纤维素载体的酶免疫测定技术

纤维素载体的酶免疫测定技术黎皓（北京生化免疫制剂中心开发室）程伯鲲（中国预防医科院流研所微生物室）自１９６６年Ｎａｋａｎｅ建立酶标记抗体技术以来,酶免疫测定技术（ＥｎｚｙｍｅＩｍｍｕｎｏａｓ－ｓａｙｓ,ＥＩＡ）在很多领域得到广泛应用,被认为是继放射免疫测定技术（ＲＩＡ）和荧光免疫测定技术（ＩＦＡ）之后的一项重要的免疫测定技术。其原理是通过化学方法将酶与抗体（或抗原）结合起来,酶标记后的抗体（或抗原）仍然保持与相应抗原（或抗体）相结合的免疫学活性以及酶的催化活性, 酶标记抗体与抗原结合后, 形成酶标抗体一抗原复合物。     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

标记抗体技术与病毒快速诊断

病毒性感染的快速诊断技术通常检测体液标本中的微量病毒抗原和早期IgM抗体,在病毒感染后几天内就可作出诊断。检测微量病毒抗原和早期IgM抗体,要求检测技术敏感性高,特异性强,重复性好,方法简便。近几年来发展起来的标记抗体技术能够满足上述要求。 荧光素、酶或核素能与抗体结合成标记抗体,标记抗体仍然具有与特异性抗原结合的免疫学特性,形成一种标记的免疫复合物。这种标记的免疫复合物可以用仪器来检测。 标记抗体技术包括免疫荧光技术、免疫酶技术和放射免疫分析。 一、免疫荧光技术 将荧光素与特异性抗体共价结合,成为荧光抗体。荧光抗体再与标本中抗原形成抗原抗体复合物,借助荧光显微镜观察标本中所显示的荧光。免疫荧光技术用于检测细胞内微量病毒抗原,对不产生细胞病变的病毒更具有诊断价值。也可用于病毒抗原的定位和检测体液中的特异性抗体。在几小时内可获得实验结果,已广泛用于病毒实验室快速诊断。 常用的荧光素有异硫氰酸荧光素和罗丹明,异硫氰酸荧光素的荧光呈黄绿色;罗丹明的荧光显红色。     

黄曲霉毒素解毒酶催化AFB1-抗体复合物中抗原变构解离初步研究

用免疫学的方法对AFB₁在黄曲霉毒素解毒酶催化前后抗原表位发生改变并从抗原抗体复合物中解离的可能性进行研究探讨。结果表明 :经黄曲霉毒素解毒酶处理后产物中有AFB1表位特征产物的量下降了 2 4 8%～ 72 8% ,而灭活酶组平均只下降203% ;活性酶组AFB1的解离率达到 88% ,灭活酶组AFB1的解离率为 8 7% ,两组比较有显著性差异 (P <0 0 1 ) ,而灭活酶组与质控组的比较无显著性差异 (P >0 0 5 )。分子的模拟计算也支持以上结果。为进一步建立特异性抗体—固定化酶催化体系提供了重要的参考数据 。     

ELISA检测奶牛乳房炎疫苗效果方法的建立

建立一种检测抗乳房炎疫苗抗体的酶联免疫吸附试验(ELISA)方法,用于免疫后具有保护性效果的抗体水平。通过对细胞抗原制备、最佳抗原包被浓度的确定、抗原包被和封闭最佳条件确定、酶标抗体的最佳工作浓度及作用时间等反应体系的筛选和确定,建立了检测抗乳房炎疫苗抗体的间接ELISA方法。结果表明,金黄色葡萄球菌(Staphylococcus aureus)、无乳链球菌(Streptococcus agalactiae)、停乳链球菌(Streptococcus dysgalactiae)抗原最佳包被质量浓度分别为5 mg.L-1、10 mg.L-1、10 mg.L-1;包被条件为4℃过夜;用2%人血白蛋白作为封闭剂最佳封闭条件是37℃封闭30 min;酶标抗体最佳作用时间为30 min。结论攻毒试验表明,疫苗免疫后,血清中抗体滴度达到或超过免疫前2倍时,奶牛即具有较好的乳腺保护效力。这一指标可作为疫苗效力检验的标准。     

超滤-高效液相色谱法检测pHLA复合物中的抗原肽北大核心CSCD

重组HLA-Ⅰ类分子/抗原肽复合物(pHLA复合物)在研究人类T细胞特异性免疫应答中有重要用途。pHLA复合物的制备以基因工程及蛋白体外稀释折叠复性技术为基础,在体外复性体系中重组HLA-Ⅰ类分子正确折叠,并结合抗原肽形成复合物。本研究建立了一种超滤-高效液相色谱法(超滤-HPLC法)定量检测重组pHLA复合物中的抗原肽,尤其针对少量制备产物中抗原肽的检测。通过将重组HLA-Ⅰ类分子和抗原肽加入到复性缓冲液中,使重组HLA-Ⅰ类分子的重链(heavy chain,HC)与轻链(β2m)复性折叠,与含锚定残基的VYF抗原肽结合形成pHLA复合物,经超滤去除未结合的游离抗原肽VYF而保留复合物,最后将pHLA复合物经酸处理破坏其相互作用从而释放抗原肽,再超滤收集VYF抗原肽并进行HPLC检测,所测得的VYF抗原肽即为重组HLA-Ⅰ类分子与抗原肽相互作用所结合的抗原肽。结果显示,制备的重组pHLA复合物可被HLA-Ⅰ分子构象特异性抗体W6/32识别,这说明重组HLA-Ⅰ类分子折叠构象正确,可鉴定为pHLA复合物;而超滤-HPLC法也可检测到pHLA复合物中含有抗原肽VYF,因此将超滤-HPLC法用于检测pHLA复合物的方法可行。与Western blotting法相比,超滤-HPLC法定量检测抗原肽浓度范围为0–9μg/mL,可根据复合物中结合的抗原肽量来优化不同结合条件,以提高HLA-Ⅰ类分子折叠效率并促进HLA-Ⅰ类分子结合抗原肽,还可根据pHLA复合物结合的抗原肽含量计算复性体系中形成pHLA复合物的制备率。因此文中所建立的超滤-HPLC法可用于pHLA复合物制备过程的质量控制,在T细胞特异性免疫研究、人工抗原呈递细胞以及特异性四聚体探针应用开发方面都具有优势。     

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。     

改进的免疫胶体铁细胞化学染色

本文以改进的方法制备了胶体铁。该胶体制备简单,稳定性高,易于抗体标记,在PH7.4时,胶体与抗体IgG稳定结合形成胶体抗体复合物的经以CM-Sephadex C-50代替AmberlightCG50进行标记物的纯化,方法可靠,所制备的胶体铁标记抗体用于免疫细胞化学染色,普鲁士蓝反应清晰地显示出标记抗体与相应抗原的特异性结合部位,与传统的免疫酶及免疫金银方法比较,具有敏感性高,背景干净,呈色鲜明等优点,结合免疫酶及免疫金银染色可用于抗原的双标及多标记。     

五种乙型肝炎放射免疫药盒的研制和中试生产

核素~(125)I标记在抗体或抗原后,成为标记抗体或标-记抗原。这种标记抗体或标记抗原,仍然具有与特异性抗原或抗体相结合的免疫学特性,形成一种标记的免疫复合物。     

梅毒螺旋体重组抗原的表达及在梅毒血清学诊断中的应用

目的：克隆、表达梅毒螺旋体（Treponema pallidum, Tp）膜蛋白TpN47、TpN17、TpN44.5和TpN15,建立双抗原夹心ELISA法,探讨其在梅毒血清学诊断中的应用。方法：应用聚合酶链反应法（PCR）从Tp全基因组中扩增4个目的片段,克隆到载体pET-HT-JKM中,在BL21(DE3)plysS菌中诱导表达,Ni-NTA亲和层析法纯化重组蛋白,辣根过氧化物酶标记,双抗原夹心酶联免疫吸附法检测人血清中的特异性IgM和IgG抗体。结果：成功表达了4种重组蛋白用作ELISA包被和酶标抗原检测国家Tp参比血清,特异性和灵敏度均为100％;检测385份临床血清样本,结果与TPPA法相比符合率达到99％（P＜0.01）,灵敏度和特异性分别为98.2%（162/165）和99.5%（219/220）。结论：表达的4种重组抗原具有很好的生物活性,为理想的梅毒的血清学诊断用抗原。以这4种表达蛋白为基础建立的双抗原夹心ELISA法灵敏度高,特异性好,而且方法简单,结果客观,易于自动化操作,值得临床大力推广。     

Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips.

We examined PCR in silicon dioxide-coated silicon-glass chips (12 microl in volume with a surface to volume ratio of approximately 17.5 mm(2)/microl) using two PCR reagent systems: (i) the conventional reagent system using Taq DNA polymerase; (ii) the hot-start reagent system based on a mixture of TaqStart antibody and Taq DNA polymerase. Quantitative results obtained from capillary electrophoresis for the expected amplification products showed that amplification in microchips was reproducible (between batch coefficient of variation 7.71%) and provided excellent yields. We also used the chip for PCR directly from isolated intact human lymphocytes. The amplification results were comparable with those obtained using extracted human genomic DNA. This investigation is fundamental to the integration of sample preparation, polynucleotide amplification and amplicate detection on a microchip.     

High-sensitivity quantitative PCR platform

Real-time PCR methods have become widely used within the past few years. However, real-time PCR is rarely used to study chronic diseases with low pathogen loads, presumably because of insufficient sensitivity. In this report, we developed an integrated nucleic acid isolation and real-time PCR platform that vastly improved the sensitivity of the quantitative detection of the intracellular bacterium, Chlamydia spp., by fluorescence resonance energy transfer real-time PCR. Determinants of the overall detection sensitivity were analyzed by extracting nucleic acids from bovine milk specimens spiked with low amounts of chlamydial organisms. Nucleic acids were optimally preserved and recovered by collection in guanidinium stabilization buffer, binding to a matrix of glass fiber fleece, and elution in low volume. Step-down thermal cycling and an excess of hot-start Taq polymerase vastly improved the robustness and sensitivity of the real-time PCR while essentially maintaining 100% specificity. The amplification of Chlamydia 23S rRNA allowed for the differentiation of chlamydial species and was more robust at low target numbers than amplification of the omp1 gene. The best combined method detected single targets per a 100-microL specimen equivalent in a 5-microL real-time PCR input. In an initial application, this high-sensitivity real-time PCR platform demonstrated a high prevalence of chlamydial infection in cattle.     

DNA probes specific for Aeromonas hydrophila (HG1)

Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.     

Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results

A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.     

Effects of PCR cycle number and DNA polymerase type on the 16S rRNA gene pyrosequencing analysis of bacterial communities

The effects of PCR cycle number and DNA polymerase type on 16S rRNA gene pyrosequencing analysis were investigated using an artificially prepared bacterial community (mock community). The bacterial richness was overestimated at increased PCR cycle number mostly due to the occurence of chimeric sequences, and this was more serious with a DNA polymerase having proofreading activity than with Taq DNA polymerase. These results suggest that PCR cycle number must be kept as low as possible for accurate estimation of bacterial richness and that particular care must be taken when a DNA polymerase having proofreading activity is used.     

PCR hot-start using duplex primers

A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.     

The Effect of the Structure of Fluorescently Labeled Nucleotide Derivatives on the Efficiency of Their Incorporation in DNA in the Polymerase Chain Reaction

The efficiency of fluorescence DNA labeling was estimated for four fluorescent 2′-deoxyuridine 5′-triphosphate derivatives differing in the orientation of the main dye axis, which passes through the polymethine chain, relative to the linker connecting the dye to the nucleotide. To estimate the polymerase chain reaction (PCR) rate, real-time PCR was run with two commercial hot-start DNA polymerases possessing 5′→3′ exonuclease activity in the presence of an intercalating dye. The efficiency of the test compound incorporation in the PCR product was estimated via a quantitative analysis of the amplification product by agarose gel electrophoresis. The fluorescently labeled product was then hybridized on a biological microchip and the ratio of signals from perfect match and mismatch duplexes was determined. The incorporation efficiency and discrimination between perfect match and mismatch duplexes were found to depend on the relative orientation of the dye and the linker between the dye and pyrimidine base, as well as on the presence of hydrophilic groups in the dye. Compounds that are efficiently incorporated in a growing DNA strand and show a high specificity in hybridization analysis were identified using biochips.     

ExCyto PCR Amplification

Background

ExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme.

Results

Because the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto cells can be transformed with a single plasmid or complex library, and then the expressed thermostable DNA polymerase can be used for PCR amplification. We demonstrate that ExCyto cells can be used to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours. Further, PCR amplification with ExCyto cells is comparable to amplification using commercial DNA polymerases. The ability to transform a bacterial strain and use the endogenously expressed protein for PCR has not previously been demonstrated.

Conclusions

ExCyto PCR reduces pipetting and greatly increases throughput for screening EST, genomic, BAC, cDNA, or SNP libraries. This technique is also more economical than traditional PCR and thus broadly useful to scientists who utilize analysis of cloned DNAs in their research.     

提高SCA2编码区CAG三核苷酸重复的PCR扩增效率

以遗传性脊髓小脑共济失调Ⅱ型基因（ｓｐｉｎｏｃｅｒｅｂｅｌｌａｒ ａｔａｘｉａ ｔｙｐｅⅡｇｅｎｅＳＣＡ２）编码区内的ＣＡＧ三核苷酸重复为研究对象（Ｇ＋Ｃ含量为６９．２％）,比较了热启动ＰＣＲ、碱基替代ＰＣＲ、添加增效剂（１％－１２．５％二甲亚砜、１％－２５％甘油、１％－１２．５％甲酰胺）与常规ＰＣＲ的扩增效率,发现热启动ＰＣＲ、碱基替代ＰＣＲ及添加增效剂（１％－１０％二甲亚砜、５％－２０％甘油、     

Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase.

DNA polymerase from Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, even when KOD DNA polymerase was used for PCR, troubles with nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3G8 and betaG1, to KOD DNA polymerase were obtained. Both of these antibodies belong to subclass IgG(1), k. K(d) values were 7.3 x 10(-8) for 3G8 and 1.1 x 10(-6) for betaG1. Nucleotide sequencing of cDNAs of these monoclonal antibodies revealed their sequences to differ in their CDRs (complementarity determining region). Exonuclease activity measurement and epitope mapping revealed that the epitope for 3G8 is located in conserved regions among alpha-like (family B) DNA polymerases (Region II), and the epitope for betaG1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was performed, the specificity of target gene amplification became much higher than in reactions without monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kbp).