Study On An Immuno-LAMP Technology For The Detection Of Phenylethanolamine A

Phenylethanolamine A?PEAA?is one of?-receptor agonists.Though it is commonly used in feed stuff to improvethe conversion rate of animal meat recently,studies have shown that phenylethanolamine A will accumulate in the human body by the transfer of food chain.When reaching definite value,it will be a great threat to human health.At present,the Ministry of agriculture published the relevant laws to prohibitthe substance from being added to feed and animal drinking water.In this paper,the enzyme linked immunosorbent assay?ELISA?was combined with real-time fluorescence quantitative PCR and loop-mediated isothermal amplification?LAMP?to develop two methods,immune-PCR and immune-LAMP,for the detection of phenylethanolamine A.The results obtained are as the following.1.Based on the de novo synthesis of PEAA,the PEAA amino derivative?PEAA-NH₂?was obtained by Raney nickel reduction method.Subsequently,combined PEAA-NH₂ with carrier proteins,the immunogen and coating antigen?PEAA-BSA and PEAA-OVA?were synthesed by diazotization reaction.According to the regular immunization protocol,New Zealand rabbits were immuned with the PEAA-BSA to get anti-PEAA polyclonal antibody.Based on the titer and sensitivity of polyclonal antibody,PEAA pAbs 1#and PEAA pAbs 5#were chosen for the methodological study on subsequent immunoassays.2.An indirect competitive enzyme-linked immune sorbent assay?ELISA?method was established by using the coating antigen,PEAA-OVA,and anti-PEAA polyclonal antibodies.Consequently,the IC₅₀ value of this method was 0.401 ng/mL,and the minimum detection limit?LOD?was 0.035 ng/m L.The result of cross-reactivity experiment was demonstrated that the anti-PEAA polyclonal antibody was highly specific for PEAA with the negligible cross-reactivity ratio with other 11?-agonist compounds?CR<0.1%?.The recovery,precision and accuracy of the developed indirect competitive ELISA were tested.The results were showed that the recovery rates ranged from 86.2%to 116.6%for intra-assay,and the coefficients of variation were both blow 10%for inter-assay and intra-assay.It was proved this method was reliable.3.In this study,an immuno-PCR?IPCR?method was developed by combining the high specificity of the ELISA and high sensitivity of the real-time fluorescent quantitation of PCR?rt-PCR?,taking replace of the enzyme marker in the antigen antibody binding step by report DNA.And then the signal of the fluorescent will be detected via rt-PCR.The anti-PEAA polyclonal antibody and report DNA labelled with biotin were prepared by using 5'-end with biotin of PCR primers and NHS method,respectively.Then the signal of antigen and antibody was amplified with the system of streptavidin and biotin.After a series of optimization,the IPCR reaction system was established for the detection of PEAA.The PCR tubes pretreated by 1%glutaraldehyde,the optimal working concentration of the coating antigen?PEAA-OVA?and biotin labeled polyclonal antibody were of 2 g/mL and 2.1 g/mL,respectively.The optimal addition of biotin labeled reporter DNA was 1.41 ng/tube.The PBST and ddH₂O were selected for the washing bufferes with 5 times repeat.The results were showed that the IC₅₀ and minimum detection limit of the assay were0.023±0.005 ng/mL and 0.0021 ng/mL,respectively,which were increased by an order of magnitude compared with the indirect competitive ELISA method.The linear detection range was between 0.0001-100 ng/m L.Compared with the indirect competition ELISA method,the linear detection range was expanded by two orders of magnitude.In this method,PEAA-polyclonal antibody was high specificity to PEAA and no cross reaction with other 11?-agonist substances?CR<0.05%?.And the coefficients of variation were less than 10%for inter-assay and intra-assay by the precision experiment,which showed the reliability of the method.4.Based on the IPCR detection technology,the LAMP was applied in this method.After designing of LAMP primers?using PCR amplified fragment as target sequence?and optimizing the LAMP system?the ratio of the external and internal primers was 1:8,the reaction temperature was 65?,and the addition of Bst polymerase was 8 U?,the immune LAMP?ILAMP?detection method was established.The experimental results were showed that the linear equation of the standard curve was y=1.1949x+42.251,R²=0.956.The linear detection range was between0.0001-100 ng/m L,which was same as the IPCR.The IC₅₀0 value and the minimum detection limit were 0.0396±0.009 ng/m L and 0.0044 ng/m L,respectively.Compared with indirect competition ELISA,the sensitivity of this method was improved one order of magnitude.The precision of the experiment results show the coefficients of variation?CV?were both blow 10%for inter-assay and intra-assay.Meanwhile,the specificity of ILAMP detection result was showed that this method was high specificity to PEAA and no cross reaction with other 11?-agonist substance?CR<0.04%?.In this study,based on the successful preparation of PEAA,PEAA-NH₂ and anti PEAA polyclonal antibody,an indirect competitive ELISA assay was established.Subsequently,combined ELISA with rt-PCR and LAMP,the two new methods,IPCR and ILAMP were developed for the detection of phenylethanolamine A.The results of sensitivity,specificity,accuracy and precision were showed that the two methods were proved to be reliable.