1. Mass production of monoclonal antibodies

There are two main methods for mass production of monoclonal antibodies:

(1) Use a rotating culture tube to culture hybridoma cells in vitro, and obtain monoclonal antibodies from a supernatant. However, this method has a low yield. Generally, the antibody content in the culture medium is 10 to 60 μg / ml. If mass production is performed, the cost is higher.

(2) Inoculate hybridoma cells in vivo to prepare ascites or serum.

2. Identification of monoclonal antibodies

The systematic identification of the prepared McAb mainly includes the following aspects:

(1) Identification of antibody specificity

In addition to the use of immunogens (antigens) for antibody detection, other antigens related to their antigen components should also be used for cross-testing. ELISA and IFA methods can be used. For example: to prepare McAbs against melanoma cells, in addition to reacting with melanoma cells, tumor cells of other organs should be cross-reacted with normal cells in order to select monoclonal antibodies specific for tumor-specific or tumor-associated antigens

(2) Identification of McAb class Ig and subclass

Generally, when screening with a secondary antibody labeled with an enzyme label or fluorescein, the Ig type of the antibody has been basically determined. If an enzyme-labeled or fluorescein-labeled rabbit anti-mouse IgG or IgM is used, the detected antibodies are generally IgG or IgM. As for subclasses, standard anti-subclass serum systems should be used for double expansion or sandwich ELISA to determine. In the double-expansion test, if the appropriate amount of PEG (3%) is added, it is more conducive to the formation of the precipitation line.

(3) Identification of McAb neutralizing activity

Animal or cell protection experiments are used to determine the biological activity of McAb. For example, if the neutralizing activity of anti-viral McAb is determined, susceptible animals or sensitive cells can be simultaneously inoculated with antibodies and viruses to observe whether the animals or cells are protected by antibodies.

(4) Identification of McAb recognition epitope

The competitive binding test and the method of measuring the addition index were used to determine the antigen sites recognized by McAb to determine whether the recognized epitopes of McAb were the same.

(5) Identification of McAb affinity

ELISA or RIA competitive binding test was used to determine the affinity of McAb to the corresponding antigen.