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。     

基于不同载体制备黄芪甲苷人工抗原的研究

目的:比较牛血清白蛋白(BSA)和匙孔型血蓝蛋白(KLH)两种蛋白载体合成黄芪甲苷人工抗原的免疫原性。方法:用高碘酸钠法将黄芪甲苷(AST)分别与蛋白载体BSA和KLH偶联成AST-BSA及AST-KLH免疫6~8周龄的BALB/c雌性小鼠,测定其血清抗体效价;以卵清白蛋白(OVA)为载体蛋白同样以高碘酸钠法合成AST-OVA作为包被抗原。多抗血清经间接酶联免疫吸附法(indirect enzyme-linked immunosorbent assay,iELISA)和间接竞争酶联免疫吸附法(indirect competitive enzyme-linked immunosorbent assay,icELISA)检测其抗体效价及特异性。结果:经AST-KLH免疫的多抗血清较之AST-BSA免疫的特异性要高,与AST进行竞争酶联免疫检测,其IC50值约为5μg/ml。两种人工抗原免疫所得血清抗体效价均为1∶51 200左右。结论:AST-KLH具有更好的免疫原性,本次实验为进一步建立黄芪甲苷的免疫学检测方法奠定了研究基础。     

百草枯人工抗原的合成及抗体的制备

目的: 制备百草枯人工抗原和抗血清, 为建立酶联免疫吸附法提供技术储备.方法: 以4, 4'-联吡啶和碘甲烷为起始原料, 于暗处通氮气保护下合成了百草枯半抗原; 混合酸酐法偶联大分子蛋白载体牛血清白蛋白(BSA)和卵清蛋白(OVA)制备免疫抗原和包被抗原; 免疫新西兰大白兔, 制备多抗.结果: 合成的半抗原经HPLC-MS、 1HNMR和IR鉴定, 初步确定合成成功; 百草枯半抗原与BSA和OVA的结合比分别为19∶ 1和14∶ 1; 抗血清经间接ELISA法检测, 效价达1∶ 2.56×104, 经饱和硫酸铵沉淀法纯化后抗体的效价达1∶ 5.12×104.结论: 合成的百草枯人工抗原具有较好的免疫原性, 为百草枯酶联免疫检测(ELISA)试剂盒的研制提供了基础.     

罗汉果甜苷V人工抗原免疫原性检测法的建立与评价

目的制备罗汉果甜苷V(mogroside V,MogV)-载体蛋白复合物人工抗原及抗血清,建立抗血清与MogV特异性反应的检测方法,为进一步制备MogV的单克隆抗体、建立快速检测MogV的ELISA法提供技术基础。方法将MogV与丁二酸酐、载体蛋白牛血清蛋白(BSA)和人血清蛋白(HSA)反应偶联,制得半抗原-载体蛋白复合物后,通过紫外扫描光谱计算出结合的半抗原数目;以此抗原免疫Balb/c小鼠,制备抗血清,并通过ELISA法检测效价和特异性。结果合成的人工抗原MogV-HS-BSA结合比约为44∶1,MogV-HS-HSA结合比约为64∶1;通过免疫小鼠得到特异性针对MogV的抗血清,血清效价较高。结论 MogV人工抗原有较好的免疫原性,建立的免疫原性检测方法简便可行。     

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。     

抗HCV HVR1模拟合成肽单克隆抗体的制备及特性鉴定

目的:制备抗-HCV高变区1(HVR1)合成肽单克隆抗体(mAb),并对其特性进行鉴定。方法:以固相合成的HCV HVR1合成肽与牛血清白蛋白(BSA)偶联后,免疫8周龄的BALB/c小鼠。采用杂交瘤技术,取免疫小鼠的脾细胞与骨髓瘤细胞融合,制备抗-HCV HVR1 mAb。经HAT、HT选择培养及有限稀释法进行克隆化后,用相关的试剂盒鉴定小鼠mAb的Ig亚类;经中和抑制后用ELISA法检测mAb的特异性;用间接ELISA法检测mAb腹水的效价及相对亲和常数。结果:获得1株可分泌抗-HCV HVR1合成肽特异性mAb的杂交瘤细胞1A9G9F11。该株mAb的Ig亚类为IgG3;腹水效价为3.125×10-5;相对亲和常数为1.0×106L/mol。该株mAb与HBsAg、HBeAg、HBcAg、HAAg、牛血清白蛋白、酪蛋白和胸腺5肽均无交叉反应,与HCV HVR1合成肽-牛血清白蛋白出现特异性反应。结论:成功地建立了1株可分泌抗-HCV HVR1 mAb的杂交瘤细胞1A9G9F11,为进一步的实验研究提供了重要的制剂。     

多菌灵人工抗原的合成及其鼠源性多克隆抗体的制备

目的 合成多菌灵人工抗原,制备多菌灵多克隆抗体并鉴定其特性。方法 利用混合酸酐法引入羧基制备多菌灵半抗原。将小分子半抗原与大分子载体牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成多菌灵人工抗原和包被抗原,利用聚丙烯酰胺凝胶电泳(SDS-PAGE)对人工抗原进行鉴定。用制备的人工抗原免疫小鼠获得抗多菌灵多克隆抗体,利用间接ELISA对抗体效价以及特异性进行测定。结果 成功制备多菌灵人工抗原。免疫小鼠后获得的抗体效价可达1∶12 800。抗体对多菌灵的半数抑制剂量(IC₅₀)为0.107μg/mL,且与苯菌灵、噻菌灵的交叉反应率均<1%。结论 成功获得高敏感性、高特异性的多克隆抗体,为多菌灵残留快速检测方法的建立奠定了基础。     

肠道病毒71 VP1基因的原核表达及其抗血清的制备

目的克隆并表达重组肠道病毒71(EV71)VP1基因,进行抗血清的制备并检测抗血清效价。方法利用原核表达系统将PCR获得的VP1片段构建成原核表达载体pET32a(+)-VP1,诱导其表达VP1融合蛋白并利用包涵体纯化方法进行重组蛋白的纯化,将纯化的重组蛋白免疫新西兰兔制备VP1抗血清,ELISA检测抗体效价。同时构建真核表达载体,利用其在真核细胞中的瞬时表达检测抗血清的特异性。结果通过克隆获得VP1原核表达载体,IPTG诱导VP1蛋白表达并纯化,SDS-PAGE结果显示重组蛋白表达且纯化浓度较高,将重组蛋白乳化后免疫新西兰兔3次,取血检测抗血清的效价,ELISA结果显示抗体效价为1∶64 000,利用所构建真核表达载体pcDNA3.1(+)-VP1表达VP1对抗血清进行检测,Western blot结果表明抗血清可较好的与VP1特异性结合。结论制备具有免疫原性的VP1蛋白及其效价较高的抗血清,为进一步研究抗EV71诊断方法、血清学诊断试剂盒及抗病毒疫苗的研制奠定基础。     

5-氟尿嘧啶多克隆抗体的制备和鉴定

目的制备5-氟尿嘧啶(5-FU)免疫原并制备其多克隆抗体。方法采用化学合成法对5-FU进行修饰制备5-氟尿嘧啶-1-基乙酸半抗原(5-FUAA),并经碳酰二亚胺盐法将其分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)连接以制备免疫原。采用5-FU与BSA结合的免疫原(5-FUAA-BSA)免疫BALB/c小鼠制备多克隆抗体,5-FU与OVA结合物(5-FUAA-OVA)包被酶标板经间接ELISA检测抗血清效价,并采用ELISA与Western blot法检测抗血清的特异性。结果成功合成半抗原5-FUAA,并分别制备5-FUAA-BSA和5-FUAA-OVA完全抗原。将5-FUAA-BSA免疫BALB/c小鼠制备的免疫血清效价达1∶1 280 000,且该多克隆抗体能特异地结合5-FU半抗原。结论成功制备了5-FU的多克隆抗体。     

去氢甲睾酮抗体的制备与鉴定

目的:制备兔抗去氢甲睾酮(DMT)抗体,为采用免疫法分析食品中该激素的残留提供基础。方法:采用碳二亚胺法将肟化后的DMT分别偶联到钥孔血蓝蛋白、鸡卵清蛋白和牛血清白蛋白蛋白载体上,制备完全抗原,并利用薄层层析法、红外和紫外光谱法对其进行确证后,免疫新西兰大白兔;通过间接ELISA法测定和比较2种兔抗血清的效价、IC50及交叉反应率。结果:成功制备了DMT的完全抗原,其抗体效价分别为1∶64000(KLH-DMT)和1∶256000(OVA-DMT),IC50值分别为0.24μg/L和5.29μg/L;抗血清与丙酸睾酮酯、群勃龙的IC50值均大于781.25μg/L。结论:制备的DMT完全抗原具有较高的免疫原性,抗体与类似物丙酸睾酮酯、群勃龙无明显的交叉反应,可用于ELISA实验。     

Measurement of immunoglobulin M, immunoglobulin G, and immunoglobulin A antibodies against Yersinia enterocolitica by enzyme-linked immunosorbent assay: comparison of lipopolysaccharide and whole bacterium as antigen

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of human immunoglobulin M (IgM), IgG, and IgA antibodies against Yersinia enterocolitica by using lipopolysaccharides as antigens is described. The results obtained with the lipopolysaccharide ELISA were compared with the results of the whole bacterium ELISA. The correlations observed were good for each immunoglobulin class. Cross-reactions between Y. enterocolitica serotypes O:3 and O:9, Yersinia pseudotuberculosis IA, and Brucella abortus were studied by human and rabbit antisera in the whole bacterium and lipopolysaccharide ELISAs and by rabbit antisera using ELISA inhibition. The greatest cross-reactivity observed was that of the anti-Brucella serum with Y. enterocolitica O:9 in the whole bacterium ELISA. In the lipopolysaccharide ELISA this cross-reaction was not demonstrable with the rabbit antiserum, but it was strong with the human antiserum. However, differential diagnosis was possible with ELISA inhibition. On the basis of our experience, we are now routinely using whole bacterium ELISA for the determination of class-specific Yersinia antibodies, and potential cross-reactions are controlled by the ELISA inhibition.     

Synthetic 6B di-, tri-, and tetrasaccharide-protein conjugates contain pneumococcal type 6A and 6B common and 6B-specific epitopes that elicit protective antibodies in mice

The immunogenicity and protective capacity of Streptococcus pneumoniae 6B capsular polysaccharide (PS)-derived synthetic phosphate-containing disaccharide (Rha-ribitol-P-), trisaccharide (ribitol-P-Gal-Glc-), and tetrasaccharide (Rha-ribitol-P-Gal-Glc-)-protein conjugates in rabbits and mice were studied. In rabbits, all saccharides conjugated to keyhole limpet hemocyanin (KLH) evoked high levels of pneumococcal (Pn) type 6B antibodies that facilitated type-specific phagocytosis. Unlike the disaccharide rabbit antisera, tri- and tetrasaccharide rabbit antisera also reacted with 6A PS in an enzyme-linked immunosorbent assay (ELISA) and promoted phagocytosis of 6A pneumococci. All these rabbit antisera passively protected mice against a Pn 6B challenge. The disaccharide conjugate-induced antiserum, however, failed to protect mice against a 6A challenge. In mice, phagocytic and protective anti-Pn 6B antibodies were only induced by the tetrasaccharide conjugate and not by PS 6B or PS 6B-protein conjugates. These antibodies did not cross-react with 6A PS in ELISA and were unable to phagocytize 6A pneumococci. In conclusion, the disaccharide and tetrasaccharide conjugates already contain epitopes capable of inducing 6B-specific, fully protective antibodies in rabbits and mice, respectively.     

An ELISA for the detection of antibodies to avian nephritis virus and related entero-like viruses

An ELISA for the detection of antibodies against avian nephritis virus (ANV) and related entero-like viruses was developed. Different antigenic preparations made from chicken kidney cells infected with the G-4260 strain of ANV were compared. Crude antigen obtained by fluorocarbon treatment of infected cells was found to be appropriate and to give reproducible results with antisera directed against ANV and three entero-like particles (ELPs): the Belgian entero PV2 and entero 3, and the Irish ELP-1. A cut-off value was determined using 30 specific pathogen-free (SPF) sera and the absence of antigenic relationships with nine different reference sera directed against avian viruses was demonstrated. The cross antigenic relationships between avian encephalomyelitis virus (AEV), ANV and the three ELPs was investigated by ELISA and confirms the classification of those fowl enteroviruses into two serotypes: the first including ANV and ELPs and the second including AEV. A strong correlation (94%) was demonstrated between ELISA and a seroneutralisation test. Using ELISA, antibodies against ANV and related ELPs were demonstrated in 13/14 breeding and 8/10 broiler flocks from Belgium.     

环丙沙星单克隆抗体的制备及其免疫学特性分析

目的:制备环丙沙星单克隆抗体,并分析其免疫学特性,为建立环丙沙星残留的免疫学检测方法服务。方法:用人工抗原环丙沙星-小牛血清白蛋白偶联物免疫BALB/c小鼠,产生预期免疫应答后,将小鼠脾细胞与瘤细胞融合,经初筛、复筛和再克隆,筛选得到1C9、3F6、6H2、6A7、6G11和8F56株分泌环丙沙星单克隆抗体的杂交瘤细胞。对单克隆抗体的亚型、纯度、亲和力、灵敏度及特异性等免疫学特性进行鉴定和分析。结果:1C9、3F6和6A7的抗体亚型为IgG2a;6H2和8F5的抗体亚型为IgG1;6G11的抗体亚型为IgG3。SDS-PAGE电泳结果显示单抗蛋白重链分子量约为50kD,轻链分子量约为25kD。6株细胞产生的单抗均具有良好的特异性和灵敏度。其中,细胞株1C9细胞培养上清和腹水效价分别为1∶6.4×102和1∶5.6×105,亲和力常数可达2.85×109L/mol,IC50值可达245.86ng/ml,最低检测限为45.25ng/ml,对氧氟沙星、双氟沙星、沙拉沙星、左氧氟沙星、孔雀石绿、氯霉素、呋喃西林等药物几乎不存在交叉反应,与恩诺沙星存在交叉反应,交叉反应率为84.6%。结论:本方法制备的环丙沙星单克隆抗体具有较好的亲和力和特异性,可用于环丙沙星残留免疫学检测。     

Monoclonal antibodies to guinea pig interferon-gamma: tools for cytokine detection and neutralization

We have generated polyclonal antisera and monoclonal antibodies against recombinant guinea pig IFN-gamma. These antibodies were used to inhibit the function of IFN-gamma in vitro and to establish a capture ELISA system for the detection and quantitation of this cytokine. Although recombinant protein expressed in E. coli was available in abundance, it was only of limited value to develop a capture ELISA which detects the native cytokine, since only a limited number of monoclonal antibodies reacted both with the recombinant and the native protein. Positive test results in an initial ELISA setup with recombinant IFN-gamma were not predictive for the detection of IFN-gamma from activated T-lymphocytes in the same assay. After evaluating several different combinations of rabbit antisera and monoclonal antibodies, an assay system was established which uses two mouse monoclonal antibodies as capture and detecting reagents. Three of the monoclonal antibodies and the rabbit antisera were able to block the function of guinea pig IFN-gamma when assayed in a luciferase reporter assay.     

A candidate vaccine against influenza virus intensively improved the immunogenicity of a neutralizing epitope

BACKGROUND: The hemagglutinin (HA) of influenza viruses is one of the major targets of the humoral response. The role of serum antibody to HA in the protection against infection has been demonstrated by long-standing observation. In previous studies, we suggested that an epitope vaccine might be a new strategy against the virus. METHODS: HA sequences of 491 H3 subtype strains from the influenza sequence database were compared and analyzed. To acquire information on the immunogenicity of the F3 epitope, F3-epitope-specific antibody levels in 81 patient sera infected with influenza virus were tested by ELISA. Based on the theory of the epitope vaccine, we designed an epitope peptide F3 (C-KAYSNCYPYDVPDY-G-KAYSNCYPYDVPDY), which contains the repeated F3 epitope KAYSNCYPYDVPDY (aa92-105) on HA (H3N2). The specificity and the titer of the antibodies induced by the epitope vaccine were determined by ELISA. The neutralizing activities of these anti-F3 antibodies were shown by inhibiting influenza virus infection of MDCK cells. RESULTS AND CONCLUSION: Comparison of HA sequences of 491 H3 subtype strains indicates that this epitope is highly conservative. Analysis of the sera from influenza virus-infected patients revealed a very low level of F3 epitope-specific antibodies, suggesting the poor immunogenicity of the F3 epitope on influenza virus. The epitope vaccine based on the F3 epitope induced high levels of F3 epitope-specific antibodies recognizing the epitope peptide F3 (antibody titer in antisera up to 1:25,600). Besides, the antisera could also recognize the natural HA in Western blotting. Interestingly, these antisera induced by the epitope vaccine could inhibit infection of MDCK cells by influenza virus (strain A/Wuhan/359/95) in the neutralization assay. These results suggest that the epitope vaccine can intensively increase the immunogenicity of neutralizing epitopes and may provide a new way to develop an effective vaccine against influenza virus.     

Caveats for the use of surface-adsorbed protein antigen to test the specificity of antibodies.

Rabbit antisera against apo-cytochrome c, which was prepared by removal of the covalently bound heme prosthetic group from yeast iso-1 cytochrome c, were tested for reactivity against native yeast iso-1-cytochrome c. When the antigen was adsorbed to a microtiter plate in a conventional enzyme-linked immunosorbent assay (ELISA), the antisera were unable to distinguish between their cognate antigen apo-cytochrome c, a random coil protein, and native cytochrome c, a small globular protein of remarkable conformational stability in solution. However, when the assay was conducted under conditions where antigen and antibody were free to associate in solution, that is in a solution-phase radioimmunoassay (RIA), the antisera were highly specific for apo-cytochrome c. Similarly, antibodies induced by native cytochrome c and discriminating strongly between native and apo-cytochrome c in a solution-phase RIA, did not distinguish between native and apo-cytochrome c in a solid-phase ELISA. This discrepancy of results obtained by different immuno assay procedures clearly indicates that adsorption to plastic alters the antigenic structure of even a conformationally stable protein such as cytochrome c. A conventional solid-phase ELISA strongly selects for those antibodies that recognize the unfolded antigen. The results presented warrant serious thoughts about previous reports on anti-peptide antibodies reacting with native whole protein molecules, as tested by those ELISA procedures that have the protein antigen adsorbed to plastic.     

Recombinant protein comprising multi-neutralizing epitopes induced high titer of antibodies against influenza A virus

In previous studies, we suggested that epitope-vaccine might be a new strategy against virus infection. Based on this hypothesis, we designed and expressed a recombinant immunogen (multi-epitope-peptide) comprising repeats of three neutralizing-epitopes (neutralizing epitopes: aa92-105, 127-133 and 183-195) of hemagglutininin (HA) of influenza virus (H3N2) in E. coli. After vaccination, the recombinant multi-epitope protein could induce a high level of antibodies with predefined multi-epitope-specificity in mice and rabbits. The epitope-specific antibodies in sera were tested using three different epitope-peptides (synthetic peptides) in ELISA assay, and the serum dilutions from 1 : 6400 to 1 : 25600 were confirmed. In western blot analysis, both the antiserum and the antibodies purified by synthetic epitope-peptide coupled sepharose columns could recognize natural HA from influenza virus particles (strain A/Wuhan/359/95 H3N2). In hemagglutination inhibition (HI) tests, these three antisera at the dilutions from 1 : 20 to 1 : 80 showed inhibitory activity. Interestingly, antisera and purified antibodies induced by the epitope-vaccine could partially inhibit plaque-formation of influenza virus (strain A/Wuhan/359/95) on MDCK cell monolayers. These results suggest that the recombinant multi-epitope vaccine can simultaneously induce multi-antiviral activities against influenza virus, which may provide a new way to develop effective vaccines against influenza virus.     

Immunogenicity of free histones and of histones complexed with RNA

Histone antibodies have been obtained by immunizing rabbits with histones H1, H2A, H2B, H3, H4 and triacetylated H4, uncomplexed to RNA. The reactivity of these antibodies was investigated by ELISA using as antigen isolated histones and chromatin as well as thirty-five different synthetic peptides covering the entire sequence of the four core histones, two peptides of H1 and two acetylated peptides of H4. The binding of these antibodies to histones was also measured in immunoblotting and in microcomplement fixation (MCF) tests. In parallel experiments using the same assays the various antigens were tested with antisera raised against histones complexed with RNA. Antibodies induced in the absence of RNA did not react with histones in MCF tests nor with chromatin in ELISA but reacted with the histones in ELISA, although the antibody titers were somewhat lower than in the case of antisera to histone-RNA complexes. Antibodies to RNA-histone complexes reacted with histones in both ELISA and MCF tests. When they were tested with peptide-coated microtiter plates in a direct binding ELISA format, antibodies induced with uncomplexed histones recognized very few fragments which were mainly located in the N- and C-terminal ends of the histones.     

An indirect sandwich ELISA utilising F(ab')2 fragments for the detection of African horsesickness virus

African horsesickness virus (AHSV), an important disease of equines is caused by an orbivirus. Because of the need to contain the spread of the disease, it is often essential to make a rapid diagnosis. For this purpose, an ELISA capable of detecting viral antigen in animal tissue and in cell culture fluid was developed. Immobilised F(ab')2 fragments prepared by digestion of AHSV-specific IgG with pepsin were used to trap virus from tissue homogenates or cell culture supernatant. After addition of intact IgG as detecting antibody, Staphylococcus aureus protein A labelled with horseradish peroxidase was added to allow visualisation of the reaction. Polyclonal antibodies directed against either whole AHSV or viral core particles were suitable as detecting antibodies. On the other hand, a monoclonal antibody that was specific for a major core protein, VP7, gave a much weaker signal in the ELISA. All known AHSV serotypes were recognised in the F(ab')2-ELISA by polyclonal antisera against either whole virus particles or viral cores. Immunoprecipitation of AHSV structural polypeptides showed that such antisera contained populations of antibodies directed against core proteins. The F(ab')2-ELISA has potential as a diagnostic technique for AHSV infections.