抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

脱落酸人工抗原合成及多克隆抗体制备

目的:为了对植物细胞中的脱落酸(ABA)进行定量和定位分析,研究了脱落酸人工抗原的合成以及多克隆抗体的制备。方法:用重氮化法将ABA分别与牛血清白蛋白(BSA)、卵清白蛋白(OVA)联结,得到ABA的免疫抗原和包被抗原,并采用紫外全波长扫描和SDS-PAGE对合成的抗原进行了鉴定。以经过鉴定的抗原免疫白兔,制备出ABA的多克隆抗体;采用间接酶联免疫法(ELISA)对抗血清进行效价检测,通过离子交换层析法获得纯化的抗体。结果:ABA与BSA的平均偶联比为5.3∶1,抗血清效价为1∶16000。结论:人工抗原和多克隆抗体制备成功,为采用ELISA和免疫胶体金技术(ICG)检测ABA提供了有效工具。     

羊口疮病毒119蛋白表达、纯化和多克隆抗体的制备

[目的]克隆表达羊口疮病毒ORFV119蛋白,以纯化重组蛋白为免疫原制备鼠多克隆抗体,并鉴定抗体的特性。[方法]PCR扩增ORFV119基因,克隆入原核表达载体p ET-28a(+)中构建重组质粒p ET28a-119。经双酶切和测序正确后,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定融合蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,之后目的蛋白免疫BALB/c小鼠,制备ORFV119多克隆抗体并对其通过中和实验进行鉴定。[结果]成功构建重组质粒p ET28a-119,在大肠杆菌BL21中ORFV119融合蛋白以部分可溶形式表达。可溶性目的蛋白纯化后作为免疫原制备鼠多克隆抗体,抗体效价达1∶12 800,中和实验显示该多抗具有保护作用,可减轻宿主细胞在病毒感染时的病变效应(中和效价66 ND50/m L)。[结论]成功诱导表达、纯化ORFV119蛋白并制备其多克隆抗体,为深入研究ORFV感染、发病机理及羊口疮疾病的临床诊断奠定基础。     

新型冠状病毒S蛋白表达、纯化及应用

[目的]克隆严重急性呼吸综合征冠状病毒2(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)刺突糖蛋白(Spike,S蛋白),纯化重组蛋白为制备鼠多克隆抗体提供免疫原,并鉴定抗体活性。[方法]依据对S蛋白的氨基酸序列解析,设计并构建了S蛋白重组质粒S713 pcDNA3.1(+)-C-6His,经酶切和测序正确后,转入中国仓鼠卵巢细胞(CHO-K1)中进行诱导表达,SDS-PAGE鉴定融合蛋白表达情况。表达产物利用Ni柱和分子筛纯化,目的蛋白混合免疫佐剂AS03免疫ICR小鼠,制备S蛋白多克隆抗体并对通过中和实验进行鉴定。[结果]成功构建重组质粒S713 pcDNA3.1(+)-C-6His,在CHO-K1细胞中S蛋白以可溶性形式表达。可溶性目的蛋白纯化后,蛋白纯度为94.4%,作为免疫原制备鼠多克隆抗体,抗体效价为1∶400,中和实验显示该多抗具有保护作用。[结论]成功诱导表达、纯化重组S蛋白并制备其多克隆抗体,为深入研究新冠病毒感染、发病机制及新型肺炎的防治奠定基础。     

DAS-ELISA法快速检测食品中副溶血弧菌TDH毒素

建立副溶血弧菌TDH毒素DAS-ELISA快速检测法,并运用于食品样品实际检测。以TDH+副溶血弧菌菌体及其TDH毒素制备免疫原,分别免疫新西兰大白兔和BALB/c小鼠,制备兔抗副溶血性弧菌多克隆抗体和鼠抗TDH毒素多克隆抗体,以鼠抗TDH毒素多克隆抗体为捕获抗体,兔抗副溶血弧菌多克隆抗体为检测抗体,建立DAS-ELISA检测法。采用间接ELISA法测定抗体效价,采用棋盘滴定法选择最佳的抗原抗体反应条件,并确定DAS-ELISA各反应条件。结果表明:鼠抗TDH多克隆抗体、兔抗副溶血弧菌多克隆抗体与TDH毒素抗原的最佳工作浓度依次为1:8 000、1:4 000和12μg/m L,酶标二抗最佳工作浓度为1:1 000,检测总时间为5 h,该法具有很强的特异性,对食品中TDH毒素的检测低限为60μg/g。与国标检测方法相比,具有检测时间短,准确度高的优点,将会得到广泛运用。     

多胺调节因子-1原核表达与多克隆抗体制备

[目的]原核表达和纯化人多胺调节因子-1(PMF-1)并以此为抗原制备多克隆抗体。[方法]PCR扩增人PMF-1编码序列并构建原核表达质粒。将此质粒转化大肠杆菌后,IPTG诱导PMF-1蛋白表达并在变性条件下用NiNTA树脂纯化。将纯化的PMF-1用作抗原免疫大耳白兔以制备抗PMF-1多克隆抗体。ELISA法检测抗体效价,免疫印迹法和细胞免疫化学法分析抗体特异性。[结果]成功构建PMF-1原核表达质粒,在大肠杆菌中IPTG可诱导该质粒高效表达PMF-1蛋白。以纯化的PMF-1蛋白为抗原免疫大耳白兔后,获得高效价的抗PMF-1抗血清(抗体效价为1∶128 000)。该抗体能与PMF-1蛋白特异性结合,并可用于PMF-1的免疫印迹和细胞免疫化学分析。[结论]成功建立了人PMF-1原核表达和纯化技术,并制备出高特异性的抗PMF-1多克隆抗体,该抗体可用于PMF-1的免疫印迹分析和细胞免疫化学分析。     

Dhori病毒核蛋白基因的重组表达及其抗体制备

[目的]表达和纯化Dhori病毒(DHOV)核蛋白(NP),并制备多克隆抗体。[方法]RT-PCR扩增NP基因,并克隆至原核表达载体pET-28a和真核表达载体pcDNA3.1中。将重组质粒pET-28a-NP转化至大肠杆菌BL21中诱导表达,SDS-PAGE分析重组蛋白NP(rNP)的表达并亲和层析纯化rNP,以抗DHOV阳性羊血清检测其抗原性,免疫新西兰兔制备抗血清,以ELISA法检测其效价。将pcDNA3.1-NP转染至Vero细胞,以间接免疫荧光法评估抗体的结合活性,Western Blotting检测抗体与重组蛋白的特异性反应能力。[结果]pET-28a-NP和pcDNA3.1-NP重组质粒构建正确,原核表达的rNP约为55.3 kDa,并能被阳性羊血清识别,制备的抗体效价为1:409 600,能特异性识别真核及原核表达产物。[结论]成功表达和纯化rNP,并制备了多克隆抗体,可用于深入研究DHOV核蛋白的生物学特性及其检测试剂。     

Carboxin抗性基因(Cbx~R)原核表达及多克隆抗体的制备

[目的]克隆CbxR基因,进行原核表达,纯化CbxR基因蛋白并制备其多克隆抗体。[方法]CbxR基因克隆至原核表达载体p ET-28a(+),转化大肠杆菌BL21(DE3)诱导表达并纯化,Western blot鉴定分析。制备CbxR蛋白的兔源多克隆抗体,运用间接ELISA方法检测多抗效价,利用Western blot印记检测抗体特异性。[结果]成功获得CbxR基因,在大肠杆菌BL21(DE3)中诱导可大量表达,成功制备CbxR蛋白兔源多克隆抗体,效价为1∶64 000,经Western blot检测表明多克隆抗体特异性良好。[结论]多克隆抗体为CbxR检测及进一步研究CbxR基因功能奠定了基础。     

除草剂2,4-D特异性多克隆抗体的制备及鉴定

目的是制备特异性的2,4-D多克隆抗体.用活性酯和混合酸酐法将半抗原2,4-D分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)共价偶联;以2,4-D-BSA作免疫原免疫两只新西兰白兔;以2,4-D-OVA包被96孔酶标板,用间接酶联免疫吸附分析(ELISA)法检测抗血清效价和竞争性ELISA法检测2,4-D.结果显示2,4-D与BSA和OVA的偶联比率分别为32∶1和18∶1,抗血清效价＞6.4×105,在优化条件下2,4-D的最低检测限达37ng/mL.实验成功地制备了2,4-D特异性多克隆抗体,为其ELISA方法的建立提供了条件.     

沙丁胺醇人工抗原的合成及抗体制备

沙丁胺醇是一种β-兴奋剂,常被很多畜禽水产养殖户非法用于动物养殖。为建立沙丁胺醇在食品中残留的快速检测方法,研究了沙丁胺醇免疫原的合成和抗体的制备方法。采用对氨基苯甲酸法合成了沙丁胺醇（SAL）免疫原SAL-cBSA,采用重氮化法合成的克伦特罗（CL）偶合物CL-cOVA作为包被抗原,用紫外光谱法分析了所合成免疫原和包被抗原。用免疫原SAL-cBSA免疫新西兰大白兔获得多克隆抗体,抗体效价达到32000。采用间接ELISA法检测抗体IC50值为8.79ng/ml,SAL的浓度在1ng/ml~100ng/ml区间时,SAL与对抗体的竞争结合力呈直线关系。表明所制备的沙丁胺醇免疫原具有良好的免疫原性,所制备的抗体拥有很高的灵敏度。     

Effect of heterologous paralytic shellfish poisoning toxin-enzyme conjugates on the cross-reactivity of a saxitoxin enzyme immunoassay

A polyclonal antiserum against saxitoxin (STX) was used in a competitive enzyme immunoassay for the detection of paralytic shellfish poisoning toxins. The extent of cross-reactions was determined from the amounts of neoSTX, decarbamoylSTX and gonyautoxin 2/3 (GTX2/3) that gave 50% inhibition in the assay. Horseradish peroxidase (HRP) conjugates of the toxins and a bovine serum albumin conjugate of STX (STX-BSA) were used. When compared with STX-BSA and STX as standard, the extent varied to which heterologous conjugates affected the binding values of the other toxins to the antibodies. The antibodies did not bind GTX2/3-HRP. By use of neoSTX-HRP or decarbamoylSTX-HRP as the labelled antigen instead of STX-HRP, the detection limit for neoSTX was improved to 100 pg ml^-1.     

In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy

Histoplasma capsulatum contains multiple antigens, among them the H antigen and M antigen, which are useful in serologic testing for histoplasmosis. We prepared 7 mouse monoclonal antibodies (5 IgG, 2 IgM) to histoplasmin, and compared these with polyclonal histoplasmin antibodies raised in rabbits and mice. Both monoclonal and polyclonal antibodies were high titered by ELISA. Colloidal gold immune electron microscopy (CGIEM) showed that polyclonal antibodies to histoplasmin or H antigen bound at multiple sites in the cell wall, cytoplasm, and nucleus of Histoplasma yeast cells. In contrast, antibodies to M antigen selectively label the cell membrane and antibodies to alkali soluble cell wall antigen label only the cell wall. Polyclonal antibodies cross reacted extensively with other fungi, both by ELISA and CGIEM. Monoclonal antibodies stained only cytoplasmic epitopes, but also cross reacted with other fungi by electron microscopy. Only periodate treated H antigen elicited polyclonal antibodies which were more specific than those of untreated H antigen or histoplasmin.     

Monoclonal antibody immunocytochemistry: novel method extending usefulness of monoclonal antibodies for antigen visualization

We describe a novel procedure combining the multiple-site reactivity of polyclonal antibodies with the defined single epitope-specificity of monoclonal antibodies. The method is based on previous findings that IgG molecules often only react with tissue-bound antigens with one of their two antigen-combining sites; thus, the remaining site is free to bind subsequently added antigen. In the procedure devised, such (undenatured) antigen is subsequently detected by a specific monoclonal antibody and the reaction is finally revealed by immunogold-silver staining. Antibody subpopulations to contaminating antigens may well be present in the polyclonal antiserum and may well bind first to tissue and then to the corresponding contaminants in the crude antigen preparation applied as second layer. Such contaminants will, however, not react with the monoclonal antibody and will therefore not be immunocytochemically detected. The method has been evaluated with one antigen which cannot be detected by monoclonal antibodies in paraffin sections (glial fibrillar acidic protein) and with another antigen (human chorionic gonadotropin) which can only be detected by the monoclonal antibody when occurring in high concentrations. In both cases the procedure resulted in strong specific staining of the antigens with no background.     

Detection of polyclonal antibody against any area of the protein-antigen using immobilized protein-antigens: the critical role of the immobilization protocol

Antigens immobilized on solid supports may be used to detect or purify their corresponding antibodies (Ab) from serum. Direct immobilization of antigens on support surfaces (through short spacer arms) may promote interesting stabilizing effects on the immobilized antigen. However, the proximity of the support may prevent the interaction of some fractions of polyclonal Ab with some regions of the antigen (those placed in close contact with the support surface). Horseradish peroxidase (HRP) was immobilized on agarose by different protocols of multipoint covalent immobilization involving different regions of the antigen surface. Glyoxyl-agarose, BrCN-agarose, and glutaraldehyde-agarose were used as activated supports. Each HRP-immobilized preparation was much more stable than the soluble enzyme, but it was only able to adsorb up to 60-70% of a mixture of polyclonal anti-HRP antibodies. On the other hand, HRP was also immobilized on agarose through a very long, flexible, and hydrophilic spacer arm (dextran). This immobilized HRP was hardly stabilized, but it was able to adsorb 100% of the polyclonal anti-HRP. The absence of steric hindrances seems to play a critical role favoring the complete recognition of all classes of polyclonal Ab. Another solution to achieve a complete adsorption of polyclonal Ab on immobilized-stabilized antigens has been also reached by using a mixture of the differently immobilized and stabilized HRP-agarose preparations. In this case, an improved storage and operational stabilities of the immobilized antigens can be combined with the complete adsorption of any class of antibody.     

重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。     

Human carcinoma-associated and salivary mucins detected by anti-bovine submaxillary mucin antibodies

Polyclonal rabbit anti-bovine submaxillary mucin antibodies, anti-BSM IgG, were analyzed by autoradiography and densitometry (after SDS electrophoresis and blotting), ELISA, and IRMA assays for reactivity against native antigen BSM, deglycosylated and desialylated BSM, and human salivary and carcinoma-associated mucins. Known human tumor marker CA19-9 antigen reacted with rabbit anti-BSM IgG under different conditions, either soluble or immobilized. As soluble antigen, in IRMA it reacted with anti-BSM antibodies used both as a catcher and a tracer, as well as in combination with monoclonal anti-CA19-9 antibodies as a catcher. MUC1 mucin is a carrier of this carbohydrate antigen, CA19-9, or sialyl-Lewis(a), as well as of CA15-3 antigen, a known breast tumor marker. Autoradiography and densitometry demonstrated binding of anti-BSM IgG to intact MUC1 in a sample of commercial standard preparation of CA19-9 antigen. The same method and analysis demonstrated binding of anti-BSM IgG to MUC1 and to smaller antigens of 85-120 kD in samples containing CA15-3 antigen: commercial standard preparation, human breast tissue, and human milk. In a sample of whole human saliva, reactions of both isoforms of MUC7 were detected by autoradiography, as bands of 85 and 115 kD, and densitogram analysis also demonstrated reaction with MUC5B. Chemical modifications performed as periodate oxidation and desialylation of the BSM demonstrated carbohydrate (i.e., sialic acid) epitope sensitivity for anti-BSM IgG. The results presented in this work indicate that polyclonal anti-BSM antibodies are specific for sialylated carbohydrate structure on mucins and could serve as a tool for investigation of human carcinoma-associated and salivary mucins.     

Human carcinoma-associated and salivary mucins detected by anti-bovine submaxillary mucin antibodies

Polyclonal rabbit anti-bovine submaxillary mucin antibodies, anti-BSM IgG, were analyzed by autoradiography and densitometry (after SDS electrophoresis and blotting), ELISA, and IRMA assays for reactivity against native antigen BSM, deglycosylated and desialylated BSM, and human salivary and carcinoma-associated mucins. Known human tumor marker CA19-9 antigen reacted with rabbit anti-BSM IgG under different conditions, either soluble or immobilized. As soluble antigen, in IRMA it reacted with anti-BSM antibodies used both as a catcher and a tracer, as well as in combination with monoclonal anti-CA19-9 antibodies as a catcher. MUC1 mucin is a carrier of this carbohydrate antigen, CA19-9, or sialyl-Lewis^a, as well as of CA15-3 antigen, a known breast tumor marker. Autoradiography and densitometry demonstrated binding of anti-BSM IgG to intact MUC1 in a sample of commercial standard preparation of CA19-9 antigen. The same method and analysis demonstrated binding of anti-BSM IgG to MUC1 and to smaller antigens of 85–120 kD in samples containing CA15-3 antigen: commercial standard preparation, human breast tissue, and human milk. In a sample of whole human saliva, reactions of both isoforms of MUC7 were detected by autoradiography, as bands of 85 and 115 kD, and densitogram analysis also demonstrated reaction with MUC5B. Chemical modifications performed as periodate oxidation and desialylation of the BSM demonstrated carbohydrate (i.e., sialic acid) epitope sensitivity for anti-BSM IgG. The results presented in this work indicate that polyclonal anti-BSM antibodies are specific for sialylated carbohydrate structure on mucins and could serve as a tool for investigation of human carcinoma-associated and salivary mucins.     

Demonstration of membrane association and surface location of Mycoplasma pneumoniae antigens using monoclonal antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.     

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens

A method is described for obtaining from plants partially purified preparations of mycoplasma-like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC-labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross-reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.     

Toxoplasma gondii: characterization of monoclonal antibodies that recognize rhoptries

We have previously reported on a series of monoclonal antibodies that recognize the rhoptries of Toxoplasma gondii and that interfere with the action of penetration enhancing factor. The antibodies immunoprecipitate several related antigens from [35S]methionine-labeled parasites that range in size from 60 to 43 kDa. By immunoblot, one of the antibodies reacts with the 60 kDa protein in the presence of protease inhibitors. Trypsin digestion of the antigen destroyed antigenic reactivity indicating that the 60 kDa antigen is a protein. The antigen was stable to periodate oxidation and failed to react with Schiff's reagent, indicating that the antigen contains little or no carbohydrate. Two-dimensional gel electrophoresis followed by immunoblot showed that the antigen recognized by Tg 49 was an acidic protein with an approximate pI of 5.8.