Hybridoma Secreting Monoclonal Antibody Research Articles

Identification and characterization of the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.

Efficient hybridoma production using previously frozen splenocytes

Splenocytes from immunized mice and rats can be frozen, thawed, and fused with myeloma cells to generate monoclonal antibody-secreting hybridoma cell lines. The hybridoma yeild per splenocyte is comparable to, and often better than, the yield when fresh splenocytes are used. Practical advantages of the technique include the ability to utilize all splenocytes from a given mouse or rat, greater scheduling flexibility, and the opportunity to perform small-scale ‘test fusions’ to monitor the immune responses of a number of immunized animals.

Monoclonal antibodies against different epitopes on colonization factor antigen I of enterotoxin-producing Escherichia coli

Hybridoma-secreting monoclonal antibodies (MAbs) against colonization factor antigen I (CFA/I) were produced by the fusion of spleen cells from immunized BALB/c mice with F/O myeloma cells. The 25 MAbs with the highest antibody titer against CFA/I, as determined by an enzyme-linked immunosorbent assay, were studied for specificity and the capacity to agglutinate CFA/I-carrying bacteria. Most of the MAbs agglutinated a majority of 16 CFA/I-positive strains tested but not any of a number of CFA-deficient mutants or strains carrying other colonization factors (CFA/II and CFA/IV). One MAb that agglutinated all the CFA/I-positive strains and one MAb that did not agglutinate any of these strains were compared for their reactivities with different preparations of CFA/I. Whereas both MAbs bound to or could be inhibited by isolated CFA/I fimbriae as well as the subunit protein, only the agglutinating MAb bound to CFA/I, as expressed on whole bacteria. The nonagglutinating MAb, on the other hand, bound considerably better than the agglutinating MAb to a peptide corresponding to the 46 N-terminal amino acid residues of the CFA/I subunit protein. These results suggest that the two MAbs are directed against different epitopes on CFA/I.

Class 1 (unique) tumor antigens of human melanoma: identification of unique and common epitopes on a 90-kDa glycoprotein.

Antibodies present in the serum of melanoma patient FD detect an antigenic determinant (FD) restricted to the autologous melanoma cell line SK-MEL-131. This cell-surface determinant is carried on a glycoprotein of 90 kDa, designated gp90. Mice were immunized with a partially purified preparation of gp90 derived from SK-MEL-131 clone 1.5, and three murine hybridomas (KF23, KF26, and KF104) secreting monoclonal antibodies (mAbs) detecting this antigen have been generated. Sequential immunoprecipitation experiments demonstrate that the three mAbs and human FD serum react with the same gp90 species. The mAbs, in contrast to FD serum, react with a broad range of cultured cells in assays for cell-surface antigens and immunoprecipitate a gp90 component from radiolabeled extracts of these cells, including autologous FD B cells. We conclude that gp90 from SK-MEL-131 has two types of determinants: restricted (detected by FD serum) and common (detected by the mouse mAbs). gp90 molecules from cell lines other than SK-MEL-131 carry only the common determinant(s). Immunoperoxidase analysis of frozen tissue sections with mAb KF23 demonstrated a restricted gp90 expression in normal tissues (capillary endothelial cells, duct epithelium in sweat glands, breast, and pancreas). Melanomas and sarcomas showed strong gp90 expression, suggesting up-regulation of gp90 synthesis in certain human cancers.

Monoclonal Antibody to Human Cross-Linked Fibrin

Cross-linked Fibrin II was prepared using Kabi grade (L) fibrinogen. Fibrin plasmic digest was separated on Sepharose CL-6B. Fragments Mr 135-300 kDa were used to immunize 6-9 weeks old female BALB-c mice. A stable hybridoma secreting monoclonal antibody (MAb) TD-1 (IgG 2a, Kappa) was prepared by fusion using myeloma cells (P3-NS1/1-Ag4-1) and immunized cells. Fibrinogen and plasmin digest of fibrinogen in serial dilutions did not compete with the immunizing antigen. To prove that TD-1 binds specifically to cross-linked fibrin, immunoprecipitation with S. aureus and affinity chromatography were performed. In both experiments, we demonstrated that TD-1 binds specifically to a protein Mr greater than 200 kDa which is found in XL-fibrin and not fibrinogen. Reduced samples showed the antibody bands (heavy and light chains) and three protein bands, Mr greater than 80 kDa (gamma-gamma dimer), Mr greater than 45 kDa (beta chain of fragment D) and Mr greater than 16 kDa (alpha chain from fragment D) were present. TD-1 reacted strongly with HPLC fraction of the immunizing antigen Mr 220 kDa (probably DD/E complex). Affinity binding constants (Scatchard Plot Analysis) were determined. The highest affinity was obtained with XL-fibrin fraction Mr 220 kDa, KD = 1.39 X 10(-8) and high molecular weight XL-fibrin fragments, KD = 1.6 X 10(-7). Fragment DD had KD of 2.8 X 10(-6). These results suggest that TD-1 is specific for the DD region of human cross-linked Fibrin II.

Production of monoclonal antibodies against RNA polymerase I from nonimmunized autoimmune MRL/lpr mice and their use in rDNA transcription analysis.

A hybridoma secreting monoclonal antibodies against RNA polymerase I was produced by the fusion of myeloma cells with spleen cells from a nonimmunized MRL/lpr mouse which is known to produce autoantibodies to RNA polymerase I. The antibodies (McAb-2D11) belong to the IgG2b subclass, reacted specifically with the second largest (120 kDa) subunit of RNA polymerase I, and inhibited accurate transcription of cloned rat rDNA in a fractionated cell extract following immunoprecipitation of RNA polymerase I. McAb-2D11 did not inhibit RNA polymerase II-mediated transcription of the mouse metallothionein-I gene. Immunocytochemical procedures with biotinylated second antibody demonstrated specific immunolocalization of RNA polymerase I in the nucleus. These studies have (a) presented direct evidence that autoantibodies to functional RNA polymerase I are produced in a murine model of systemic lupus erythematosus, (b) demonstrated specificity of the monoclonal antibody for RNA polymerase I, and (c) provided a useful tool for the purification of RNA polymerase I and/or transcription factor(s) associated with RNA polymerase I.

Monoclonal antibody specific for T cell-derived human IgE binding factors.

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.

Establishment and characterization of a human hybridoma secreting monoclonal antibody with high titers of sperm immobilizing and agglutinating activities against human seminal plasma

Peripheral blood lymphocytes isolated from an infertile woman possessing strong sperm immobilizing and agglutinating antibodies were stimulated by culturing with poke-weed mitogen (PWM) and spermatozoa from a healthy donor for 5 days. The stimulated lymphocytes were fused with mouse myeloma NS-1 by PEG-1000. Fused growing hybrid cells were observed in 58 of 96 wells, and 22 of these showed the production of human immunoglobulin. Among the 22, one hybridoma clone (H6-3C4) was found to produce human IgM (λ) with strong sperm immobilizing and agglutinating activities. The supernatant from the culture medium contained approximately 1.5 μg IgM/ml and the antibody titers were 5000 SI 50 units on sperm immobilization and 1:1600 dilutions on sperm agglutination. The hybridoma H6-3C4 has continuously produced high titers of antibody exhibiting sperm immobilizing and agglutinating activities over 8 months and contains chromosomes of acrocentric type from mouse and metacentric type from human. The monoclonal antibody (Mab) H6-3C4 reacted specifically to human seminal plasma, ejaculated spermatozoa and male accessory gland but not to testis, any other somatic tissues, or secreted fluids tested. Immunofluorescence staining indicated that the antigen corresponding to Mab H6-3C4 was present over the surface of ejaculated spermatozoa. The binding of Mab H6-3C4 to human spermatozoa was blocked by the serum of the patient from whom the lymphocytes were obtained for cell fusion.

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 × 10 −6 was obtained, with 22% of the resulting hybridomas secreting human immunoglobulin. Fusions with EBV-transformed cells gave fusion frequencies of 1.0 × 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 μg ml −1 of human immunoglobulin. Mouse immunoglobulin. M 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.

Monoclonal antibody against non-histone chromosomal protein high mobility group 1 Co-migrates with high mobility group 1 into the nucleus.

Non-histone chromosomal protein high mobility group 1 (HMG-1) rapidly migrates into the nucleus when injected into the cytoplasm of bovine fibroblasts and HeLa cells by red cell-mediated microinjection (Rechsteiner, M., and Kuehl, L. (1979) Cell 16, 901-908). We isolated hybridomas secreting monoclonal antibodies against HMG-1. One of these monoclonal antibodies, FR-1, inhibited in vitro binding of 125I-HMG-1 to chromatin isolated from FL cells. When 125I-HMG-1 was co-introduced with antibody FR-1 by red cell-mediated microinjection, antibody FR-1 did not prevent the accumulation of 125I-HMG-1 in the nucleus. When 125I-antibody FR-1 or fluorescein isothiocyanate antibody FR-1 was introduced into the cytoplasm of FL cells, most of the antibody did not accumulate in the nucleus. But when 125I- or fluorescein isothiocyanate antibody FR-1 was co-introduced with HMG-1 into the cytoplasm of FL cells, it did migrate into the nucleus.

A murine monoclonal antibody recognising a single glycoprotein within a human cytomegalovirus virion envelope glycoprotein complex.

Nonionic detergent solubilised polypeptides from highly purified human cytomegalovirus virions were used as immunogens to produce murine monoclonal antibody secreting hybridomas. One monoclonal antibody was shown, by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), to precipitate three glycoproteins with molecular weights 52, 95, and 130 (all X 10(3)) and one minor component with a molecular weight of 50 X 10(3). When virion envelope components were first separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes, this monoclonal antibody recognised two related components with molecular weights 50 and 52 (both X 10(3)). Immunofluorescence studies suggested that these viral antigens were associated with membrane systems of virus-infected cells and were particularly abundant late in infection.

A rapid and efficient immunization protocol for production of monoclonal antibodies reactive with autoantigens

A simple, time-saving and efficient immunization method suitable for the production of mouse monoclonal antibody secreting hybridomas is described. Draining lymph nodes isolated 9 days after a primary immunization were used as the source of antibody producing cells. No systemic spread of antibody producing cells or specific antibodies could be detected. The present protocol was employed to generate a panel of collagen type II reactive monoclonal antibodies. Most of the monoclonals so generated were found to be of the IgG class.

A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas

In this study the exposure period of the lymphocyte-myeloma cell mixture to the fusogen was evaluated for its influence upon the yield of total hybridoma colonies and those which secreted monoclonal antibodies. Sp2/0 and FOX-NY myeloma cells were fused for varying periods with murine splenic lymphocytes immunized with sheep red blood cells. The optimal fusion period consisted of adding the fusogen (5.0 ml Kodak 1450 PEG, 0.5 ml dimethylsulfoxide, and 4.5 ml of phosphate-buffered saline, pH 7.0) to the cell mixture over a 45 s period at 37°C. The fusion process was stopped by gradually diluting the mixture in 50 ml of RPMI-1640. After 10 min, the cells were centrifuged, resuspended in selective medium with feeder macrophages and cultured. In comparison to common, longer fusion techniques, this procedure produces approxiamately a 5-fold increase in the number of hybrids produced when using the Sp2/0 cells and a 30-fold increase in the number of hybrids produced when using the FOX-NY cells as the fusion partner. In both cases, virtually all the wells contain monoclonal antibody-secreting hybridoma colonies. This high efficiency fusion technique can be used most advantageously to produce monoclonal antibodies against weak immunogens or to reduce the time needed for immunonization with stronger immunogens.

Clinical applications of monoclonal antibodies.

Immunology has come a long way in the hundred or so years since the general concepts were first enuciated by Metchnikoff, Ehrlich, Von Bebring and others, One of the landmarks in this progress was the invention and development of monoclonal antibody secreting hybridomas by Milstein and bis co-workers in Cambridge. Unlike most modern inventions of this importance that of monoclonal antibody production was made available to the scientific community tbroughout the world unimpeded by patent protection. This may explain tbe unusual rapidity witb which it has been applied to the benefit of mankind in general. This book, representing as it does the proceedings of tbe first International Symposium to be held on the clinical appli- cations of monoclonal antibodies, shows just how much bas been achieved within the space of little more than a decade. The enormaus promise of monoclonal antibody technology, which became apparent soon after its discovery, has already progressed a long way towards fulfillment. The contributors to tbis volume, all of whom are actively engaged in monoclonal antibody development and application, represent the state of the art. Professor Vincent Marks V INTRODUCTION It has been some twelve years since the pioneering experiments of Kohler and Milstein led to the discovery of monoclonal antibodies. Single molecular species antiborlies with desired specificities could be produced by the fusion of antibody - producing cells with neoplastic cells.

A Human Monoclonal Antibody Reactive with Human Prostate

A human immunoglobulin M monoclonal antibody secreting hybridoma, termed MHG7, has been isolated and characterized for its reactivity against human prostate cells. Lymphocytes isolated from a regional draining lymph node of a patient with prostate carcinoma were fused with murine P3-NS1-Ag4-1 myeloma cells. Supernatants from the generated mouse-human somatic cell hybrids were first screened for human immunoglobulin production by an enzyme immunoassay. The identified human immunoglobulin-secreting hybridomas were expanded for further analysis and their supernatants screened by enzyme immunoassay against a panel of prostate cell lines. The human immunoglobulin M monoclonal antibody MHG7, in addition to reacting with prostate cell lines, also reacted with prostate carcinoma cells and benign prostatic hypertrophy cells on both frozen and paraffin embedded tissue sections. These data suggest that regional draining lymph nodes of prostate carcinoma patients can be used as a source of human lymphocytes for generating human immunoglobulin-secreting hybridomas reactive with human prostate cells.

Monoclonal antibodies reactive with different sites of N and H antigenic particles of poliovirus.

We established three hybridomas (designated 2A12, 4C4 and 6H8) secreting monoclonal antibodies which react with N and H(heated) antigenic particles of poliovirus type 1 (Mahoney strain). One of these monoclonal antibodies, 4C4, had virus-neutralizing activity, while the other two were non-neutralizing. A solid-phase radioimmunoassay and a neutralization test indicated that the specificity of the neutralizing monoclonal antibody 4C4 was the same as that of the monospecific antibody HN31, previously prepared by us by absorbing poliovirus antiserum with an equine serum inhibitor-resistant mutant (M-HN31) of poliovirus. Furthermore, by means of immune electron microscopy, it was revealed that the combining site of the 4C4 neutralizing monoclonal antibody is on or around vertices of the N and H antigenic particles, while the other two monoclonal antibodies are directed to the entire surface of the antigens.

Production of IgA secreting hybridomas: a monoclonal rat antibody of the IgA class with specificity for RT1 c

We report a method for the production of rat hybridomas secreting IgA antibodies with biological activity. By fusing the rat myeloma Y3.Ag.1.2.3 with mesenteric lymph node cells taken from animals that had been immunised via the Peyer's patches with allogeneic cells we have obtained a hybridoma secreting monoclonal antibodies of the IgA class with specificity for RT1 c.

Replica plating of cultured mammalian cells.

A simple device is described which permits the replica plating of lymphocytes as well as substrate adherent fibroblasts. The replica plating device consists of an 8 X 12 array of cotton-tipped swabs (Q-tips). The principle of the replica plating system involves the use of an array of absorbent cotton-tipped swabs to transfer nonadherent lymphocytes (or conditioned medium) to corresponding wells of 96-well microtiter plates. Replica plating of substrate adherent cells requires one additional step; i.e., dislodgement of adherent cells by rotating the cotton-tipped swabs against the culture surface of each well. Once adherent cells are dislodged, replica plating of adherent cells can be carried out in an identical fashion to the replica plating of nonadherent lymphocytes. Application of the Q-tip replicator in the isolation of monoclonal antibody secreting hybridomas is also presented.

Preparation, characterization, and primate testing of monoclonal antithymocyte globulin.

Monoclonal antibodies specific for T cells from both the human and rhesus primate species were detected by their ability to inhibit T cell rosette formation with sheep erythrocytes. The antibodies were shown by fluorescence techniques to react with all thymocytes and peripheral blood T cells but not to B cells, monocytes, platelets, or erythrocytes. Rosette inhibition titers of these antibodies were 30-fold lower when rhesus, rather than human T, cells were used as the rosette-forming cell in assay. Nonetheless, two monoclonal antibodies, of the IgG3 isotype, termed antithymocyte monoclonal (ATM) e.1 and 2.2, were shown to depress selectively circulating T cells to nondetectable levels following single dose administration to rhesus primates and to prolong skin allograft survival in a rhesus primate given a 6-dose course of treatment. The rhesus primates suffered no ill effects and no peripheral blood cellular component other than T cells was depressed. Monoclonal antibody secreting hybridoma cells are capable of producing ATM 3.1 and 3.2 in quantity when grown as peritoneal tumors in selected mouse hybrids. Purification of ATM 3.1 or 3.2 is easily accomplished by affinity chromatography on protein A. These properties suggest that ATM antibodies may become useful immunosuppressive agents in clinical transplantation.

A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2.

A hybridoma-secreting monoclonal antibody was produced from the spleen cells of a mouse immunized with human thymocytes. This hybridoma antibody, termed OKT5, was reactive by indirect immunofluorescence with 80% of human thymocytes but only 20% of peripheral blood T cells. Moreover, OKT5 was unreactive with normal B cells, null cells, and macrophages at any dilution tested. A similar pattern of reactivity was seen with an equine antiserum to human thymocytes termed anti-TH2. Fluorescence-activated cell sorting demonstrated that the OKT5 antibody reactivity on peripheral T cells was restricted to the majority of the previously defined TH2+ subpopulation. In functional studies, the OKT5+ subset, like the TH2+ subset, proliferated well to the mitogen Con A and to alloantigens, and contained cytotoxic effector cells after sensitization in MLC, and suppressor effector cells after activation with Con A. In addition, like the TH2+ T cell, the OKT+ T cell was virtually unresponsive to soluble antigen. Thus, the OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset. OKT5 should provide an important probe to assess the status of suppressor cells in human disease.