| In recent years,food safety problems occur frequently,among which veterinary drug residue is a serious food safety problem.Long-term eating food with residual antibiotics and insect-resistant veterinary drugs will not only cause harm to human health,but even lead to drug resistance,threatening human survival.Thus,China as a big user of veterinary drugs,must strengthen the supervision of veterinary drug residues in animal food.Compared with the instrument detection method,immunoassay has the advantages of rapid,high efficiency,low cost,high throughput and high sensitivity,and is more suitable for the initial screening of a large number of samples.This study aiming to product nine highly sensitive and affinitive mAbs against E1,E2,E3,DEX,HDS,DIC,TOL,TCD and AS drugs,and develop the fast immunoassays for these drugs based on these mAbs.（1）According to the chemical structures of E1,E2 and E3,the antigens capable of exposing their characteristic groups in the vivo were designed and synthesized,and then the specific mAbs to E1,E2 and E3 were prepared.The subtypes of mAbs against E1,E2 and E3 were IgG2b,IgG2b and IgG3 respectively,the affinity constants were 8.23?109,6.68?109 and 9.44?109,the IC50s were 0.46 ng/mL,0.36 ng/mL and 0.39 ng/mL,the antibody response to other hormonal drugs without cross reaction.By optimizing the working point and ic-ELISA working buffer,ic-ELISAs for detecting E1,E2 and E3 were successfully established.The detection range of testing E1 by ic-ELISA was 0.111.96 ng/mL,and the recovery rate was 81.087.9%in milk sample.The detection range of testing E2 was 0.121.07 ng/mL,and the recovery rate was86.899.0%in milk sample.And the detection range of testing E3 was 0.082.03 ng/mL,and the recovery rate was 87.093.0%in milk sample.The qualitative immunochromatographic assays for detecting E1,E2 and E3 were established by optimizing the synthesis of gold nanoparticle-labeled antibody,antigen and antibody dosage,etc.The cut-off values of E1,E2and E3 in milk all were 5ng/mL.（2）The mAbs against DEX and HDS were successfully prepared based on new DEX and HDS haptens.The subtypes of mAbs against DEX and HDS were IgG2b and IgG2a respectively,the affinity constants were 6.34?109 and 5.72?109,the IC50s all were 0.95ng/mL,anti-DEX mAb has a CR with 5.1%to betamethasone,and anti-HDS mAb has a CR with4.7%to fludrocortisone,the CRs to other drugs are less than 2%.By optimizing the working point and ic-ELISA working buffer,ic-ELISAs for detecting DEX and HDS were successfully established.The detection range of testing DEX by ic-ELISA was 0.0340.265ng/mL,and the recovery rate was 92.4102.8%in milk sample.The detection range of testing HDS was 0.0290.301 ng/mL,and the recovery rate was 92.098.5%in milk sample.The qualitative immunochromatographic assays for detecting DEX and HDS were established by optimizing the synthesis of gold nanoparticle-labeled antibody,antigen and antibody dosage,etc.The cut-off values of DEX and HDS in milk were 0.5ng/mL and 2 ng/mL respectively.（3）The haptens and antigens of DIC,TOL and TCD were successfully designed and synthesized,and the antibodies of these drugs were successfully developed.The subtypes of mAbs against DIC,TOL and TCD were IgG2a,IgG1 and IgG2a respectively,the affinity constants were 2.24?1010,8.68?109 and 5.46?109,the IC50s were 0.36 ng/mL,2.19 ng/mL and 1.49 ng/mL.Each antibody only has the cross reaction to the drug and its metabolites,and no cross to other drugs.By optimizing the working point and ic-ELISA working buffer,ic-ELISAs for detecting DIC,TOL and TCD were successfully established.The detection range of testing DIC by ic-ELISA was 0.121.07 ng/mL,and the recovery rate was 97.4107.8%in chicken sample.The detection range of testing TOL was 0.568.57 ng/mL,and the recovery rate was 94.9102.4%in chicken sample.And the detection range of testing TCD was0.375.91 ng/mL,and the recovery rate was 99.1101.4%in bovine muscle sample.The quantitative immunochromatographic assays for detecting DIC,TOL and TCD were established by optimizing the synthesis of gold nanoparticle-labeled antibody,antigen and antibody dosage,resuspending buffer and incubation time.Using this assay,the limit of detection（LOD）of DIC in chicken sample was 1.08μg/kg,and the recovery rate was96.96%110.21%;the LODs of TOL-SO2 in egg and chicken samples 1.19 and 0.78μg/kg,respectively,and the recovery rates were 90.6%96.6%and 87.1%89.4%,respectively;the LOD of TCD in bovine muscle sample was 0.11μg/kg,and the recovery rate was92.0%107.3%.（4）Basing on the nucleus structure of TMP,DVD,OMP and BQP,a new hapten of antibacterial synerists（AS）was synthesized and a high sensitive and wide broad-spectrum mAb to AS were developed.The subtypes of the antibody was IgG2b,the affinity constant was 5.46?1010,the IC50s were 0.09 ng/mL for TMP,0.12 ng/mL for DVD,0.21 ng/mL for OMP and0.25 ng/mL for BQP.By optimizing the working point and ic-ELISA working buffer,ic-ELISA for detecting ASs was successfully established.The detection range of testing Ass by ic-ELISA in bovine sample were 0.0250.739 ng/mL,and the recovery rate was 95.7%101.8%.The qualitative immunochromatographic assay for detecting ASs was established by optimizing the synthesis of gold nanoparticle-labeled antibody,antigen and antibody dosage,etc.The cut-off values of TMP,DVD,OMP and BQP in bovine muscle samples were 0.25,0.5,1 and 1μg/kg,respectively.（5）In this study,a gold nanoparticle based multiple immunochromatographic strip assay was developed.The strip contained three detection lines that could simultaneously detect 17hormones in milk samples,which could be divided into three categories:NR and its analogues,DEX and its analogues,and HES and its metabolites.Multi-ICS was assembled by optimizing the concentration of coated antigen,the amount of gold nanoparticle-labeled antibody,resuspending buffer.The multi-ICS was successfully applied to the simultaneous qualitative detection of 17 hormones in milk,and the minimum cut-off value was 1 ng/mL.With the help of a hand strip scanner and the standard inhibition curves,the quantitative detection of 17hormones can be realized.The LOD of NR and its metabolites were 0.064.85 ng/mL;the LOD of DEX and its analogues were 0.0052.85 ng/mL;and the LOD of HES and its analogues were0.032.14 ng/mL.The recovery rate were 78.8%99.4%in milk,which meets the test requirements of the real sample. |
PDF Full Text Request