Preparation Of Monoclonal Antibody Against Tenuazonic Acid And Development Of Its High-sensitive Immunoassay

Tenuazonic acid（TA）is a toxic metabolite mainly produced by the Alternaria genus,with a molecular weight of 197.231.It was confirmed to have strong acute and subacute toxicity,potential carcinogenicity and cytotoxicity.Long-term exposure or eating may cause tissue hemorrhage and circulatory system failure,motor dysfunction and death,especially for dogs,mice and chickens.TA extensively pollutes grain,food and feed,especially fruits and vegetables and their products.The detection rate of fruit and vegetable products in individual areas reached 100%.Therefore,it needed to establish high-sensitively rapid detection method for TA,which it is extremely important for food safety.This study aimed to obtain stable monoclonal antibodies with high affinity and specificity through hybridoma technique and establish high-sensitive immunoassay.It was successfully prepared complete antigen TA-O-KLH and TA-O-BSA,identified by agarose gel electrophoresis and UV-vis spectroscopy.TA-O-KLH was used as immunogen,and TA-O-BSA was used to detect the serum titer of immunized mice.After three times of immunization,the titer reached 1:1000,and TA-O-KLH could effectively recognize TA.The IC₅₀of two mice reached 32.4 ng/m L and 66.8 ng/m L,respectively.After repeatedly immunization,the serum titer of them reached 1:32k and1:16k with the OD value was higher than 1.0,respectively,which met the requirements for cell fusion.It is indicated that the preparation of anti TA antigen can stimulate the body to produce TA specific antibody.Spleen cells immunized with TA-O-KLH and SP2/0 cells were artificially induced to fuse by PEG1450.The average fusion rate was94.38%and the positive rate was 6.15%by i ELISA.The positives with high titer(OD_450nm≥1.20)were selected for ic ELISA.Twenty strains were sub-cloned.After three rounds of sub-cloning,six strains with good specificity were selected to enlarge cultivation.Finally,four specific hybridoma secreting TA antibodies were successfully screened,named3H10,4H6,6D5 and 9G6,respectively.The titer of 6D5 was the highest,and its isotype was Ig G2b,and the chromosome number was within 107±3,which was formed by the fusion of spleen cell and SP2/0 cells.Therefore,the 6D5 cell lines was chosen for succedent experiment.Ascites were prepared in vivo seduction method.The antibodies was extracted successively by octanoic acid ammonium sulfate method and Protein G chromatography.Identified by SDS-PAGE,highly purified m Ab was obtained at a concentration of 1.56 mg/m L,and the titer was over 1.024×10⁶.The hybridoma was stable after 90 days of continuous culture and cryopreservation.The titer of purified antibody was stable after 90 days of storage.The affinity was 1.72×10¹⁰L/mol,and it was the highest of reported.The IC₅₀was 2.5 ng/m L.It was the highest sensitivity of reported.An ELISA method was developed with linear range of0.49-13.39 ng/ml and LOD of 0.17 ng/m L.The average recoveries of intra-and inter-assays in apple juice were 88.227±2.078%and 86.022±3.858%,respectively,and the coefficients of variation（CV）were 2.356%and 4.453%,respectively.The average recoveries of tomato ketchup were 93.404±3.183%and 85.704±5.175%respectively,and the CVs were 3.440%and 6.214%,with good precision.The detection results of real samples are similar to UPLC-MS/MS,which is accurate and reliable.The 20 nm colloidal gold nano-particle（GNP）was prepared by sodium citrate reduction method.The peak of UV Vis absorption spectrum was519.3 nm,and the average particle size was 17.52 nm.TEM observation showed that the morphology was nearly spherical and the particle size was highly uniform.Using sodium chloride titration,the results showed that the optimal p H was 25μL 0.1M K₂CO₃and the optimal amount of antibody was 7.5μL（1.56 mg/m L）6D5 in 1m L GNP.The test line（T line）was coated 1/4 diluted antigen（3.20 mg/m L）with 1.25μL/cm,while the control line（C line）was coated 1/4 diluted GAMA（1 mg/m L）with 1.25μL/cm,dried at 37℃for 24h.The probe was diluted with 2/5 on conjugate pad and freeze-dried.It showed the lateral flow immunoassay（LFIA）strip had high specificity.T-line was completely eliminated at 100ng/m L,and the visual limit of detection（visual LOD）was 12.5 ng/m L,with good reproducibility and an assay time of 15 min.Using 20 nm colloidal gold as seeds via hydroquinone mediated seed growth method,50 nm flower-like gold nanoparticles（Au NF）were prepared.The dark blue,clear and transparent colloid was obtained.The wave peak was near 580 nm.TEM showed that the colloidal was well dispersed,and the morphology which was flower-like was obvious.With sodium chloride titration,the optional condition was determined,with 5μL 0.1M K₂CO₃and 7.5μL（1.56 mg/m L）6D5 in 1m L Au NF.The T-line was coated 1/8 diluted antigen（3.20 mg/m L）with 1.5μL/cm,and C-line was coated 1/4 diluted GAMA（1 mg/m L）with 1.5μL/cm,dried at 37℃for 24 h,while the probe was diluted with 2/5,freeze-dried.The results showed that the Au NF-based LFIA strip had high specificity.The T-line was eliminated completely at 50 ng/m L,and visual LOD was 0.78 ng/m L.The process was 15 min,which was convenient,rapid and reproducible.A novel strategy for competitive LFIA strip was presented in this work.Gold@platinum hybrid nanopartical（Au@Pt HNP）was prepared with 20nm colloidal gold as seed.With controlling the molar ratio of Platinum to Gold,a series of Au@Pt_rHNPs with different molar ratio（r）was obtained.The colloids were clear,transparent and well dispersed,and its color gradually deepens with the increase of r.When r was from 0 to 0.4,the OD450nm tends to increase linearly in TMB/H₂O₂tests of catalytic performance.After crossing 0.4,the OD450nm tends to be flat,thus,Au@Pt_rHNP with r≈0.4,denoted by Au@Pt_0.4HNP,was obtained.It retained most of properties of the colloidal gold,while it obtained catalytic property and deeper color.The Au@Pt_0.4HNP-LFIA strip was work by catalyzing precipitation-type TMB and gathering sediments as signal,with lateral flow in 15min and colouration in 15-45 min.It showed well reproducibility without cross-reaction in the sample test.The T line was eliminated at 25 ng/m L,while the visual LOD was 0.39 ng/m L.In summary,this study screened monoclonal antibodies with high specificity and high affinity,and then established ELISA method and developed three kinds of LFIA test strip based on colloidal gold,Au NF and Au@Pt HNP.The monoclonal antibody（m Ab）was the most specific and highest affinity m Ab against TA so far.Through contrast,the sensitivity of ic ELISA was improved by an order of magnitude;and the sensitivities of three kinds of LFIA test strip were respectively increased5 folds,84 folds,167 folds,while the threshold were respectively reduced by more than 30 folds,64 folds,128 folds.These results were to solve the problem of low sensitivity for TA rapid immunoassay,to realize the rapidly trace determination of tenuazonic acid,so as to ensure the effective monitoring and prevention of Alternaria tenuazonic acid.It has an importantly referential significance for the monitoring and controlling for food safety and for food security in import and export trade.