The Development Of Sulphamethazine Residues In Edible Food By Enzyme-linked Immunosorbent Assay (ELISA)

Sulphamethazine(SMZ) is one kind of sulphonamide which is widely used to treat bacterial diseases in human and veterinary medicine and to promote growth in cattle,sheep, pigs and poultry.However,it has potential menace to human health.To determine sulphamethazine(SMZ) residues in edible animal foods(pig muscle,chicken muscle,egg, fish,milk and liver),a competitive direct enzyme-linked immunosorbent assay(ELISA) method was established.The anti-SMZ antibody produced in this project was specific for SMZ,and then an antibody-based simple,rapid and convenient direct competitive enzyme-linked immunosorbent assay was developed.The sensitivity of the method was high,and the limit of detection for determination of SMZ residues in different matrix was far below the maximum residue levels(MRLs),which was 5μg kg^-1 in liver,2μg kg^-1 in other five samples.So the method could satisfy the international standardization detection demand. After liquid extraction with methanol,sample extracts diluted with buffer were analyzed by direct competitive enzyme-linked immunosorbent assay directly.Matrix effects could be avoided when a 1:20 dilution of pork,chicken,egg,fish and milk samples and a 1:50 dilution of liver samples with selected buffer.A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed with recoveries between 70%-120%.To compare the effect of homologous assay and heterologous assay on the ELISA sensitivity and specificity,two enzyme conjugates were synthesized.The result indicated that heterologous format showed higher sensitivity.The specificity of the antibody for SMZ was determined by testing 14 kinds of sulphonamides.In general,the developed immunoassay is highly specific for SMZ.All tested sulphonamides showed low cross-reactivity(＜0.5%) except sulphamerazine(SMR), which of chemical structure is one methyl less than SMZ,showing cross-reactivity of 13.3%Traditional analytical methods for the determination of SMZ in edible foods were high-performance liquid chromatography.In my project,a HPLC method was also developed for the validation of the developed ELISA method.Sample extracts were analyzed by ELISA and HPLC,and results obtained by two methods correlated well,the correlation was greater than 0.9.