IMMUNOASSAY

IMMUNOASSAY

Any substance possessing the properties of antigens — immunogenic or non-immunogenic (haptens) — can be quantitatively measured with the help of enzyme immu-noassay (EIA). The EIA requires purified antigen, the specific antibody, an enzyme which serves as a label for the antigen or for the antibody and a tool for enzyme activity determi-nation. High sensitivity, rapidity and reliability are characteristic of the EIA.

Any substance, domain-en-giving properties tigena, full or defective (hapten) can be quantified using enzyme-linked immunosorbent assay (ELISA). To perform the ELISA must be purified antigen specific antibody, an enzyme as a label for an antigen or antibody, and means for recording the activity of the enzyme. IFA is a high sensitivity Flow rate, speed and reliability.

VD Samuel

Moscow State University. MV Lomonosov

INTRODUCTION

Is it possible to quantification of a component in the blood serum, growth-enforcement juice, the culture fluid from mikroor-isms, cell homogenates, drawn from a sample of soil, containing hundreds or thousands of different components? Maybe, but on one condition: the analyzed component must have the properties of the antigen, ie the ability in humans or animals the synthesis of specific proteins — antibodies with high specificity con-binding antigen. The solution of such problems — the sphere of enzyme immunoassay (EIA). However, you must first obtain a purified antigen and the corresponding antibody to choose an enzyme as a label for an antibody or antigen.

And then you can spend immunochemical D shares: AAH + Lam * -► A (Ar-Ar *) + (b — a) Am * (1) or AAG + Lam + sagas -► (2)

-► A (Ar * -AT) + e (Ar-Ar) + (a — a) Ag + (a — f) Ar

In the first embodiment (Equation (1)) of Ax antigen present in the test sample, and a labeled antibody Am *, added in a concentration greater than the concentration of Ag, Ag-complexed Am *. Quantitative analysis of the reaction (1) can be carried out by forming a complex Ar-Ar *, detected via an enzymatic label, or Ar *, remaining in a free state. This non-competitive ELISA. The WTO-rom version (equation (2)) limiting the sub-stratum is an antibody Am. The labeled antigen Ag *, being added to the sample to be analyzed in a known concentration, binds to antite-scrap, forming an Ar * -AT. In this reaction, Ar * will compete with unlabelled antigen Ar contained in the sample. The concentration of the Ar * -AT is inversely proportional to the concentration of Ag. In this way, according to a previously known concentration of Ar * can determine the unknown concentration of Ag. It’s a competitive ELISA. In both vari-antah IFA is necessary to solve an important problem — to share the bound fraction of the labeled reagent (Ar-Ar or Ar * * -AT) from the unbound free agent

(AT * or Ar *) in order to determine the proportion of specific binding.

Sets immunochemical reagents for defined-division called antigens diagnosticums. To create them, necessary to solve the problem of obtaining an antigen, antibody, antigen or antibody with an enzyme to establish registration im munohimicheskoy reaction activity of enzyme used as the label. Along with enzymes as labels for antigens using radioactive and fluorescent compounds: respectively radioimmunoassay and fluorescence-immunoassay (RIA and FIA). ELISA uses preferences, since it does not require complicated measurement apparatus and the use of radioactive compounds, and therefore it is safe, useful in the field.

Immunochemical analysis is not limited to ELISA, RIA and FIA, based on a direct interaction of the antigen with the antibody. There are methods to detect and quantify antigens by changing the physical state of the interaction with antibodies. For example, if the antigen is on the surface of bacterial cells, the antibodies cause their adhesion (agglutination). This principle is based on the determination of blood groups: red blood cells in the interaction of bonding surface antigens with added antibodies (hemagglutination). Antibodies are added in a specific ratio to the solution makromole-molecular-antigens, causing their precipitation — the formation of large, visually detectable aggregates of antigen molecules associated antibodies. There are methods, based on the reaction of precipitation in gels: in the formation of immune complexes Ag-Al gel having a line of precipitation, in the form of these lines can be judged on the number and immunologically related antigens. For the identification of proteins is widely used im munoblotting: first protein mixture separated by electrophoresis on a gel, then the gel to a nitrocellulose membrane, and imposes on it electrophoretically transferred (subjected to electron-roblottingu) separated proteins are iden-fied by means of labeled antibodies. The labeled antibody is widely used in the study of the localization of cell components and tissue — it immunotsito- techniques and immunohistochemistry. The cells were labeled with fluorescent antibodies can be separated from unlabeled cells — by flow of qi-tofluorimetrii. Chromatographic column with a sorbent which is covalently bound an antigen (or antibody) used in affinity chromatography — separation of appropriate antibody (or antigen) from mixtures resulting in the formation of immune complexes (see. Section "Antibody"). Another use of immunochemical analysis — immunosensors: piezocrystal coated with antigen (antibody) the binding of antibodies

(antigen) and increases its mass changes the frequency of resonance oscillations in alternating electric field that allows you to record the change in mass of about 10 piezoelectric"’12

Thus, the EIA — is just one of the ways to determine the antigens received wide practical application due to the possibility of quantitative determinations of high sensitivity and commercial availability. The on-scientific and research these possibilities immunochemical method is always used together with IFA and even with the same reagents. Consideration of the totality of these methods is physically impossible in one article. Therefore, this work is limited to ELISA.

ANTIGEN

As the antigen may be cell-mikroor-organisms, viruses, proteins and polysaccharides. This half-fledged antigens. Antibodies generated against the whole molecule is not a protein or a bacterial cell, but to a small area on the surface — the antigenic determinants (epitopes). Such sites in proteins include at least five amino acid residues. Antigenic determinants branched molecules polysaccharide chain of four to six residues.

There are also defective antigens — haptens. Antibodies to haptens are formed only at their landing on the polymer matrix, which are used as proteins, bacterial cell wall polymers, synthetic polyelectrolytes. In this manner, antibodies can be raised to antibiotics and other medicinal compounds, peptide and steroid hormones, vitamins, pesticides, and even such simple compounds as 2,4-dinitrophenol. Haptens are not only low molecular weight compounds. Thus, the nucleic acids themselves are non-immunogenic. However, antibodies to nucleic acids are synthesized when they are complexed with proteins. The antigenic determinants of nucleic acids — tri- and tetranucleotides.

Often the isolated and purified antigen, such as a viral protein, it can be difficult. After all, it must be borne in appreciable quantities when it comes to creating a diagnostic kit. In such cases, the antigen used for the synthesis of genetically engineered (transgenic) bacteria or yeast.

ANTIBODY

Antibodies are obtained by immunizing animals with antigens: This in vitro mice, guinea pigs, rabbits, Production sheep, goats, horses. Suitable chickens: antibodies following immunization are isolated from the yolks of eggs.

Immune response (see. [1, 2]) for the introduction of the antigen accompanied by the synthesis of antibodies, pour-sponding to the structure and functional pre-destination (immune system Eshel-nated: There are several lines of defense against the introduction of the antigen). This immunoglobulin (Ig) classes G, D, E, A, M, of which the first three can bind two molecules of an antigen that is divalent, four- or octavalent IgA, IgM desyativalentny. Given that the antigen used to immunize an animal, contains not one but several antigenic determinants, one can imagine a multitude of antibodies produced in the organism to the introduction of antigen. These antibodies are called polyclonal. They Goethe Rogen affinity to antigens. Their composition is not constant and depends on the type of animal, its genetic characteristics and physiological state.

For purification and standardization of polyclonal antibodies using affinity chromatography. Serum from the immunized animal is passed through a column of polysaccharide beads to which is covalently ligated antigen. Specific antibodies bind antigen, all other proteins, including antibodies with low affinity for the antigen, fall through the column, followed by the specifically bound antibody is washed off the column with an acidic or alkaline solution: association of antigen with antibody noncovalently. One cycle of affinity chromatography allows to purify proteins to 1000 times or more.

However, even this method of treatment can not completely get rid of the heterogeneity of the antibody preparation. Out of this difficulty — in obtaining a single specificity antibody reactive with a single antigenic determinant. These monoclonal antibodies are referred to (see [3].). They are prepared by techniques of cell engineering by hybridization of immunocompetent lymph node B-cells and tsitov myeloma tumors are capable of rapid proliferation, an unlimited number of divisions (in contrast to the majority of non-tumor cells in which the number of divisions is limited). Monoclonal antibody preparations are characterized by the constancy of the composition and physico-chemical properties, low probability of cross-reaction with "strangers" antigens. This high-tech product. Its deficiency — often relatively low substrate affinity, low affinity.

Of enzymes as labels in IFA

It is necessary to select such an enzyme which retains its activity is long, does not lose its binding operation when the antigen or antibody has a high substrate specificity. Widely used horseradish peroxidase, alkaline phosphatase, P-galactosidase and Escherichia coli. Promising firefly luciferase and luminescent bacteria. The enzyme activity is detected by a change in optical density, fluorimetriche ski and electrochemically. Luciferase reaction accompanied by light emission of photons, so they register for bioluminescence. A few examples.

Peroxidase catalyzes the reaction

AH2 + H2O2 -► A + 2H2O

As AH2 can be different compounds. So restored colorless o-phenylenediamine in the peroxidase reaction is transformed into oxidized colored form with a maximum absorbance at 435 nm recorded photo metrically.

Alkaline phosphatase catalyses the hydrolysis of esters Sforno FD-4-nitrophenyl phosphate is converted into 4-nitrophenol, detected by absorbance at 405 nm; 4-Methylumbelliferyl Phosphate converted into 4-methylumbelliferone fluoresce with a high quantum yield at 450 nm (fluorescence excited at 365 nm).

P-galactosidase catalyzes the hydrolysis of lactose to form glucose and galactose. If, instead of the natural substrate take-4-methylumbelliferyl P-D-galactoside, galactose formed by hydrolysis of 4-methylumbelliferone and registered by fluo-rimetricheski.

The luciferase reaction is detected biological-cence:

Luciferase

,T. ,. fireflies

ATP + luciferin + O2 ►

Bacterial luciferase

-► AMP + Fn + oxyluciferin + hn 560 nm,

FMNN2 + O2 + R-CHO

— FMN + R-COOH + H2O + hV49o nm where ATP — adenosine, AMP — adenosine monophosphate Pi — orthophosphate, FMN and FMNN2 — oxidized and reduced flavinmononuk-leotid, R-CHO and R-COOH — fatty aldehyde and the appropriate acid.

Binding enzyme label

How to link the enzyme to an antibody or antigen without loss of enzyme activity and properties of the antibody and disorders antigen? To do this, there are three groups of methods: biochemical, immunological and genetic engineering.

The biochemical methods used cross-linking enzyme E with an antibody or antigen with the participation of the free reactive groups: -NH2, -COOH, -SH, -OH. For example,

E-NH2 + HOOC-Ar -► E-NH-CO-Ar + H2O

If direct communication can not be realized, in the course are bifunctional crosslinking agents: glutardialdehyde and benzoquinone. Functional groups of the reactants can be activated. For example, hydroxy groups in the carbohydrate component of horseradish peroxidase may be oxidized using sodium periodate to aldehyde, which is then sewn through the antigen or antibody with the assistance of amino to form a Schiff base:

E-CHO + BjN-A E-CH = N-Ar ^ -► E-CH = N + H-Ar

Vitamin Biotin binds strongly only with egg white avidin — an inhibitor of the B-otinsoderzhaschih enzymes: binding constant in the reaction of avidin (A) + biotin (B) -► AB complex, is determined from the equation Kj = [AB] / [A] • [B] is 1015 M-1. If prepare complexes Ar-biotin and avidin-E and then to mix them, a complex is formed Ar-biotin-avidin-E.

Immunological methods for the preparation of antigens or antibodies labeled with enzymes that are based on the use of antibodies or as a component of crosslinking units. Antibodies all five classes are built on a single plan. Fig. 1 shows the immunoglobulin G. The antibody is constructed of four polypeptide chains: two heavy (H from the English. Heavy — heavy) and two light (L from the light — light) with molecular masses of 50 and 25 kDa. Heavy chains are linked to each other by two disulfide bonds through residues of cis-theine. Light heavy chains form one disulfide bond. Both circuits consist of domains by ~ 110 amino acid residues in each of: a heavy chain — four in the light — the two domains. N-terminal domains of H- and L-chains, designated VH and VL (variable from V — variable) determine antibody specificity and their primary structure is variable. The remaining domains are characterized by the constancy of the structure and are designated CH1, CH2, CH3 and CL (C of constant — constant). The form of the antibody molecule resembles the letter Y, which is associated with foot spikes hinge region.

In a study of the structure of antibodies used proteolytic enzymes. Papain cleaves a molecule into three fragments. Two of them are identical, the reaction of a monovalent antigen binding Fab-and named fragments (Fab by antigen binding Fragment — fragment antigen binding). A third fragment does not bind the antigen is easily crystallized, and therefore called Fc-fragment (Fc from Fragment crystallizable — crystallizable fragment). Pepsin cleaves the antibody molecule differently. In this form the bivalent F (ab ‘) 2 frag-ment and a few fragments, Fc fragments, the largest of which is named pFc’-fragment.

Method of hybrid antibodies for the antigen crosslink with the enzyme involves several steps (Fig. 2) Preparation of F (ab ‘) 2 fragments to the antigen and the enzyme by treating the corresponding antibody with pepsin, their separation by reduction of di-sulphide bonds mercaptoethanol, mixing the obtained Fab- fragments, anti-merkaptoe methanol dialysis, leading to re-association to form hybrid F (ab ‘) 2 fragments, and antigen binding enzyme into a single complex.

Fc-fragments of the antibodies form a strong complex with protein A Staphylococcus. In the presence of antibodies to an enzyme and a conjugate protein A-antigen complex can receive E-Ar-Ar-A protein.

Genetic engineering method for producing a labeled antigen is based on the synthesis of fusion proteins using microorganisms. This method using the transgenic E. coli, in the laboratory ES Severin produce fusion proteins comprising the complete amino acid sequence of the bacterial P-galactosidase protein and specific sequence of the human immunodeficiency virus or hepatitis B virus

Saving the native structure and function of sshi-Vai reagents depends on their individual CCA-tures, so there can not be applied to any standard method. The specific method of crosslinking is selected individually.

Separate free and bound reagents enzyme-labeled

Quantitative analysis of immunochemical reactions (see. Eq (1) and (2)) can be carried out on products (Ar-Ar and Ar * * -AT) or reaction substrates remaining in a free state (Ar *, and Ar *). For this purpose, two basic approaches.

The first approach — directed influence on the enzymatic activity of the complex Ar-E is bound Am means leading to the inhibition or, conversely, to increase the rate of enzymatic reaction (see. Section "Homogenous ELISA"). Such an ELISA can be implemented in a single phase system — in solution and is therefore called homogeneous or liquid phase. This ELISA without physical separation of labeled League-Dov (Ar * or Ar *) from their immunochemical complexes — products of the reactions (1) and (2).

The second approach — the physical separation by the solid phase, labeled binding reagent. This heterogeneous or solid-phase ELISA. As the solid walls of the vessels used in which the analysis. These vessels are designed as special plates of transparent, non-porous polymer materials: polystyrene, polyvinyl chloride, polymethacrylate. Proteins (antigens or antibodies) are adsorbed on the surface of the plastic material — and in this framework ELISA. To increase the strength of the binding proteins plastic is modified (processing performed glutaraldehyde, poly ^ -lysine, toluene-2,4-diisocyanate; process modification — the know-how of the manufacturer) in such a manner that the active surface becomes capable of covalently bonding to proteins. By the modification of the regime strict requirements: the homogeneity of the binding properties of the surface of the plastic, its optical transparency (ELISA results are recorded photometrically). After the procedure, binding to the surface of the plastic, the unbound labeled reagent is simply washed away from the surface of the tablet. In English literature ELISA is called ELISA (enzyme-linked immunosorbent assay) — determination of enzyme linked immunosorbent with.

Under both approaches, there are many all-Rianta. Consider a few examples.

HOMOGENEOUS IFA

Antibody forming a complex with the antigen inhibits the activity of bound enzyme: Ag-E complex, like free E, catalyzes the conversion of a substrate S in the reaction product P

S -P, while the complex Al-Ag-E loses activity

Al-Ar-E S ^ *

P

The loss of activity can be caused by a change in the conformation of the enzyme, leading to disruption of the structure of its active site. Another reason — the enzyme may not be active as an antibody to block access of the substrate to the active site of the enzyme.

If you add the free antigen, it is competing for Am, is the regeneration of the Ag-E, the enzyme activity appears a (Al-Ag-E) + bA —

-► With (Al-Ag-E) + d (Ar-Ar) + e ^ -E) + fA

If there is a calibration curve with known concentrations of labeled and unlabelled antigen (the schedule will be a linear relationship between the concentration of Ag and the enzymatic activity of Ag-E), this method (competitive, homogeneous EIA) can determine the concentration of antigen in the sample.

In addition to enzymes in ELISA as homogeneous mark may be used modulators (M) enzymes — substances able to inhibit or induce the activity of enzymes:

Am + (Ar-M-E)

Ar + Ar

— (Am-Ar-M-E), (Al-Ag)

S

P

Ar-S-M E P

Al-Ar-M-E

The complex Ar-M-E catalytically active and being associated with Al, is unable to catalyze the reaction. Free Ai, who is in the test sample competes with Ag-M-E for binding to AT, added in a limited quantity, leading to increased concentrations of Ag-M-E and thus promotes the enzymatic reaction. This embodiment with a positive enzyme modulator. Conversely, a negative modulator of the enzyme activity will decrease with increasing free Ar in the test sample.

There are many other modifications homogenous EIA. We say that three of them: the use of the mark as a prosthetic group of enzymes, covalently bonded to Ar; complexes fluo-Roguin substrates (S) with Ar enzyme (as opposed to complex Ar-S-Ar Ar-S can not serve as a substrate of the enzyme, the enzymatic reaction produces intensely fluorescent product); The use of antibodies being bound to the active site of the enzyme to inhibit its activity. Time for which the homogeneous EIA is conducted not more than 5 minutes. Although homogeneous ELISA inherent rapidity and low labor input, it is characterized by a low sensitivity in comparison with heterogeneous ELISA.

HETEROGENEOUS IFA

Am Antibodies bind to the walls of the wells of a polystyrene plate (Fig. 3a). At excess wash buffer. Remaining unoccupied binding sites on the polystyrene surface blocked with irrelevant protein such as bovine serum albumin; the surface is washed again (in Fig. 3, not shown). The sample containing the antigen Ag in an unknown concentration, mixed with a certain amount of added labeled antigen and antibodies, fixed on a polystyrene substrate. Ar and Ar-F competitively bind to the AT. The surface of the polystyrene again washed to remove unbound Ag and Ag-E. Thereafter, the enzyme activity in the well of a polystyrene plate, which is inversely proportional to the concentration of antigen in the test sample. It is a heterogeneous competitive ELISA.

Fig. 3b shows another embodiment of the heterogeneous ELISA — non-competitive, so-called sandwich method. On the surface of polystyrene adsorb antibodies Ab1 binding Ar (it must have at least two valencies) of the test sample. Then a second antibody AT2 crosslinked enzyme E. After removing the excess AT2 E well polystyrene plate was added reaction substrate and the enzyme activity was determined to be linearly dependent on the concentration of Ag in the sample.

With a special spectral device results in the enzymatic reaction can be measured by polystyrene plates for one minute in 96 wells. However, in general, the heterogeneous and

+ 7At

+ 3At

Washing off

+ 5 (AG-E) + 5Ag

-AT-AG-E -At-AG AT-AG-E AT-AG

, Washing off

+ 3 (AG-E) + * ■ 3Ag

AT-AG-E-AH AT AT-AT E-AH-AH B

+ 2Ag

AT1 AT1

At1-AG

At1 AG-AT1

| -At1-AG-E-At2 -At1 -At1-AH-AH-At2 -At1-E-AG

Washing off

At1-AG-E-At2 | -At1 At1-AH-AH-At2-E

Fig. 3. Competitive (a) and noncompetitive (b) a heterogeneous enzyme immunoassay

ELISA longer homogeneous. Its implementation requires 3-4 hours, because at every step of the operation should be a balance, and only then can spend washing immuno-nosorbenta, which also takes time.

Application ELISA

The sensitivity of the ELISA is that certain substances in concentrations of 10" 9-10 ~ 12 M, protein micro-programs Partial-nanogram in 1 ml — is commonplace de lo. Just as the human eye detects a single light quantum, IFA can usover-shenstvovat so that it cascaded systems using amplification allows detection of single molecules of the analyte.

Possibility of increasing the sensitivity og ranichivayutsya background analyte (i.e., its presence not only in the sample, but in the reagents and solvents used), and substrate specificity of the enzyme affinity antibodies. Limitations ELISA relates the presence of test samples cofactors, inhibitors, stimulators and enzyme activity. Another drawback — EIA not distinguish between native proteins and their biologically inactive fragments that retain antigenic determinants. ELISA hindrance may be a change in the catalytic activity of the enzyme when conjugated to an antigen. A limitation of the ELISA is its applicability only to the well-studied systems, where there is a purified antigen and highly specific antibodies us.

High sensitivity in conjunction with the speed of the analysis (from several minutes to several hours), to simultaneously test a large number of samples and the absence of a specific need of preliminary operations for purification and concentration of the analyte in the sample ELISA impart advantages over other analytical methods. So today ELISA is widely used in health care, the areas of agriculture, industrial biotechnology, environmental protection and scientific research.

Any disease of humans and animals can be quickly and accurately diagnose by the identification of the pathogen, its individual antigenic components of antibodies to these components or substances which are not peculiar to a healthy body and synthesized by its pathological conditions (cancer, cardiovascular and endocrine diseases). Clinical examination of the population, epidemiological surveys, detection of poisoning, the availability of drugs in the blood, determination of drug compounds in the tissues, viral diseases of plants, certain antibiotics, vitamins and other biologically active compounds in the selection of active producing strains in industrial biotechnology, quality control of medicines from donated blood for viruses, pathogens of AIDS and hepatitis B — this is just a small list of the practical application of ELISA. Modern funda-mental studies in biochemistry, cellular physiology and immunology, microbiology, Viru-sologii, cancer is difficult to imagine without the IFA. Reagents for IFA today become someone-commercial products and can be purchased through catalogs known companies.

REFERENCES

1. Abelev TN Basics about immunity // Soros-razovatelny Journal. 1996. № 5. C. 4-10.

2. Galaktionov VG How does the immune system // Ibid. 1997. № 12. C. 2-9.

3. Abelev TN Monoclonal antibodies // Ibid. 1998. № 1. pp 16-20.

4. Igor Berezin et al. Biotechnology / Ed. NS Egorov, VD Samuel. M .: Higher. HQ., 1987. Kn. 8: Engineering enzymology.

5. Egorov AM and others. The theory and practice of immunoassays-element-analysis. M .: Higher. wk., 1991.

6. ELISA / Ed. TT Ngo, G. Lenhoff. M .: Mir, 1988.

* * *

Vitaliy D. Samuels, Sc.D., Professor, Department of Cell Physiology and Immunology, Moscow State University Research interests — bioenergetics, microbiology, biotechnology, immunology. Author of more than 200 articles, monographs and three textbooks.

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