Preparation Of Monoclonal Antibodies Against Aflatoxin B₁ And Study On Immunoassay Methods

Aflatoxins（AFT）are the most toxic toxicants of mycotoxins and harmful to humans.Among them,Aflatoxin B₁（AFB₁）is common and has the highest toxicity and high detection rate in foodstuffs.Therefore,Administration of Quality Supervision,Inspection and Quarantine of the People’s Republic of China stipulate the limited standard of AFB₁ in the most foodstuffs.There are two single-analysis methods detect AFB₁,comprising instrumental methods and immunoassay methods.By Comparison of the instrumental methods,the immunoassay methods have the prominent advantages,such as high sensitivity,high specificity,high throughput and low cost.In this paper,AFB₁ is the research object and studied via immunoassay method.The main research results are as follows:The spleen cells of BALB/c mice immunized with AFB₁-BSA and SP2/0 myeloma cells were fused,screened and subcloned.The obtained hybridoma cells（4D₉）were able to stably secrete monoclonal antibodies against AFB₁.Enzyme-linked immunosorbent assays（ELISA）were used to identify antibodies（including antibody subtypes,sensitivity,detection limits,specificity,accuracy,stability）.The immunoglobulin subtypes of the monoclonal antibody was shown as IgG3 with kappa light chains.Using the optimized experimental conditions,the limits of detection（LOD）and semi-inhibitory concentrations(IC₅₀)of antibody were 0.044μg/kg and0.254μg/kg respectively.And the detection range was 0.044-1.35μg/kg.The maximum cross-reactivity rate with structural analogs was 3.95%.The results showed that the antibody has strong affinity and specificity for AFB₁.And the sensitivity also meets the national limited standard.An ELISA（TSA-ELISA）method based on tyramine signal amplification was established.Under the optimal experimental conditions,the LOD and IC₅₀0 were 0.004μg/kg and 0.039μg/kg respectively.And the detection range was 0.004-0.414μg/kg.The maximum cross-reactivity rate of the analogues was 3.48%.The recovery of AFB₁ was between 81.4%and 118.8%in the three edible oil samples,and the coefficient of variation was between 3.8-9.0%.The results of TSA-ELISA and high performance liquid chromatography（HPLC）for the detection of AFB₁ in edible oils were highly correlated（R²=0.9898）.The TSA-ELISA and HPLC positive results in the 18 authentic samples were between 0.031-4.73μg/kg and 0.027-4.32μg/kg respectively.The results showed that the method has high sensitivity,strong specificity and high accuracy.A high-efficiency AFB₁ analysis method based on immunomagnetic beads（IMB）was established.The LOD and IC₅₀0 were 0.0335μg/kg and 0.606μg/kg respectively.And the detection range was 0.0690-5.319μg/kg.The biotin-streptavidin（B-SA）system was used to amplify the results.The LOD and IC₅₀0 were measured to be 0.00579μg/kg and 0.573μg/kg respectively.And the detection range was 0.0183-17.946μg/kg.The immunoassay method based on B-SA with IMB have more advantages and was chosen for the following studies.The maximum cross-reactivity rate was 3.97%.The recovery rate of AFB₁ in the agricultural samples and soil samples were between 89.6%and 118.2%,the coefficient of variation was 3.4-13.2%.The correlation coefficient with the HPLC method was 0.9928.The established IMB homogeneous analysis method based on B-SA was simple,efficient,sensitive,specific.And it has a wide detection range.It can be used for high-efficiency and rapid screening of AFB₁contamination in environmental samples.