Michael W. Steward

The importance of antibody affinity in the performance of immunoassays for antibody

(I) Introduction Over the last 25 years or so, a wide range of new antibody immunoassay techniques has been developed to add to the historically accepted methods such as precipitation, agglutination and complement fixation. These newer techniques involve the use of electrophoresis, fluorescence, radioactivity, laser nephelometry, solid-phase immunoadsorbents and enzyme conjugates. In general, all tests for antibody-antigen interaction exploit the fact that following combination with antigen antibodies will agglutinate cells or particles coated with antigen, form precipitates with the antigen which can be visualized in gel or solution or otherwise quantified, or can potentiate measurable biological reactions. Not surprisingly, there is a wide range of sensitivity of such tests which depends in part on the particular property of the antibody which is being exploited. Thus tests which depend solely on the primary binding of antibody to antigen (e.g., equilibrium dialysis, fluorescence quenching, radioimmunoassays and enzyme-linked immunoabsorbent assays) are more sensitive than those requiting secondary manifestations of binding (e.g., precipitation, agglutination and complement fixation) (Minden et al., 1968). However, it is becoming increasingly clear that the affinity of an antibody for the corresponding antigen markedly influences its biological activity. Thus to characterize an antiserum solely in terms of its antibody content or titre is less th~n adequate and therefore a knowledge of the affinity of the antibody is of crucial importance (Steward and Steensgaard, 1983). It is also apparent that the affinity of the antibody being measured is itself very likely to influence the efficiency of the assay employed for its quantification. In this review, the importance of antibody affinity in the performance of a number of immunoassays is discussed. In particular, emphasis is placed upon the influence of antibody affinity on the enzyme-linked immunosorbent assays (ELISA) which are enjoying considerable international popularity.

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.

An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity

A method to measure the relative affinity of antibodies using an inhibition enzyme immunoassay is described. It is validated using monoclonal antibodies of defined affinity characteristics and by comparison with conventional methods of affinity measurement. The method allows measurement of the relative affinity of low levels of antibody, and the calculation of an empirical estimate of the heterogeneity of affinity in antibody populations.

Nasal delivery of chitosan-DNA plasmid expressing epitopes of respiratory syncytial virus (RSV) induces protective CTL responses in BALB/c mice.

Respiratory syncytial virus (RSV), an important pathogen of the lower respiratory tract, is responsible for severe illness both in new born and young children and in elderly people. Due to complications associated with the use of the early developed vaccines, there is still a need for an effective vaccine against RSV. Most pathogens enter the body via mucosal surfaces and therefore vaccine delivery via routes such as the nasal, may well prove to be superior in inducing protective immune responses against respiratory viruses, since both local and systemic immunity can be induced by nasal immunisation. Previously we have shown that intradermal immunisation of a plasmid DNA encoding the CTL epitope from the M2 protein of RSV induced protective CTL responses. In the present study, the mucosal delivery of plasmid DNA formulated with chitosan has been investigated. Chitosan is a polysachharide consisting of copolymers of N-acetylglucosamine and glucosamine that is derived from chitin, a material found in the shells of crustacea. Intranasal immunisation with plasmid DNA formulated with chitosan induced peptide- and virus-specific CTL responses in BALB/c mice that were comparable to those induced via intradermal immunisation. Following RSV challenge of chitosan/DNA immunised mice, a significant reduction (P<0.001) in the virus load was observed in the lungs of immunised mice compared to that in the control group. These results indicate the potential of immunisation with chitosan-formulated epitope-based vaccines via the intranasal route.

Synthetic peptides: a next generation of vaccines?

Of the strategies being considered in the search for new kinds of vaccine, one of the most powerful is the use of synthetic peptides. Here, Michael Steward and Colin Howard discuss recent insights into the nature of antigenicity which influence the use of such peptides, and review the value of peptide immunogens in a variety of diseases.

Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans

Amino acid residues 288 to 302 of the fusion protein of measles virus were predicted by a variety of methods to represent a putative T cell epitope. This sequence was synthesized and the peptide was injected into mice of six inbred strains to test this possibility. Lymphocytes from peptide-immunized mice from all six H-2 disparate strains were able to mount a proliferative response following in vitro culture with the peptide. In addition, lymphocytes from three strains also proliferated in the presence of live measles virus. The peptide also behaved as a B cell epitope in that immunization with free peptide in adjuvant resulted in anti-peptide antibody production in all mouse strains. However, these antibodies did not react with the virus in either a solid-phase immunoassay or a virus neutralization assay. Peripheral blood lymphocytes from 10 laboratory personnel with a prior history of exposure to measles virus were tested in a proliferation assay with the peptide and with the virus. Lymphocytes from all 10 individuals proliferated in response to culture with the virus and those from eight responded to the peptide. These results give further support to the concept of permissive interaction of antigenic peptides with a wide range of class II major histocompatibility complex molecules both in mice and man and indicate the possibility of designing peptides that could be used as components of a synthetic vaccine for use in man.

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.

Immune responses in mice following immunization with chimeric synthetic peptides representing B and T cell epitopes of measles virus proteins.

The immunogenicity of chimeric peptides produced by collinear synthesis to contain both T and B cell epitopes from the fusion protein and the haemagglutinin of measles virus was studied in mice. The T cell epitope used was from the fusion protein (residues 288 to 302), which has been shown to be promiscuous in its binding to mouse major histocompatibility complex molecules. This epitope was coupled by (i) a glycine-glycine spacer to a B cell epitope from the fusion protein (residues 404 to 414) and (ii) either its amino or carboxy terminus to a neutralizing antibody epitope from the haemagglutinin (residues 188 to 199). The results obtained show that such chimeric peptides can indeed function as complete immunogens in a range of mouse strains of different H-2 haplotype, and can induce the production of antibodies which bind to the fusion protein and to measles virus. Furthermore, it was shown that the orientation of the T cell epitope with respect to the B cell epitope had a significant effect upon the immunogenicity and antigenic specificity of the chimera. This work gives further support to the concept of rationally designed synthetic peptide vaccines.

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.

The affinity of IgG antibodies to gag p24 and p17 in HIV‐1‐infected patients correlates with disease progression

The affinity of anti‐gag antibody was studied for up to 9 years (1984–1993) in sera from 15 HIV‐1+ patients with haemophilia. On the basis of their 1993 clinical status patients were divided into two groups: (i) patients who remained asymptomatic (n= 9); and (ii) those who progressed to AIDS between late 1987 and 1993. The affinity constants of antibody for p24 and p17 were determined by a double isotope fluid‐phase radioimmunoassay; and the relationships between antibody affinity and titre, patient clinical course, CD4 cell counts and p24 antigenaemia were analysed. The affinity of p24‐ and p17‐specilic antibody was up to 100 times greater in asymptomatic patients than in patients who progressed to AIDS. Patients who developed AIDS either lost or failed to develop high‐affinity antibodies early in the infection. Asymptomatic patients maintained high‐affinity antibodies for several years; however, in some of these patients the affinity of anti‐p24 and p17 antibodies subsequently fell later in the study period. The presence of low‐affinity antibody and progressive reduction in the titre of specific antibody were earlier predictors of disease onset than CD4 cell counts. The failure to either develop or maintain high affinity gag‐specific antibody suggests an early impairment of T helper function in individuals who progressed to AIDS. The presence of antibody of high affinity could be essential in controlling virus replication and the onset of AIDS.