Preparation and Purification of Mice Monoclonal Antibody

The preparation and purification of mice antibody can be divided into cell fusion, screening and monoclonalization of positive hybridoma cells, and production and purification of monoclonal antibodies.

Cell fusion

The method of animal immunization is the same as that of antiserum. In order to ensure that the obtained B cells secrete strong antibody activity, intravenous enhanced immunization should be carried out three days before fusion. Mice myeloma cells (such as SP2/0-Ag14 cell line) should grow well in the logarithmic division stage when fused. B cells from spleen of mice obtained under sterile condition should be washed twice or three times with serum-free medium to remove mice production serum. The ratio of spleen cells to myeloma cells is generally 5:1 to 10:1. The commonly used fusion agent is 50% polyethylene glycol. After fusion, PEG is removed by centrifugation after slow dilution of culture medium. Then the cells are inoculated into 96-well plate by HAT medium (RPMI1640 contains 10%-20% fetal or calf serum and HAT suspension). Normally, clone growth can be seen under light microscope on the 3rd and 4th day after fusion, and screening can be carried out after the 10th day.

PEG is commonly used because of its low fusion efficiency, simple method and low cost, although the fusion rate of electrofusion is high, the number of cells in one fusion is small, and special equipment is needed, so it is seldom used. In the process of cell culture after fusion, the abdominal cavity cells of the same animal (i.e. feeder cells) can be added at the same time. The phagocytes can remove the debris of dead cells and contribute to the growth of hybridoma cells. Commercial hybridoma growth factor can also promote the growth of hybridoma cells.

Screening and Monoclonalization of Positive Hybridoma Cells

ELISA is the most commonly used screening method for hybridized cells after 10 to 14 days of culture (1 to 2 times of medium replacement). The positive cloned cells were subcloned by limited dilution method to ensure that the antibody secreting cells came from a single cell. Because the chromosomes of fused cells are easily lost, it is generally necessary to subclone them several times until all subclones from the same clone are positive, indicating that the cloned antibody-coding genes have been relatively stable and can be expanded for cultivation and construction.

Expanded production of monoclonal antibodies

The method of monoclonal cell line expansion culture and antibody preparation depends on the actual needs. At present, there are three commonly used methods to produce large quantities of monoclonal antibodies: mouse ascites preparation, bottle culture and hollow fiber reactor. The former is mostly used for laboratory preparation, and the latter two are suitable for industrial production. Ascites preparation method is low cost, but it is easy to inactivate antibodies due to the high amount of impurity proteins in ascites. Therefore, ascites should be purified as soon as possible after collection to prevent the degradation of antibodies. The supernatant obtained from flask culture was large in volume, but the concentration of antibody was low, and the cost of culture and purification of antibody was high. Compared with large bottle culture, the production of monoclonal antibodies using hollow fiber reactor is a more economical method, but the cost of equipment and equipment is larger.

For laboratory research or non-drug antibody production in mice, the antibodies obtained by ascites preparation and purification of protein A or protein G can basically meet the needs. The concentration of antibodies in the supernatant of hybridoma culture is generally in the order of microgram per milliliter, which can be directly used in immunoblotting and immunoprecipitation experiments. Using special serum-free medium for hybridoma culture can easily obtain the antibody requirements of general experiments.