Analysis of monoclonal antibodies reacting with molecules expressed on gammadelta T-cells.

Twenty-six monoclonal antibodies (mAbs) selected after the first round of analysis in the Third International Swine Workshop were grouped with additional mAbs from the first and second workshops and mAbs under study for further evaluation. Preparations of peripheral blood leukocytes were used in single and multicolor flow cytometric (FC) analyses. Six mAbs did not react with gammadelta T-cells. Two were negative for all tested specificities. Seven mAbs recognized molecules expressed on gammadelta T-cells that were not lineage restricted. One of these from the first workshop (2B11) yielded a pattern of labeling identical to a mAb under study (PGB73A). Ten mAbs were characterized in previous workshops and known to react with the gammadelta TCR or molecules expressed on subsets of gammadelta T-cells. One belonged to SWC4, two to SWC5, and one to SWC6. Two mAbs from the second workshop recognized a molecule or molecules expressed on subsets of gammadelta T-cells. A new mAb (PPT16) added late to the workshop following a request by the workshop chairs appeared to recognize a determinant expressed on the gammadelta TCR/CD3 molecular complex.     

Preparation and characterization of monoclonal antibodies against bovine B lymphocyte surface antigens

Two monoclonal antibodies (MoAbs; BLMo-4 and BLMo-10) were prepared by immunizing with a cell line established from peripheral blood mononuclear cells (PBMC) of enzootic bovine leukosis (EBL) cattle. The specificities of these MoAbs were assayed using bovine PBMC. BLMo-4 reacted with all surface immunoglobulin-positive cells (SIg+ cells; B lymphocytes) and also recognized monocytes, but did not react with T lymphocytes. BLMo-10 recognized a majority, although not all, B lymphocytes, but did not react with either T lymphocytes or monocytes. The antigens recognized by BLMo-4 and BLMo-10 were not Ig, Fc or C3 receptors on the surface of B lymphocytes. The reactivity of the MoAbs with mononuclear cells from the lymphoid organs of adult cattle was studied. BLMo-4 and BLMo-10 did not react with any bone marrow cells. BLMo-10 reacted with 7.4% of thymocytes, and stained the medulla of the thymus in the immunoperoxidase assay. In the case of PBMC, spleen and lymph node cells, the percentage of cells positive for BLMo-4 was slightly higher than that of SIg+ cells, but BLMo-10 showed a slightly lower value.     

磺胺间甲氧嘧啶单克隆抗体的制备

磺胺间甲氧嘧啶(Sulfamonomethoxine,SMM)与牛血清白蛋白(BSA)偶联,制备完全抗原(BSA-SMM),并以BSA-SMM免疫Balb/c小鼠,应用杂交瘤技术将免疫鼠脾细胞与NSO细胞融合,建立分泌SMM单克隆抗体的杂交瘤细胞株。通过对杂交瘤细胞培养上清液的检测、鉴定,筛选出12株高亲和力的杂交瘤细胞株,其中4B9、1H10、2E9的腹水ELISA效价为1×10-7、5.1×10-6和1×10-7。该单克隆抗体可用于饲料和动物源性食品中磺胺间甲氧嘧啶残留检测的免疫学快速检测方法的建立。     

Characterization of monoclonal antibodies to bovine IgG1

Monoclonal antibodies were developed to bovine IgG1. In addition, production of monoclonal antibodies to bovine light chain is also reported. Monoclonal antibody specificities were initially determined by solid-phase enzyme immunoassay. The monoclonal antibovine IgG1 was shown by a specificity-independent isotyping solid-phase enzyme immunoassay to be mouse IgG1 with kappa light chains. Ascites derived monoclonal antibovine IgG1 antibodies were linked to cyanogen bromide-activated Sepharose and used for affinity isolation of bovine IgG1. The bovine IgG1 eluted from the affinity column was characterized using immuno-electrophoresis, acrylamide gel electrophoresis, isoelectric focusing and solid-phase enzyme immunoassay. Affinity chromatography using monoclonal antibodies provided both a verification of monoclonal antibody specificity and a rapid technique for the isolation of bovine IgG1. This technique may also be employed to remove IgG1 contaminants during purification of bovine IgA.     

Study of bovine herpesvirus type 1 strains with monoclonal antibodies.

Fourteen strains of bovine herpesvirus type 1 (BHV-1, IBRV) representing all three groups of BHV-1 (BHV-1.1, BHV-1.2, BHV-1.3) were studied by ELISA using 106 monoclonal antibodies (Mabs) produced against BHV-1. On the basis of the ELISA, the Mabs could be divided into three groups. The first group (40 Mabs, 38%) reacted with all strains, the second group (43 Mabs, 41%) with the respiratory and genital strains (BHV-1.1 and BHV-1.2) while the third group (23 Mabs, 22%) only with the respiratory strains. Only 5 out of the antibodies neutralized respiratory and genital strains, and none of them neutralized the encephalitogenic strains (1.3). Three Mabs selected from each of the 3 groups, and the above five neutralizing strains were studied by Western blot. Antibodies of groups 1 and 3, and two neutralizing antibodies bound to a 90k protein (gpIII), whereas members of group 2 and 3 neutralizing antibodies reacted with a 74k and a 130k protein (both gpl). The results indicate that reactivity with monoclonal antibodies is as suitable for the classification of BHV-1 strains as is restriction endonuclease (RE) analysis but it cannot distinguish between subgroups within the groups.     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

Antigenic analysis of feline and bovine Chlamydia psittaci with monoclonal antibodies.

Monoclonal antibodies were established for antigenic analysis of feline and bovine Chlamydia psittaci. The monoclonal antibodies recognized lipopolysaccharide (LPS), 56-64, 84 or 86 kDa antigens. At least 5 antibody-binding sites were detected on LPS with the monoclonal antibodies. The 56-64 kDa antigen was suggested to have both polypeptide and carbohydrate antibody binding sites. Immunoblotting analysis of cat and cattle sera indicated that the 56-64 kDa antigen is an important antigen in host immune response. The monoclonal antibodies are extremely useful tools to analyse the structure and function of chlamydial antigens.     

吉氏巴贝斯虫单克隆抗体制备的研究

应用淋巴细胞杂交瘤技术制备杂交瘤细胞株 ,经间接萤光抗体法筛选和克隆 ,获得了 5株能稳定分泌吉氏巴贝斯虫特异性抗体的杂交瘤细胞株 ,分别命名为C3B5、M8B7、E9C5、G6 D8、H2 A7。经过鉴定 ,这 5株杂交瘤细胞分泌的单克隆抗体亚类及相对分子质量分别为IgG2b,1 8× 1 0 4 ;IgG2a,1 8× 1 0 4 ;IgG1 ,1 8× 1 0 4 ;IgM ,3 2× 1 0 4 ;IgM ,1 8× 1 0 4 。腹水效价为 1∶1 0 4 ～ 1∶1 0 5。其中E9C5杂交瘤细胞株分泌的单克隆抗体是一种保护性抗体 ,经对实验感染吉氏巴贝斯虫小鼠体内虫体的杀虫试验证明具有较强的杀灭作用。     

重组牛朊蛋白特异性抗体的制备及鉴定

以原核表达后经纯化的重组牛朊蛋白为免疫原,免疫prnp-/-基因敲除鼠。4次免疫后,利用淋巴细胞杂交瘤技术,取脾细胞和SP2/0骨髓瘤细胞进行细胞融合。间接ELISA方法筛选出阳性杂交瘤细胞,采用有限稀释法对阳性杂交瘤细胞进行3次克隆,用间接ELISA筛选出了稳定分泌针对牛重组朊蛋白特异性单克隆抗体的杂交瘤细胞株,命名为5C9D6。Western blotting鉴定结果表明,5C9D6均能特异性识别重组牛朊蛋白、健康牛、BALB/c脑组织匀浆中的PrPc,不识别prnp-/-基因敲除鼠脑组织匀浆液。本试验制备了可与牛、BALB/c鼠反应的单克隆抗体,同时也为牛海绵状脑病的研究及其诊断方法的建立奠定了基础。     

抗牛病毒性腹泻病毒单克隆抗体的制备及双抗夹心ELISA检测方法的建立

为建立牛病毒性腹泻病毒(BVDV)双抗夹心ELISA检测方法,本研究将BVDV 890病毒株(BVDV-2)浓缩并纯化后免疫BALB/c小鼠,经常规技术进行细胞融合并筛选得到一株稳定分泌IgG的杂交瘤细胞株,命名为3F9。以BVDV单克隆抗体(MAb)IgM为捕获抗体,生物素标记的3F9为检测抗体,初步建立BVDV特异的双抗体夹心ELISA检测方法(BAS-ELISA)。采用建立的BAS-ELISA方法检测35份临床病牛血清样品,检出阳性样本13份;与RT-PCR的符合率达到94.29%;表明本研究所建立的BAS-ELISA方法可用于BVDV感染的临床诊断,为BVDV的免疫学研究奠定了基础。     

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.     

Monoclonal antibodies reacting specifically with Francisella sp

Twenty two hybridoma strains producing monoclonal antibodies against Francisella tularensis ATCC 6223, var. tularensis, were characterized. In an enzyme-linked-immunosorbent-assay (ELISA) using formaldehyde fixed bacteria as antigens, neither cross-reactions with six different Brucella spp., with Yersinia enterocolitica 0:9 nor with two biotypes of Yersinia pseudotuberculosis could be detected. The antibodies gave comparable titres with the three strains of F.tularensis tested. ELISA binding studies indicated that fifteen of the antibodies bound with high affinities to their epitopes of the three Francisella strains, while the others each seemed to bind with low affinity to at least one of the antigens. Immunoblot analysis showed that six of the antibodies were directed to epitopes on the core moiety of the lipopolysaccharide molecule, while the other 16 antibodies bound to O side chain components.     

Cross-reactivity of monoclonal antibodies to bovine immunoglobulins with immunoglobulins of other species.

Monoclonal antibodies (Mabs) to bovine immunoglobulin heavy chain of the four major isotypes gamma 1, gamma 2, alpha, mu and the light chains (combined kappa and lambda) were produced and found to cross-react in enzyme-linked immunoassay (ELISA) with immunoglobulins of some other animal species despite the discrete specificity associated with an antibody derived from a single clone. This cross-reactivity, particularly amongst ruminants, could be utilized in serological testing for the diagnosis of disease in these species. For example, Mabs produced against bovine immunoglobulin light chain cross-react with bison immunoglobulin light chain and were used successfully in serological testing as the secondary detection antibody in an indirect ELISA for the diagnosis of Brucella abortus in bison herds in north-western Canada.     

抗猪细小病毒单克隆抗体的制备

为了建立一种快速准确的猪细小病毒(PPV)病诊断体系,本研究制备了抗PPV的单克隆抗体(MAb)的杂交瘤细胞株.将PPV免疫的BALB/c小鼠脾细胞与SP2/0细胞在聚乙二醇作用下融合,用ELISA方法筛选,得到8株分泌抗PPV MAb细胞株,这8株MAb亚类均为IgG1,轻链均为κ型.交叉实验等特异性分析发现这些MAb只特异地与PPV发生反应,而不与猪圆环病毒Ⅱ型(PCV2)、猪伪狂犬病毒(PRV)、猪繁殖和呼吸综合征病毒(PRRSV)等发生交叉反应.将杂交瘤细胞注射BALB/c小鼠制备的腹水抗体效价介于1:2 560～1:20 480之间.Western blot结果显示,8株MAb中2株针对VP1发生反应,5株针对VP2、VP3发生反应,但其中有1株呈阴性反应,该MAb可能识别的是PPV的构象表位.     

阿维菌素单克隆抗体的制备与鉴定

将与牛血清白蛋白（BSA）偶联的阿维菌素人工合成抗原AVM-BSA免疫8周龄雌性BALB/c小鼠,利用杂交瘤技术获得了2株分泌抗阿维菌素特异性抗体的杂交瘤细胞株,分别命名为2F2和2H10.生物学特性鉴定的结果表明,2株杂交瘤细胞株诱生腹水的效价为1∶6 400,分泌的抗体亚型均为IgM;间接竞争酶联免疫吸附试验结果表明,抗体的50%抑制质量浓度（IC50）为101 ng/mL,2株单克隆抗体与伊维菌素、多拉菌素、红霉素和白霉素均无交叉反应,证实单克隆抗体2H10具有很强的特异性,可用于阿维菌素药物残留免疫分析方法的建立.     

Diagnosis of bovine viral diarrhea virus infection using monoclonal antibodies

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.     

禽脑脊髓炎病毒单克隆抗体制备

禽脑脊髓炎病毒（ＡＥＶ）在患鸡体内含量很低,且细胞培养的复制水平也很低,故对该病毒的研究一直不够深入,国外有关ＡＥＶ单克隆抗体的报告首见于１９９４年［１］。本研究制备了３株抗ＡＥＶ单克隆抗体,为ＡＥＶ的实验室诊断提供了一种简单、快速、可靠的方法,同时...     

阿维菌素类药物单克隆抗体的制备和鉴定

将阿维菌素与牛血清白蛋白偶联,免疫Balb／c小鼠,用杂交瘤技术获得了1株分泌抗阿维菌素特异性抗体的杂交瘤细胞株。用间接ELISA测定腹水的效价为1：160000,抗体的50％抑制药物浓度（IC50）为2．5ng／ml,抗体与伊维菌素的交叉反应为12．5％,与多拉菌素、莫西菌素均无交叉反应。     

Production and characterization of monoclonal antibodies specific for bovine gamma-interferon

Nine stable hybridoma cell lines were established which secreted specific monoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-gamma). Specific binding of each of the MAbs to recombinant BoIFN-gamma (rBoIFN-gamma) was demonstrated in an indirect ELISA, whilst none of the MAbs bound to rBoIFN-alpha or rBoIFN-beta. In a Western blot the MAbs reacted with the 16 kDa and 32 kDa polypeptides present in rBoIFN-gamma preparations. Competitive ELISA's showed that four MAbs bound to one epitope on rBoIFN-gamma, and the other five MAbs bound to a separate epitope. Two MAbs, each recognising different epitopes, were shown to neutralise the anti-viral activity of natural BoIFN-gamma.