Search PubMed⌕ Search

Biomedical subjects

N C Hughes-Jones

Publications and source records attributed to N C Hughes-Jones.

At least 19 recordsLinked to original sources
PubMed1999

Synthesis of Rh Fv phage-antibodies using VH and VL germline genes.

Antibodies to the D antigen of the Rh system use a restricted set of immunoglobulin V and J gene segments, especially VH DP50 and DP63, JH6, Vlambda DPL16 and Jlambda 2/3. These gene segments may confer a natural affinity on the antibodies for the D antigen and this hypothesis has been tested by constructing two single-chain Fv phage-antibody libraries based on the germline gene segments DP50 and DP63; structural variability was obtained by insertion of 11 amino acids in random sequence in the VHCDR3. 10 anti-D antibodies were selected from these libraries, each with a unique VHCDR3. In contrast, selections with the CcEe antigens produced antibodies reacting with the Rh polypeptide molecules but without strict blood group specificity. One of these latter DP50-based antibodies was converted into 12 different antibodies with specificity for E by replacing the original germline light chain with chains from a rearranged L chain library. The CDR1 and CDR2 sequences of the DP50-based antibodies were common to both anti-D and anti-E molecules; differentiation between D and E specificity was dependent on VHCDR3 sequences and their correct pairing with an appropriate L chain.

Antibody Specificity↗
PubMed1995

Transcript analysis of D category phenotypes predicts hybrid Rh D-CE-D proteins associated with alteration of D epitopes.

The RH blood group locus from RhD-positive donors is composed of two closely related genes, RHCE and RHD, encoding the Cc/Ee and D antigens, respectively. The major Rh antigen, D, is serologically defined as a mosaic of at least nine determinants (epD1 to epD9), and the lack of expression of some of these D epitopes at the surface of variant red blood cells defines the D category phenotypes. In this report, we have analyzed the Rh transcripts from reticulocytes of different D category phenotypes (DIVa, DIVb, DVa, and DFR). Although Southern blot analysis did not sow obvious deletions within the RHD gene, sequence analysis of the RhD transcripts indicated that, in all cases studied, the lack of D epitopes is associated with substitutions, in the deduced polypeptides, of amino acids specific of the RhD protein by those encoded at the equivalent position by the RHCE gene. These results strongly suggested that the D category phenotypes resulted from segmental DNA replacement between RHD-specific fragments and their equivalents in the RHCE gene. The regions involved in the DIVa, DIVb, DVa, and DFR phenotypes were shown to encompass all or part of the exons 3 and 7, exons 7 to 9, exon 5, and exon 4, respectively. All protein variants encoded by these rearranged RH genes represent new CE-D-CE hybrid molecules that retain only some of the nine D epitopes. Because segmental DNA replacements have been previously identified in other Rh variant genomes, we postulate that such genomic rearrangements between different regions of the RHCE and RHD genes should be one of the most frequent events involved in the extreme polymorphism of the RH blood group system.

Base Sequence↗
PubMed1995

Prediction of the severity of haemolytic disease of the newborn. Quantitative IgG anti-D subclass determinations explain the correlation with functional assay results.

Sera containing anti-D, taken from 44 RhD-negative women with RhD-positive infants, were tested in antibody-dependent cellular cytotoxicity (ADCC) and monocyte monolayer assays (MMA) which used similar target and effector cell populations. In addition, the anti-D concentration was measured in the Auto Analyzer and the number of IgG1 and IgG3 anti-D molecules bound to the target red cells was measured by flow cytometry. The results of the functional assays and Auto Analyzer quantitation were examined for correlation with IgG subclass quantitation and all results were compared for their ability to predict the severity of haemolytic disease of the newborn (HDN). ADCC correctly predicted HDN in 39/44 (88.6%) cases, Auto Analyzer quantitation in 35/44 (79.5%) and the MMA in 32/44 (72.7%). For all three assays, the number of correct predictions was highest when the maternal serum contained both IgG1 and IgG3 anti-D. ADCC activity and HDN were correlated with the number of cell-bound IgG1 molecules (r > or = 0.58), but MMA activity was most closely correlated with the number of cell-bound IgG3 molecules (r = 0.68). Hence the superior predictive value of ADCC is due to its ability to reflect the IgG1 component of maternal anti-D, which has a better correlation than IgG3 anti-D with the severity of HDN.

Autoanalysis↗
PubMed1994

Characterization of human blood group scFv antibodies derived from a V gene phage-display library.

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

ABO Blood-Group System↗
PubMed1993

Human anti-self antibodies with high specificity from phage display libraries.

Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V-genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V-genes were shuffled at random and cloned for display as single-chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V-genes, with high specificities of binding to human self-antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)-10(6) M-1s-1, k(off) = 10(-2)s-1 and Ka = 10(7) M-1). The kinetics of association are typical of known Ab-protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.

Amino Acid Sequence↗
PubMed1993

Human antibody fragments specific for human blood group antigens from a phage display library.

We have isolated human antibody fragments with binding specificities against the blood group antigens of the ABO and I blood group systems (B and HI), of the Rh system (D and E) and of the Kell system (Kpb), by selecting a phage-antibody library on human red cells. The fragments, expressed in bacteria, were antigen-specific and effective in assays including agglutination and immunohistochemistry. Selection of phage-antibody libraries using intact cells seems to offer a promising alternative to hybridoma technology for the production of antibodies against cell surface molecules.

ABO Blood-Group System↗
PubMed1993

Quantitation of D sites on selected 'weak D' and 'partial D' red cells.

Measurements have been made of the number of available sites on 10 examples of red cells in which the only abnormality appeared to be a quantitative reduction in the expression of D (weak D cells); these estimates were carried out using three monoclonal anti-D antibodies, Fog-1, Brad-3 and Los-2. The values varied with the monoclonal antibody that was used and fell within the range of 170-1,870 sites/cell. A further 3 examples of weak D cells which had brought about immunisation following transfusion were found to have between 390 and 1,470 sites per red cell. The implications of the D site density on the immunogenicity of weak D cells are discussed. The number of sites on red cells with structurally abnormal D (partial D cells) were also estimated, using the antibody Fog-1. Four of the 5 examples of cells of category IVa (probable phenotype Ror) were found to have a high expression of D (range 29,300-41,300), but the available D sites of categories DVa, DVIa, and DVII were considerably reduced (< 500, < 500 and 2,400-7,500 sites/cell, respectively). As a working hypothesis, it is suggested that there are two types of genetic abnormality leading to an abnormal expression of D. First, a defect in genomic DNA leading only to a quantitative reduction in the number of available D sites; this genomic lesion should be termed 'weak D'. Secondly, genomic defects leading to amino acid sequence abnormalities and structural change in the D polypeptide; these lesions should be collectively known as 'partial D'.

Antibodies, Monoclonal↗
PubMed1992

Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. Evidence for affinity maturation by somatic hypermutation and repertoire shift.

To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.9 point mutations in the V gene segments of the four IgM antibodies (Ka = 1-4 x 10(7)/M-1) compared with 19 point mutations in the V gene segments of the 10 IgG antibodies. The four highest affinity antibodies (Ka = 0.9-3 x 10(9)/M-1) averaged 25.5 point mutations. The use of repertoire shift and somatic hypermutation in affinity maturation of human alloantibodies is similar to data obtained in inbred mice immunized with haptens.

Antibodies, Monoclonal↗
PubMed1991

Differential exposure of epitopes on the human red cell antigen D (Rh).

The D polypeptide of the human Rh blood group system has a number of different epitopes on its surface. It is also known that there is considerable variation in the number of D antigen sites available to different human monoclonal anti-D antibodies. For instance, certain monoclonal antibodies recognise only a small number of sites on the red cell surface (about 9,000 sites/red cell on R1R2 cells) whereas other antibodies recognise a high number (about 20,000-30,000 sites/red cell). It has been found that cholesterol enrichment of the red cell membrane increases the number of sites available to those antibodies which recognize only a few sites but has no effect on those recognising many sites. The results are consistent with the view that access to some of the D epitopes is partially hindered by neighbouring molecules in the membrane and that alteration of the lipid content of the membrane changes it in such a way as to allow increased access to these obstructed epitopes.

Antibodies, Monoclonal↗
PubMed1991

Relative functional binding activity of IgG1 and IgG3 anti-D in IgG preparations.

The intention to replace polyclonal IgG anti-D with human monoclonal antibody in the prophylaxis of haemolytic disease of the newborn requires knowledge concerning the relative content of IgG1 and IgG3 anti-D in prophylactic IgG preparations that are in present use. This has been carried out using a functional assay in which the absolute amount of IgG1 and IgG3 anti-D present on red cells was determined after incubation with IgG preparations. The assay was carried out by flow cytometry on 17 samples; expressed as a percentage of the total, the average value for the amount of IgG3 anti-D on the cells was 8% (range 1-18%). Similar measurements were also made on the anti-D present in 18 samples of antisera; IgG3 anti-D formed a larger fraction of the total, the average value being 17% (range 0-60%) confirming previously reported estimates. It is suggested that some of the low values found for IgG3 in IgG preparations may be due do preferential loss during production.

Cell Separation↗
PubMed1990

Clearance of Rh D-positive red cells with monoclonal anti-D.

Two human monoclonal antibodies, one IgG3 and one IgG1, with anti-Rh D specificity, were tested for their ability to clear red cells. Samples of red cells from 12 D-positive subjects were sensitised in vitro with various amounts of antibody, the number of antibody molecules bound to the cells was estimated, and the cells were reinjected into the donor's circulation. Both antibodies mediated clearance but substantially fewer IgG3 than IgG1 antibody molecules were required to produce a given rate of clearance. The IgG3 antibody was slightly more effective than polyclonal anti-D, as judged by comparison with previously published results. Since there is believed to be an association between the rate of red cell clearance by anti-D and the ability of the antibody to suppress D immunisation, the IgG3 monoclonal antibody may prove suitable for the immunoprophylaxis of Rh D haemolytic disease.

Antibodies, Monoclonal↗
PubMed1990

Nucleotide sequences and three-dimensional modelling of the VH and VL domains of two human monoclonal antibodies specific for the D antigen of the human Rh-blood-group system.

The nucleotide sequences were determined for the VH and VL domains of two human IgG1 antibodies, Pag-1 and Fog-B, specific for the D antigen of the Rh-blood-group system. The VH-region genes of the two antibodies were derived from separate germ-line genes within the VH-IV gene family, but both antibodies used the same JH6 gene. The D-region genes differed from each other, and no similarity was found to known D regions. The light chain of Fog-B belongs to the V lambda-I subgroup and that of Pag-1 probably belongs to the V lambda-V subgroup; both light chains used the J2/3 gene. Three-dimensional models of the variable domains were made, based on those of known crystallographic structure. The surface contours at the combining sites are clearly different, consistent with the evidence that the antibodies recognize different but overlapping epitopes. Some details of the molecular modelling of hypervariable regions have been deposited as Supplementary Publication SUP 50155 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction.

Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.

Amino Acid Sequence↗