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Biomedical subjects

M W Steward

Publications and source records attributed to M W Steward.

At least 91 records · Page 5Linked to original sources
PubMed1986

Antibody affinity maturation in selectively bred high and low-affinity mice.

The affinity of serum antibodies produced by selectively bred lines of mice [high affinity, low affinity, low nonmaturing (N/M)] injected with T-dependent [human serum albumin (HSA), dinitrophenylated bovine gamma-globulin (DNP-BGG)] and T-independent (DNP-Ficoll) antigens in saline and adjuvant has been determined. The lines of mice differ significantly in the affinity of antibody produced to T-dependent antigens injected in saline but not to the T-independent antigen. Unlike mice of the high and low-affinity lines, low-affinity N/M mice failed to show affinity maturation to HSA and DNP-BGG injected in Freund's incomplete adjuvant. However, low-N/M mice responded to DNP-Ficoll injected in adjuvant by the production of antibody of affinity comparable to that produced in the other lines and with a similar maturation in affinity. Carrier priming resulted in the suppression of anti-hapten antibody affinity in all lines but low-N/M mice showed significantly greater suppression late in the response to challenge. Low doses of cyclophosphamide produced a significant increase in affinity in low-N/M mice. These results suggest that the failure of low-N/M mice to show affinity maturation results from increased suppressor T cell activity. The availability of the selectively bred mice provides a useful model for the detailed study of the cellular basis of the control of antibody affinity maturation.

Adjuvants, Immunologic↗
PubMed1985

Circulating antibody-antigen complexes following Trypanosoma musculi infection in mice genetically selected to produce high or low affinity antibody.

Mice from lines genetically selected for the production of either high or low affinity antibody to protein antigens and which differ in their susceptibility to chronic immune complex disease were infected with Trypanosoma musculi. The parasite became patent in both lines by day 5 and no significant differences in the levels of parasites during the infection were observed between the two lines. Serum levels of both antigen non-specific and T. musculi antigen specific immune complexes were determined during the infection by the solid phase conglutinin and Clq binding assays. In both lines, antigen non-specific complexes were detected by day 15 after infection with maximum levels observed by day 30. At this time, low affinity line mice had significantly higher levels than did high line mice as determined by the Clq assay but not by the conglutinin assay. The deposition of immune complex like material in glomeruli, assessed by immunofluorescence, was associated with the clearance of the parasite and the presence of circulating antigen specific complexes. The pattern of localization of complexes in both lines was predominantly mesangial with some deposition in the capillaries. The intensity of fluorescence increased during the infection. Initially (day 10) only IgM was observed in the glomeruli but IgG1 and IgG2b were detected from day 20 to day 40. IgG2a was only detected on day 40. However, in none of the animals was this deposition of complexes associated with proteinuria. Hence, the data presented here show that the low affinity line mice produce higher levels of smaller circulating complexes following T. musculi infection than do high affinity mice. However, this does not result in significant differences in localization and induction of renal disease as seen following chronic antigen injection.

Animals↗
PubMed1984

Determination of the affinity of antibodies to hepatitis B surface antigen in human sera.

The measurement of the affinity of anti-HBs antibody in human sera using 3 HBsAg-related antigens is described. The antigens used were (i) a synthetic linear peptide corresponding to amino acids 139-147 of the major polypeptide of HBsAg, (ii) a cyclical form of this same peptide and (iii) a polypeptide complex of a 28,000 MW glycoprotein and a 23,000 MW protein from purified HBsAg. The method was established with a pooled human anti-HBs immunoglobulin preparation and a monoclonal anti-HBs antibody reactive to the 'a' determinant of HBsAg. The results indicate that both these antibody preparations effectively bind the 3 antigens with affinity values of between 2 X 10(6) to 9 X 10(7) litres/mole. However, the affinity of both antibody preparations for the cyclical form of the peptide was higher than for the linear form. The level of antibody (expressed as Abt, molar antigen binding sites) in the pooled human immunoglobulin for each of the 3 antigens was similar. Measurements of anti-HBs antibodies in the sera of recovered acute hepatitis B patients and from HBsAg negative chronic liver disease patients showed that the cyclical form of the antigen was bound with a higher affinity than the linear form. Affinity values of antibody in the sera of the latter group of patients was significantly lower (3 X 10(5) to 2.7 X 10(6) litres/mole) than those observed in sera from other individuals. The implication of these results in determining the importance of the measurement of affinity in the assessment of the efficacy of vaccines is discussed.

Amino Acid Sequence↗
PubMed1984

Influence of antibody affinity on the performance of different antibody assays.

The effect of antibody affinity on the performance of 5 commonly used assays was studied. The assays used were measurement of antigen binding capacity in a Farr type assay, haemagglutination, solid-phase radioimmunoassay (SP-RIA), solid-phase ELISA and precipitation. The first 4 assays were all more sensitive for high affinity antibodies. Precipitation was not related to affinity, suggesting that factors secondary to antigen-antibody binding may be more important in determining the level of precipitate formation. The effect of epitope density of the antigen was also investigated in the SP-RIA and ELISA. Affinity dependence was more marked when antigen of low epitope density was used and this dependence could be reduced in the ELISA by choosing a low OD endpoint. Thus, the most reliable way to estimate antibody content by the ELISA may be to determine a low OD endpoint titre against antigen of high epitope density. When epitope density per molecule cannot be increased, an alternative approach to the problem is to increase epitope density by covalent coupling of antigen to the solid-phase rather than by adsorption.

Animals↗
PubMed1984

Affinity of antibody responses in man to hepatitis B vaccine determined with synthetic peptides.

The affinity and level of antibody to hepatitis B surface antigen (anti-HBs) in recipients of a plasma-derived hepatitis B vaccine were determined with three different antigens. The first two antigens were prepared by chemical synthesis, to represent linear or cyclical forms of aminoacid sequences 139 to 147 of the major hepatitis B surface antigen (HBsAg) polypeptide. The binding of antibodies to these synthetic peptides was compared with that to a third antigen, prepared by solubilisation of the naturally occurring HBsAg, the basic component of the currently licensed hepatitis B vaccine in the United Kingdom. Antibody levels, expressed as total antibody combining sites (Abt) in fixed volumes of immune sera, increased throughout the course of immunisation and correlated with the development of antibody as measured by a commercially available radioimmunoassay. Abt values were similar for both forms of the synthetic peptide, although higher affinity values were found with the cyclical structure, which illustrates the importance of protein conformation in antibody responses to HBsAg. Antibody affinity for the three antigens increased progressively throughout the immunisation schedule but the pattern of affinity maturation varied according to the peptide used as an antigen probe and between subjects. Most subjects showed a significant rise in antibody affinity after the third (booster) dose of vaccine given at six months. The use of synthetic peptides allowed a quantitative and qualitative assessment of antibody responses to hepatitis B vaccine and confirmed that selected peptides corresponding to relevant HBsAg epitopes may be useful as alternative hepatitis B vaccines.

Antibody Affinity↗
PubMed1984

Localization of covalent immune complexes on the epithelial side of the glomerular basement membrane in mice.

The site of immune complex localization in human glomerulonephritis is important in determining the kind of disease that may develop. Immune complex deposits found on the subepithelial side of the glomerular basement membrane are characteristic of membranous nephropathy, a common and often severe form of human renal disease. The pathogenesis of this disease remains controversial and arguments have recently been put forward that the subepithelial deposits arise from an in situ mechanism. This mechanism involves the initial localization of antigen allowing the antibody to subsequently combine, i.e. the immune complex is formed locally. This mechanism is thus in contrast to that in which immune complexes formed in the circulation are localized in the glomeruli. Covalent immune complexes do not dissociate and hence are ideal tools for studies to investigate whether complexes are able to cross the glomerular basement membrane. Covalent immune complexes of defined size and antigen-antibody ratio were prepared with a monoclonal antibody and photoaffinity labelling antigen, 4-azido-2-nitro-phenylated bovine serum albumin. When these complexes were injected into mice, complexes of 550,000 MW molecular weight (but not those of 800,000 MW) were shown by electron microscopic autoradiography to localize on the subepithelial side of the glomerular basement membrane. It is thus proposed that small circulating immune complexes may be important in the pathogenesis of subepithelial deposits and there is a need for devising reliable tests for measuring small immune complexes.

Animals↗
PubMed1984

Hepatitis B surface antigen containing immune complexes occur in seronegative hepatocellular carcinoma patients.

IgG, IgM and hepatitis B surface antigen (HBsAg) containing immune complexes (IC) were detected by the Clq and conglutinin solid phase assays in both HBsAg+ and HBsAg- groups of patients with primary hepatocellular carcinoma (HCC). No differences were observed between the two patient groups either in the levels of antigen non-specific and HBsAg specific complexes or in the immunoglobulin isotype in the complexes. The results show that HBsAg can occur in an IC form in the sera of patients classified as HBsAg- by sensitive commercial assays and provides evidence of a further association of hepatitis B virus (HBV) and HCC in antigen negative patients. Furthermore, the HBsAg IC in HCC patients differ from those in other HBV infected subjects in that they are preferentially detected by the Clq assay.

Antigen-Antibody Complex↗
PubMed1984

Glomerulonephritis: the use of grafted hybridomas to investigate the role of epitope density, antibody affinity and antibody isotype in active serum sickness.

Two experimental models were used to investigate the role of antigen epitope density, antibody isotype and antibody affinity in immune-complex glomerulonephritis. In both models, two antigens were used: dinitrophenylated bovine serum albumin of high and low substitution ( DNP19BSA and DNP4BSA , respectively). In the first, acute actively induced model, a single intravenous injection of either of the two antigens was given to mice bearing hybridomas that secreted one of five monoclonal antibodies--four IgGl with varying affinities for the dinitrophenol hapten, and an IgM antibody with a similar affinity to that of the lowest affinity IgGl. The second, chronic actively induced model was similar, except that mice were immunosuppressed to reduce interference by the hosts' antibody responses and that 15 daily intraperitoneal injections of antigen were given. The immunofluorescent findings in both models were similar. Glomerular deposition of antibody and antigen in mice with high affinity IgG or low affinity IgM antibodies was mesangial when DNP4BSA or DNP19BSA was injected. However in mice with the lowest affinity IgG antibodies, deposition was extensive and predominantly capillary when DNP19BSA was injected but was minimal when DNP4BSA was injected. The relevance of these findings to the pathogenesis of glomerulonephritis is discussed.

Acute Disease↗
PubMed1984

Glomerulonephritis induced by pre-formed immune complexes containing monoclonal antibodies of defined affinity and isotype.

The work presented here represents the first report of the induction of experimental immune complex (IC) disease in mice using monoclonal antibodies (MoAb) derived from somatic cell hybridization. IC were formed using two antigens of either high (DNP19BSA) or low (DNP4BSA) epitope density and five MoAb (four IgGl with varying affinities for the dinitrophenol hapten and one IgM with a similar affinity to that of the lowest affinity IgGl). Circulating levels and sizes of IC were dependent on the affinity of the antibody component of the complex. When antigen of high epitope density was used, the glomerular localization of injected IC was diffuse mesangial for the IgM antibody, focal mesangial for the highest affinity IgG and diffuse, and predominantly capillary for the low affinity IgG antibodies. Subepithelial electron dense deposits were observed only with IC made with the low affinity IgG antibodies. When IC containing antigen of a lower epitope density were injected, localization was only observed with IC made near equivalence. Deposition of these IC was less prominent than that found when IC containing antigen of higher epitope density were injected. The relevance of these findings to the pathogenesis of glomerulonephritis is discussed.

Animals↗
PubMed1984

Failure of affinity maturation leads to increased susceptibility to immune complex glomerulonephritis.

Mice, previously selected for the production of low affinity antibody after four injections of protein antigens in saline, fell into two groups on the basis of their antibody response after injection of adjuvantized antigen. One group produced antibody of sequentially rising affinity but the other produced only low affinity antibody. Mice from the latter group were interbred to produce low affinity non-maturing mice (low N/M mice). Daily injections in these mice produced a more rapid and severe glomerulonephritis than that observed in mice of the original low affinity line. Male low N/M mice were more severely affected than female low N/M mice. Susceptibility to the disease were associated not only with an inability to produce the maturational transition from low affinity to high affinity antibody with time but also with the production of low levels of antibody. It is suggested that these quantitative and qualitative defects in the antibody response may lead to increased susceptibility to immune complex disease.

Animals↗
PubMed1983

The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody.

The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-epsilon-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.

Animals↗
PubMed1983

Chronic liver disease: the detection and characterization of circulating immune complexes.

Circulating immune complexes containing IgG, IgM and hepatitis B surface antigen (HBsAg) in sera from groups of patients with various liver diseases were detected by both the C1q and conglutinin solid phase assays. Elevated levels of antigen non-specific immune complexes were observed in sera from all groups and complexes containing IgG were present to a greater extent than were IgM-containing complexes. Higher levels of complexes were generally obtained using the conglutinin assay than the C1q assay and the two assays were shown to preferentially bind complexes of different size ranges and antigen-antibody ratios. Only sera from HBsAg-positive patients had complexes containing HBsAg, and although serum HBsAg titres and levels of HBsAg-containing complexes were correlated, the correlation coefficient was low. The mean levels of immune complexes and the frequency of positive sera varied between different disease categories, but there was little correlation between levels of the three types of complexes detected by the two tests. Assay of immune complexes in sequential serum samples from an individual patient revealed considerable variation in the levels of the three complex types, demonstrating that the measurement of complexes in single serum samples is of limited value in assessing the potential significance of circulating immune complexes in hepatitis B.

Antigen-Antibody Complex↗
PubMed1983

Circulating immune complexes in hepatitis B: levels, immunoglobulin class of antibody and the presence of hepatitis B surface antigen.

Circulating immune complexes containing IgG, IgM and HBsAg in sera from groups of patients with various liver diseases were detected by the Clq and conglutinin solid phase assays. Elevated levels of antigen-non-specific complexes were observed in sera from all groups and complexes containing IgG were present to a greater extent than were IgM-containing complexes. However, only sera from HBsAg-positive patients had significant levels of complexes containing HBsAg.

Antigen-Antibody Complex↗
PubMed1983

Trypanosome infection of mice depresses antibody affinity and delays affinity maturation.

Infection with either a pathogenic species of trypanosome (Trypanosoma brucei brucei) or a non-pathogenic trypanosome (Trypanosoma musculi) had differing effects on the response of mice to a soluble protein antigen (human serum albumin, HSA) injected in either Freund's incomplete adjuvant or in saline. T. brucei suppressed the response to HSA to a level undetectable by ammonium sulphate globulin precipitation, irrespective of the mode of immunization, whereas T. musculi did not suppress the amount of antibody produced in response to either form of antigen presentation. The affinity of the antibody produced in response to antigen in adjuvant was unaffected, but antibody affinity was significantly reduced in infected animals in which the antigen was given in saline. This depression of antibody affinity was related to the period of infection and arose as a result of a delay in the normal maturation of affinity. Furthermore, the depression was only observed when infection preceded the exposure to antigen. Possible mechanisms which may lead to a depression of affinity without a corresponding effect upon antibody levels are discussed in context of current knowledge of immunosuppression in trypanosome infections.

Animals↗