人μ链单克隆抗体的研制及临床应用

IgM是人血清中重要的免疫球蛋白之一,血清、组织及细胞中IgM检测与临床许多疾病的诊断有密切关系。特异性IgM抗体的检测对细菌、病毒感染早期快速诊断有重要价值。为此,我们用杂交瘤技术制备了抗人μ链单克隆抗体(MCAb),并已初步应用于临床。其主要步骤为: 一、人μ链McAb制备 从巨球蛋白血症患者血清中提取、纯化lgM(1.2g/L)免疫BALB/c小鼠,取免疫小鼠脾细胞与同系小鼠SP2/0骨髓瘤细胞融合,用PHA及ELISA八二种方法筛选,结果获二个分泌抗人μ链杂交瘤细胞株(2D6、E4),具抗体效价分别为2~(-5)和蔼~(-4)(PHA)。选取2D6制备小鼠腹     

抗人μ链单克隆抗体的制备、鉴定及初步应用

目的:制备高效价的鼠源性抗人μ链单克隆抗体(mAb),并建立可用于感染性疾病早期血清学诊断的ELISA捕捉法。方法:以人IgM全分子免疫BALB/c小鼠,按常规方法进行细胞融合,用间接ELISA法筛选及克隆化,建立可稳定分泌抗μ链mAb的杂交瘤细胞株。mAb的特性(效价、Ig亚类、特异性及相对亲和力)采用ELISA及Westernblot法鉴定。以纯化的mAb包被建立ELISA捕捉法,并用于可疑乙脑患者标本中特异性IgM抗体的检测。结果:筛选到1株可稳定分泌抗人μ链mAb的细胞株2E5。mAb腹水的ELISA效价为1×10-6,Ig亚类(型)为IgG1(κ),相对亲和力为1×10-5。Westernblot结果显示mAb2E5仅与IgM的μ链结合,Mr为75000。以辛酸硫酸铵法纯化的mAb2E5包被,建立了ELISA捕捉法,用于30份乙脑患者血清IgM的检测,敏感性及特异性良好。结论:成功地制备1株抗人μ链mAb2E5,建立了可用于感染性疾病早期血清学诊断的ELISA捕捉法。     

兔抗B16黑素细胞多克隆抗体的制备及对黑素细胞增殖的影响

目的:制备兔抗B16黑素细胞多克隆抗体,研究其对黑素细胞增殖的影响。方法:①用B16黑素细胞颗粒抗原免疫家兔得到兔抗B16黑素细胞多克隆抗体;试管凝集法检测抗血清效价;G蛋白亲和层析纯化抗血清;②SDS-PAGE检测纯化抗体分子量大小;MTT法检测纯化抗体IgG对B16黑素细胞增殖的影响。结果:兔抗B16黑素细胞抗血清效价高达1:1280;亲和层析纯化抗血清获得的免疫球蛋白IgG,经SDS-PAGE电泳显示重链分子量大小约为66.2kD;MTT比色法显示IgG对黑素细胞增殖有抑制作用,与非IgG和未纯化血清差异显著。结论:成功制备并鉴定了高效价的兔抗B16黑素细胞多克隆抗体,纯化后所获得的高纯度IgG,对B16黑素细胞增殖具有抑制作用,为进一步研究此抗体对黑素细胞生长及色素代谢的影响,以及色素减退性疾病白癜风的发病机制奠定了基础。     

日本血吸虫成虫抗原和虫卵抗原检测先天性感染仔兔血清抗体的动态变化

目的　观察仔兔血清抗体动态变化 ,探讨家兔日本血吸虫病垂直传播的体液免疫反应规律。方法　 5只怀孕晚期母兔分别人工感染 5 0 0条日本血吸虫尾蚴 只 ,仔兔出生后 4 3d起每隔 2周采血收集血清 ,至仔兔发育成熟 (约出生后113d)。分别运用日本血吸虫成虫抗原 (AWA)和可溶性虫卵抗原 (SEA) ,ELISA法检测血清中特异性IgG、IgM抗体 ,观察动态变化。结果　仔兔先天性感染率为 6 0 % (12 2 0 ) ,其中 5只双性感染 ,7只单性感染。 2 0只仔兔 ,抗AWA IgG、抗AWA IgM抗体检测始终呈阴性 ;抗SEA IgG检测 ,5只双性感染仔兔有 4只于出生后 5 7d起陆续呈阳性 ,其余 16只始终为阴性 ;2 0只仔兔抗SEA IgM也始终呈阴性。结论　先天性感染日本血吸虫病的仔兔呈低免疫应答状态 ,可能存在免疫耐受     

类风湿关节炎患者血清IgG-IgM型类风湿因子免疫复合物检测的临床意义

目的 探讨血清IgM型类风湿因子与变性IgG抗原形成的复合物定量检测对类风湿关节炎诊断的临床意义.方法 收集类风湿关节炎(RA)患者36例、非RA患者41例和体检健康者40名作为研究对象.用包被有鼠抗人μ链抗体的ELISA孔板及HRP标记的兔抗人IgG对血清中IgM型类风湿因子(RF)与变性IgG抗原形成的免疫复合物水平进行检测,同时用胶乳凝集法检测RF,并对二者结果进行相关性分析;用ELISA试剂盒进行抗环瓜氨酸肽抗体(抗CCP抗体)的定性检测,并比较其与IgG-IgM型RF免疫复合物对于RA诊断的敏感性和特异性.结果 待检血清最佳稀释倍数为100倍.IgG-IgM型RF免疫复合物对于RA的敏感性为72.2％,特异性为95.3％.IgG-IgM型RF免疫复合物OD值与RF阳性程度相关系数为0.687(P ＜0.01).结论 血清IgG-IgM型RF免疫复合物水平对RA具有较高的敏感性和特异性,可作为RA诊断的参考指标.     

沙鼠IgG兔抗血清的制备及其在IgG抗体检测中的应用

目的用纯化的蒙古沙鼠血清IgG制备相应兔抗血清,并用于幽门螺杆菌（Helicobacter pylori,Hp）疫苗免疫后沙鼠血清中抗原特异性IgG的检测。方法采用饱和硫酸铵沉淀法和Q sepharose high performance阴离子交换层析法纯化蒙古沙鼠血清IgG,免疫日本大白耳兔制备兔抗沙鼠IgG抗血清并进行rProtein A亲和纯化,建立间接ELISA方法用于检测Hp疫苗注射免疫蒙古沙鼠后血清抗原特异性IgG水平。结果以纯化的蒙古沙鼠血清IgG（纯度大于98％）免疫日本大白耳兔获得相应兔抗血清,经亲和层析后其纯度大于90％,回收率约为85％,双扩效价为1：16,ELISA效价为1：320000;ELISA检测结果显示Hp疫苗注射免疫蒙古沙鼠于第2次免疫后产生高水平IgG,第4次免疫后第7天IgG水平达到高峰,随后几周仍然保持高水平。结论所制备的兔抗沙鼠IgG抗体可以用于蒙古沙鼠血清中抗原特异性IgG的检测。     

用免疫荧光法筛选抗人μ链单克隆抗体(McAb)的研究

用人血清IgM球蛋白重链-μ链免疫的BALB/c小鼠脾细胞与鼠骨髓瘤Sp 2/0细胞在PEG作用下融合,用免疫荧光法筛选出6种分泌McAb的杂交瘤细胞,其中5株确定为抗人μ链McAb,一株未定。杂交瘤细胞经四次克隆,半年多传代,接种BALB/c鼠可稳定的产生腹水抗体。     

用HRP——抗IgM法快速诊断流行性出血热

本文报道用流行性出血热(EHF)病毒抗原片,以酶标抗人IgMu链单克隆抗体染色法(简称HRP-抗IgM)检测患者血清中的IgM抗体,从而对EHF进行早期诊断。为确证本方法的可靠性,同时与间接免疫荧光IgG法(IFA-IgG)进行了比较.经阻断试验和2-巯基乙醇耐性试验证明,HRP-抗IgM检出的抗体系EHF特异性抗体.用双盲法检测了51份血清,结果全部符合。实验结果表明,HRP-抗IgM特异性强.敏感性高,用普通显微镜便可观察,结果清晰.4小时内可获得诊断结果,可以用作早期诊断.     

辣根过氧化物酶标记的兔抗鼠IgM抗体的制备

我室制备的数株抗大肠癌单抗已用于大肠癌的免疫组化、血清学检测、放射免疫定位及导向治疗等研究,其中涉及多株IgM单抗。需要使用酶标兔抗鼠IgM抗体,国内尚无酶标抗IgM抗体供应。现以本室制备的鼠IgM单折为免疫原,采用皮内多点注射免疫     

应用单克隆抗HBc和抗人IgM(μ链特异)检测血清抗HBcIgM试剂盒的研究

本试剂盒以自制羊抗人IgM(μ链特异)为包被抗体,鼠单克隆抗HBcIgG_3的酶结合物为指示抗体,并采用HBsAg携带者非肝炎死者肝脏提取的HBcAg。经166份病人及健康人血清1:1,000稀释后,用丹麦Dako公司和美国Sigma公司抗人IgM(μ链特异产品)包被微板进行比较,以Dako公司抗人IgM检测结果为相对标准,初步证明:自制抗人IgM与Sigma公司产品不论是相对灵敏度,相对特异性,相对预报率还是相对符合率等四个指标,X~2测定结果并无显著差别。己初步用于临床实验室诊断,结果满意。     

Immunoglobulin binding by Tritrichomonas foetus.

A better method for diagnosis of bovine trichomoniasis is needed because culture is slow and somewhat lacking in sensitivity. Immunodiagnosis of Tritrichomonas foetus infection usually involves detection of antigen-antibody reactions with an anti-immunoglobulin conjugate. However, nonspecific immunoglobulin (Ig), bound to the surface of T. foetus, would also be detected by an anti-Ig conjugate and thus would interfere with the specificity of the immunoassay. The goals of this study were to define the binding of bovine immunoglobulins to T. foetus. To determine whether nonimmune binding of Ig to T. foetus occurs, we immunized rabbits with organisms that had been grown in medium containing normal bovine serum and vigorously washed three times with phosphate-buffered saline. The rabbit antiserum had similar titers to T. foetus and to normal bovine serum by enzyme-linked immunosorbent assay (ELISA). Furthermore, two bovine serum proteins were immunoprecipitated by the rabbit antiserum in an immunoelectrophoretogram. One of the serum proteins had a distribution characteristic of IgG2. The rabbit antiserum was then shown to react with purified bovine IgG and IgM by ELISA. Reactivity to IgG was greater. To identify the IgG subisotypes bound and to confirm nonimmune binding of Ig, we grew T. foetus in agammaglobulinemic fetal calf serum and reacted it with IgG1, IgG2, and IgM specific for dinitrophenol (DNP) in ELISA. The binding of IgG2 was greatest, that of IgG1 was next, and that of IgM was least. Little competitive inhibition by DNP was detected, indicating that binding of DNP-specific antibodies was predominantly nonimmune rather than antigen-specific Ig binding. We also demonstrated that T. foetus grew well in medium containing agammaglobulinemic fetal calf serum or serum made agammaglobulinemic by ammonium sulfate precipitation of Igs. This may overcome the problem of low specificity in diagnostic assays as a result of antigen with Ig bound by nonimmune mechanisms.     

Detection of human and marmoset immunoglobulin heavy chain by a polyclonal antiserum to a marmoset immunoglobulin-related T cell product

We show that a polyclonal rabbit antiserum raised against a purified monoclonal T cell component from a lower primate (cotton-topped marmoset) reacts by immunoblot transfer (Western Blot analysis) with serum immunoglobulin of man and marmoset. The antigenic component had an approximate mass of 68 kilodaltons and was isolated by immune-affinity chromatography from culture fluid in which the marmoset T cell leukemia 70-N2 had been grown. The reaction with human serum immunoglobulin is with a subset of the IgG molecules and is localized to the gamma heavy chain. The reaction with marmoset serum immunoglobulin is predominantly directed against the heavy chain, but slight reactivity is also noted against the light chains. These results substantiate reports of serological cross-reactions between immunoglobulin-like T cell receptors and classical immunoglobulins and illustrate the similarity between immunoglobulins of man and those of a distantly related New World primate.     

Passive immunization against Pseudomonas with a ribosomal vaccine-induced immune serum and immunoglobulin fractions.

Passive protection of mice against Pseudomonas aeruginosa using specific antisera and immunoglobulin fractions induced by immunizing rabbits with a ribosomal vaccine is reported. The results demonstrated that protection by the ribosomal vaccine against challenge with live organisms can be serum mediated. Previous work has shown that the vaccine can be separated into two components on the basis of molecular weight and that both higher (peak A)- and lower (peak B)-molecular-weight fractions were capable of inducing active immunity in mice. The present report indicates that both fractions are also capable of eliciting the production of mouse-protective antibody in rabbits. Agar gel diffusion with antisera to peaks A and B or unfractionated vaccine indicated a common antigenic component among them in addition to an extra antigen in unfractionated vaccine not present in peak B. Passive hemagglutination with antisera to peaks A and B demonstrated high-titer agglutinating antibody only with antiserum to peak A when a method of erythrocyte sensitization for lipopolysaccharide antigens was used. Also, passive hemagglutination was greatly inhibited by small amounts of lipopolysaccharide prepared from the same organism from which the vaccine was made. Both antisera to peaks A and B fixed complement with either A or B antigens. Antisera to peaks A and B, when reacted with peak B antigen, had about the same complement fixation titer (as determined by a quantitative complement fixation test). However, when peak A antigen was used, antiserum to peak A had about twice the complement fixation titer that antiserum to peak B had. These results are consistent with previous observations which suggest that the ribosomal vaccine contains lipopolysaccharide in addition to an unidentified immunogenic principle associated with ribosomes. Furthermore, this immunogen was present in both peaks A and B, but detectable amounts of lipopolysaccharide were present only in peak A. The relative importance of the immunoglobulin G (IgG) and IgM classes of antibodies was also compared. The results indicated that both IgG and IgM isolated from immune rabbit serum are protective in mice. Only IgG precipitated with the vaccine in agar gel diffusion, but both IgG and IgM were active in passive hemagglutination and in complement fixation. The passive hemagglutination titer of the IgM was higher than that of the IgG, but the complement fixation titer of the IgG was higher than that of the IgM. The mouse-protective capability of the IgG and IgM was about the same.     

Same Idiotypc of B-Lymphocyte Membrane IgD and IgM. Formal Evidence for Monoclonality of Chronic Lymphocytic Leukemia Cells

Idiotypic antisera were raised in rabbits against serum M-cnmponents in two patients with chronic lymphocytic leukemia (CLL) In one of the patients the leukemic cells had membrane-bound IgG. in the other IgM and IgD. The M-components were IgG and IgM. respectively. By immunofluorescence technique the CLL cells were shown to be positive when stained with idiotypic antiserum prepared against the corresponding M component, whereas normal lymphocytes, as well as cells from other CLL patients, were negative. These findings represent strong evidence for the monoclonality of the malignant B cells in CLL The fact that the CLL cells had membrane-bound Ig with the same idiotypic determinants as the corresponding serum M-component indicated that the latter had been synthesized and secreted by the malignant clone of B cells. In further experiments redistribution of membrane-bound Ig on CLL cells bearing both IgM and IgD was induced with idiotypic antiserum This indicated that IgM and IgD on the same cells share idiotypic specificity and therefore have the same variable region and. presumably, the same antibody specificity     

A peculiar immunoglobulin M (IgM) identified in eggs of chum salmon (Oncorhynchus keta).

A protein reacting with antibody against chum salmon serum IgM was found in salmon egg yolk. The protein tentatively named IgM-like protein was partially purified from egg yolk extract by removal of euglobulin from the extract by dialysis against a low ionic strength buffer following DEAE ion-exchange chromatography. The IgM-like protein was antigenically identical with serum IgM, showing a complete fusion of precipitin line of the IgM-like protein with that of serum IgM on double immunodiffusion with antiserum IgM. The molecular weight of intact IgM-like protein was assessed by 3% SDS-PAGE to be 495 kDa, smaller than that of serum IgM (750 kDa). Upon 10% SDS-PAGE with mercaptane and subsequent Western blotting with antiserum IgM, the IgM-like protein separated into three components with molecular weights of 68 kDa, 51.5 kDa, and 23 kDa. The 68 kDa, the most minor component, and the 23 kDa, the smallest molecular weight component, were identified, respectively, as H and L chain in view of their molecular weights identical to those of serum IgM. The 51.5 kDa, the main component, was also identified as H chain, since it reacted with antiserum IgM absorbed with purified L chain. From these results, it was concluded that the IgM-like protein in egg yolk, having a lower molecular weight than that of serum IgM, consists of H chain of smaller molecular weight as well as H and L chain of ordinary molecular weight.     

Immunochemical and biochemical study of an abnormal heavy chain of bovine IgG1

An abnormal heavy chain (HC) protein of IgG1 was isolated from the serum of a leukemic cow by gel filtration on a Sephadex G-200 column with the following ion-exchange chromatography. The HC protein migrated electrophoretically in the anodic region. The molecular weight of the untreated HC protein was about 48,000. Enzymatic treatment with papain and reduction with beta-mercaptoethanol showed no effect on the protein. Antigenic analysis of the HC protein using a specific rabbit antiserum against bovine IgG1 showed a complete identity with the Fc fragment and a partial identity with the intact bovine IgG1. Antigenic determinants of the light-chain were not found. The HC protein consisted of only one polypeptide chain.     

Separation of cell-dependent antibody (CDA) and inhibitory antibody by protein-A affinity chromatography and the effect of fractions on antibody-dependent cellular cytotoxicity (ADCC).

The nature of cell-dependent antibody (CDA) and the mechanism of inhibition of antibody-dependent cellular cytotoxicity (ADCC) were studied in the ADCC assay system in which culture cells of methylcholanthrene-induced rat fibrosarcoma (KMT-50) were used as target cells, xenogeneic antiserum (rabbit anti-KMT-50) as the CDA, and human peripheral blood leucocytes (PBL) as effector cells, respectively. By using protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-KMT-50 serum, CDA was shown to bind protein A. Complement dependent-cytotoxicity (CDC), however, was demonstrated in both the adsorbed fraction (eluate) and the non-adsorbed fraction (effluent) to protein A from the same affinity column chromatography. These data confirmed that CDA was IgG with an intact Fc portion. Inhibition of ADCC occurred by pretreatment of effector cells with rabbit anti-effector (human PBL) serum even with extremely small amounts of antiserum. Such inhibition was demonstrated with the eluate but not with the effluent from protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-effector serum. F(ab')2 fragments of the same eluate (IgG) did not inhibit the ADCC activity. These data showed that the inhibition of ADCC was induced by the blocking of Fc receptors of effector cells with the Fc portions of IgG in anti-effector serum. The data obtained indicate the usefulness of protein A in separation and analysis of CDA and in investigation of the inhibitory mechanisms of ADCC.     

Studies of human anti-IgM anti-IgG cryoglobulins. I. Patterns of reactivity with autologous and isologous human IgG and its subunits.

Monoclonal human anti-IgG preparations purified from mixed IgM-IgG cryoglobulins were tested for their antigenic specificity by haemagglutination-inhibition assay. A panel of fourteen IgG preparations of the four gamma chain subclasses were prepared from myeloma sera and used as inhibitors of haemagglutination. Each of six IgM anti-globulins demonstrated different reactivity profiles with these IgG preparations. In addition, the fraction of the serum IgG which had bound to and cryoprecipitated with the IgM preparations, termed 'antigen-IgG', was purified and assayed for subclass content. The gamma chain subclasses found in the 'antigen-IgG' fractions showed that each IgM cryoprecipitated an IgG from serum which had different quantities of the subclasses present. These 'autologous' reactivity patterns were in instances different from the specificities expected from the results obtained with the myeloma proteins. When all antigen-IgG pools were tested with each IgM, some antiglobulins showed stronger reactivity with isologous than with their own, antigen-IgG pools. The IgM anti-IgG preparations were also compared in reactivity with IgG and its subunits in order to localize the antigenic determinant(s) with which these autoantibodies react. Heavy chains showed far greater reactivity than Fc fragment for 5/6 IgM preparations. Light chains, F(ab')2, pFc' and Fab were non-reactive. A relationship between the length of papain digestion and Fc reactivity was demonstrated. Based on the data, possible locations for the antigenic determinant(s) were considered.     

Identity of a T-lymphocyte inhibitor with mouse immunoglobulin in the serum of tumour-bearing mice.

A component of the serum of tumour-bearing mice has been shown to be inhibitory to the immunological function of normal mouse T cells. This factor fractionates with monomeric immunoglobulin upon gel filtration. Studies were carried out utilizing goat antisera to the major immunoglobulin chains of mouse Ig (kappa, gamma, alpha and mu) mixed with the immunoglobulin-rich fraction of serum from normal mice and tumour bearers and passed through immunoadsorbent columns prepared with rabbit anti-goat immunoglobulin. Such studies showed that the inhibitory activity in tumour-bearing serum could be removed after treatment with anti-kappa, anti-gamma and anti-mu chain antisera but not by treatment with either anti-alpha chain or goat immunoglobulin. That the inhibitory activity of tumour-bearing immunoglobulin could be attributed to simple quantitative differences in the levels of IgM and IgG in the test samples was discounted by quantification of the amounts of immunoglobulins in normal and tumour-bearing sera.     

Lack of evidence for IgM-induced ADCC: studies with monoclonal and polyclonal antibodies.

Different kinds of IgM antibodies were tested for their activity in antibody-dependent cellular cytotoxicity (ADCC): firstly an anti-benzylpenicilloyl (BPO) IgM antibody from immune rabbit serum purified by affinity, ion exchange, and molecular-sieving chromatography, secondly two monoclonal rat anti-BPO IgM antibodies and thirdly a human antidextran antibody prepared from a patient showing restriction of anti-dextran antibodies to the IgM class. Human lymphocytes or purified monocytes served as effector cells. While the two monoclonal rat and the human IgM antibodies showed no ADCC-mediating capacity, ADCC was induced by the rabbit anti-BPO IgM antibody when high antibody concentrations were used. This activity was abolished by further purification using an anti-rabbit IgG (Fc) immunosorbent. The initially observed activity was shown to be likely due to traces of aggregated anti-BPO IgG, which cannot be detected by the methods commonly used. Preincubation of lymphocytes for 24 hr increased the number of EA (IgM)] rosette forming cells but failed to induce IgM-mediated ADCC. Furthermore, evidence for amplification of low-dose IgG-ADCC by IgM could not be found.