MONOCLONAL-ANTIBODIES TO VRV-PL-VIIIA, A BASIC MULTITOXIC PHOSPHOLIPASE-A(2) FROM VIPERA-RUSSELLI VENOM

VRV-PL-VIIIa, the most basic phospholipase A(2) (PLA(2)) from the veno m of Vipera russelli, induces multiple toxic effects, including neurot oxicity, myotoxicity, edema and hemorrhage. Rabbit polyclonal antiseru m was raised against VRV-PL-VIIIa. The antiserum cross-reacted in enzy me-linked immunosorbant assay (ELISA) with two other PLA(2) from the s ame venom, VRV-PL-V and VRV-PL-VI, and with ammodytoxin A, caudoxin an d crotoxin. Twenty-two hybridoma cell lines secreting monoclonal antib odies against VRV-PL-VIIIa were isolated. The monoclonal antibodies ex hibited apparent binding affinities in ELISA with VRV-PL-VIIIa ranging over two orders of magnitude. Most of the monoclonal antibodies cross -reacted moderately with VRV-PL-V and weakly with VRV-PL-VI. None of t he antibodies cross-reacted with ammodytoxin, caudoxin or crotoxin. Re ducing the disulfide bonds of VRV-PL-VIIIa lowered the ELISA signals o f each monoclonal antibody to nonspecific levels, suggesting that all the antibodies recognize conformational epitopes. Four of the 22 antib odies neutralized the enzymatic activity of VRV-PL-VIIIa. Interestingl y, two of the four exhibited the lowest affinities of the monoclonal a ntibody library for VRV-PL-VIIIa in ELISA, while the other two exhibit ed the highest. Each of the monoclonal antibodies was biotinylated and spatial binding relationships were evaluated by competition ELISA.