Protein separation by differential drainage from foam.

Commercially available beer, which is a dilute solution containing components of yeast, malt, and hop used in the manufacture of the beer, was used as a model system to demonstrate the potential of foam fractionation beyond the primary foaming stage. Most of the components present in the beer concentrated in the initial foam, but they drained differentially in the subsequent collapsed foam collected over a period of 30 min. This resulted in further enrichment, in particular, of components which were present in low concentration in the original beer, Preferential drainage from foam, hence, might provide a novel way of fractionating further the proteins concentrated initially in the liquid films of foam. (c) 1994 John Wiley & Sons, Inc.

Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery.

The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth.

Ligand location and biochemical productivity of silica-based immunoaffinity adsorbents.

Ligand location within particles, detected by immunogold labelling, was shown to influence the biochemical productivity of a silica-based solid phase, Sorbsil C-500, using a model ligand-biomolecule system (immobilised human immunoglobulin G-anti-human immunoglobulin G monoclonal antibody). The distribution of the ligand was in turn affected by the initial ligand challenge used to prepare the immunoadsorbents. Maximal productivity was achieved with adsorbents prepared with an initial challenge of about 3 mg human immunoglobulin G per ml: the ligand in these cases was shown to be more uniformally distributed within the adsorbent particles than adsorbents, exhibiting low productivity, prepared with either low (1 mg/ml) or high (9 mg/ml) concentrations of human immunoglobulin G. The ligand in the latter was restricted to the periphery of the particles.

Silica-based solid phases for affinity chromatography: Effect of pore size and ligand location upon biochemical productivity.

The influence of pore size and surface chemistry upon the productivity in affinity chromatography of three silica-based solid phases, Sorbsil C-200, C-500, and C-1000 (40-60 microm particle diameter and the corresponding pore diameters of 20, 50, and 100 nm), was studied using three model ligand/biomolecule systems of varying molecular masses. These studies revealed two unique parameters, biochemical productivity and maximum physical capacity, of the matrix as generically essential in the successful design and operation of productive affinity chromatography systems. Biochemical productivity, the molar ratio of the amount of product recovered per unit volume of adsorbent and ligand concentration, utilized the expected stoichiometry of binding of the two molecules to assess the efficacy of the adsorbent. This parameter, determined by equilibrium binding in batch suspensions and by saturation binding capacities and recoveries in fixed beds, yielded the optimum ligand concentration required for maximal performance. Maximum physical capacity, of the adsorbent to accommodate the biomolecules, was calculated from pore and molecular dimensions assuming that there was no steric hindrance to access. Using an immobilized human-IgG (Hu-IgG)/anti-Hu-IgG monoclonal antibody (MCAB) system, in which both the ligand and the product are of the same size (150 kDa), it was shown that the physical capacity of C-200 was only 16% of the theoretically expected amount. This capacity increased to 70 and 90% of the expected value with C-500 and C-1000, respectively, as the steric hindrance to protein penetration induced by pore dimensions decreased. The distribution of immobilized Hu-IgG within individual particles, visualized by immunofluorescence and immunogold labeling, showed that the ligand was restricted to the peripheral 3 microm of the C-200 particles (12% radius). In contrast, it was present throughout the C-1000 particles, indicating that there was no hindrance to access in this solid phase. The C-200 was suitable for use in a small ligand/biomolecule system studied (immobilized trypsin-inhibitor binding trypsin; 22.1 and 23.3 kDa, respectively) for which more than 60% of the maximum physical capacity was available for interactions. The C-500 proved satisfactory for the Hu-IgG/MCAB model system but showed steric limitations when an immobilized anti-beta-galactosidase MCAB (anti-beta-gal) was used to purify a larger product (beta-galacosidase; 460 kDa). The binding capacity and overall productivity of Hu-IgG- and anti-beta-gal-C-1000 was equivalent to that of Sepharose CL-4B. Selection of matrices with pore sizes appropriate to the dimensions of the ligand and product was, therefore, important. Finally, the Sorbsil silicas packed easily into beds and were used successfully with conventional chromatography equipment for low-pressure affinity chromatography. They therefore offer an ideal alternative to silica-based high-performance liquid affinity chromatography and soft-gel supports.

Determination of purity and yield.

The dual concepts of protein purity and yield are so basic in protein chemistry that it is easy to forget that both of them are almost impossible to define in absolute terms. A protein is totally pure only when it 1s known to contain only a single species uncontaminated with salts or adventitious water. Samples of such a "pure" protein will yield a single band after electrophoresis on a one- or two-dimensional SDSPAGE gel, will elute from a gel filtration, HPLC, or ion exchange column as a single symmetrical absorbance peak, will yield a single set of mass spectrometric, NMR, or W absorbance spectral signals, and where appropriate, will be free of contaminating enzyme activities. However, many tests are used to assess the purity of a product. Ultimately, they all share one property: Rather than prove that the preparation is absolutely pure, each additional test simply provides another example of the failure to detect any contaminating species that might be present. Since absolute purity can never be established, a simple criterion of purity is used routinely, namely, the inability to detect more than a single band of protein after SDS-PAGE. Since onedimensional SDSPAGE is technically far easier than two-dimensional SDS-PAGE, this technique will be described in detail.

Passive release of monoclonal antibodies from hybridoma cells.

Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed. Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted.

Acetyl-coenzyme A carboxylase from avocado (Persea americana) plastids and spinach (Spinacia oleracea) chloroplasts.

Preparations of acetyl-CoA carboxylase [acetyl-CoA-carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] have been obtained from the plastids of avocado (Persea americana) fruit mesocarp and from spinach (Spinacia oleracea) chloroplasts. Both preparations required bovine serum albumin, HCO3-, citrate and glycerol for stabilization. The molecular weight of the avocado enzyme was about 6.5 X 10(5) on the basis of 1 mol of biotin/mol of enzyme, the behaviour of both enzymes on gel filtration being in accord with such a value. Removal of the stabilizing bovine serum albumin resulted in the loss of a biotin-containing fragment from the avocado enzyme. Citrate stabilized the enzyme at 10 mM and activated it optimally at 3.0 mM, effecting an approx. 2-fold increase in Vmax. It is suggested that in vivo the enzyme may be located within the chloroplast lamellae.

Effect of dextran and ammonium sulphate on the reaction catalysed by a glucosyltransferase complex from Streptococcus mutans.

The highly aggregated proteins precipitated by (NH4)2SO4 from the culture fluid of three strains of Streptococcus mutans gradually released less aggregated glucosyltransferase activities - dextransucrase and mutansucrase - which catalysed the synthesis of water-soluble and insoluble glucans from sucrose. Mutansucrase was eluted from a column of Sepharose 6B before dextransucrase. This activity was lost during subsequent dialysis and gel filtration, but there was a corresponding increase in dextransucrase activity which catalysed the formation of soluble glucan when incubated with sucrose alone, and insoluble glucan when incubated with sucrose and 1.55 M-(NH4)2SO4. Relative rates of synthesis of soluble and insoluble glucan in the presence of 1.55 M-(MH4)2SO4 were dependent upon the enzyme concentration: high concentrations favoured insoluble glucan synthesis. Insoluble glucans synthesized by mutansucrase or by dextransucrase in the presence of 1.55 M-(NH4)2SO4 were more sensitive to hydrolysis by mutanase than by dextranse, but soluble glucans were more extensively hydrolysed by dextranase than by mutanase. Partially purified dextransucrase sedimented through glycerol density gradients as a single symmetrical peak with an apparent molecular weight in the range 100000 to 110000. In the presence of 1.55 M-(NH4)2SO4, part of the activity sedimented rapidly as a high molecular weight aggregate. The results strongly suggest that soluble and insoluble glucans are synthesized by interconvertible forms of the same glucosyltransferase. The aggregated form, mutansucrase, preferentially catalyses (1 leads to 3)-alpha bond formation but dissociates during gel filtration to the dextransucrase form which catalyses (1 leads to 6)-alpha bond formation.

Sero-diagnosis of invasive aspergillosis: attempts to determine antigen and antibody relevance to infection.

Attempt was made to define antigens and antisera which might prove useful in diagnosis of invasive aspergillosis in man. A convalescent antiserum (serum from rabbits after liver infection with Aspergillus fumigatus conidia) which might be more representative of immunological reaction to fungal growth in vivo, did not react in enzyme-linked immunosorbent assay with commerical antigens which are used at present in attempts to detect antibody response in systemic infections in man. However, this convalescent antiserum reacted with antigens from a range of fungal extracts. Antigens from young culture filtrates in particular the 24th culture filtrate are advocated as the standard antigens for antibody detection using conventional immunoprecipitation techniques. For the detection of circulating antigens, the use of convalescent antiserum in enzyme-linked immunosorbent assay might be promising in the early diagnosis of invasive aspergillosis.