For immunization, we use the inbred strain of mice BALB/c (Pushchino Nursery for Laboratory Animals). The immunization scheme is developed with an account of the nature of the antigen for which the monoclonal antibodies are to be obtained, and its immunogenicity. If an antigen is toxic, then a non-toxic dose of the antigen shall be determined first. The immunization scheme shall be developed taking into account the peculiarities of antigen metabolism and the antigen immune properties. Under different immunization schemes, at first, the chosen dose of antigen usually introduced subcutaneously in the hind paw of an animal in the quantity of 30-50 µl using Complete Freund’s Adjuvant (CFA)  1:1 with phosphate salt buffer (PSB). Then Incomplete Freund’s Adjuvant (IFA) is used for immunization. We can use other adjuvants. During further immunizations, the used quantity of the antigen in PSB is introduced intraperitoneally in the quantity of 200-300 µl. In doing so, the titer of the antibodies produced in the animal serum is controlled by blood sampling from supraorbital sinus or from vena caudalis and by indirect solid-phase enzyme-linked immunosorbent assay (ELISA). When the titer of antibodies reaches the required values, intraperitoneal boosting of the mouse with the antigen in FSB is performed within 3 days. Then the animal with a good titer of antibodies is killed by cervical dislocation and the spleen is extracted.

Taking spleen samples and splenocytes from it is carried out in aseptic conditions inside a laminar flow cabinet by adding antibiotics in the media. The splenocytes are washed out from the spleen stroma and their quantity is counted using Goryaev counting chamber.

To get antibody-producing hybridomas, the splenocytes of immunized mice of BALB/c line and Sp2/0-Ag14 myeloma cells are used. The myeloma cells and splenocytes in the ratio 1:1 are treated with 40-50 % polyethyleneglycol solution (PEG, Sigma). After the fusion, the selection is carried out from non-fused cells complete, selection by using DMEM growth media with 15 % fetal bovine serum and addition of hypoxanthine, aminopterin, and thymidine (HAT). The cells are dispersed onto culture 96-well plates. Then the selection is performed in the media with the addition of hypoxanthine and thymidine (GT).

Cloning of hybridomas from positive wells in several days after the fusion procedure, a screening of the cell supernatant is performed to identify the presence of the required antibodies by indirect ELISA.

The cells from the wells with positive ELISA signal are further cloned onto 96-well plates, 200 cells per plate, to confirm the monoclonality of the culture. Once all the clones of the assumed monoclonal culture are tested, the monoclonality of the culture is considered confirmed when all the subclones are positive. The confirmed monoclonal cultures are released in the mass culture.

The most widely used today are two methods of accumulation of hybridoma cells and monoclonal antibodies: accumulation on culture media inside CO2-incubator and in the ascite fluids of mice. Each of them has its own advantages and disadvantages. Accumulation of antibodies on culture media is a lengthier and more labor-intensive process. To yield a large quantity of required antibodies, we use animals (BALB/c line mice). One week prior to introduction of the cells, Pristane, an adjuvant stimulating the formation of ascite tumors, is introduced intraperitoneally in the BALB/c line mice in the amount of 200 µl. Then the hybridoma cells, which have been washed out in FSB, are introduced intraperitoneally in mice in the amount of 500 thous. to 1 million of cells. In two-four weeks, the ascite, which contains monoclonal antibodies, is extracted from the abdominal cavity of mice.

From ascitic fluid collected from mice, monoclonal antibodies are isolated by precipitation sequentially 50 %, then 40 % ammonium sulfate. Then it is purified on a DEAE column by anion exchange chromatography, followed by purification on G-protein. All in all, the obtainment of monoclonal antibodies is quite a lengthy and labor-intensive process. However, a great advantage of monoclonal antibodies is their high specificity to a certain antigen, which is very important when it comes to the development of enzyme-linked immunosorbent assay.

The monoclonal antibodies that we obtained are further used as basic components of our test systems, which ensures a high quality of the company’s products and the plasticity of the production process.