检测外周血MUC1蛋白双抗体夹心ELISA方法的建立及初步应用

目的为提高外周血MUC1蛋白检测的敏感度,建立了抗MUC1多克隆抗体的夹心ELISA试剂盒,并对其进行了初步测试。方法首先成功构建-表达并纯化了MUC1-GST和MUC1-MBP融和蛋白;通过免疫家兔和大鼠,获得抗MUC1血清,经饱和硫酸铵沉淀、Protein A/G纯化及抗GST和MBP抗体吸收的进一步纯化获得纯的家兔抗人及大鼠抗人MUC1多克隆抗体;通过凝血酶溶解MUC1-GST获得MUC1标准品。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MUC1抗体作为检测抗体的双抗体夹心试剂盒,敏感度可达到0.2 ng/ml。结果对32例乳腺癌患者和20例健康受试者外周血血清中MUC1蛋白水平的检测结果显示,乳腺癌患者的阳性检出率达到53.1%(17例/32例),而健康对照组检出率为0。结论本研究建立的抗MUC1多克隆抗体双夹心试剂盒有望应用于临床诊断。     

双抗体夹心ELISA法检测人血清弓形虫抗体

本文报告了双抗体夹心竞争ＥＬＩＳＡ法检测人血清弓形虫抗体，并与间接ＥＬＩＳＡ法进行了比较。结果二法阳性符合率为９１．７％。夹心ＥＬＩＳＡ法特异性、稳定性优于间接ＥＬＩＳＡ法，间接ＥＬＩＳＡ法阳性检出率略高于夹心法，但无统计学差异（Ｐ＞０．０５）。     

一种双抗体夹心ELISA检测热休克融合蛋白方法的建立

MUC1蛋白在乳腺癌、卵巢癌、胰腺癌等多种肿瘤细胞都呈现高水平表达。MUC1蛋白的表位肽VNTR，是诱生MUC1特异性免疫应答的靶点；采用MUC1 VNTR多肽研制出的生物制剂能刺激小鼠抑制表达MUC1肿瘤细胞的生长。     

间接ELISA检测抗核抗体及其临床应用

用小鼠肝细胞核包被聚苯乙烯反应板建立了间接ＥＬＩＳＡ法检测抗核抗体。通过特异性、精密度试验，达到方法学要求。对临床５１例病人检测，与间接荧光抗体法比较，两种方法检出率无明显差异（ｘ２＝３．６７Ｐ＞０．０５）。可进行半定量分析及大批量操作。     

人内皮唾液酸蛋白改良双抗体夹心ELISA检测方法的建立

为建立肿瘤内皮标志物1(tumor endothelial marker 1,TEM 1)ELISA检测方法,依据TEM 1基因胞外区片段序列及原核表达载体pMAL-p5x多克隆位点设计引物,采用PCR技术从含TEM 1全长基因载体中扩增目的片段,将双酶切的目的基因和载体经胶回收连接后插入表达载体并经测序鉴定,在转入宿主菌后用IPTG诱导目的蛋白表达,用分离纯化后的目的蛋白分别免疫鼠和兔以制备多克隆抗体并用ELISA和流式细胞术对抗体进行鉴定,采用兔抗TEM1为捕获抗体、鼠抗TEM 1为检测抗体建立双抗体夹心ELISA检测方法,棋盘滴定法探索各抗体最佳工作浓度,倍比稀释TEM 1,检测建立方法的灵敏度。成功构建的重组表达质粒pMAL-p5x-hCD248,经纯化复性后获得目的蛋白,免疫后获得兔多抗抗体效价为1∶1 024 000、鼠多抗抗体1∶512 000,棋盘法确定捕获抗体、检测抗体及酶标抗体最佳浓度分别为1∶500、1∶16 000和1∶20 000,经检测证实该方法测定下限为18.31ng/mL;检测50例恶性肿瘤患者与50例健康人血清TEM 1含量,发现差异无统计学意义。     

ELISA方法检测血清抗心磷脂抗体

抗心磷脂抗体与SLE及其它自身免疫性疾病的关系十分密切,但其检测方法很多,敏感性及特异性差别很大。本文报告经正交试验设计确定的实验条件、用ELISA检测血清ACA的方法。81例正常人血清IgG·ACABI为<1.6,IgM-ACA BI为<1.9。活动期SLE患者30例中,血清IgG-ACA BI超过正常值者占50%。IgM-ACA BI高于正常值者为37%。结果表明,本法检测ACA敏感性较高,特异性强,重复性好。实验流程短,方法简便。     

双抗体夹心ELISA法检测相思子毒素

目的：建立相思子毒素（abrin）的酶联免疫检测方法, 为abrin临床诊断、中毒治疗、法医学鉴定等应用领域提供技术基础和参考依据.方法：采用双抗体夹心ELISA法来检测微量abrin.结果：该法检测abrin线性范围为0.25 ～125 μg/L, 线性回归方程为Y=0.51015X＋0.4153（r=0.9880, n=10,P＜0.0001）, 检测下限为0.25 μg/L.不同浓度蓖麻毒素（ricin）、葡萄球菌肠毒素B（SEB）对检测结果基本无干扰.该法能用于abrin毒素污染水样、土样、食品、血液等模拟样品的分析, 相对标准差为3.52% ～4.86%, 具有较好的重现性.结论：成功地建立了双抗体夹心ELISA法检测abrin, 将多克隆抗体的强富集能力和单克隆抗体（mAb）的特异性结合起来, 提高检测的灵敏度和特异性, 可适用于各种微量abrin样品的分析.     

建立一种用重组蛋白检测抗口蹄疫病毒抗体的间接ELISA方法

目的研究以重组蛋白作为包被抗原检测口蹄疫病毒（FMDV）感染后的动物血清中特异性抗体的可能性,为建立一种非病毒颗粒的ELISA检测试剂盒提供实验依据。方法利用自行构建表达的O型口蹄疫病毒VP1表位肽重组蛋白（VP1epi）作为包被抗原,采用间接ELISA方法确定抗原的最佳工作浓度和包被方法,优化各项实验条件,并以FMDV感染后的豚鼠血清作为标准阳性血清确定ELISA方法的特异性和灵敏度。结果FMDV感染后的阳性豚鼠血清可以很好地识别VP1epi重组蛋白,用此蛋白包被检测抗FMDV抗体的灵敏度可达1∶3200,并证明所检测的抗体是FMDV特异性的。结论VP1epi重组蛋白可以替代FMDV颗粒用于建立检测抗FMDV抗体的ELISA试剂盒。     

Development of an indirect competitive ELISA for the detection of doxycycline residue in animal edible tissues

A synthetic hapten doxycycline (DOX) with a spacer-arm (para-aminobenzoic acid (PABA)) was attached to bovine serum albumin (BSA) or ovalbumin (OVA) by the diazonium coupling reaction and mixed anhydride methods. Then, DOX–PABA–OVA conjugate was used as a coating antigen in enzyme-linked immunosorbent assay (ELISA), while DOX–PABA–BSA was used as an immunogen to produce polyclonal antibodies. A reliable and sensitive indirect competitive enzyme-linked immunosorbent assay was developed and applied to the quantitative determination of DOX residue in muscle and liver samples. After the optimisation of the main parameters, 50% inhibition was 8.74 µg/l and the limit of detection was 1.96 µg/l. A weak cross-reactivity (CR) was also observed with other structurally related compounds, such as oxytetracycline (10.71%), tetracycline (4.10%) and chlortetracycline (1.89%). The CRs with other antibiotics were all below 0.1%. With the ELISA method, the recoveries were demonstrated to be from 80.19 to 89.41% in liver samples and 83.98–94.75% in muscle samples. The mean of the coefficients of variation with the intra-assay test were 5.75 and 7.53% in liver and muscle samples, respectively. For the inter-assay test, the average of the coefficients of variation was 5.92% in liver and 7.21% in muscle samples. The ELISA method is accurate and reliable for the detection of DOX residue in edible foods of animal origins.     

Optimisation of a peptide-based indirect ELISA for the detection of antibody in the serum of HIV-1 seropositive patients

A modified peptide-based indirect ELISA technique for the detection of HIV-1 specific antibodies in the sera of HIV-1 seropositive individuals is described. We found that the reduction of non-specific binding of HIV-1 seropositive sera to the ELISA plate was essential for the reliable detection of serum antibodies in the peptide based indirect ELISA. Optimal results were obtained using Immulon microtitre plates, different concentrations of denatured, purified grade of casein in the blocking (1%) and washing (0.25%) solutions and by diluting HIV-1 seropositive sera 1 in 1600. These conditions reduced non-specific binding and improved assay sensitivity. We show that the inclusion of a control peptide is essential to reducing the incidence of false positive and false negative results. Taken together, the modifications described in this report improve reliability of the peptide-based indirect ELISA without compromising its sensitivity and have particular relevance for those wishing to apply the peptide-based indirect ELISA technique to serum samples which exhibit high levels of non-specific binding. To illustrate this, levels of antibody in the sera of HIV-1 seropositive and seronegative donors that are specific for peptides derived from a conserved region of HIV-1 gp120 sharing homology with the FAS apoptosis antigen were analysed using this technique.     

MHC-restricted presentation of a single repeat of MUC1 mucin

Major histocompatibility complex (MHC) restriction of antigen presentation of a single mucin1 (MUC1) variable number of tandem repeats peptide (VNTR1) was examined by generating cytotoxic T lymphocytes (CTL) derived from peripheral blood mononuclear cells (PBMC) stimulated with a single repeat MUC1 peptide presented by allogeneic (MHC-independent) or autologous (MHC-dependent) Epstein-Barr Virus (EBV) immortalized B lymphocytes. The ability to generate greater CTL activity against MUC1-expressing tumor cells by stimulation with autologous versus allogeneic EBV-B supports the hypothesis that presentation of a single repeat of MUC1 peptide is MHC-restricted (MHC-dependent).     

Detection of Campylobacter species in foods by indirect competitive ELISA using hen and rabbit antibodies

The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 10⁴–3.2 10⁶ cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assay's detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp.

A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath® Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.     

Detection of Campylobacter species in foods by indirect competitive ELISA using hen and rabbit antibodies

The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 10⁴-3.2 10⁶ cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assay's detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp.

A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath® Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.     

Development and application of one-step ELISA for the detection of neomycin in milk

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin (NEO) in milk was developed. Two conjugates were synthesized as the immunogen and coating antigen. Four BALB/c female mice were immunised and the antisera were screened by indirect competitive ELISA (icELISA). The icELISA was further optimised using different coating methods, pH values, ionic strengths of assay buffer and reaction times. After optimised, the indirect competitive ELISA (icELISA) could be finished in 85min with an IC₅₀ value of 0.74±0.05 ng/mL. One-step indirect competitive ELISA (icELISA) exhibited similar sensitivity with an IC₅₀ value of 0.73±0.03 ng/mL, which was sensitive enough for NEO detection and could be finished in 55 min. No cross-reactivity of the antibody was observed with other aminoglycosides based on icELISA, indicating that the antibody is highly specific for neomycin. Milk samples were further analyzed with recovery ranging from 85 to 110% at spiked level of 0.25–10 ng/mL, and the detection limit was 0.08 ng/mL.     

Development and validation of a sandwich ELISA for quantification of peanut agglutinin (PNA) in foods

In this study, two anti-peanut agglutinin (PNA) polyclonal antibodies were successfully prepared. Using mouse anti-PNA polyclonal antibody as the capture antibody and rabbit anti-PNA polyclonal antibody as the detection antibody, a novel sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify PNA. The detection limit of the ELISA method was low (0.49 ng/mL), and the linear dynamic range was between 0.78 and 100 ng/mL. The recovery ranged from 93.86% to 139.3%, whereas the intra- and inter-assay coefficients of variation were less than 10.25% and 12.06%, respectively. Sample analysis verified this method as a reliable tool for the detection of PNA in processed foods.     

Potential for novel MUC1 glycopeptide-specific antibody in passive cancer immunotherapy

Abstract

MUC1 is an important target for antibodies in passive cancer immunotherapy. Antibodies against mucin glycans or mucin peptide backbone alone may give rise to cross reactivity with normal tissues. Therefore, attempts to identify antibodies against cancer-specific MUC1 glycopeptide epitopes havebeen made. We recently demonstrated that a monoclonal antibody against the immunodominant Tn-MUC1 (GalNAc-α-MUC1) antigen induced ADCC in breast cancer cell lines, suggesting the feasibility of targeting combined glycopeptide epitopes in future passive cancer immunotherapy.     

MUC1/Y基因真核表达载体构建及MUC1、MUC1/Y体外修饰DC诱导特异性抗肿瘤免疫应答

目的　构建MUC1 Y全长cDNA真核表达载体 ,并以MUC1及MUC1 Y真核表达载体修饰树突状细胞 (DC)诱导特异性抗肿瘤免疫。方法　构建MUC1 YDNA真核表达载体 ,选取 10例HLA A2乳腺癌患者 ,体外IL 4、GM CSF联合诱导DC ,并以pCDNA3.1 MUC1和pCDNA3.1 MUC1 Y转染DC ,同时以空载体为对照 ,以pIRES2 EGFP MUC1 Y观察转染效率 ,转染后DC与自体T细胞混合培养 ,诱导CTL。以MCF 7为特异性靶细胞 ,Raji为非特异性靶细胞 ,通过乳酸脱氢酶释放实验检测杀伤活性 ,以annexinⅤ FITC检测特异性CTL诱导靶细胞凋亡的情况。以ELISA法测定基因修饰后DC刺激自体T细胞分泌IFN γ的能力。结果　酶切鉴定及测序结果表明成功构建了MUC1 Y真核表达载体pIRES2 EGFP MUC1 Y和pCDNA3.1 MUC1 Y。pIRES2 EGFP MUC1 Y转染效率基本为 10 %左右 ,DC中MUC1表达率为 8%。杀伤实验表明T DC pCDNA3.1 MUC的杀伤活性为 6 4 % ,T DC pCDNA3.1 MUC1 Y为 4 6 %。这两组间差异有显著性 ,同时与对照组比较差异均有显著性。annexinⅤ FITC标记实验显示 ,T DC pCDNA3.1 MUC1对靶细胞的杀伤和诱导凋亡的能力显著高于T DC pCDNA3.1 MUC1 Y及对照组。基因修饰DC刺激自体T细胞分泌IFN γ的能力显著升高。结论　构建的真核表达载体pIRES2 EGFP MUC1 Y可用