Human immunoglobulin G-glucose oxidase conjugates (HIgG-GO) were studied in detail to establish optimum conditions under which maximum enzyme activities and immunological reactivities can be retained. These properties determine the analytical utility of these conjugates. Reactions of four different coupling reagents (glutaraldehyde, p-benzoquinone, sodium periodate and N-succinimidyl 3-(2-pyridyldithio) propionate) with glucose oxidase (GO) and human immunoglobulin G (HIgG) were investigated. Conjugates made with p-benzoquinone were found to be far superior since they retained maximum enzyme and/or immunological reactivities. Enzyme activities were determined by spectrophotometric measurements of the active flavin content of GO. This method was simple, convenient and correlated well with the standard peroxidase procedure. Rate nephelometry was established as a reliable method to measure apparent immunological reactivities of the conjugates at μg/ml levels. Hill plot treatment of the rate nephelometric data shows that the immunoprecipitin reactions may be cooperative, with two antigenic sites or steps in operation. This may explain the need for mixtures of monoclonal antibodies to observe immunoprecipitin reactions. Angular and concentration dependence of the scattered intensity during immunoprecipitin reactions was measured for the conjugated and unconjugated HIgG. From these data the weight-average-molecular weight (Ṁ) of the transient scattering complexes were estimated using Zimm plots. The Ṁ values were in the range of 2.8 x 10⁸ - 11.0 x 10⁸ daltons and were in good agreement with the theoretically predicted values. Equilibrium binding constants of the conjugates were not significantly different from that of the unconjugated HIgG (K´ₒ = 6.0 x 10⁷), showing that under equilibrium conditions the binding properties are similar. However, heterogeneity indices obtained via Sip's transformation were higher for the conjugates (a = 0.68-1.0). Such a behavior may be due to some restriction of the number and type of antigenic sites recognized by the immobilized antibodies in the presence of an enzyme label on the antigen.

Thesis (M.S.)--Rochester Institute of Technology, 1995. / Typescript. Bibliography: leaf 23.

Thesis (doctoral)--University of Jyväskylä, 1983. / At head of title: The Department of Clinical Sciences, University of Tampere, the Department of Cell Biology, University of Jyväskylä. Includes bibliographical references.

Thesis (doctoral)--University of Jyväskylä, 1983. / At head of title: The Department of Clinical Sciences, University of Tampere, the Department of Cell Biology, University of Jyväskylä. Includes bibliographical references.

Recent clinical studies have highlighted the effectiveness of immunotherapy for Gram-negative bacteraemia in humans. Studies in America, undertaken on patients with Gram-negative bacteraemia, have shown that mortality was reduced by virtually 50% in patients who received specific antiendotoxin antiserum. In India, mortality from pseudomonas septicaemia was significantly reduced by the administration of small quantities of a anti-pseudomonas immunoglobulin. The antibodies in those studies were raised by vaccination of healthy volunteers with heat-killed Gram-negative bacteria or vaccines containing endotoxin. Adverse side effects in volunteers as well as logistic and legal problems make it difficult to produce antiserum on a large scale, in this manner. In Israel, S.L. Gaffin and coworkers found that approximately 7% of plasma units in a blood bank had antiendotoxin antibody concentrations of 40 ).1g/m1 or greater. This high titre human plasma significantly protected cats from lethal endotoxic shock secondary to haemorrhage. The immunoprecipitin technique used by them to measure antiendotoxin antibody concentrations was unsuitable for screening large numbers of blood samples. To overcome this problem we have devised an enzyme-linked imounosorbent assay (ELISA) for determining the level of antiendotoxin immunoglobulin G in human plasma. The assay, which is suitable for large scale use, was found to be specific for antiendotoxin antibodies. It was calibrated using a serum sample of specific antibody concentration as determined by an ilununoprecipitin assay. Serum samples found to be high in antiendotoxin titres (> 40_ug/m1) were tested for their specificity towards endotoxins from 12 bacterial iv strains and species. While each sample was found to have its own characteristic specificities, most were found to react strongly with Sh. flexneri, S. typhimurium and S. enteritidis. The Natal Blood Transfusion Service has found that in Natal, blood units containing high concentrations of specific antibodies occur with a frequency of 3,6% among all White donors and 10,35% among all African donors. They found that African females, in turn, had almost twice the frequency of high titre serum as African males. In this study, Indian female hospital patients did not have a statistically higher frequency of high-titre serum than Indian male patients. Blood units donated to the Natal Blood Transfusion Service are now routinely screened by ELISA for antiendotoxin antibodies and those units with high concentrations (> 40 ug/ml) of antibody were pooled and fractionated to obtain a gamma globulin, Lot LG-l. The binding capacity of the LG-1 antibodies towards 12 endotoxins was examined. Binding was found to be highest with endotoxin from Sh. flexneri, S. abortus equi and S. typhimurium and intermediate with S. enteritidis and E.coli 026:B6. Binding with the other endotoxins tested was relatively low. Differential absorption experiments showed that LG-1 was made up of a mixture of cross-reacting as well as specific antibodies For example, the antibodies binding Sh. flexneri endotoxin were mainly specific. Those binding E. coli 026:B6 endotoxin were specific and cross-reacting in almost equal proportions. Antibodies to the endotoxins from the salmonella strains tested were mainly cross-reacting. The specificities of the LG-1 antibodies towards endotoxins from the various Gram-negative bacteria did not in most cases reflect the incidence of these organisms in blood cultures taken from hospital patients. V The activity of LG-1 antibodies was compared to that of normal human immunoglobulin preparations obtained from the National Blood Fractionation Centre, Pinetown and to an anti-pseudomonas immunoglobulin prepared by Wellcome Laboratories, England. The binding capacity of the antibodies in the standard globulin preparations towards most of the endotoxins tested was less than 15% of that of the LG-1 antibodies. The anti-pseudomonas immunoglobulin was shown to bind poorly to most of the endotoxins tested in comparison with binding by LG-1 antibodies. / Thesis (MMedSc.)-University of Natal, Durban, 1984.

Thesis (M.Sc.) -- University of Adelaide, Dept. of Microbiology and Immunology, 2000? / Bibliography: leaves 106-128.

Thesis (M.S.)--University of Michigan, 1986.

Thesis (M.S.)--University of Michigan, 1986.

Thesis--M. Med. Sc., University of Hong Kong, 1986. / Photocopy from typescript.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences