Studies On Colloidal Gold Immunochromatographic Rapid Multi-Detecting Technology For Sulfonamides Residues

Sulfonamides is a class of broad-spectrum synthetic antibiotics, which content 4-N-sulfasulfonamide, was widely used for prophylactic or therapeutic purposes with bacterial and protozoon disease, because of its curative effect and available, However, the irrational usages caused their residues in eatable animal foodstuffs and pose a potential health threat to consumer. Nowadays, the methods, which were widely and frequently used to assay such residues, were high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). Those methods include fussy operation, require special equipment and well trained analysts, which limited their extensive uses, especially for screen and detect numerous samples.Colloidal gold immunochromatographic assay (G1CA) is a novel and quickly detecting method, which was widely used in human clinic and self-test, recently be used in dopes detection. Because of it's easy to store, to take along, to operate, to judge, and low cost, very suitable for screening large number of samples in spot.This research was based on the former study of GICA for single residues of sulfonamides in our laboratory, though utilizing more than one monoclonal antibody and more detect line to develop GICA for simultaneous detection of sulfonamides. The results of this study were as following.1. Production of artificial antigen and monoclonal antibody to sulfamethazineSulfamethazine was coupled with carrier proteins by diazotization to prepare conjugate of HSA-SM2 and OVA-SM2. The results of UV-Visible spectra showed two conjugates were obtained successfully. Conjugate HSA-SM2 was used as immunogen to immunity BALB/c to inducate antibody; OVA-SM2 was used as the competitor of sulfamethazine to detect antibody titer of serum and cell supernatant. After five panels of limiting dilution, eight hybridoma cell lines were obtained, which produced antitbodies to sulfamethazine. Cell line 4D6 and 2G6 exhibiting high affinity to SM2, the antibody was purified and identitied after mass producted by ascites.2. Development and application of the immunochromatographic assay for rapid detection of sulfamethazineThe monoclonal antibody 4D6 was conjugated with colloidal gold to serve as tracer; artificial antigen HSA-SM2 and rabbit anti-mouse IgG polyclonal antibody were immobilized on the nitrocellulose membrane as test line and control line for the preparation of colloidal gold immunochromatographic assay to sulfamethazine. Intra-day and inter-day coefficient variation were 5.9%-8.4% and 6.3%-9.3%, lower than 15%. The result of animal experiment shows that this method has good agreement with high performance liquid chromatography (HPLC):95.1% (21/22) for positive samples,97.7% (42/43) for negative samples, and 96.9% (62/64) for all animal experiment samples. The result of market sample detection shows that the agreements of the two methods were 90% (9/10) for positive samples,99.7% (350/351) for negative samples, and 99.4% (358/360) for all samples.3. Development and application of the immunochromatographic assay for rapid detection of sulfamethazine, sulfadiazine and sulfaquinoxalineThe monoclonal antibody 4D6, which anti-SM2; SD4, which anti-SD and 3A9, which anti-SQX were conjugated with colloidal gold to serve as tracer, artificial antigen HSA-SM2 (1:6), BSA-SD (1:10) and BSA-SQX (1:10) were immobilized on the nitrocellulose membrane as test lines (T1, T2 and T3) and rabbit anti-mouse IgG polyclonal antibody were immobilized as control line for the preparation of colloidal gold immunochromatographic assay to sulfamethazine, sulfadiazine and sulfaquinoxaline. Intra-day and inter-day coefficient variation were 3.5%-7% and 3.5%-7%, lower than 15%. The result of animal experiment shows that this method and high performance liquid chromatography (HPLC) have 99.0%(95/96) for positive samples,98.9%(89/90) for negative samples, and 98.6%(142/144) for all samples. The result of chicken breast market sample detection shows that the agreement of the two methods were 93.3% (14/15) for positive samples,99.8% (545/546) for negative samples, and 99.6% (558/560) for all the market chicken breast samples.4. Development and application of the immunochromatographic assay for rapid detection of six kinds of sulfonamidesThe monoclonal antibody 2G6, which anti-SM2 and SDM; SD4, which anti-SD; 3A9, which anti-SQX; and SDL4, which anti-SMM and SMD were conjugated with colloidal gold to serve as tracer, artificial antigen HSA-SM2 (1:6), BSA-SD (1:10), BSA-SQX (1:10) and BSA-SDL(1:10) were immobilized on the nitrocellulose membrane as test lines (T1, T2, T3 and T4) and rabbit anti-mouse IgG polyclonal antibody were immobilized as control line for the preparation of colloidal gold immunochromatographic assay to sulfamethazine, sulfadiazine, sulfaquinoxaline sulfamonomethoxine, sulfadimethoxine, and sulfametoxydiazine. Intra-day and inter-day coefficient variation were 3.5%-9% and 3%-10.2%, lower than 15%. The result of animal experiment shows that this method and high performance liquid chromatography (HPLC) have 99.0% (95/96) for positive samples,98.9%(89/90) for negative samples, and 98.9%(182/184) for all animal experiment samples. The result of chicken breast market sample detection shows that the agreement of the two methods were 93.3%(14/15) for positive samples, 99.8%(545/546) for negative samples, and 99.6%(558/560) for all the market chicken breast samples.