A Multiple Lateral Flow Immunoassay Based On AuNPs For The Simultaneous Detection Of 5 Kinds Of Harm Factors In Milk

Aminoglycosides,quinolones,sulfonamides and tetracyclines are widely used antibiotics,which are often used for the prevention and treatment of animal diseases and also as additives to promote the growth of animals.The improper use or excessive use of antibiotics can cause the accumulation of antibiotics in animals,resulting in excessive residues in animal food,and further cause food safety problems.Aflatoxin M₁ is a toxin produced by animal metabolism after eating feed contaminated by aflatoxin B₁.It is often detected in milk,also known as“milk toxin”.Antibiotic residues and mycotoxin contamination in food,collectively known as harm factors,can cause harm to humans and affect health.At the same time,these chemical contaminants may coexist in the same food samples.Therefore,it is of great significance to establish an efficient and rapid multiple detection method.The traditional lateral flow immunoassay method focuses on the detection of one contaminant.There may be one or more kinds of contaminant in food,and the detection of only one of them cannot guarantee the safety of food.Multiple lateral flow immunoassay（M-LFIA）has multiple detection bands,that is multi-pathway assay,and can be used for simultaneous field detection of multiple pollutants in food samples,which has the advantages of reducing sample size and saving detection time and cost.Make it more and more extensive in the field of field detection.By preparing highly sensitive m Ab,this study established M-LFIA for simultaneous detection of five chemical pollutants in milk.The main research methods and conclusions are as follows:（1）Gentamicin（GEN）and carrier protein were combined into complete antigen by glutaraldehyde method,and the complete antigen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis.Mice were immunized with the successfully synthesized immunogen,and the serum of the mice was monitored by indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）.After the fifth immunization,the mice with high positive rate and good inhibition rate were selected for cell fusion.Mouse spleen cells and myeloma cells were fused by polyethylene glycol method.After a series of hybridoma cell screening and subcloning steps,three hybridomas capable of secreting m Ab were finally obtained.Three kinds of m Ab（4G7,4H9 and 4H11）were obtained by ascites preparation and purification.The half-maximal inhibitory concentrations(IC₅₀)of the m Abs were 0.339,0.469 and 0.625ng/m L,respectively.The cross-reaction rate of anti-GEN-4G7 m Ab with sisomicin was0.23%,indicating good specificity.Therefore,the anti-GEN m Ab prepared in this study has high sensitivity and good specificity,which lays a foundation for the subsequent establishment of M-LFIA.（2）Based on anti-GEN m Ab,anti-enrofloxacin（ENR）m Ab,anti-sulfamethazine（SMZ）m Ab,anti-tetracycline（TC）m Ab and anti-AFM₁ m Ab established a colloidal gold nanoparticles（Au NPs）LFIA for the detection of five chemical contaminants in milk.The p H,m Ab labeling amount of Au NPs coupled with m Ab,complete antigen concentration on T line,probe addition amount and immune reaction time were optimized.Under the optimal parameter,LFIA was used to qualitatively detect five harm factors in PBS.The limits of detection（LODs）of GEN,ENR,SMZ,TC and AFM₁ were 25,1,2.5,2.5 and 0.25 ng/m L,respectively.Further,LFIA was used to qualitatively detect five chemical contaminants in milk,and the LODs were basically the same as that in PBS,indicating that matrix effects had little interference with LFIA.The established M-LFIA was used for qualitative analysis and quantitative detection of five harm factors in milk.The LODs were 25,1,2.5,2.5 and 0.25 ng/m L,respectively.According to the established standard curves,the calculated LODs were 0.385,0.078,0.059,0.082 and 0.0064 ng/m L.The actual recoveries of GEN,ENR,SMZ,TC and AFM₁ in milk samples were 99.8%～113.0%,76.5%～127.3%,96.8%～113.5%,107.1%～166.6%and 93.5%～130.3%,respectively.The coefficients of variation（CVs）were less than 15%.Therefore,the M-LFIA established in this study can quickly and sensitively detect the five chemical contaminants in milk,which provides a beneficial tool for the rapid screening of chemical contaminants in milk.