Partial purification and characterization of immunoglobulin production stimulating factor derived from namalwa cells

Summary We screened for immunoglobulin (Ig) production stimulating factor (IPSF) which enhanced Ig production of human-to-human hybridomas in serum-free culture, and found that culture supernatant and lysate of human lymphoblastoid Namalwa cells stimulated proliferation and Ig production of human-to-human hybridoma HB4C5 cells. The IPSF in Namalwa lysate was partially purified with DEAE-Toyopearl 650M, hydroxylapatite and Superose 6HR 10/30 column chromatographies. The partially purified IPSF was a macromolecule of about 500 000 dalton containing 72 000 dalton protein as a major component. The activity was stable at pH 6 to 12, but inactivated partially by heating over 40° C (60% decrease) and completely by trypsin digestion. These results suggest that the IPSF activity is due to its protein and heat-stable components. The Namalwa IPSF stimulated proliferation of human-to-human hybridomas but not that of mouse-to-mouse hybridomas. The IPSF also stimulated Ig production of human-to-human hybridomas derived from NAT-30 cells, but not that of other human-to-human or mouse-to-mouse hybridomas. NAT-30 is a human fusion partner derived from Namalwa cells. These results suggest that the Namalwa IPSF is an autocrine factor that stimulates proliferation and Ig production of hybridomas derived from NAT-30 cells. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture (Japan) and by Sapporo Bioscience Foundation.     

PPD-induced immunoglobulin production in human peripheral blood lymphocytes. I. Necessary conditions for inducing the response.

The ability of PPD to induce Ig production in human PBL was investigated. PPD proved to be a good B cell activator for inducing polyclonal Ig production in PBL from healthy Japanese. Comparative studies of this response with PWM-induced Ig production showed that the cellular mechanisms involved in the two responses were different. First, PBL from an atypical individual with a deficient IgM production to PWM responded normally to PPD with IgM production as well as IgG production. Secondly, in IgG production, the effects of the two mitogens (PPDand PWM) were additive. An analysis of the cellular requirements in PPD-induced Ig production clearly demonstrated that T cells played a role in this response as well as in the PWM-induced response. However, the head-to-head comparative study on the titration curves of helper T cells in the two responses showed that PWM-induced helper activity was 2 to 5 times more effective than PPD-induced helper activity. Moreover, PPD-induced helper activity was shown to be more sensitive to ionizing radiation than was PWM-induced helper activity. Thus, this system of PPD-induced Ig production may provide a useful tool for understanding the human antibody production system as well as the PWM-induced response.     

Protein production and purification

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.     

RS virus components. I. Isolation and purification.

RS virus was centrifuged in zonal rotor on 55% sucrose cushion. Three layers were collected: light (RS-LL) containing complement-fixing antigen, medium (RS-ML) containing both complement-fixing and virus particle antigens, and heavy (RS-HL) containing antigen associated with the virus particle. RS-LL was chromatographed on Sephadex G-200 column. Two peaks were obtained, containing complement-fixing activity (RS-LL-1 and RS-LL-2). After sonication (20 Hz, 30 min) RS-ML and RS-HL also were chromatographed on Sephadex G-200 column. Two protein peaks were obtained from each layer (RS-ML-1 and RS-ML-2 from medium, and RS-HL-1 and RS-HL-2 from the heavy layer), corresponding to RS virus proteins.     

Technological and economic aspects of coal biodesulfurisation

The sulfur found in coal is either part of the molecular coal structure (organically bound sulfur), is contained in minerals such as pyrite (FeS₂), or occurs in minor quantities in the form of sulfate and elemental sulfur. When pyrite crystals are finely distributed within the coal matrix, mechanical cleaning can only remove part of the pyrite. It can, however, be removed by microbial action requiring only mild conditions. The process involves simple equipment, almost no chemicals, but relatively long reaction times, and treatment of iron sulfate containing process water. Different process configurations are possibly depending on the coal particle size. Coal with particle sizes of less than 0.5 mm is preferably desulfurised in slurry reactors, while lump coal (> 0.5 mm) should be treated in heaps. Investment and operating costs are estimated for different process configurations on an industrial scale. Concerning the organically bound sulfur in coal there is up to now no promising biochemical pathway for the degradation and/or desulfurisation of such compounds.     

Protein A immobilized affinity cartridge for immunoglobulin purification

Recombinant Protein A was immobilized on a cellulose and acrylic composite matrix through Schiff base formation. Various factors that could affect the binding of immunoglobulin by the Protein A molecules immobilized on the solid matrix were studied to achieve optimum affinity purification. The spacer arm length and ligand concentration of Protein A were verified as factors crucial to optimized IgG purification. Liquid-phase environmental conditions such as pH and salt concentration also play important roles in adsorption capacity by affecting the molecular interaction between IgG and the immobilized Protein A. The rate of interaction between Protein A and IgG is rather fast, with minimal differences observed at 10-fold increases in the cartridge loading rate. This paper describes a cellulose/acrylic composite matrix for immobilizing Protein A, at an optimized ligand concentration, installed on a spacer arm of adequate length, to purify immunoglobulins from animal plasma. The fast-flow property of the cartridge made from such a matrix and its simplicity in operation provide effective means for purifying immunoglobulins on a relatively large scale.     

Somatic cell genetics of immunoglobulin production

Tissue culture lines of mouse myeloma cells have been used to study the somatic cell genetics of immunoglobulin production. Assays have been developed to identify and quantify mutants that have undergone changes in either the synthesis or structure of the immunoglobulin molecule. All of the classical types of mutants have been identified. What is unusual is that these mutants arise at a very high frequency. This genetic instability seems to be restricted to immunoglobulin genes. The fusion of mutant and wild-type cells allows the study of interaction of genes and gene products.