IgA hybridomas: A method for generation in high numbers

An immunization regimen has been developed which yields a high frequency of hybridomas producing IgA isotype, antigen-specific antibody when spleen cells from immunized mice are fused with non-immunoglobulin secreting murine myeloma cells. Germfree BALB/c mice were carrier-primed with sheep erythrocytes (SRBC) by gastric intubation (GI) for 2 consecutive days followed 1 week later by GI with trinitrophenyl (TNP)-haptenated SRBC. After 7 days, spleen cells were fused with non-immunoglobulin secreting myeloma cells (X63-Ag8.653), and 2–3 weeks later, culture wells were scored for hybrid clones. Of 240 culture wells plated, 157 wells (65.4%) exhibited clones producing anti-TNP antibodies as determined by enzyme-linked immunosorbent assay. A total of 50 specific cell lines were established, of which 27 clones (54%) produced IgA isotype anti-TNP antibodies, while the anti-TNP antibodies produced by the remaining 23 clones were approximately equally distributed between the IgM and IgG isotypes. The IgA and IgM monoclonal antibodies were more effective in hemagglutinating TNP-SRBC than were IgG isotype antibodies. This study describes a method for production of a high number of antigen-specific IgA hybridomas which will allow production of IgA monoclonal antibodies to important antigens on mucosally-associated pathogens, and thus allow elucidation of functions of IgA antibody at mucosal surfaces.     

Mink-mouse interspecific hybridomas

Mink-mouse interspecific hybridomas were produced by fusion of the american mink spleen cells with the NSO cells. Seven cloned lines of the mink-mouse hybridoma were isolated, and their functional mink Ig secretion and karyological characteristics are given. During cytogenetic analysis of mink-mouse hybridoma cell lines, we observed the elimination of mink chromosomes, and inter- and intralineral variability of the numbers of the cells with particular quantities of mink DNA. We did not find that the characteristic peculiarities of mink DNA distribution in the hybridoma cell lines had any bearing upon the secretion or non-secretion of mink Ig. There was no synthesis of lambda-L-chains of mink Ig in line 7 cells because the line lost the lambda-gene. With the aid of in situ hybridization with 3H-labeled total mink DNA, a considerable transformation of hybridoma cell karyotype was observed. Multiple integration of the mink DNA into mouse chromosomes and the appearance of chromosomes not characteristic for either the mink or mouse parent cells were noted. Increasing numbers of cells with translocations of mink chromosomes fragments into mouse chromosomes were found in the hybridoma lines cultivated for lengthy periods.     

Establishment of gerbil-mouse heterohybridoma secreting immunoglobulin of Mongolian gerbil (Meriones unguiculatus) establishment of gerbil-mouse heterohybridoma

Hybridomas, generated by fusing myeloma cells with B lymphocytes, secrete monoclonal antibodies (mAbs) specific for an antigen. To establish hybridomas that secrete Mongolian gerbil (Meriones unguiculatus) mAbs, we immunized gerbils with keyhole limpet hemocyanin (KLH) and fused splenocytes from them with a non-secreting mouse myeloma cell line (P3-X63-Ag8.653). We obtained hybridomas that secreted immunoglobulin (Ig) against KLH. These hybridomas had more chromosomes than either parent and retained several gerbil chromosomes. Therefore, these cells were heterohybridomas with both gerbil and mouse chromosomes. They expressed gerbil Ig heavy-chain constant (IGHC) region mRNA, but not mouse gamma (γ) 1 IGHC. SDS-PAGE and Western blot analyses indicated that the cells secreted whole Ig molecules of gerbil origin. Moreover, the cells continued secreting Ig for several months.     

Production and characterisation of anti-theophylline monoclonal antibodies suitable for immunoassay

Spleen cells from BALB/c mice immunised with KLH-theophylline conjugate were fused with a mouse myeloma cell line P3-X63-Ag8.653, and antibody-producing hybrids were identified by enzyme immunoassay. Three cell clones were obtained, each capable of producing a unique monoclonal antibody to theophylline. Using these monoclonal antibodies, an immunoassay system for theophylline was developed.     

Production of monoclonal antibody with anti-B specificity, to be used as reagent for blood classification

The purpose of this research was to produce murine monoclonal antibodies with anti B specificity to be used as reagents in red cells typing. Balb/c mice were immunized with substance B from saliva of a donor secreting B antigen, in order to obtain spleen cells or splenocytes producing antibodies with anti B specificity. These cells were fused with mouse mycloma cells P3-X63.Ag8.653 by using the PEG technique. After seeded, the hybridomas secreting the putative antibodies were cloned through the limiting dilution technique to obtain monoclonality. Afterwards, the antibodies were evaluated using immunological and immunochemical methods. BMS-4 clone secreting antibodies IgM-kappa with anti B specificity were obtained, which displayed high potency, excellent avidity, great sensibility and long time stability. We think that this clone can be used to produce a red cell typing reagent.     

The efficient production of stable, human monoclonal antibody-secreting hybridomas from EBV-transformed lymphocytes using the mouse myeloma X63-Ag8.653 as a fusion partner

The mouse myeloma X63-Ag8.653 was fused to peripheral blood lymphocytes (PBL) from apparently healthy individuals, autoimmune patients and volunteers immunised with Rhesus (D) positive erythrocytes. Fusions were performed with or without prior transformation of PBL with Epstein-Barr virus (EBV). Using untransformed PBL, under the best conditions a mean fusion frequency of 8.4 X 10(-6) was obtained, with 22% of the resulting hybridomas secreting human immunoglobulin. Fusions with EBV-transformed cells gave fusion frequencies of 1.0 X 10(-4), with 85-90% of hybridomas secreting human immunoglobulin. The heterohybridomas formed in both cases cloned efficiently and had doubling times of 24-30 h. The heterohybridomas secreted human IgM, IgG and IgA of both kappa and lambda isotypes and culture supernatants contained up to 50 micrograms ml-1 of human immunoglobulin. Mouse immunoglobulin was not detected in the culture supernatants. 28 hybrids were selected for vigorous growth and antibody production by repeated cloning. Immunoglobulin synthesis was stabilised in 26 of these hybridomas after two or three cloning steps. The heterohybridomas have been successfully grown in large volumes for periods up to 15 months. It is concluded that the mouse myeloma X63-Ag8.653 is a suitable fusion partner with EBV-transformed B cells in the efficient production of human monoclonal antibodies.     

New antigenic clusters on human thyroglobulin defined by an expanded panel of monoclonal antibodies.

Twenty-seven hybridomas secreting monoclonal antibodies (mAb) directed against new antigenic clusters on human thyroglobulin (hTg) were obtained by fusion of the mouse myeloma P3-X63-Ag8 653 with spleen cells from BALB/c mice immunized with a mixture of hTg and six anti-hTg mAb with the aim of masking the corresponding antigenic clusters previously reported. Fourteen mAb were selected, produced in ascitic fluid and characterized. All these mAb were of the IgG1 subclass. Five new antigenic clusters on the hTg molecule were defined by the 14 mAb, extending the initial antigenic map of hTg to 11 clusters. These mAb were used in an attempt to probe the interaction between hTg and the autoantibodies from patients with Hashimoto's thyroiditis who do not recognize antigenic cluster II, a cluster whose recognition by anti-hTg autoantibodies is significantly associated with thyroid disorders.     

Production of human monoclonal IgG and IgM antibodies with anti-D (rhesus) specificity using heterohybridomas

Heterohybridomas secreting human IgM and IgG anti-D antibodies of the rhesus blood group system have been established by fusion of EBV-transformed anti-D secreting cells with the mouse myeloma cells X63-Ag8.653. Both classes of antibody reacted with all Rh-positive cells, some Du cells but not with Rh-negative or DB cells. Concentrations of both antibodies reached between 25 micrograms/ml and 50 micrograms/ml in the culture supernatants. The cell lines have been maintained in culture for 14 months and have been shown to be suitable for large-scale production of antibody.     

Production and partial characterization of monoclonal antibodies against 3,3',5-triiodo-L-thyronine

Spleen cells of Biozzi HL mouse (selection V) immunized with bovine albumin-triiodothyronine conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Thirteen monoclonal antibodies, selected by an enzyme-linked immunosorbent assay and a radioimmunoassay, were produced in mouse ascites fluid, purified and analyzed. All of them were IgG1 (kappa). The cross-reactivity of all these monoclonal antibodies with thyroxine (T4) was less than 0.2%. The association constant determined by Scatchard analysis ranged from 1.5 x 10(9) M-1 to 2.7 x 10(10) M-1.     

乙型脑炎病毒prM蛋白单克隆抗体的制备

目的 克隆日本乙型脑炎病毒(Japanese encephalitis virus,JEV)前膜蛋白(prM)编码基因,原核表达、纯化prM蛋白,以纯化产物为免疫原制备单克隆抗体(mAb);方法从感染JEV病毒的鼠脑中克隆编码JEV prM蛋白的基因并将其克隆入原核表达载体pET32a,转化大肠埃希菌BL21(DE3)LvsS后以IPTG诱导表达.表达产物经Ni-NTA纯化后进行SDS-PAGE分析.用纯化的蛋白免疫BALB/c小鼠,经细胞融合和亚克隆后筛选出能分泌识别prM蛋白的mAb的杂交瘤细胞株.用Western Blot和免疫组化方法检测单克隆抗体的特异性.结果 从鼠脑中克隆出约500 bp的JEV prM基因,将其克隆入原核表达载体中,在大肠埃希菌中获得了较好表达.纯化的prM蛋白免疫BALB/c小鼠,经传统细胞融合及筛选方法制备出单克隆抗体,抗体滴度为105.ELISA、Western Blot和免疫组化检测结果证实该株单抗具有较好的特异性.结论 成功的表达和纯化了日本乙型脑炎病毒的prM蛋白,并完成了单克隆抗体的制备,为乙型脑炎病毒感染的早期诊断及预防的研究奠定了良好的基础.     

Monoclonal antibodies specific for Corynebacterium sepedonicum, the causative agent of potato ring rot

BALB/c mice were immunized with Corynebacterium sepedonicum, and spleen cells from the immunized animals were fused with cells of the mouse myeloma line P3-X63-Ag8.653. Several hybridoma cell cultures were selected for further study. Monoclonal CS-B-5 was specific for C. sepedonicum and did not react significantly with other closely related phytopathogenic corynebacteria in an enzyme-linked immunosorbent assay (ELISA). As few as 10(3) organisms could be detected. This approach should prove useful for developing improved diagnostic procedures for a number of bacterial plant pathogens.     

Quantitation and purification of the Pasteurella multocida toxin by using monoclonal antibodies.

Pasteurella multocida toxin (PMT), derived from a toxigenic strain of P. multocida originally isolated from a pig with clinical atrophic rhinitis, was used to immunize BALB/c mice. Ninety-two hybridomas secreting monoclonal antibodies (MAbs) against PMT were produced by fusion of spleen cells from these mice with P3-X63-Ag8.653 myeloma cells. The specificity for PMT of the MAbs was ascertained by enzyme-linked immunosorbent assay and immunoblotting analysis. The interrelationship of a panel of 10 MAbs and their respective epitopes was characterized by a competitive enzyme-linked immunosorbent assay based on the biotin-avidin system and by an in vitro neutralization assay based on the cytopathic effect of PMT on embryonic bovine lung cells. In vivo neutralization of the lethal effect of PMT in mice was obtained by passive immunization with an anti-PMT MAb 2 days before challenge with PMT. PMT was quantified by a sandwich enzyme-linked immunosorbent assay with a lower detection limit of approximately 50 pg of PMT. Application of supernatant or bacterial extract from cultivation of toxigenic P. multocida to an affinity column containing immobilized MAb resulted in purification of PMT with a yield of 78 to 93% of the PMT applied.     

Application of monoclonal antibodies in the avian leukosis virus GS-antigen ELISA

Five hybridoma cell lines which secrete antibodies to avian leukosis/ sarcoma (ALV) group-specific (gs) antigens (gag gene products) have been established. The hybrid cells resulted from fusion of P3/X63-Ag8.653 myeloma cells with splenocytes of BALB/c mice which had been immunised with purified avian myeloblastosis virus (AMV). Screening of supernatant fluids was performed by an indirect double antibody sandwich enzyme-linked immunosorbent assay (IDAS-ELISA) and the immunoelectroblotting technique. Three hybrid clones secrete monoclonal antibodies (MCA) to 27,000 dalton polypeptides (p27) and two hybridomas produce monoclonal antibody directed to 19,000 dalton phosphoprotein (pp19). All five monoclonal antibodies belong to mouse immunoglobulin isotype IgG(1). MCAs directed to different ALV-p27 epitopes can replace polyclonal rabbit or hamster anti-gs sera in the DAS-ELISA in use for the detection of congenitally ALV-shedding hens. In albumen samples a 16- to 32-fold increase of sensitivity of ALV gs-antigen detection was obtained as compared to the DAS-ELISA employing polyclonal sera. Gs-antigens of a purified AMV preparation were detectable up to minimal concentrations of 13 pg/ml.     

Human monoclonal antibodies against blood group antigens. Preparation of a series of stable EBV immortalized B clones producing high levels of antibody of different isotypes and specificities

The EBV immortalization technique was used to produce stable clones, from B lymphocytes, secreting human monoclonal antibodies to Rh(D), Rh(G), Rh(c), Rh(E), Kell, A and A1 blood group antigens. These clones were obtained from peripheral blood lymphocytes of hyperimmunized plasmapheresis donors or from spleen lymphocytes of immunized patients. Mean levels of antibody concentration varied between 4 and 50 micrograms/ml. The antibodies obtained were of IgG1, IgG2, IgM or IgA class. Most of the clones have been stable for growth and antibody production during long periods of continuous culture, extending upto 4 years. Hybridization of two clones was effected with the human lymphoblastoid cell line KR-4 and with the mouse myeloma X63-Ag8.653, but did not result in any marked improvement of clone characteristics. One of the anti-Rh(D)-producing EBV-transformed clones was used to produce an anti-Rh(D) typing reagent which has proved satisfactory for 2 years in routine blood typing in several laboratories.     

Monoclonal antibodies to K88ab, K88ac and K88ad fimbriae from enterotoxigenic Escherichia coli

K88ab, K88ac and K88ad fimbriae derived from enterotoxigenic Escherichia coli strains involved in porcine colibacillosis were used to immunize BALB/c mice. Several hybridomas secreting monoclonal antibodies (MAbs) against the three intact K88 fimbriae subtypes were produced by fusion of spleen cells from these mice with P3-X63-Ag8.653 myeloma cells. Hybridomas producing MAbs with affinity for all 3 E. coli K88 subtypes proved to be the most frequent (248/303), but subtype-specific monoclonals (39/303) as well as MAbs reacting with two but not with the third subtype (16/303) were also produced. The antibody-containing culture supernatants from 71 selected hybridomas were characterized by enzyme-linked immunosorbent assay (ELISA) titrations, ELISA inhibition experiments and further examined by immunoblotting. Derivation of several MAbs specific for one of the E. coli fimbrial antigens, K88ab, K88ac or K88ad, was of interest in view of the extensive sequence homology in their primary structures. Specific binding of the MAbs to fimbriae on the surface of K88-positive E. coli strains was indicated by agglutination tests and visualized by immuno gold labeling and electron microscopy. The present MAbs against K88 fimbriae have potential veterinary applications for diagnosis and treatment of porcine colibacillosis. Preliminary results indicate the therapeutic value of oral administration of murine ascitic fluid containing anti-K88 MAbs to piglets experimentally infected with E. coli K88.     

Monoclonal antibodies to cyanogen bromide fragments of glycophorin A

Eleven mouse monoclonal antibodies directed against epitopes on CNBr peptides of the major sialoglycoconjugate of the human red blood cell, glycophorin A, have been produced by hybridomas derived from P3-X63-Ag8.653 myeloma cells and spleen cells from BALB/c mice immunized with purified glycophorin. The monoclonal antibodies could be divided into four groups according to their reactivities with CNBr peptides in a direct ELISA assay: one antibody (6B5) that binds solely to the aminoterminal octapeptide (CNBr3); two antibodies (8F10 and 9C3) that bind to CNBrl (residues 9-81); two antibodies (3D2 and 4C6) that are reactive with CNBr2, The C-terminal portion of the molecule (residues 82-131); six antibodies (1B4, 4C3, 4E7, 7B10, 7C11 and 9D6) which are cross-reactive with an epitope on both CNBr1 and CNBR3 glycopeptides. This cross-reactive epitope(s) appears to involve both carbohydrate and protein residues.     

Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG₁ subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.     

Production of a monoclonal antibody with specificity for the pellicle ofPneumocystis carinii by hybridoma

This study reports the establishment of cloned hybridomas between mouse myeloma cells (P3-X63-Ag8, 6·5·3) and spleen cells from BALB/c mice immunized with lung homogenates of nude mice (BALB/c nu/nu) heavily infected withPneumocystis carinii. A hybridoma subclone, designated 1E12-8, produced antibodies of an IgG₁ subclass. Using indirect immunofluorescent antibody techniques, the monoclonal antibody was found to be directed toward the pellicle antigen of air-driedP. carinii both in trophozoite and cyst forms, and to recognize theP. carinii antigen not only from nude mice but also from rats. The antibody did not cross-react with other components of the infected host lung tissue and showed little cross-reactivity with fungi examined. The monoclonal antibody should be useful in the isolation and identification of corresponding antigenic substances ofP. carinii and may provide a useful tool in epidemiology, taxonomy, and diagnosis of this organism.     

Production and characterization of specific monoclonal antibodies of the human thyroid stimulating hormone

Spleen cells from BALB/c mice immunized with human thyroid stimulating hormone (beta-subunit) were fused with mouse myeloma cells (P3/X63-Ag8) and five hybridomas secreting monoclonal antibodies (MAbs) were obtained. These hybridomas specifically recognize (hTSH) and do not cross-react with the other human glycoprotein hormones such as: luteinizing hormone (LH), follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hcG). The MAbs were of the IgG1 subclass and ascitic fluid from these hybridomas was purified by affinity chromatography on Protein A-sepharose CL-4B column to isolate the IgG1 active fraction. The affinity constant of these MAbs ranged from 3.2 x 10(10) to 1.5 x 10(11) M(-1).     

Production and use of monoclonal antibodies for the detection of apple chlorotic leaf spot virus

Stable hybridoma cell lines secreting antibodies specific for the apple chlorotic leaf spot virus (CLSV) were produced by fusing spleen cells of a Biozzi mouse immunized with CLSV P863 strain, with the non-secretory P3 X63 Ag8.653 myeloma cell line. Two hybridoma clones producing monoclonal antibodies of the IgG1 subclass were obtained. These monoclonal antibodies were used for virus detection by enzyme-linked immunosorbent assay (ELISA). In contrast to polyclonal antisera to CLSV, which always contain some antibodies to host components, monoclonal antibodies are highly specific for the virus. It was thus possible to develop a detection assay which is more sensitive and specific than the assays using polyclonal antibodies. Using monoclonal antibodies, it was possible to detect less than 0.1 ng/ml of purified virus. In addition, these two monoclonal antibodies recognize 17 strains or isolates maintained in our laboratory and representing most of the known CLSV strains.