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W C Hanly

Publications and source records attributed to W C Hanly.

35 records · Page 2Linked to original sources
PubMed1978

Identifying immunoglobulin isotypes, allotypes and idiotypes by a hemolytic assay in gel.

We developed a hemolytic radial immunodiffusion assay for identifying immunoglobulin (Ig) isotypes, allotypes and idiotypes by using gels containing erythrocytes coated with anti-Ig antibody or erythrocytes coated with Staphylococcal protein A. These indicator cells lysed specifically when treated sequentially with Ig antigen, the appropriate anti-Ig antiserum (developer) and complement. To identify these Ig subpopulations, we used monospecific indicator cells, e.g. erythrocytes coated with antibody specific for an Ig isotype, and developers with broader specificities ('multispecific'), e.g. antiserum to Fab. Alternatively, we used 'multispecific' indicator cells, e.g. erythrocytes coated with antibody to Fab and monospecific developers, e.g. antiserum to Ig idiotype. To identify Ig subpopulations specifically, either the indicator cells or the developer need to be monospecific. When both the indicator cells and the developer were monospecific, e.g. to allotype and to isotype, the specificity was determined by both reagents and ultimately restricted by the reagent with the narrower specificity, that is, reacting with the smallest Ig subpopulation. This sensitive hemolytic assay may be used to quantitate subpopulations of Ig molecules and may be modified into a reverse plaque forming cell assay to count lymphocytes secreting a given Ig class, type, allotype and idiotype.

Agar↗
PubMed1977

Additional rabbit IgA allotypes, f69, f70, g76,g77: control by the C alphaf and C alphag loci.

Genetic and immunochemical studies have led to the identification of four additional rabbit IgA allotypes controlled by the Calphaf and Calphag loci. The folowing linkage combinations of the VHa and the 'new' alleles were observed among the populations of rabbits studied: a1f70g76, a1f69g77, and a2f69g77. Cross-reactions among g74, g76, and g77 molecules with various anti-g anti-allotype antisera indicate that the IgA-g allotypic specificities are comprised of multiple antigenic determinants. These studies provide a basis for further understanding of the evolution and gnetic control of the immunoglobulin heavy chain chromosomal region.

Alleles↗
PubMed1975

Cellular distribution of rabbit IgA allotypes.

Intestinal tissue from rabbits heterozygous at one or both of the alpha-chain subclass loci, i.e., f or g, was examined with mixtures of rhodamine-labeled and fluorescein-labeled antibodies each directed against one of the allelic products of the f or g locus. Individual plasma cells were stained by only one fluorescent reagent and thus exhibited both allelic exclusion and sublcass exclusion. In contrast, the epithelial cells of the Lieberkükhn's glands sid not exhibit allelic exclusion. Studies with rabbits heterozygous at the f and g loci revealed that the ratio of the number of cells expressing maternal-type f cells to paternal-type f is equal to the ratio of the number of maternal-type g cells to paternal-type g cells. These data are used to suggest that the expression of each of the genes in the heavy chain chromosomal region of a particular allogroup is under the same control.

Absorption↗
PubMed1974

Recombinant rabbit secretory immunoglobulin molecules: alpha chains with maternal (paternal) variable-region allotypes and paternal (maternal) constant-region allotypes.

A population of IgA molecules having heavy chains coded by two parental chromosomes in trans position has been identified in rabbits heterozygous at both the V(H)a locus, which controls allotypic specificities on the variable part of heavy chains, and the C(alpha)g locus, which controls allotypic specificities on the constant part of alpha chains. These recombinant molecules have alpha-chain allotypic specificities controlled by both the maternal V(H)a gene and the paternal C(alpha)g gene or conversely, the paternal V(H)a gene and the maternal C(alpha)g gene. These recombinant molecules were found in F(ab)(2alpha) fractions obtained after passage of F(ab)(2alpha) preparations through immunosorbent columns designed to remove one population of F(ab)(2alpha) molecules, i.e., g74- or g75-type molecules. The effluent F(ab)(2alpha) fractions were then examined by radioprecipitation methods for allotypic specificities controlled by the V(H)a and C(alpha)g loci. About 40% of the g75 F(ab)(2alpha) molecules from each of three rabbits with the a(1)g(74) and a(2)g(75) allogroups were alg75 recombinants. These alg75 recombinant molecules represented from 2.5-5.6% of the total unfractionated F(ab)(2alpha) sample. The F(ab)(2alpha) fractions from two rabbits with the a(1)g(75) and a(3)g(74) allogroups had from 1.8-8.2% recombinant molecules: some were alg74 recombinants and some were a3g75 recombinants. Somatic recombination as a mechanism responsible for the synthesis of polypeptide chains in which part of the information is obtained from one chromosome and part from the homologous chromosome is discussed.

Alleles↗
PubMedDate not supplied

Covalent disulfide binding of human IL-1 beta to alpha 2-macroglobulin: inhibition by D-penicillamine.

Secreted human IL-1 beta is known to have two free SH groups due to unpaired cysteines (positions 8 and 71). Alpha 2-Macroglobulin (alpha 2-M) has internal thioester bonds between cysteine and glutamate residues. Free SH groups may be generated at these alpha 2M residues through the action of proteinases, amines such as methylamine, or at a slow rate, by H2O ("aging" of alpha 2M). Thus, the possibility that IL-1 beta forms a disulfide bond with alpha 2M was investigated. 125I-labeled human rIL-1 beta (15 kDa) was incubated with fresh normal human serum or with purified alpha 2M, treated or not with methylamine. The mixtures were submitted to nondenaturing and denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. IL-1 beta bound to commercially purified "aged" alpha 2M and to alpha 2M in methylamine-treated serum but not to native serum alpha 2M. It did not bind detectably to any other serum proteins. The addition of D-penicillamine (D-pen) during the reaction of [125I]rIL-1 beta with serum or purified alpha 2M blocked the covalent binding of rIL-1 beta to alpha 2M. [125I]rIL-1 beta was removed from alpha 2M by 2-mercaptoethanol in SDS. Thus, disulfide bonds were formed between the free SH groups on [125I]rIL-1 beta and those resulting from the cleavage of the internal thioester bonds of alpha 2M. "Cold" rIL-1 beta and a Cys71----Ser71 rIL-1 beta mutant effectively competed with [125I]rIL.1 beta for binding sites on alpha 2M. When complexes of rIL-1 beta or the mutant rIL-1 beta and alpha 2M were subjected to nonreducing SDS-PAGE and subsequent Western blot analysis, the rIL-1 beta molecules were found to be present in the alpha 2M bands in a dose-dependent manner. rIL-1 beta attached to alpha 2M in the presence or absence of D-pen showed similar biological activity in the mouse thymocyte-assay. Thus, rIL-1 beta attached noncovalently to alpha 2M is biologically active. The lack of inhibition of rIL-1 beta activity by binding to methylamine-treated alpha 2M in the absence of D-pen suggests, but does not prove, that the covalently bound rIL-1 beta is also active. We concluded that human rIL-1 beta binds to alpha 2M through the Cys at position 8 and that D-pen inhibits this binding. We speculate that this inhibitory effect may contribute to the therapeutic benefits of D-pen in patients with rheumatoid arthritis.

Animals↗