以合成多肽为抗原检测EB病毒抗体的酶免疫测定法

用固相合成肽方法合成了３ 个ＥＢＶ多肽抗原， 建立了测定ＥＢ病毒抗体的酶免疫测定法。共测定４５ 份鼻咽癌病人血清标本， 阳性率为８９％ 。本方法观察结果方便， 操作简单， 试剂稳定。     

Detection of AB antigen in blood stain using enzyme-linked immunosorbent assay

A new method for detection of AB antigen from blood stain using enzyme-linked immunosorbent assay (ELISA) is described. Flat-bottomed-wells of polystylen plate coated with human anti-B or rabbit anti-A were sensitized with AB antigen which was extracted from blood stain with 1% octyl-glucopyranoside in 0.1 M phosphate buffer pH 8.0. Mouse monoclonal anti-A or anti-B, and peroxidase conjugated anti-mouse immunoglobulin were added to the wells, respectively. Subsequently, the substrate was dropped into the wells, and the absorbance of the solution was measured. By this method, we could distinguish AB group blood stain from the mixed stain of A and B group bloods. When rabbit antiserum was used as the first antibody, differentiation between these antigens was unsuccessful presumably because of non-specific adsorption.     

Measurement of human G-CSF by enzyme-linked immunosorbent assay using monoclonal antibody

An IgG monoclonal antibody to recombinant human granulocyte colony-stimulating factor (G-CSF), designated HG1, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. This HG1 was capable of neutralizing the colony-stimulating activity of G-CSF in vitro, and it did not cross-react with human granulocyte/macrophage colony-stimulating factor (GM-CSF). An enzyme-linked immunosorbent assay (ELISA) for measurement of G-CSF was developed using HG1 and a polyclonal antibody against G-CSF raised in a rabbit. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml of recombinant G-CSF. This assay system therefore warrants further attention.     

竞争抑制法检测抗-HBc总抗体的影响因素及对策

目的 研究酶联免疫吸附试验（ELISA）待测血清标本与辣根过氧化物酶（HRP）标记的乙型肝炎病毒（HBV）核心抗体（抗-HBc-HRP）加入间隔时间对抗-HBc总抗体检测结果的影响。方法 选择HBV标志物检测结果为阴性的标本分A、B、C3组：A组,加样后延长室温放置不同时间再加入抗-HBc-HRP;B组,加样同时加入抗HBc-HRP延长室温放置不同时间;C组,A组方式测定的阴性转阳性标本再用ELISA试剂2和化学发光（CLIA）试剂重复测定,确认阴、阳性结果。结果 A组加样方式对抗一HBc总抗体检测结果影响明显,且加样和加入抗-HBc-HRP的间隔时间越长,标本的假阳性越多;B组加样方式对抗-HBc总抗体检测结果基本无影响;C组ELISA试剂1和试剂2有72．8％的检测结果一致;20例ELISA阴性转阳性标本经过CLIA方法验证仍为阴性。结论 A组的不公平竞争在不同时间内可出现程度不等的抗-HBc总抗体假阳性;B组加样方式可最大限度地减少抗-HBc总抗体假阳性,保证检验质量。     

探讨ELISA法检测血清结核杆菌抗原对肺结核病的诊断价值

目的探讨检测结核杆菌抗原对肺结核病的诊断价值。方法应用酶联免疫吸附试验检测血清中的结核杆菌分泌蛋白desA抗原。结果 139例活动性肺结核患者血清结核抗原的阳性检出率为84.1%;64例非结核性肺部疾病组结核抗原阳性率7.81%,29例健康对照者均为阴性。特异性为92.19%。结论检测血清中结核杆菌分泌蛋白desA抗原对诊断肺结核病有较高的应用价值。     

探讨ELISA法检测血清结核杆菌抗原对肺结核病的诊断价值

目的 探讨检测结核杆菌抗原对肺结核病的诊断价值.方法 应用酶联免疫吸附试验检测血清中的结核杆菌分泌蛋白desA抗原.结果 139例活动性肺结核患者血清结核抗原的阳性检出率为84.1%:64.例非结核性肺部疾病组结核抗原阳性率7.81%,29例健康对照者均为阴性.特异性为92.19%.结论 检测血清中结核杆菌分泌蛋白desA抗原对诊断肺结核病有较高的应用价值.     

竞争抑制法检测抗-HBc总抗体的影响因素及对策

目的研究酶联免疫吸附试验(ELISA)待测血清标本与辣根过氧化物酶(HRP)标记的乙型肝炎病毒(HBV)核心抗体(抗-HBc-HRP)加入间隔时间对抗-HBc总抗体检测结果的影响。方法选择HBV标志物检测结果为阴性的标本分A、B、C 3组:A组,加样后延长室温放置不同时间再加入抗-HBc-HRP;B组,加样同时加入抗-HBc-HRP延长室温放置不同时间;C组,A组方式测定的阴性转阳性标本再用ELISA试剂2和化学发光(CLIA)试剂重复测定,确认阴、阳性结果。结果A组加样方式对抗-HBc总抗体检测结果影响明显,且加样和加入抗-HBc-HRP的间隔时间越长,标本的假阳性越多;B组加样方式对抗-HBc总抗体检测结果基本无影响;C组ELISA试剂1和试剂2有72.8%的检测结果一致;20例ELISA阴性转阳性标本经过CLIA方法验证仍为阴性。结论A组的不公平竞争在不同时间内可出现程度不等的抗-HBc总抗体假阳性;B组加样方式可最大限度地减少抗-HBc总抗体假阳性,保证检验质量。     

Molecular properties of the Ro/SSA antigen and enzyme-linked immunosorbent assay for quantitation of antibody.

Antibodies to the Ro/SSA antigen occur in patients with systemic lupus erythematosus and Sjögren's syndrome. An immunoaffinity method for the preparation of electrophoretically homogeneous Ro/SSA antigen is described. Several molecular properties of the antigen have been determined. The native RNA protein particle has a molecular weight of approximately 100,000 D determined by gel filtration. Sodium dodecyl sulfate-analysis of the purified Ro/SSA antigen and analysis by staining of bands with silver and Coomassie Blue, Western blotting, and RNAase treatment leads to a hypothesis for the structure of the particle in which an antigenic 60,000 protein is bound to 24,000-27,000 RNA molecules which are not antigenic. An enzyme-linked immunoabsorbent assay method for assay of anti-Ro/SSA is also described which sensitively measures antigen binding at dilutions of sera containing anti-Ro/SSA precipitins up to 10(7) fold. Normal sera on average have 10(3) less binding activity.     

An enzyme-linked immunosorbent assay for measurement of protein S

Protein S is an important component of the haemostatic balance mechanism, and deficiency of this protein predisposes to thrombotic risk. We describe an inexpensive and reliable enzyme-linked immunosorbent assay for measurement of protein S, which is suitable for use in routine hospital laboratories.     

Evaluation of a new enzyme-linked immunosorbent assay for detection of Chagas antibody in US blood donors

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, represents a serious blood safety problem due to increasing immigration from Latin America. The Food and Drug Administration recently recommended implementation of Chagas antibody screening for US donors as soon as a suitable assay is licensed. An anonymized preclinical study of a prototype T. cruzi lysate-based enzyme-linked immunosorbent assay (ELISA) developed by Ortho-Clinical Diagnostics was conducted. STUDY DESIGN AND METHODS: Two populations of specimens were evaluated: 1) 10,192 sequential donations from blood donors residing in the El Paso, Texas, area and 2) 178 specimens from South America which were presumptively positive for antibodies to T. cruzi and purchased from commercial vendors. RESULTS: A total of 10,189 (99.97%) of the 10,192 screened donor specimens did not react, whereas 3 (0.03%) tested initially reactive. The 3 initially reactive specimens tested repeat reactive and were confirmed by radioimmunoprecipitation analysis (RIPA). Based on antibody profile analysis, 2 of the 3 Chagas-positive specimens were from the same donor. Observed specificity of the test was therefore 100 percent. Of the specimens from South America, 173 of 178 were reactive by the prototype ELISA. Of the 5 nonreactive specimens, all did not react by indirect fluorescence assay, but 4 were positive by RIPA. Therefore, calculated sensitivity of the ELISA was 97.7 percent (173/177). CONCLUSIONS: These studies indicate that the prototype ELISA has excellent sensitivity and specificity for detection of antibodies to T. cruzi in donors. Moreover, among donations from a geographically selected collection region of the United States, observed seroprevalence was 0.03 percent.     

Evaluation of a human pancreas-specific antigen by enzyme-linked immunosorbent assay

A sensitive sandwich-type enzyme immunosorbent assay has been developed for quantitation of a new human pancreas-specific antigen (PaA). With this method, PaA at a concentration as low as 0.8 ng/ml can be detected. The assay was reproducible as shown by the coefficients of variation for within (4.8%) and between (6.1%) assays. Serum PaA levels from 51 healthy persons ranged from less than 4 to 34 ng/ml. Using 21.5 ng/ml (the upper 97.5 percentile of normal controls) as an upper limit, 42 of 60 pancreatic cancer (70%), 3 of 14 pancreatitis (21%) and 2 of 6 cholelithiasis (33%) had an elevated PaA. Very few patients with other cancers were shown to have an elevated PaA: 3/40 (8%) of lung cancer, 2/43 (5%) of colorectal cancer, 3/40 (8%) of prostate cancer and 1/39 (3%) of breast cancer. The sensitivity and specificity of the serum PaA test for the detection of pancreatic cancer were calculated to be 70% and 95%, respectively. These results indicate that PaA may be useful as an adjunctive tool in immunodiagnosis of pancreatic cancer.     

Quantitation of antibody to Candida mannan by enzyme-linked immunosorbent assay

Early clinical diagnosis of invasive candidiasis is difficult. To facilitate rapid diagnosis of Candida infections, we developed an ELISA to quantitate levels of antibody to Candida mannan. The test was standardized by analysis of a nonselected inpatient population to determine a cutoff point defining the upper 5% of such a population as test-positive. Passively acquired sera from patients in intensive care units, patients with neoplastic disease or recent renal allografts, and other patients were analyzed. There was no significant difference between the number of positive tests obtained from patients in whom candidiasis was considered but cultures were negative and from the nonselected inpatient population. Positive tests were obtained from 18.5% of patients with Candida mucocutaneous colonization or infection and 40% and 63.6% of patients with probable and proven invasive candidiasis, respectively. Patients with neoplastic disease had lower test sensitivity than patients in other test categories. These results demonstrate the usefulness of a simple, rapid, standardized test for quantitation of levels of antibody activity to Candida mannan in the serodiagnosis of candidiasis.     

微阵列酶联免疫吸附试验在临床输血检测中的应用

目的探讨微阵列酶联免疫吸附试验（Array-ELISA）的临床应用价值。方法用ELISA和Array-ELISA同时检测150例样本的人类免疫缺陷病毒抗体（抗-HIV）、梅毒螺旋体抗体（抗-TP）和HBsAg,观察对比两种检测方法的结果。结果两种方法对150例样本的检测结果经卡方检验证明差异无统计学意义。除了HBsAg检测结果的总符合率为96.7%,其余指标检测结果的总符合率均为100.0%。结论Array-ELISA与ELISA试剂盒有极高的符合率,初步证明其可以满足临床输血前检测的要求。     

An enzyme-linked immunosorbent assay for the quantification of serum platelet-bindable IgG

An enzyme-linked immunosorbent assay (ELISA) using F(ab')2 peroxidase-labeled antihuman immunoglobulin and o-phenylenediamine dihydrochloride (OPD) as a substrate was developed to measure serum platelet bindable IgG (S-PBIgG). The assay was made quantitative by standardizing the number of normal "target" platelets bound to microtiter plate wells, and by incorporating quantitated IgG standards with each microtiter plate tested to prepare a standard calibration curve. By this method, S-PBIgG for normal individuals was 3.4 +/- 1.6 fg per platelet (mean +/- 1 SD; n = 40). Increased S-PBIgG levels were detected in 36 of 40 patients with clinical autoimmune thrombocytopenia (ATP), ranging from 7.0 to 85 fg per platelet. Normal S-PBIgG levels were found in 34 of 40 patients with nonimmune thrombocytopenia. This method showed a sensitivity of 90 percent, specificity of 85 percent, and in the sample population studied, a positive predictive value of 0.86 and a negative predictive value of 0.90. This assay is highly reproducible (coefficient of variation was 6.8%) and appears useful in the evaluation of patients with suspected immune-mediated thrombocytopenia.     

Competitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of apolipoprotein A-I using a monoclonal antibody

A semi-automated competitive enzyme-linked immunosorbent assay for human plasma apolipoprotein (Apo) A-I has been developed which utilizes nondelipidated samples, microtiter plates, commercially available monoclonal antibodies and alkaline phosphatase conjugated second antibody. The working range of the assay is 5-100 ng of Apo A-I. The range of plasma concentrations for plasma Apo A-I was 1.21 +/- 0.34 g/l for a random sample of 40 healthy adults. Intra- and inter-assay coefficients of variation (CV) were 4 and 7%, respectively. There was a good correlation between this assay and a radial immunodiffusion assay (r = 0.96). The assay is suitable for measurement of apolipoprotein A-I in either normal or pathological plasma, lipoprotein density classes, and for cell biological and molecular biological investigations.     

Anti-endothelial cell antibodies: detection and characterization using a cellular enzyme-linked immunosorbent assay

Anti-endothelial cell antibodies (AECA) have been detected in autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma (PSS) but their role in pathogenesis is unknown. Immunofluorescence, immunohistochemistry, complement-dependent antibody lysis, and radioimmunoassay have been used in the past to detect AECA. We have developed a rapid, sensitive, and quantitative cellular enzyme-linked immunosorbent assay (ELISA) to detect and characterize AECA. Sera were obtained from 28 normal volunteers, 28 patients with SLE, and 14 patients with PSS. We also performed studies in 47 patients with various monoclonal gammopathies. Endothelial cells (EC) were obtained from human umbilical veins by standard methods and subcultured on 96-well tissue culture plates without fixation. EC were then sequentially incubated with sera, peroxidase-conjugated goat anti-human Ig (IgG, IgM, or IgA), and substrate. Optical density readings were converted to arbitrary units by developing a standard curve. Heavy-chain specific antibodies were used to determine the class of AECA binding to EC. IgG was purified by using protein A columns and digested with pepsin to obtain F(ab')2 fragments. The mean units of AECA from normals were 19.3 for IgG and 12.5 for IgM. SLE sera showed significant levels of IgM AECA (37 units, P less than 0.001) but not IgG (29 units, P less than 0.1). PSS sera showed significant levels of both IgM AECA (38 units, P = 0.001) and IgG AECA (42.7 units, P less than 0.005). IgA AECA were not detected in normal, SLE, or PSS sera. Blocking Fc receptors with rabbit IgG did not affect the titer of IgG or IgM AECA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serodignosis of herpes simplex encephalitis by antibody capture enzyme-linked immunosorbent assay

The sensitivity of a herpes simplex virus (HSV) IgG capture enzyme-linked immunosorbent assay was tested in 103 cerebrospinal fluid (CSF) and serum samples from 47 patients with herpes simplex encephalitis (HSE). Significant differences between CSF and serum absorbances were found in all HSE patients; earlier in three out of 30 patients by the capture technique than by the indirect ELISA. In follow-up samples with exceedingly high anti-HSV activity, maximal absorbance values were found in CSF and serum resulting in falsely-negative absorbance differences. Reanalysis with a lower concentration of capture anti-IgG solved the problem. This lower anti-IgG concentration was found suboptimal for analysis of early specimens. The specificity was tested in specimens from 115 patients without intrathecal anti-HSV production as determined by indirect ELISA. Five out of 73 patients with focal encephalitis of non-HSV origin had low level positive capture results. Four of them had varicella zoster (VZ) meningoencephalitis. Severe blood-CSF barrier damage did not interfere in seven patients tested. The IgG-capture was found advantageous to the indirect technique as the results were more sensitive and clearcut. It is less labour intensive and may be completed within five to six hours.     

An enzyme-linked immunosorbent assay for the evaluation of thrombocytopenia induced by heparin

Five patients with heparin-associated thrombocytopenia (HAT) were evaluated by platelet aggregation and quantitation of immunoglobulin binding to intact target platelets in both the presence and absence of heparin. These patients developed thrombocytopenia (12,000 to 70,000 platelets/microliter) 7 to 15 days and embolic and hemorrhagic complications 9 to 15 days after the initiation of heparin therapy. Platelet aggregation after the addition of heparin was demonstrated in two of four HAT serum samples, whereas normal serum samples showed no significant platelet aggregation. The five HAT serum samples showed normal to elevated baseline serum platelet-bindable immunoglobulin (SPBIg) with a range of 4.3 to 11.4 fg/platelet (normal less than or equal to 1.0 to 6.5 fg/platelet). When HAT sera were incubated with target platelets and heparin (5 U/ml), the SPBIg increased to 8.5 to 37.5 fg/platelet, a mean increase of 148% in the presence of heparin. Normal and control serum samples (from 10 normal laboratory volunteers, nine patients without thrombocytopenia receiving heparin, nine patients with autoimmune thrombocytopenic purpura, and nine patients with nonimmune thrombocytopenia not receiving heparin) showed only a slight increase in SPBIg of 0 to 2.8 fg/platelet above baseline, a mean increase of 15% after heparin incubation with the serum samples. The measurement of SPBIg of washed platelets incubated with test serum samples in the presence and absence of heparin is potentially a specific and sensitive in vitro test for the diagnosis of HAT and may prove more sensitive than platelet aggregation studies with heparin.