ENZIM-IMMUNOLOGICHESKIY the METHOD (a synonym reaction an enzyme-sword-nykh of antibodies) — the serological test based on interaction of antigen with the antibody which is chemically connected with enzyme (enzyme). The antigen formed a complex — an antibody gains enzymatic activity and splits the corresponding substrate with color or luminescent effect.
The enzyme-immunological method is 10 — 100 times more sensitive than classical serological tests (see. Serological researches), on sensitivity it approaches a ra-dioimmunologichesky method (see). Enzim-immuno the logical method can be used not only for titration of antibodies, but also for detection of antigens, microbes, napr, viruses or viral antigens.
AA. - and. m apply at a lab. to diagnosis of a brucellosis, cholera, tuberculosis, rickettsioses, measles, rubellas, flu, a tick-borne encephalitis, and also parasitic infections (malaria, a schistosomatosis, a trypanosomiasis, toxoplasmosis) since others it is gray l. methods at these diseases are a little effective.
For a tag of antibodies use peroxidase of horse-radish and an alkaline phosphatase more often, and also (Z-B-galaktozi-Dazu, glucosidase, glucoamylase, acetylcholinesterase, a penicillinase and other enzymes. With immunoglobulin (antibody) apply glutaric dialdehyde or periodate of sodium to conjugation of enzyme. As substrate for immunoglobulins, marked peroxidase, serves the ortho-phenylenediamine, during the splitting to-rogo Wednesday is painted in brown color. For immunoglobulins, marked an alkaline phosphatase, take 4 nitrophenyl phosphate, products of its splitting give green coloring. Except the called chromogenic substrates exist lyumo-gene, to-rye later splitting shine in UV rays.
Distinguish a straight line E. - and. m when antigen reacts with a marked antibody, and indirect E. - and. the m, at Krom a complex antigen — an antibody comes to light marked antiimmunoglobuli-new (anti-IgG) serum. The direct method is used by hl. obr. for indication of antigen, and indirect — for identification and titration of antibodies.
At express diagnosis of rage prints of a brain of sick animals on a slide plate fix acetone and apply on drug marked with peroxidase antirabic serum. In 1 hour of an incubation at t ° 37 ° apply substrate on drug — the sulfate-diamino-benzidine prepared on the tris-buffer (pH 7,4) with addition of hydrogen peroxide. In 5 min. drugs wash water and investigate by means of a light microscope. At a positive take in cytoplasm of cells find inclusions, specific to rage — Negri's little bodies painted in brown color (see Rage). At an indirect method drug is processed at first not marked rabbit antirabic serum, takes up edges with antigen, and then this complex is revealed by addition of marked antikro-lichy globulin and substrate.
For titration of antigens and antibodies in fluid medium one of reagents is immobilized on the firm carrier, napr, on a surface of holes of microtablets from polystyrene. This modification of reaction is called a solid-phase immunoenzyme method (TIFM), to-ry in foreign medical literature it is known as ELISA (enzyme linked immunosorbent assay). For identification, e.g., about-tivokorevykh antibodies bring the purified antigen of a virus of measles in holes of microtablets and leave for adsorption on 18 — 20 hours at f 4 °. Nesvya-zavshiysya antigen is washed, add the studied serum in cultivation to holes 1:50 and left for
2 hours at f 37 °. Then holes carefully wash out and bring in them serum, marked peroxidase immune to globulins of the person. Then again wash and add substrate — an orthophenylenediamine. At a positive take (existence of antibodies in the studied serum) substrate changes color on yellowy-brown, holes of microtablets with serums are left colourless without antibodies. Reaction can be estimated with the naked eye, but it is better by means of the spectrophotometer. Applying marked anti-IgM and anti-IgG of serum, it is possible to find at the same time antibodies and to define a class of immunoglobulins, to the Crimea they belong. Holes of microtablets cover with immunoglobulin, antibody-containing to the studied antigen, napr, to a virus; then bring test, in a cut look for antigen (virus). If in test there is an antigen, then it contacts antibodies. At the subsequent addition of marked serum, specific to required antigen, she unites to free receptors of a virus and decomposes the substrate brought after that.
Bibliography: Voller A., Bidu-
ell D. and Bartlett of A. Immunofer-mentativnye of reaction in diagnostic medicine, Bulletin WHO, t. 53, No. 1, page 38, 1977, bibliogr. S. Ya. Gaydamovich.