| Domoic acid(DOM)is a marine biological toxin with strong neurotoxicity.It is a crystalline amino acid and soluble in water.Its molecular formula is C15H21NO6 with molar mass of 311.33 g/mol,and contains three carboxyl groups.There are numerous global poisoning events caused by DOM that affected marine wildlife,human health,and the prenatal exposure in rodents to DOM showed strong developmental effects.DOM is produced by diatomic algal genus Pseudo-nitzschia.It is an analogue of kainic acid,causes amnesic shellfish poisoning(ASP)that includes temporary or permanent memory loss,neural necrosis,seizures,and fatal at high doses.DOM from Pseudo-nitzschia is accumulated in shellfish,sardines and anchovies in global waters.Therefore,the detection of DOM with high specificity and high sensitivity is crucial.Domoic acid is a characteristic hapten and cannot induce immuno-response after injection.Therefore,it was conjugated with larger carrier proteins(BSA or OVA)by active ester method.The BALB/c mice were immunized with domoic acid-BSA as complete antigen emulsified in Freund's complete adjuvant.Subsequently,five immunizations were done by using Freund's incomplete adjuvant.The mouse 1 had the highest serum titer of 1:256,000 from all immunized mice that was determined by indirect noncompetitive ELISA.The mouse 1 was used for the cell fusion and euthanized in aseptic environment.Spleens were isolated from the immunized mouse 1,and fused with Sp2/0 myeloma cells to develop antibody producing hybridoma cell line,and incubated at 37?,5%CO2,and 95%humidity in the presence of RPMI-1640 medium(10%FBS)for 7 to 10 d.Then,hybridoma cells were screened by indirect noncompetitive ELISA for positive clone.Finally,a positive hybridoma cell named 1C3 was successfully obtained.The chromosome number of 1C3 was counted,and the result showed that its number was 102±4,indicating that the hybridoma cell 1C3 was really originated from the fusion of Sp2/0 myeloma(39-40 chromosomes)and spleen cells(62-68 chromosomes).Hybridoma clone 1C3 was further expanded for the production of higher quantities of mAb as ascites.The mAb obtained from ascites was purified with caprylic acid/ammonium sulfate(NH4)2SO4 precipitation method.The concentration of mAb was 1.63 mg/mL determined by bicinchoninic acid assay.Characterization of mAb showed that it belongs to IgG3 subtype with high OD.The antibody affinity constant was 2.5 x 108 L/mol determined by indirect noncompetitive ELSIA using different coating concentration(0.25,0.5,1 and 2?g/mL)of domoic acid-ovalbumin as a coating antigen.Standard curves were drawn for establishment of indirect competitive ELISA immunoassay,and the LOD was 0.006 ng/mL.The presence of domoic acid in harvest shellfish samples from marine waters is harmful for the health of animals and humans living at coastal areas specifically.Therefore,the established indirect competent ELISA was applied for the detection of domoic acid in the real shellfish samples,and the results showed that very low or no toxin was detected out respectively,so the samples collected in this study were not contaminated with DOM and safe for human consumption. |
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