人N端脑钠肽前体(NT-ProBNP)单抗制备及双抗体夹心ELISA检测方法的建立

目的获得人脑钠肽前体N端(NT-pro BNP)的高亲和力、高特异性的抗体,建立双抗体夹心ELISA法检测人血浆中NT-pro BNP。方法通过Discovery Studio4.0软件预测抗原表位,合成其表位肽偶联载体蛋白免疫Balb/c小鼠,采用杂交瘤细胞融合技术获得分泌抗体的阳性杂交瘤细胞株,制备腹水型单抗。通过SDS-PAGE、ELISA等方法对获得的抗体性质进行鉴定,建立了双抗体夹心ELISA检测方法,并对120例临床血液样本中NT-pro BNP进行快速检测。结果获得6株能稳定分泌抗人NT-Pro BNP抗体的杂交瘤细胞株,其中4株单抗的腹水型效价高于1×10~(-6)。共筛选出4对能双抗夹心ELISA配对的抗体,其中Ab1与Ab5具有较高的灵敏度和较大的线性范围,其亲和力分别为1.0×10~(10)L/mol与5.9×109L/mol,检测线性范围:300~9 600 pg/ml。样本检测结果显示,自制试剂盒的阳性检出率为97.5%(78/80),阴性检出率为100%(40/40)。结论筛选获得1对高亲和力、高特异性的配对抗体,建立了双抗体夹心ELISA的检测方法,亦为新的快速免疫学检测方法的建立奠定了基础。     

抗HIV-1 p24 单克隆抗体的制备、特性鉴定及初步应用

目的：制备抗HIV-1p24单克隆抗体（mAb）及特性鉴定。方法：采用细胞融合技术制备可稳定分泌抗HIV-1p24mAb的杂交瘤细胞。选择高效价、识别不同抗原表位的mAb纯化后，分别包被ELISA板及作为酶标记mAb，建立双mAb夹心ELISA法，检测400份正常人血浆，20份抗HIV抗体阳性（p24抗原阴性）的血浆，20份HIV-1感染的细胞培养上清及HIV-1p24抗原国家参考品，并同时用进口p24抗原检测试剂盒进行对比检测。结果：经细胞融合、筛选、克隆化，共获得10株可分泌高效价mAb的杂交瘤细胞。纯化的腹水mAb经配对试验，选A值≥2．50的2株mAb建立的双mAb夹心ELISA法，敏感性可达2．5ng／L，且与进口试剂盒检测结果的一致性良好。结论：建立了具有高度敏感性和特异性的双mAb夹心ELISA法，为研制可用于检测HIV-1p24抗原的试剂盒奠定了基础。     

抗青霉素单克隆抗体的制备及初步应用

目的:制备抗青霉素的单克隆抗体(mAb)并建立双抗体夹心ELISA检测方法,对临床上引起青霉素过敏反应的过敏原青霉噻唑蛋白进行研究。方法:将半抗原青霉素和载体蛋白偶联后免疫BALB/c小鼠,应用杂交瘤技术建立稳定分泌抗青霉素mAb的杂交瘤细胞株。常规制备腹水,用辛酸-硫酸铵法纯化,并对纯化的mAb进行特异性鉴定。通过对不同抗体组合的分析和条件的优化,建立检测过敏原的双抗体夹心ELISA方法。结果:经细胞融合、筛选及克隆化,共获得9株稳定分泌抗青霉素mAb的杂交瘤细胞株,其中5株亲和力较高。建立了双抗体夹心ELISA相对定量检测方法,该方法灵敏度达到870 U/L,平均回收率为107.81%,批内变异系数平均为6.7%,批间变异系数平均为9.3%,可用于A群链球菌制剂中青霉噻唑蛋白的检测。结论:成功地制备了抗青霉素的mAb,并建立了相对定量检测青霉噻唑蛋白的双抗体夹心ELISA法。     

抗HIV-1 gp120单克隆抗体的制备及其初步应用

目的：制备抗HIV-1 gp120单克隆抗体（mAb），并建立一种可用于检测gp120的ELISA法。方法：采用基因工程抗原HIV-1gp120免疫BALB／c小鼠，通过B细胞杂交瘤技术制备抗HIV-1gp120的mAb。用ELISA法答定mAb的Ig亚类、效价及特异性。用饱和硫酸铵（SAS）纯化mAb，并用HRP标记后建立双mAb夹心ELISA法。结果：筛选出10株稳定分泌抗HIV-1gp120 mAb的杂交瘤细胞株，9株为IgG，其中4株的腹水效价为32X10～256X10所得mAb与HIV-1gp41、HIV-1p24及HIV一2gp36Ag均无交义反应，仅与HIV-1gp120产生特异性反应。用mAb 6H9和9H12，建立了双夹心ELISA法，检测gp120抗原的灵敏度是10μg／L。结论：获得10株特异性强、效价高的机HIV-1gp120的mAb，并建立了灵敏度良好的双mAb夹心ELISA法.     

一种快捷、定量检测烟曲霉GM抗原双mAb夹心ELISA的方法

目的 :建立一种快速诊断烟曲霉病的双mAb夹心ELISA法。方法 :应用 4株抗烟曲霉GM单克隆抗体 (mAb) ,分别包被和制备HRP mAb ,用双mAb夹心ELISA法配对试验 ,选择捕获及检测mAb。结果 :经筛选得到捕获及检测mAb的最佳组合 ,并建立了双mAb夹心ELISA法。该法检测GM抗原的灵敏度为 0 .1μg/L ,测出范围在 0 .1～ 10 μg/L之间。连续 6d用ELISA法检测同一份样品 ,所获CV的平均值为 ( 7.2± 3.8) %。结论 :建立了一种可快速、定量检测烟曲霉GM抗原的双mAb夹心ELISA法 ,灵敏度高、重复性好 ,对研制试剂盒应用于烟曲霉病早期诊断和防治 ,具有重要的临床应用价值     

抗铜绿假单胞菌外膜蛋白F单克隆抗体制备及夹心ELISA检测方法的建立

目的:制备抗铜绿假单胞菌外膜蛋白F(OprF)的单克隆抗体(mAb),并建立双mAb夹心ELISA检测方法。方法:利用分子生物学方法克隆OprF基因,诱导表达并纯化OprF。用OprF免疫BALB/c小鼠后通过杂交瘤技术制备特异性的mAb,并用ELISA法测定mAb的免疫活性;建立检测铜绿假单胞菌的双mAb夹心ELISA方法,并对其敏感性、特异性进行初步评价。结果:成功地克隆OprF基因,经诱导表达获得铜绿假单胞菌的OprF。通过杂交瘤技术筛选出4株mAb:6D8F7、2H2B6、3C2F5和7B5D9,经鉴定mAb 6D8F7可作为捕获抗体,mAb 7B5D9被HRP标记后可作为检测抗体,以这2株mAb建立的双mAb夹心ELISA方法重复性好、特异性强,敏感性高达到1×103集落形成单位(CFU/mL)。检测的线性范围为1×103～108CFU/mL。与传统的细菌分离方法比较,检测临床标本的符合率高达94.7%。结论:通过杂交瘤技术制备抗铜绿假单胞菌OprF的mAb,并建立了高特异性、高敏感性检测铜绿假单胞菌抗原的双mAb夹心ELISA方法,可用于临床检测铜绿假单胞菌的感染。     

抗烟曲霉菌单克隆抗体鉴定和初步应用

目的 :制备抗烟曲霉菌单克隆抗体 (McAb) ,建立一种快速检测烟曲霉菌抗原方法。方法 :用基因重组烟曲霉菌半乳糖甘蛋白 (AFMP1)抗原 ,免疫BALB/c小鼠 ,制备单克隆抗体 ,选择针对不同抗原决定簇单抗配对 ,建立双抗夹心ELISA法检测烟曲霉菌抗原。结果 :筛选出 3株稳定分泌抗烟曲霉菌单抗杂交瘤细胞株 ,IgG亚类鉴定分别为IgG1、IgG2a、IgG2b ,抗体亲和常数分别为 1 2× 10 10 、4 5 6× 10 9和 1 81× 10 10 mol/L ,免疫印迹证实单抗特异性识别烟曲霉菌培养上清和细胞裂解产物 ,相加试验表明 3株单抗是针对不同抗原决定簇 ,组成配对双抗夹心ELISA法 ,检测最高灵敏度为 0 1ng/ml,可测范围为 0 1～ 6 0ng/ml。结论 :3株杂交瘤细胞株特异性好、亲和力高 ,组成配对夹心ELISA法可用于快速检测烟曲霉菌抗原。     

用于定量ELISA方法的抗人β2微球蛋白单克隆抗体的制备及鉴定

目的制备抗人β2微球蛋白(β2-microglobulin,β2-MG)特异性单克隆抗体并建立其双抗体夹心定量ELISA免疫检测方法。方法以高纯度的人β2-MG作为免疫原,采用常规免疫和脾内免疫相结合的方法免疫纯系Balb/c小鼠,通过杂交瘤技术制备抗人β2-MG单克隆抗体并鉴定,以此为基础建立双抗体夹心ELISA检测方法。结果制备出5株稳定分泌抗人β2-MG的单克隆抗体,经鉴定5株单抗皆为IgG1类,均为β2-MG抗原特异性抗体。经抗体配对试验筛选出1对可用于ELISA检测的配对抗体,建立了定量ELISA检测方法。结论制备出抗人β2-MG单克隆抗体并建立了双抗体夹心定量ELISA免疫检测方法,与国外试剂盒检测结果相关性良好。     

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。     

抗人心脏型脂肪酸结合蛋白单克隆抗体的制备、表位鉴定及其应用

目的 制备抗人心脏型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),鉴定其基本特性并通过配对检测临床样本血清,对已配对的mAb进行表位鉴定.方法 通过已制备的H-FABP抗原和合成肽免疫BALB/c小鼠,常规融合筛选后进行抗体亚型鉴定、效价和亲和力检测,纯化mAb后通过间接ELISA、Western blot法对mAb进行特异性检测;将获得的mAb分别作为捕获和检测抗体,经过配对建立检测重组H-FABP的ELISA体系,并初步用于临床血清检测;采用生物信息学分析设计2段HFABP表位,通过原核表达获得其短肽并纯化,通过Western blot法对配对的mAb进行表位鉴定.结果 成功获得4株抗HFABP特异性mAb,亚类分别为IgG2a和IgG2b,抗体效价为1:51 200～1:1024 000,亲和力最高可达到9.02×109 mol/L;经Western blot法和间接ELISA鉴定均能够特异检测重组H-FABP蛋白,经过配对发现mAb 3-H5和1-F10能够检测重组H-FABP,并在其临床血清样本检测中发现正常组和急性心梗组之间H-FABP水平具有显著性统计学差异;通过表位鉴定发现未知表位的mAb 1-F10能够特异识别位点为H-FABP的第86到第133位氨基酸.结论 制备了4株高特异性和高亲和力的抗H-FABPmAb,成功建立了检测临床样本血清中的ELISA体系,并对其表位进行了分析鉴定.     

A mathematical model to predict the effects of temperature on timed ELISA

Timed ELISA is a protocol that enables quantitative ELISA without the aid of sophisticated laboratory equipment. It is likely be used outside of a temperature‐controlled environment and the response of this system to temperature is therefore significant. A model of Timed ELISA, based on the expected performance of its individual reactions, was able to predict the performance of the whole system between 15 and 25°C. Both the model and experimental results confirmed that Timed ELISA possessed an element of temperature compensation, although antigen standards were still required for accuracy. Interrogating the model with regards to a more thermostable conjugate and increased specific activity predicted that the latter would be most suited to extending the temperature range of this system.     

Diagnostic significance of the human antibody response to a major cytoplasmic antigen of C. albicans

In contrast to the ubiquitous presence of IgG antibodies to mannan or to crude antigen preparations of C. albicans (CA), antibodies to a purified cytoplasmic antigen (SSF) of CA were detected by ELISA only in patients with candidiasis. The differences of mean absorbancy values found in different groups of sera can allow the distinction of normal sensitized controls from patients with candidiasis and can also distinguish between patients with different degrees of C. albicans invasion.     

Antibody to Aspergillus fumigatus Antigens in Normal Sera: Influence on Positive-Negative Discrimination in ELISA

The response of precipitin-negative sera from non-selected asthmatic patients against a range of somatic and culture filtrate antigens of Aspergillus fumigatus in ELISA for anti-A. fumigatus IgG is described. Antibody to the various antigens was widely distributed but the precise distribution was dependent upon antigen type. To determine how the selection of negative reference sera from precipitin-negative sera can influence the discrimination between precipitin-positive and -negative sera in ELISA, panels of 10 sera were chosen from the extremes of the frequency distribution and by random selection. The closest agreement between precipitin testing and ELISA was seen when randomly-selected negative reference sera were incorporated in the assay, although the degree of correlation was dependent upon the detector antigen. These findings demonstrate the requirement for careful selection of sera included as negative reference sera in ELISA for anti-A. fumigatus IgG.     

抗牛碱性成纤维细胞生长因子单克隆抗体的制备及初步鉴定

目的：制备抗牛碱性成纤维细胞生长因子(bFGF)单克隆抗体(mAh)，并鉴定其亚类，建立ELISA检测bFGF含量的方法。方法：应用基因重组的牛bFGF免疫BALB／c小鼠，通过细胞融合，建立分泌抗bFGF mAh的杂交瘤细胞株。应用免疫沉淀技术鉴定抗bFGF mAh的亚类；应用基因重组的牛bFGF免疫青紫蓝兔，制备抗bFGF的多抗血清；将抗bFGF mAb及兔抗血清用Protein A亲和层析纯化后，建立检测bFGF含量的ELISA方法。结果：共获得3株稳定分泌抗bFGF mAb的杂交瘤细胞株；它们所分泌的mAb均为IgG1；采用夹心ELISA法检测bFGF的敏感性达ng水平。结论：抗bFGF mAb(IgG1)和多克隆抗体制备为临床应用及相关研究提供了必要的试剂。     

An investigation of the use of urease-antibody conjugates in enzyme immunoassays

The development of urease (E.C.3.5.1.5) as a label for enzyme immunoassay (EIA) procedures is described and the use of such conjugates illustrated with examples. Urease catalyzes the hydrolysis of urea to carbon dioxide and ammonia. The production of ammonia may be detected readily by a pH shift which we have found best indicated by the vivid colour change (yellow to purple) of bromocresol purple incorporated in the substrate solution. This enzyme-substrate system offers a number of important advantages. The substrate in aqueous solution is stable, titration end points are sharp and readily visible and the enzyme is not inhibited by sodium azide. Thus, test reagents may be prepared with this preservative and stored ready to use. Urease of high specific activity is commercially available and because it does not occur in mammalian tissues, it is suitable for use in EIA test to detect cell-associated antigens and their antibodies. Finally, the enzyme reaction may be stopped by the addition of organomercurial preservatives, thus allowing storage of developed test for later examination.     

2009-2011年重庆市输入性血吸虫病监测及SWOT分析

目的分析2009-2011年重庆市输入性血吸虫病的监测结果,为进一步制订和调整血吸虫病防治的目标和重点提供理论依据。方法应用描述流行病学方法,分析重庆市2009-2011年市级血吸虫病监测点血吸虫病的流行情况;应用SWOT法分析血吸虫病疫情监测工作所处的内部和外部环境,并提出相应的策略和措施。结果 2009-2011年共调查流动人口4591人,疫区返乡和疫区来渝人员的血清阳性率分别为2.49%（76/3048）和1.04%（16/1543）,差异有统计学意义（χ2=11.06,P〈0.05）;不同年份间疫区返乡人员血清学阳性率比较差异有统计学意义（χ2=6.82,P〈0.05）。不同年份间疫区来渝人员血清学阳性率比较差异有统计学意义（χ2=0.85,P〉0.05）。重庆市血吸虫病疫情监测工作所面临的优势和机会分别为监测内容不断深入和政府支持,所面临的劣势和威胁分别为缺乏长期完善的监测机制和当前严峻的血防形势。结论重庆市血吸虫病防治工作依然严峻,应更进一步加强血吸虫病防治工作,不断提高血防工作能力。     

Determination of IgA antibodies to human cytomegalovirus by enzyme-linked immunosorbent assay (ELISA)

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies cytomegalovirus (CMV). The antigen consisted of a sonicated extract of CMV infected human embryo cells. The tested sera were absorbed with staphylococcus aureus (strain Cowan 1) before analysis. Rabbit antihuman IgA peroxidase conjugate was used to detect human IgA bound to viral antigen. In parallel, Igm and IgG antibodies to CMV were studied by ELISA and by the immunoperoxidase antibody to membrane antigen (IPAMA) technique, respectively. CMV IgA was detected in high titers by ELISA in eight of nine CMV patients. The maximal IgA titers were generally lower than the IgM titers detected by ELISA. No CMV IgA antibodies (titer less than 20) were detected by ELISA in 57 control sera (healthy adults, hospitalized patients with various other diseases), paired sera of five patients with acute herpes simplex, infection, two patients with Epstein-Barr infections, one patients with varicella, and one with zoster infections. The potential application of the ELISA CMV IgA technique in serodiagnosis of CMV infections is discussed.     

恶性肿瘤患者的抗Koc自身抗体

目的:为了探讨恶性肿瘤患者的抗Koc自身抗体,研究其临床意义。方法:在优化重组Koc表达和提纯条件的基础上,以重组Koc为包被抗原,采用所建立的检测IgM、IgG、IgA类抗Koc自身抗体的ELISA技术。结果:发现抗Koc的阳性率与恶性肿瘤的类型有密切关系,以肝癌、直肠癌和肺癌较高,胃癌居中,而食道癌最低,其中后者与健康对照无明显差异。以IgM、IgG、IgA中任一类Ig抗Koc对阳性为抗Koc阳性标本,肝癌、直肠癌和肺癌的阳性率均分别＞50．0%,胃癌为25．5%,食道癌为15．8%,均明显较单一阳性率为高。结论:提示Ig类型抗体同步检测对于提高阳性率具有重要意义,抗Koc不失为恶性肿瘤诊断的一个良好新指标。     

An ELISA method for the detection of autoantibodies to adrenal cortex

An enzyme-linked immunosorbent assay (ELISA) is described for autoantibodies to adrenal cortex. Microsomes were prepared from fresh human adrenal glands, and microtitre ELISA plates were incubated at 4 degrees C overnight with 25 micrograms antigen/ml, the optimal concentration for the system. Optimal dilution of patient's serum was 1/500. Peroxidase-labelled anti-human IgG and IgM sera were used in separate tests and o-phenylenediamine and H2O2 served as substrate. Intra-assay variance of optical density units was 4.5%, and inter-assay variance was negligible when antigen preparations from 2 different adrenal glands were compared. All sera positive or negative at first test gave the same qualitative result in a second. Non-organ-specific binding of sera containing mitochondrial or ribosomal antibodies was eliminated by a blocking ELISA system where the antigen-coated plates were incubated with test sera, and in a second step, peroxidase-labelled IgG from an adrenal antibody-positive control serum was added. In this test, optimal antigen concentration for coating of plates was 6.25 micrograms/ml and optimal serum dilution 1/50. The ELISA proved more sensitive and reproducible than indirect immunofluorescence. Adrenal antibodies detected by ELISA were usually of IgG class alone and only 1 of the 30 positives also contained IgM specificity. 30 out of 38 sera (79%) from patients with 'idiopathic' Addison's disease were positive whereas immunofluorescence revealed only 23 (61%) at first testing and another 4 positives when sera were tested on different adrenal glands. The ELISA described is useful for both scientific work and clinical diagnosis of autoimmune adrenalitis.