1998;352:21C26. by CIPPIA, and the results showed good correlation ( 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA process combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins. Cyanobacteria (blue-green algae) produce a wide range of secondary metabolites which are hazardous to humans, livestock, and wildlife (2). Among these are a group of potent hepatotoxins, the microcystins and nodularins. Several bloom-forming cyanobacterial genera are capable of producing these toxins; these genera include (23), to dephosphorylate the chromogenic substrate (23), was diluted in buffer made up of 50 mM Tris-HCl, 1 mM Na2EDTA, 2 mM MnCl2, 0.5 g of bovine serum albumin per liter, and 0.1% (vol/vol) -mercaptoethanol, the pH was adjusted to 7.4, and 10 l was added to each well. for 10 min in an Eppendorf 5415 centrifuge, and the producing supernatants were evaluated by CIPPIA and HPLC with Biotin sulfone DAD (13). RESULTS Preincubation of polyclonal microcystin-LR antiserum with purified microcystin-LR was found to completely neutralize the inhibitory effect of this toxin when PP1 was added up to a microcystin-LR concentration of 100 g liter?1 (Fig. ?(Fig.1a).1a). The ability of microcystin-LR antiserum to bind microcystin-LR and thereby safeguard PP1 from subsequent inhibition by the toxin was dependent upon the antiserum concentration. Preincubation of microcystin-LR with preimmune serum did not prevent inhibition of the PP1 enzyme. At a higher microcystin-LR concentration (500 g liter?1), microcystin-LR antiserum at a 1/200 dilution was not able to completely prevent inhibition of PP1 activity by the toxin. However, the PP1 activities at this concentration of microcystin-LR were still higher if preparations were preincubated with a 1/200 dilution of microcystin-LR antiserum compared to toxin at comparative concentrations preincubated with preimmune serum (Fig. ?(Fig.1a).1a). The high microcystin-LR concentrations were beyond the linear detection range of the standard colorimetric protein phosphatase inhibition assay (1, 22). Calculating the differences in PP1 activity between assays that included microcystin-LR incubated in the presence of preimmune serum and assays that included microcystin-LR incubated in the presence of microcystin-LR antiserum and multiplying the values by the microcystin-LR comparative concentration used to inhibit PP1 revealed that there is a dose-response relationship between total theoretical protection of PP1 and total theoretical inhibition of PP1 (Fig. ?(Fig.1b).1b). At microcystin-LR concentrations greater Biotin sulfone than 100 nM, the calculated functions for PP1 and microcystin-LR showed values Rabbit polyclonal to USP33 of 46,000, 28,000, 10,000, 4,000, and 0 for total theoretical protection, 1/100, 1/200, and 1/500 dilutions of Biotin sulfone antiserum, and theoretical total inhibition, respectively (Fig. ?(Fig.1b).1b). Open in a separate windows FIG. 1 Neutralization of the inhibitory effect of microcystin-LR (MC-LR) on PP1 by preincubation of purified microcystin-LR with microcystin-LR antiserum. (a) Microcystin-LR was preincubated with microcystin-LR antiserum at 1/100 (), 1/200 (?), and 1/500 (?) dilutions, and the results were compared to results obtained after preincubation of microcystin-LR with preimmune serum at a 1/100 dilution (). Preparations were incubated for 1 h at 37C before analysis by the colorimetric protein phosphatase inhibition assay. The vertical error bars indicate standard deviations (= 3). (b) Mean delta PP1 activities (PP1) were Biotin sulfone calculated (%ActAS ? %ActNS) at each microcystin-LR concentration in the presence of antiserum at 1/100 (), 1/200 (?), and 1/500 (?) dilutions and were multiplied by the microcystin-LR equivalent concentration, and the results were compared with the complete theoretical protection (?) and total theoretical inhibition ().