检测丙型肝炎病毒F抗体的一种间接ELISA的初步评价

目的评价以丙型肝炎病毒（HCV）F蛋白为包被抗原的间接酶联免疫吸附试验（ELISA）的准确性和可靠性。方法以基因工程重组HCV F抗原包被酶标微孔板,检测HCV感染者血清抗-F滴度,建立阳性判断值,计算HCV感染者血清抗-F阳性率。与某市售HCV ELISA试剂同时检测HCV感染者和非感染者抗-F阳性率,计算该ELISA的敏感性和特异性等技术指标。结果该间接ELISA检测的敏感性为66.7%,特异性为96.7%,正确指数为63.3%,一致率为81.7%,Kappa值为0.63,变异系数（CV）为6.23%。结论该间接ELISA初步评价指标符合准确性和可靠性要求,具有潜在补充检测HCV感染的扩展应用价值。     

定量检测人狂犬病毒中和抗体间接酶联免疫吸附测定方法的建立

目的 建立定量检测人狂犬病毒中和抗体间接酶联免疫吸附测定(ELISA)方法.方法 选择包被抗原和酶标二抗的最适浓度,用人狂犬病毒抗体标准品标化系列工作标准品,对临床收集的各类血清标本进行检测和分析.结果 间接ELISA定量检测中和抗体的最佳包被糖蛋白抗原浓度为10μg/L,酶标二抗的工作浓度为1:4 000,临床检验,间接ELISA定量方法灵敏度为100Vo.特异度为98.2%,总符合率99.0%;与快速荧光灶抑制实验呈(RFFIT)高度相关性(r=0.981).结论 本研究建立的定量检测狂犬病中和抗体方法,可以用于人狂犬病毒中和抗体的定量检测.     

狂犬病毒核蛋白抗原ELISA定量法的建立及初步验证

目的建立检测狂犬病疫苗各工序产品中核蛋白抗原含量的双抗体夹心ELISA法。方法以兔抗狂犬病毒多抗为包被抗体,辣根过氧化物酶标记的抗狂犬病毒核蛋白单抗作为酶标记抗体,对狂犬病毒核蛋白抗原含量进行定量测定,并对该方法进行初步验证。结果此方法线性相关系数大于0.97;最佳线性范围为0.000625～0.01IU/ml,定量限度为0.000625IU/ml;准确性为102%～109%;变异系数(CV)为7.2%～9.4%;与小牛血清、牛血清清蛋白(BSA)、卵清蛋白(OVA)、流感疫苗纯化液、乙脑疫苗纯化液、甲肝疫苗纯化液等均无交叉反应。结论该方法特异性强,灵敏度高,准确性、重复性和稳定性好,可用于狂犬病疫苗各工序产品中核蛋白抗原的定量检测。     

融合抗原ELISA检测EB病毒抗体的临床价值

IgG阳性血清均发生反应,与8份两种抗体均阴性血清不发生反应.(2)临床检验:融合抗原EIJSA和IFA同时检测上述442份标本.以IFA为标准,融合抗原检测VCA IgG的敏感度、特异度和准确性分别为97.3%(392/403)、97.4%(38/39)和97.3%(430/442);检测VCA IgM的分别为94.2%(65/69)、99.5%(371/373)和98.6%(436/442).结论 融合抗原ELISA特异性强,灵敏度高,可用于临床EBV感染诊断及流行病学调查.     

Development of an ELISA for measurement of BCAR1 protein in human breast cancer tissue

BACKGROUND: High concentrations of breast cancer anti-estrogen resistance 1 (BCAR1) protein measured by Western blotting in primary breast tumor cytosols are associated with early disease progression and failure of tamoxifen therapy. The aim of the present study was to develop an ELISA to measure BCAR1 quantitatively in extracts of human breast cancer tissue. METHODS: A recombinant fragment of BCAR1 (the human homolog of murine p130Cas) was produced in bacterial M15 cells, purified, and injected into chickens and rabbits. The generated antibodies were affinity-purified and used for the construction of an ELISA. After validation, the results obtained with the ELISA were compared with Western blot findings on primary breast tumors. RESULTS: The detection limit the BCAR1 ELISA was 0.0031 microg/L, and the within-run imprecision (CV) was <20% at concentrations down to 0.004 microg/L. The within-run imprecision (CV) was 1.0-7.2%, and the between-run CV was 3.6-5.4%. There was no cross-reactivity with family member HEF1. The assay exhibited parallelism of results between serial dilutions and a mean recovery (range) of 96 (79-118)%. CONCLUSIONS: The ELISA measures BCAR1 in human breast cancer cytosols with high sensitivity and specificity. The assay can be used to confirm and to quantitatively extend previous semiquantitative Western blot data on the prognostic and predictive value of BCAR1 in human breast cancer; it can also be applied for other diseases.     

间接酶联免疫吸附试验检测血清抗凝血酶原抗体

目的 建立人抗凝血酶原抗体IgG、IgM的间接酶联免疫吸附试验(ELISA)检测方法.方法 纯化人凝血酶原浓度10 μg/ml包被于高吸附活性的聚氯乙烯微孔中,用山羊血清封闭,血清在Tirs盐酸缓冲生理盐水并含有1%牛血清清蛋白的溶液中按1∶100稀释,第2抗体为辣根过氧化物酶标记的山羊抗人IgG或IgM,稀释度为1∶10 000,以四甲基联苯胺为底物,1 mmol/L HCl为终止液建立的ELISA反应体系检测血清中的抗凝血酶原抗体(aPT)-IgG与IgM.结果 本实验建立检测血清中aPT-IgG、IgM的ELISA法,稳定性、特异性及重复性佳,与国外商品试剂盒有很好的相关性,有较好的准确度及精密度.结论 本实验应用的间接ELISA半定量检测aPT,适合于实验研究及临床应用.     

Cloning and expression of an envelope gene of West Nile virus and evaluation of the protein for use in an IgM ELISA

West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and epidemiologic control in areas where multiple flaviviruses are endemic. The coexistence of WNV along with other members of flaviviruses like dengue and Japanese encephalitis in India has complicated the serodiagnosis due to cross-reactive antigens. In the present study, the development and evaluation of a highly sensitive and specific IgM enzyme-linked immunosorbent assay (ELISA) using the recombinant envelope protein (rWNV-Env) for rapid, early, and accurate diagnosis of WNV are reported. The gene coding for the envelope protein of WNV was cloned and expressed in pET 28a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rWNV-Env protein was optimized having no cross reactivity with healthy human serum. Furthermore, the specificity of this assay was confirmed by cross checking with serum samples obtained from patients with dengue and Japanese encephalitis viruses. The comparative evaluation of this rWNV-Env protein–specific IgM ELISA with plaque reduction neutralization test assay using 105 acute phase of clinical samples revealed 95% concordance with sensitivity and specificity of 92% and 97%, respectively. The positive and negative predictive values of recombinant-based Env ELISA were 94% and 96%, respectively. The recombinant envelope protein–based WNV-specific ELISA reported in this study will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks.     

双抗原夹心与间接ELISA法检测抗HCV对比研究

[目的]用研制的双抗原夹心ELISA法试剂与间接ELISA法试剂对比检测抗HCV。[方法]利用基因重组技术表达的HCV多表位抗原分别包被和标记，收集献血员样本，二种国产（试剂A，B）及雅培间接抗HCV试剂同时检测，对一种试剂单独阳性和3种试剂检测均为阳性的样本，再用研制的双抗原夹心法进行检测。[结果]试剂A检测单独阳性的样本12份，试剂B单独检测阳性20份样本中，双抗原夹心检测为阴性 ；而三家试剂检测均为阳性9份样本，双抗原夹心检测均为阳性。[结论]双抗原夹心检测抗HCV特异性较间接法好，灵敏度二者相当。     

Clear detection and typing of herpes simplex virus types 1 and 2 by an indirect ELISA assay: Comparison with three different combined methods—capture ELISA,restriction enzymes,and polymerase chain reaction

The severity and recurrences of Herpes Simplex Virus (HSV) infection depend on the type of the infectious agent (HSV-1 or HSV-2), which induces the necessity of a nonambiguous detecting typing. The commonly used capture ELISA technique has to be often supported by DNA analysis to confirm the detection and the typing of HSV viruses in exposed patients. In this report, we describe a rapid and cheap indirect ELISA method using anti-HSV monospecific polyclonal antibodies prepared in the laboratory. The typing of the studied samples was clear, did not need series of dilution, and allowed the immediate classification of viruses without further control examination. We tested 51 specimens, which were typed 25 HSV-1 and 26 HSV-2 strains. The comparison with capture ELISA, restriction enzyme and polymerase chain reaction analysis definitely allowed our method to be assessed as a useful tool for a routine diagnostic. J. Clin. Lab. Anal. 11:146–153, 1997. © 1997 Wiley-Liss, Inc.     

以合成肽作抗原的酶联免疫吸附法诊断戊型肝炎病毒感染

建立了用合成多肽抗原测定戊型肝炎病毒（ＨＥＶ）抗体的酶联免疫吸附法。合成多肽Ｅ３３（ｃ）（２μｇ／ｍｌ）、Ｅ１５（Ｍ）（１μｇ／ｍｌ）混合包被，测定了４８份ＨＥＶ抗体阳性血清，阳性符合率为９５．８％。４１份阴性血清均为阴性。此方法灵敏度高、特异性强、简便快速，适合临床应用。     

Recombinant OMP28 antigen-based indirect ELISA for serodiagnosis of bovine brucellosis

Brucellosis is a zoonosis of both public health and economic importance in many developing countries including India. Early detection and segregation of the infected animals are important in order to control the disease. Serodiagnostic tests for brucellosis is mainly based on detection of antibodies developed against lipopolysaccharide (LPS) component of cell. In this study we evaluated a protein antigen, 28 kDa outer membrane protein (OMP28), of Brucella melitensis as an alternative to LPS. Recombinant OMP28 was produced in Escherichia coli system. The efficacy of purified OMP28 was studied in an indirect enzyme-linked immunosorbent assay (ELISA) for diagnosis of brucellosis in field sera collected from different regions of country. Using known negative and known positive serum samples it was found that OMP28 is immunoreactive to Brucella infected cattle, sheep, goat and dog sera. Three hundred and eighty two cattle sera were screened by OMP28 antigen-based ELISA and the results were compared to rose Bengal plate agglutination Test (RBPT). Recombinant OMP28 antigen-based ELISA has shown sensitivity of 88.7%, specificity of 93.8% and accuracy of 92.9%. It was concluded that recombinant B. melitensis OMP28 could be used as a protein antigen for diagnosis of brucellosis in domestic animals.     

Potential of recombinant flagellin fragment from Burkholderia thailandensis as an antigen for melioidosis antibody detection by indirect ELISA

Non-pathogenic Burkholderia thailandensis may be used as a model for Burkholderia pseudomallei due to the genetic similarity of these species. Moreover, the experimental manipulation of B. thailandensis is safer. In this study, we constructed recombinant flagellin protein fragments of B. thailandensis E264 (FLAG300, FLAG600, FLAG900, and FLAGFL fragments) and used fragments as the antigen to detect melioidosis antibodies by an indirect enzyme-linked immunosorbent assay (indirect ELISA). The serum samples consisted of serodiagnostic melioidosis sera (N = 52), septicemic sera caused by other bacteria (disease control) (N = 16) and healthy donor sera (N = 40). The area under the receiver operating characteristic (ROC) curve at optimal value (mean plus standard deviation) of all constructed fragments to melioidosis antibodies detection ranged from 0.654 to 0.953. The highest area under the ROC curve (AUROCC, 0.953) for FLAG300 fragment at 1600 serum titer revealed the best performance of melioidosis diagnosis. The indirect ELISA using this fragment as the antigen possessed 82.7% sensitivity and 94.6% specificity.     

Development and evaluation of an ELISA for human trefoil factor 3

BACKGROUND: The three trefoil factors (TFF1, TFF2, and TFF3) are small peptides believed to cross-link mucous glycoproteins and to play a role in the maintenance and repair of the gastrointestinal mucosa. To define the physiologic and potential diagnostic values of TFF3, assays able to measure TFF3 are warranted. METHODS: An ELISA was developed that uses two antibodies from rabbits immunized with recombinant human TFF3 and a calibrator (3-100 pmol/L) prepared from recombinant human TFF3. RESULTS: The ELISA had a detection limit of 3.0 pmol/L. The imprecision (CV) was 5-9% for mean concentrations of 13-65 pmol/L, corresponding to serum concentrations of 65-330 pmol/L. There was no cross-reaction toward human TFF1 and TFF2 (40 nmol/L). Neither food intake nor the menstrual cycle influenced the values of TFF3 significantly. The central 95% reference interval for TFF3 in serum from healthy blood donors (n = 300) was 91-250 pmol/L and showed no variation with age and limited variation with sex. TFF3 was increased in serum from patients (n = 12) with inflammation and/or ulceration of the upper gastrointestinal tract (P <0.05), whereas in serial measurements of serum from three patients with severe exacerbation of chronic inflammatory bowel disease restricted to the colon, normal concentrations and only minor variations during treatment and tapering were observed. CONCLUSIONS: The ELISA measures TFF3 in human serum and represents a specific and precise method for measurement of TFF3, which will be of value for further studies of TFF3 in health and disease.     

Demonstration of anti-mitochondria antibodies by ELISA. Comparison with the indirect immunofluorescence technique on organ sections

We determined the optimal conditions for the detection of anti-mitochondria antibodies by an enzyme linked immunosorbent assay (ELISA). Pig heart mitochondria was used at 5 mg/ml to coat polystyrene microplates and a serum dilution of 1/200 to carry out testing. This procedure detects anti-M1, M2, M4 and M6 antibodies and is more sensitive than indirect immunofluorescence. Anti-endoplasmic reticulum and anti-ribosome antibodies give negative results but anti-ds-DNA at high titres are a possible cause of false positivity.     

抗瓜氨酸化蛋白抗体特异性免疫复合物ELISA法的建立与临床初步应用

目的建立检测血清抗瓜氨酸化蛋白抗体特异性免疫复合物(ACPA-IC)的ELISA技术,探讨ACPA-IC对类风湿关节炎(RA)的临床意义。方法建立检测血清ACPA-IC的ELISA技术,并对方法的特异性、稳定性和重复性进行评估。采用PEG6000沉淀法自RA患者血清中提取IC。用建立的ACPA-IC ELISA法对92例RA患者包括46例ACPA阳性(ACPA+)患者和46例ACPA阴性(ACPA-)患者、46例SLE患者及47例健康献血员血清ACPA-IC水平进行检测。并分析RA患者血清ACPA-IC水平与ACPA和类风湿因子(RF)的相关性。结果经方法学评估,建立的ACPA-IC ELISA法在特异性、稳定性和可重复性方面均达到检测要求。ACPA+患者ACPA-IC水平高于ACPA-患者、SLE患者及健康对照组(P0.01)。当cut off值设为0.9时,该法测得RA患者血清ACPA-IC的阳性率为45.65%(42/92),显著高于SLE(6/46,13.04%)及健康对照组(2/47,4.26%),χ2分别为14.376和24.632,P均0.01。46例ACPA+RA患者ACPA-IC阳性者36例(78.26%),而ACPARA患者ACPA-IC阳性者仅6例(13.04%),差异有统计学意义(χ2=39.429,P0.01)。ACPA-IC与ACPA和RF的Spearman相关分析结果表明,RA患者血清ACPA-IC水平分别与血清ACPA(r=0.708,P=0.000)、RF(r=0.571,P=0.000)呈正相关。结论成功建立检测血清ACPA-IC水平的ELISA技术。RA患者尤其是ACPA+患者血清ACPA-IC水平显著升高。ACPA-IC在RA患者血清中出现可能具有重要的致病意义。     

Development of antigen-specific ELISA for circulating autoantibodies to extracellular matrix protein 1 in lichen sclerosus

Lichen sclerosus is a common, acquired chronic inflammatory skin disease of unknown etiology, although circulating autoantibodies to the glycoprotein extracellular matrix protein 1 (ECM1) have been detected in most patients' sera. We have examined the nature of ECM1 epitopes in lichen sclerosus sera, developed an ELISA system for serologic diagnosis, and assessed clinicopathological correlation between ELISA titer and disease. Epitope-mapping studies revealed that lichen sclerosus sera most frequently recognized the distal second tandem repeat domain and carboxyl-terminus of ECM1. We analyzed serum autoantibody reactivity against this immunodominant epitope in 413 individuals (95 subjects with lichen sclerosus, 161 normal control subjects, and 157 subjects with other autoimmune basement membrane or sclerosing diseases). The ELISA assay was highly sensitive; 76 of 95 lichen sclerosus patients (80.0%) exhibited IgG reactivity. It was also highly specific (93.7%) in discriminating between lichen sclerosus and other disease/control sera. Higher anti-ECM1 titers also correlated with more longstanding and refractory disease and cases complicated by squamous cell carcinoma. Furthermore, passive transfer of affinity-purified patient IgG reproduced some histologic and immunopathologic features of lichen sclerosus skin. This new ELISA is valuable for the accurate detection and quantification of anti-ECM1 autoantibodies. Moreover, the values may have clinical significance in patients with lichen sclerosus.     

Effect of wearing an N95 filtering facepiece respirator on superomedial orbital infrared indirect brain temperature measurements

To determine any effect of wearing a filtering facepiece respirator on brain temperature. Subjects (n = 18) wore a filtering facepiece respirator (FFR) for 1 h at rest while undergoing infrared thermography measurements of the superomedial periobital region of the eye, a non-invasive indirect method of brain temperature measurements we termed the superomedial orbital infrared indirect brain temperature (SOIIBT) measurement. Temperature of the facial skin covered by the FFR, infrared temperature measurements of the tympanic membrane and superficial temporal artery region were concurrently measured, and subjective impressions of thermal comfort obtained simultaneously. The temperature of the skin under the FFR and subjective impressions of thermal discomfort both increased significantly. The mean tympanic membrane temperature did not increase, and the superficial temporal artery region temperature decreased significantly. The SOIIBT values did not change significantly, but subjects who switched from nasal to oronasal breathing during the study (n = 5) experienced a slight increase in the SOIIBT measurements. Wearing a FFR for 1 h at rest does not have a significant effect on brain temperatures, as evaluated by the SOIIBT measurements, but a change in the route of breathing may impact these measurements. These findings suggest that subjective impressions of thermal discomfort from wearing a FFR under the study conditions are more likely the result of local dermal sensations rather than brain warming.     

Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range

BACKGROUND: C-reactive protein (CRP), a marker of systemic inflammation, has been proposed to predict outcome in patients with unstable angina; and elevated levels of CRP were found to be associated with an increased risk of coronary events. METHODS: Two enzyme-linked immunosorbent assays (ELISA) of different sensitivities were developed on microplate for CRP. Both ELISA established used Dako polyclonal anti-CRP antibody for coating and Dako horse radish peroxidase (HRP)-conjugated polyclonal anti-CRP antibody for detection. RESULTS: The sensitivity of the high and regular sensitivity ELISA was 0.16 and 0.6 mg/l, respectively. Our assays demonstrated an excellent correlation with commercial CRP assays performed on a Behring Nephelometer Analyzer II (BNII) at both regular and ultrasensitive levels, with both correlation coefficients above 0.98 and slopes of approximately 1. Using our microplate assays, we established normal reference value for serum CRP. Based on ANOVA statistical test, we found that the mean +/- S.D. was 1.3 +/- 1.27 mg/l (n=202) for normal individuals of 50-80 years and 0.43 +/- 0.42 mg/l (n=148) for the group of 20-50 years. CONCLUSIONS: The normal serum CRP mean concentrations for two age groups were distinctively different (p value<0.001). Our study suggests two different normal cutoffs of serum CRP to be employed for individuals in different age groups.     

Development of an ELISA for quantification of tumstatin in serum samples and tissue extracts of patients with lung carcinoma

BackgroundTumstatin, an angiogenesis inhibitor with anti-tumor activity in mice, is the bioactive NC1 domain of Col IVa3, the potential significance of tumstatin as an endogenous angiogenesis inhibitor in human is not yet completely understood. This study aimed to develop an enzyme-linked immunoassay (ELISA) for tumstatin to accurately measure its concentrations in biological samples.MethodsRecombinant tumstatin as an immunogen was expressed by E. coli. The purified tumstatin was injected into mice for antibody generation. An ELISA was developed with the monoclonal antibodies.ResultsThe detection limit of the ELISA was 1.4 ng/ml, and the intra- and inter- assay CVs were within 10%. Recovery of tumstatin added to sera was 92.7–115%. Patients with metastases had serum tumstatin levels 50% lower than patients without metastases (P = 0.039). Tumstatin levels in poorly differentiated tumor tissues were significantly lower than in nontumorous tissues and well-differentiated tumor tissues (P < 0.001).ConclusionsThe development of a highly sensitive and reliable ELISA method capable of quantifying tumstatin in human serum samples and tissue extracts is reported. This assay for tumstatin in biological samples may be helpful in evaluating the therapeutic and/or prognostic value of tumstatin in cancer patients.