Preparation Of Monoclonal Antibody Against Brevetoxin And Research Of Detection Method

Brevetoxins are marine biotoxins,which mostly produced by planktonic dinoflagellate species of the genus Gymnodinium breve.Brevetoxins have extended shelf life and can exist in the seafood products for a long time rather than undergoing gradual degradation.These toxins are able to transmit into the humans by the consumption of contaminated seafood,and result in a number of adverse health effects.Therefore,it is essential to develop rapid detection methods of BTXs for they are widely distributed in various waters across the globe.Moreover,they have a pivotal implication in the country's principle seafood import and exports.In the current study,a monoclonal antibody(mcAb)was developed and further characterized for the establishment of immunoassays against brevetoxin-1(BTX-1).BTX-1 was conjugated with larger carrier proteins such as ovalbumin(OVA)and bovine serum albumin(BSA)by mixed anhydride method.BTX-1-OVA was used for administering animals,and BTX-1-BSA was used for coating antigen.The cell fusion was subsequently carried out after serum titer reached 1:8000.Finally,a stable cell line that can secret the mcAb against BTX-1 named 6C6 was screened out after four times sub-cloning.The subtype of the mcAb was IgG1,and the affinity constant was 1.055×108 L/mol.The mcAb showed high specificity and no cross-reactivity with other toxins.In this study,the purified mcAb was obtained from ascites by caprylic acid-ammonium sulfate purification method and titer of antibody reached 1:32000.Indirect competent ELISA was established with the mcAb against BTX-1.The logistic equation of standard curve was y=0.08779+0.011138/[1+(x/58.00982)^1.58278],with a correlation coefficient R2 of 0.98078.The half inhibitory concentration of BTX-1 binding to anti-BTX-1 mcAb was 58 ng/mL.The detection range from 20%to 80%of the standard curve was 25 to 208 ng/mL,with the lower limit of detection of 14 ng/mL.The recovery rate of BTX-1 by ELISA from the spiked samples between batch to batch was high,and coefficient of variation was less than 5%.So,the ELISA kit for detection of BTX-1 was successfully established.Moreover,colloidal gold test strip was developed based on this mcAb.The sensitivity of the strip was 200 ng/mL,and the strip exhibited high detection specificity to BTX-1.The ic-ELISA and colloidal gold test strip was successfully established based on the mcAb against BTX-1.The establishment of sensitive immunoassay have significant applications for the detection of marine biotoxins and pathogens.Additionally,they are vital for rapid detection and monitoring of the seafood products for the safety of human health.