Pub Date : 2016-03-10DOI: 10.1080/15321819.2015.1126600
Anmol Kumar, K. Pulicherla, K. S. Rao
The relatively short circulatory half-life (2–3 min) of staphylokinase is a major drawback in the development of SAK- (staphylokinase) based thrombolytic drug. A rapid and sensitive method, based on indirect competitive ELISA, was developed and validated for quantitative determination of SAK in rabbit plasma. The dynamic range of the assay varied between 0.41 ± 0.16 μg/L and 9.03 ± 0.38 μg/L (R2 = 0.98) for SAK in rabbit plasma. There were no dilution linearity issues apparent with this assay. The precision (% CV) ranged from 4.6–9.7% for the intraassay and from 17.1–19.3% for interassay. This validated method was successfully employed for evaluation of various pharmacokinetic parameters of SAK in rabbit.
{"title":"Development and validation of an indirect competitive ELISA for quantification of recombinant staphylokinase in rabbit plasma: Application to pharmacokinetic study","authors":"Anmol Kumar, K. Pulicherla, K. S. Rao","doi":"10.1080/15321819.2015.1126600","DOIUrl":"https://doi.org/10.1080/15321819.2015.1126600","url":null,"abstract":"The relatively short circulatory half-life (2–3 min) of staphylokinase is a major drawback in the development of SAK- (staphylokinase) based thrombolytic drug. A rapid and sensitive method, based on indirect competitive ELISA, was developed and validated for quantitative determination of SAK in rabbit plasma. The dynamic range of the assay varied between 0.41 ± 0.16 μg/L and 9.03 ± 0.38 μg/L (R2 = 0.98) for SAK in rabbit plasma. There were no dilution linearity issues apparent with this assay. The precision (% CV) ranged from 4.6–9.7% for the intraassay and from 17.1–19.3% for interassay. This validated method was successfully employed for evaluation of various pharmacokinetic parameters of SAK in rabbit.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"10 1","pages":"228 - 242"},"PeriodicalIF":0.0,"publicationDate":"2016-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77694263","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
ABSTRACT A two-step dual-label TRFIA was developed for the simultaneous detection of human epididymis protein-4 and cancer antigen 125 in a single run. The performance of this assay was first evaluated using clinical serum samples, and then compared with commercialized kits. The sensitivity of this assay for cancer antigen 125 detection was 0.5 U/mL (dynamic range, 0–1400 U/L), and the sensitivity for human epididymis protein-4 detection was 1 pM (dynamic range, 1–900 pM). High correlation coefficients (R) were obtained between the present dual-label TRFIA and commercially available kits (R = 0.99). The present dual-label TRFIA has high sensitivity, specificity, and accuracy in clinical sample analysis. It is a good alternative to the single-label diagnostic methods.
{"title":"The development of dual-label time-resolved fluorescence immunoassay (TRFIA) for screening of ovarian cancer based on simultaneous detection of human epididymis protein-4 and cancer antigen 125","authors":"Wengji Sun, Yan Wang, Xiao-zhu Liu, Lai-qing Li, Cuicui Chen, Licheng Zhang","doi":"10.1080/15321819.2015.1106950","DOIUrl":"https://doi.org/10.1080/15321819.2015.1106950","url":null,"abstract":"ABSTRACT A two-step dual-label TRFIA was developed for the simultaneous detection of human epididymis protein-4 and cancer antigen 125 in a single run. The performance of this assay was first evaluated using clinical serum samples, and then compared with commercialized kits. The sensitivity of this assay for cancer antigen 125 detection was 0.5 U/mL (dynamic range, 0–1400 U/L), and the sensitivity for human epididymis protein-4 detection was 1 pM (dynamic range, 1–900 pM). High correlation coefficients (R) were obtained between the present dual-label TRFIA and commercially available kits (R = 0.99). The present dual-label TRFIA has high sensitivity, specificity, and accuracy in clinical sample analysis. It is a good alternative to the single-label diagnostic methods.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"35 1","pages":"453 - 462"},"PeriodicalIF":0.0,"publicationDate":"2016-02-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76972989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-26DOI: 10.1080/15321819.2016.1146757
A. Arfaoui, S. Mejri, R. Belhaj, W. Karkni, M. Chebil, S. Rammeh
ABSTRACT We studied epidermal growth factor receptor (EGFR) expression profile in urothelial bladder carcinoma (UBC) which is a complex and heterogeneous disease with a large spectrum of histological aspects and deadly potential. Using immunohistochemistry (IHC), all GI tumors and pTa cases showed a low expression profile of EGFR. However, we note that when the stage of disease is advanced, tumors over-express EGFR. Indeed, 5% and 25% of GII and GIII tumors over-expressed EGFR, respectively. Further, 0% of pTa, 9,5% of pT1, 15% of pT2, 50% of pT3, and 90% of pT4 tumors were shown to be high EGFR expression (HEE). Moreover, we found a statistically significant correlation between the EGFR over-expression and grade and stage (P < 0.05). Thus, EGFR over-expression could be a potential prognostic marker to predict poor outcome in Tunisian patients with UBC.
{"title":"Prognostic value of immunohistochemical expression profile of epidermal growth factor receptor in urothelial bladder cancer","authors":"A. Arfaoui, S. Mejri, R. Belhaj, W. Karkni, M. Chebil, S. Rammeh","doi":"10.1080/15321819.2016.1146757","DOIUrl":"https://doi.org/10.1080/15321819.2016.1146757","url":null,"abstract":"ABSTRACT We studied epidermal growth factor receptor (EGFR) expression profile in urothelial bladder carcinoma (UBC) which is a complex and heterogeneous disease with a large spectrum of histological aspects and deadly potential. Using immunohistochemistry (IHC), all GI tumors and pTa cases showed a low expression profile of EGFR. However, we note that when the stage of disease is advanced, tumors over-express EGFR. Indeed, 5% and 25% of GII and GIII tumors over-expressed EGFR, respectively. Further, 0% of pTa, 9,5% of pT1, 15% of pT2, 50% of pT3, and 90% of pT4 tumors were shown to be high EGFR expression (HEE). Moreover, we found a statistically significant correlation between the EGFR over-expression and grade and stage (P < 0.05). Thus, EGFR over-expression could be a potential prognostic marker to predict poor outcome in Tunisian patients with UBC.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"28 1","pages":"359 - 367"},"PeriodicalIF":0.0,"publicationDate":"2016-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91428421","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-26DOI: 10.1080/15321819.2016.1151439
I. Shittu, L. Sulaiman, D. Gado, A. Egbuji, M. Ndahi, E. Pam, T. Joannis
ABSTRACT Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens with outbreaks resulting in high economic losses due to increased mortality and drop in egg production. This study reports a survey of ILT virus antibody conducted in nine local government areas (LGAs) of Plateau State involving 67 randomly selected commercial poultry flocks. In all, 938 sera were tested using the Agar Gel Immuno-diffusion (AGID) technique. Overall prevalence of 1.2% (N = 11) was recorded. ILT virus antibody was found in 2.5% (n = 9) and 7.1% (n = 2) of the tested sera from Jos South and Langtang North LGAs, respectively. No detectable ILT virus antibody was found from the other seven LGAs. This is the first report of ILT infection in poultry from the North central part of Nigeria. It is therefore recommended that the economic implication of ILT infection in Nigerian poultry population be conducted in order to know if vaccination should be adopted for control.
{"title":"Sero-epizootiological investigation of infectious laryngotracheitis infection in commercial poultry of Plateau State, north central Nigeria","authors":"I. Shittu, L. Sulaiman, D. Gado, A. Egbuji, M. Ndahi, E. Pam, T. Joannis","doi":"10.1080/15321819.2016.1151439","DOIUrl":"https://doi.org/10.1080/15321819.2016.1151439","url":null,"abstract":"ABSTRACT Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens with outbreaks resulting in high economic losses due to increased mortality and drop in egg production. This study reports a survey of ILT virus antibody conducted in nine local government areas (LGAs) of Plateau State involving 67 randomly selected commercial poultry flocks. In all, 938 sera were tested using the Agar Gel Immuno-diffusion (AGID) technique. Overall prevalence of 1.2% (N = 11) was recorded. ILT virus antibody was found in 2.5% (n = 9) and 7.1% (n = 2) of the tested sera from Jos South and Langtang North LGAs, respectively. No detectable ILT virus antibody was found from the other seven LGAs. This is the first report of ILT infection in poultry from the North central part of Nigeria. It is therefore recommended that the economic implication of ILT infection in Nigerian poultry population be conducted in order to know if vaccination should be adopted for control.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"9 1","pages":"368 - 375"},"PeriodicalIF":0.0,"publicationDate":"2016-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81344205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-22DOI: 10.1080/15321819.2015.1120745
M. Mondal, M. Karunakaran, K. K. Baruah
Metastin, also known as kisspeptin-10, is a potent stimulator of gonadotropin-releasing hormone (GnRH) neurons in the central nervous system. Recently, it has been emerged as a key player in the regulation of reproduction in mammals. Blood concentrations of metastin during different physiological stages in bovine species in general and mithun (Bos frontalis) in particular are not available. Lacking of such information may probably be due to non-availability of simple assay procedure to measure the peptide. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for metastin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique. Biotin was coupled to metastin and used to bridge between streptavidin-peroxidase and the immobilized metastin antiserum in the competitive assay. The EIA was conducted directly in 150 μL of unknown mithun plasma. Metastin standards ranging from 0.01–51.2 ng/150 μL/well were prepared in hormone-free plasma. The lowest detection limit was 0.07 ng/mL plasma. Plasma volumes for the EIA, viz., 75, 150, and 200 μL did not influence the shape of standard curve even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun metastin with metastin standard used. It showed good parallelism with the metastin standard curve. For the biological validation of the assay, metastin was measured in (a) blood samples collected from 12 pregnant mithun cows during different stages of pregnancy, (b) in blood from seven early pregnant and 12 non-pregnant mithuns, and (c) in follicular fluid obtained from different types of follicle. It was found that the plasma metastin concentrations increased (P < 0.001) from first through last trimester of pregnancy. Plasma metastin levels were much higher (P < 0.001) in early pregnant than non-pregnant cows. Follicular fluid metastin concentrations were found to increase (P < 0.001) as the follicles grow and the highest levels were recorded in preovulatory follicles. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine metastin levels in mithun. A wide range of metastin concentrations can be detected during different physiological stages in mithun using this metastin-EIA procedure.
{"title":"Development and Validation of a Sensitive Enzymeimmunoassay for Determination of Plasma Metastin in Mithun (Bos frontalis)","authors":"M. Mondal, M. Karunakaran, K. K. Baruah","doi":"10.1080/15321819.2015.1120745","DOIUrl":"https://doi.org/10.1080/15321819.2015.1120745","url":null,"abstract":"Metastin, also known as kisspeptin-10, is a potent stimulator of gonadotropin-releasing hormone (GnRH) neurons in the central nervous system. Recently, it has been emerged as a key player in the regulation of reproduction in mammals. Blood concentrations of metastin during different physiological stages in bovine species in general and mithun (Bos frontalis) in particular are not available. Lacking of such information may probably be due to non-availability of simple assay procedure to measure the peptide. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for metastin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique. Biotin was coupled to metastin and used to bridge between streptavidin-peroxidase and the immobilized metastin antiserum in the competitive assay. The EIA was conducted directly in 150 μL of unknown mithun plasma. Metastin standards ranging from 0.01–51.2 ng/150 μL/well were prepared in hormone-free plasma. The lowest detection limit was 0.07 ng/mL plasma. Plasma volumes for the EIA, viz., 75, 150, and 200 μL did not influence the shape of standard curve even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun metastin with metastin standard used. It showed good parallelism with the metastin standard curve. For the biological validation of the assay, metastin was measured in (a) blood samples collected from 12 pregnant mithun cows during different stages of pregnancy, (b) in blood from seven early pregnant and 12 non-pregnant mithuns, and (c) in follicular fluid obtained from different types of follicle. It was found that the plasma metastin concentrations increased (P < 0.001) from first through last trimester of pregnancy. Plasma metastin levels were much higher (P < 0.001) in early pregnant than non-pregnant cows. Follicular fluid metastin concentrations were found to increase (P < 0.001) as the follicles grow and the highest levels were recorded in preovulatory follicles. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine metastin levels in mithun. A wide range of metastin concentrations can be detected during different physiological stages in mithun using this metastin-EIA procedure.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"36 1","pages":"201 - 216"},"PeriodicalIF":0.0,"publicationDate":"2016-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84083923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
ABSTRACT Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.
{"title":"Development and application of monoclonal antibodies for detection and analysis of aquareoviruses","authors":"Xiao-chan Gao, Zhong-yuan Chen, Jia Liu, Qi-ya Zhang","doi":"10.1080/15321819.2016.1151440","DOIUrl":"https://doi.org/10.1080/15321819.2016.1151440","url":null,"abstract":"ABSTRACT Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"24 1","pages":"376 - 389"},"PeriodicalIF":0.0,"publicationDate":"2016-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85274638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-18DOI: 10.1080/15321819.2016.1152276
Cristina Mambet, L. Matei, L. Necula, C. Diaconu
ABSTRACT The current understanding of BCR-ABL1 negative myeloproliferative neoplasms pathogenesis is centred on the phenotypic driver mutations in JAK2, MPL, or CALR genes, and the constitutive activation of JAK-STAT pathway. Nonetheless, there is still a need to better characterize the cellular processes that are triggered by these genetic alterations, such as apoptosis that might play a role in the pathological expansion of the myeloid lineages and, especially, in the morphological anomalies of the bone marrow megakaryocytes. In this article we will explore the connection between the driver mutations in MPN and the abnormal apoptosis that might be translated in new therapeutic strategies.
{"title":"A link between the driver mutations and dysregulated apoptosis in BCR-ABL1 negative myeloproliferative neoplasms","authors":"Cristina Mambet, L. Matei, L. Necula, C. Diaconu","doi":"10.1080/15321819.2016.1152276","DOIUrl":"https://doi.org/10.1080/15321819.2016.1152276","url":null,"abstract":"ABSTRACT The current understanding of BCR-ABL1 negative myeloproliferative neoplasms pathogenesis is centred on the phenotypic driver mutations in JAK2, MPL, or CALR genes, and the constitutive activation of JAK-STAT pathway. Nonetheless, there is still a need to better characterize the cellular processes that are triggered by these genetic alterations, such as apoptosis that might play a role in the pathological expansion of the myeloid lineages and, especially, in the morphological anomalies of the bone marrow megakaryocytes. In this article we will explore the connection between the driver mutations in MPN and the abnormal apoptosis that might be translated in new therapeutic strategies.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"30 1","pages":"331 - 345"},"PeriodicalIF":0.0,"publicationDate":"2016-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82518774","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-01DOI: 10.1080/15321819.2015.1135160
Y. T. Redda, G. Venkatesh, S. Kalaiyarasu, S. Bhatia, D. S. Kumar, S. Nagarajan, A. Pillai, S. Tripathi, D. D. Kulkarni, S. C. Dubey
ABSTRACT The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.
将PCR扩增的H5N1 (H5HA1)禽流感病毒(AIV)血凝素基因HA1片段克隆到pET28a(+)表达载体中，在Rosetta Blue (DE3) pLysS细胞中表达。包涵体SDS-PAGE与H5N1血清特异性反应后，采用被动凝胶洗脱纯化重组H5HA1 (rH5HA1)蛋白。建立了以rH5HA1蛋白为包被抗原的AIV H5亚型特异性间接酶联免疫吸附试验(iELISA)。与血凝抑制试验相比，灵敏度为89.04%，特异性为95.95%。Kappa值为0.842，表明试验结果完全一致。开发的iELISA可用于鸡的禽流感血清监测。
{"title":"Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA","authors":"Y. T. Redda, G. Venkatesh, S. Kalaiyarasu, S. Bhatia, D. S. Kumar, S. Nagarajan, A. Pillai, S. Tripathi, D. D. Kulkarni, S. C. Dubey","doi":"10.1080/15321819.2015.1135160","DOIUrl":"https://doi.org/10.1080/15321819.2015.1135160","url":null,"abstract":"ABSTRACT The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"14 1","pages":"346 - 358"},"PeriodicalIF":0.0,"publicationDate":"2016-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81026743","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-01-08DOI: 10.1080/15321819.2015.1135161
S. Sakamoto, R. Nagamitsu, Yurino Matsuura, Y. Tsuneura, H. Kurose, Hiroyuki Tanaka, S. Morimoto
We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugates in situ in the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81 μg/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten.
{"title":"A new approach of indirect enzyme-linked immunosorbent assay for determination of D-glutamic acid through in situ conjugation","authors":"S. Sakamoto, R. Nagamitsu, Yurino Matsuura, Y. Tsuneura, H. Kurose, Hiroyuki Tanaka, S. Morimoto","doi":"10.1080/15321819.2015.1135161","DOIUrl":"https://doi.org/10.1080/15321819.2015.1135161","url":null,"abstract":"We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugates in situ in the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81 μg/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten.","PeriodicalId":15987,"journal":{"name":"Journal of Immunoassay and Immunochemistry","volume":"19 1","pages":"296 - 306"},"PeriodicalIF":0.0,"publicationDate":"2016-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84424070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-01-08DOI: 10.1080/15321819.2015.1132230
Oluwatosin Akende, O. Akanbi, A. Oluremi, I. Okonko, O. Opaleye
Cytomegalovirus (CMV) is one of the predominant viral infections that lead to congenital diseases and teratogenic risks during the perinatal stage. There is paucity of seroepidemiological data on anti-CMV IgG antibody in pregnant women in Osogbo, Osun State, Nigeria. This study was aimed at determining the seroprevalence of Cytomegalovirus IgG antibody among pregnant women visiting antenatal clinic, LAUTECH Teaching Hospital, Osogbo, Nigeria. One hundred and seventy-four sera from the pregnant women were screened by Enzyme linked Immunosorbent Assay (ELISA) for cytomegalovirus (CMV) IgG antibody. Data analysis was done using SPSS software. In this study, 105 of the 174 pregnant women were seropositive for CMV IgG antibodies giving an antibody prevalence of 60%. There was no association found between CMV IgG seropositivity and the subjects’ demographic characteristics, however, the 60.0% prevalence of CMV-IgG antibody observed amongst pregnant women in this study demands for vaccines and regular testing for the presence of CMV and its related risk factors in antenatal clinic.
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