Joseph C. Moore

A europium fluoroimmunoassay for measuring binding of antigen to class II MHC glycoproteins.

A dissociation-enhanced lanthanide fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was developed to measure binding of biotin-labeled peptides to class II MHC proteins. Binding of biotin-peptides as measured by this assay was saturable and inhibited in the presence of unlabeled peptide. Background fluorescence was minimal and there was a direct relationship between signal and biotin-peptide/class II complex concentration from 1.3 pmol to less than 1 fmol total class II. The sensitivity of the assay and the ability to selectively capture specific class II proteins from detergent lysates of cells with solid phase mAb made it possible to measure formation peptide/class II complexes in live APC cultured with biotin-labeled insulin. This assay is expected to be useful for routine measurement of peptide/class II binding and biochemical analysis of Ag processing events.

A europium fluoroimmunoassay for measuring peptide binding to MHC class I molecules.

A fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was used to measure binding of biotin-labeled peptides to H-2Dk molecules. A fluorescein-labeled octameric peptide from the middle T (MT) protein of mouse polyoma virus was identified that specifically binds to purified Dk using a previously-established assay based on size exclusion chromatography. A europium immunoassay was adapted to measure binding of a biotin-derivative of this peptide to purified Dk. The assay was found to be sensitive and highly specific. Binding was optimal at pH 5.0 and 24-27 degrees C, and it was not enhanced in the presence of additional beta2-microglobulin (beta2m). Excellent results were also obtained in experiments with fixed cells that express Dk. This assay is expected to be useful for high volume, routine analysis of peptide binding to MHC class I molecules.

Intact proteins can bind to class II histocompatibility molecules with high affinity

The ability of intact protein antigens to bind to purified class II histocompatibility molecules was investigated. Intact bovine ribonuclease (RNase) inhibited peptide binding to DR1 with a potency similar to that of a high affinity peptide or irreversibly denatured RNase. Similarly, horse myoglobin (Mb) was a potent inhibitor of peptide binding to I-E(k). I-E(k)-Mb complexes were directly visualized as a distinct band with reduced mobility on SDS PAGE. Direct binding experiments with biotin-labeled proteins demonstrated that Mb and RNase bind to class II molecules through the peptide-binding groove with high affinity, and that binding occurs in the absence of detergent. The possibility that HLA-DM can catalyse the binding of intact protein antigens was supported by the observation that DM enhances the binding of biotin-RNase to DR1. Our results provide further support for the hypothesis that intact, partially unfolded protein antigens can act as ligands for initial interaction with class II molecules.

Peptide binding to mixed isotype AβdEαd class II histocompatibility molecules

Previous studies have demonstrated that mixed isotype A beta(d) E alpha(d) molecules are expressed in transfected cell lines and that the level of expression is very low in normal B cells from H-2(d) mice. T-cell responses restricted by A beta(d) E alpha(d) are induced in H-2(d) mice immunized with the synthetic peptides YL2 and FL2 or with sperm whale myoglobin, despite the low concentration of mixed isotype molecules expressed on antigen-presenting cells. In the present study, the peptide binding behavior of A beta(d) E alpha(d) was investigated. A peptide from the cytoplasmic domain of invariant chain, I(1-18), was observed to bind with high affinity to purified A beta(d) E alpha(d). Binding was optimal at pH 5, indicating that these molecules prefer to bind peptide in the acidic environment of endosomal compartments similar to other murine class II proteins. YL2 and FL2 bind to A beta(d) E alpha(d) with slightly lower affinity. The selective restriction of YL2- and FL2-specific T cells to mixed isotype molecules was accounted for by the observation that these peptides do not bind to either I-E(d) or I-A(d). By contrast, myoglobin peptides bind to both parental and mixed isotype molecules. None of the A beta(d) E alpha(d)-restricted peptide determinants bind to A beta(d) E alpha(d) with extremely high affinity. Thus it is unlikely that these peptides occupy an unusually high fraction of mixed isotype molecules during antigen presentation in vivo. It is more likely that the presence of a subpopulation of high-affinity T cells capable of being stimulated by very low concentrations of A beta(d) E alpha(d)/peptide complexes is responsible for the unusual A beta(d) E alpha(d)-restricted response observed with some antigens.