Preparation Of Aflatoxin M₁ Monoclonal Antibody And The Development Of Aggregation-induced Luminescence Immunochromatography Strip

In the process of transportation,storage and processing of agricultural products such as grain and feed,aflatoxin residue is easily caused by aspergillus flavus and aspergillus parasiticus,which seriously endangers human health.There are more than20 kinds of aflatoxin and its derivatives,among which aflatoxin B₁（AFB₁）is the most common.AFB₁can be converted into aflatoxin M₁（AFM₁）by hydroxylation after it enters the animal body through feed and AFM₁is excreted with milk.With the improvement of our living standard,the intakes of rich nutritious milk and dairy products gradually increases.AFM₁has stable chemical structure,high temperature resistance,and is difficult to decompose in the process of food processing.Therefore,monitoring AFM₁in milk and dairy products is of great significance to maintain the health of consumers,especially infants.At present,the detection methods of AFM₁mainly rely on the instrumental analysis method of large equipment.Although such detection methods are highly sensitive,they are unable to achieve large-scale on-site screening due to expensive non-portable equipment required,complex sample pre-processing process and high requirements on operators.On the other hand,Lateral Flow Immunochromatography（LFIA）is suitable for rapid field assay because it is easy to operate and can interpret results with naked eyes.The immunochromatographic performance is closely related to the antibody performance and the labeling material.Among them,the sensitivity and specificity of immunoassay can be effectively improved by using antibodies with appropriate immune affinity and strong specificity.The new probe material with strong signal output ability can replace the traditional Au NPs with weak optical signal,which can effectively improve the sensitivity of LFIA and meet the increasingly strict requirements of food safety detection.Aggregation-induced Emission（AIE）refers to a class of molecules that do not emit light or emit light weakly in dilute solution,but emit light strongly in the state of aggregation or solid state.AIE fluorescent molecules have the advantages of large Stokes shift,strong photobleaching resistance and high stability.Aggregation-induced Emission Fluorescence Microspheres（AIEFMs）prepared from AIE molecules applied in LFIA can significantly improve the sensitivity of the strips.Therefore,in this study,the preparation of AFM₁monoclonal antibody and the research and development of AIEFMs-LFIA were mainly carried out as follows:（1）Preparation of AFM₁monoclonal antibody.AFM₁haptens containing carboxyl group were synthesized by modifying AFM₁with CMO.Then it was coupled with carrier protein KLH/OVA by EDC/NHS method to synthesize complete antigen with both immunogenicity and reactivity.A hybridoma cell 5G12 producing AFM₁monoclonal antibody was obtained by cell fusion,hybridoma cell screening and cloning.ELISA was used to evaluate the performance of the antibody produced by 5G12.The results showed that the Ka of the antibody reached 5.39×10⁸L/mol.When AFM₁-OVA was used as the detection antigen,the IC₅₀and LOD were 0.458ng/m L and 0.135 ng/m L respectively.This antibody has a high cross-reaction rate with aflatoxin such as AFB₁,AFB₂and AFG₁,and is a broad-spectrum antibody.Based on the high cross-reaction rate with common aflatoxins,this study further evaluated the performance of AFB₁-BSA as an alternative to the expensive AFM₁-OVA for antigen detection.The results showed that the IC₅₀and LOD were0.475 ng/m L and 0.120 ng/m L respectively when AFB₁-BSA was used as the detection antigen,which were similar to the results obtained when AFM₁-OVA was used as antigen.Therefore,immunoassay based on 5G12 antibody can use AFB₁-BSA,which has a lower price,as an alternative detection antigen to reduce the detection cost and facilitate the mass production and market application of detection products.Since milk does not contain AFB₁,AFB₁-BSA as detection antigen will not cause non-specificity of AFM₁detection.（2）Development of AIEFMs-LFIA for the detection of AFM₁.A probe was prepared by coupling the AFM₁antibody with AIEFMs.By optimizing the amount of antibody labeling,the amount of probe,the T-line antigen AFB₁-BSA coupling ratio and the spraying concentration of AFB₁-BSA,AIEFMs-LFIA method without sample pretreatment was established for the rapid detection of AFM₁in milk.The results showed that the regression equation of AFM₁in milk quantitatively detected by AIEFMs-LFIA was y=-0.177ln（x）+0.2672（R²=0.9915）,the IC₅₀and LOD values were 0.2684μg/kg and 0.0280μg/kg,respectively.The linear range is 0.0492～1.5625μg/kg.Its LOD is 1/17.8 of the AFM₁national limit（0.5μg/kg）for milk and dairy products.The recoveries of whole milk,skimmed milk and raw milk were85.6%～101.7%,92.0%～107.8%,81.1%～109.7%,and the coefficients of variation were less than 10%.These results indicate that AIEFMs-LFIA can achieve high sensitivity,high specificity detection of AFM₁in milk,and the method is accurate and reliable.