DeveLopment And Application Of MonocLonaL Antibodies Against Microcystins-LR

Microcystin-LR(MC-LR)is one of the most harmful toxin produced by cyanobacteria.It has multiple organ toxicity,hereditary toxicity and carcinogenicity.Due to the serious eutrophication of water in China,cyanobacteria bloom occurs frequenty.In addition,the properties of MC-LR are quite stale,the degradation rate of MC-LR in water is slow.Human health is seriously threatened by MC-LR contaminated drinking water and aquatic products.At present,the conventionaL detection methods for MC-LR are bioassays,chemicaL anaLysis and immunoassays.Due to its high sensitivity and high detection throughput,immunoassay has become a routine screening methods for MC-LR.In this paper,MC-LR was used as the research object.Firstly,immunogens and coating antigens were synthesize directly by coupLing the free carboxyl group of MC-LR with carrier protein.Sebsequently,the monocLonaL antibody against MC-LR was deveLoped by the immunogens.Finally,an indirect competitive ELISA method for the detection of MC-LR were estabLished based on the monocLonal antibodies.The main resuLts were as follows:1.Synthesis and identification of artificial antigens of microcystin-LR:MC-LR is a hapten with the molecullar weight less than 1000 Da.It is necessary to be coupLed with a carrier protein to stimuLate the immune response.In this study,MC-LR was conjugated with keyhoLe hemocyanin(KLH)and bovine serum aLbumin(BSA)by carbodiimide method and used as immunogens and coating antigens,respectively.Subsequently,the immunogens and coating antigen were identified by UV absorption spectra,SDS-PAGE and matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS).The resuLts showed that the UV absorption spectra of the two artificial antigens has been changed comparing with the correspondent carrier proteins.SDS-PAGE showed that the molecular weight of MC-BSA was higher than BSA.Finally,the molecular weight of MC-BSA was determined to be 76515.84 Da by MALDI-TOF MS.The calculated ratio of MC-LR to BSA was 10:1.In this study,the immunogens and coating antigens were successfully synthesized,which Laid a foundation for the preparation of monoclonal antibodies against MC-LR.2.DeveLopment of monoclonal antibodies against MC-LR:Three balb/c mice were immunized with MC-KLH and Freund's adjuvant.After 5 times of immunization,the antiserum titer and sensitivity of antiserum were detecteded by ELISA.The resuLts showed that the titer of No.l mouse antiserum has reached 1:32000,and the half inhibiton concentration(IC50)of antiserum was 0.53 ?g/mL.Then,the spleen of No.1 mouse were selected to fused with SP2/0 cells under PEG 1500,and the proportion of the spleen cells to SP2/0 was 10:1.The hybrid were cuLtured with selective medium.When the area of hybrid was estimated to be 1/10 of the well,the supernatant was detected by indirect non-competitive ELISA and indirect competitive ELISA.The positive cLones were seLected by the finite diLution method,and the cell lines named 2A9 could stably secrete monoclonal antibodies recognizing MC-LR.Subsequently,ascites of 2A9 was prepared in vivo,and purified by saturated ammonium suLfate and protein G column.After purification,the subtype of antibody was determined to be IgG2b type by indirect ELISA,The antibody affinity constant is 2.4 × 107 L/mol,which belongs to the middle and higher affinity antibody.3.Establishment of indirect competitive ELISA for MC-LR:The optimal working concentration of coating antigens and ascites of 2A9 was determined to be 2.5?g/mL and 1:500 diLution by chessboard titration.Under the optimaL working concentration,an indirect ELISA.method was estabLished.The resuLts show that IC50 and the detection Limit of the antibodies were 3.42 ?g/mL and 0.10?g/mL,respectively.In addition,the cross-reactivities of antibodies to MC-RR was 66.25%.Finally,lake water and tap water without MC-LR were spiked with three different levels,the recoveries and CVs were between 74.22-92.23%,1.87-9.31%,respectively.The results were verified by HPLC chromatography.These ELISA method can be used to detect MC-LR with high concentration in spires heaLth food and 2A9 were aLso potentiaL to be empLoyed to produce immunoaffinity columns.