K.A. Kelly

Kinetic studies on antibody-hapten reactions-I: Reactions with antibodies and their univalent Fab′ fragments☆

Abstract Rate constants and equilibrium constants were determined for reactions of antibodies specific to the 2,4-dinitrophenyl determinant and their univalent Fab′ fragments with the haptens, ϵ-DNP-lysine and 1-hydroxy-4-(2,4-dinitrophenylazo)-2,5 napthalene disulfonate. Binding constants were established by spectrophotometric and fluorescence quenching techniques, and the rate constants with the temperature-jump relaxation and stopped-flow methods. The Fab′ fragments had a slightly higher affinity for the haptens than the intact antibodies, which was primarily a consequence of an increase in the rate constants, by a factor of almost two, for the association reactions. This increase in rate constants for the univalent fragments was attributed to a greater accessibility of their combining sites.

Bovine superoxide dismutase: A radioimmunoassay

Abstract Rabbit antibodies to bovine superoxide dismutase have been produced and used to develop a double-antibody solid phase radioimmunoassay for the enzyme. The assay is sensitive and highly specific for the bovine enzyme, showing no cross-reactivity with the murine or human superoxide dismutases. It has been applied to the quantitation of exogenous enzyme in serum and extracts of mouse cells and tissues.

The development of a radioallergosorbent test (RAST) for murine IgE antibodies

A purified monoclonal IgE preparation, isolated from the ascitic fluid of mice bearing a hybridoma secreting IgE with specificity to ovalbumin, was used for the production of goat anti-murine IgE (GAME) antiserum, which was then rendered monospecific for the epsilon chain. Another monoclonal hybridoma IgE preparation with specificity for the 2,4-dinitrophenyl group was isolated from ascitic fluid in a relatively pure state by affinity chromatography and used in the form of an immunosorbent to isolate antibodies from the monospecific goat serum. The GAME antibodies were 125I-labeled and used to develop a radioallergosorbent test (RAST) for the quantitation of murine IgE antibodies specifically adsorbed onto antigen-coupled paper discs. The RAST was specific for antibodies of the IgE class only and was as sensitive as and more accurate than PCA assay. RAST results on sera of mice treated with tolerogenic conjugates indicated a reduction in the affinity and concentration of the IgE antibody populations on suppression of the IgE response. The effect of interference by non-IgE antibody populations on the RAST curves has been discussed.

Standardization of radioimmunoassay: Differences in reactivity of rat IgEs with antibodies to a monoclonal IgE

Abstract Examination of antisera raised against a rat monoclonal IgE 162 revealed that antibodies to its myelotypic determinants had a much higher affinity for IgE 162 than antibodies to its class-specific determinants. Moreover, in competitive inhibition radioimmunoassays designed for the quantitation of total IgE in rat sera, using immunospecifically purified antibodies to the class-specific IgE determinants, it was found that purified IgE standards isolated from other myeloma protein sources were not as efficient as IgE 162 in inhibiting the binding of 125 I-IgE 162 ; these results suggest the existence of subclasses of IgE. In these assays, it was found imperative, therefore, to use antibodies against class-specific determinants, i.e. anti-IgE antibodies devoid of anti-myelotypic antibodies, in conjunction with 125 I-labelled purified IgE other than IgE 162 which had been used as the immunizing antigen.

Interaction of superoxide dismutase with phospholipid liposomes. An uptake, spin label and calorimetric study.

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu2+, Zn2+ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7 degrees C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.

Suppression of IgE antibody production in sensitized mice and rats by tolerogenic conjugates of synthetic hydrophilic polymers with antigen or hapten: effect on antigen-induced histamine release from peritoneal mast cells.

IgE antibodies were produced in mice and rats by immunization with ragweed pollen extract (RAG) or dinitrophenylated ovalbumin (DNP3-OA). Treatment of these animals with tolerogenic conjugates of (i) the antigen (RAG or OA) with monomethoxypolyethylene glycol (mPEG), or (ii) DNP with polyvinyl alcohol (DNP-PVA) resulted, within 7-14 days, in a fall in circulating IgE antibodies and in mast cell sensitivity, as assessed by the radioallergosorbent test (RAST) and in the in vitro antigen-induced histamine release (HR) test, respectively. The reduction in responsiveness was more marked in mice than rats; 10 days after a booster immunization, the IgE antibody titres in the RAG-mPEG-treated group of mice were approximately 10-fold lower than in the saline-treated group, with a 100-fold difference in cell sensitivity. DNP-PVA treatment of mice produced a more than 10-fold reduction in IgE anti-DNP titres with a substantial reduction in histamine release.

Properties of bovine colostral IgG1 anti-DNP antibodies--I. Isolation and binding studies.

Abstract Bovine IgG 1 anti-DNP antibodies were isolated from the colostrum of two animals using a combination of DEAE-cellulose and Bio-Gel chromatography as well as an immunosorbent. Binding studies with ϵ-DNP-lysine yielded conventional results while those with the dye-hapten 1-hydroxy-4-(2,4-dinitrophenylazo)-2,5-napththalene disulfonate (1N-2,5S-4DNP), revealed the presence of two kinds of combining site present in about equal concentration, as determined by computer analysis. Titration of one antibody preparation with 1N-2,5S-4DNP detected only a maximum of 1.2 combining sites per intact IgG 1 molecule. However, upon removal of the Fc component by pepsin, the expected 1.8 binding sites per F(ab′) 2 were found.