Site-specific antibodies distinguish single amino acid substitutions in position 57 in HLA-DQ beta-chain alleles associated with insulin-dependent diabetes

TY - JOUR

T1 - Site-specific antibodies distinguish single amino acid substitutions in position 57 in HLA-DQ beta-chain alleles associated with insulin-dependent diabetes

AU - Atar, D

AU - Dyrberg, T

AU - Michelsen, Birgitte

AU - Karlsen, Alan E

AU - Kofod, Hans

AU - Mølvig, J

AU - Lernmark, A

PY - 1989/7/15

Y1 - 1989/7/15

N2 - The HLA-DQ beta-chain gene shows a close association with susceptibility or resistance to autoimmune insulin-dependent diabetes mellitus (IDDM) and it has been suggested that the amino acid in position 57 may be of pathogenetic importance. To study the expression of the IDDM associated HLA-DQ beta-chain alleles, we immunized rabbits with 12 to 13 amino acid long peptides representing HLA-DQw7 and -DQw8 allelic sequences, differing only by one amino acid in position 57 being aspartic acid (Asp) and alanine (Ala), respectively. Immunoblot analysis of lymphoblastoid cells showed that several antisera recognized a 29-kDa protein, equivalent to the expected molecular size of the HLA-DQ beta-chain to yield two antisera specific for HLA-DQw7 (pos. 57Asp) and three antisera for HLA-DQw8 (pos. 57Ala) positive cells. Analysis of HLA-DR 3/4 positive IDDM patients (n = 24) and controls (n = 19) showed that all (100%) patients were positive for pos. 57Ala antiserum compared to 13 of 19 (68%) of the controls. The remaining six controls reacted with the pos. 57Asp antisera, whereas none of the patients did. We have therefore successfully been able to generate site-specific antibodies that distinguish single amino acid substitutions in predetermined positions of allelic HLA-DQ beta-chain gene products. Such sera should become useful to detect and investigate HLA associated susceptibility to autoimmune diseases in man.

AB - The HLA-DQ beta-chain gene shows a close association with susceptibility or resistance to autoimmune insulin-dependent diabetes mellitus (IDDM) and it has been suggested that the amino acid in position 57 may be of pathogenetic importance. To study the expression of the IDDM associated HLA-DQ beta-chain alleles, we immunized rabbits with 12 to 13 amino acid long peptides representing HLA-DQw7 and -DQw8 allelic sequences, differing only by one amino acid in position 57 being aspartic acid (Asp) and alanine (Ala), respectively. Immunoblot analysis of lymphoblastoid cells showed that several antisera recognized a 29-kDa protein, equivalent to the expected molecular size of the HLA-DQ beta-chain to yield two antisera specific for HLA-DQw7 (pos. 57Asp) and three antisera for HLA-DQw8 (pos. 57Ala) positive cells. Analysis of HLA-DR 3/4 positive IDDM patients (n = 24) and controls (n = 19) showed that all (100%) patients were positive for pos. 57Ala antiserum compared to 13 of 19 (68%) of the controls. The remaining six controls reacted with the pos. 57Asp antisera, whereas none of the patients did. We have therefore successfully been able to generate site-specific antibodies that distinguish single amino acid substitutions in predetermined positions of allelic HLA-DQ beta-chain gene products. Such sera should become useful to detect and investigate HLA associated susceptibility to autoimmune diseases in man.

KW - Alleles

KW - Amino Acid Sequence

KW - Antibody Specificity

KW - Diabetes Mellitus, Type 1

KW - Enzyme-Linked Immunosorbent Assay

KW - HLA-DQ Antigens

KW - Humans

KW - Immune Sera

KW - Immunoblotting

KW - Molecular Sequence Data

KW - Molecular Weight

KW - Organ Specificity

KW - Peptide Fragments

M3 - Journal article

C2 - 2738402

SN - 0022-1767

VL - 143

SP - 533

EP - 538

JO - Journal of Immunology

JF - Journal of Immunology

IS - 2

ER -