An enzyme linked immunosorbent assay for leucocyte and platelet antibodies

An enzyme linked immunosorbent assay (ELISA) method for the detection of antibodies to platelets and leucocytes is presented. The method can be used for large numbers of samples. The method is objective when photometers are used.Approximately 50% of all cases of possible autoimmune thrombocytopenic purpura (A.T.P.) and unexplained neutropenia showed positive results. The results obtained using ELISA and standard tests for leucocyte and platelet antibodies are compared. The ELISA tests may also detect immune complexes.     

Development of heterologous enzyme linked immunosorbent assay for dehydroepiandrosterone in serum

Homologous and heterologous combinations of enzyme conjugate with immunogen in steroid enzyme immunoassay (EIA) influence labeled steroid recognition by an antibody that affects the sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) enzyme linked immunosorbent assay (ELISA), antibodies were generated against dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) and dehydroepiandrosterone-7-carboxymethyloxime- bovine serum albumin (DHEA-7-CMO-BSA). Two dehydroepiandrosterone (DHEA) horse radish peroxidise (HRP) enzyme conjugates were prepared using two dehydroepiandrosterone derivatives (DHEA-17-CMO and DHEA-7-CMO). Four combinations of homologous and heterologous assays were evaluated. The use of heterologous combination improved the sensitivity of the assay.     

Heterologous enzyme linked immunosorbent assay for measurement of testosterone in serum

In steroid enzyme immunoassay (EIA), homologous and heterologous combinations of enzyme conjugate with immunogen influences labeled steroid recognition by antibodies that affect sensitivity of the assay. To develop testosterone enzyme linked immunosorbent assay (ELISA), antibodies were generated against testosterone-3-carboxymethyloxime-bovine serum albumin (T-3-CMO-BSA), testosterone-11-hemisuccinate-bovine serum albumin (T-11-HS-BSA), testosterone-17-hemisuccinate-bovine serum albumin (T-17-HS-BSA), testosterone-17-glucuronide-bovine serum albumin (T-17-G-BSA), and testosterone-19-carboxymethylether-bovine serum albumin (T-19-CME-BSA). Testosterone horseradish peroxidase (HRP) enzyme conjugate were prepared using carboxyl derivatives of 11-keto-testosterone (11-keto-T) and 1-dehydrotestosterone (1-Dehydro-T). Ten combinations of heterologous assays were evaluated. The data of the present study revealed that the use of the T-11-HS-BSA antibody in antigen plus site heterologous assay that employed 11-keto-testosterone-3-CMO-HRP as the label showed binding and displacement, and led to the development of sensitive and specific assay.     

Comparison of enzyme linked immunosorbent assay and enzyme linked fluorescence immunoassay for detection of antibodies against Chlamydia trachomatis.

An enzyme linked fluorescence immunoassay (ELFA) has been evaluated for the detection of antibodies against Chlamydia trachomatis. Reticulate bodies and elementary bodies from C trachomatis L2/434 strain were used as antigens. An enzyme linked immunosorbent assay (ELISA) has also been evaluated using the same antigens. Results obtained by ELISA and ELFA for human sera with these two antigens were compared with each other and with the results obtained by a micro-immunofluorescence (micro-IF) test. Serum IgG antibodies against C trachomatis L2 reticulate bodies and elementary bodies were found in 32 (20.0%) and 11 (6.9%), respectively, of 160 serum samples from pregnant women by the micro-IF test (titre greater than or equal to 1/32). All of these 32 pregnant women had IgG antibodies to C trachomatis reticulate bodies (titre greater than or equal to 1/100), whereas 20 (12.5%) had IgG antibodies to elementary bodies in the ELISA. On the other hand, 25 (15.6%) and 19 (11.9%) of them had IgG antibodies to C trachomatis L2 reticulate bodies and elementary bodies, respectively, by the ELFA (titre greater than or equal to 1/500).     

One step enzyme linked immunosorbent assay for direct estimation of serum cortisol

One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol in human serum is described. Cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) was used as an immunogen and cortisol-21-hemisuccinate-horse radish peroxidase (cortisol-21-HS-HRP) was used as a tracer. To the cortisol antibody coated microtiter wells, standards or serum samples (25 microl) along with cortisol-HRP conjugate (100 microl) were incubated for 2 hours at 37 degrees C. Bound enzyme activity was measured by, using TMB/H2O2 as a substrate. In this new strategy, chilled acetone stripped pooled human serum and sodium salicylate were used for preparing the standards and blocking the cortisol binding globulin (CBG), respectively. The sensitivity of the assay was .28 microg/100ml. The intraassay and interassay coefficient of variations (CVs) were ranged from 1.3% to 9.3% and 6.8% to 12.3 %, respectively. The analytical recoveries were 94% to 101.5%. The serum cortisol values, obtained by this method were correlated well with those, obtained by radioimmunoassay; r=0.95 (n=52).     

One step enzyme linked immunosorbent assay for direct estimation of serum testosterone

One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of testosterone in human serum is described. Testosterone-3-O-carboxymethyl-oxime-bovine serum albumin (testosterone-3-O-CMO-BSA), was used as immunogen and testosterone-3-O-carboxymethyl-oxime-adipic-acid dihydrazide-horseradish peroxidase (testosterone-3-O-CMO-ADH-HRP) was used as tracer. To the testosterone antibody coated microtiter wells, standard or serum samples (100 microL), along with testosterone-3-O-CMO-ADH-HRP conjugate (100 microL) were incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using tetra methyl benzidine/hydrogen peroxide (TMB/H2O2) as a substrate. In this new strategy, charcoal stripped pooled human serum spiked with non-cross reactive C18, C19, C21, and C27 steroids, used for preparing the standards and blocking the sex hormone binding globulin (SHBG)/and other steroid binding globulins (SBG). The sensitivity of the assay was 0.015 ng/mL. The intra-assay and inter-assay coefficients of variation (CVs) were ranged from 7.8 to 11.8 and 4.8 to 10.4, respectively. The serum testosterone values, obtained by this method, were correlated well with those obtained by radioimmunoassay r = .98 (n = 100).     

A two‐site enzyme‐linked immunosorbent assay for wheat gliadins

A two site enzyme‐linked immunosorbent assay for wheat gliadins has been developed. Polyclonal antibodies from chicken eggs were used as the capture agent adsorbed to the surface of the microtitration plate. Murine monoclonal antibodies bound to captured gliadin were detected by addition of enzyme‐labelled, species‐specific antibodies. The assay was developed for the purposes of screening monoclonal antibodies specific for conformational epitopes on gliadins. The properties of a panel of 6 anti‐gliadin antibodies in the assay were assessed and compared to properties exhibited under conditions where conformational epitopes were less likely to be present. Three of the monoclonal antibodies appeared to be specific for conformational epitopes.     

A non-denaturing enzyme linked immunosorbent assay with protein preadsorbed onto aluminum hydroxide

A method has been developed which prevents denaturation of proteins used for coating of plastic surfaces in enzyme linked immunosorbent assays (ELISA). The system takes advantage of the use of aluminum hydroxide (Al(OH)₃) as an adsorbent for proteins. A model protein has been analyzed, and monoclonal antibodies specific for either the native form or the denatured form of the protein were used to monitor the extent of denaturation. Adsorption of the proteins to Al(OH)₃ in carbonate buffer, pH 9.3, before coating the ELISA plate abolished the denaturation otherwise observed after direct adsorption of protein to plastic surfaces. The protection against denaturation was dependent on the buffer system and was not observed when phosphate buffers were used, due to elution of protein from Al(OH)₃ or lack of binding to Al(OH)₃ in the presence of phosphate. There is evidence that protein adsorbed onto the Al(OH)₃ is required for binding of Al(OH)₃ onto the plastic surface. This system may be useful in assay systems where discrimination between the native and denatured forms of proteins is important.     

An enzyme‐linked immunosorbent assay for pyridoxamine and its comparison with alternative analytical procedures

A pyridoxal‐protein conjugate was used as an immunogen to raise antisera in rabbits. All sera recognized pyridoxamine strongly and specifically, except one which also recognized pyridoxol. An enzyme‐linked immunosorbent assay (ELISA) for pyridoxamine was set up using the microtitation plate format. Comparison of results obtained with the ELISA on 11 different food samples (ELISA limit of detection 7 pg per well) with those obtained with high performance liquid chromatography (HPLC) showed good agreement except for beef and chicken samples. Comparison of HPLC results with those obtained by microbiological procedures also showed good agreement except for potato and cabbage samples. Food table data for samples at higher levels of B6 did not show good agreement with data derived by HPLC or microbiological procedures.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.     

诺如病毒酶联免疫检测试剂盒效果的研究

目的 比较RIDASCREEN Norovirus 3rdGeneration kit(R-biopham AG,darmstadt,Germany)(必发试剂盒)与IDEIA NLV kit(DAKOCytomation.,Ely,UK)(dako试剂盒)两试剂盒检测诺如病毒效果.方法 用此两种试剂盒以及RT-PGR方法 对长春和广州两地的308份腹泻患儿粪便标本各检测一遍.对RT-PCR阳性标本进行基因测序、序列对比以确定型别.结果 以RT-PCR方法 为金标准,必发试剂盒的灵敏度和特异度分别是96.10%和93.51%,阳性预测值和阴性预测值是83.14%和98.63%;dako试剂盒的灵敏度和特异度分别是95.83%和95.76%,阳性预测值和阴性预测值是87.34%和98.69%.结论 必发试剂盒因为具有满意的灵敏度和较好的特异度,应该成为ELLSA检测的首选.