RU2399383C1 - Method for preparing reference serum for anticardiolipin antibodies diagnosis in quantitative microprecipitin test for syphilis - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to preparing diagnostic products. The method is implemented by intratesticular infections of rabbits producers of 3.0-3.5 kg with pathogenic Nichols strain T. Pallidum pallidum. 7-8 days after the infection, the animals are additionally twice intravenously immunised with purified treponema proteins ("ОПТ"-antigen) recovered from "КСТ"-antigen of cultural Treponema Palladium in dosage 0.023 g, every 7-8 days. It is followed with exsanguination 30 days after the infection. The prepared serums are analysed for anticardiolipin antibodies titre in quantitative cardiolipin antigen microprecipitation test.

EFFECT: method allows increasing anticardiolipin antibodies titre, standardising a procedure of quantitative cardiolipin microprecipitation test for syphilis, and halving the findings analysis time.

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Description

The invention relates to biotechnology, in particular to the production of diagnostic preparations, and can be used in the production of diagnostic sera to control the quantitative reaction of microprecipitation with cardiolipin (CRMP-K) for syphilis.

The invention solves the problem of organizing the production of inexpensive diagnostic serum for monitoring the results of CRMP-K for syphilis.

With syphilis, immunity is significantly different in nature from immunity in other infectious diseases of humans and animals. The course of the disease is accompanied by the development of two types of antibodies: nonspecific - anticardiolipin (reagins) and specific - anti-treponemal. Reagins react in vitro with a cardiolipin antigen (diphosphatidiglycerin) extracted from bovine heart muscles and are detected in the complement binding reaction (CSC - Wasserman reaction), a quantitative microprecipitation reaction with cardiolipin (CIRMP-K), a flocculation reaction for rapid diagnosis with (EMF). Reagins are produced and determined in the serum of all patients with syphilis, but do not have a protective effect against infection, however, their presence together with anti-treponem antibodies indicates syphilis and the presence of vegetative forms of pathogenic T. Pallidum pallidum (the causative agent of syphilis) in the body. Specific anti-treponemal antibodies (IgY and IgM) are produced under the influence of protein antigens of the syphilis pathogen. Recovering from an infection during treatment can become infected and become ill again. The syphilis immunity is “non-sterile” - “infectious” and is characterized by cellular and humoral defense reactions that cause the formation of the primary chancre, generalization of infection against the background of the production of both types of antibodies in the patient’s blood (O.K. Pozdeev under the editorship of V.N. Pokrovsky. Medical Microbiology. - Moscow: GEOTAR-MED, 2001, pp. 433-434, A.I. Korotyaev, S.A. Babichev, Medical Microbiology, Immunology and Virology. - St. Petersburg: Special Literature, 2002, p. .486-478).

Currently, syphilis has become epidemic in both developed and developing countries. The timely diagnosis of infection is of primary importance, however, the industrial production of domestic inexpensive control standardized diagnostic drugs for syphilis for use in serological laboratories of various levels is not well established.

There is a method of obtaining an anti-syphilitic diagnostic control serum for CSC (Pharmacopoeia article "Serum for the diagnosis of syphilis control, positive, dry, for CSC" FS 42-12 BC-91, approved by the Pharmacopoeia Committee of the Ministry of Health of the USSR 07.07.91,). The disadvantage of this method is the high cost of serum associated with low titers of anticardiolipin antibodies (from 1:12 to 1:15 before drying and 1:10 after drying of sera), and the use of large amounts of rabbit with experimental syphilis in this regard in the production of production rabbits to obtain the required production volumes.

Closest to the claimed invention is a method for producing control serum for the diagnosis of syphilis in RSK (RF Patent No. 2185856 "Method for producing control serum for the diagnosis of syphilis." Pozharskaya V.O., Gurtovoy E.A., Ermolov A.V. Publ. 27.07 .2002 Bull. No. 21). The method is carried out by intratesticular infection of rabbits weighing 3.0-3.5 kg with a pathogenic Nichols strain T. Pallidum pallidum, 7-8 days after infection, the animals are immunized twice intravenously with treponem cell structures (KST antigen) from culture pale treponemas (CBT), followed by bleeding of animals 30 days after infection. The pale treponem biomass is grown on an artificial thioglycol medium from a complete set of strains of three antigenic groups, followed by ultrasonic disintegration of the cells, centrifugation, separation of the undisturbed treponem, centrifugation of the supernatant, isolation of the treponeme wall sediment and the supernatant (RF Patent No. 2141339 for the production of anti-trepanem cultures from ". Pozharskaya VO Publ. November 20, 1999 Bull. No. 32). The method allows to increase the average titers of anticardiolipin antibodies in CSC from a dilution of 1:15 to 1:40 in the blood of bloodless animals.

According to the orders of the Ministry of Health of the Russian Federation No. 87 dated March 26, 2001 “On improving the serological diagnosis of syphilis” (Cl. 2.1. Appendix No. 1) and No. 327 dated July 25, 2003 “On the approval of the Syphilis protocol for the management of patients (based on Methodical guidelines of the Central Research and Design Institute for the Prevention and Treatment of Syphilis. M .: 1999) the KMPP-K test was introduced into the complex of serological reactions (DAC) for syphilis to monitor the presence and dynamics of titers of anticardiolipin antibodies (reagins) for the diagnosis of syphilis in serological laboratories all levels: regional GOVERNMENTAL, regional, district and for monitoring the effectiveness of the specific treatment of syphilis infection in conjunction with the haemagglutination test (RGA) - treponemal syphilis. However, the control serum for the diagnosis of anticardiolipin antibodies in CRMP-K for syphilis is not produced and their daily titration is required before placing CRMP-K according to the same order of the Ministry of Health of the Russian Federation No. 87 dated 03/26/2001 (p. 4-6), which doubles the time of setting and recording the results of the studied sera for syphilis in this reaction.

The aim of the invention is the possibility of organizing the industrial production of control serum for the diagnosis of anticardiolipin antibodies in CRMP-K for syphilis and halving the time for setting the reaction in serological laboratories at all levels by half, and also reducing the cost of its production by increasing titers of anticardiolipin antibodies by immunizing rabbits with experimental syphilis purified treponem proteins (OPT) obtained from KBT KBT, and a corresponding decrease in the number of used lively tn-producers.

This goal is achieved in that rabbits weighing 3.0-3.5 kg are intratesticularly infected with a pathogenic Nichols strain T. Pallidum pallidum. 7-8 days after infection, the animals are additionally immunized intravenously at a dose of 0.023 g twice with an interval of 0.023 g of purified Treponem proteins (OPT) obtained from CTB KBT, then the animals are bled for 30-31 days after infection, serum is obtained, titers are determined anticardiolipin antibodies in CRMP-K, standardize, pour and dry the finished product.

An OPT antigen for immunization of rabbits with experimental syphilis is obtained from the CTF antigen by freeing it from general group antigens (RF Patent No. 2156467 “Enzyme-linked immunosorbent assay for detecting antibodies to the causative agent of syphilis.” Shavyrina MV, Ermolov VV , Pozharskaya V.O., Telbuh V.P. Publish. 09/20/2000 Bull. No. 26).

In relation to the above prototypes of the claimed method has the following distinctive features.

Additional immunization after infection of rabbits with the pathogenic Nichols strain T. Pallidum pallidum is carried out by an OPT antigen twice at a dose of 0.023 g, which leads to a higher level of increase in the titer of antibodies to a nonspecific cardiolipin antigen as a complement of binding (in CSC) and precipitins (CMPP-K )

If according to the prototype immunization with KST antigen, positive (4+) titers to the anticardiolipin antigen in the CSC are detected at a dilution of 1:40, and at KMPP-K at a dilution of 1: 8, then according to the proposed method (immunization with an OPT antigen), the titers of anticardiolipin antibodies are detected in DSCs are detected at a dilution of 1: 50-1: 60, and in KRMP-K at a dilution of 1: 32-1: 40.

The obtained results say that the OPT antigen significantly increases the titer of not only complement-binding antibodies, but also precipitins, which justifies its use in obtaining control positive sera for the formulation of CRMP-K for syphilis.

According to the requirements of the pharmacopeia article, the released dry control positive sera with cardiolipin antibody in CSCs must be positive no lower than in 1:10 dilution.

According to the order of the Ministry of Health of the Russian Federation No. 87 dated 03/26/01, "On improving the serological diagnosis of syphilis" (Cl. 2.1. Appendix No. 1, Clause 2, Clause 4-6) in the preparation of control positive sera in laboratories by daily titration in As a control, positive sera in dilutions of not lower than 1: 8 are used.

The possibility of practical use of the proposed method is confirmed by examples of specific performance.

The basis for further studies described above is the standardization of the used KST and OPT antigens by dry weight (g) in 1 ml and administered to producer rabbits with experimental syphilis obtained by the standard method 7 and 14 days after infection with their bleeding for 30 days (terms of maximum titers of anticardiolipin antibodies), serum was prepared according to a known method and titers of anticardiolipin antibodies were determined in it in CSC and in KMPP-K.

30 animals with experimental syphilis were injected intravenously on the 7th day after infection with 0.0115 g of KST antigen, on the 14th day - 0.023 g of KST antigen. In rabbit blood serum, on day 30, the average titers of anticardiolipin antibodies were 1: 8 in RSK and 1: 8 in KRMP-K (RF Patent No. 2185856 “Method for the production of control serum for the diagnosis of syphilis.” Pozharskaya V.O., Gurtova E .A., Ermolov A.V., published on July 27, 2002, Bull. No. 21).

Example No. 1. Control group. 30 animals with experimental syphilis were injected intravenously on the 7th and 14th day after infection with 0.023 g of KST antigen. On day 30 in the blood serum of rabbits, the average titers of anticardiolipin antibodies were 1: 43 ± 2.1 in RSK, and 1: 9 ± 1.3 in KRMP-K. The mixture of sera obtained from 30 rabbits amounted to a titer of anticardiolipin antibodies in CSC - 1:45, in CLCP-K 1:10. Thus, the comparative titers of the obtained anticardiolipin antibodies in CSCs and in KMPP-K turned out to be 1.012 and 1.25 times more, respectively, and are the standard background for comparing the same amount of OPT antigen introduced (0.023 g).

Example No. 2. The proposed method. 30 animals with experimental syphilis on the 7th and 14th day after infection were injected intravenously with 0.023 g of OPT antigen. On day 30 in serum of rabbits, the average titers of anticardiolipin antibodies were 1: 57 ± 2.8 in CSCs and 1: 36 ± 3.8 in CRMP-K. A mixture of sera obtained from 30 rabbits, when titrated, showed a titer of anticardiolipin antibodies in CSC 1:55 in KRMP-K 1:34.

The obtained comparative results allow us to establish a reliable P <0.01 increase in the titer of precipitating anticardiolipin antibodies. When immunizing rabbits with an OPT antigen in CRMP-K, the proposed method of immunization, which exceeded the control titer by 3.4 times, at the same time, comparative titers of anticardiolipin antibodies in CSCs exceeded the control by 1.2 times, which indicates higher activation of anticardiolipin antibody production in rabbits with experimental syphilis with the introduction of an OPT antigen.

In the production of control sera, standardization (bottling, drying), taking into account production losses of up to 20%, required to have titers of anticardiolipin antibodies in KMPP-K brought to 1:10 when the serum was bottled.

The obtained control series of the production release of sera showed titers of anticardiolipin antibodies in CRMP-K at a dilution of 1: 8. Taking into account titers of precipitins in serums when setting up KMPP-K, it is possible to determine the degree of dilution as 1: 2 or 1: 2.5, which significantly increases the amount of serum produced, ensuring their lower cost (reducing the number of serum producers), as well as halving the time of obtaining the results of the study on syphilis in KRMP-K.

The control production series of the drug were tested at the bases of the Branch of the Scientific-Production Unitary Enterprise “Microgen” of the Ministry of Health of the Russian Federation “Allergen” in the city of Stavropol and the Stavropol Regional Dermatovenerologic Dispensary (serological laboratory).

The production series of control positive sera for the diagnosis of anticardiolipin antibodies in CRMP-K for syphilis and the documentation for them for approving the release of the drug are being prepared.

Thus, the invention is feasible, its use in biotechnology will provide for the first time in the Russian Federation the industrial standard production of low-cost control sera for CRMP-K for syphilis, which will increase the effectiveness of monitoring the diagnosis of syphilis, and reducing the time to obtain research results will reduce the labor costs for setting up CRMP-K on average 40-60%.

Claims (1)

A method of obtaining a control serum for the diagnosis of anticardiolipin antibodies in a quantitative microprecipitation reaction for syphilis, including intratesticular infection of rabbits weighing 3-3.5 kg with a pathogenic Nichols strain T. Pallidum pallidum, bloodless animals, serum production, determination of anticardiolipin antibodies in a quantitative anti-cardiolipin antigen reaction with cardi , characterized in that the infected rabbits after 7-8 days are additionally intravenously immunized twice with purified treponem proteins , isolated from the CST antigen of the cultured pale treponema, at a dose of 0.023 g with an interval of 7-8 days, the blood is bled from animals 30 days after infection.