Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with porcine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

Summary The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with poreine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.Supported by the Deutsche Forschungsgemeinschaft, Carvas SFB 90     

Monoclonal antibodies against human beta-glucocerebrosidase

Monoclonal antibodies were obtained against the membrane-bound lysosomal enzyme beta-glucocerebrosidase (acid beta-glucosidase), which is deficient in Gaucher's disease. BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against beta-glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme. Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine-T procedure whereas a second one is not.     

Monoclonal antibodies identify individual determinants on mouse mammary tumor virus glycoprotein gp52 with group, class, or type specificity

Hybrid cell lines producing monoclonal antibodies against the C3H strain of mouse mammary tumor virus (C3H MMTV) were prepared by the fusion of mouse myeloma cells with the lymphocytes of BALB/c mice that were immunized with C3H MMTV. Approximately 10% of the hybrid cells initially plated after cell fusion produced immunoglobulins that reacted in antibody-binding assays with C3H MMTV; 40 of these cells were cloned, and 6 eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from serum and ascites fluid of syngeneic mice inoculated with the hybrid cells. All of the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the immunoglobulin M subclass and three produced immunoglobulin G2a. The monoclonal antibodies showed limited charge heterogeneity in light and heavy chains when analyzed by high-resolution, two-dimensional gel electrophoresis. Three serologically distinct specificities were observed when these ascites fluids were tested against different strains of MMTV. The antigenic determinants detected were the following: (i) a type-specific determinant unique to the C3H strain of MMTV; (ii) class-specific determinants shared between C3H and GR MMTVs; and (iii) a group-specific determinant found on C3H, GR, RIII, and the endogenous C3H (C3Hf) MMTVs. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the three patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule and also clearly show that one strain of MMTV can be distinguished from other strains.     

Production and characterization of monoclonal antibodies with P1 specificity

Four monoclonal antibodies, with P1 specificity were obtained after fusion of myeloma cells and spleen cells from mice immunized with turtle dove ovomucoid. Immediately after the fusion, the culture supernatants were studied for specificity with panels of erythrocytes and red blood cells sharing rare phenotypes (P1K, P2K, p) in the P system. After cloning, four monoclonal antibodies were produced, these antibodies strongly agglutinate P1 red blood cells, specially when they are used with 3% of dextran or with a 350 mmol/l concentration of sodium.     

Specific monoclonal antibodies to Opisthorchis viverrini.

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.     

Isolation and analysis of monoclonal antibodies against various gangliosides

Female BALB/c mice were immunized with human melanoma (Mewo) cells containing ganglioside GD3 as a surface antigen. Immune splenocytes were fused with syngeneic P3-X63.Ag 8 myeloma cells. Antibodies produced by hybrid clones were analyzed by solid phase immunoassay. B, C, D and Q clones producing antibodies against Raja clavata brain gangliosides were obtained. Monoclonal B and C antibodies bound monosialogangliosides. Monoclonal D antibody bound a number of gangliosides but reacted predominantly with GD1a. Monoclonal Q antibody reacted selectively with GQ1c. It is assumed that ganglioside GQ1c is expressed on the melanoma cell surface and may be found only in the early stage of ontogenesis of high vertebrates.     

抗黄曲霉尿酸氧化酶单克隆抗体的制备和初步鉴定

目的:制备抗黄曲霉尿酸氧化酶单克隆抗体。方法:采用杂交瘤技术,获得2株针对重组黄曲霉尿酸氧化酶(rUOX)的杂交瘤细胞McAb17和McAb3;采用盐析和A蛋白亲合层析柱纯化该抗体。结果:McAb17和McAb3的腹水效价达到1∶512000,纯化后获得纯度大于95%的单抗,抗体亚类(型)分别为IgG1/k型和IgG2a/k型;ELISA结果显示制备的单抗与rUOX和Rasburicase(商品化rUOX)可发生特异反应。结论:制备了抗黄曲霉尿酸氧化酶单克隆抗体McAb17和McAb3,为检测rUOX在动物体内的代谢变化提供了良好的实验基础。     

Advantages of using swine serum in culturing animal and human cell lines

Using the culture of lymphoid human cells, mice myeloma cells and hybridoma (the latter was obtained by fusion of myeloma cells with those of mice spleen immunized with phage lambda), the fetal serum was shown to be surpassed in growth activity by that from adult swines. This fact is especially pronounced at small serum concentrations. When hybridoma cells were cultured there were no differences in titre of monoclonal antibodies specific to phage lambda with the use of both sera. The possibility of substitution of swine serum for human one was demonstrated using the culture of lymphocytes from human peripheral blood.     

Rat monoclonal antibody specific for prostaglandin E structure

Six different monoclonal antibodies directed against prostaglandin E2 were obtained from hybrid myelomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a rat immunized with bovine serum albumin conjugates of prostaglandin E2. Four of them were of the IgG2a subclass and the other two were an IgG2b and an IgG2c. Affinities of antibodies for prostaglandin E2 were in the range 5.8 X 10(6)-6.7 X 10(8) M-1. Cross-reactivity experiments showed that one monoclonal antibody was directed almost exclusively against the prostaglandin E structure. The specific monoclonal antibody purified from ascites fluid was used for enzyme immunoassay, and as little as 30 pg of prostaglandin E1 and 100 pg of prostaglandin E2 were detected, which values are comparable to those obtained by radioimmunoassay. These results reveal that the hybridization technique is a reliable way to obtain prostaglandin E-specific antibody and that monoclonal antibodies can be valuable reagents for immunoassays.     

Monoclonal antibodies against ovalbumin

Fusion of cells of the NS-1 mouse myeloma line with spleen cells from $\text{BALB}\text{c}$ mice immunized against ovalbumin produced hybrid cells which continuously secrete antibodies specific for ovalbumin. One of these cells was used to establish a cloned line. Studies of its antibody obtained either from ascites fluid or from medium from hybridoma cultures showed high titer and specificity against ovalbumin using the double antibody technique with rabbit anti-mouse immunobeads; the antibody proved to be of the IgG₁ (kappa) subclass and type.     

Monoclonal antibodies to bovine blood group antigens

Hybridomas were made by fusing mouse myeloma cells with spleen cells from mice immunized with bovine red cells. Sixteen cloned lines which secreted haemolytic monoclonal antibodies reacting with antigens in the A, B, F, Z and S blood group systems were established; one of the antibodies identified a new factor in the B system. Extensive tests on red cells from 1000 animals indicated that several of the antibodies are suitable for use in routine blood typing; others are of potential use for genetic studies of the bovine blood group systems.     

TT病毒重组蛋白单克隆抗体的制备

采用杂交瘤技术,获得了4株稳定分泌抗TTV重组蛋白的单克隆抗体杂交瘤细胞株,1株属IgG2bλ链、1株属IgG1κ链、2株属IgG2aκ链。4株杂交瘤细胞培养上清液效价为1：80-1：1280,腹水效价为1：32万-1：160万。     

Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis

Antibody-secreting hybrid cells have been derived from a fusion between mouse myeloma cells and spleen cells from a mouse immunized with membrane from human tonsil lymphocyte preparations. Hybrids secreting antibodies to cell surface antigens were detected by assaying culture supernatants for antibody binding to human tonsil cells. Six different antibodies (called W6/1, /28, /32, /34, /45 and /46 were analyzed. These were either against antigens of wide tissue distribution (W6/32, /34, and /46) or mainly on erythrocytes (W6/1 and W6/28). One of the anti-erythrocyte antibodies (W6/1) detected a polymorphic antigen, since blood group A1 and A2 erythrocytes were labeled while B and O were not. Antibodies W6/34, /45 and /46 were all against antigens which were mapped to the short arm of chromosome 11 by segregation analysis of mouse-human hybrids. Immunoprecipitation studies suggest that W6/45 antigen may be a protein of 16,000 dalton, apparent molecular weight, while W6/34 and /46 antigens could not be detected by this technique. Antibody W6/32 is against a determinant common to most, if not all, of the 43,000 dalton molecular weight chains of HLA-A, B and C antigens. This was established by somatic cell genetic techniques and by immunoprecipitation analysis. Tonsil leucocytes bound 370,000 W6/32 antibody molecules per cell at saturation. The hybrid myelomas W6/32 and W6/34 have been cloned, and both secrete an IgG2 antibody. W6/32 cells were grown in mice, and the serum of the tumor-bearing animals contained greater than 10 mg/ml of monoclonal antibody. The experiments established the usefulness of the bybrid myeloma technique in preparing monospecific antibodies against human cell surface antigens. In particular, this study highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymorphisms.     

Monoclonal antibodies to bradykinin inhibit smooth muscle contractile action of bradykinin

Four hybridoma cell lines have been established that secrete monoclonal antibodies to nonapeptide bradykinin. Bradykinin coupled to ovalbumin, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, was used to immunize BALB/c mice. Spleen cells from the immunized animals were fused to P3-X63-AG8-653 mouse myeloma cells. The resultant hybrid cells were screened by enzyme-linked immunoassay for production of antibodies to bradykinin. Hybrids from four positive wells were subcloned by limiting dilution and expanded as ascites tumor into pristane-primed mice. All the four hybrids secreted monoclonal antibodies of IgG1 (k) isotype. Unlabeled peptides bradykinin, lysyl-bradykinin (kallidin) and methionyl-lysyl-bradykinin competed with the radiolabeled [Tyr1]kallidin for monoclonal antibody binding sites. These antibodies recognized preferentially either NH2- or COOH-terminals of the nonapeptide bradykinin and can distinguish between des-Arg1-bradykinin and des-Arg9-bradykinin. Bradykinin fragments smaller than eight residues were not recognized by these antibodies. Monoclonal antibodies BK-D6A5, BK-B6C9 and BK-A3D9 neutralized the smooth muscle contractile activity of bradykinin. An enzyme-linked immunoassay developed using these monoclonal antibodies showed the effective range of bradykinin determination between 5 and 150 ng.     

Light chain myeloma plasma cells induce a strong cell-mediated immune response mainly directed against the monoclonal light chain determinants in a murine experimental model

An important goal for the treatment of human B cell malignancies lies in the induction of an active immune response directed against the tumoral clone, and more particularly against antigenic determinants of the monoclonal immunoglobulin (Ig). The presence of idiotype-reactive T-cells in patients with multiple myeloma has been previously reported, and strategies to increase their responsiveness towards the malignant plasma cells are being actively explored. Light chain (LC) myeloma is often an aggressive form of the disease, regarding which antitumoral immunity has not yet been studied. Here, we investigated in an experimental murine model a secreted monoclonal LC that may behave as a strong tumor antigen after immunization of animals with mitomycin C-treated malignant plasma cells producing monoclonal Ig. Non-dividing plasma cells were utilized as a cell suspension to immunize the mice intraperitoneally (i.p.). The immunized mice produced anti-Ig LC antibodies, mounted a cell-mediated response mainly directed against the monoclonal LC determinants, and survived tumor challenge with significant frequency. These results suggest that plasma cells are capable of presenting antigenic determinants derived from a secreted monoclonal LC in an MHC class I context, and of predominantly inducing a monoclonal Ig-specific T-cell response which can contribute to tumor rejection.     

Monoclonal antibodies to yeast catalase T

Hybridoma cell lines secreting antibodies directed against $\text{Saccharomyces cerevisiae}$ catalase T were constructed by fusing spleen cells of mice immunized with catalase T with P3×63Ag8 mouse myeloma cells. Culture supernatants were assayed for specific antibodies by incubation with ³⁵S-labelled yeast extracts, adsorption of the immune complexes to Protein A — carrying $\text{Staphylococus aureus}$ cells and analysis of the adsorbed yeast proteins by sodium dodecylsulfate gel electrophoresis. Two hybrid clones were isolated mediating adsorption of a protein with electrophoretic mobility of catalase T; one of them, showing considerably higher activity, was characterized further. Anti-bodies produced by this clone belong to the IgG class of immuniglobulins; they can be used for immunoadsorption, but not for direct immunoprecipitation and recognize authentic catalase T as well as catalase T apoprotein.     

小鼠抗食蟹猴IgG单克隆抗体的制备

目的制备抗食蟹猴、恒河猴等非人灵长类实验动物免疫球蛋白二级抗体,开展对其传染病血清学快速诊断方法的建立。方法采用饱和硫酸铵盐析、Agarose-Protein G亲和层析技术,从食蟹猴血清中提纯IgG。经SDS-PAGE电泳鉴定,采用常规法免疫C57BL/6小鼠,三次免疫后取脾细胞与Sp2/0-Ag14骨髓瘤细胞通过PEG4000融合制备杂交瘤细胞,利用间接ELISA、Western blot等方法进行筛选、鉴定。结果得到5株阳性杂交瘤,分别命名为2B6、2B7、2D9、3B2、5E4,并且5株杂交瘤分泌的抗体均与恒河猴的IgG或血清发生交叉反应,而与其他物种如东北虎、犬等动物的IgG或血清无交叉反应。结论 5株杂交瘤产生的单克隆抗体（McAb）具有较好免疫活性,且能长期、稳定地分泌抗体。此项研究工作为后续研究食蟹猴、恒河猴传染病血清学诊断方法奠定基础。     

牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清白蛋白(BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。