tetrodotoxin

Abstract

Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 × 108 L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006–0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56 ± 2.8% with the coefficient variation of 0.01–0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.

2.4. Subclass and Chromosome Count of 1C3 Hybridoma Cell Line

The hybridoma cell line 1C3 against DA was analyzed for subclass using a commercial isotyping kit (IgM, IgG1, IgG2a, IgG2b, IgA, and IgG3). The results of the subclass analysis showed that the mAb producing 1C3 hybridoma cell had the IgG3 subclass (A). The chromosome count analysis by Geimsa staining exhibited that the chromosome number of 1C3 hybridoma cell line was 102 ± 4 (B) among the experiment replicates, and was produced by cell fusion between splenocytes with chromosome number 66 ± 4 and Sp2/0 myeloma cells with chromosome number 39 ± 1, respectively []. Therefore, the results indicated that the 1C3 hybridoma clone was produced by successfully fusing splenocyte and the Sp2/0 cell.

2.8. Preparation of the Standard Curve

The standard curve and recovery test of the 1C3 mAb was analyzed by competitive inhibition ELISA. The standard curve was prepared and the data was analyzed by Microcal OriginPro 9.1 to test the relationship of the concentration of DA with its inhibition. The standard curve was analyzed by the logistic curve and the equation was y = 0.447/(1 + (x/0.0349)0.795), and the correlation coefficient (R2) was about 0.98 (A). The linear equation was y = 0.2507 − 0.0452x with the correlation coefficient (R2) 0.99 (B). In the present study, the half inhibitory concentration (IC50) was 0.03 ng/mL, and the linear range of detection was 0.006–0.2 ng/mL with a lower LOD of 0.006 ng/mL.

2.9. Analysis of Recovery Test and Sample Detection by ic-ELISA Using 1C3 mAb

Recovery test was carried out to assess the quality of the established ic-ELISA, Non-contaminated shellfish extract was diluted properly to minimize the matrix effect and was spiked with DA at various concentrations (0.5, 5, 100, 1000 ng/mL) (). The results demonstrated that the recovery range was from (95.03 ± 5.4)% to (106.18 ± 1.7)% with an average recovery of (100.56 ± 2.8)%, and its coefficient of variation (CV) was ranged from 0.01 to 0.1% with an average CV of 0.03% (). Real shellfish samples (abalone, sea mussel, fresh oyster, giant clam, king scallop, razor clam, fresh water mussel and triangular clam) were randomly purchased from the seafood market for sample analysis. After extraction, the samples were assayed independently in duplicates to check the working of the established ic-ELISA for DA detection in complex matrix. The results showed that four samples from all shellfish samples were DA positive with levels under 30 ng/mL (120 ng/g), and the extracts of razor clam and giant clam showed the highest levels of DA at 6.70 ng/mL (26.80 ng/g) and 2.89 ng/mL (11.56 ng/g), respectively. Moreover, extracts of abalone (0.76 ng/mL) and fresh oyster (0.02 ng/mL) exhibited least detection, demonstrated DA levels lower than 1.0 ng/mL (4 ng/g). Similarly, all other shellfish samples were DA free with no contamination compared with the control in ic-ELISA at OD 450 nm using 1C3 mAb in the study ().