Optimizing antibody production in batch hybridoma cell culture.

Optimizing productivity by hybridoma cells relies partly on developing suitable methods for screening and selection of high producing cultures and on understanding regulation of antibody production. In this study, the behavior of hybridoma cells in batch culture was investigated using flow cytometry, and a simple model for antibody production was used to explain production data obtained from these cultures. Surface antibody fluorescence values were found to closely follow the decreasing trend of specific antibody secretion rate over the course of several batch cultures. Therefore, for the hybridoma cell lines studied here (ATCC HB124 and TIB138), surface immunofluorescence levels can be used to select high producing cells as well as to monitor culture productivity. Surface and intracellular antibody fluorescence values were also found to be correlated for cells exhibiting a bimodal distribution with respect to intracellular antibody content. The population of cells containing a bimodal distribution with respect to intracellular antibody content. The population of cells containing lower levels of intracellular antibody was determined to secrete significantly less antibody than the population possessing high intracellular antibody concentrations. Factors which influence antibody production rates and possible strategies for optimizing monoclonal antibody yield are discussed.

Manipulation of heterogeneous hybridoma cultures for overproduction of monoclonal antibodies.

In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124) to obtain increased production of monoclonal antibodies (IgG2a), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface-associated antibodies and a fluorescence-activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry.

Comparative analysis of Haemophilus influenzae type b and Escherichia coli J5 lipopolysaccharides.

Haemophilus influenzae type b (HIB) and Escherichia coli J5 (J5) lipopolysaccharides (LPS) were examined to explore the basis of previously observed cross-protection. HIB-LPS and J5-LPS contained ketodeoxyoctonate, glucose, glucoheptose and glucosamine as common carbohydrate moieties, and laurate, myristate, beta-hydroxymyristate and palmitate as common fatty acids, although in different ratios. J5-LPS was five times more lethal than HIB-LPS for chick embryos. Weak serological cross-reactivity was observed by haemagglutination and two-dimensional immunoelectrophoresis. No significant cross-reactivity was demonstrated by enzyme-linked immunosorbent or toxicity-neutralisation assays. The cross-reactivity observed between HIB-LPS and J5-LPS was probably due to common components in the core glycolipid.

Pasteurella haemolytica: purification of saline-extractable proteins by isoelectrofocusing.

A procedure was developed for separating antigens associated with a saline extract of Pasteurella haemolytica serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the carbohydrate antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of mice with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent P. haemolytica was seen in mice injected with antigen Groups 2 and 4. Six calves were immunized with saline extract. These calves had greater resistance to experimental pneumonic pasteurellosis than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated calves by an enzyme-linked immunosorbant assay.

Immunogenicity of potassium thiocyanate extracted and electrofocused Pasteurella multocida X-73I antigens in chickens and mice.

The immunogenicity of antigenic fractions obtained by the extraction of Pasteurella multocida strain X-73 (serotype 1) with potassium thiocyanate (KSCN) was determined in chickens and mice. The initial KSCN extract was centrifuged at 105 000 X g, and the antigens were separated into a particulate fraction (40p) and a soluble supernatant fraction (40s). The ultracentrifuged fractions were further resolved by preparative electrofocusing. The 40p fraction was resolved into two subgroups having isoelectric points of 3.5-3.9 and 5.5-6.0; the 40s fraction was resolved into five subgroups ranging in isoelectric points from 4.4 to 9.0. The 40p fractions were antigenically similar and contained lipopolysaccharide (LPS) and protein. The 40s fractions were antigenically distinct from the 40p fractions and from each other; they contained proteins and polysaccharides but no LPS. The 40p antigens were strongly immunogenic in mice and chickens, whereas the 40s antigens were weakly immunogenic in chickens and not immunogenic in mice. The incorporation of Freund's complete adjuvant increased the immunogenicity of the 40s antigens in chickens. The 40p antigens induced greater frequencies of serological responses in chickens than the 40s antigens as detected by counterimmunoelectrophoresis and immunodiffusion. This suggested that the increased protection associated with the 40p antigens may have been the result of better antibody response. The toxicity of all the fractions was evaluated by determination of lethality for 10-day-old chicken embryos because of the sensitivity and reliability of the test. The 40p fraction had an LD50 = 0.38 micrograms, and the 40s fraction had an LD50 = 2.5 micrograms. Since the 40s fraction contained no detectable LPS, it is likely that two toxins are present, one which contains LPS and one which does not.

Purification of Pasteurella multocida antigens by ultracentrifugation and isoelectrofocusing.

A procedure was developed to purify Pasteurella multocida X-731 antigens extracted by potassium thiocyanate. The crude extract was centrifuged at 105 000 x g; the antigens were then separated into a particulate (40p) fraction and a soluble (40s) fraction consisting of proteins and polysaccharides. These fractions were antigenically different. The ultracentrifuged antigens were resolved further by preparative isoelectrofocusing. The 40p antigens focused in a pH range of 3.0 to 6.0; distinctive proteins focused at pH's of 3.5, 3.6, and 3.8. The electrofocused 40p antigens were antigenically similar. The 40s antigens were initially electrofocused in a broad pH range and were found within a pH range of 4.6 to 9.0. The process was repeated with a narrower pH range and antigens that were focused in a narrower pH range could be separated and unique antigenic activities identified. Specific antigens from defined pH ranges were pooled and examined further by immunoelectrophoresis, analytical electrofocusing, and sodium dodecyl sulphate--polyacrylamide gel electrophoresis.

Immunoelectrophoresis employing avian antisera for the detection and quantitation of Pasteurella multocida antigens.

Immunoelectrophoresis with various buffer systems at high and low pH was examined for suitability to detect and quantitate Pasteurella multocida antigens with turkey or chicken anti-P. multocida sera. Counterimmunoelectrophoresis was used to develop a buffer system for one-dimensional, two-dimensional, and rocket immunoelectrophoresis. The effects of pH, buffer, and molarity on resolution of immunoprecipitates were determined; 0.05 M sodium acetate-acetic acid buffer at pH 5.6 was the most suitable buffer. This buffer could be used in counterimmunoelectrophoresis with turkey or chicken sera to detect minute amounts of P. multocida protein antigens (4.3 ng/test) or lipopolysaccharide (3.12 micrograms/test). One-dimensional immunoelectrophoresis with the acetate buffer system required treatment of the gels with a 17% NaCl solution to induce immunoprecipitation of P. multocida lipopolysaccharide. Other techniques using the acetate buffer system did not require the high salt treatment. In two-dimensional immunoelectrophoresis, antisera migrated in the second dimension at pH 8.6, but did not migrate at pH 5.6. Rocket immunoelectrophoresis with the acetate buffer system was effective for quantitating P. multocida antigens.

Chemotaxis of fowl monocytes to Pasteurella multocida and associated antigens.

The interaction of monocytes and serum from Pasteurella multocida-immunized and non-immunized turkeys and chickens was studied for their chemotactic activity to lipopolysaccharide, free endotoxin, and P. multocida whole cells. Chemotactic factors were released from the interaction between either normal sera or antisera and the bacterial antigens. Heating of the sera affected their ability to interact and release chemotaxins. Heated normal sera were ineffective and heated antisera were less effective than unheated sera. Monocytes collected from immunized and non-immunized fowls responded to the chemotactic factors. This occurrence of heat-labile and heat-stable chemotaxigens in the sera suggests that antibody and complement are involved in the stimulation of chemotaxis.