抗体捕获ELISA检测包虫病人特异性IgM抗体及其应用的研究

用标记抗原、标记抗体、标记抗抗体及ABC四种形式的抗体捕获ELISA法检测包虫病人血清中特异性IgM抗体,结果以ABC-捕获ELISA法阳性率最高为52.5％,标记抗抗体法次之为39.6％,标记抗体法为22.5％,标记抗原法阳性率最低为20.7％。四种方法检测健康人血清均为阴性。手术前的包虫病人血清中特异性IgM抗体的阳性率明显高于手术后者,分别为52,2％和20％。不同寄生部位包虫病人特异性IgM抗体的阳性率没有明显差别。在入院就诊的病人中阳性率为35.4％,而在普查中发现的无症状病人中阳性率达70.5％,说明特异性IgM的检测在包虫病中也具有早期诊断的意义。     

双烯雌酚人工抗原的合成及特异性抗体的制备

目的制备抗双烯雌酚(DIEN)特异性抗体,为进一步研制DIEN免疫检测试剂盒打基础。方法采用4-溴丁酸乙酯对DIEN进行活化,以活泼酯法与BSA偶联制备免疫原(DIEN-CP-BSA);经紫外扫描和飞行时间质谱扫描鉴定偶联情况;以DIEN-CP-BSA免疫Balb/c小鼠制备特异性抗体,间接(竞争)ELISA评价抗血清效价及特异性。结果本试验获得较高效价的DIEN抗血清,其效价达1∶64 000,双烯雌酚半抑制浓度(IC50)为62 ng/ml;抗血清与结构类似物己烷雌酚的交叉反应率仅为0.34%,与己烯雌酚和17-β雌二醇无交叉反应;利用该抗体建立的间接竞争ELISA检测法,双烯雌酚在10～300 ng/ml呈线性关系。结论本研究制备了抗双烯雌酚(DIEN)特异性抗体,为研究畜产品中DIEN残留及开发DIEN免疫检测试剂盒奠定了基础。     

改变半抗原与载体间隔臂的长度对环丙沙星抗体特性的影响

目的在用乙二胺(ethanediamine,EDA)化牛血清白蛋白(BSA)为载体合成环丙沙星(ciprofloxacin,CIP)免疫原已制备了高特异性抗体(IC50达1 ng/ml)的基础上,尝试延长间隔臂长度,以己二胺(hexamethylenediamine,HMD)进一步修饰BSA合成免疫原制备抗血清,探索其对环丙沙星抗体灵敏度及特异性的影响。方法以己二胺修饰BSA,合成环丙沙星免疫原,免疫Balb/c小鼠制备多克隆抗体,同时以天然、乙二胺及己二胺化鸡卵清白蛋白(OVA)为载体合成包被原CIP-OVA、CIP-EDA-OVA和CIP-HMD-OVA,用间接竞争酶联免疫检测方法(ic-ELISA)对抗血清进行评价。结果获得了更高特异性的环丙沙星抗血清,IC50达0.1 ng/ml,抗体的交叉反应性也明显降低,且与CIP-EDA-OVA、CIP-HMD-OVA包被原相比,以CIP-OVA为包被原可显著提高环丙沙星ELISA灵敏度。结论改变抗原间隔臂长度对特异性抗体的产生有较大影响,且改变包被原间隔臂的结构可显著提高ELISA的灵敏度。     

双特异性单链抗体

双特异性单链抗体是新型基因工程抗体，在疾病诊断，治疗上有巨大临床应用潜力。目前已有较多各种双特异性单链抗体成功构建的报道。本文对其概念、制作方法及应用前景进行了综述。     

HAV噬菌体抗体特异性的鉴定

ＨＡＶ噬菌体抗体特异性的鉴定万泽生王海涛姜绍谆近年出现的噬菌体抗体库技术，使人们能够不经过细胞融合，采用基因工程的方法，从人外周血淋巴细胞ＩｇｍＲＮＡ直接制备抗体。我们利用这一技术，已成功地构建了噬菌体抗体库〔１〕。本文报道从库中筛选具有抗甲肝病毒（...     

前列腺特异性抗原EIA试剂盒的研制及应用

目的 建立可定量测定人血清中前列腺特异性抗原（ＰＳＡ）含量的夹心ＥＬＩＳＡ法,研制ＰＳＡ－ＥＩＡ检测试剂盒。方法 从健康男性精液中提取并纯化ＰＳＡ,分别免疫Ｂａｌｂ／ｃ小鼠和山羊制备特异性单克隆抗体和多克隆抗体,并以纯化的ＰＳＡ为标准品,建立可定量测定血清中ＰＳＡ含量的夹心ＥＬＩＳＡ法。在此基础上组装ＰＳＡ－ＥＩＡ试剂盒,对该试剂盒的特异性、灵敏度、精密度、正确性和稳定性等多项指标进行评价。应用该     

化学偶联双特异性抗体的制备及鉴定

用化学偶联的方法,制备了抗鼠IgM-抗破伤风类毒素双特异性抗体复合物,以此作为研究B细胞抗原递呈作用的工具及模拟抗原特异性B细胞的高效抗原递呈作用。经ELISA鉴定以及通过ELISA竞争抑制试验和中和抑制试验证实,此抗体复合物确有识别两种不同抗原的双特异性。     

20例SARS患者特异性抗体变化规律

目的：了解严重急性呼吸道综合征(SARS)特异性IgM和IgG抗体的变化规律。方法：采用间接酶联免疫吸附试验(ELISA)检测20例SARS患者系列血清中特异性IgM和IgG抗体，系列血清包括患者发病后1周，2周，3周，4周，8周，12周所采集的样本。结果：20例患者发病后第1周IgM和IgM抗体均为阴性；第2周时16例IgM抗体阳性，17例IgM抗体阳性；第3周后所有患者IgG抗体阳性并持续至第12周，而IgM抗体阳性患者逐渐减少，至第12周时所有患者均为阴性。结论：SARS特异性IgG抗体消失较早，其存在是近期感染的标志；IgG抗体的持续存在可能是获得病后免疫力的标志。     

双特异性抗体的制备和应用

双特异特抗具有两个不同的抗原结合，可同时识别连接两种细胞或结构而将效应细胞或其偶连的药物，酶、放射性核素等富集到靶部位，目前可通过杂交－杂交瘤技术，化学偶连和基因工程方法制备ＢｓＡｂ，应用于肿瘤的免疫诊断与治疗，激活化疗原药，定向溶栓等方面，能够提高特异性，减小副作用。     

Development of polyclonal antibodies against Fusarium verticillioides exoantigens

Exoantigens from Fusarium verticillioides strains were analysed by polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and Western blot and were used for production of polyclonal antibodies. Rabbits were immunized with exoantigens from strains 97K, 162A and 113F. Polyclonal antibodies against F. verticillioides 97K exoantigen showed the highest titre in ELISA (1:12.800) and were used in further studies. The cross-reactivity of anti-97K antibodies with 14 species from 7 other fungal genera was low, ranging from 4.5-14.7%; the highest reactivities (51.2-76.0%) were observed in Fusarium genera. The detection limit of exoantigen by indirect competitive ELISA was 0.27 µg/ml. Western blot analysis showed two immunodominant antigens (67 kDa and 113 kDa) common to all the F. verticillioides strains analysed. The results suggest that polyclonal antibodies raised against exoantigens from F. verticillioides 97K are promising for Fusarium sp. immunodetection.     

Development of sensitive indirect enzyme-linked immunosorbent assays for specific detection of antibodies against fowl adenovirus serotypes 1 and 4 in chickens

Conventional serological methods for detection and differentiation of antibodies against fowl aviadenoviruses (FAdVs) are laborious and time-consuming, therefore ELISAs based upon recombinant proteins were developed in the present study to overcome this limitation for clinically relevant serotypes FAdV-1 and FAdV-4. In order to develop serotype-specific ELISAs, the two distinct fibers, fiber-1 (fib-1) and fiber-2 (fib-2), characteristically present only in FAdV-1 and FAdV-4, were applied separately as coating antigens. Sera raised against each recombinant fib-1 and fib-2 of FAdV-1 and FAdV-4 did not react with any of the heterologous fiber ELISAs, as anticipated by the low degree of amino acid identity between those FAdV fibers (23.1–41.2%), indicating that heterologous fibers do not share common epitopes. Testing of 172 monospecific sera, raised against all FAdV serotypes (1–8a and 8b–11), retrieved specificities between 99.3% and 100.0% for the ELISAs, further substantiating the serotype-specificity of fibers. Investigating sera from chickens experimentally inoculated with different FAdV-1 or FAdV-4 strains revealed that ELISAs were equally or more sensitive than the virus-neutralization (VN) test. Furthermore, strong correlations were demonstrated between fiber antibody titres and neutralization activity. Particularly, sera directed against live virus showed a pronounced fiber antibody response, which might be explained by an excessive production of fibers during infection. Application of the newly developed fiber ELISAs on field sera with heterogeneous serological status demonstrated high sensitivity and serotype-specificity of this test system, providing for the first time a diagnostic tool for mass screening of chicken flocks against FAdV serotypes, namely FAdV-1 and FAdV-4.     

Competitive ELISA employing monoclonal antibodies specific for dothistromin

Dothistromin (DOTH) is a fungal toxin occurring in Pinus radiata needles contaminated with Dothistroma pini. Monoclonal antibodies (MAbs) of high affinity were prepared against DOTH and incorporated into competitive ELISAs. MAbs were secreted by hybri‐doma prepared from mice immunized with DOTH conjugated, through the aromatic ring of the anthraquinone, to bovine serum albumin. DOTH could be quantitated at 2–60 ng ml^‐1 (0.1–2 ng/assay) and at 8–300 ng ml^‐1 (0.4–15 ng/assay) using peroxidase‐labelled MAb 10C12A5 and MAb 6A2D4 respectively in indirect competitive ELISA. The corresponding limits of detection of DOTH were < 300 pg and 1 ng ml^‐1 respectively. The furan ring was an important structure in the epitope recognized by both MAbs.     

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。     

雪卡毒素单克隆抗体的制备及鉴定

目的制备雪卡毒素的单克隆抗体并对其生物学特性进行鉴定。方法以雪卡毒素类似物莫能菌素为半抗原,分别采用混合酸酐法将其与载体牛血清白蛋白(BSA)偶联合成免疫抗原M-BSA,碳二亚胺法与卵清白蛋白(OVA)偶联合成包被抗原M-OVA。以M-BSA免疫Balb/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合制备杂交瘤。以自制雪卡毒素提取物为竞争抗原,间接竞争ELISA法筛选稳定分泌雪卡毒素抗体的特异性细胞株。小鼠体内诱生腹水,辛酸硫酸铵沉淀法进行纯化。采用抗体亚型鉴定试剂盒鉴定所得抗体的亚型,间接竞争ELISA法鉴定抗体的特异性和交叉性。结果获得3株稳定分泌抗雪卡毒素抗体的杂交瘤细胞株1D5、2G11、3G11,其Ig亚型均属于IgG1亚型。交叉反应结果表明3株单克隆细胞产生的抗体除与莫能菌素有较高的交叉反应外,与其他雪卡毒素类似物均无明显交叉反应。结论成功筛选到了分泌雪卡毒素抗体的细胞株,为雪卡毒素免疫分析方法的进一步研究奠定基础。     

Competitive ELISA employing monoclonal antibodies specific for coronafacoyl amino acid conjugates

Coronatine (COR) is composed of two structural components, coronafacic acid (CFA) and the amino acid coronamic acid (CMA), linked by an amide bond. Monoclonal antibodies (MAbs) were prepared against COR and incorporated into competitive ELISAs. MAbs were secreted by hybridoma cells which had been prepared from mice immunized with COR and conjugated, through the free carboxyl group on CMA, to ovalbumin, the available amino groups of which had been increased by derivatization with propyl diamine via free carboxyl groups. COR and coronafacoyl valine could be quantified at 6.800 ng ml^‐1(0.34–40 ng/assay) using peroxidase‐labelled MAb 8H3G2 in an indirect competitive ELISA. The corresponding limit of detection was 1 ng ml～’. Reduction and subsequent acetylation of the ketone oxygen of the CFA moiety of COR reduced the affinity of MAb 8H3G2 some 250 times, whereas methylation of the free carboxyl group on COR slightly enhanced the affinity four‐fold. These and other results suggest that CFA and the amide bond are important structural features of the epitope recognized by MAb 8H3G2.     

Development of antibodies against the fluoroquinolone sarafloxacin and molecular modeling studies of cross‐reactive compounds

Polyclonal antibodies were prepared against the fluoroquinolone sarafloxacin. Sarafloxacin was conjugated directly to cationized bovine serum albumin (BSA) and ovalbumin. Balb/c mice were immunized with the sarafloxacin‐BSA conjugate (cBSA‐saraflox) and sarafloxacin‐reactive sera (1–5) were obtained from these mice. Serum from mouse 1 (Abl) exhibited the lowest IC₅₀ for free sarafloxacin using an indirect competitive inhibition ELISA (ci‐ELISA). Other structurally related quinolones, including difloxacin, enrofloxacin, norfloxacin, trovafloxacin and nalidixic acid, demonstrated cross‐reactivity with the sarafloxacin antibodies as determined by ci‐ELISA. In an effort to correlate antibody binding with three‐dimensional properties of the cross‐reactive compounds, all of the fluoroquinolones as well as nalidixic acid were modeled, and global energy minima were determined using molecular mechanical and quantum mechanical methods. The results demonstrate that the three‐dimensional models can yield information that explains observed cross‐reactivity data. These models are particularly helpful when the chemical structure of an analog varies greatly from the immunogen yet the IC₅₀ value for the compound is not vastly different. Furthermore, conformational and electronic data from this study can be used to predict whether other fluoroquinolones will exhibit good cross‐reactivity in this ELISA.     

人幽门螺杆菌尿素酶抗体诊断试剂盒的研制

幽门螺杆菌 (Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ,Ｈ .ｐｙｌｏｒｉ)为慢性胃炎、消化性胃溃疡的致病菌 ,并且与胃癌的发生密切相关 ,世界卫生组织 (ＷＨＯ)将其列为一级致癌因子 ,早期诊断Ｈ .ｐｙｌｏｒｉ感染并对其治疗和预后的观察具有重要意义。目前 ,临床上用于诊断Ｈ .ｐｙｌｏｒｉ感染的方法主要有胃镜下组织活检、细菌培养、13 Ｃ呼气试验等 ,前者需活检组织 ,病人痛苦大 ,且阳性率低 ,后者需特殊性设备 ,不易推广 ,细菌培养则阳性检出率低 ,而血清学诊断具有灵敏、快速、准确、简便、费用低等优点 ,易在临床上广泛使用。但是 ,…     

Immunologic tests of specific antibodies to organic acid anhydrides

The outcome of immunologic tests of antibodies directed against hapten conjugates of organic acid anhydrides and human serum albumin (HSA) has been studied in workers exposed to phthalic anhydride (PA), methyltetrahydrophthalic anhydride (MTHPA), hexahydrophthalic anhydride (HHPA), and methylhexahydrophthalic anhydride (MHHPA). HSA conjugates of PA, MTHPA, HHPA, MHHPA, and maleic anhydride (MA) have been prepared and used in the tests. The hapten densities (HD) of the conjugates were varied by different molar ratios of hapten and macromolecule in the preparative procedure. Skin prick reactions to MTHPA-HSA increased with rising HD over the range 6-13 mol/mol. The achieved HD was tested by spectrometric and gas chromatographic methods. In RAST of IgE antibodies MTHPA-HSA with HD six and 25 showed significantly lower bindings than conjugates with intermediate HD. There was a good correlation between skin prick tests and RAST. Of 234 workers tested [MTHPA (n = 145), and HHPA (n = 89)], 45 had a skin prick reaction greater than or equal to 50% of the histamine reaction (1 mg/ml). All but two of these were RAST positive (RAST value greater than 0.3%; 0.3% upper range in 147 controls; MTHPA, n = 63; HHPA, n = 84). Nine RAST positive workers had no obvious skin prick reaction. However, their RAST values were low (less than 0.8%). In exposed workers, the ELISA value of specific IgG antibodies to MTHPA-HSA showed optimal values when tested with the HD 13 conjugate.(ABSTRACT TRUNCATED AT 250 WORDS)