Preparation Of Anti-Florfenicol Amine IgY Antibody And Development Of Elisa For The Rapid Detection Of Florfenicol Amine

Florfenicol (FF) is a broad spectrum antibacterial of the amphenicol family. Comparedwith chloromycetin (CAP) and thiamphenicol (TAP), it exhibits stronger activity and does notcarry the risk of inducing human aplastic anemia. Due to the ban of CAP, FF has been widelyused in the veterinary clinical settings. However, the residues owning to widely using of FFmay cause potential risks to human health.It has been reported that florfenicol amine (FFA) is the main metabolite of FF in mostedible animal tissues. FF and its metabolites are also converted to FFA by acid hydrolysis.Therefore, FFA is defined as one of the residue markers of FF in many countries, and themaximum residue limit (MRL) of the total residues of FF and FFA has been set. To date, thecommercial ELISA kits all aimed at detecting FF. In this study, we aimed at preparation ofanti-FFA IgY antibody and development a rapid ELISA detection method for quantification ofFFA in edible tissues of animals. The total residue of FF and FFA can be detected by the FFAELISA kits at the same time. The paper includes three parts as follows:1. The synthesis and identification of FFA artificial antigenSince FFA contains a free amino group in its structure, FFA was linked to carrier proteinsBSA and OVA that can induce a five carbon atoms spacer arm through glutaradehyde method.The results of UV, SDA-PAGE and NAGE showed that the artificial antigen FFA-BSA andFFA-OVA were successfully synthesized.2. Preparation of anti-FFA IgYAfter the immunization of immunogen FFA-BSA to laying hens, the IgY antibody wasextracted from egg yolk by the PEG-6000precipitated method. The development of the IgYantibody titer was monitored by indirect ELISA. The result showed that the IgY antibody wasgenerated at10day time after the first immunization. The titer was gradually increased andreached its peak (1:8192) at60day time. The high titer of the IgY antibody maintainedthrough the whole observation period. The sensitivity of anti-FFA IgY antibody was detectedby the competitive blocking ELISA. The IC50of anti-FFA IgY antibody was147.91ng/ml(Chicken No.1) and19.95ng/ml (Chicken No.2), indicating the IgY antibody obtaining from chicken No.2was more sensitive.3. Development of icELISA detection method of FFAAccording to the optimal working conditions, the rapid icELISA detection method of FFAwas established. The linearity of the standard curve showed good, the liner regressionequation was y=-13.71x+64.95with R~2=0.945. The FFA icELISA detection method exhibitedhigh specificity and the cross-reactivity with FF and TAP was1.15%and0.13%respectively.In this study, artificial antigen of FFA was synthesized and high yield of anti-FFA IgYantibody was obtained. The icELISA method based on anti-FFA IgY antibody specially aimedat the detection of FFA has been developed, which can be further applied to the developmentof ELISA kits for FFA detection. However, the results of the titer and sensitive of the IgYantibody are not satisfied, it is necessary to further optimize the antigen preparation andantibody generation methods.