HLA Class I Biochemistry:
Definition and Frequency Determination of Subtypes by
One-Dimensional Isoelectric Focusing and Immunoblotting

Frenz, Gabriele; Doxiadis, Ilias I.N.; Vögeler, U.; Grosse‐Wilde, H.

doi:10.1159/000460960

Cited by 4 publications

(11 citation statements)

References 14 publications

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“…native molecules, molecules with no transmembrane portion and molecules with deleted transmembrane portion and cytoplasmic tail [9]) do not alter the isoelectric points of the single HLA gene product. The analysis of 16 individSample Preparation for 1D-IEF Neuraminidase-treated whole lysates or immunoprecipitated class I proteins were subjected to 1D-IEF as already described [5,12]. Two-dimensional gel electrophoresis (2D-Gel) was performed as described by O'Farrell [13],…”

Section: Resultsmentioning

confidence: 99%

“…In short: EDTA-plasma of serologically HLA-typed individuals and families was collected by high-speed centrifugation in order to remove contaminating platelets and used directly or stored at -80 "C. PBL of the same individuals were isolated by Ficoll gradient centrifugation using the usual methods. 5xl06 cells were either stored at -80"C as a dry pellet or subjected to Triton-X-114 phase separation as described elsewhere [5]. kindly provided by Dr. R. Mierau, Rheumaforschungsinstitut, Rheu ma-Klinik Aachen, FRG, were coupled to 30 |il sheep anti-mouse Ig activated polystyrene magnetobeads (Dynal, Oslo, Norway).…”

Section: Plasma and Cellsmentioning

confidence: 99%

“…Fifteen microliters of MAb beads were incubated with 50-250 pi serum at 4 °C for 60 min, followed by application of the vial to a magnet (Dynal, Norway, Oslo) and washed 10 times with NET buffer (0.5 M NaCl, 20 mMEDTA, Tris-HCl, pH 7.4, 0.5% NP-40), by resuspend ing the beads in buffer and applying the vial to the magnet. The immunoprecipitated molecules were then neuraminidase-treated as described elsewhere [5,12],…”

Section: Plasma and Cellsmentioning

confidence: 99%

“…Recent data, however, showed that biochemi cal characterization of class I heavy chains on isotopelabelled transformed cells or PBL by one-dimensional isoelectric focusing (1D-IEF) [1][2][3][4] and immunoblotting [5] increased the serologically defined class I polymor phism.…”

Section: Introductionmentioning

confidence: 99%

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Typing for HLA Class I Gene Products Using Plasma as Source

Doxiadis¹,

Grosse‐Wilde²

1989

Vox Sanguinis

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Soluble HLA class I proteins have been found in serum or plasma of healthy and diseased individuals. Here we present evidence that these molecules can be readily used for determination of the HLA type by biochemical methods. Immunoprecipitation of the soluble class I gene products using monomorphic monoclonal antibodies coated to immunobeads and one-dimensional isoelectric focusing followed by immunoblotting represents a feasible and reproducible technique for typing. Analysis of these gene products in families (n = 12, with a total of 62 individuals) as well as in the population (n = 82) showed that all serologically defined antigens tested to date were present in plasma. A reference chart established primarily for the membrane-bound antigens could also be used for the soluble ones.

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Section: Resultsmentioning

confidence: 99%

Section: Plasma and Cellsmentioning

confidence: 99%

Section: Plasma and Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Typing for HLA Class I Gene Products Using Plasma as Source

Doxiadis¹,

Grosse‐Wilde²

1989

Vox Sanguinis

Self Cite

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“…After three washes with PBS containing 0.05% Tween 20, filters were dried and autoradiographed at Ϫ70°C using Hyperfilm-MP (Amersham). One-dimensional isoelectric focusing (1D-IEF) of HLA class I Ags using detergent-solubilized B lymphoblastoid cell line lysates was performed as described by Neefjes et al (13), followed by immunoblotting with mAb TP25.99 or with rabbit anti-HLA class I heavy chain antiserum (23).…”

Section: Binding Assaysmentioning

confidence: 99%

Structural Relatedness of Distinct Determinants Recognized by Monoclonal Antibody TP25.99 on β2-Microglobulin-Associated and β2-Microglobulin-Free HLA Class I Heavy Chains

Desai

Wang

Noronha

et al. 2000

The Journal of Immunology

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The association of HLA class I heavy chains with β2-microglobulin (β2m) changes their antigenic profile. As a result, Abs react with either β2m-free or β2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both β2m-associated and β2m-free HLA class I heavy chains. The reactivity with β2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194–198 in the α3 domain. The reactivity with β2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239–242, 245, and 246 in the α3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.

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Bone marrow transplantation from partially HLA‐matched related donors in adults with leukaemia: The Experience at the University Hospital of Essen, Germany

et al. 1996

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The Seattle group has demonstrated that bone marrow transplantation (BMT) using partially HLA-mismatched related donors is feasible in principal. However, it was unclear whether these results can also be achieved at smaller-sized BMT units. Therefore a matched pair analysis enrolling 52 BMTs from partially HLA-mismatched relatives and 52 control BMTs from HLA-identical siblings was performed at the University Hospital of Essen. Overall survival (OS) and incidence of graft-versus-host disease (GVHD) did not differ significantly after study and control BMTs (OS: 52% v 63% 1 year, 46% v 48% 5 years post transplant; acute GVHD: 37% v 35%, chronic GVHD: 67% v 55%). After study BMTs, however, therapy-related mortality (P=0.018) and incidence of graft failure (P=0.002) were increased, whereas relapse rate was reduced (11% v 27%). Two or three mismatches in HVG direction implied the same risk of graft failure as one mismatch. Therefore, (i) OS after BMT from one HLA antigen mismatched relative and from HLA-identical siblings does not differ significantly even when performed at a 'smaller' centre; (ii) two or even three HLA mismatches in host-versus graft (HVG) direction might be acceptable in family BMT for leukaemia.

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HLA Class I Biochemistry: Definition and Frequency Determination of Subtypes by One-Dimensional Isoelectric Focusing and Immunoblotting

Cited by 4 publications

References 14 publications

Typing for HLA Class I Gene Products Using Plasma as Source

Typing for HLA Class I Gene Products Using Plasma as Source

Structural Relatedness of Distinct Determinants Recognized by Monoclonal Antibody TP25.99 on β2-Microglobulin-Associated and β2-Microglobulin-Free HLA Class I Heavy Chains

Bone marrow transplantation from partially HLA‐matched related donors in adults with leukaemia: The Experience at the University Hospital of Essen, Germany

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