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
Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar carcinoma （HCC）, and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patien...Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar carcinoma （HCC）, and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say （ELISA）. Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody （mAb） designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls （P = 0.0036）. Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls （P = 0.0172）.展开更多
A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for ...A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA.The MAb,designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study.The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein.The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits.The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India,1,2,15,17,18 and 23.The assay could detect BTV antigen as early as day 8 in blood.It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood,washed RBCs,buffy coat and plasma.A total of 102 field samples from animals,suspected of being infected with BTV,were tested and 29.42% were positive.The blood samples were also amplified in cell culture which improved the sensitivity of the assay.Results confirmed that the sELISA is rapid and specific.展开更多
Kallikrein-related peptidases (KLKs) have been proposed as potential cancer biomarkers. Kallikrein-related peptidase 5 (KLK5) is a secreted trypsin-like protease of the KLKs. Until now, detection of KLK5 in both biolo...Kallikrein-related peptidases (KLKs) have been proposed as potential cancer biomarkers. Kallikrein-related peptidase 5 (KLK5) is a secreted trypsin-like protease of the KLKs. Until now, detection of KLK5 in both biological fluids and tissues has been described frequently due to the potential of being a new cancer biomarker. Our objective was to prepare KLK5 antibodies and establish an ELISA method for KLK5 to study the possible clinical application of KLK5 as a biomarker for malignancies. In this study, recombinant KLK5 protein was produced and purified using a prokaryotic expression system, and then used as immunogen to generate antibodies. High titers of specific antibodies were measured in serum of rabbits after the forth booster injection. And the titer of the antiserum reached 1:106. We have also generated monoclonal antibodies using hybridoma technology and the titer reached 1:105. The activity of KLK5 antibodies was characterized by Western blot and immunohistochemistry. To quantitatively examine KLK5 in serum samples, we established double antibody sandwich ELISA method using mouse mAb as capture and rabbit pAb as tracer antibody. We have detected KLK5 levels in ovarian cancer serum to ensure that our sandwich ELISA measurement to have high sensitivity and specificity. The ranges of linearity reached by the standard curves of the newly developed ELISA were 0.45 ng/mL to 125 ng/mL. The detection limit of the method, defined as the concentration of KLK5 can be distinguished, was 0.20 ng/mL. Median serum KLK5 levels were 3.77 ng/mL and 0.86 ng/mL in ovarian cancer patients and normal female, respectively (P ELISA assay for KLK5. Our preliminary findings prompt that KLK5 may be a new potential biomarker for the diagnosis and prognosis in patients with ovarian.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supported by the Science and Technology Foundation of Nanjing Medical University (No. 07NMUZ005).
文摘Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar carcinoma （HCC）, and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say （ELISA）. Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody （mAb） designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls （P = 0.0036）. Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls （P = 0.0172）.
文摘A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA.The MAb,designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study.The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein.The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits.The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India,1,2,15,17,18 and 23.The assay could detect BTV antigen as early as day 8 in blood.It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood,washed RBCs,buffy coat and plasma.A total of 102 field samples from animals,suspected of being infected with BTV,were tested and 29.42% were positive.The blood samples were also amplified in cell culture which improved the sensitivity of the assay.Results confirmed that the sELISA is rapid and specific.
文摘Kallikrein-related peptidases (KLKs) have been proposed as potential cancer biomarkers. Kallikrein-related peptidase 5 (KLK5) is a secreted trypsin-like protease of the KLKs. Until now, detection of KLK5 in both biological fluids and tissues has been described frequently due to the potential of being a new cancer biomarker. Our objective was to prepare KLK5 antibodies and establish an ELISA method for KLK5 to study the possible clinical application of KLK5 as a biomarker for malignancies. In this study, recombinant KLK5 protein was produced and purified using a prokaryotic expression system, and then used as immunogen to generate antibodies. High titers of specific antibodies were measured in serum of rabbits after the forth booster injection. And the titer of the antiserum reached 1:106. We have also generated monoclonal antibodies using hybridoma technology and the titer reached 1:105. The activity of KLK5 antibodies was characterized by Western blot and immunohistochemistry. To quantitatively examine KLK5 in serum samples, we established double antibody sandwich ELISA method using mouse mAb as capture and rabbit pAb as tracer antibody. We have detected KLK5 levels in ovarian cancer serum to ensure that our sandwich ELISA measurement to have high sensitivity and specificity. The ranges of linearity reached by the standard curves of the newly developed ELISA were 0.45 ng/mL to 125 ng/mL. The detection limit of the method, defined as the concentration of KLK5 can be distinguished, was 0.20 ng/mL. Median serum KLK5 levels were 3.77 ng/mL and 0.86 ng/mL in ovarian cancer patients and normal female, respectively (P ELISA assay for KLK5. Our preliminary findings prompt that KLK5 may be a new potential biomarker for the diagnosis and prognosis in patients with ovarian.
