| Sulfonamides are used widely in animal feeds as an antimicrobial for growth stimulation and for prevention and treatment of diseases. They are commonly administered in conjunction with antibiotics. But, they all have some poisonousness and they are potentially harmful to food hygiene and to human health. Therefore, their residues in the edible tissues of food-producing animals such as meat, milk and egg et al. are paid more and more attention. It is stipulated in our country that the total sulfonamide residues should be under 300 ng/ml. At present, the chemical methods for detecting sulfonamide residues are mainly as follows: high performance liquid chromatography ( HPLC ), thin layer chromatography ( TLC ) and gas chromatography ( GC ) et al.. Though all these methods are very sensitive and very accurate, they are not suitable for screening a large number of samples, becausse they are time-taking, costly and highly skilled. Enzyme-linked immunosorbent assay ( ELISA) as a rapid, idiosyncratic and sensitive biological methods is gradually applied to detecting sulfonamide residues. It is necessary to establish ELISA for determining sulfamethoxydiazine (SMD) residue in that it is often used as antiplasmodial drug for domestic animals and fowls. So, the present research is launched to prepare artificial SMD antigens served as immunogen and coating antigen in ELISA.In this paper, bovine serum albumin (BSA) and ovalbumin (OVA) were used as protein carriers respectively to couple with semiantigen SMD by glutaraldehyde method, and complete antigens SMD-BSA and SMD-OVA were prepared. SMD-BSA will act as immunogen, while SMD-OVA will act as coating antigen in ELISA. The solutions of SMD-BSA, SMD-OVA and SMD as well as BSA were scanned with ultraviolet ray at the range of 200 nm~500 nm, and their spectrum suggest that it is succeessful to link between SMD and protein carriers. In addition, the lyophilized SMD-BSA , SMD solid and BSA solid were tested by IR spectrum, and the results prove that the combination is successful also.In order to study the law of antibody formation, and to screen the antibody with strong idiosyncrasy and high efficiency, and to increase the sensitivity and selectivity of ELISA, many kinds of SMD-BSA with different combination ratios were synthesized. On thecondition that the use level of BSA was 200 mg and the use level of glutaraldehyde (25 %) was 0.20 ml, SMD with mass of 50 mg, 100 mg, 150 mg and 200 mg were added respectively, and as a result, combination ratios of the synthesized SMD-BSA were 10, 19, 11 and 13 correspondingly. While on the condition that the use level of SMD was 100 mg and the use level of BSA was 200 mg, glutaraldehyde with volume of 0.10 ml, 0.20 ml, 0.30 ml, 0.40 ml and 0.50 ml were added respectively, and as aresult, combination ratios of the synthesized SMD-BSA were 14, 19, 9, 7 and 10 correspondingly. It can be seen that all the above combination rayios are within the range of 5~20 and they accord with the required molecular combination ratio of BSA carrier with semiantigens. Therefore, all the prepared SMD-BSA above can be used to immunize rabbits to obtain antiserum and anti-SMD antibody.On thr basis of the present researches, ELISA can be further set up for detecting SMD residue, and moreover reagent box can be developed to put into use in market and import or export quarantine. In addition, a great many of samples can be detected and screened in short time by coating microtiter plate in laboratory beforehand and adding samples, antibodies and enzyme-labelled secondary antibody in situ followed by incubating, washing and adding substrate et al.. In a word, it is of extensively applied prospect to go on the present study. |
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