RU2356578C2 - Antigen for anticancer antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to antigen used for qualitation and quantification of anticancer antibodies in the analysed material. According to the invention, antigen is prepared of stable nonreversible L-bacteria No.118-L2 being in antigenic affinity with malignant growth cells. The L-bacteria are recovered from the soil biothermally disinfected.

EFFECT: antigen is intended for detecting anticancer antibody titres in blood to reveal risk groups among the population and evaluate effectiveness of anticancer immunisation.

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Description

The invention relates to medicine and relates to the serological determination of anti-cancer antibodies.

In the diagnosis of infectious diseases, such as cholera ("Microbiology and laboratory diagnosis of cholera", edited by M. S. Drozhevkina and V. N. Milyutin. From Rostov, GPCHI, 1975), brucellosis (P. A. Vershilov. Brucellosis. M .: Medicine, 1972; KP Studentsov. Animal brucellosis. Kainar, Alma-Ata, 1975), in serological practice specific antigens made from microbial cells and their components are widely used. Strictly specific cancer antigens for the determination of cancer antibodies have not been developed. Therefore, serological diagnostic methods for mass prophylactic examination in oncology do not exist.

The objective of the invention is the development of antigen, with which it would be possible to determine anti-cancer antibodies in the blood serum.

This goal is achieved by the fact that in the preparation of a diagnostic cancer antigen, bacteria are used in a stable, irreversible L-form. This is justified by the fact that the cytoplasmic membranes of L-forms of bacteria are related in antigenic structure to the cytoplasmic membranes of cancer cells, and in the cross-adsorption of immune sera against the L-form of bacteria and against cancer cells by antigens from L-forms and cancer cells, antibodies are depleted in these sera . The vaccine against cancer from the L-form of bacteria that we previously proposed is based on the same antigenic relationship (Application for invention No. 2003124662, publ. February 27, 2005).

The proposed strain in the L-form No. 118 L2, has lost the cell wall, all species characteristics and can not be attributed to any species according to the "Bergey" bacterial determinant, the strain was isolated in 1982 by planting extracts from soil subjected to biothermal disinfection on selective medium containing 100000 PIECES of penicillin and 80 mg of ammonium chloride in 1 ml of medium, since 1982, is in the collection of the author and stored on nutrient media without changing its properties.

Cultural and morphological signs - coccal cells of the size of 0.5-2.5 microns motionless, gram-negative. In an isotonic solution and with a decrease in atmospheric pressure, they lyse, grow on all nutrient media for heterotrophic microorganisms at a temperature of 15 to 45 ° C. The broth retains viability for more than a year at a temperature of (+2) - (+ 8) ° С. Giant creeping colonies are formed on the agar, and the growth energy exceeds the growth of other L-forms of bacteria obtained from lactic acid bacteria, No. 19, No. 16/4 vaccine and tularemia vaccine strain.

Physiological signs - have the same high glycolytic activity under aerobic and anaerobic conditions, do not absorb oxygen, have high catalase activity, thin the gelatin, have urease activity, and produce hydrogen sulfide.

Virulent properties - do not possess infectious and invasive properties. When administered subcutaneously to rabbits and guinea pigs, they exhibit toxigenic properties in the form of local necrosis of the skin area with the introduction of more than 10 billion microbial cells. With the introduction of less than 10 billion microbial cells, inflammatory swelling of the skin area is formed without colonization and the formation of pathological processes in the internal organs and on the skin.

Antigenic properties - with subcutaneous administration of less than 10 billion microbial cells to laboratory animals of strain No. 118-L2, specific antibodies are produced in their blood that are completely adsorbed from the sera when cancer antigen obtained by precipitation by centrifugation of pleural and ascites fluids taken from patients is added to them cancer in 4 stages. Conversely, antibodies obtained by immunizing animals with a cancer antigen are completely adsorbed by the antigen from strain No. 118-L2 form. This property makes it possible to determine the titer of anti-cancer antibodies in diluted blood sera using antigens prepared from cancer cells or better from L-forms of bacteria, because their reproduction is possible in unlimited quantities on nutrient media, and the same reproduction of cancer cells is impossible.

Preparation of antigen for the formulation of the agglutination reaction.

A tank grown on nutrient media. the mass is precipitated and killed with ethyl alcohol, mixing by volume 1: 2. Shake well and leave for 1 day, during which another 3-4 times mix. Then the tank. the mass is precipitated, the alcohol is removed and the precipitate is diluted to a concentration of 10 billion microbial cells in 1 ml of saline containing 0.5% phenol. The resulting suspension of bacteria is stained with a crystal violet and used to set the agglutination reaction.

Determination of anticancer antibody titer

The determination of the titer of anti-cancer antibodies in blood serum is carried out using the agglutination reaction on transparent plates with wells in a volume of 0.5 ml. The reaction is put with the test sera in five dilutions, and with the control serum to the limit titer. For this purpose, 0.9 ml of physiological saline (0.85% sodium chloride solution with 0.5% phenol added) is pipetted into the first well, and 0.5 ml of the same saline solution is added to all subsequent ones. Then in the first hole with 0.9 ml of physical. 0.1 ml of the test serum is added to the solution and is also supplied with the control positive serum, mixed with a pipette and 0.5 ml of the mixture is transferred to the 2nd well, etc. until the last dilution and from the last 0.5 ml is discarded. The result was a series of dilutions of serums: in the first 1:10, and then 1:20, 1:40, 1:80, 1: 160, etc. As a negative control, 0.5 ml of the same saline solution is poured into two wells. As a positive control, use serum obtained from animals immunized with a cancer or L-antigen. Then, in each hole, one drop of L antigen is introduced (shake the vial with antigen first). The contents of the wells are mixed by shaking the plates, covered with a film or other material that prevents drying of the contents of the holes. And left at room temperature (+20) - (+ 28) ° C for 20-24 hours. Then take into account the results of the reaction. In positive cases, the resulting agglutinate falls to the bottom of the hole in the form of an openwork, translucent umbrella, and in negative cases, the entire antigen collects in the center of the hole in the form of an opaque dark spot.

Assessment of agglutination reaction: A blood serum test is performed to identify risk groups and monitor the effectiveness of anti-cancer immunization. For this purpose, studies are carried out before vaccination and then 3-4 weeks after vaccination. In individuals with a high level of immunity, anti-cancer antibodies are found in blood serum at a titer of 1:40 and above. In these cases, preventive cancer vaccination is not required.

In elderly people and having immune deficiency, the titers of these antibodies are 1:20 and lower until the absence. Persons with a titer of 1:10 and a zero value are at risk and are subject to mandatory immunization with a cancer vaccine.

Examples

1. Patient E.I., 68 years old. Dz: Cancer of the right breast in the initial stage. In the first study on the agglutination reaction (RA) did not react, i.e. the number of anti-cancer antibodies corresponded to zero. In a blood test 25 days after vaccination, the titer in RA was 1:20, 5 months after revaccination, the titer in RA was 1: 160, which corresponded to a sharp improvement in overall health.

2. Patient LI, 63 g. D-s: myoma 3-4 weeks. RA before vaccination 1:10, after vaccination 1: 160.

3. Patient L.P., 44 g. D-s: myoma 4-5 weeks. RA before vaccination 1:20, after 1: 320.

4. Patient V.Ya., 56 years old. Dz: mastopathy. RA before vaccination negative, after pref. 1:80.

Claims (1)

Antigen for determining anti-cancer antibodies, characterized in that bacteria are used in its preparation in a stable, non-reversible L-form of strain No. 118-L2, which has antigenic affinity for malignant tumor cells and is isolated by selective nutrient media from soil subjected to biothermal disinfection.