甲胎蛋白的酶联免疫吸附分析法

本文介绍用酶联免疫吸附分析法,测定人体血清甲胎蛋白的技术。采用了双抗体夹心法,以聚苯乙烯管为包盖抗体的支持物,用亲和层析提纯的马抗甲胎蛋白抗体,与辣根过氧化物酶交联制得酶标抗体,以邻苯二胺为底物,显色后进行定量分析测定,可获得较高的敏感度(5毫微克/毫升以下)。本文还对影响实验的一些因素及这项技术的发展前景进行了讨论。     

甲胎蛋白的酶联免疫吸附分析法

本文介绍用酶联免疫吸附分析法,测定人体血清甲胎蛋白的技术。采用了双抗体夹心法,以聚苯乙烯管为包盖抗体的支持物,用亲和层析提纯的马抗甲胎蛋白抗体,与辣根过氧化物酶交联制得酶标抗体,以邻苯二胺为底物,显色后进行定量分析测定,可获得较高的敏感度(5毫微克/毫升以下)。本文还对影响实验的一些因素及这项技术的发展前景进行了讨论。     

小鼠17β-hsd10的克隆、表达及双抗夹心ELISA方法的建立

原核表达小鼠17β-羟基类固醇脱氢酶(17β-hsd10),制备抗17β-hsd10多克隆抗体,建立17β-hsd10双抗夹心酶联免疫检测方法 (Enzyme linked immunosorbent assay,ELISA)。采用RT-PCR技术克隆得到小鼠肝脏17β-hsd 10基因,构建原核表达体系p ET15b-17β-hsd10/Escherichia coli BL21(DE3),IPTG诱导重组蛋白表达,并优化表达条件;目的蛋白用His融合蛋白纯化柱(His-Binding-resin)纯化并免疫BALB/c小鼠和新西兰大耳白兔,免疫血清总Ig G采用硫酸铵沉淀法纯化,用获得的高效价鼠源和兔源两种多克隆抗体,建立17β-hsd10双抗夹心酶联免疫检测方法。表达蛋白约为29.5 k Da,纯化后浓度为1.5 mg/m L,鼠源和兔源多克隆抗体效价分别为1.25×104和2.5×104,建立的检测方法对于多种羟基类固醇脱氢酶无交叉反应,特异性良好,可检出含量约为0.05-0.1μg/m L的阳性血清。建立的17β-hsd10双抗夹心ELISA方法,简便、快速、敏感,适合实验室研究科研广泛应用。并可能为检测阿尔茨海默病提供新思路和方法。     

MDCK细胞宿主蛋白含量双抗体夹心ELISA检测方法的建立

目的建立检测犬肾细胞(madin-darby canine kidney, MDCK)宿主细胞蛋白(host cell protein, HCP)含量的双抗体夹心ELISA。方法从MDCK细胞中提取细胞总蛋白,免疫新西兰兔制备兔抗MDCK细胞蛋白多克隆抗体,抗体经辛酸-硫酸铵沉淀和Protein A层析纯化后,采用SDS-PAGE分析抗体纯度,Western blot检测抗体特异性。用纯化的多克隆抗体作为包被抗体,并采用改良过碘酸钠标记法制备酶标抗体,建立ELISA并确定包被抗体浓度和辣根过氧化物酶(HRP)标记抗体稀释度等最适条件。确定该方法较佳的线性范围及检测限,并对该法特异性、准确度、精密性和重复性进行验证。最后,用该方法分别对接种流感病毒的MDCK细胞上清收获液和纯化样品进行MDCK细胞蛋白含量检测,初步验证其在纯化工艺开发中的适用性。结果通过免疫新西兰兔制备了高滴度的兔抗MDCK细胞蛋白多克隆抗体血清,滴度可达1∶8 000。纯化后的兔抗MDCK细胞蛋白多克隆抗体纯度>90%,并可与MDCK细胞蛋白特异性结合。建立的双抗体夹心ELISA的理想包被抗体质量浓度为10μg/mL,酶标抗体的工作浓度为1∶500稀释。该方法的线性范围为50~2 500 ng/mL,检测限为50 ng/mL;该方法对Vero细胞、293T细胞和Mrc-5细胞等其他细胞HCP无交叉反应,特异性良好;不同浓度的MDCK细胞HCP回收率在98.5%~111.9%之间,变异系数均<10%。接种流感病毒的MDCK细胞培养上清经多步纯化后MDCK细胞蛋白质量浓度逐渐降低至<900 ng/mL,纯化工艺可有效去除MDCK细胞蛋白残留。结论建立双抗体夹心ELISA检测MDCK细胞残余HCP含量的方法,可用于基于MDCK细胞培养的流感疫苗下游工艺开发中宿主细胞残留HCP含量监测。     

基于磁性纳米微球的γ干扰素酶联免疫检测方法建立

目的:建立及评价使用磁性纳米微球作为固相载体的人γ干扰素(Interferon-gamma,IFN-γ)双抗体夹心酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)检测方法。方法:以杂化细乳液合成法制备磁性纳米微球,将其作为免疫检测的固相载体。将磁性微球与IFN-γ抗体进行偶联,建立基于磁性微球的ELISA检测方法,检测人IFN-γ,绘制IFN-γ标准曲线并进行方法学评价。结果:获得包被有人IFN-γ抗体的免疫微球,抗体偶联率为54.5%。用它建立IFN-γ的双抗体夹心的ELISA检测方法,检测范围为0-1000 pg/m L,相关系数为0.9996,灵敏度23.2 pg/m L,功能灵敏度0 pg/m L,批内和批间变异系数(Coefficients of Variance,CVs)8%,检测总共需要2小时。结论:成功制备了IFN-γ免疫微球并建立了定量检测人IFN-γ的双抗体夹心磁珠酶联免疫方法。     

双抗体夹心ELISA检测HIV-1 gp41抗原方法的建立

目的:建立检测HIV-1gp41抗原的双抗体夹心ELISA,并探讨其临床应用的可行性。方法:用饱和硫酸铵(SAS)纯化抗HIV-1gp41-5单克隆抗体(mAb),用HRP标记后建立双抗体夹心ELISA法,对其灵敏度及特异性进行检测,并用该方法对40份HIV-1阳性血清进行了检测。结果:用mAbE12(5μg/mL)为包被抗体,2H6为酶标记抗体(1∶900)建立了双抗体夹心ELISA法,检测gp41-5多肽的灵敏度是100pg/mL。对HIV-1阳性血清中gp41抗原的检出率为67.5%(27/40)。结论:建立了特异性强、灵敏度良好的检测HIV-1gp41抗原的双抗体夹心ELISA法。     

抗SEA鸡卵黄抗体用于血吸虫循环抗原检测的初步研究

目的:探讨抗可溶性虫卵抗原(SEA)鸡卵黄抗体(Immunoglobulin Y,IgY)用于血吸虫循环抗原检测的可行性。方法:以纯化的鸡抗SEA卵黄抗体为捕捉抗体,以酶标抗SEA单克隆抗体NP28-5B进行双抗体夹心酶联免疫吸附试验法(S-ELISA)检测急、慢性血吸虫病病人和健康人血清,并与常规检测抗体的酶联免疫吸附试验(SEA-ELISA)法做比较。结果:S-ELISA法测得SEA浓度(y)与A450值(x)呈明显的正相关(r=0.9481,y=459.22x-108.14)。S-ELISA法检测急性血吸虫病人循环抗原的阳性率为100%,慢性血吸虫病人循环抗原的阳性率为84.40%,健康人的特异性为96%。两种方法对血吸虫病的检出率差异无统计学意义(P>0.05)。结论:S-ELISA可用于血吸虫病的免疫诊断。     

应用病毒感染细胞酶联免疫吸附试验检测肾综合征出血热病毒抗原和抗体

应用病毒感染细胞酶联免疫吸附试验(VIC-ELISA)检测肾综合征出血热病毒(HFRSV)感染性滴度比双抗体间接ELISA和间接免疫荧光法(IFA)分别敏感10倍和100倍。VIC-ELISA检测兔抗HFRSV抗体的滴度比双抗体间接夹心ELISA和IFA分别敏感1.6倍和8倍。VIC-ELISA能敏感、快速、有效地检测HFRSV抗原和抗体。     

破伤风类毒素抗体酶联双抗原夹心法定量检测试剂的研制及评价

研制破伤风类毒素抗体酶联双抗原夹心法定量检测试剂,用于破伤风免疫血浆抗体效价检测。以精制破伤风类毒素经Sephacryl S-300柱层析纯化后作为包被抗原,用辣根过氧化物酶以改良过碘酸钠法标记精制破伤风类毒素作为酶标记抗原,以破伤风人免疫球蛋白国家标准品采用小鼠中和试验法标定试剂盒定量标准品,制备双抗原夹心法定量检测试剂;进行试剂盒检测范围、特异性、重复性、精密度及稳定性考核,并与小鼠中和试验法、琼脂双扩散法及国外破伤风类毒素抗体酶联试剂盒进行比较。结果显示,试剂盒的检测范围为10~150mIU/ml,灵敏度为10mIU/ml,线性好(r>0.996),板内孔间变异度小(CV<8%),特异性强(100%),重复性好(CV<13%),于37℃放置6天测定结果无明显差异,与小鼠中和试验法、英国Biding Site酶联试剂有良好的一致性。试验证明所研制的试剂盒适用于破伤风免疫血浆中的破伤风抗体效价定量检测。     

小鼠神经生长因子直接竞争酶联免疫检测方法的建立

目的:利用小鼠神经生长因子(mNGF)和纯化的兔抗mNGF IgG,建立mNGF直接竞争酶联免疫检测方法。方法:以亲和纯化兔抗mNGF IgG作为包被抗体,封闭、洗涤后加入用稀释液稀释的不同浓度的标准mNGF或样品液和生物素标记的mNGF混合液,于37℃孵育80 min或室温孵育120 min,洗涤后加入辣根过氧化物酶标记的链酶亲和素,洗涤后加入显色液显色,测定D450nm值,通过标准曲线计算待测样品中mNGF含量。结果与结论:建立了mNGF直接竞争酶联免疫检测方法;该方法的灵敏度约20 ng,变异系数为批内≤10%、批间≤15%,回收率为85%～115%;利用该方法检测了3批样品中mNGF的含量。     

Detection of Listeria monocytogenes using polyclonal antibody

Polyclonal antibody sensitive to Listeria was assayed for the detection of Listeria using two different methods, direct and sandwich enzyme linked immunosorbent assays (ELISAs). The direct ELISA uses anti-goat IgG antibody conjugated with horse-radish peroxidase, while the sandwich ELISA uses two antibodies both specific to Listeria antigens, one coated onto the microtitre plate and the other conjugated to horse-radish peroxidase. The results obtained show that the direct ELISA is superior to the sandwich ELISA in two distinct ways: (i) with direct ELISA the non- Listeria gave readings <0.2, whereas with sandwich ELISA it gave readings of 0.3–0.4; (ii) the direct ELISA is more cost-effective than the sandwich ELISA.     

A New ELISA to Overcome the Pitfalls in Quantification of Recombinant Human Monoclonal Anti-HBs,GC1102, by Commercial Immunoassays

Several methods for the quantification of human anti-HBs, an antibody to hepatitis B surface antigen (HBsAg), have been developed based on enzyme reaction, chemiluminescence, fluorescence, and radioactivity for application to human serum or plasma. Commercial anti-HBs immunoassay kits use a sandwich method in which a bridge is formed by the anti-HBs between a HBsAg immobilized solid matrix and the labeled HBsAg. However, this direct sandwich enzyme-linked immunosorbent assay (ELISA) is insufficient to accurately evaluate the activity of the human monoclonal anti-HBs, GC1102. As an alternative, we developed an indirect anti-HBs ELISA (anti-HBs qELISA_v.1) that improved detection of anti-HBs. In this current study, we further optimized this indirect method to minimize nonspecific binding of human serum, by employing incubation buffers containing animal serum, Tween 20, skim milk, and a low pH washing buffer. This new and improved method, termed anti-HBs qELISA_v.2, showed accurate quantification of plasma-derived hepatitis B immune globulin (HBIG) and was comparable to results obtained with commercial ELISA (r?=?0.93) and RIA (r?=?0.85) kits. Further, the GC1102 in human serum could be precisely measured using the anti-HBs qELISA_v.2 without limitations of nonspecific binding.     

Characterization of New Alternaria alternata–Specific Rat Monoclonal Antibodies

In this study, three different rat hybridoma cell lines secreting monoclonal antibodies (mAbs) recognizing the spores from Alternaria alternata, a plant pathogenic fungus, contaminant of food products and important cause of both allergic rhinitis and asthma, have been characterized. These three mAbs are all of IgM isotype. Two antibodies, A1 and F10, were cross-reactive antibodies recognizing spores from Alternaria, Cladosporium, Penicillium, Aspergillus and Stachybotrys genera, but not the yeasts Saccharomyces cerevisiae or Candida albicans. Competitive and sandwich assays demonstrated that these two mAbs were directed against the same or very close repetitive(s) epitope(s). A1-based sandwich ELISA efficiently detected this epitope in various mould (but not yeast)-soluble extracts prepared from strains grown in the laboratory. Moreover, this A1-based sandwich ELISA detected its cognate epitope in air and dust samples obtained from dwellings. The third antibody, E5, recognized only the spores of Alternaria and the phylogenetically very close Ulocladium botrytis. This E5 antibody is directed against a repetitive epitope found in Alternaria and Ulocladium laboratory extracts and can be used in a sandwich assay for the quantification of these moulds. Therefore, E5 antibody is a promising tool for the development of Alternaria-Ulocladium-specific immunoassays, while A1 and F10 could be interesting tools for the quantification of the total mould biomass.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

检测HSA—GLP-1融合蛋白双抗体夹心ELISA方法的建立

目的：为检测血清中HSA-GLP-1融合蛋白整体分子的浓度,建立一种特异、灵敏的定量检测食蟹猴体内HSA-GLP-1融合蛋白浓度的双抗体夹心ELISA的方法。方法：采用双抗体夹心ELISA方法,以GLP-1单克隆抗体为包被抗体、HSA-GLP-1融合蛋白为夹心抗原、anti-HSA-Biotin为检测抗体,用Streptavidin-HRP进行免疫放大,TMB显色。结果：建立了检测HSA-GLP-1融合蛋白的ELISA方法,其线性范围为15.6~1000 ng/mL,最低检测限为15.6 ng/mL,与GLP-1、HSA、IL2-HSA均无交叉反应,板内和板间精密度均小于15%,准确度为±15%,冻融稳定性和稀释稳定性良好。结论：建立的HSA-GLP-1蛋白检测方法符合新生物制品临床前药代动力学研究指导原则要求,可用于HSA-GLP-1融合蛋白在临床前药代动力学试验的定量检测。     

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Quantification of capsular polysaccharide of Streptococcus pneumoniae serotype 14 in culture broth samples

Streptococcus pneumoniae is a major cause of mortality in underdeveloped countries, where more than one million people die from pneumococcal disease every year. Vaccines are the most efficient method for preventing the infection and are based on the capsular polysaccharide (PS) protection. The serotype 14 is the most frequent in pediatric infections worldwide. This study aimed to establish a quantification protocol for PS present in culture broth samples of S. pneumoniae serotype 14 (PS14) and use this protocol for selection of the best PS14 producer strain. Phenol-sulfuric, HPSEC, competitive ELISA, and sandwich ELISA methods were tested for PS14 quantification. Sandwich ELISA was the method with the best reproducibility and sensitivity and the least susceptible to interferences. The quantification limit and detection limit of this method were 0.99 and 0.57 ng/mL, respectively. Statistical analysis was performed to calculate the coefficient of variation (CV) intraassay (1-3% intraplate and 2-6% interplate) and interassay (11-15%) and the reproducibility in different days (CV<20%). The sandwich ELISA allows us to select, among six strains evaluated, the strain 5287 as the best PS14 producer (11.68 mg PS14/biomass) and it was shown to be the best choice for measurement of pneumococcal polysaccharides in culture broth samples.     

抗Trx单克隆抗体用于血吸虫病诊断的研究

目的：建立双抗体夹心ELISA 法检测日本血吸虫硫氧还蛋白（Thioredoxin,Trx）。方法：用重组日本血吸虫Trx（rTrx）蛋白免疫BALB/c 小鼠,筛选高滴度、高特异性的单克隆抗体建立双抗体夹心ELISA法。通过检测日本血吸虫排泄- 分泌物（excretorysecretions,ES）与rTrx的浓度评价该方法的敏感性;通过对健康人血清的检测确定其特异性;通过对布氏姜片吸虫病、华支睾吸虫病、卫氏并殖吸虫病、囊虫病患者血清进行交叉反应试验,评价该方法的特异性。结果：获得2 株稳定分泌抗rTrx 蛋白单克隆抗体的杂交瘤细胞株,命名为McTrx1 和McTrx2。以McTrx1 为包被抗体,HRP-McTrx2 为酶标抗体,建立的双抗体夹心ELISA 可检测出ES的最低浓度为4.8 μg/ml,检测出rTrx 的最低浓度为1.2 滋g/ml。该方法的特异性为96 %。结论：以抗rTrx 蛋白单克隆抗体McTrx1 与McTrx2为基础建立的双抗体夹心ELISA 法具有较高的特异性。     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。     

口蹄疫病毒单克隆抗体的制备及检测应用

用纯化的口蹄疫病毒(Footandmouthdiseasevirus,FMDV)免疫BALB/C小鼠,将免疫鼠的脾细胞与SP2/0骨髓瘤细胞融合,采用有限稀释法进行克隆,经筛选获得多株能稳定分泌抗FMDV单抗的杂交瘤细胞株。选择其中一株(2G12)用于下列实验,其细胞培养上清液的效价是1:256,腹水效价是1:1280;以自行制备的兔抗FMDV高免血清IgG为捕获抗体包被酶联免疫吸附试验微量反应板,以单抗2G12为第二抗体,建立了快速检测FMDV抗原的双抗体夹心ELISA,该方法能检出90ng病毒,而且只与FMDV发生特异性反应,与猪瘟病毒(HCV)、猪蓝耳病病毒(PRRSV)、伪狂犬病毒(PRV)、猪细小病毒(PPV)和乙脑病毒(JEV)均不发生反应。本研究为检测口蹄疫病毒抗原提供了灵敏和特异的方法。