Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.
By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.
de Haard
H J
HJ
Biosciences Research Unit, Organon Teknika, Boxtel, Maastricht, The Netherlands. HANS-DE.HAARD@UNILEVER.COM
Kazemier
B
B
Koolen
M J
MJ
Nijholt
L J
LJ
Meloen
R H
RH
van Gemen
B
B
Hoogenboom
H R
HR
Arends
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018604:Epitope Mapping; D015723:Gene Library; D015483:HIV Antibodies / Q000235:genetics / Q000276:immunology; D015488:HIV Antigens / Q000276:immunology*; D016655:HIV Core Protein p24 / Q000276:immunology*; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D016056:Immunodominant Epitopes; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D017422:Sequence Analysis, DNA
D015483:HIV Antibodies; D015488:HIV Antigens; D016655:HIV Core Protein p24; D016056:Immunodominant Epitopes; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins