Study On An Enzyme-Linked Immunosorbent Assay For The Detection Of Tyramine

Tyramine, one of the biogenic amines (BAs), forms mainly through microbial decarboxylation of free amino acids. Because its content can objectively reflect the spoilage process of meat, it can be an important index in quality assurance of meat. We have developed a highly selective enzyme-linked immunosorbent assay (ELISA) for the determination of the tyramine in food.The tyramine protein conjugates (bovine serum albumin for immunogen, ovalbumin for coating antigen) were prepared by using formaldehyde and glutaraldehyde as linker, respectively. The antiserum with high titer and specificity was obtained by immunizing New Zealand rabbits, and purified by Protein A Sepharose-4B affinity chromatography to obain a polyclonal antibody. We established indirect competition ELISA method, the influences of experimental factors, including antigen incubation concentration, dilution ratio of antibody, dilution ratio of HRP-conjugated secondary antibody, blocking buffer, were studied. The standard curve with the highest sensitivity and stability was obtained when antigen incubation concentration was0.1μg/well, dilution ratio of antibody was1:500, dilution ratio of HRP-conjugated secondary antibody was1:15000, the blocking buffer was0.5%skim milk powder/PBS, and incubation temperature was25℃. The sensitivity and the detection limit of the developed method was0.20±0.015mg/L and0.02±0.004mg/L, respectively. And the antibody possessing high specificity didn’t exhibited any cross reaction with tyrosine, phenethylamine,histamine, tryptamine.The matrix effect could be eliminated through optimizing the conditions of sample preparation. The obtained method with merits of simplicity and feasibility was further applied to determine the tyramine in pork, beef, squid and cod, and the detection limit of samples were1.20mg/kg. When the different levels of12,24,60mg/kg of tyramine were spiked in the samples. The recoveries ranged from80.71to101.12%with relative standard deviation (n=3) of less than10%. The quantitative results were in good agreement (R2=0.9893) with those obtained by high-performance liquid chromatography (HPLC), confirming accuracy of the developed method. The method can rapidly detect tyramine in food samples with a simple sample preparation procedure.