the cofactors, carbohydrates and other structural elements of non-protein nature, which makes methodically simple genetic engineering conjugation. The ends of amino acid chains barnase and barstar not participate in the formation of intermolecular complex, so you can use them for conjugation with markers and immunoreagents without loss of activity (S.M. Deyev, Yazynin S.A., Kuznetsov D.A., Jukovich M., R.W. Hartley Ribonuclease-charged vector for facile direct cloning with positive selection. Mol. Gen. Genet. 1998; 259 (4): 379-382). Moreover, it is shown that genetically engineered joining barnesy for antibodies to increase their stability, and because of this - and the reproducibility of the analysis results when storing reagents (Martsev S.P., Tsybovsky Y.I., Stremovskiy O.A., Odintsov S.G., Balandin THUS, Arosio P., Kravchuk Z.I., S.M. Deyev Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stability. Protein Eng. Des. Sel. 2004; 17 (1): 85-93). The binding constant of the 1014M-1provides quantitative formation of complexes and the absence of any significant dissociation at the time of analysis. The advantage of this system is the possibility of obtaining conjugates standard stoichiometry and chemical synthesis, because the molecule Barnaby does not contain cysteine groups, as introduced by site-specific mutagenesis in the terminal portion of the molecule of cysteine allows to synthesize the conjugates of composition 1:1.

Thus, the difference h is aemula approach from the prototype is that the accession of the enzyme label to detectivemisa immune complex is carried out not by the interaction of the Biotin-(strept)avidin, and through interaction barnase-barstar, which allows to obtain intermolecular conjugates for analyses of genetic engineering methods and unify the composition of the conjugates, thereby ensuring high reproducibility of the analysis results.

The feasibility and effectiveness of the proposed approach confirms the examples below immunoassay system using barnase-barstar introduced into the composition detectable immune complexes by chemical (Example 1) or genetically engineered (Example 2) conjugation.

Example 1. Enzyme-linked immunosorbent assay herbicide atrazine.

The analysis scheme is presented in figure 1. 100 μl of the conjugate solution of atrazine with bovine serum albumin (0.5 μg/ml, FB - 50 mm K-phosphate buffer, pH 7.4, with 0.1 M NaCl) was added to the wells of polystyrene microtiter plate ELISA plate and incubated over night at 4°C. then the wells are washed four times FBT - FB with 0.05% detergent Triton X-100. 50 μl atrisinajumi samples and 50 ál of chemical conjugate barnase with a monoclonal antibody E/A12 against atrazine (0.2 ág/ml, FBT) was added to the wells simultaneously and incubated for 1 hour at 7° C. the tablet Then again washed four times, was added to each well 100 µl of the chemical conjugate barstar-peroxidase (1 mg/ml, in FBT) and incubated 1 hour at 37°C. After that, the tablet thrice washed FBT and once with distilled water. To register catalytic activity contacting surface of the tablet to the enzyme label, the wells were added to 100 μl of 100 mm Na-citrate buffer, pH 6.0, containing 3,3',5,5'-tetramethylbenzidine (0.1 mg/ml) and hydrogen peroxide (3.3 mm). After 15-minute incubation at room temperature the reaction was stopped by adding 100 μl of 0.1 M sulfuric acid. The optical density of the formed colored product was detected at 450 nm. The obtained calibration curve competitive immunodetection presented in figure 2.

Example 2. Enzyme-linked immunosorbent assay protein marker p185HER2.

The analysis scheme is presented on figure 3. 100 μl of the solution RHER2(0.1 ág/ml, FB) was added into the wells of the microplate, incubated 2 hours at 37°and four laundered FBT. 50 μl samples containing RHER2and 50 μl of recombinant conjugate barnase or barstar with mini-antibody 4D5 against RHER2(in both cases at a concentration of 8 ng/ml, FBT) were incubated together for 20 min at room temperature and then added to the wells of the Board is as. After 1 hour incubation at 37°and subsequent washing was added to 100 ál conjugate barstar-peroxidase to detect barnase or barnase-peroxidase to detect barstar (1 μg/ml, FBT) and incubated 1 hour at 37°C. a Final wash and registration peroxidase activity was carried out as described in Example 1. The obtained calibration curves competitive immunodetection presented on figure 4, 5.

Method enzyme immunoassay detection of antigens, including the formation on the surface of the carrier complexes between molecules of antigen and specific antibodies attach to complexes of the enzyme label and the detection of the formation of the product of the enzymatic reaction, wherein attaching the enzyme label is carried out through the interaction of two proteins - bacterial ribonuclease barnase and its inhibitor barstar, one of the interacting proteins covalently or genetically engineered attached to immunoreagent, and the other to the enzyme label.



 

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