Design And Study Of A Column Of Ofloxacin Immunoaffinity Chromatography

In this study, ofloxacin(OFL) were linked respectively to two kinds of carrier proteins , by which the two kinds of artificial antigens were synthesized successfully.Using the artificial immunogens,rabbits were immunized to obtain the antiserum against ofloxacin. Then,using this purified antiserum,we produced the column of ofloxacin immunoaffinity chromatography which was evaluated by HPCE.1.Synthesis and Identification of the Artifical Antigens for Ofloxacin.Ofloxacin was coupled respectively to bovine serum albumin (BSA) by Carbodiimide method, Ofloxacin was also coupled respectively to ovalbumin(OVA) by Carbodiimide method.by which the Conjugates OFL-BSA and OFL-OVA were prepaired.The Conjugates were identified by colour reaction of FeCl3 and UV Spectrum.The results demonstrated that the four kinds of artificial antigens were synthesized successfully.2.Preparation and dentification of Antisera.OFL-BSA was used as immunogens to immunise rabbits, by which the antiserum against ofloxacin was obtained.The titer of the antiserum against ofloxacin was 1:128×103. The antiserum against ofloxacin whose titer still was 1:128×103, was purified from the antiserum by a series procedure of precipitation with saturated ammonium sulfate and chromatography with DEAE-Sephadex A-50. Using the antiserum against ofloxacin to establish Ci-ELISA methods for ofloxacin ,the working concentrations of the coating antigen (OFL-OVA) was 2.5μg/ml, the working concentrations of the antisera was 1:32×103, the working concentrations of Ab-HRP was 1:10000. By determinating the cross-reactivity of OFL, cross-reactivity rates were in the range from 1.80% to 4.52% between OFL and the fluoroquinolones. But OFL had no cross-reactivity with sulfadiazine, chloramphenicol, terramycin or penicillin. In this study the affinity constant was 7.91×107 L /mol.3.The preparation for the column of ofloxacin immunoaffinity chromatography and the dynamic capacity of IAC was evaluated.The regression equation of OFL in HPCE was y=241.21x-158.03, and the coefficient was 0.9981. the limits of detect was 2ug/ml. After evaluating the column,we have determined the coupling rate of 93%,the dynamic capacity of 2204ng/mL and the absolute capacity of 473.98ng/mL for ofloxacin. Using 5mL methanol as elution buffer,the recoveries were 94 %,92.6 %,95.5 %and C.V. was 5.32%,3.17%,3.84% at levels of 200ng/mL,300ng/mL,400ng/mL in 5mL standard working liquid detected by HPCE for six times.