Development And Evaluation Of Monoclonal Antibodies Of Olaquindox For ELISA Deteciton

Olaquindox （OLA）, a derivative of quinoxaline-1,4-dioxide, is one of the most commonantimicrobial growth promoter owing to its broad-spectrum antibacterial and protein assimilation.However, some researches showed that excessive utilization of OLA in animal feeds could result incumulative toxicity, residued in animal food and caused aberration and mutagenicity. Therefore, OLA asfeed additive has been banned in EU and USA, and can be only used in feed for pigs less than35㎏inChina. Recently, the main method to detect OLA residues has been high performance liquidchromatography （HPLC）, but it is difficult to be widely applied. And ELISA has been applied to detectdrug residues for its sensitivity, specificity and high-throughput. However, ELISA kits made in Chinafor determination of OLA in market have high cross-reactivities to its analogues. In this study, antigenesof OLA was synthesized, production and evaluation of monoclonal antibodies against OLA were carriedout. The study provides a scientific basis to improve the specificity of OLA kit.1. The active ester method was applied to prepare immune antigen of OLA （OLA-NHS-BSA） andits detective antigen （OLA-NHS-OVA）. Their concentration were4.69㎎/mL and8.85㎎/mL detectedby BCA kit. The characterizations of the two conjugates were examined by UV, and the coupling ratioof BSA and OVA to OLA-HS were1:4.9,1:6.4.2. Balb/C mice were respectively immunized with50μg,100μg or200μg OLA-NHS-BSA to onemouse and titers of their serum were detected after the4th immunization of mice. The titers of serumwere1:16000at dose of100μg per mouse, higer than other doses. Two stable hybridoma lines of5B10and3B6were established by PEG-induced cells fusion. The isotypes of them respectively were IgM/κand IgG2a/κ. The titer of5B10was1:1.28×102in supernatant and1:1.28×10⁴in ascites. And the titers of3B6were1:5.12×10⁴in supernatant and1:1.6×10⁷in ascites. The affinity constant （Ka） of3B6was5.47×10⁹L/mol, IC₅₀was14.74ng/mL. It was observed that there was a low cross-reactivity tocarbadox of3.62%and to mequindox of4.16%and no cross-reactivities to either MQCA、QCA or otherdrugs.3. The best coating concentration of the detective antigen was0.188μg/mL,the optimal workingdilution of OLAmAb and goat anti-mouse IgG-HRP respectively was1:256000and1:10000.The bestreaction condition between OLA and OLAmAb was37℃and0.5h. In the concentration of0.51³70.37ng/mL, the regression equation of the standard curve by the indirect competitive ELISAwas Y=-0.3699x+0.9322（R²=0.9885）.4. The study was carried out to evaluate the effect of trehalose as biological preservative agent onthe stability of ELISA by adding2.5%、5%、10%and20%trehalose to sealing liquid. The result showedthat trehalose had protective effects on the activity of antigen coated in ELISA and2.5%trehaloseadded was best.