Human Fab fragments specific for the Haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression.
To determine whether the human antibody (Ab) repertoire to the Haemophilus influenzae type b capsular polysaccharide (Hib PS) could be studied at the molecular level with phage display technology, we constructed a phage Fab library by using peripheral blood from a vaccinated adult. Phage were selected based on Hib PS binding. Two distinct Hib PS-specific phage clones were identified whose Fab fragments used the same V(H) region paired with two different V(L) regions. The V(L) regions were derived from two independent rearrangements of the A2c gene with Jkappa1, and both contained a nontemplated arginine codon at the V-Jkappa junction. The two A2 V gene segments differed from the A2c germ line sequence in 0 and 5 bases. The V(H) region consisted of the V(H)26 gene segment having 98% identity to the germline nucleotide sequence, a D region of 9 bases, and J(H)4b1. Usage of V(H)26 in combination with A2 V regions containing a junctional arginine is a predominant configuration of naturally occurring Hib PS-specific Abs. Liquid- and solid-phase assays showed that phage-derived Fab reacted with Hib PS and expressed HibId-1, an idiotype associated with the kappaII-A2 V region. These findings extend the database of V region polymorphisms that can contribute to the Hib PS repertoire and demonstrate that Hib PS-specific Fab fragments isolated from combinatorial phage libraries use V gene combinations which mirror the natural repertoire.
Reason
D C
DC
Children's Hospital Oakland Research Institute, California 94609, USA.
Wagner
T C
TC
Lucas
A H
AH
D000328:Adult; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D006193:Haemophilus influenzae / Q000145:classification / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007130:Immunoglobulin Idiotypes / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D019151:Peptide Library; D011135:Polysaccharides, Bacterial / Q000276:immunology*; D011995:Recombination, Genetic
D007140:Immunoglobulin Fab Fragments; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011135:Polysaccharides, Bacterial