In two cases the VAV curves did not have a well-defined maximum which is shown for H. stephensii venom with TSAV and A. antarcticus venom with DAAV, which
had broad VAV peaks with two maxima ( Fig. 2D and E). T. carinatus with TSAV and P. porphyriacus with both BlSAV and TSAV had distinct maxima in the VAV curve (data not shown). E. carinatus and E. ocellatus venoms (250 ng/ml) were incubated with Indian polyvalent antivenom and applied to a plate coated with anti-E. ocellatus antibodies and D. russelii venom (250 ng/ml) was incubated with Indian polyvalent antivenom and applied to a plate coated with anti-D. russelii antibodies. Detection was with labelled anti-horse antibodies. Fig. 4 shows a clear VAV peak for D. russelii venom signaling pathway but not for E. carinatus venom. The antivenom concentration where there was a peak in absorbance due to VAV increased with increasing venom concentration and was determined using the fitted curves. Fig. 5 shows the linear relationship between the antivenom concentrations for the VAV peak versus venom concentration over the venom range of 50 ng/ml to
500 ng/ml. The slope of the lines can then be interpreted as the ratio of antivenom to venom where there is http://www.selleckchem.com/products/Adriamycin.html a peak in absorbance from venom–antivenom complexes. This varied between 0.04 and 0.15 mU/ng for all Australian commercial venoms (Table 1, Fig. 5A) and was 0.09 U/μg (95%CI: 0.07–0.12 U/μg) for P. textilis venom, 0.04 U/μg (95%CI: 0.035–0.05 U/μg) for N. scutatus venom and 0.08 U/μg (95%CI: 0.06–0.10 U/μg) for O. scutellatus venom. For D. russelii venom the slope of the line was 180 ng AV/ng ( Fig. 5B). To examine the behaviour of individual venom components, we collected four well-defined fractions from the HPLC of N. scutatus venom ( Fig. 6). Each fraction comprised 6–8% of the total
area of the HPLC trace. The fractions were characterised acetylcholine by mass spectrometry and matched to previous structures as: Fraction I – notexin (13,544 Da), fraction II (16,742 Da), fraction III (14,002 Da) and fraction IV – notecarin (46,678 Da). Fraction II could not be matched to a previously isolated structure. Fraction III matched to a phospholipase A2 toxin in Acanthophis sp. (acanmyotoxin-3 [fragment]). The prothrombin activator Notecarin has previously been isolated in this manner, and shown to consist of two peaks corresponding to two isoforms ( Rao et al., 2003). Fraction II and Notecarin bound poorly to the rabbit anti-N. scutatus antibodies used to coat the plate so VAV measurement with these fractions was not possible. Notexin and fraction III produced VAV curves similar to whole venom, but with maxima displaced to higher or lower TSAV concentrations compared to whole venom ( Fig. 7). RVVFX, the FX-activating component from RVV, was mixed with Indian polyvalent antivenom and assayed for VAV. A set of VAV curves was obtained at RVVFX = 50, 100, 250 and 500 ng/ml, showing a concentration of AV at VAVmax as 8, 18, 36 and 66 μg/ml (Fig. 3).