In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal...In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1：3 200 and 1：160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1：10 000. The positive standard value was 0.247 （OD450.m）. The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL^-1. The ELISA had no cross-reaction with Newcastle disease virus （NDV）, Avian influenza virus （AIV）, Infectious bronchitis virus （IBV）, Infectious bursal disease virus （IBDV）, Duck hepatitis virus （DHV）, and Gosling plague virus （GPV）. Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.展开更多
CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains...CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.展开更多
In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentra...In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4000,a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase(HRP)of 1:1000,a sample incubation time of 60 min and a detection antibody reaction time of 60 min.The specificity,sensitivity,repeatability and stability of this assay were detemmined.The limit of detection for beef and 1amb skeleta1 muscle troponin I was 45 mg/kg,the inter-assay and intra-assay recovery rates ranged from 80.4%to 115.7%,the coefficients of variation were below 13.6%,and the cIoss reaction rates of the tissue components of chicken,duck and fish were below 13.4%.The sandwich ELISA method established in this study is stable and has high accuracy.The test results were consistent with the polymerase chain reaction(PCR)method at 50 and 100 g/kg-Therefore,this ELISA method can be used to quantitatively detect beef and 1amb components in meat products.展开更多
基金supported by the National Natural Science Foundation of China (31172345)the Jiangsu Provincial Agricultural Science and Technology InnovationFoundation, China (cx(11)4039)
文摘In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1：3 200 and 1：160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1：10 000. The positive standard value was 0.247 （OD450.m）. The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL^-1. The ELISA had no cross-reaction with Newcastle disease virus （NDV）, Avian influenza virus （AIV）, Infectious bronchitis virus （IBV）, Infectious bursal disease virus （IBDV）, Duck hepatitis virus （DHV）, and Gosling plague virus （GPV）. Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.
基金funded by the National Natural Science Foundation of China (31201891)the Ph D Programs Foundation of Ministry of Education of China (20125103120012)+3 种基金the Innovative Research Team Program in Education Department of Sichuan Province, China (2013TD0015)the National Key Technology R&D Program of China (2015BAD12B05)the Integration and Demonstration of Key Technologies for Duck Industrial in Sichuan Province, China (2014NZ0030)the China Agricultural Research System (CARS-43-8)
文摘CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.
基金This research was funded by Hebei Provincial Department of Science and Technology(21375501D)the Hebei Academy of Sciences(2019Q01).
文摘In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4000,a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase(HRP)of 1:1000,a sample incubation time of 60 min and a detection antibody reaction time of 60 min.The specificity,sensitivity,repeatability and stability of this assay were detemmined.The limit of detection for beef and 1amb skeleta1 muscle troponin I was 45 mg/kg,the inter-assay and intra-assay recovery rates ranged from 80.4%to 115.7%,the coefficients of variation were below 13.6%,and the cIoss reaction rates of the tissue components of chicken,duck and fish were below 13.4%.The sandwich ELISA method established in this study is stable and has high accuracy.The test results were consistent with the polymerase chain reaction(PCR)method at 50 and 100 g/kg-Therefore,this ELISA method can be used to quantitatively detect beef and 1amb components in meat products.
