Development of a Hemolytic Plaque Assay for Glutamic Acid, Lysine-Containing Polypeptides: Demonstration that Nonresponder Mice Produce Antibodies to These Peptides When Conjugated to an Immunogenic Carrier

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Fowl gamma-globulin, when chemically conjugated to GLO or GL, functions as a T-dependent immunogenic carrier and stimulates anti-GLO and anti-GL antibody production in nonresponder mice. The conjugation procedure utilizes the Schiff base reaction. The anti-GL and anti-GLO responses were detected by hemagglutination and hemolytic plaque assays by using GL-coated erythrocytes. The coupling of GL to erythrocytes utilizes a novel procedure in which a palmitoyl derivative of GL is adsorbed onto red blood cells. The optimal conditions for preparing the palmitoyl derivative and for coupling to SRBC are presented. With the hemolytic plaque assay, we have verified that GLO responder animals make both IgM and IgG responses, whereas nonresponder mice fail to make either IgM or IgG plaque-forming cells.