ELISA测定正常人和吸入型哮喘患者血清IgE

本实验用酶联免疫吸附试验(ELISA)中双抗体夹心法,测定上海正常人和吸入型哮喘患者血清IgE水平。实验中经棋盘滴定选择了包被抗体和酶结合物最适浓度,并对血清稀释度、血清和酶结合物不同保温条件作了比较。每块微量反应板上带有IgE工作标准曲线和空白对照,调整待测血清稀释     

山西汉族可溶性HLA-Ⅰ类抗原检测

目的:定量检测正常人血清中可溶性人白细胞抗原-Ⅰ(sHLA-Ⅰ),以探讨山西汉族人群的正常参考值.方法:ELISA法检测血清sHLA-Ⅰ类抗原水平.将不同浓度的标准品和待检血清分别加入包被有特异性sHLA-Ⅰ抗体的酶标板,与生物素化的sHLA-Ⅰ、辣根过氧化物酶(HRP)标记的链霉亲和素共同孵育,弃上清并用洗涤液洗涤,加底物应用液四甲基联苯胺(TMB)显色,以酶标仪450nm测定吸光度值,根据不同浓度标准品显色后测得的吸光度值绘制标准曲线,测定60例健康山西人的sHLA-Ⅰ含量.结果:ELISA法能够准确检测sHLA-Ⅰ含量,60例健康山西人的sHLA-Ⅰ含量为(382.62±106.68)ng·ml-1.结论:山西汉族人群sHLA-Ⅰ正常值为(382.62±106.68)ng·ml-1.     

白介素8酶联免疫分析方法及其初步应用

建立高灵敏度和特异性的白介素8(IL-8)ELISA法.用人工重组的IL-8多次免疫兔,获取高效价抗体.纯化抗体经生物素标记,同辣根过氧化酶(HRP)标记的亲和素可特异结合.采用纯化的抗体包被96孔板,形成固相抗体.加入不同浓度标准品后,反应1.5h,洗净后再加入生物素标记抗体,以酶标记亲和素作为探针,经酶底物显色反应可获得良好的ELISA标准曲线.该方法测定范围为0.2-10.8ng/mL,最低测出值为0.02ng/mL,批内和批间CV小于8%和10%.用该法测得18名正常人血浆中IL-8水平为0.15±0.06ng/mL.18份高低不同浓度IL-8水平的血清样品经本法和RIA测定其均值为0.54±0.35ng/mL和0.63±0.59ng/mL,相关性强(r=0.819,t=5.89).在U937细胞系体外培养液中,LPS刺激24h和48h可测出IL-8水平明显上升;而经TNF-α刺激24h和48h后同对照没有明显变化.该方法操作简便,具有较强灵敏度和特异性,可满足人血清和细胞培养液中IL-8水平的检测.     

用间接ELISA检测抗H9亚型AIV独特型单克隆抗体的研究

目的为制备抗H9亚型A型流感病毒(AIV)独特型杂交瘤细胞系,运用异种动物间共有独特型理论建立的间接ELISA进行检测.方法本试验采用兔抗H9亚型低致病力AIV IgG作为检测抗原,通过预试验确定了包被抗原的工作浓度为800μg/mL,以间接ELISA方法检测融合细胞培养上清液,筛选到产生抗AIV鸡和兔种间共有独特型抗体的杂交瘤细胞.结果间接ELISA试验中P/N值达12,效价达到2-4,证明杂交瘤细胞株分泌目的单克隆抗体的能力强.结论该检测方法排除了分泌抗鸡同种型表位以及超变区其他表位抗体的杂交瘤细胞,从而成为筛选分泌抗H9亚型AIV独特型单克隆抗体杂交瘤细胞株的可靠方法.     

检测人博卡病毒抗体间接酶联免疫吸附法的建立及临床应用

目的 建立间接酶联免疫吸附法(ELISA)检测人博卡病毒(HBoV)抗体,探讨其临床应用的可行性.方法 以重组表达的HBoV融合蛋白VP2作为包被抗原,建立检测HBoV抗体的间接ELISA,优化该检测方法的最佳条件,观察其精密度、敏感度和特异性,并与荧光定量PCR方法对比检测40份临床样本,同时采用免疫印迹法(Western blot)确认其符合率.结果 所建立的间接ELISA最佳抗原包被浓度为2 mg/L,酶标二抗工作浓度为1∶5000,血清标本最佳稀释度为1∶200.该方法平均批内变异系数为6.87%,平均批间变异系数为4.67%.40份临床样本间接ELISA检测结果与荧光定量PCR及免疫印迹结果一致,符合率100%.结论 利用VP2融合蛋白作为抗原,建立的检测血清HBoV抗体间接ELISA方法特异性强、重复性好,可用于HBoV抗体的定量和定性检测.     

山西汉族可溶性HLA-I类抗原检测

目的：定量检测正常人血清中可溶性人白细胞抗原-I（sHLA-I）,以探讨山西汉族人群的正常参考值。方法：ELISA法检测血清sHLA-I类抗原水平。将不同浓度的标准品和待捡血清分别加入包被有特异性sHLA-I抗体的酶标板,与生物素化的sHLA-I、辣根过氧化物酶（HRP）标记的链霉亲和素共同孵育,弃上清并用洗涤液洗涤,加底物应用液四甲基联苯胺（TMB）显色,以酶标仪450nm测定吸光度值,根据不同浓度标准品显色后测得的吸光度值绘制标准曲线,测定60例健康山西人的sHLA-I含量。结果：ELISA法能够准确检测sHLA-I含量,60例健康山西人的sHLA-I含量为（382．62±106．68）ng·ml-1。结论：山西汉族人群sHLA-I正常值为（382．62±106．68）mg·ml-1。     

TpN17重组抗原的表达及在梅毒血清学诊断中的初步应用

目的用基因工程技术表达梅毒螺旋体TpN17重组抗原,并建立操作简便、特异性好、敏感性高的梅毒血清抗体间接酶联免疫吸附试验(ELISA)检测。方法采用PCR技术扩增梅毒螺旋体TpN17基因,再进行T-A克隆及测序,然后亚克隆到原核表达载体中,以亲和层析法纯化重组蛋白。将重组蛋白包被于反应板,建立检测血清中梅毒抗体的ELISA间接检测法,利用该法与梅毒螺旋体血凝试验(TPHA)、甲苯胺红不加热血清试验(TRUST)同时检测血清样品,对其结果进行比较研究。结果TpN17重组蛋白在大肠杆菌BL21中得到稳定表达,并成功建立了检测血清中梅毒抗体的ELISA间接检测法及试剂。对135例梅毒可疑患者血清进行检测,ELISA、TPHA和TRUST的检出阳性率分别为82.96%、98.52%和71.11%,而TRUST测出的8例可疑样品及31例阴性样品中,TPHA结果全部阳性、而ELISA只有2例阴性。结论TpN17重组抗原ELISA试剂在特异性和敏感性上明显优于TRUST法。     

间接ELISA法检测轮状病毒

本文报道检测急性小儿腹泻患者粪便中轮状病毒的间接 ELISA 法。分别用兔抗 SA_(11)轮状病毒血清和正常兔血清的粗提 IgG 包板,检测抗体为豚鼠抗 SA_(11)抗体。酶标抗体为辣根过氧化物酶标记的抗豚鼠 IgG 抗体。根据正常人标本结果为阴性,阻断试验成立,以及阳性标本用正常豚鼠血清检测为阴性,说明本试验具特异性。分析比较50份标本的电镜检查结果和间接 ELISA 试验结果,说明本试验的敏感性好。初步比较了抗牛轮状病毒(NCDV)抗体和抗 SA_(11)抗体检测的结果,两者基本一致。用硫酸铵粗提的 IgG 代替兔全血清,可减少非特异性显色。     

分泌型ICOS-mIg重组融合蛋白定量检测方法的建立和应用

目的:建立双抗体夹心ELISA法,定量检测重组可诱导共刺激分子(ICOS)-mIg融合蛋白的分泌型表达,并对其灵敏度、特异性及线性检测范围进行评价。方法:用分子生物学技术制备重组ICOS-mIg融合蛋白。以羊抗小鼠IgG为包被抗体,HRP标记的马抗小鼠IgG为检测抗体,通过配对试验、方阵滴定试验及绘制mIgG浓度与A450值的标准曲线,建立定量检测重组ICOS-mIg融合蛋白的双抗体夹心ELISA法。结果:建立了双抗体夹心ELISA法,检测的线性范围为7.8~500μg/L,标准曲线的回归方程为:y=-0.7864 1.1635log(x),R2=0.9911,P<0.0001。应用该方法可快速检测哺乳动物细胞表达的重组ICOS-mIg融合蛋白的分泌量。结论:建立了一种可快速定量检测重组ICOS-mIg融合蛋白分泌表达的双抗体夹心ELISA法。该法灵敏、准确、快速、实用性好,对优化ICOS-mIg融合蛋白表达细胞的筛选和大规模制备具有重要价值。     

检测人博卡病毒抗体间接酶联免疫吸附法的建立及临床应用

目的 建立间接酶联免疫吸附法(ELISA)检测人博卡病毒(HBoV)抗体,探讨其临床应用的可行性.方法 以重组表达的HBoV融合蛋白VP2作为包被抗原,建立检测HBoV抗体的间接ELISA,优化该检测方法的最佳条件,观察其精密度、敏感度和特异性,并与荧光定量PCR方法对比检测40份临床样本,同时采用免疫印迹法(Western blot)确认其符合率.结果 所建立的间接ELISA最佳抗原包被浓度为2 mg/L,酶标二抗工作浓度为1∶5000,血清标本最佳稀释度为1∶200.该方法平均批内变异系数为6.87%,平均批间变异系数为4.67%.40份临床样本间接ELISA检测结果与荧光定量PCR及免疫印迹结果一致,符合率100%.结论 利用VP2融合蛋白作为抗原,建立的检测血清HBoV抗体间接ELISA方法特异性强、重复性好,可用于HBoV抗体的定量和定性检测.     

ELISA of streptomycin in buffer and milk: Effect of reagents' structure and analysis format on assay performance

The influence of immunoreagents' structure on assay performance was investigated and a range of ELISAs for streptomycin in direct and indirect format was developed. Streptomycin was conjugated with proteins (bovine serum albumin (BSA) for immunization and ovalbumin (OVA) for immobilization on a plate) by two different methods. Streptomycin was derivatized with carboxymethoxylamine (CMO) and then coupled to a protein or the protein was activated with adipic acid dihydrazide (ADH) and then coupled with streptomycin. A conjugate with horse-radish peroxidase was synthesized using streptomycin-ADH derivative. With the indirect ELISA the most sensitive assay for polyclonal antisera against streptomycin-oxime-BSA in combination with homologous and heterologous conjugates (limit of detection 2.5 ng ml^-1) was developed, whereas for a combination 'antisera against streptomycin-ADH-BSA/heterologous conjugate' higher background level of a calibration curve was observed. Besides the level was very high (about 60%) for homologous conjugate. In a direct ELISA similar sensitivity was achieved only for antisera against streptomycin-oxime-BSA (limit of detection 3.0 ng ml^-1). Chemiluminescent detection allowed to increase the assay sensitivity by several times (limit of detection 0.5 ng ml^-1) but led to the worse reproducibility (CV 16%). A sensitive and simple direct ELISA for analysis of streptomycin in milk products without preliminary sample preparation was developed (limit of detection 3.2 ng ml^-1). In the indirect ELISA an influence of fat content of a milk product on assay performance was observed.     

A comparative indirect ELISA for the detection of henipavirus antibodies based on a recombinant nucleocapsid protein expressed in Escherichia coli

The indirect ELISA is a simple and useful method for detection of pathogen-specific antibodies in animal sera. However, non-specific or background binding is often a problem, especially when recombinant proteins from Escherichia coli are used. In this study, a comparative indirect ELISA in which the total reactivity and the background binding were determined simultaneously on the same ELISA plate was reported. The background was determined by incubation of the test sera with excess free antigen to block specific binding. The sample was considered positive only when its total reactivity reading was higher than a pre-determined cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Using this approach, an antibody assay for henipaviruses using a recombinant Nipah virus nucleocapsid protein expressed in E. coli was developed. A total of 919 negative serum samples were tested in this assay and the specificity was 95.8%. In addition, eight positive experimental serum samples all tested positive. The use of recombinant protein as the ELISA antigen, instead of inactivated virus antigens, will be of significant advantage for countries where there is no facility of Biosafety level 4 to handle this group of zoonotic viruses.     

Lipopolysaccharide-based enzyme-linked immunosorbent assay for experimental use in detection of antibodies to Lawsonia intracellularis in pigs

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.     

双抗体间接夹心ELISA法检测乳腺癌患者外周血MUC1蛋白水平的评价

目的优化双抗体间接夹心ELISA试剂盒,并探讨其在乳腺癌患者中检测MUC1黏蛋白水平的应用价值。方法用基因重组MUC1-GST和MUC1-MBP融和蛋白免疫家兔和大鼠,获得抗MUC1血清,并对其纯化,获得纯化的家兔抗人及大鼠抗人MUC1多克隆抗体;经不同的筛选确立了以家兔抗人MUC1抗体作为包被抗体、大鼠抗人MUC1抗体作为检测抗体的双抗体间接夹心试剂盒,敏感度可达到0.2 ng/ml。结果应用建立的试剂盒对40例乳腺癌,18例乳腺良性疾病和120健康对照者血清中MUC1蛋白水平的进行检测,检测结果绘制ROC曲线,分析得出以2.75 ng/ml为乳腺癌患者与乳腺良性疾病患者的临界值,以1.86 ng/ml为乳腺疾病与正常人为临界值,检测结果表明本研究对乳腺癌诊断的阳性率高达97.5%,乳腺良性疾病的阳性率为66.7%,正常人特异性为96.7%。对于乳腺癌同一病例样本用酶联免疫法CA15-3诊断试剂盒进行对比检测,其检出率为3.33%,特异度为100%。绘制ROC曲线对比显示,本研究所建立的双抗体夹心ELISA方法对乳腺癌诊断的准确度明显高于CA15-3试剂盒。结论本研究成功建立了特异性强,灵敏度良好的双抗体间接夹心ELISA试剂盒,有望开发为临床辅助诊断的常规试剂盒,尤其有望应用于乳腺癌的大规模筛查及早期诊断。     

Indirect enzyme-linked immunosorbent assay for detection of antibody to a 110,000-molecular-weight hemolysin of Actinobacillus pleuropneumoniae.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect swine antibody to a 110,000-molecular-weight hemolysin (110K hemolysin) of Actinobacillus pleuropneumoniae. Affinity-purified rabbit polyclonal or mouse monoclonal immunoglobulin G to the hemolysin of A. pleuropneumoniae serotype 5 strain J45, followed by hemolysin-rich concentrated culture supernatant, was used to bind swine antibody to hemolysin to microdilution plates. Sixty-nine serum samples from swine that were clinically normal, presented with clinical evidence of pleuropneumonia, were experimentally immunized or challenged, or were free of pleuropneumonia were tested, and their ELISA titers were compared with complement fixation (CF) titers. On the basis of serum samples from swine that were clinically normal and negative by CF, an ELISA titer of 1:320 or greater was considered positive. In comparison with CF, the sensitivity of the ELISA was 98.1% and the specificity was 90%. The two samples negative by CF and positive by indirect ELISA were, however, also positive for antibody to serotype 5 capsule by ELISA. Immunization of normal pigs with whole cells or purified hemolysin boosted titers 4- to 128-fold within 4 weeks. Immunoblotting demonstrated that the affinity-purified immunoglobulin G to hemolysin used for capture in the assay recognized only a 110K protein of A. pleuropneumoniae serotypes 1 to 7, although the reactivity was quantitatively variable between serotypes. Therefore, the indirect ELISA is capable of identifying animals infected with or exposed to most, if not all, serotypes of A. pleuropneumoniae. If an indirect ELISA titer of 1:320 or greater is considered positive, the assay can be a valuable diagnostic tool in both clinical and research laboratories.     

Use of Saccharomyces cerevisiae-expressed recombinant nucleocapsid protein to detect Hantaan virus-specific immunoglobulin G (IgG) and IgM in oral fluid

Hantaan virus is the causative agent of severe hemorrhagic fever with renal syndrome. Clinical surveillance for Hantaan virus infection is unreliable, and laboratory verification is essential. The detection of virus-specific immunoglobulin M (IgM) and IgG in serum is most commonly used for the diagnosis of hantavirus infection. Testing of oral fluid samples instead of serum offers many advantages for surveillance. However, commercial tests for hantavirus-specific antibodies are unavailable. For the detection of Hantaan virus in the oral fluid of humans, we have developed a monoclonal antibody-based capture enzyme-linked immunosorbent IgM assay (IgM capture ELISA) and indirect enzyme-linked immunosorbent IgG and IgM assays (indirect IgG and IgM ELISAs) for paired serum and oral fluid samples using the Saccharomyces cerevisiae yeast-expressed nucleocapsid protein of the Hantaan-Fojnica virus. The sensitivity and specificity of the oral fluid IgM capture ELISA in comparison with the results of the serum Hantaan virus IgM assay were 96.7% and of 94.9%, respectively. Thus, data on the overall performance of the oral fluid IgM capture ELISA are in close agreement with those of the serum IgM assay, and the method exhibits the potential to serve as an easily transferable tool for large-scale epidemiological studies. Data on the indirect IgM ELISA also showed close agreement with the serum IgM assay data; however, the indirect IgG ELISA displayed a lower sensitivity and a lower specificity. In conclusion, the IgM capture ELISA can be used with oral fluid instead of serum samples for the diagnosis of Hantaan virus infection.     

Lipopolysaccharide-Based Enzyme-Linked Immunosorbent Assay for Experimental Use in Detection of Antibodies to Lawsonia intracellularis in Pigs

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.     

Development of a blocking enzyme-linked immunosorbent assay for the serological diagnosis of chicken anaemia virus

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibody to chicken anaemia virus (CAV) are described. This test depends on the abilities of CAV-specific antibodies present in convalescent chicken serum to block the reaction between virus antigen, adsorbed to the ELISA plate. and a CAV-specific mouse monoclonal antibody (MAb), 2A9, that has been conjugated to horseradish peroxidase. The 2A9 MAb has been shown to react with 10 geographically different field isolates of CAV, a finding which indicates that the test will find worldwide application. In comparative experiments involving 525 serum samples from specific pathogen free and commercial breeder flocks, there was 98.5% agreement between the results obtained with the blocking ELISA and those obtained with an indirect ELISA developed previously in this laboratory. The blocking ELISA was found to have advantages in terms of speed and cost compared with the indirect ELISA format.     

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.     

Enzyme-linked immunosorbent assay for Potomac horse fever disease.

An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs.