| Animal-derived food safety issues are closely related to our lives.Tetrodotoxin(TTX)naturally exists in the body of pufferfish.As a natural non-protein neurotoxin,if plays an important role in the safety of animal origin.Even now,many scholars have focoused on the detection of TTX.The immunological detection is a simple,rapid and sensitive method.And it commonly used in recent years.To establish a simple and rapid method for the detection of TTX in animal-derived foods,a preliminary study was conducted to establish an ELISA assay for TTX in this study.1.To obtain artificial antigen,formaldehyde was used as a coupling agent to couple TTX with keyhole limpet hemocyanin(KLH)or bovine albumin(BSA).The products,carrier proteins and hapten were detected by UV scanning and gel electrophoresis respectively.The results suggested that the synthesis of the coupled antigen was preliminarily determined.By immunizing Balb/c mice,the mouse tail serum titer was tested to prove the immunogenicty of the prepare completely artificial antigen,further proving the success of artificial antigen coupling.2.The immunization of Balb/c mice was carried out by routine immunization protocol.The antibody titer of rat tail serum was greater than 1:4×105.Monoclonal antibody were screened by cell fusion ELISA and other techniques,and three cell lines A3D6 F10G4 D9F3 with stable secre tion of TTX antibody were obtained.Among them,the A3D6 cell lines with the highest inhibition rate were used and purified to prepare monoclonal antibodies by injecting into rat.SDS-PAGE revealed that the content of miscellaneous protein decreased significantly,and the ascites was purified completely.The ascites protein concentration was 34.84 mg/ml detected by BCA method,and ascites concentration was 0.98mg/m L after the ascites was purified by ammonium sulfate precipitation affinity chromatogrephy.Th ascites protein recovery rate was 9.51%.Antibody subtype detection showed that the heavy chain of Mc Ab was Ig G1 and light chain was Kappa type.Antibody affinity detected by ELISA method showed that the antibody affinity of this strain was 4.8×108L/mol,with a high antibody affinity.3.Indirect ELISA and indirect competitive ELISA were used to establish a TTX ELISA assay for purified A3D6 monoclonal antibody.The optimized conditions were: 1:2500 dilution of coating antigen,1 hour of coating at 37 °C,1% BSA blocking at 37 °C for 1 h,the monoclonal antibody 1:6000 diluted,mixed with the TTX standard sample,added to the enzyme plate for 30 min at 37 °C,and incubated with a 1:1×104 diluted secondary antibody at 37 °C for 30 min,color development 15 min,terminated by 2M sulfuric acid terminated,enzyme atandard meter reading OD450 value.The ic ELISA detection method was performed by this condition,and the standard curve was linear at 5-200 ng/ml.Under this restriction the linear equation was y=0.4071 x – 0.1671,R2=0.9909,IC50=42.52 ng/ml,LOD=3.03ng/ml.The sample matrix interference test results showed that the interference of the sample matrix could be basically removed when the snail meat was diluted 10 times and the shellfish meat was diluted 5 times.The addition recovery test revealed that the average spike recovery is 90% and the average coefficient of variation is 10%.Based on the above results,complete artifical antigens KLH-TTX and BSA-TTX were successfully prepared in this research.KLH-TTX was used as immunogen to effectively generate antiserum in immunized mice,and spleen cells were taken for cell fusion to screen out TTX monoclonal antibody cell strain A3D6 with stable secretion and high specificity and intibition rate.According to A3D6 cell line,the TTX ELISA dection method was pre liminarily established,with a good linear range of the standard curve,laying a good foundation for the detection and application of TTX in production and life. |
