Titering Antibodies

unit 4.1

The antibodies used for immunophenotyping are primarily monoclonal, derived from hybridoma fusions of mouse cells immunized against the appropriate antigen. They are almost always of the IgG class, most frequently IgG1, IgG2a, and IgG2b. IgM is a less commonly used class of monoclonal antibody; because it is approximately five times the molecular weight of IgG, it can be more troublesome. Polyclonal antibodies derived from sera of immunized animals are less frequently used as a primary antibody directed against the desired antigen, but are almost always used as the second fluorochrome-conjugated antibody. The F(ab')2 fragment should always be used, to minimize Fc receptor binding of polyclonal antibodies. F(ab')2 fragments of monoclonal antibodies are not readily available because rodent-derived F(ab')2 fragments are difficult to prepare in high yield.

This unit will define the strategy for titering antibodies to give the highest discrimination of positive cells from negative cells. Once titered, antibodies may be combined for providing simultaneous evaluation of multiple antigens expressed by cells.

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