RU2303783C1 - Method for immunoferment detection of antigens - Google Patents

Abstract

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.

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Description

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

Enzyme immunoassay methods for the analysis of various substances are widely used in modern practice (Gabaldon JA, Maquieira A., Puchades R. Current trends in immunoassay-based kits for pesticide analysis. Crit. Rev. Food Sci. Nutr. 1999; 39 (6): 519- 538; Stefan RL, van Staden JF, Aboul-Enein HY Immunosensors in clinical analysis. Fresenius J. Anal. Chem. 2000; 366 (6-7): 659-668; Lee NA, Kennedy IR Environmental monitoring of pesticides by immunoanalytical techniques : validation, current status, and future perspectives. J. AOAC Int. 2001; 84 (5): 1393-1406; Luppa PB, Sokoll LJ, Chan DW Immunosensors - principles and applications to clinical chemistry. Clin. Chim. Acta. 2001 ; 314 (1-2): 1-26), since the combination of antibodies and enzymes to form detectable intermolecular complexes leads to at the same time to the specificity of the analysis provided by highly selective antibodies, and its sensitivity provided by highly active enzyme labels. Enzyme-linked immunosorbent assays (ELISA) are divided into direct, in which the label is directly attached to the immunoreagent (standard antigen preparation or specific antibody), and indirect, in which the introduction of the label into the immune complex is an additional stage of analysis (Egorov AM, Osipov A.P., Dzantiev B. B., Gavrilova E. M. Theory and practice of enzyme-linked immunosorbent assay. // M .: Higher school. 1991). Despite the long duration of indirect immunoassay, in a large part of cases its use is preferable. Indirect labeling prevents the enzyme from contacting the components of the sample being analyzed, which could potentially cause its inactivation. In addition, in indirect immunoassay, an enzyme-containing conjugate is a universal reagent suitable for the determination of a wide variety of antigens. Indirect labeling eliminates the need for each new task to synthesize specific conjugates and characterize the properties of the products obtained in the synthesis.

When implementing indirect ELISA in modern practice, two main methods of labeling are used:

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

- modification of specific antibodies with biotin and their subsequent detection using the conjugate avidin-enzyme or streptavidin-enzyme.

Although the first method allows you to work with native specific antibodies without resorting to any processing of them, interaction with antivirus antibodies introduces a significant element of uncertainty into the analysis, since the interaction between them can vary significantly depending on the properties of antivirus and specific antibodies. In this regard, the biotin- (strept) avidin reaction is certainly more promising, since it has a high binding constant - about 10 ¹⁵ M ^-1 (6-7 orders of magnitude higher than the reaction with antispecies antibodies), unchanged for all interacting molecules and independent on the individual properties of the drugs used (Hevey RC, Malmros M.K. Methods for the detection and determination of ligands. US Patent 4228237. October 14, 1980; Wilchek M., Bayer EA Applications of avidin-biotin technology: literature survey. Methods Enzymol . 1990; 184: 14-45; Diamandis EP, Christopoulos T.K. The biotin- (strept) avidin system: principles and applications in biotechnology. Clin. Chem. 1991; 37 (5): 625-636; Lindq vist Y., Schneider G. Protein-biotin interactions. Curr. Opin. Struct. Biol. 1996; 6 (6): 798-803). Currently, both biotin derivatives (activated esters that allow fast and gentle conditions to biotinylate antibodies or other proteins) and streptavidin conjugates with highly active enzyme labels such as peroxidase, alkaline phosphatase, etc. (Swanson PE, Wick MR Commercial avidin- biotin-peroxidase complex kits. Am. J. Clin. Pathol. 1989; 91 (4, Suppl. 1): S43-44; Selvanayagam ZE, Gopalakrishnakone P. Tests for detection of snake venoms, toxins and venom antibodies: review on recent trends (1987-1997). Toxicon. 1999; 37 (4): 565-586; Wilbur DS, Pathare PM, Hamlin DK, Stayton PS, To R., Klumb LA, Buhler KR, Vessella RL 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 enzyme-linked immunosorbent assay using the biotin- (streptavidin) system in the most common variants 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) prior to analysis, biotinylation of specific antibodies is carried out with the separation of the synthesis product — the conjugate containing one antibody molecule and an undetermined (varying for different conjugate molecules within the same synthesis) number of biotin molecules — 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 order of which is selected based on the characteristics of the antigen and antibodies), an immobilized complex containing antigen molecules (native or modified), biotinylated specific antibodies and, if necessary, additional reagents is formed on the surface of the ELISA plate second specific antibodies, etc.);

B) molecules not included in the immobilized complexes are removed by washing the plate;

D) a streptavidin-enzyme conjugate solution is added to the wells. After incubation, the plate is washed, which separates unreacted conjugate molecules from those bound to the carrier. (If necessary, accelerate the analysis of the conjugate steptavidin-enzyme can be introduced at the stage of formation of specific complexes.);

E) a substrate is added to the wells, which is transformed by the enzyme molecules in the composition of the immobilized complexes with the formation of products detected by photometric, fluorimetric, electrochemical or other means;

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

One of the options for implementing this approach is described in the article Song X.H., 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. The following is a summary of the analysis methodology proposed by the authors of this article, which is considered as a prototype in this application.

The antigen solution was added to the wells of microtiter ELISA plates and incubated overnight at 4 ° C. After blocking unoccupied sites of sorption, the plates were washed from unreacted molecules. Test samples potentially containing the detectable antigen, or dilutions thereof, were added to the wells simultaneously with specific antibodies against the detected antigen and incubated. The amounts of immobilized antigen and dilutions of specific antibodies used were optimized based on a comparison of the analysis results for successive dilutions of both reagents (the so-called “chess titration”). After incubation, the wells were washed, biotinylated anti-species antibodies were added, re-incubated and the plate was washed. Next, the formed complexes were incubated with a streptavidin-peroxidase conjugate solution and the plate was washed again. Wells with a peroxidase substrate solution were incubated, the reaction of its transformation was stopped with sulfuric acid, and the absorption of the product was recorded using a microtiter plate photometer. Antigen concentration was calculated using a standard calibration curve obtained for samples with known amounts of antigen added.

Note that the introduction of the enzyme label through the interaction of biotin- (stept) avidin has significant drawbacks that have not been overcome in the currently known works.

1. During the chemical conjugation of antibodies with biotin, a mixture of products of different stoichiometry and, accordingly, different properties is formed. The ratio of products depends on both controlled (initial reagent concentrations, duration of interaction, temperature, composition of the reaction medium) and uncontrolled factors (presence of impurities, degree of retention of reactivity by modified biotin, conformational state of antibodies, number and degree of availability of reactive groups on their surface).

2. If it is necessary to contact the avidin-enzyme conjugate (strept) with the sample, the analysis results are distorted when the sample matrix contains biotin, which is characteristic, in particular, of meat samples (Kopinski JS, Leibholz J., Bryden WL 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 urine analysis, which is most likely due to the biotin metabolites present in it (Raguseo RM A direct streptavidin-binding assay does not accurately quantitate biotin in human urine. J. Nutrition, 2001; 131 (8): 2208-2214), as well as the immunochemical characterization of helminths (Romaris F., Iglesias R., Garcia LO, Leiro J., Santamarina MT, Paniagua E., Ubeira FM 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, the avidin present in the sample (White N.V. 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 steps are carried out sequentially and the enzyme conjugate is not in contact with the sample.

The ability to work with intermolecular conjugates of standard composition (and thus, uniform properties) arises when switching from chemical conjugation to genetic engineering (Witkowski A., Daunert S., Kindy MS, Bachas LG Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein. Anal. Chem. 1993; 65 (9): 1147-1151; Grigorenko V., Andreeva I., Borchers T., Spener F., Egorov A. A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays. Anal. Chem. 2001; 73 (6): 1134-1139; Rau D., Kramer K., Hock B. 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, due to its non-protein nature, this approach is not applicable.

The objective of the invention is to develop a method for enzyme-linked immunosorbent detection of antigens, in which the indirect introduction of the enzyme label into the detected immune complexes is carried out by protein-protein interaction with a high affinity of the reaction, which significantly exceeds the immunochemical interactions in binding constant, and the possibility of genetic engineering to obtain intermolecular conjugates of standard composition.

Applicants are invited to introduce the enzyme label to use the reaction between the bacterial enzyme ribonuclease (barnase) and its inhibitor barstar. This interaction is characterized by high affinity (the binding constant of the complex is 10 ¹⁴ M ^-1 ) and specificity, occurs in a wide range of parameters of the reaction medium and does not depend on the presence of any competing or inhibiting factors (Hartley RW Barnase-barstar interaction. Methods Enzymol . 2001; 341: 599-611). Both barnase and barstar are small proteins (weighing 12 and 10 kDa, respectively) without cofactors, carbohydrates, and other structural elements of a non-protein nature, which makes their genetic engineering conjugation methodically simple. The terminal sections of the amino acid chains of barnase and barstar do not participate in the formation of the intermolecular complex, which allows them to be used for conjugation with markers and immunoreagents without loss of activity (Deyev SM, Yazynin SA, Kuznetsov DA, Jukovich M., Hartley RW Ribonuclease-charged vector for facile direct cloning with positive selection. Mol. Gen. Genet. 1998; 259 (4): 379-382). Moreover, the genetic engineering addition of barnase to antibodies has been shown to increase their stability and, therefore, the reproducibility of analysis results during storage of reagents (Martsev SP, Tsybovsky YI, Stremovskiy OA, Odintsov SG, Balandin T.O., Arosio P. , Kravchuk ZI, Deyev SM 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 10 ¹⁴ M ^-1 provides the quantitative formation of complexes and the absence of any significant dissociation during the analysis. An advantage of the system is the possibility of obtaining conjugates of standard stoichiometry in chemical synthesis, since the barnase molecule does not contain cysteine groups, and cysteine introduced by site-specific mutagenesis allows the synthesis of conjugates of 1: 1 composition.

Thus, the difference between the proposed approach and the prototype one is that the attachment of the enzyme label to the detected immune complex is carried out not through the interaction of biotin- (strept) avidin, but through the interaction of barnase-barstar, which allows to obtain intermolecular conjugates for analysis by genetic engineering methods and unify the composition of conjugates, thereby ensuring high reproducibility of the analysis results.

The feasibility and effectiveness of the proposed approach is confirmed by the following examples of enzyme-linked immunosorbent assay using the Barnase-Barstar system introduced into the detected immune complexes through chemical (Example 1) or genetic engineering (Example 2) conjugation.

Example 1. Enzyme-linked immunosorbent assay of atrazine herbicide.

The analysis scheme is presented in figure 1. 100 μl of a solution of atrazine conjugate with bovine serum albumin (0.5 μg / ml, in FB 50 mM K-phosphate buffer, pH 7.4, with 0.1 M NaCl) was added to the wells of the polystyrene ELISA microplate and incubated for nights at 4 ° C. After this, the wells were washed four times with PBT - FB with 0.05% Triton X-100 detergent. 50 μl of atrazine-containing samples and 50 μl of a chemical conjugate of barnase with monoclonal antibody 1E4 / A12 against atrazine (0.2 μg / ml, in PBT) were added to the wells simultaneously and incubated for 1 hour at 37 ° C. The plate was then washed four times four times, 100 μl of the barstar peroxidase chemical conjugate (1 μg / ml, in PBT) was added to each well and incubated for 1 hour at 37 ° C. After this, the tablet was washed three times with PBT and once with distilled water. To record the catalytic activity of the enzyme label bound to the surface of the plate, 100 μl of 100 mM Na-citrate buffer, pH 6.0, containing 3.3 ', 5.5'-tetramethylbenzidine (0.1 mg / ml) was added to the wells hydrogen peroxide (3.3 mmol). After a 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 resulting colored products was recorded at 450 nm. The resulting calibration curve of competitive immunodetection is presented in figure 2.

Example 2. Enzyme-linked immunosorbent assay of the protein tumor marker p185 ^HER2 .

The analysis scheme is presented in figure 3. 100 μl of p185 ^HER2 solution (0.1 μg / ml, in PBS) was added to the wells of the microplate, incubated for 2 hours at 37 ° C, and the PBT was washed four times. 50 μl of samples containing p185 ^HER2 , and 50 μl of the recombinant conjugate of barnase or barstar with 4D5 mini-antibody against p185 ^HER2 (in both cases at a concentration of 8 ng / ml, in PBT) were incubated together for 20 min at room temperature and then added to the wells of the tablet. After 1 hour of incubation at 37 ° C and subsequent washing, 100 μl of barstar peroxidase conjugate was added to detect barnase or barnase peroxidase to detect barstar (1 μg / ml, in PBT) and incubated for 1 hour at 37 ° C. The final washing and registration of peroxidase activity was carried out as described in Example 1. The obtained calibration curves of competitive immunodetection are presented in figure 4, 5.

Claims (1)

An enzyme immunoassay for determining antigens, including the formation of complexes between antigen molecules and specific antibodies on the surface of the carrier, attachment to the enzyme label complexes and its detection by the formation of an enzymatic reaction product, characterized in that the enzyme label is attached by the interaction of two proteins - bacterial barnase ribonuclease and its a barstar inhibitor, while one of the interacting proteins is covalently or genetically engineered to the immunore agent, and the other to the enzyme label.

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