Antibodies Of Different Affinities Research Articles

Molecular form of pancreatic elastase 1 in human plasma.

In order to study the molecular forms of immunoreactive pancreatic elastase 1 (IRE) in human plasma, we investigated the characteristics of proelastase 1, elastase 1, and their alpha 1-protease inhibitor (alpha 1-PI) complexes on anion-exchange (Mono Q) chromatography, gel (Superose 12) chromatography, and enzyme immunoassay systems with monoclonal antielastase 1 antibodies of different affinities for alpha 1-PI-elastase 1 and alpha 1-PI-proelastase 1 complexes. The rate of complex formation between proelastase 1 and alpha 1-PI was dependent on temperature of incubation and concentration of alpha 1-PI. At 37 degrees C, 90% of proelastase 1 was bound to alpha 1-PI during 2 h of incubation of purified human proelastase 1 with human alpha 1-PI. Similar results were also obtained by 2 h of incubation of proelastase 1 with human plasma at 37 degrees C. Therefore, under physiological conditions, neither free proelastase 1 nor elastase 1 can be detected in the human blood stream, and most IRE exists as the complex of proelastase 1 with alpha 1-PI in human plasma.

Radiolocalisation and imaging of stably HPLAP-transfected MO4 tumours with monoclonal antibodies and fragments

Immunotargeting of PLAP-expressing tumours was studied for two radioiodinated, highly specific anti-PLAP monoclonal antibodies, 7E8 and 17E3, differing 10-fold in affinity, as well as for 7E8 F(ab')2 fragments. An anti-CEA monoclonal antibody or anti-CD3 F(ab')2 fragments were used as controls. Specific and non-specific targeting was examined in nude mice simultaneously grafted with PLAP-positive tumours derived from MO4 1-4 cells, and CEA-positive tumours, derived from 5583-S cells. Results indicated that (1) MO4 1-4 tumours, with a stable expression of PLAP on the plasma membrane, represent a useful new in vivo model for immunodirected tumour targeting; (2) differences in antibody affinity for PLAP in vitro are not reflected in antibody avidity for tumour cells in vivo; and (3) excellent selective and specific localisation of the PLAP-positive tumours is achieved when 7E8 F(ab')2 fragments are used. The high tumour/blood ratios (10.7 +/- 3.9 at 46 h after injection) were due to a much faster blood clearance of 7E8 F(ab')2 fragments. At this time point, the mean tumour/non-tumour tissue ratio was as high as 34.5, and the mean specific localisation index was 29.0. As expected, the F(ab')2 fragments provided high tumour imaging efficiency on gamma camera recording. These data imply important potentials of the PLAP/anti-PLAP system for immunolocalisation and therapy in patients, but also emphasise that in vitro criteria alone are not reflected in in vivo tumour localisation capacities of antibodies.

Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens

The long-standing hypothesis that tolerance to self antigens is mediated by either elimination or functional inactivation (anergy) or self-reactive lymphocytes is now accepted, but little is known about the factors responsible for initiating one process rather than the other. In the B-cell lineage, tolerant self-reactive cells persist in the peripheral lymphoid organs of transgenic mice expressing lysozyme and anti-lysozyme immunoglobulin genes, but are eliminated in similar transgenic mice expressing anti-major histocompatibility complex immunoglobulin genes. By modifying the structure of the lysozyme transgene and the isotype of the anti-lysozyme immunoglobulin genes, we demonstrate here that induction of anergy or deletion is not due to differences in antibody affinity or isotype, but to recognition of monomeric or oligomeric soluble antigen versus highly multivalent membrane-bound antigen. Our findings indicate that the degree of receptor crosslinking can have qualitatively distinct signalling consequences for lymphocyte development.

The influence of epitope density on the estimation of the affinity of antibody for complex antigens

The influence of the epitope density of the antigen on antibody affinity values determined by fluid- and solid-phase immunoassays was assessed. The affinity of the interaction of a panel of monoclonal anti-DNP antibodies of different affinities (as determined by equilibrium dialysis) for DNP-protein conjugates of various hapten substitution ratios was used as the test system. The results obtained showed that the epitope density of the antigen markedly influences the observed affinity values obtained by both experimental approaches. However, the monoclonal antibodies were ranked in affinity terms by both assays in a similar order to that given by equilibrium dialysis. It is concluded that provided due care is exercised in choosing an appropriate epitope density for the test antigen, these methods can be used to provide rapid estimations of average antibody affinities.

Theoretical considerations of the ability of monoclonal antibodies to detect antigenic differences between closely related variants, with particular reference to heterospecific reactions

In theory monoclonal antibodies can be used to analyse antigenic determinants in great detail by correlating differences in antibody affinity for variant antigens with their amino acid differences. In particular, heteroclitic antibodies should be detected, which would normally be masked in a polyclonal antiserum. Recognition of such antibodies may be important for our understanding of the scope of antibody repertoires particularly when the immunogen is closely related to a component of the immunised animal. In practice the immunoassays commonly used to measure affinity differences between different antigens fall short of these capabilities. Mathematical studies were carried out to identify factors controlling the sensitivity of 4 types of assay to differences in affinities for different antigens. The most important factors controlling assay sensitivity were found to be the ratio of antibody affinity ( K) to epitope density in direct binding assays, the ratio of K to antibody concentration in liquid phase competition assays, and the ratio of solid phase to liquid phase values of K for solid-phase competition assays. It is predicted that a combination of solid-phase competition assay with high epitope density and direct binding assay with low epitope density would result in optimal detection of (1) heteroclitic antibodies and (2) small differences in antibody affinity for cross-reactive antigens.

Antibody affinity and immune complexes after immunization with tetanus toxoid in protein-energy malnutrition

Protein-energy malnutrition reduced the affinity of antibody to tetanus toxoid, particularly after primary immunization. The effect on antibody affinity was more marked in patients with hypoproteinemia than in those with marasmus. Hemagglutinating antibody levels were comparable in undernourished and well-nourished groups. Circulating immune complexes were detected in eight of 21 children with protein-energy malnutrition and in one of the controls. Differences in antibody affinity in malnutrition may be an important determinant of altered host resistance and of complications of disease in nutritional deficiency.

The effect of different antibody affinities on ELISA absorbance and titer

Twenty-one rabbit serum samples were assayed for IgG antibodies against 3-iodo-5-nitrophenyl-ϵ-amino-n-caproic acid (NIP-cap) by equilibrium dialysis and enzyme-linked immunosorbent assay (ELISA). The results of ELISA, expressed either as absorbances or titers, did not correlate to antibody concentration or average affinity measured by the equilibrium dialysis. Affinity distributions of the antibodies within each sample were determined by equilibrium dialysis in 30 antigen concentrations. Thus, it was possible to study the correlation of ELISA results with separate ranges of antibody affinities. ELISA absorbances measured at a low sample dilution (1:80) correlated with high affinity antibodies (affinity>8 × 10 6 M −1). Use of high antigen density increased this correlation. End-point titers obtained at a low cut-off absorbance level tended to correlate to a larger range of affinities (affinity>6×10 4 M −1 ). ELISA measures antibodies of different affinities depending on how the results are expressed. This can be utilized in qualitative characterization of the measured antibodies.

Comparative catabolic rates of anti-hapten IgG antibodies of different affinities

Comparative catabolic rates of anti-hapten IgG antibodies of different affinities

Comparative catabolic rates of anti-hapten IgG antibodies of different affinities

Rabbit IgG anti-fluorescyl antibodies were purified from immune antisera by immunoadsorption into subpopulations on the basis of affinities for the homologous ligand. Low and high affinity populations from individual rabbits were radioiodinated in dual-isotope studies. Antibodies, in approximately equal concentrations, were passively administered simultaneously (intravenously) to the same rabbit from which the antibodies were derived (i.e. autologous). Rate of decay studies showed that high affinity antibodies were catabolized 2–3 times faster than low affinity antibody molecules. Pepsin digestion experiments inferred that the F c fragment was responsible for different catabolic rates. It is proposed that differences in the carbohydrate content of F c fragments of IgG molecules with differing affinities for the fluorescyl ligand, are important in determining catabolic rates. This proposal is consistent with previous studies which showed that low and high affinity antibody molecules differ qualitatively and quantitatively in their carbohydrate content (Mitchell et all., 1976).

In vitro target cell lysis mediated by normal human lymphocytes. Influence of molecular properties and affinity of inducing antibody.

Lysis of DNP-coated chicken erythrocytes by human blood lymphocytes (K cells) was induced by means of rabbit anti-DNP antibodies. Antisera were prepared by injecting the animals with DNP-conjugated proteins emulsified in Freund's complete adjuvant. An ammonium sulphate precipitation technique was used for assay of antibody concentration and affinity. Sephadex G-200 chromatography indicated that 90% of the DNP antibodies were 7S in the bleedings on days 10-16, whereas 99.8% were 7S in later bleedings. 7S antibodies induced K-cell lysis at high dilutions, whereas 19S antibodies were essentially negative. Antibody fractions obtained by DEAE- or CM-cellulose chromatography were used to establish possible heterogeneities in the capacity of 7S antibodies to induce either K-cell- or complement-mediated target cell lysis. No such heterogeneities were founnd. Fifteen IgG preparations containing antibodies of different affinities were compared with regard to their capacity to induce K-cell-mediated lysis. A statistically significant correlation was found between antibody affinity and efficiency in K-cell-mediated lysis. In a similar study of complement-mediated lysis the correlation was not significant at the 5% level but was significant at the 10% level.

Relationship between macrophage clearance of PVP and affinity of anti-protein antibody response in inbred mouse strains.

MACROPHAGES are important in immunity on their own, in delayed-type hypersensitivity, in phagocytosis of immune complexes, and in the afferent limb of antibody responses. Evidence for the latter includes the increased immunogenicity of macrophage-processed antigen1, and the requirement for macrophages in antibody responses involving cooperation between T and B lymphocytes2. In addition, antibody titres can be increased or decreased by agents, such as adjuvants3 or colloidal carbon4, which probably act on macrophages. Changes in either quantity or quality (affinity) of antibody, or both, may occur: carbon5 reduces the affinity of mouse anti-protein antibody but does not affect quantity, whereas adjuvant6 increases both. Differences in macrophage activity could therefore be responsible for differences in antibody affinity between mouse strains7. Carbon clearance differs between strains5,8, and has been found to be loosely related to affinity5. We have used a new test of macrophages which shows differences of clearance function which relate closely to differences of antibody affinity.

Immune reactions in polysaccharide media. Experiments with specific antibodies of different affinities for serum albumin.

Rabbit antibody fractions of different affinities for human serum albumin were prepared by an immunosorbent technique. The fractions were used in studies on the enhancement of the precipitin reaction by polymers. Dextran increased the immune precipitation to about the same extent regardless of whether antibodies with high and low affinities were used. The effect should considerably facilitate the detection of antibodies with low precipitating ability in immunological assays. The results are discussed in terms of a steric-exclusion mechanism.