Production and characterization of monoclonal antibodies against stonustoxin from Synanceja horrida.
Stonustoxin (SNTX), a lethal factor purified from the venom of stonefish Synanceja horrida, is a protein (148,000 mol. wt) existing as a dimer comprising two subunits (alpha and beta) of mol. wts 71,000 and 79,000, respectively. Its LD50 (i.v.) is 17 ng/g in mice and it causes haemolysis of rat and rabbit erythrocytes in vitro. Eight monoclonal antibodies (Mabs) against SNTX have been developed using the Balb/C mouse. These Mabs have been purified by Protein G affinity membrane disc chromatography. They were all classified as IgG1 with half of them having kappa and the rest lambda light chains. They had affinity constants ranging from 3.75 x 10(-9) to 9.74 x 10(-9) M. Six were able to protect mice from a challenge of a lethal dose of SNTX. However, not all protective Mabs were able to neutralize the haemolytic effect in vitro. Only four Mabs (31A, 32B, 38A and 46A) could inhibit rat and rabbit erythrocyte haemolysis, while one Mab (43D) offered partial inhibition and another Mab (8A) did not inhibit haemolysis at all. The non-protective Mabs (43B and 44G) were also incapable of neutralizing haemolysis. 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. Thus these results suggest that the domain on the SNTX molecule responsible for lethality is probably distinct from the domain important for in vitro haemolytic activity.
Yuen
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R
Department of Biochemistry, National University of Singapore.
Cai
B
B
Khoo
H E
HE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000917:Antibody Formation; D002846:Chromatography, Affinity; D015941:Complement Hemolytic Activity Assay; D000939:Epitopes; D004912:Erythrocytes / Q000187:drug effects*; D005397:Fish Venoms / Q000276:immunology / Q000378:metabolism / Q000633:toxicity*; D005399:Fishes; D006460:Hemolysin Proteins / Q000276:immunology / Q000378:metabolism / Q000633:toxicity*; D006461:Hemolysis / Q000187:drug effects; D006825:Hybridomas; D007928:Lethal Dose 50; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005397:Fish Venoms; D006460:Hemolysin Proteins; C073228:stonustoxin