Studies On Preparation Of Monoclonal Antibody Against Doxycycline And Its Preliminary Application

Doxycycline (DC) belongs to the tetracycline family of antibiotics. It is widely used in animal husbandry and aquculture. However, the DC residues in aquatic products may cause great threat to human health. And this problem had attracted great attention of many people. Therefore, it is necessary to establish a sensitive and rapid method to monitor DC residues in aqutic tissues.The work in this study included the synthesis of artificial antigen, preparation of DC PcAb and McAb, development and evaluation of indirect competition ELISA method(ci ELISA), development of glass slide protein microarray for detection of DC. The results were as follows:1.The synthesis and verification of immunogen and coating antigenThe immunogen(DC-BSA) and coating antigen(DC-OVA) were synthesized by the improved carbodiimide method. UV scanning analysis and animal immune test were carried out. The conjugation ration of DC to BSA was about3:1and the DC PcAb had a higher titer and good sensitivity. This results showed that the artifical antigens were prepared successfully.2.Preparation and evaluation of DC McAbBALB/C mice were immunized with DC-BSA, and the spleen cells of the mouse with highest titer and best sensitivity were fused with SP2/0cells. Then through detecting, coloning and selecting, one kind of hybridaization tumor cells that can secrete the momoclonal antibodies against DC were selected. The mice were injected with the hybridaization tumor cells named3D1-H4to produce ascites which with DC McAb in it. Then, the immunological traits of McAb were identified by the indirect ELISA and the indirect competitive ELISA method. The titers of the McAb were1:8×102and1:5.12×105in supernatant and ascites, respectively. The McAb belonging to IgG1was screened by the indirect ELISA method. The McAb showed an IC50of44.46ng/mL and had8.87%, 5.86%and2.74%cross-reactivity with tetracycline, oxytetracycline and chlotetracycline, respectively.3.Development and evaluation of the ciELSA methodAn indirect competitive ELISA method(ciELISA) was developed with the DC McAb. The standard curve equation was y=-0.3591x+1.0602(R2=0.9919). The ciELISA method had a linear detection ranging from1.58～199.53ng/mL, a sensitivity of2.88ng/mL and an IC50of36.31ng/mL. The recoveries of DC spiked in channel catfish muscle and liver were85.13%and79.69%, respectively. Both the inter-assay and intra-assay coefficient variation were less than15%. These results suggested that a ciELISA method was developed for detecting DC residues.4.Development of glass slide protein microarrayDC-OVA was spotted on the glass slide as capture protein. The DC McAb was used as the fist antibody and the goat antibody labeled with a fluorophore was as the second antibody. Several key factors that may influence the results were tested. We found that, when0.5mg/mL DC-OVA was spotted on the slide, bloked with0.1mol/mL Tris for30min, and then reacted with DC McAb(1:800) for1h, then second antibody(1:6) for1h, presented strong fluorescence signal, and without nonspecific signal.