Experimental glomerulonephritis induced by immune complexes of monoclonal antibodies produced by immunoglobulin class-switch variants

Experimental glomerulonephritis was induced in mice by injecting performed antigen-antibody complexes composed of monoclonal anti-dansyl antibody of switch-variant origin and dansyl-conjugated bovine serum albumin. A comparison was made of the ability of two kinds of monoclonal antibodies, IgE and IgG2a, of the same variant origin to induce glomerulonephritis. Both types of immune complexes (IC), (IgE-IC and IgG2a-IC), given daily elicited exudative proliferative glomerulonephritis accompanied by proteinuria. Significant glomerular hypercellularity including invasion by polymorphonuclear leukocytes was observed as early as 2 days and was prominent at 14 days after the start of daily injections. Deposits of IgE-IC and IgG2a-IC plus the third complement component were observed mainly in mesangial areas early in the experiment (3 to 8 days) and additionally along peripheral loops at a later stage (9 to 14 days). By electron microscopic examination, immune deposits were detected in the mesangial areas as well as in the subepithelial aspect of the peripheral loops. These results reveal that the isotype of the antibody used to prepare IC does not influence the form or severity of glomerulonephritis.     

Production of a human monoclonal antibody to HLA by human-human hybridoma technology. A preliminary report.

An Epstein-Barr-virus-transformed lymphoblastoid cell line (ECEBV) was derived from a multiply transfused renal dialysis patient. ECEBV was shown to secrete specific antibody in a cellular enzyme-linked immunosorbent assay (CELISA) and was hybridized with the mutagenized human fusion partner G M1500 resistant to 6-thioguanine and ouabain. Hybridomas surviving hypoxanthine-aminopterin-thymidine (HAT) and ouabain selection were cloned by limiting dilution. The hybridomas continue to secrete antibody which reacts with some human cells but not with others after 14 months in culture. None reacts with K562 (no HLA-A, -B, -C or -DR) or with Daudi (no HLA-A, -B, or -C). This is a preliminary report of the production of a human monoclonal antibody to HLA. Application of this technique could result in the large-scale production of human monoclonal antibodies for HLA typing, the production of anti-idiotype antibodies for use in transplant patients to prevent acute rejection, and for the study of the structure and function of HLA in man.     

Production of human monoclonal antibody in mouse ascites

Human B-cell hybridomas were grown in nude mouse ascites. The growth of cells in ascites requires prior subcutaneous (SC) adaptation, but the amount of antibody harvested is 100-fold that produced in in vitro culture. The relative merits of in vitro versus in vivo production of human monoclonal antibodies are discussed.     

Production and characterisation of a monoclonal antibody to human papillomavirus type 16 using recombinant vaccinia virus.

A monoclonal antibody was raised against the major capsid protein L1 of human papillomavirus type 16, using a recombinant vaccinia virus that expresses the L1 protein, as a target for screening. This antibody, designated CAMVIR-1, reacted with a 56 kilodalton protein in cells infected with L1-vaccinia virus, and the protein was present in a predominantly nuclear location. The antibody also detects the HPV-16 L1 antigen in formalin fixed, paraffin wax embedded biopsy specimens and on routine cervical smears. The antibody reacts strongly and consistently with biopsy specimens containing HPV-16 or HPV-33, but very weak reactions were occasionally observed with biopsy specimens or smears containing HPV-6 or HPV-11. The potential advantages of using a vaccinia recombinant are (i) the target protein is synthesised in a eukoryotic cell so that its "processing" and location are normal; (ii) cells infected with vaccinia recombinants can be subjected to various fixing procedures similar to those used for routine clinical material. This greatly increases the probability that an identified antibody will be useful in a clinical setting.     

重组人血管生长素及其单克隆抗体的特性鉴定

目的 鉴定自制重组人血管生长素rhANG)及其单克隆抗体（mAb)。方法 采用凝胶扫描法分析rhANG的纯度;ELISA等方法检测抗rhANC mAb的特异性,鸡胚绒毛尿囊膜实验检测rhANG及其mAb的生物学活性,并与商品化标准品的免疫结合性进行对比研究。结果 rhANG的纯度达96％,具有促进血管生长的生物学活性;抗ANG mAb具有很好的特异性和抑制血管生长的活性;而且二者较标准品均具有良好的免疫结合活性。结论 该rhANG及其mAb可用于进一步的实验和临床应用研究。     

抗rhEPO单克隆抗体的制备及初步应用

目的 :研制抗人促红细胞生成素的特异性单克隆抗体 (mAb) ,对其生物学特性进行初步鉴定 ,并用于转基因羊乳中重组人促红细胞生成素 (rhEPO)的提纯。方法 :以自制的rhEPO粗品免疫BALB/c小鼠 ,制备抗rhEPO的mAb。以Westernblot和间接ELISA法 ,对所获mAb进行初步鉴定。以纯化的mAb同预活化的Sepharose 4B偶联 ,制得mAb亲和层析柱 ,并用于转基因羊乳中rhEPO的提纯。结果 :获得两株可分泌特异性抗rhEPO的mAb的杂交瘤细胞株 (1E7和 2E6 )。Ig亚类 (型 )的鉴定分别为IgG1和IgG2b ,轻链均为κ型。用Westernblot证实 ,获得的mAb可特异性地识别rhEPO。用自制的免疫亲和层析柱用于转基因羊乳中rhEPO的提纯 ,具有很好的吸附作用 ,回收率达 70 %。结论 :成功地获得两株可分泌特异性抗rhEPO的mAb的杂交瘤细胞。用自制的免疫亲和层析柱提纯转基因羊奶中的rhEPO具有较高的吸附效率     

抗人HPPCn单克隆抗体的制备及鉴定

目的:制备高灵敏度的抗人HPPCn单克隆抗体(mAb),以用于HPPCn的功能研究及其疾病相关性研究。方法:用重组蛋白免疫雌性BALB/c小鼠,采用常规杂交瘤技术进行细胞融合,经ELISA法进行阳性克隆筛选、通过有限稀释法进行亚克隆,获得稳定分泌抗人HPPCn mAb的细胞株。采用间接ELISA、Western blot、Ig亚类快速定性试纸分析法等鉴定抗体的生物学特性;通过细胞免疫荧光实验观察了HPPCn蛋白的细胞定位。通过噬菌体肽库技术分析抗体所识别的抗原表位。结果:得到了1株稳定分泌抗人HPPCn抗体的杂交瘤细胞株,命名为W2-D5。经Ig亚类分析确定,该细胞株分泌的抗体属于IgG1亚类。间接ELISA检测表明,该抗体检测HPPCn的极限为0.1μg/L;Western blot结果显示,该抗体能特异性识别HPPCn。细胞免疫荧光实验,证实了HPPCn蛋白定位在细胞核。通过肽库筛选及表位分析认为,该抗体识别的表位可能为HPPCn7-13(IHLELRN)。结论:成功地获得了抗人HPPCn的特异性mAb。     

Production of a monoclonal antibody against a human osteosarcoma xenograft

Monoclonal antibodies (MAbs) against a human sarcoma xenograft carried in BALB/c nu/nu mice were produced by immunizing BALB/c mice with tumor cells and fusing their spleens with the SP2/O-Ag 14 mouse myeloma cell line. Hybridoma supernatants were screened using cryostat tissue sections and an immunoperoxidase reaction for ability to stain osteosarcoma xenograft tumor cells but not tonsil lymphocytes. Of 73 supernatants tested, 19 reacted with both osteosarcoma tumor cells and lymphocytes, while three reacted only with osteosarcoma. One of the latter hybridomas was cloned by limiting dilution to establish a line producing an IgG1 MAb (OS-1). By immunoperoxidase, this MAb stained tumor cells in surgical biopsies of primary (6 of 7) and metastatic (1) osteosarcomas and showed limited cross-reactivity with other tumors. It also cross-reacted with some basement membranes, endothelium and muscular media of blood vessels, and smooth muscle, but not with parenchymal cells of various normal tissues. This MAb may prove useful for the immunohistochemical confirmation of a diagnosis of osteosarcoma in surgical pathology.     

Recombinant monoclonal antibody technology.

With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.     

Production of monoclonal human antibody to HLA-DR5 (DRw11) by mouse/human heterohybridomas

We describe here the production of a human monoclonal antibody to the HLA-DR5 antigen. A human B-cell line secreting cytotoxic antibody that reacted preferentially with DR5-positive targets was fused to the mouse myeloma P3X63Ag8.653 and the resulting heterohybridomas cloned twice. The clones secreted human IgM (lambda light chain), which showed specificity for the DR5 antigen in cytotoxicity assays and reacted with DRw11-positive but not DRw12-positive targets. These results demonstrate the potential of this approach to the production of human monoclonal antibodies to transplantation antigens.     

抗重组人表皮生长因子单克隆抗体的制备及生物学特性鉴定

目的　制备出高效价的抗重组人表皮生长因子 (EGF)单克隆抗体。方法　以重组人表皮生长因子作为抗原 ,免疫Balb/c小鼠 ,以未免疫的Balb/c小鼠脾细胞为饲养细胞 ,运用细胞杂交瘤技术制备 ,间接ELISA法筛选产生针对人表皮生长因子的单克隆抗体细胞株 ;以体内诱生法产生腹水 ,并采用ProteinA Sepharose柱对其纯化 ,快速定性试纸鉴定McAb的Ig亚类 ,采用间接ELISA法相加实验鉴定抗原识别表位。结果　获得 3株产生针对人表皮生长因子的单克隆抗体细胞株EGF B2 、EGF C7、EGF A8,Ig亚分别为IgG1κ型 ,IgG1λ型 ,IgG3 κ型 ,亲和力常数分别为 2 .76× 10 10 、3.2× 10 9、1.4 5× 10 9。结论　成功制备 3株稳定分泌抗rhEGF的杂交瘤细胞株 ,产生的McAb特异性好 ,亲和力高 ,为探讨EGF的作用机制及临床应用奠定了基础 ,为肿瘤的诊断与治疗提供具有实用价值的研究工具 ,此外 ,为EGF的纯化提供实验材料     

Production and characterization of anti-recombinant human erythropoietin (rhEPO) monoclonal antibody

Anti-rhEPO McAb is valuable for the determination of recombinant human erythropoietin (rhEPO) levels for diagnosis of renal anemia and for doping control analysis. In this paper, anti-rhEPO hybridoma was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse, using an enzyme-linked immunosorbent assay (ELISA) method to screen the positive hybridoma. The purified McAb was characterized by ELISA, SDS-PAGE, and Western-blotting. Experimental results showed that the subclass and the light chain of anti-rhEPO McAb was IgG1 and kappa light chain. The molecular weight of anti-rhEPO McAb was 166,000 Daltons. The affinity constant (K(aff)) of anti-rhEPO McAb with coated antigen was 5.0 x 10(5)L/mol.     

Immunoassay for serologic diagnosis of influenza type A using recombinant DNA produced nucleoprotein antigen and monoclonal antibody to human IgG

Influenza type A nucleoprotein (NP) derived from the full length cloned gene expressed in E. coli was evaluated in a solid phase enzyme immunoassay (EIA) for detection of human antibody to influenza. Monoclonal antibody to human IgG was used for detection. Direct and indirect assays were developed and sera were tested in serial and single dilution formats. Preliminary results indicated that recombinant-and virion-derived NP antigens were comparable in binding ability to plastic and binding human antibody. Eighty-seven paired sera from influenza patients were tested. The most sensitive assay (indirect-serial dilution) detected 56 (64%) rises and the simplest assay (direct-single dilution) detected 43 (49%) rises, compared to 36 (41%) for complement fixation. Paired sera from 18 control patients showed no evidence of antibody rises by any of the assays. Forty-nine paired sera from influenza B infected patients were negative for antibody rises except for one borderline rise by the indirect-serial dilution assay. These results indicate that the use of recombinant DNA derived nucleoprotein for immunoassay is feasible. The sensitivity of immunoassays using NP adsorbed to the solid phase and monoclonal antibody specific for human IgG to detect bound antibody exceeded that of conventional complement fixation testing for establishing serologic evidence of influenza type A infection.     

抗人VEGF受体Ⅱ胞外Ⅲ区单克隆抗体的制备

目的 制备人血管内皮细胞生长因子受体Ⅱ(Kinase insert domain tontaining receptor,KDR)胞外Ⅲ区的单克隆抗体(McAb)，探讨其抑制VEGF诱导的体外活性。方法 在大肠杆菌中表达重组KDR胞外Ⅲ区融合蛋白。融合蛋白经抗E—tag柱分离纯化后免疫Balb/c小鼠，采用传统杂交瘤技术制备抗KDR胞外Ⅲ区McAb，采用ELISA和Western免疫印迹鉴定其亚类和抗原结合特异性，并测定单克隆抗体对VEGF165刺激脐静脉内皮细胞株ECV304增殖的中和活性。结果 成功筛选出一株能稳定分泌KDR胞外Ⅲ区单克隆抗体的杂交瘤细胞株(1D3)，其分泌抗体亚类为IgGl。该McAb可明显阻断VEGF165对脐静脉内皮细胞株ECV304的刺激增生作用。结论 抗人血管内皮细胞生长因子受体KDR胞外Ⅲ区McAb可通过封闭KDR而抑制VEGF活性，McAb 1D3具有潜在治疗肿瘤的活性。     

Production and characterization of a monoclonal antibody to human saposin C.

A murine IgG1 monoclonal antibody, termed 68-12, was produced against purified human saposin C. Immunoprecipitation and binding analysis indicated that the antibody reacted only with saposin C. Dot blotting and Western analysis demonstrated that antibody 68-12 also reacted with prosaposin and a higher molecular weight protein(s) in murine spleen and cerebral grey matter. Solid phase competitive radioimmunoassay against 125I labeled saposin C (0.25 micrograms/ml) showed no cross reactivity for saposin A, B and D up to 15 micrograms/ml. At a concentration of 50 micrograms/ml saposin A, B and D cross reacted 21, 1.5, and 49% respectively. Monoclonal antibody 68-12 appears to have potential utility in the purification, detection and quantitation of human saposin C and its precursor.     

Production of stable mouse x human hybridomas secreting HBs antigen-specific human monoclonal antibody by using in vitro sensitization

The stable mouse x human hybridomas were established by cell fusion of a mouse myeloma cell line, a P3-X63-Ag8-653 (P3-653) subclone, with human peripheral blood mononuclear cells (PBMs) from normal volunteers for production of human monoclonal antibody against a predefined, clinically important antigen of viral origin, hepatitis-B surface (HBs) antigen. For successful mouse x human fusions, it was extremely important to preselect volunteers and to sensitize their PBMs by using in vitro sensitization. The resulting mouse x human hybridomas have continuously secreted a relatively high amount of human monoclonal antibodies to HBs antigen over nine months without cloning.     

Immunomodulation using the recombinant monoclonal human B7-DC cross-linking antibody rHIgM12

A patient with Waldenstrom's macroglobulinaemia expresses a high titre IgM antibody in serum that binds both mouse and human dendritic cells (DC) in a B7-DC (PD-L2)-dependent manner. We have reported previously that purified antibody from patient serum activates immature and mature DC in vitro, enhancing the ability of these professional antigen-presenting cells to activate naive T cells, take up antigen, resist a cytokine-depleted environment and secrete immunomodulatory cytokines, such as interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha. Systemic treatment of experimental animals with this antibody induces potent anti-melanoma immunity and modulates protectively the recall response against antigen challenge through the airway in an experimental model of inflammatory airway disease. Here we describe a monoclonal IgM antibody derived from this serum immunoglobulin that recapitulates each of these earlier observations, providing direct evidence that M protein from the Waldenstrom's patient mediates these potent immunomodulatory effects. Furthermore, cell lines expressing this recombinant form of the human antibody provide the basis for developing this reagent for clinical application.     

Isolation of low-frequency class-switch variants from rat hybrid myelomas

Class-switch variants have been isolated from rat-rat hybrid myelomas by sib selection using a simple assay based on red cell-labelled antiglobulins. The variants detected are consistent with the gene order deduced from molecular cloning. They appear to arise spontaneously at a rate approximately ten-fold lower than for mouse cell lines but the rate of switching back to the parental isotype is substantial in comparison. An IgG2b variant antibody having the same specificity as CAMPATH-1 for human lymphocytes and monocytes is active in antibody-dependent cell-mediated killing (unlike the parental IgG2a) and may prove to be a valuable therapeutic antibody for immunosuppression and treatment of leukaemia and lymphoma.