Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies.
Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRK-RIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum KD was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed "linear" epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design.
Seligman
S J
SJ
Department of Medicine, SUNY Health Science Center, Brooklyn 11203, USA. SSELIGMAN@NETMAIL.HSCBKLYN.EDU
Binley
J M
JM
Gorny
M K
MK
Burton
D R
DR
Zolla-Pazner
S
S
Sokolowski
K A
KA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D015483:HIV Antibodies / Q000737:chemistry*; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; D010456:Peptides, Cyclic