Four PLA₂ isoenzymes (N. nigricollis CM-PLA₂I-IV) were purified from Naja nigricollis (N. nigricollis) venom using Sephadex G-50 gel filtration, and ion exchange chromatography on CM-Sephadex C-25. The characterization of the isolated PLA₂ isoenzymes revealed similarities in molecular weights, and differences in the isoelectric points, the optimum temperature, optimum pH, optimum Ca⁺²concentration and metal ion requirements. A good correlation (r   <0.7) for the in vitro neutralization of enzymatic PLA₂s activity and the ELISA titers was found for sera collected at one week from each boosting of the rabbits. The found correlations were particularly high (r>0.9) when the purified Seph-PLA₂ and CM-PLA₂II were used rather than the whole venom. The established correlations show the importance of the purified PLA₂ enzymes as immunogens for raising therapeutic antisera and as diagnostic reagents for in vitro determination of the potency of the therapeutic antisera.