[Objective] This study aimed to increase the sensitivity and specificity of enzyme-linked immunosorbent assay （ELISA） through analyzing the effects of different blocking buffers and sample diluents and their differe...[Objective] This study aimed to increase the sensitivity and specificity of enzyme-linked immunosorbent assay （ELISA） through analyzing the effects of different blocking buffers and sample diluents and their different concentrations on the result of ELISA. [Method] Different types of blocking buffer [casein, gelatin, BSA, goat serum （GS）, horse serum （HS） and rabbit serum （RS）]and sample diluent （PBST, casein, gelatin, BSA, GS, HS and RS） as well as their different concentrations were tested in ELISA to screen the optimal combination of blocking buffer and sample diluent. [Result] The results showed that 2% BSA had better effect on blocking than 1% and 3% BSA, and both 2% and 1% casein had better blocking effect than 3% casein; 8% and 10% RS showed better blocking effects than 6%RS and 7%RS; compared to BSA and casein, RS had the best effect on blocking, and 8% RS performed best as the blocking buffer and sample diluent. [Conclusion] A good combination of blocking buffer and diluent can effectively reduce the non-specific reaction and improve the sensitivity and specificity of ELISA. This study provides an important reference for the development of a perfect ELISA method.展开更多
Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA...Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.展开更多
基金Supported by the Jiangsu Agriculture Sience and Technology Innovation Fund(JASTIF)[CX(13)3076]Jiangsu Provincial Postdoctoral Sustentation Fund(6511201,1202055C)~~
文摘[Objective] This study aimed to increase the sensitivity and specificity of enzyme-linked immunosorbent assay （ELISA） through analyzing the effects of different blocking buffers and sample diluents and their different concentrations on the result of ELISA. [Method] Different types of blocking buffer [casein, gelatin, BSA, goat serum （GS）, horse serum （HS） and rabbit serum （RS）]and sample diluent （PBST, casein, gelatin, BSA, GS, HS and RS） as well as their different concentrations were tested in ELISA to screen the optimal combination of blocking buffer and sample diluent. [Result] The results showed that 2% BSA had better effect on blocking than 1% and 3% BSA, and both 2% and 1% casein had better blocking effect than 3% casein; 8% and 10% RS showed better blocking effects than 6%RS and 7%RS; compared to BSA and casein, RS had the best effect on blocking, and 8% RS performed best as the blocking buffer and sample diluent. [Conclusion] A good combination of blocking buffer and diluent can effectively reduce the non-specific reaction and improve the sensitivity and specificity of ELISA. This study provides an important reference for the development of a perfect ELISA method.
基金supported by the National Natural Science Foundation of China(32273041)the Key R&D Program of Shaanxi Province,China(2022NY-104)the Natural Science Foundation of Shaanxi Province,China(2022JC-12)。
文摘Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.
