IMMUNOFLUORESCENCE (Latin immunis free, saved from something + fluorescence) — a luminescence in an ultraviolet light of a microscope of the biological object containing the studied antigen after its pretreatment by specific antibodies, marked flyuorokhromy.
The microscopic technique based on the phenomenon (process) And., carries the name of a method of Koons, a method I., a method of the luminescing antibodies (serums) it is also applied in practice as an express method at mikrobiol. to diagnosis of infectious diseases. It is applied also during the studying of a pathogeny of infectious diseases, the mechanism of an antibody response, the antigenic analysis biol, objects.
The immunofluorescent method is the universal immunochemical method combining rather exact morfol, the analysis with specificity and high resolving power immunol, methods. It is based on use of the phenomenon of a luminescence for identification of reaction antigen — an antibody, occurring on a surface of cells or on cuts of fabric.
Big advantage of a method I. consists in its simplicity, the high sensitivity surpassing some other serol, methods and also in speed of obtaining results. It is probably possible to localize and identify any substance having antigenic (gaptenny) properties irrespective of its nature and function. Besides, method I. enters into a round of researches particle antigens of fabrics which are unavailable to studying by many immunol, methods, in particular immunodiffusion.
Method I. it was offered in 1942 by A. Coons with coauthors and gained further development in the 50th after synthesis of the best of the existing flyuorokhrom — isothiocyanate of a flyuorestsein (FITTs). In the same years in the USSR researches on use of this method and its improvement were begun.
Success of a method I. in many respects depends on quality of the luminescing antibodies (serums) which receive by chemical reaction between the specific antibodies which are contained in immune serum and fluorescent dye — flyuorokhromy (see). The received reaction product is called a conjugate. In some cases preparation of specific and active conjugates — a difficult task. Usually with fluorescent dyes conjugate not whole immune serums, but fractions of serum proteins, antibody-containing. And than cleaning of ballast proteins is better, especially the qualitative conjugate can be received afterwards. The best results are yielded by use for a tag of drugs of pure antibodies, but it, unfortunately, is not always possible. Quality of a conjugate is defined also by purity and activity of dye, its quantity at a tag, concentration of protein and antibodies, the size pH at a tag, time and temperature of conjugation, and also extent of cleaning of ballast proteins and excess of dye and existence of hetero logical and normal antibodies.
Flyuorokhroma are such dyes which are capable to absorb light and to radiate it through a short time term (10 - 6 — 10 - 9 sec.). Intensity of fluorescence is proportional to intensity of exciting radiation, and at small concentration of substance it is possible to define quantitatively fluorescent substance on microscopic or tsitol, drug.
The following main requirements are imposed to the flyuorokhroma intended for a tag of specific protein: color of their fluorescence shall differ from an autoflyuorestsention of an object and contrast with a background; they shall have high intensity of fluorescence after accession to protein and shall not change physical significantly. - chemical and serol, properties of antibodies.
In practice use the flyuorokhroma having a flavovirent, yellow and red luminescence. Except FITTs, apply sulphochloride of rhodamine 200B (PCX), sulphonic-acid fluoride of rhodamine 200B (RSF), tetramethylrhodamine isothiocyanate (MRITTs), dikhlortriazinilaminoflyuorestsein (DHTAF), etc.
Conjugation of protein with flyuorokhromy is chemical reaction therefore new connection is formed, in Krom dye is attached to protein by a covalent bond. In reaction also trailer amino groups of a proteinaceous molecule participate in the basic of a ε-amino group of a lysine.
Method I. it is applied in three main modifications. At a direct method [Koons, Kaplan (M. of Kaplan), 1950] apply the specific luminescing serum (antibody) on the drug containing required antigen. After reaction drug is washed out and studied under a luminescent microscope. Advantage of this method is its one-staging and use of a small amount of kontroly reaction.
At an indirect method [Weller (T. Weller), Koons, 1954] process the drug containing required antigen specific not marked serum, not communicated proteins wash and apply the luminescing serum to globulins of not marked serum. In this case the antibodies of the first serum connected by drug — not marked act as antigens. Advantage of this modification is big sensitivity, than at a direct method, and a possibility of use of limited set of the luminescing antibodies.
The indirect method with a complement [Goldvasser, Shepard (V. of Goldwasser, S. of Shepard), 1958] is three-stage. This option consists in use of marked anticomplementary serum which joins a complement of a complex antigen — an antibody. In such statement marked serum appears even more universal.
K. A. Lebedev with coauthors (1971) described a possibility of carrying out an indirect method with use of two luminescing serums (both antibodies against the revealed antigen, and antibodies against immunoglobulins of that species of animals from which the first specific serum is received).
The direct method of identification of antibodies by means of marked antigen for the first time applied Mellors (V. of Mellors) with soavt, in 1959 to the proof of presence of a rhematoid factor at certain cells of an organism. This option of a method has limited use.
In all options Immunochemical, the essence of a method remains invariable: localization of antigen in drug is found on specific fluorescence in the place of reaction antigen — an antibody.
The technique of preparation of drugs is various and depends on type of the studied drug. Subject to studying smears, smears prints from bodies, cuts of bodies, culture of fabric and so forth. Use the fixers to each antigen. Drugs study under a luminescent microscope (ML-2, MLD-1, LYuMAM-2, etc.).
Because in reaction And. many components participate, assessment of reliability and accuracy of results is an obligatory part of a research irrespective of the nature of antigen, properties and the characteristic of antibodies. Therefore carry out control immunol, specificity of the luminescence observed on drug and are consistently convinced of specificity of marked serum, quality of its cleaning, prove immunol. character of the found luminescence, carry out control of a background luminescence with normal serum, are convinced of lack of reaction at the expense of antibodies to foreign substances and define immunol, specificity of observed reaction. Only after that do the conclusion about specificity of observed reaction.
For the purpose of increase in specificity of an indirect method I. at identification of fabric antigens apply as a reactant the marked pure antibodies or conjugates with a high antiserum capacity adsorbed by previously fabric powders or homogenates, free of antigen, related to a specific antibody.
At identification of bacteria, viruses, rickettsiae, the elementary use methods of contrasting of a nonspecific luminescence. For this purpose use bull (or other) albumine, marked rhodamine, Evans's blue, congo red and some diazodyes. During the use of a method of contrasting create the luminescing background on color contrasting with a specific luminescence of a body of interest.
The possibility of definition and differentiation of required antigen in mix with other antigens allowed to create the accelerated diagnostic methods of identification of causative agents of infectious diseases. Method I. it is used for definition of localization and identification of various corpuscular antigens (protozoa, bacteria, rickettsiae, viruses) in the true and mixed cultures (tsvetn. fig. 1 — 3), in culture of cells, in contact preparations, cuts of bodies and fabrics, patol, material from patients. Test-sensitivity at a research of smears from a suspension of bacteria — within 10 4 — 5*10 4 cells in 1 ml. He allows to find causative agents of those infections which laboratory diagnosis is based on studying of antigenic properties of activators (viral diseases, rickettsioses, colienterites). Method I. it is effective at detection of activators (plague, a tularemia, salmonelloses, etc.) in blood of the person, in bodies and tissues of animals (see. Identification of viruses , Identification of microbes ).
Method I. it is widely used in a serodiagnosis of syphilis and immunomorbid conditions. At inspection of patients with diseases of connecting fabric it is used for detection of antinuclear factors, anti-cardial antibodies, immunoglobin receptors of a cell and for identification immunol, factors in sites of the damaged fabric.
Studying of antigens of tumor cells which have superficially localized specific antigens which are absent in normal cells is of special interest. To studying of tumoral antigens, to their role in the course of a malignancy of a cell and in relationship of a tumor and the owner it is devoted many researches using a method I.
By means of a method I. also studying of genetic markers of immunoglobulins of the person is carried out.
See also Luminescence .
Bibliography: Goldin R. B., etc. Immunolyuminestsention in medicine, M., 1977; Zubzhitsky Yu. N. A method of luminescent microscopy in microbiology, virology and immunology, L., 1964, bibliogr.; Immune marker analysis, under the editorship of L. A. Zilber, page 202, M., 1968; Levina E. N and d river. The luminescing antibodies (in studying of pathogenic microorganisms), M., 1972, bibliogr.; Mikhaylov I. F. and Dyakov S. I. Luminescent microscopy, M., 1961, bibliogr.; Goons A. N and. lake of Demonstration of pneumococcal antigen in tissues by use of fluorescent antibody, J. Immunol., v. 45, p. 159, 1942; G o 1 d m a n M. Fluorescent antibody methods, N. Y. — L., 1968, bibliogr.; Kawamura A. Fluorescent antibody techniques and their applications, Tokyo, 1969; Nairn R.G. Fluorescent protein tracing-, Edinburgh, 1964; Wagner M. Fluoreszierende Anti-korper und ihre Anwendung in der Mikro-biologie, Jena, 1967. Bibliogr.
K. L. Shakhanina