Detection Of Artificial Antigens Of Three Small-Molecule Chemicals Using Polyclonal Antibodies And Protein Microarrays

Although the massive use of veterinary medicines and chemical additives has made tremendous contributions in terms of the livestock disease preventions and proliferations, it also causes the problem of excess chemical residues in our daily food that becomes alarming concerns of human beings. Most of the veterinary drugs are small molecular chemicals (MW<1000D), their residues can cause great damages to the different systems of a human body, such as drug accumulation, drug resistance, body malformation, and cancer induction, etc. Therefore, it is getting more and more important to develop a novel and efficient assay to detect these drug residuals in our daily food. In this study, we selected three commonly used small molecular chemicals (Clenbuterol CL, Sulfamethazine SM₂ and Sulfaquinoxaline SQ) to develop their corresponding artificial antigens and polyclonal antibodies using molecular biology and immunology method. These artificial antigens were then detected using ELISA and protein microarrays.Due to the lack of immunogenicity of small molecule chemicals, we coupled CL, SM₂ and SQ with bovine serum albumin (BSA), human serum albumin (HSA) and ovalbumin (OVA) by diazotization. The complete artificial antigens were confirmed by ultraviolet (UV) scanning spectrum, and the molecular ratios of drugs to protein were as follows. CL-BSA 23:1, CL-OVA 16:1, SM₂-HSA 4:1, SM₂-OVA 3:1, SQ-HSA 14:1, SQ-OVA 5:1. The conjugate of CL-BSA, SM₂-HSA and SQ-HSA antigens were then used to immunize rabbits. The titer and specificity of antisera from those rabbits were tested with competitive indirect ELISA (ci-ELISA). The results showed that the conjugate of artificial antigens possessed immunogenicity and the sensitivity to the corresponding CL, SM₂ and SQ polyclonal antibodies were 174.57ng/ml, 159.46ng/ml and 3.532μg/ml. The same antigens were further tested using protein microarrays with fluorescent (CY3) labeled anti-rabbit IgG as detection probe. The results showed more sensitive detection levels of CL-BSA, SM₂-HSA and...