High yield monoclonal antibody production in ascites

BALB/c males were mated with Swiss Webster/HPB females to produce a first generation cross. Hybridoma cells derived from fusing SP2/0 myeloma cells and histocompatible spleen cells were injected intraperitoneally into these mice to induce formation of ascites tumors and production of ascitic fluid containing large quantities of monoclonal antibody. Mice, 80 days old weighing between 28 g to 35 g, were treated with 0.5 ml of pristane 18 days before inoculation of 3.2 X 10(6) hybridoma cells. The first generation crosses, (BALB/c male X SW/HPB female)F1, produced up to 4 times more ascitic fluid of equally high antibody level over a longer period compared to the BALB/c parent. This first generation cross is a cost effective means for monoclonal antibody production.     

Purification of the fifth component of murine complement from ascites fluid

The murine complement component C5 was purified on an affinity column using a monoclonal anti-mouse C5 antibody. We describe in this paper that ascites fluid from normal (C5-sufficient) mice contains almost as much C5 protein as mouse serum. Since ascites fluid is much easier to obtain in large quantities it is a convenient source for the purification of this mouse serum protein.     

Parameters affecting ascites tumour formation in mice and monoclonal antibody production

Hybridoma cells injected intraperitoneally into mice induce formation of ascites tumours and production of ascites fluid containing high levels of monoclonal antibody. Several parameters affecting the growth of the immunoglobulin-producing tumours have been studied in order to define optimal conditions for ascitic fluid formation and monoclonal antibody production. Using hybridomas produced by fusing SP2/0 myeloma cells with immunized mouse spleen cells we have shown: (1) that the optimal number of hybridoma cells required to induce an ascites tumour was between 6 and 32 X 10(5) cells; (2) that each mouse should be treated with a maximum of 0.5 ml of pristane; (3) that the priming period for pristane should be 14 days prior to the injection of cells; (4) that ascites formation and monoclonal antibody production is significantly better in males; and finally (5) that the age of mice used should range between 43 and 78 days. Under these conditions each mouse produces on average 7-10 ml of ascites fluid, containing a high level of antibody, over a maximum period of 6 days. The animals should start producing between the 5th and 9th day and usually survive 11-16 days after being injected with the tumour cells.     

Monoclonal antibody production by hybridoma growth in Freund's adjuvant primed mice

Incomplete Freund's adjuvant was used as a priming agent prior to the injection of hybridoma cells in mice to expand monoclonal antibody production. Two hybridoma cell lines, FDO28B (IgG1) and FDO31C (IgM), which produce monoclonal antibodies reactive towards human placenta, were used. Monoclonal antibody was detected in the ascites fluids by agarose gel electrophoresis. It was found that the time interval between adjuvant priming and cell injection could be reduced to 1 day, allowing collection of ascites fluid containing monoclonal antibody within 2 weeks of priming. In addition, as few as 1 X 10(5) hybridoma cells were needed to collect approximately 5-7 ml of ascites fluid containing antibody detectable by gel electrophoresis. Thus priming with incomplete Freund's adjuvant enables production of large amounts of monoclonal antibody in a short time using a low number of hybridoma cells.     

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

Quality control of murine hybridoma secretory products was performed using two variations of the isoelectric focusing affinity immunoblot analysis. The first approach employed antigen-coated nitrocellulose placed on top of an acrylamide gel containing isoelectrically focused ascites to bind antigen specific monoclonal antibody (MoAb). Murine antibody bound to the insolubilized antigen was then detected with enzyme-conjugated anti-mouse IgG. In a second variation, focused ascites proteins were passively blotted onto nitro-cellulose and specific monoclonal antibody was detected with enzyme-conjugated antigen. Several batches of ascites containing anti-human IgG antibodies that were produced by 6 hybridomas over a 1-3 year period were assessed by IEF-affinity immunoblot analysis. Both immunoblot approaches permitted effective monitoring of immunoreactive antibody for pI microheterogeneity. IEF-affinity immunoblot patterns of unprocessed ascites displayed specific MoAb banding patterns with narrow pI ranges (less than or equal to 0.6 pH units), in contrast to the reported 5.5-8.0 pI range of polyclonal mouse IgG. Banding patterns obtained in the IEF affinity immunoblot typically displayed 3-5 major dense bands flanked by 2-4 minor fainter bands. Batches of ascites obtained years apart produced similar immunoblot patterns, indicating constant antibody production and confirming the stability of these hybridoma clones. Minor bands appeared in 2 earlier lots of ascites, suggesting possible modification of antibody during storage. IEF affinity immunoblot analysis is a useful tool for monitoring MoAb pI microheterogeneity as an indicator of antibody quality without the need for isolation of monoclonal antibody from culture medium or ascites.     

Specific immunoglobulin production and enhanced tumorigenicity following ascites growth of human hybridomas

Human X human hybridomas constructed with the B6 lymphoblastoid clone, which produces antitetanus toxoid (TT) antibody, and the lymphoblastoid cell line KR-4 or human hybrid myeloma KR-12, were adapted to growth as ascites in pristane-treated BALB/c nude mice by a single prior passage as a solid subcutaneous (s.c.) tumor in irradiated nude mice followed by in vitro culture. Both B6 X KR-4 and B6 X KR-12 hybrids produced anti-TT antibody and phenotypically resembled the lymphoblastoid KR-4, or the hybrid myeloma KR-12 parent, respectively. Growth as ascites increased the tumorigenicity of both hybrids in nude mice as measured by tumor incidence and rate of tumor growth. The observed increase in tumorigenicity of these hybrid cells after ascites growth was associated with a substantial loss of chromosomes. Passage of the B6 X KR-4 lymphoblastoid hybrid resulted in several reversible morphological changes characteristic of myeloma cells. These changes correlated with increased human Ig production. These observations provide a system for greatly amplifying human monoclonal antibody production.     

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.     

A modified method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells

Immunized BALB/c mice were injected with 10(6) viable Sp2/0-Ag14 cells to induce ascites fluid concurrently with maximum serum antibody activity. Ascites fluids developed in a systematic manner, yielded large amounts of specific polyclonal antibody even when the mice were injected with relatively small amounts of immunogen. A combination of an initial pristane injection prior to 3 intraperitoneal immunizations of immunogen emulsified in Freund's complete adjuvant (total volume of 0.1-0.2 ml per injection), followed 4 days later by injection of 10(6) viable Sp2/0-Ag14 cells, resulted in a consistent production of ascites fluid with substantial specific antibody activity.     

Growth of rat-mouse hybridomas in nude mice and nude rats

Athymic (nude) mice and rats were inoculated intraperitoneally with rat-mouse hybridoma cells secreting monoclonal antibodies to rat MHC class I antigens in order to improve the yield of antibodies. Pristane priming and subsequent intraperitoneal injection of the hybridoma cells in to nude mice yielded ascites which contained antibody in high concentration (10-15 mg/ml). Complete Freund's adjuvant, mineral oil, pristane or antibody-antigen complexes were used to induce ascites in nude rats, but only pristane priming did so consistently. The hybridoma cells in the ascitic fluid failed to secrete antibody, although they contained intracellular antibody. However, when the pristane-primed nude rats received 250-500 rads of total body radiation prior to injection with the hybridoma cells, they produced large amounts of antibody. When the nude rats were splenectomized and injected with the hybridoma cells, they also produced antibody in high titers. There was no in vitro inhibition of antibody formation by the hybridoma cells cultured in medium containing 15% serum from nude rats, but co-culture of the hybridoma cells with splenic lymphocytes from normal or nude rats markedly inhibited antibody production. These results indicate that the defect in antibody secretion by the hybridoma cells in the ascites of nude rats is due to the presence of radiation-sensitive suppressor cells in the spleen.     

Spontaneous release of cytotoxic alloantibody from viable cells sensitized in excess antibody

When viable cells sensitized in excess cytotoxic alloantibody are washed and resuspended in antibody-free medium, they spontaneously release appreciable quantities of antibody. The amount released is directly proportional to the concentration of alloantibody during sensitization. Spontaneous release was observed from all cell types tested (thymocytes, lymphocytes, leukaemia cells and ascites sarcoma cells) and with all alloantibodies tested (H-2, θ and Ly-B). In preliminary tests with radio-labelled H-2 antibody, the quantity of antibody released in a period of 2½ hours was 29 per cent of the antibody originally absorbed. Dissociation at 37° was greater (or more rapid) than at 1°. When washed sensitized cells were suspended in antibody directed to an antigen closely adjacent on the cell surface to the site of attachment of the first antibody, release of the first antibody was impeded.     

Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites

A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria parva, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetically radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/mmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. parva sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens.     

Dialysis-based bioreactor systems for the production of monoclonal antibodies--alternatives to ascites production in mice

Two commercially available bioreactor systems, CELLine and miniPERM, were evaluated for their ability to support the production of monoclonal antibody (mAb) from a variety of murine hybridoma cell lines. Production and purity of mAbs were compared between the two systems and with mouse ascites tumour fluid generation. The quality and purity of the mAb generated by each method was analysed on SDS-PAGE gels and the antibody immunoreactivity in each case was quantified by indirect ELISA tests. The relative benefits of conventional growth medium (Dulbecco's modified Eagle's media, DMEM) and serum-free medium (hybridoma serum-free media, H-SFM) using the miniPERM system were also analysed, in terms of the amount of antibody produced, cell concentration and specific antibody titre. In all cases, the CELLine units tested gave higher protein concentrations compared to the miniPERM system under the same conditions (means and 95% confidence limits are 4.2+/-0.8 and 2.1+/-0.8 mg/ml, respectively), yet the miniPERM system yielded greater total amounts over a similar culture period (428.7+/-243.3 mg compared to 183.3+/-100.9 mg in the CL-350 CELLine unit). When defined by specific ELISA titre, both bioreactor systems yielded mAb levels that compared favourably with those derived from ascites. In addition, SDS-PAGE analysis indicated that the bioreactor antibody product was relatively free of contaminating protein, whereas ascites tumour fluid preparations displayed significant levels of extraneous protein. This study has shown that both bioreactor systems are acceptable in vitro alternatives to the in vivo production of mAbs in mice.     

Ascites produced in recalcitrant hybridomas by backcrossing to a parental myeloma

Hybridoma lines frequently lose their ability to produce ascites upon extended cultivation in vitro. We have reintroduced genes for tumor formation into three independent hybridoma lines by backcrossing them to the parental myeloma. The parental myeloma cells were eliminated by a brief in vitro selective period in HAT medium, after which the cells were inoculated into pristane-primed mice. In two out of three cases the resultant lines were able to produce ascites without further subcloning or other manipulations. The antibody retained its specificity as judged by immunoblotting. This method is a rapid and efficient approach for the reestablishment of ascites production in hybridoma lines.     

Production of antibodies to human interferons in mice.

Neutralizing antibodies were raised in mice that had been inoculated repeatedly with moderate quantities of human leukocyte interferon highly purified by affinity chromatography on immobilized anti-interferon globulins. Interferon preparations of lesser purity sensitized the mice to subsequent inoculations of interferon and almost invariably caused death before anti-interferon titers developed. Antibody-purified interferon stabilized by sodium dodecyl sulfate was a superior antigen to interferon that had received mouse serum albumin as an additive. The amount of antibody could be augmented by experimental induction of ascites. The antibodies specifically neutralized leukocyte and lymphoblastoid interferons but not those interferons obtained cultures of human foreskin fibroblasts, embryonic kidney cells, and amnion cells.     

VEGF/Flk-1 interaction, a requirement for malignant ascites recurrence.

Vascular endothelial growth factor (VEGF) plays an important role in the production of ascitic fluid associated with malignant tumor growth. In an experimental model for malignant ascites formation, mice were inoculated intraperitoneally with syngeneic mouse sarcoma tumor cells. Ascites development was not prevented by administering tumor necrosis factor (TNF) simultaneously with the tumor cell inoculation. When the malignant ascites was first drained and renewal of ascites was monitored, however, a TNF dose-dependent inhibition of ascitic fluid accumulation was observed. Northern blot analyses indicated transient downregulation by TNF on the expression of VEGF mRNA in tumor cells. Monoclonal antibody, (mAb) DC101 generated against the mouse VEGF receptor Flk-1 prevented the recurrence of malignant ascites in mice similar to TNF inhibition. In addition, exogenous soluble human Flt-1 used as an inhibitor of endogenous VEGF binding also inhibited ascites recurrence. These data demonstrate that the observed inhibitory effect of TNF on reestablishment of malignant ascites can be achieved equally by inhibition of the interaction of VEGF with its receptor Flk-1.     

Serum antibody responses in mice to intermittent inhalation of ovalbumin dust

Serum antibody production and induction of antibody tolerance were monitored in mice following intermittent inhalation of ovalbumin dust (mass concentration = 4.2 mg m-3; diameter of 80% of particles less than 2.6 microns). Repeated inhalation of microgram quantities of dust stimulated serum antibody production and development of tolerance. The sequence of these responses is analogous to that following oral presentation of antigen, but is produced by a much lower dose when antigen is presented via the respiratory tract.     

Growth of rat-mouse hybridoma cells in immunosuppressed hamsters. An easy and effective method to prepare monoclonal antibodies from heterohybridoma cell lines

Rat-mouse hybridoma cells producing anti-mouse IgE antibodies were intraperitoneally or subcutaneously inoculated into newborn or suckling hamsters receiving rabbit anti-hamster thymocyte globulin from the day of birth twice a week for at least 3 weeks. The hybridoma cells were found to grow in the abdominal cavity of the hamsters as ascites tumor or in subcutaneous tissue as solid tumor without loss of antibody-secreting activities. For the production of ascites, 2-week-old hamsters were preferable to newborn hamsters. In 3-week-old hamsters, the hybridoma cells could scarcely survive. The antibody titers of the ascites were determined to be 10(5)-10(6) in the ELISA and in the ability to neutralize the skin-sensitizing capacity of mouse IgE antibodies. The rat monoclonal antibodies were easily separated from ascites, serum or cell culture supernatant with affinity chromatography using Affigel protein A-Sepharose and anti-hamster IgG-Sepharose columns. The described method could be efficiently applicable for the proliferation of other hybridomas, such as human-human, human-mouse or hamster-mouse, etc.     

Delayed antibody synthesis in mice after transfer of immune peritoneal fluid cells

Ascites fluid, rich in bacteriophage-neutralizing antibody, was produced when mice were treated first, with lethal or near-lethal whole body X-radiation; secondly, intravenous injection of spleen cells from donor mice immunized against bacteriophage; and thirdly, with an intraperitoneal injection of bacteriophage in Freund's adjuvant. The `immune ascites cells' were washed and transferred to other mice without further addition of antigen. The production of phage-neutralizing antibody in recipient mice showed the following properties.

(1) The highest rate of antibody synthesis occurred between the 5th and the 11th day after cell transfer. In contrast, spleen cells similarly transferred gave rise to antibody formation with the maximum rate of synthesis immediately after transfer.

(2) The antibody formation occurred essentially only in isologous recipients, not in homologous ones, whether the latter were pre-immunized against cells of the donor strain or not. With spleen cells, antibody synthesis was not impaired in homologous hosts for about 4 days after transfer, if the hosts were not pre-immunized against the donor strain.

(3) Freezing and thawing of the donor cells prior to injection into the hosts abolished subsequent antibody synthesis.

(4) Irradiation of the cells with 650 R. abolished antibody formation after transfer.

(5) Whole-body irradiation of the recipient mice resulted in increased antibody formation.

(6) When immune ascites cells were injected into newborn mice, high levels of antibody were found 13 days afterwards.

It is concluded (a) that the population of immune ascites cells carries both the specific information and the stimulus for antibody synthesis, and (b) that the antibody-forming apparatus is not yet present in a functional state at the time of transfer, but develops several days afterwards in the host mice.

     

Development and characterization of a monoclonal antibody against the tube precipitin antigen of Coccidioides immitis.

Primary infection with Coccidioides immitis is commonly accompanied by the production of an immunoglobulin M precipitin antibody which is detected by the tube precipitin (TP) assay or by the immunodiffusion assay for TP antibody (IDTP assay). In the present investigation, spleen cells from spherulin-immunized BALB/c mice were fused with SP2/O Ag14 myeloma cells, and the resulting hybridomas were screened for antibody to the IDTP antigen by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned by limiting dilution and injected into pristane-primed mice for ascites production. Characterization of antibody reactivity was accomplished with the IDTP assay, two-dimensional immunoelectrophoresis, and immunoblotting. An immunoglobulin G1 monoclonal antibody which reacts with the IDTP antigen of C. immitis is described. The epitope that is recognized by the monoclonal antibody is also present, but to a lesser extent, on a second coccidioidal antigen which has been designated antigen 2. The monoclonal antibody was not reactive in immunoblots of histoplasmin or blastomycin, indicating that the epitope recognized by this antibody may be specific for C. immitis.     

Protection against experimental pseudomembranous colitis in gnotobiotic mice by use of monoclonal antibodies against Clostridium difficile toxin A.

The pathogenicity of Clostridium difficile is due to the production of two toxins (toxins A and B). We prepared monoclonal antibodies against toxin A and determined whether axenic mice passively immunized with the monoclonal antibodies were protected against C. difficile disease. The mice were kept in an isolator and were given ascites fluid intravenously prior to challenge with a toxinogenic strain of C. difficile. Control mice and mice receiving ascites fluid devoid of toxin antibody died within 2 days and had high levels of toxins A and B in their feces. Mice that received ascites fluid containing high amounts of toxin A monoclonal antibodies directed against the repeating units of the toxin survived. In protected mice, toxin B levels were similar to those in dying mice, but toxin A levels were greatly reduced. These data show that passive immunity induced by monoclonal antibodies against toxin A was effective against pseudomembranous cecitis.