Preparation Of Monoclonal Antibody Against Nodularin And Its Application In ELISA

NOD is a cyclic pentapeptide hepatotoxin produced by the cyanobacterial genus Nodularia. NOD is a hazardous algal toxin, and it is believed to be potent tumor promoter and initiator. The main target organs of NOD is liver. The toxicity problem of NOD to animals and people is of increasing concern, with the incidence of algal blooms grows. Concern regarding the presence of NOD in water and their possible contamination in food has been raised the attention, it is absolutely necessary to develop a fast, economical and sensitive method to determine NOD.The common method to determine NOD is chromatography, but it is only used for the corroborative analysis because of the complicated sample preparation and the needing of expensive instruments. So a simple and reliable analytical method for this toxin is required.The complete antigens of NOD were prepared and the monoclonal antibody (McAb) against NOD was produced. An indirect competitive inhibition enzyme-linked immunosorbent assay (ic-ELISA) was developed for detection the NOD in water and animal edible tissues. The study will provide foundations for further study of the test kits for detection of NOD in water and animal food samples.NOD was conjugated to Immunglobulin G (IgG) and bovine serum albumin (BSA) by Glutaralaehyde method. The conjugates NOD-IgG and NOD-BSA were analyzed by nondenaturing gel electrophoresis and ultraviolet spectrophotometry. The results demonstrated that the two kinds of artificial antigens were synthesized successfully, the molecule conjugate ratios of NOD to IgG and BSA were 15:1 and 10:1 respectively.Female BALB/c mice were immunized by foot pad injection with the emulsive mixture of NOD-IgG and CFA/IFA adjuvant. 15 days after priming, serum titers of immunized mice were determined by indirect ELISA. The mice, whose serum titers were 4000 or higher were selected to be popliteal lymph nodes donors for hybridoma production.Lymph-node cells from mice immunized with NOD-IgG were fused with myeloma cells SP2/0. Two hybridomas cell lines named 2B2 and 1C6, which secret McAb against NOD were selected,. The characteristics of the McAb secreted by 2B2 and 1C6 were studied, the titers of ascites were 1:1.02×106 and 1:2.56×105, respectively, the subclasses and the affinity constant were IgG1, IgG2a, 5.24×109 M-1and 2.68×109 M-1. The monoclonal antibody did not cross-react with MC-LR, Okadaic Acid and Tetrodotoxin.The McAb against NOD secreted by 2B2 was used to deveop the ELISA. An ic-ELISA was developed for the quantitative detection of NOD and the optimized condition as follow: The NOD-BSA was added to microtiter plates at a concentration of 1.0μg/mL and incubated overnight at 4℃. The plates were washed three times with PBST for 30 seconds, and 100μL 1% NH4Cl was added to each well to eliminate nonspecific binding by blocking the plastic surface where protein was not bound. After 1h of incubation at 37℃. The plates were washed three times with PBST, the NOD-McAb (1:16000) and varying concentrations of standard NOD (50μL/well) were added. After 30 min of incubation at 37℃. The Plates were washed three times with PBST, and 100μL/well goat anti-mouse IgG-HRP (1:4000) was added and incubated for 1h at 37℃. The plates were washed four times, and 100μL/well OPD substratae solution was added, followed by the addition of stopping solution (2M H2SO4) after 10 minutes of incubation in the dark at 37℃. Absorbance values at 490nm were determined by an enzyme immunoassay reader. The inhibiting rate (A/A0×100%) was calculated from the absorbance value obtained in the presence (A) and absence (Ao) of NOD. A linear dose-response standard curve was prepared by plotting log[NOD] versus inhibiting rate.The regression equation of the standard curve was y=--16.224x+63.052. The correlation coefficient, the lower limit detection and a linear range were R2=0.9975, 0.16 ng/mL and 0.16 ng/mL～100 ng/mL respectively.Water samples were collected from our laboratory and NanHu Lake, and each water sample was divided into five groups, the standard NOD ( 0.5, 5, 10, 20 and 50 ng/mL) were added to each group respectively. The ic-ELISA was used to determine the sample. The results were as follow: The average recoveries of NOD were 88.02% and 85.2%, and the coefficients of variation were 2.34% and 2.42%. We also collected fish organ samples (muscle, innards) which were sold on the local market, each sample was divided into five groups (5 g/group), NOD (0.5, 5, 10, 20 and 50 ng/mL ) was added to each group. The sample was extracted twice with the tissue homogenizer and the phosphate buffered solution (pH7.0), The results were as follow: The average recoveries of NOD from muscle and innards were 80.5% and 80.2%, and the coefficients of variation were 3.04% and 3.48%.The recoveries in the sample of water and fish organs were more than 80%, which corresponded with the common regulation of ELISA. It was no report that the McAb against NOD was applied to the ELISA for dection of NOD in water and food animal edible tissues. This experiment will provide foundations for further study of the test kits for detection of NOD and the detection research of other toxins.