DEVELOPMENT AND ADAPTATION TO LARGE-SCALE PRODUCTION IMMUNOENZYMATIC TEST SYSTEM INCREASED SENSITIVITY FOR EARLY DIAGNOSIS OF HIV INFECTION

Abstract

 Actuality - more than 25 years the problem of the spread of the human immunodeficiency virus (HIV) continues to be relevant for the whole world. The timely detection of HIV infection, the introduction of new ones and the improvement of existing diagnostic methods remain the main direction in carrying out measures to prevent the spread of the disease. The problem of early diagnosis of the human immunodeficiency virus can be solved by introducing into the laboratory practice the 4th generation high-sensitivity diagnostic immunoenzyme test systems. The advantage of this combination analysis is the possibility of simultaneous detection in the blood serum of HIV-1 p24 antigen and HIV-specific focal antibodies (IgG, IgM, IgA), which allows detecting infection in the early stages of development. The fourth generation ELISA test system is constructed on the basis of purified recombinant proteins HIV-1, HIV-2 and monoclonal antibodies, which is adapted to large-scale production. Indicators of informativity of the enzyme immunoassay system "DIA-HIV-Ag / Ab" were analyzed, the analytical sensitivity according to the AVI standard was 2.5 pg / ml, and according to the NIBSC standard - 1 IU.

In recent years, Ukraine has developed an unfavorable epidemic situation with respect to the infection caused by the human immunodeficiency virus (HIV), which has become the most important and most urgent medical and social problem of our country [10]. Ukraine has one of the first places among the countries of Europe in terms of the spread of HIV / AIDS .

For the period 1987 - 2016 in Ukraine, 503 413 positive HIV tests were detected according to the data of the CEM (seroepidemiological monitoring of the spread of HIV), 297 424 cases of HIV infection were registered officially among Ukrainian citizens, including 92 897 cases of AIDS, and 41 710 deaths from AIDS-related illnesses.

ELISA for detection of antibodies to HIV- specific proteins is at the center of the HIV diagnostic program. Antibodies (Ab) HIV detected in the serum of almost 99 % of those infected. Early diagnosis of HIV/AIDS is an important measure in the health system. Recent research suggests that early detection of HIV infection, early antiretroviral therapy (ART) can significantly reduce the rate of HIV/AIDS and reduce mortality from the disease. Therefore, the development of diagnostic tools for the early detection of HIV is an urgent task for modern medicine, biology and biotechnology.

RESULTS AND DISCUSSION

Recombinant polypeptides.  Recombinant antigens Env1 and Env2 - analogs proteins gp120, gp41 of HIV-1 and gp105, gp36 HIV-2 is designed to pEX or pET expression system E. coli by cloning specific sequences in the corresponding vectors. These recombinant polypeptides Env1 Env2 and was used for the preparation of imunosorbentu for test systems "DIA-HIV-Ag/Ab", and recombinant proteins Trx-Env1 and Trx-Env2 - for producing conjugates with  HRP.

Getting monoclonal antibodies. A distinct advantage for the application of mAbs in ELISA is the ability to select only those clones of hybridomas that produce high affine antibodies with defined specificity, which can significantly increase the sensitivity and specificity of diagnostic test kits. In addition, the identity of mAbs from batch to batch reproducibility provides analysis procedure.

After the two-stage affinity chromatography using protein A consistent and protein G Sepharose, quality cleaning mAbs was assessed by electrophoresis in 10% PAGE-SDS. In the preparation of mAbs electrophoresis in reducing conditions revealed heavy (about 50 kDa) and light (approximately 25 kDa) chains of immunoglobulins. These mAbs used in this way as part imunosorbentu or conjugate with biotin.

Optimal dose and adsorption buffer mAbs to p24 of HIV-1 and recombinant antigens Env1 and Env2. Detection of HIV p24 antigen is due to use in highly specific mAbs imunosorbentu while immobilized recombinant Ag Env1 and Env2 provide detection present in the sera of antibodies to the parasite.

MAbs to HIV-1 p24 adsorbed at concentrations of 1 to 4 mkg/ml in carbonate buffer (CB) and carbonate-bicarbonate buffer (CBB) using as control antigen p24 HIV (ABI) in concentrations ranging from 5 to 80 pg / ml and 30 negative sera. The optimal sorption mAbs dose is 4 mkg/ml CSC, with a decrease in the concentration of specific mAbs observed decrease in the ratio of  s/co.

MAbs to HIV-1 p24 adsorbed as described above, sorption recombinant Ag performed at CSC in the following concentrations : Env1 - 3, 1.5 and 0.7 mkg / ml , Env2 - 1,5 and 0,7 mkg / ml. Based on the results, recombinant AG Env1 and Env2 at concentrations of 1.5 and 0.7 mkg / ml, respectively.

Determination of the sensitivity of the test system created "DIA-HIV-Ag/Ab" for p24 antigen of HIV-1 was carried out using international standards of HIV p24 ABI and NIBSC. The sensitivity of the developed test system standard ABI was 2.5 pg / ml, and the standard NIBSC - 1 U/ml.

The sensitivity of the test system when testing it on seroconversion panels PRB-927 and PRB-928 4, panels PRB 927 and PRB 928, only the first 928 sera were found positive, since they, according to passport date, no specific antibodies, p24 antigen and HIV RNA. Recent serum composed of panels (№ 2-5 PRB927, № 2-5 PRB928 detected as positive with relatively high CO / Cut off.

CONCLUSIONS

Test system "DIA-HIV-Ag/Ab", which is developed based on recombinant polypeptides mAbs to HIV and HIV p24 may simultaneously in a single analysis to detect both HIV hypertension and blood pressure against it. Point feature set is to use additional signal amplification by introducing a biotin-streptavidin amplifier that increases the sensitivity of the test system and detect up to 2.5 pg/ml p24 antigen 3-5 days earlier than with other diagnostic kits for HIV infection.