CALCULATIONS FOR RADIOIMMUNQASSAY 



PbTx-3 



Unh concentrations ^ OJOl nMoies/bter to 100 nMoles/lher, or OJOl pMol/mL to 100 pMoI/mL 

 50% displacement (EDjg) of tmiated toxin occurs at IjO pMol/mL (0.895 pgrams) 



Unknowns 



Unit concentration « 125 fiL of iOOfd, original san^le (125%)« 100 Units 

 1 Unit » 0JQ12S% of sample 



Figure 1 



( #) 2505-9 fraction 4^ [plate 3] adjudged incondusive 

 Iy) 2505-9 fraction 73 [plate 3] adjudged incondusive 

 (A) 2505-9 HPLC peak 50% di^lacement at 11 Units or 0.1375 % at ehited peak (11 z 0i)125%) 



Therefore, 0.1375% * 895 pgram equivalents in HPLC peak fay oonqurison with standard curve for PbTx-3. 



100% / 0.1375% - 72727. Yielding (895 x 727)- 650,655 pg. or 650 ng in total peak. 



The peak assayed was 50 mL/IOOO^L total, wfaidi « 650 ngx (1000/50)- 10 ug PbTx-3 equivalents in sample 

 prior to HPLC 



Back calculations to original amount: 



three plates, using 42% or each plate for bioassay - 87i^ toodn remaining at HPLC stq>. 



(87.9% X 10 Mg equivalents calculated against standards) - apppiujum ately 11 ^g brevetoxin in sample 2505-9. 



'Based on 46.639 g of liver supplied, appi o xim ately 024 fig toodn per g liver was presem in the sanq>le.* 



Figure 2 



( • ) 2505-6 fraction 3 [plate 2] 50% displacement at 20 units or 0253% of sample at this stage (20 x 

 0i)125%). 



Therefore, 0253% - 895 pgram equivalents in fraction 3 from the TLC plate by comparison with standard 

 curves for PbTx-3. 



100%/D253% - 39526, Yielding (895 x 39526) - 353,757 pg. or 353 ng in total sample. 



That 353 ng is equivalent to 91.8% of the total sanqile (100% z 0i^8 z 0.958 - 91 J%). 



Therefore, the sample was calculated to contain 385 ng brevetoxin (353 x 100%A)1.8%). 



'Based on a liver we^t of 41255 g, appfoiim a tel y 933 ng taadn per g fiver was present* 



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