Qualitative Imminoassay For The Determination Of Allergic Degradation Product Of Benzylpenicillin

In this paper, a colloidal gold immunochromatographic assay (CGIA) and a immunoaffinity test column assay (IATCA) for rapid detection of the allergic degradation product of benzyl penicillin (Benzylpenicilloic acid, BPA) were developed.Colloidal gold particles were prepared via sodium citrate reduction method. The optimal reaction pH was9and the optimal quantity of BPA antibody labeled with colloidal gold was36μg. The BPA-OVA and goat anti-rabbit IgG were dispensed on the NC membrane as test line and control line respectively and the polyclonal antibody was solidified to glass fibers to prepare immunochromatographic strips. The optimal dilution fold of the coated antigen, the gold-labeled antibodies and the goat anti-rabbit IgG were1:1,1:5and1:100respectively. The NC membrane was Millipore HF180without any treatment, while the treatment buffer of the conjugate pad was PB containing0.5%TritonX-100,0.1%PVP,2%BSA and5%sucrose. The standard solution was5%methanol/PB.The detection limit of the CGIA is defined by visually significant differences of the detection line of BPA compared with the control line as10μg/L within10min. The CGIA of BPA developed in this paper has a high specificity (no cross-reactivity with chloramphenicol, melamine and some other drugs). Milk and milk powder samples were centrifuged for the CGIA to determine the detection limit as the results of10μg/L and40μg/kg.The immunoaffinity test column. assay was developed with CNBr-activated SepharoseTM4B as the solid phase support. The anti-BPA gel was prepared via coupling with CNBr-activated SepharoseTM4B and antibodies as the detection layer, while the anti-HRP gel was prepared as the control layer.The working conditions were optimized to establish the IATCA of BPA. The optimal fold of the anti-BPA gel, the anti-HRP gel and the BPA-HRP were1:10,1:200and1:10000respectively. The standard solution was PBS, pH5.7. While the volume of the washing buffer was5mL.The detection limit of the IATCA is defined by visually disappearance of the color of the detection layer as10μg/L, and whole detection process could be completed within10min. The IATCA of BPA developed in this paper has a high specificity (no cross-reactivity with chloramphenicol and some other drugs). Milk samples were centrifuged for the IATCA to determine the detection limit as the results of10μg/L.The accuracy of the developed methods as CGIA and IATCA was validated by the enzyme-linked immunosorbent assay(ELISA). A good agreement was observed among the detection results of the CGIA, IATCA and ELISA, indicating the validity of the CGIA and IATCA.The process of CGIA and IATCA is simple, rapid and convenient for the qualitative and semi-quantitative detection of BPA. The results can be visually estimated by eyes without instrumentation. The stability and high sensitivity and simplicity make it feasible to be used as an effective screening tool of penicilloic acid residues in milk samples.