Screening by Immunoprecipitation and Polyacrylamide Gel Electrophoresis

It is customary to identify positive hybridoma wells by simple binding assays, and then identify the antigen biochemically. However, Brown et al. { 1980) have shown that it is feasible to reverse this order and identify the antigen as part of the screening procedure. The method is unlikely to be the first choice in most circumstances, but when antibodies against a particular polypeptide in a complex mixture are sought, it could be considered.

In order to minimize the number of supernates to be tested, two strategies were adopted. First, all supernates were screened for IgG production by testing their ability to compete with [l25I]-IgG for binding to protein A-con-taining staphylococci. About 25% of wells with growth contained IgG. The second strategy involved pooling a series of horizontal rows (1-12), and pooling a series of vertical columns (A-H), with each primary well contributing 10 |il supernatant. The pools were held for 1 h at 0°C with 100-200 nl 125I-labelled lysate of the immunizing cell (c. 108 c.p.m.), and then 1 mg of heat-killed and formalin-fixed protein A-bearing Staphyloccus aureus was added. (One could substitute 50 |xl of 10% v/v protein A-Sepharose for the intact bacteria.) The staphyloccoci, to which the immune complexes were bound, were harvested by centrifugation and washed twice. Bound antigen was released by heating the bacteria to 100°C in SDS sample buffer, and identified by SDS-polyacrylamide electrophoresis and autoradiography. If, for example, and Mr 50000 polypeptide was present only in row pool C and column pool 11, the relevant antibody would be found in primary well C11. The cells in this well could then be cloned and grown in large numbers. A more detailed account of immunoprecipitation analysis can be found in Chapter 10.

8.10.15 Other Assays

A great many other assays are possible. Indeed, any way in which antibodies have been used may be considered. For example, antibodies against hormones could be identified by standard radioimmunoassay techniques in which 125I- or 3H-labelled hormone is precipitated. In many cases, biological assays might be used. For example, antibodies to an enzyme or hormone might be identified by inhibition of its activity (Frackelton and Rotman, 1980; Secher and Burke, 1980). Antibodies which cause passive cutaneous anaphylaxis (PCA) may be identified by subcutaneous injection into rats (Böttcher et al., 1978; Eshhar et al., 1980).

0 0

Post a comment