Biomedical subjects
G W Jack
Publications and source records attributed to G W Jack.
A comparison of the leakage of a monoclonal antibody from various immunoaffinity chromatography matrices.
Antibody leakage from immunoaffinity chromatography (IAC) matrices could reduce the working life of the IAC matrix and/or contaminate parenteral products, purified by IAC. There is therefore a need to measure the leakage of antibody from IAC matrices and to reduce such leakage. Using sensitive ELISAs it was found that the type of activated matrix, the buffer, the presence of proteases in the feedstock and the storage of IAC matrices between runs could all effect antibody leakage.
Development of enzyme-linked immunosorbent assays (ELISAs) for the detection of monoclonal antibody leakage from immunoaffinity chromatography matrices.
Antibodies can leak from immunoaffinity chromatography (IAC) matrices, reducing the working life of the IAC matrix and/or compromising the purity of the product, purified by IAC, for therapeutic use. There is therefore a need to monitor the leakage of antibody from IAC matrices. Antibody leakage from a model IAC system was measured using two-site 'non-competitive' ELISAs. Two assays were developed to measure the leakage of intact and fragmented antibody from the IAC matrix. By measuring the leakage of intact and fragmented antibody the mechanisms underlying antibody leakage from solid-supports could be elucidated.
Immunoaffinity chromatography.
The basic procedure of immunoaffinity chromatography (IAC) is described. The insoluble support matrices available for IAC and their activation chemistries, including some of the most recently introduced, are reviewed. Means of selecting the most appropriate monoclonal antibody (MAb) are described, although an empirical approach is still required for the final choice of antibody. Precise methods of running IAC columns are surveyed including the binding, washing, and elution stages, although no precise recommendations can be made particularly for the elution step since this is unique to a particular MAb and antigen. All IAC sorbents lose activity with time through a combination of MAb inactivation and ligand leakage. The relative importance of the two phenomena is discussed, and suggestions are made to minimize the problem along with an indication of the relative stabilities of a range of coupling chemistries. A sample of the proteins purified by IAC is given together with pointers to the future of the technique.
Recombinant protein A from Escherichia coli JM83.
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In-vivo studies of a human-thyrotrophin preparation.
The effects on thyroid function of a new preparation of human thyrotrophin (hTSH) were studied in four subjects whose endogenous production of TSH had been suppressed by administration of thyroxine (T4). The hTSH, prepared from human cadaveric pituitary glands and highly purified using a monoclonal antibody technique, was given as an intravenous bolus of 2 i.u. hTSH. Serum TSH levels rose rapidly to a maximum of about 150 mu./l and then declined exponentially with a half-life of 100 min. After injection, the hTSH distributed rapidly in a volume averaging about 13 litres which corresponded approximately to the expected extracellular fluid volume of the subjects. Serum free tri-iodothyronine and free T4 rose significantly, reaching a maximum between 4 and 8 h after injection of hTSH; serum thyroglobulin was not altered significantly. The rise of thyroid pertechnetate uptake, a measure of the thyroid iodide uptake, occurred later, being only slightly increased at 8 h after administration of hTSH and reaching a maximum at 24 h.
Differential sensitivity of normal and leukaemic haemopoietic cells to methionine deprivation by L-methioninase.
The in vitro sensitivity of bone marrow cells from patients with leukaemia and from patients with non-malignant diseases to L-methionine removal by L-methioninase (L-methionine-alpha-deamino-gamma-mercaptomethane-lyase, EC 4.4.1.11) was determined using the incorporation of [methyl-3H]thymidine into acid-insoluble material as an index of survival. When compared with controls growing in medium containing 10 micrograms/ml of L-methionine, leukaemic cells showed a lower incorporation of [methyl-3H]thymidine after 24 h in the presence of 0.1 (normal 78 +/- 24%; leukaemic 26 +/- 18%, p less than 0.01) or 0.05 (normal 84 +/- 15%; leukaemic 50 +/- 21%, p less than 0.01) units of L-methioninase per ml. A similar differential sensitivity of leukaemic cells to L-methioninase was seen after 48 h of incubation. There was little effect on [methyl-3H]thymidine incorporation in the presence of boiled enzyme. Attempts to reverse L-methioninase toxicity with D-homocystine did not result in a differential effect on the normal cell population. The effects of L-methionine removal with L-methioninase were similar to those observed in L-methionine-depleted culture medium supplemented with 0.1 mM L-homocysteine. After 24 h in such depleted media leukaemic cells showed a lower incorporation of [methyl-3H]thymidine into acid-insoluble material (normal 88 +/- 17%; leukaemic 35 +/- 14%, p less than 0.01) and there was an elevation of the L-methionine-dependent enzymes: methionine adenosyltransferase, tRNA methyltransferase and S-adenosylmethionine decarboxylase. These results suggest the possibility of trying L-methioninase in the treatment of suitable leukaemias.
The effect of histidine ammonia-lyase on some murine tumours.
The histidine ammonia-lyase from bacterial strain CAMR 5315 was partially purified to assess its effect on the growth of murine tumours. This strain was selected as the source after an extensive screening programme for histidine ammonia-lyases. The enzyme was partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Sephadex G-150. The enzyme reduced circulating L-histidine levels in Wistar rats and in mice persisted with a half-life of 6-7 h. Neither LDH virus nor chemical modification with ethylacetimidate increased the half-life as observed with L-asparaginase and L-glutaminase. The enzyme was tested in mice against Ehrlich carcinoma, L5178Y lymphoblastic leukaemia, Mc/S sarcoma, B16 melanoma, P8157 mastocytoma, P1798 lymphosarcoma and the Gardner 6C3HED lymphosarcoma. The only tumours to show sensitivity to the enzyme were the Mc/S sarcoma against which a 65% increase in life span was observed at the highest enzyme dose, 1000 U/kg on alternate days over 14 days and the Ehrlich ascites carcinoma where cures were obtained at 250 U/kg on alternate days over 14 days, but only at inocula levels of 10(5) and 10(3) cells/animal respectively.
The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis.
Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75 000 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65 000-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65 000-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.
A bacterial phenylalanine aminotransferase lacking pyridoxal 5'-phosphate as cofactor.
A bacterium has been isolated from soil which metabolises phenylalanine initially through the action of a phenylalanine aminotransferase. This enzyme has been purified by conventional techniques and affinity chromatography and shown to be unusual among aminotransferases in not containing pyridoxal 5'-phosphate as cofactor.
A novel bacterial phenylalanine aminotransferase [proceedings].
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Precipitation of nucleic acids with polyethyleneimine and the chromatography of nucleic acids and proteins on immobilised polyethyleneimine.
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The purification and some properties of a -lactamase sensitive to inhibition by p-chloromercuribenzoate.
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Mechanisms of drug resistance. The beta-lactamases of gram-negative bacteria including pseudomonads.
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A comparative study of eight distinct beta-lactamases synthesized by gram-negative bacteria.
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The -lactamases of gram-negative bacteria and their transfer by R-factors.
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The beta-lactamases of Gram-negative bacteria.
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