Specific Radioimmunoassay for the Detection of a Purified Porcine Zona Pellucida Antigen (PPZA)

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Abstract
A radioimmunoassay (RIA) utilizing a biochemically purified porcine zona pellucida antigen (purified porcine zona antigen, PPZA) and rabbit antibodies raised against PPZA is described and characterized. PPZA was used for 125I labeling and as reference material in the RIA. Anti-PPZA activity was titered in serial dilutions of sera collected at weekly intervals for 11 wk following the initial series of immunization injections and for 5 wk following a single booster injection. This antiserum was of high titer, high affinity and devoid of significant reactivity against the porcine tissues tested; a measurable but low level of cross reactivity was observed when tested against pig serum and pig follicular fluid. In response to the initial immunization series, peak titer was obtained in 6 wk at which time 30% of [125I]-PPZA was precipitated by antibody diluted to 1:400,000. There was no further increase in antibody titer following the booster immunization. Details of PPZA iodination, stability of [125I]-PPZA, RIA methodology and assay validation for sensitivity, precision, accuracy, specificity and other parameters are described.