PRODUCTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES AGAINST STONUSTOXIN FROM SYNANCEJA-HORRIDA

Stonustoxin (SNTX), a lethal factor purified from the venom of stonefi sh Synanceja horrida, is a protein (148,000 mol. wt) existing as a dim er comprising two subunits (alpha and beta) of mol. wts 71,000 and 79, 000, respectively. Its LD(50) (i.v.) is 17 ng/g in mice and it causes haemolysis of rat and rabbit erythrocytes in vitro. Eight monoclonal a ntibodies (Mabs) against SNTX have been developed using the Balb/C mou se. These Mabs have been purified by Protein G affinity membrane disc chromatography. They were all classified as IgG(1) with half of them h aving kappa and the rest lambda, light chains. They had affinity const ants ranging from 3.75 x 10(-9) to 9.74 x 10(-9) M. Six were able to p rotect mice from a challenge of a lethal dose of SNTX. However, not al l protective Mabs were able to neutralize the haemolytic effect in vit ro. Only four Mabs (31A, 32B, 38A and 46A) could inhibit rat and rabbi t erythrocyte haemolysis, while one Mab (43D) offered partial inhibiti on and another Mab (8A) did not inhibit haemolysis at all. The non-pro tective Mabs (43B and 44G) were also incapable of neutralizing haemoly sis. Five epitopes were recognized by the eight Mabs. Four Mabs (31A, 32B, 38A and 46A) were found to have similar epitope specificity while the rest were directed at different epitopes on the SNTX molecule. Th us these results suggest that the domain on the SNTX molecule responsi ble for lethality is probably distinct from the domain important for i n vitro haemolytic activity.