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
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
Rice ragged stunt virus(RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein(CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21(...Rice ragged stunt virus(RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein(CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21(DE3) using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody(MAb) against RRSV was obtained by fusing mouse myeloma cells(Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay(ACP-ELISA), a dot enzyme-linked immunosorbent assay(dot-ELISA), and immunocapture-RT-PCR(IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1 280 and 1:655 360(w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12 800 and 1:1 600(an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.展开更多
To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect Eocoli O157 in foods. Methods Spleen...To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect Eocoli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with routine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized miLk were tested to confirm efficiency of the method. Results MAb 3A5 specific for E.coli O157 and O 113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1× 10^6. No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1: 1× 10^5 with E.coli O 157. The detection limit of this sandwich ELISA was 10^3- 10^4 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion MAb 3A5 specific for E.coli O 157 and O 113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.展开更多
Objective To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids. Methods rHuE...Objective To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids. Methods rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked irmnunosorbent assay (ELISA), SDS-PAGE and Western blot. Results The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1x10s L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.展开更多
The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on i...The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.展开更多
A monoclonal antibody was first prepared by fusion of mouse myeloma cells (SP2/0-Ag-14) with spleen cells isolated from male BALB/ c mice immunized with spermidine-bovine serum albumin conjugate (SPD- BSA). The hybrid...A monoclonal antibody was first prepared by fusion of mouse myeloma cells (SP2/0-Ag-14) with spleen cells isolated from male BALB/ c mice immunized with spermidine-bovine serum albumin conjugate (SPD- BSA). The hybridoma cell line producing antibody specific for spermidine was cultured in vitro and after i. p. into mice, the ascitic fluid gave suitably high dilution titres (1: 106) by enzyme immunoassay. This monoclonal antibody is of IgG1 class and the bimolecular compleex with molecular weight of 52KD and 27 KD. The monoclonal antibody was clearly specific to spermidine comparing with spermine or putriscine. Monclonal antibody may prove to be useful in the rapid diagnosis and evaluation of patients with cancer.展开更多
Aggregate amyloid beta protein1-42 (Aβ1-42) can typically be found in the early stage of Alzheimer’s disease (AD). Aβ1-42 self-assembles and is highly toxic to neurons. Thus, recognizing aggregated Aβ1-42 is very ...Aggregate amyloid beta protein1-42 (Aβ1-42) can typically be found in the early stage of Alzheimer’s disease (AD). Aβ1-42 self-assembles and is highly toxic to neurons. Thus, recognizing aggregated Aβ1-42 is very important for elucidation of Aβ1-42 structure and for the diagnosis of AD. In this study, the specificity of the 79-3 monoclonal antibody against soluble aggre- gate Aβ1-42 was measured by sandwich Enzyme-Linked Immuno Sorbent Assay (ELISA). Eight monoclonal antibodies against both soluble aggregates and amorphous aggregates were used as primary antibodies. Soluble aggregates and amorphous aggregates were used as antigen. As secondary antibody, HRP was labeled with the 79-3 monoclonal antibody. The reactivity of the 79-3 monoclonal antibody against soluble aggregates was confirmed in all combinations, but little reactivity against amorphous aggregates was found. Furthermore, we performed the above sandwich ELISA using the 37-11 antibody, which is reactive against large oval aggregates (LOA) that occur in micro aggregates, instead of the 79-3 antibody. The 77-3 antibody is 1 of the 8 monoclonal antibodies against soluble aggregates;amorphous aggregates also reacted with the 37-11 antibody. These results indicated that soluble aggregates are specifically recognized by a combination of different antibodies. The combined use of these antibodies can be applied to the diagnosis of AD and to defining the structure of the Aβ1-42.展开更多
A monoclonal antibody specific to sea bass(Lates calcarifer) vitellogenin(VTG) was developed,for use as a tool for monitoring endocrine disrupting chemicals(EDCs). VTG was induced in sea bass by intramuscular injectio...A monoclonal antibody specific to sea bass(Lates calcarifer) vitellogenin(VTG) was developed,for use as a tool for monitoring endocrine disrupting chemicals(EDCs). VTG was induced in sea bass by intramuscular injection of 17β-estradiol(E_2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry(LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41(MAb-sea bass VTG 41)was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay(ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E_2-treated sea bass and others EDCs(Nonylphenol, Benzo[a]pyrene and CdCl_2). Moreover, cross-reactivity was also found in E_2-treated coral grouper(Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.展开更多
High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Tr...High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy(FT-IR)and UVVis.Competitive enzyme-linked immunosorbent assay(ELISA)for quantitative detection of cadmium in aqueous sample was developed.The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99%cross-reactivity with other 11 metals.The limit of detection was 0.10μg·L^(-1),and the effective linear range was 10^(-1)-10^(3)μg·L^(-1).The intra-and inter-assay coefficient variations were 1.5%-6.3%and 3.2%-7.4%,respectively.The spike recovery in different water samples were between 98.5%and 110.3%.The detection limit of this assay was well below the allowable concentration of cadmium(3μg·L^(-1)),and the working range was wider than that of other methods which showed the range of 2.19-86.38 and 0-10^(3)μg·L^(-1).The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples.The results will lay a solid foundation for construction of an immunoassay kit for cadmium.展开更多
Based on the preparation of an anti-diethylstilbestrol(DES) monoclonal antibody,a simple and convenient indirect competitive enzyme-linked immunosorbent assay(ELISA) method for DES detection has been developed.The mon...Based on the preparation of an anti-diethylstilbestrol(DES) monoclonal antibody,a simple and convenient indirect competitive enzyme-linked immunosorbent assay(ELISA) method for DES detection has been developed.The monoclonal antibody demonstrated high sensitivity to DES with an IC50 value of 275 pg mL-1 and detection limit(LOD) of 90 pg mL-1.The specificity of the assay was studied by measuring cross-reactivity of the antibody with structurally related compounds of ethinyl estradiol(<7%),estrone(<0.1%),estriol(<0.1%),and diethylstilbestrol benzoate(<0.1%).Chicken,fish,shrimp,urine and bile spiked with different concentration of DES were detected by the developed method,and the recovery rates were greater than 79.5%.Intra-and inter-assay variations were about 6%.This method exhibited high stability with a coefficient of variation less than 10% in buffer and in real samples.The LODs in fish/shrimp,liver,feed and urine spiked with DES were 600,600,4800 and 600 pg mL-1,respectively.These results confirmed that the antibody to DES was successfully produced and could be used to establish ELISA methods for DES detection in food producing animals.展开更多
19-Nortestosterone (NT) has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme...19-Nortestosterone (NT) has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme linked immunosorbent assay (ELISA) kit. For this purpose, hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mouse. Noncompetitive and competitive indirect ELISA were used to screen positive cell clones. To optimize the indirect competitive ELISA (icELISA) method, various methanol concentrations in assay buffer were evaluated. Matrix effects in urine and spiking test were also investigated. Finally, five hybridoma cell lines named NT-1, NT-2, NT-3, NT-4 and NT-5 were screened out. The corresponding monoclonal antibodies (mAbs) were of the IgG 1 isotype with a k light chain, and the antibody affinity of all mAbs were between 2.6×10 9 and 4.7×10 9 L/mol. The titer and IC 50 values of purified ascites were in the range of 0.64×10 5 2.56×10 5 and 0.55-1.0 ng/mL, respectively. Based on the NT-1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT in cattle urine. The dynamic range was from 0.004 to 85.8 ng/mL, with a detection limit for the assay and IC 50 values of 0.002 and 0.55 ng/mL, respectively. Except for a high cross-reactivity (62%) to α-NT, negligible cross-reactivity to other compounds was observed. After optimization, 10% of methanol was used in the assay buffer, and a 20-fold dilution in cattle urine gave an inhibition curve almost the same as that in phosphate buffered saline. The correlation coefficient between the established icELISA and LC-MS/MS method was 0.9871. The results showed that the established heterologous icELISA method provides an excellent alternative for the detection of NT residues in food producing animals.展开更多
采用两种识别不同抗原决定簇的抗尿激酶(UK)单克隆抗体建立了测定人 UK 含量的夹心法ELISA.本法灵敏度为0.15ng/ml,批内 CV4.3%,批间 CV8.7%,平均回收率98%.应用本法测定了部分正常细胞和肿瘤细胞株培养上清中的 UK 含量,肿瘤细胞培养上...采用两种识别不同抗原决定簇的抗尿激酶(UK)单克隆抗体建立了测定人 UK 含量的夹心法ELISA.本法灵敏度为0.15ng/ml,批内 CV4.3%,批间 CV8.7%,平均回收率98%.应用本法测定了部分正常细胞和肿瘤细胞株培养上清中的 UK 含量,肿瘤细胞培养上清 UK 量明显高于正常细胞.测定82名正常人血浆中 UK 含量,其结果为1.31±0.6ng/ml.展开更多
Objective This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumonioe) antigens in humans with the variable domains (VD) 2 and 3 of the major oute...Objective This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumonioe) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPvD2-VD~) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors). Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.展开更多
Malachite green(MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms(LMG) that may reside in fish muscles for a long perio...Malachite green(MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms(LMG) that may reside in fish muscles for a long period, thus being harmful to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay(ELISA) to detect MG and LMG specifically. The monoclonal antibody(m Ab) to LMG was generated using a hybridoma technique. The obtained m Ab showed good cross-reactivity(CR) to malachite green(MG), but not to crystal violet(CV) and Brilliant Green(BG). The m Ab was used to develop a fast detecting ELISA of MG and LMG in fish. By introducing the conjugation LMG-HRP, the detection capability was 0.37 ng m L-1 for MG and LMG. The mean recovery from spiked grass carp tissues ranged from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. The stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG.展开更多
Objective To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for detection of genital C. trachomatis infecti...Objective To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for detection of genital C. trachomatis infections. and the Methods The pORF5 protein was expressed in Escherichia coil and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA. Results Two hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6). Conclusion Two DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.展开更多
Objective:To optimize the ELISA for the determination of tetrodotoxin.Methods:A corn petitive enzyme-linked immunosorbent assay(ELISA) was used.In the ELISA.100μl antigen(1.0μg/ml) was coated on the microtiter plate...Objective:To optimize the ELISA for the determination of tetrodotoxin.Methods:A corn petitive enzyme-linked immunosorbent assay(ELISA) was used.In the ELISA.100μl antigen(1.0μg/ml) was coated on the microtiter plate for 60 min at 37℃or over night at 4℃.The plate was then washed 3 times with PBS-T for 3-5 s each time.The optimal incubation time for monoclonal antibody (mAb),goat anti-mice IgG peroxidase conjugate and OPD were 30 min.20 rain and 10 min at 37℃,re- spectively.Results:The detection limit is 0.05 ng in each well.The curve was linear for TTX doses be tween 5 5 000 ng/ml (0.25 250 ng for every assay).The linear regress equation was Y=0.30 88X-- 0.17 41 (R=0.99 01).The average callback for TTX of muscles and gonads were 99.74% and 100.30%, respectively.The sensitivity of optimization ELISA was 5 times than traditional method and the time of 1.8 h were saved.Conclusion..The optimized ELISA is an ideal method for the determination of tetrodotoxin.展开更多
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.
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supported by the National Basic Research Program of China(2010CB126203)the special fund for Agro-scientific Research in the Public Interest,China(201003031)+1 种基金Earmarked Funds for Modern Agro-industry Technology Research SystemZhejiang Provincial Natural Science Foundation of China(Z3090039)
文摘Rice ragged stunt virus(RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein(CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21(DE3) using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody(MAb) against RRSV was obtained by fusing mouse myeloma cells(Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay(ACP-ELISA), a dot enzyme-linked immunosorbent assay(dot-ELISA), and immunocapture-RT-PCR(IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1 280 and 1:655 360(w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12 800 and 1:1 600(an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.
文摘To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect Eocoli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with routine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized miLk were tested to confirm efficiency of the method. Results MAb 3A5 specific for E.coli O157 and O 113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1× 10^6. No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1: 1× 10^5 with E.coli O 157. The detection limit of this sandwich ELISA was 10^3- 10^4 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion MAb 3A5 specific for E.coli O 157 and O 113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.
基金This work was supported by the National Natural Science Foundation of China (No.20675006).
文摘Objective To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids. Methods rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked irmnunosorbent assay (ELISA), SDS-PAGE and Western blot. Results The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1x10s L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.
文摘The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.
文摘A monoclonal antibody was first prepared by fusion of mouse myeloma cells (SP2/0-Ag-14) with spleen cells isolated from male BALB/ c mice immunized with spermidine-bovine serum albumin conjugate (SPD- BSA). The hybridoma cell line producing antibody specific for spermidine was cultured in vitro and after i. p. into mice, the ascitic fluid gave suitably high dilution titres (1: 106) by enzyme immunoassay. This monoclonal antibody is of IgG1 class and the bimolecular compleex with molecular weight of 52KD and 27 KD. The monoclonal antibody was clearly specific to spermidine comparing with spermine or putriscine. Monclonal antibody may prove to be useful in the rapid diagnosis and evaluation of patients with cancer.
文摘Aggregate amyloid beta protein1-42 (Aβ1-42) can typically be found in the early stage of Alzheimer’s disease (AD). Aβ1-42 self-assembles and is highly toxic to neurons. Thus, recognizing aggregated Aβ1-42 is very important for elucidation of Aβ1-42 structure and for the diagnosis of AD. In this study, the specificity of the 79-3 monoclonal antibody against soluble aggre- gate Aβ1-42 was measured by sandwich Enzyme-Linked Immuno Sorbent Assay (ELISA). Eight monoclonal antibodies against both soluble aggregates and amorphous aggregates were used as primary antibodies. Soluble aggregates and amorphous aggregates were used as antigen. As secondary antibody, HRP was labeled with the 79-3 monoclonal antibody. The reactivity of the 79-3 monoclonal antibody against soluble aggregates was confirmed in all combinations, but little reactivity against amorphous aggregates was found. Furthermore, we performed the above sandwich ELISA using the 37-11 antibody, which is reactive against large oval aggregates (LOA) that occur in micro aggregates, instead of the 79-3 antibody. The 77-3 antibody is 1 of the 8 monoclonal antibodies against soluble aggregates;amorphous aggregates also reacted with the 37-11 antibody. These results indicated that soluble aggregates are specifically recognized by a combination of different antibodies. The combined use of these antibodies can be applied to the diagnosis of AD and to defining the structure of the Aβ1-42.
基金financially supported by the Research grant of Burapha University through the National Research Council of Thailand (No.79/2558)Graduate study of faculty of Science Burapha University
文摘A monoclonal antibody specific to sea bass(Lates calcarifer) vitellogenin(VTG) was developed,for use as a tool for monitoring endocrine disrupting chemicals(EDCs). VTG was induced in sea bass by intramuscular injection of 17β-estradiol(E_2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry(LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41(MAb-sea bass VTG 41)was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay(ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E_2-treated sea bass and others EDCs(Nonylphenol, Benzo[a]pyrene and CdCl_2). Moreover, cross-reactivity was also found in E_2-treated coral grouper(Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.
基金This work was supported by the National High Technology Research and Development Program of China(No.2006AA06Z411)China Postdoctoral Science Foundation(No.20100471297)Jiangsu Planned Projects for Postdoctoral Research Funds(No.0902065C).
文摘High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy(FT-IR)and UVVis.Competitive enzyme-linked immunosorbent assay(ELISA)for quantitative detection of cadmium in aqueous sample was developed.The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99%cross-reactivity with other 11 metals.The limit of detection was 0.10μg·L^(-1),and the effective linear range was 10^(-1)-10^(3)μg·L^(-1).The intra-and inter-assay coefficient variations were 1.5%-6.3%and 3.2%-7.4%,respectively.The spike recovery in different water samples were between 98.5%and 110.3%.The detection limit of this assay was well below the allowable concentration of cadmium(3μg·L^(-1)),and the working range was wider than that of other methods which showed the range of 2.19-86.38 and 0-10^(3)μg·L^(-1).The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples.The results will lay a solid foundation for construction of an immunoassay kit for cadmium.
基金supported by the Shandong Natural Science Foundation (Y2008B31)the National High-Tech Research and Development Program of China (07AA10Z435, 2007AA06A407)+1 种基金the National Natural Science Foundation of China (20675048)the Fundamental Research Funds for the Central Universities (65011121)
文摘Based on the preparation of an anti-diethylstilbestrol(DES) monoclonal antibody,a simple and convenient indirect competitive enzyme-linked immunosorbent assay(ELISA) method for DES detection has been developed.The monoclonal antibody demonstrated high sensitivity to DES with an IC50 value of 275 pg mL-1 and detection limit(LOD) of 90 pg mL-1.The specificity of the assay was studied by measuring cross-reactivity of the antibody with structurally related compounds of ethinyl estradiol(<7%),estrone(<0.1%),estriol(<0.1%),and diethylstilbestrol benzoate(<0.1%).Chicken,fish,shrimp,urine and bile spiked with different concentration of DES were detected by the developed method,and the recovery rates were greater than 79.5%.Intra-and inter-assay variations were about 6%.This method exhibited high stability with a coefficient of variation less than 10% in buffer and in real samples.The LODs in fish/shrimp,liver,feed and urine spiked with DES were 600,600,4800 and 600 pg mL-1,respectively.These results confirmed that the antibody to DES was successfully produced and could be used to establish ELISA methods for DES detection in food producing animals.
基金supported by the Eleventh Five-Year Plan for National Science and Technology of China (2006BAK02A21/1)Henan Innovation Project for University Prominent Research Talents (2010HASTIT026)
文摘19-Nortestosterone (NT) has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme linked immunosorbent assay (ELISA) kit. For this purpose, hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mouse. Noncompetitive and competitive indirect ELISA were used to screen positive cell clones. To optimize the indirect competitive ELISA (icELISA) method, various methanol concentrations in assay buffer were evaluated. Matrix effects in urine and spiking test were also investigated. Finally, five hybridoma cell lines named NT-1, NT-2, NT-3, NT-4 and NT-5 were screened out. The corresponding monoclonal antibodies (mAbs) were of the IgG 1 isotype with a k light chain, and the antibody affinity of all mAbs were between 2.6×10 9 and 4.7×10 9 L/mol. The titer and IC 50 values of purified ascites were in the range of 0.64×10 5 2.56×10 5 and 0.55-1.0 ng/mL, respectively. Based on the NT-1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT in cattle urine. The dynamic range was from 0.004 to 85.8 ng/mL, with a detection limit for the assay and IC 50 values of 0.002 and 0.55 ng/mL, respectively. Except for a high cross-reactivity (62%) to α-NT, negligible cross-reactivity to other compounds was observed. After optimization, 10% of methanol was used in the assay buffer, and a 20-fold dilution in cattle urine gave an inhibition curve almost the same as that in phosphate buffered saline. The correlation coefficient between the established icELISA and LC-MS/MS method was 0.9871. The results showed that the established heterologous icELISA method provides an excellent alternative for the detection of NT residues in food producing animals.
文摘采用两种识别不同抗原决定簇的抗尿激酶(UK)单克隆抗体建立了测定人 UK 含量的夹心法ELISA.本法灵敏度为0.15ng/ml,批内 CV4.3%,批间 CV8.7%,平均回收率98%.应用本法测定了部分正常细胞和肿瘤细胞株培养上清中的 UK 含量,肿瘤细胞培养上清 UK 量明显高于正常细胞.测定82名正常人血浆中 UK 含量,其结果为1.31±0.6ng/ml.
基金supported by grants from the National Natural Science Foundation of China (Grant No. 30901352)Innovative Research Team in University of Hunan Province (Number: [2008] 51)Hunan Provincial Innovation Foundation for Postgraduate and Hunan Provincial Training and Innovation Base for Post-graduate
文摘Objective This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumonioe) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPvD2-VD~) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors). Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.
基金supported by the National High Technology Research and Development Program of China (Granted no. 2011AA10A216)Special Fund for Agroscientific Research in the Public Interest (Granted no. 201203085)
文摘Malachite green(MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms(LMG) that may reside in fish muscles for a long period, thus being harmful to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay(ELISA) to detect MG and LMG specifically. The monoclonal antibody(m Ab) to LMG was generated using a hybridoma technique. The obtained m Ab showed good cross-reactivity(CR) to malachite green(MG), but not to crystal violet(CV) and Brilliant Green(BG). The m Ab was used to develop a fast detecting ELISA of MG and LMG in fish. By introducing the conjugation LMG-HRP, the detection capability was 0.37 ng m L-1 for MG and LMG. The mean recovery from spiked grass carp tissues ranged from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. The stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG.
基金supported by grants from the National Natural Science Foundation of China (30970165 and 81102230)Hunan Provincial Natural Science Foundation of China (09JJ3059)Team Project for the Technology Innovation of Higher Education of Hunan province, China, 2010
文摘Objective To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for detection of genital C. trachomatis infections. and the Methods The pORF5 protein was expressed in Escherichia coil and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA. Results Two hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6). Conclusion Two DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.
基金the grants from PhD Priming Foundation of Jilin University(430505010276)
文摘Objective:To optimize the ELISA for the determination of tetrodotoxin.Methods:A corn petitive enzyme-linked immunosorbent assay(ELISA) was used.In the ELISA.100μl antigen(1.0μg/ml) was coated on the microtiter plate for 60 min at 37℃or over night at 4℃.The plate was then washed 3 times with PBS-T for 3-5 s each time.The optimal incubation time for monoclonal antibody (mAb),goat anti-mice IgG peroxidase conjugate and OPD were 30 min.20 rain and 10 min at 37℃,re- spectively.Results:The detection limit is 0.05 ng in each well.The curve was linear for TTX doses be tween 5 5 000 ng/ml (0.25 250 ng for every assay).The linear regress equation was Y=0.30 88X-- 0.17 41 (R=0.99 01).The average callback for TTX of muscles and gonads were 99.74% and 100.30%, respectively.The sensitivity of optimization ELISA was 5 times than traditional method and the time of 1.8 h were saved.Conclusion..The optimized ELISA is an ideal method for the determination of tetrodotoxin.