Site-specific antibodies distinguish single amino acid substitutions in position 57 in HLA-DQ beta-chain alleles associated with insulin-dependent diabetes.
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.
Atar
D
D
Hagedorn Research Laboratory, Gentofte, Denmark.
Dyrberg
T
T
Michelsen
B
B
Karlsen
A
A
Kofod
H
H
Mølvig
J
J
Lernmark
A
A
D000483:Alleles; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006683:HLA-DQ Antigens / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D010446:Peptide Fragments / Q000276:immunology
D006683:HLA-DQ Antigens; D007106:Immune Sera; D010446:Peptide Fragments