Establishment Of Novel Enzyme-linked Immunosorbent Assay And Development Of Colloidal Gold Immunochromatographic Assay Card For Detection Of Amantadine

Amantadine(AMD)is a human antiviral drug that was firstly used for the prevention and treatment of influenza.It has also been used as a drug for bird flu,swine flu and gastroenteritis and was widely used in livestock and poultry farming.However,excess AMD accumulates in the human body through the food chain,may cause neurotoxicity to humans and result in severe viral resistance,so it has been banned as a veterinary drug now.In this paper,a novel enzyme-linked immunosorbent assay(ELISA)and a colloidal gold immunochromatography assay(CG-ICA)were established to achieve a qualitative detection of AMD.These methods had the advantages of high accuracy,good specificity,un-instrumented,and were suitable for on-site detection.In the second chapter,the AMD hapten was firstly synthesized and the AMD artificial antigen was obtained by EDC/NHS mediated method.The successful synthesis of hapten and artificial antigens were confirmed by mass spectrometry and UV spectroscopy,respectively.The mice were immunized with artificial antigens,and the mouse antisera titers and inhibitive situation were evaluated by indirect competition ELISA.In the third chapter,a novel ELISA method based on DNA hybridization reaction and non-crosslinking gold nanoparticles(AuNPs)aggregation was developed for the detection of AMD by naked eye.Primer 1-AuNPs-mAb(monoclonal antibody)was prepared by modifying Primer 1 and mAb on the surface of AuNPs for subsequent ELISA detection.Accordingly,the Primer 1-AuNPs-mAb could be captured by AMD artificial antigen on ELISA wells.Primer 2,which was complementary paired to Primer 1,was eventually added into the ELISA wells.After the hybridization reaction,the free Primer 2 in the supernatant was mixed with AuNPs and NaCl and induced a rapid color change of AuNPs.The ssDNA can effectively stabilize AuNPs and enhance resistance to salt-induced aggregation,and the degree of dispersion of AuNPs had a positive correlation with the concentration of ssDNA.The lack of AMD in the sample resulted in capturing a substantial Primer 1-AuNPs-mAb complex and limited free Primer 2 in the supernatant.After adding NaCl,the color of AuNPs turned blue with limited Primer 2.The optimized parameters were as follows: the concentration of AMD artificial antigen,Primer 1,AMD mAb,Primer 2 was 1.0 ?g/m L,0.3 ?M,8 ?g/m L,and 0.2 ?M,respectively,the volume of resuspended solution of Primer 1-AuNPs-mAb complex was 1 m L,the size and the concentration of AuNPs was 13 nm and 5.8 nM,respectively.With the optimal conditions,as low as 0.033 ?M AMD can be detected by naked eye,and no cross reaction with AMD structural analogues and other common veterinary drugs was observed,exhibited excellent sensitivity and specificity.In the forth chapter,the CG-ICA was established to qualitatively detect AMD in chicken meat.The AuNPs-mAb probes were prepared and AMD artificial antigen and goat anti-mouse secondary antibody were sprayed on the nitrocellulose membrane as test line and control line,respectively.The optimal conditions were determined as follows: the coupling pH of AuNPs and mAb was 6.5;the concentration of antibody,artificial antigen and secondary antibody was 4 ?g/m L,1.5 mg/m L,0.6 mg/m L,respectively;the volume of AuNPs-mAb was 6 ?L/cm.Under the optimal conditions,the sensitivity of this method was 1.5 ng/m L in extract solution and 5 ?g/kg in real sample.And no cross reaction with AMD structural analogues and common veterinary drugs was observed,exhibited excellent sensitivity and specificity.