Preparations of toxic components from Naja kaouthia venom by selective heat denaturation.

A simple procedure to prepare the toxic components from Naja kaouthia venom for use as immunogens has been studied. The aim was to produce serum rich in antitoxins. By heating the venom (1-6 mg/ml) at 100 degrees C for 10 min at pH 5.0, at least 10 proteins with MW greater than 25,000 daltons were precipitated and removed. The toxic components, i.e., postsynaptic toxins, Direct Lytic Factor (DLF), and phospholipase A2 were relatively stable to this treatment; however, their activities were progressively lost as the heating time was prolonged. The LD50S of the heated (100 degrees C, 10 min) and the unheated venom were 0.37 and 0.325 mg/kg, respectively. As compared to the unheated venom, immunization of rabbits with the heated venom resulted in a 3.38-fold increase in precipitable antibodies against N. kaouthia toxin 3 and a 1.85-fold increase in neutralizing capacity. This toxin preparation should be useful as an immunogen or as a starting material for chemical modification prior to immunization in the production of potent therapeutic antiserum.

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography.

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30-50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab')2 fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl) A peak containing primarily the F(ab')2 antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.

Immunoassays of amphetamines: immunogen structure vs antibody specificity.

Various immunoassays have been developed for the detection of amphetamines. These have varying degrees of cross-reactivity to other drug and food components. Information on the immunogen structures used, and the specificities of the antibodies obtained, have allowed formulation of a "structure-specificity" pattern delineated on the basis of immunochemistry and stereochemistry. The 'structure-specificity' relationship should be useful to future developments of these immunoassays. Specifically, immunoassays intended to detect either amphetamine or methamphetamine with minimal cross-reaction, should employ immunogens with amphetamine (or methamphetamine) derivatized via the para position of the phenyl ring. Such assays should show minimal cross-reaction with other secondary (or tertiary) amines but should strongly cross-react with phenyl ring substituted analogs. On the other hand, assays intended for detection of both amphetamine and methamphetamine should employ amphetamine (rather than methamphetamine) derivatized via its amino group as an immunogen. Such assays should show minimal cross-reaction with other tertiary amines and phenyl-substituted amphetamine/methamphetamine.