| Chloramphenicol is broad-spectrum antibiotic, They have been applied to treatment bacteroidal desease widely. But chloramphenicol has strong side-effect, and have been forbid to applying in animal food productin. But traditional detecting is based on such methods as biochemistry,instrumental analytical,etc. And the operation is complicated and time consuming So it is very important to detect the residue rapidly and accurately. Immunochromatography colloidal gold strip are sensitive analytical methods for detection of antigens and antibodies. It is a fast, sensitive and selective method compared with conventional analysis procedures. They are fit for on-site screening of large numbers of samples. In this dissertation ,the main purpose is to develop Immunochromatography colloidal gold strip for rapid detection of chloramphenicol in food.The actived ester reaction and diazotization reaction were employed respectively to synthesize the immunogen and coatingen of chloramphenicol ,and identified with UV-scanning and Infrared spectrometry(IRS). The results demonstrated that molar extinction coefficient of hapten to carrier proteins of bovine serum albumin (BSA) and ovalbumin (OVA) was164523,49402 ;372723,46601 respectively,and exceed hapten and carrier protein obviously.The antigen of chloramphenicol was synthesized successfully.The couple ratio of hapten to BSA and OVA was 12,2 with the actived ester reaction, and was 30,1 with diazotization reaction ,respectively.Then,antiserum was raised against the immunogen of hapten -BSA. ELISAs showed that the cross-reactivity with chloramphenicol succinate sodium salt is 180%,and the cross-reactivity with streptomycin,tetracycline, oxytetracycline, penicillin, clenbuterol are all bellow 0.01%. The sensitivity for detection of chloramphenicol was0.01μg/L. This antiserum had specificity and high affinity to chloramphenicol. It can be used for sensitive and selective Immunochromatography colloidal gold test strip of chloramphenicol.Two kinds of colloid gold with different diameter were prepared by trisodim cicitrate reduction. According to the exosyndrome of color, UV-scanning curve and scanning electron microscope, the colloid gold with 16nm in diameter was used to label antibody. The optimun pH for labeling was 7.6, and the concentration of antibody was 24μg/mL. Immunogold (colloid gold labeled antibody complex) was purified at high speed and low temperature and it was proved alive by using dot immunogold filtration assay.Nitrocellulose membrane, gold conjugate pad, sample pad and absorbing pad were assembled to immunochromatographic test strip. The gold conjugate pad was freeze dried after soaked immunogold to glass membrane. The HF13504 of Millipore corporation were used to prepare the test strip,in which the sheep anti-rabbit IgG coated as control line, the concentration of sheep anti-rabbit IgG dilutionin 1:10, the polycolne antibody coated at 0.225mg/mL as the test line. The materials with high hydroscopicity were used as the sample pad and absorbent pad.The sensitivity of strips was 1μg/mL.The positive samples were detected with 100% positive result by the strips compared with ELISA when the concentration above 1μg/mL. The result could be read clearly in 6 minutes. Sealed in plastic bag with drier, after 4 months stored in ordinary temperature,the sensitivity and test time of the strip was no significant difference.The strip has good stability.In a word, the strip is prepared to detect chloramphenicol. The strip is used to detect the chloramphenicol, And it shows that the strip is time saving, sensitive, easy to be justified and suitable for the on-site detection. These key techniques will help us research and develop a great strip to detect chloramphenicol on-site more quickly.
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