Subtype-specificity of antipeptide antibodies raised against unique sequences of human interferons-alpha.
A strategy for the production of human interferon-alpha (IFN-alpha) subtype-specific antibodies, based on immunizing rabbits with short unique synthetic peptides coupled to protein carriers, has been validated. These peptides correspond to amino acid residues 99-111 of IFN-alpha 1, 50-57 and 103-116 of IFN-alpha 2, and 37-50 of IFN-alpha 4. The antipeptide antibodies [anti-IFN alpha 1(99-111), anti-IFN alpha 2(50-57C), anti-IFN alpha 2(103-116) and anti-IFN alpha 4(C37-50)] were tested by ELISA and Western blotting for their reactivity with immunoaffinity-purified recombinant human IFN-alpha 1, -alpha 2b and -alpha 4a. The anti-IFN alpha 1(99-111) and anti-IFN alpha 2(50-57C) reacted with their corresponding IFN-alpha and did not crossreact with the other IFN subtypes. The anti-IFN alpha 2(103-116) reacted with IFN-alpha 2b and also crossreacted slightly with the other subtypes. The anti-IFN alpha 4(C37-50) reacted well with IFN-alpha 4a, crossreacted with significantly lower affinity with IFN-alpha 1 and did not bind IFN-alpha 2b. Residues 104-107 and 108-111 are the major components of the epitopes recognized by anti-IFN alpha 1(99-111) and anti-IFN alpha 2(103-116), respectively, as determined by ELISA against overlapping octapeptides.
Sattayasai
N
N
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Marzuki
S
S
McMullen
G L
GL
Geysen
H M
HM
Mason
T J
TJ
Hibbs
A R
AR
Overall
M
M
Hertzog
P J
PJ
Linnane
A W
AW
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001667:Binding, Competitive; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid
D016898:Interferon-alpha; D011994:Recombinant Proteins