抗体芯片的应用进展

抗体芯片作为蛋白芯片的一种,具有微型化、集成化、高通量化的特点,可以用于检测某一特定的生理或病理过程相关蛋白的表达丰度,目前主要用于信号转导、蛋白质组学、肿瘤及其他疾病的相关研究。本文就抗体芯片的原理、基本技术环节、检测内容、应用领域及存在的问题等方面进行综述。     

组织芯片微量抗体的免疫组化染色

组织芯片(tissue chip)又称组织微阵列(tissue microarrav)技术，是由Kononen等于1998年首先建立并报道，一般是将数十至上百个甚至更多小的组织整齐有序地排列在一张载玻片上而制成缩微组织芯片，即组织切片。组织芯片蜡块可连续切片100～200张，这样用同一套组织芯片即可对上百种生物分子标记(如抗体、DNA和RNA)进行分析、检测，     

抗体芯片技术研究进展

抗体芯片是蛋白质组学的新兴技术,属于蛋白芯片的一个分支,具有高通量、高特异性、平行性分析的优点。目前抗体芯片技术在癌症标记物发现、药物发展、毒素检测、细胞因子研究、信号转导、表达产物分析、RNAs检测以及其它的基础和应用蛋白质组学研究等领域具有广泛的应用,并且在临床上有补充或取代传统免疫学方法的趋势。     

抗体芯片技术研究进展

抗体芯片是蛋白质组学的新兴技术,属于蛋白芯片的一个分支,具有高通量、高特异性、平行性分析的优点.目前抗体芯片技术在癌症标记物发现、药物发展、毒素检测、细胞因子研究、信号转导、表达产物分析、RNAs检测以及其它的基础和应用蛋白质组学研究等领域具有广泛的应用,并且在临床上有补充或取代传统免疫学方法的趋势.     

抗体芯片技术研究进展

抗体芯片是蛋白质组学的新兴技术,属于蛋白芯片的一个分支,具有高通量、高特异性、平行性分析的优点.目前抗体芯片技术在癌症标记物发现、药物发展、毒素检测、细胞因子研究、信号转导、表达产物分析、RNAs检测以及其它的基础和应用蛋白质组学研究等领域具有广泛的应用,并且在临床上有补充或取代传统免疫学方法的趋势.     

玻璃载体微阵列芯片法在ENA抗体检测中的应用

为评价以玻璃为载体的微阵列芯片法对ENA抗体检测的灵敏度和特异度,及其在常见自身免疫性疾病中的临床应用价值,我们以免疫印迹法(Western blotting,WB)和以玻璃为载体的微阵列芯片法(Microarray)分别对508例自身免疫性疾病患者和155例健康体检者的血清样本进行了检测,并与WB比较。结果显示:与WB方法相比,玻璃载体微阵列芯片法检测的结果如下:Sm/nRNP灵敏度为98.95%,特异度为99.82%;Sm灵敏度为97.18%,特异度为99.49%;SSA灵敏度为95.29%,特异度为98.73%;SSB灵敏度为96.10%,特异度为99.83%;Scl-70灵敏度为98.48%,特异度为99.66%;PM-Scl灵敏度为100%,特异度为99.49%;Jo-1灵敏度为100%,特异度为99.67%;CENP-B灵敏度为98.39%,特异度为99.67%;PCNA灵敏度为98.33%,特异度为99.83%;dsDNA灵敏度为95.28%,特异度为99.64%;核小体灵敏度为96.70%,特异度为99.30%;组蛋白灵敏度为100%,特异度为98.16%;核糖体P蛋白灵敏度为92.50%,特异度为99.83%;AMA-M2灵敏度为92.31%,特异度为99.67%。从检测结果来看,阳性一致率为97.11%,阴性一致率为99.49%。微阵列芯片法与WB结果具有高度的一致性。用微阵列芯片法可同时检测14项ENA抗体谱,操作简单、速度快、灵敏度高、特异性强,具有完成多种项目同时检测的作用,结果可用仪器和软件来自动判读,值得临床推广应用。     

抗体的选择——从传统免疫分析到蛋白质芯片

随着后基因组时代（post—genome era）的到来和人类蛋白质组计划（human proteome project，HPP）的启动，蛋白质组学的研究开始受到越来越多的关注。蛋白质微阵列是蛋白质组研究中的一项重要技术，由三大紧密联系的技术支撑，即固相载体的选择与修饰，捕获分子和检测系统的选择。其中，捕获分子的选择和表征是蛋白质微阵列发展的关键技术之一，这里就蛋白质芯片构建过程中抗体的选择作一综述。     

用于制备抗体芯片的抗人甲胎蛋白单克隆抗体的制备及特性分析

目的：制备抗人甲胎蛋白（AFP）单克隆抗体（mAb），并用于制备抗体芯片。方法：利用杂交瘤技术建立能稳定分泌抗人AFPmAb的杂交瘤细胞株；对抗体的亲和力、特异性等特性进行分析；利用抗体相加实验，双抗体夹心ELISA、胶体金免疫层析实验以及抗体芯片技术对抗体表位及其配对情况进行分析研究。结果：共获得16株稳定分泌抗人AFPmAb的杂交瘤细胞株，从中筛选出多组性能优良的配对抗体，结合抗体芯片技术，最终筛选出适合制备抗体芯片的配对抗体。结论：当抗体包被材料或包被方法发生改变时，会引起mAb构象发生变化，从而影响抗体的配对效果。因此，筛选适合制备抗体芯片的配对mAb时，需要对mAb的特性进行综合分析，选取特异性好，亲和力高的mAb进行配对筛选。     

细胞因子抗体芯片筛选脓毒症患者血清蛋白标记物

目的:应用细胞因子抗体芯片(CAC)分析脓毒症患者血清中炎性细胞因子表达的变化.方法:采用CAC技术平台,测定9例脓毒症患者(脓毒症组)和4例正常健康人(对照组)血清中79种细胞因子.以两组样本中每组至少有4个样品的杂交信号值超过400为有表达的蛋白,筛选出有表达的蛋白38个.应用芯片差异显著性分析(SAM)软件对38个有表达的蛋白进行差异表达分析.结果:筛选到胰岛素样生长因子结合蛋白-1( IGFBP-1)、肝细胞生长因子(HGF)、骨桥蛋白(Osteopotin)、胰岛素样生长因子结合蛋白-4(IGFBP-4)、干扰素诱导蛋白-10( IP-10)和B淋巴细胞趋化因子(BLC)等6个在脓毒症患者血清中高表达的细胞因子,以及血小板衍生生长因子-BB(PDGF-BB)、脑源性神经营养因子(BDNF)、巨噬细胞炎性蛋白(MIP-11β)、白细胞介素-8( IL-8)、表皮生长因子(EGF)和白介素-1β(IL-1β)等6个在脓毒症患者血清中低表达的细胞因子.聚类分析表明,上述这12个差异表达的细胞因子能够区分脓毒症和正常健康对照.结论:CAC是一种高效、快速的蛋白质组学技术,利用CAC从血清中筛选脓毒症相关蛋白分子标记物是可行的.     

利用蛋白质芯片技术筛查多发性硬化患者的自身抗体

目的用高通量蛋白质芯片技术筛选多发性硬化(Multiple Sclerosis,MS)患者血清中可能存在的自身抗体,为筛选新的疾病辅助性诊断的指标奠定基础。方法 15例MS患者作为疾病组,选取同样数量MS阴性并无其他自身免疫疾病的性别和年龄相匹配的健康人15位作为健康对照组。两组组内随机分为3个小组,血清充分混匀后,采用高通量蛋白质芯片法检测MS患者血清中可能存在的自身抗体。结果通过高通量蛋白质芯片技术检测及与对照组分析比对,共筛出MS患者血清27种自身抗体。其中阳性率较高为SLC16A4、NOL3和DTX1,提示其可能是MS患者血清中特异性自身抗体的靶蛋白。阳性蛋白数量上,MS组高于HC组。功能聚类结果显示,自身抗体主要针对神经相关的蛋白质。结论采用高通量蛋白质芯片技术,可以较为有效筛选出MS血清中的自身抗体的靶抗原,为进一步验证MS相关自身抗体的功能及作用机制奠定了基础。     

Enzyme immunoassay with high sensitivity and accuracy for specific antibody to neocarzinostatin

A quantitative enzyme immunoassay (EIA) for specific antibody to neocarzinostatin (NCS) is described which uses enzyme-labeled anti-rabbit IgG antibody, solid-phase NCS and standard purified specific antibody to NCS. The dose of the standard was determined by sandwich EIA for rabbit IgG. The lower detection limit was 3 ng of the specific antibody per tube. The accuracy of the assay was excellent and a comparative study with the sandwich EIA for rabbit IgG showed good correlation. The antiserum to NCS of the highest titer was found to contain 0.6 mg and 40 mg per ml of specific antibody to NCS and of normal IgG, respectively. The accuracy of the assay results and the purity of the standard was established by 2 recovery tests for anti-NCS antibody.     

The detection of antibodies to cytomegalovirus in the sera of renal transplant patients by an igm antibody capture assay

An IgM antibody capture assay for detection of cytomegalovirus (CMV) IgM antibody (MACRIA) was developed. It was shown to be of similar sensitivity to the indirect immunofluorescence test but has the advantage that rheumatoid factor does not react in it and pretest fractionation of serum is not required. It does, however, give false results with some Paul Bunnell-positive sera. The assay was used to measure the IgM response in 28 renal transplant patients followed prospectively. Seven patients (100%) with primary infections and six of 13 (46%) patients with secondary infections developed IgM by MACRIA. Nine of 13 (69%) patients with CMV IgM-positive sera had symptoms other than pyrexia associated with CMV infections, while only one of seven (14%) IgM-negative infections were symptomatic. Four of seven irreversible rejection episodes were associated with CMV IgM. The possible significance of CMV IgM production is discussed.     

Production and characterization of a monoclonal antibody to chloramphenicol

A monoclonal antibody to chloramphenicol (CAP) was produced. After immunization of BALB/c mice with CAP base coupled to human serum albumin and incubation of the stimulated splenocytes in vitro in the presence of antigen for three days, these splenocytes were hybridized with X63‐Ag8·653 myeloma cells. The antibody, designated 6A10, proved to be IgG2b, and it had a detection limit for CAP of 10 ng/ml (0·5 ng/assay) in the direct enzyme immunoassay using horseradish peroxidase‐labelled CAP. The cross‐reactivities with CAP base, p‐nitrobenzyl alcohol, and p‐nitrophenol were 5·0, 0·94, and 0·007%, respectively. No cross‐reactivities were observed with penicillin, tetracycline and thiamphenicol, respectively.     

Development of an improved monoclonal antibody‐based Elisa for fumonisin b1–3 and the use of molecular modeling to explain observed detection limits

Monoclonal antibodies were prepared against the fumonisins, a group of mycotoxins produced by the plant pathogen, Fusarium moniliforme. Splenic lymphocytes, from Balb/c mice immunized with fumonisin B₁‐ovalbumin conjugate, were fused with SP2/O myeloma cells, and 14 hybridomas were selected. In a competitive enzyme‐linked immunosorbent assay, fumonisin B₁‐bovine serum albumin and free fumonisin B₁ (FB₁) competed for the monoclonal antibody. The concentrations of FB₁ required to inhibit 50% antibody binding (IC₅₀) ranged from 300 to 670 ppb. Antibodies also cross‐reacted with fumonisins B₂ and B₃ (FB₂, FB₃), and the hydrolyzed backbone of fumonisin B₁ (HB‐FB₁). None of the 14 monoclonal antibodies recognized the sphingolipids, sphingosine and sphinganine, that are structurally similar to the backbone of the fumonisins. Three‐dimensional computer models of FB₁, FB₂ and FB₃ show the amine backbone folding with the two esterified trimethyl‐propane‐1,2,3‐tricarboxylic acid side‐chains to form a cage into which the hydroxyl and acid groups of these fumonisins extend. The HB‐FB₁ molecule, with the two trimethyl‐propane‐1,2,3‐tricarboxylic acid esterified moieties at carbons 14 and 15 removed, does not possess two of the three branches which are folded together with inter‐hydrogen bonding to formulate the three‐dimensional structure that makes up the cage feature of FB₁, FB₂ and FB₃. Because of the unexpected folding of the three arms to make a cage of FB₁, attachment of the protein to the amine group, which is close to, or appears to be pari of, the epitope, may have allowed the immune system of the mouse to produce antibodies more specific for the FB₁‐protein conjugate than to free FB₁.     

噬菌体抗体微阵列的研究策略

抗体微阵列技术是近年来发展起来的一项蛋白质组学研究技术,广泛应用于蛋白质组学研究、医学基础与临床诊断等研究领域.抗体微阵列构建过程中的关键技术为高通量抗体制备技术及抗体固定技术.噬菌体抗体微阵列技术能很好地解决抗体来源问题及抗体固定问题,是抗体微阵列的发展方向.本文简要介绍了噬菌体抗体微阵列技术的研究策略及研究进展情况.     

Addition of polyethylene glycol 6000 improves the sensitivity of the protein A plaque assay for detection of human immunoglobulin secreting cells

A technical modification of the protein A plaque assay is described in which the addition of polyethylene glycol 6000 results in increased sensitivity in detection of human immunoglobulin secreting cells. Optimal conditions for use of this modification are described which result in improved detection and visualisation of haemolytic zones and have, with continued use, been shown to improve the reproducibility and reliability of this assay.     

A comparison of antibody capture radio- and enzyme immunoassays with immunofluorescence for detecting IgM antibody in infants with congenital rubella

IgM antibody capture radioimmunoassay (MACRIA) and enzyme immunoassay (MACEIA) were compared with immunofluorescence (IF) for detecting specific IgM antibody in 99 sera from 76 infants with confirmed congenital rubella, and 61 sera from a comparative group of 59 infants who had miscellaneous abnormalities but in whom congenital rubella was not confirmed.All of 35 specimens collected from confirmed cases within 12 weeks of birth were positive by all three methods and all but one of 17 specimens collected after the age of 18 months were uniformly negative. At intermediate ages discrepancies occurred in 18 specimens, of which eight were positive and 10 negative by IF. Three of these 18 specimens were negative by both antibody capture procedures but showed weak fluorescence; the other 15 were negative by MACEIA, but positive by MACRIA which appears to be the more sensitive of the antibody capture methods. Sera from five infants in the comparative group were clearly positive by all three methods. These five infants were probably congenitally infected with rubella. Sera from the other 54 infants were negative, except for one that gave a weakly positive result by MACRIA alone.Antibody capture procedures offer several advantages over previous methods for detecting IgM antibody. Although MACRIA was found to be slightly more sensitive than MACEIA, the greater stability of the enzyme label, together with the possibility of both visual and quantitative assessment, could make MACEIA the method of choice for detecting rubella-specific IgM.     

A competitive inhibition test of enzyme immunoassay for the anti-nRNP antibody

A competitive inhibition test for anti-nRNP antibody was developed, using horseradish-peroxidase-labelled anti-nRNP IgG derived from the serum of a patient with mixed connective tissue disease. In this test, the anti-nRNP antibody was clearly distinguished from the anti-Sm antibody in the patients' sera, and the sensitivity of the assay for anti-nRNP antibody was close to 50 ng/ml.     

Quantitative IgE antibody assays in allergic diseases

During the past several years, immunoassays for specific IgE antibodies have been refined to permit reporting results in mass units. Thus quantitative immunoassays for IgE antibodies may be an adjunct to skin tests. In cases of food allergy among children with atopic dermatitis, cutoff values for IgE antibody concentrations to egg, milk, peanut, and fish have been derived to provide 95% positive and 90% negative predictive values. Food-specific IgE antibody determinations can also be used to predict which food allergies are resolving spontaneously. Elevated egg-specific IgE antibody levels in infancy are associated with significantly increased risk for development of inhalant allergies later in childhood. In cases of inhalant allergy, specific IgE antibody levels correlate closely with results of inhalation challenge studies in cat-sensitive persons. Also, mite-specific IgE antibody levels correlate significantly with the mite allergen contents of reservoir dust in the homes of mite-sensitive persons. Immunoassays for quantitation of specific IgE antibodies may be used to document allergen sensitization over time and to evaluate the risk of reaction on allergen exposure. However, immunoassays and skin tests are not entirely interchangeable, and neither will replace the other in appropriate circumstances. (J Allergy Clin Immunol 2000;105:1077-84.)