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Characterization of the Primary IgM Response to Gat and GT: Conditions Required for the Detection of IgM Antibodies¹

From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Abstract

Primary IgM antibody responses to synthetic linear copolymers of L-glutamic acid, L-tyrosine, and L-alanine were investigated. The appearance of primary IgM anti-GAT antibodies was detected in BALB/c mice by using a solid phase radioimmunoassay (SPRIA) procedure. This finding was verified for GAT in responder mice and GAT-MBSA and GT-MBSA in nonresponder mice in an indirect plaque forming cell (PFC) assay by using a rabbit antiserum directed against the µ myeloma protein, MOPC 104E. Facilitated IgM PFC could be inhibited by a purified µ myeloma protein, TEPC 183. Maximal facilitated IgM plaque response was found to precede the IgG response by several days. A direct plaque assay was developed for the detection of IgM anti-GAT plaques using poly-L-lysine (PLL) to couple GAT to sheep erythrocytes (SRBC). GAT-SRBC coupled by the PLL method optimally couple 4 to 5 times less antigen to the indicator cell surface than does the CrCl₃-coupling method routinely employed in our laboratory. These findings were extended to a conventional antigen, chicken gamma globulin (CG). We found that a less dense epitope coat on the indicator cell surface favors detection of direct IgM PFC, whereas a more densely coated indicator cell favors the detection of facilitated IgM and IgG PFC responses.

Footnotes

¹ This work was supported by Grant AI-09920 from the National Institutes of Health.

² Supported by Grant CA-01930 from the United States Public Health Service.

³ Supported by an Arthur Sachs grant from Harvard University and by the Pasteur Institute, Paris.