Substitution at the same position in the A18b gene-encoded Fab, Fab3-23/A18b, with the A2a gene-encoded glutamine could not, however, confer the ability to bind HibCP (Fab3-23/A18bGln93) (Fig. units (20, 23, 38). Antibodies Arimoclomol maleate to the ends of the polysaccharide have not been described. Antibodies to HibCP are predominated by molecules (mostly immunoglobulin G [IgG]) carrying a kappa light chain encoded by the variable (V) region VII A2 gene (Immunogenetics database [IMGT] nomenclature, IGKV 2D-29) rearranged to one of the joining (J) genes, J1, J2, or J3 (47). The VJ genes are only slightly mutated and have extended third complementarity-determining regions (CDR) (10 amino acids, codons 89 to 97) with a characteristic arginine in the place of VJ recombination (codon 95A; nomenclature according to Kabat and colleagues Rabbit polyclonal to IL7R [27]) (1, 3, 6, 31, 46). Two highly homologous alleles at the A2 locus, A2a and A2c, have been used. The corresponding heavy chain is encoded by one of the highly homologous heavy chain V genes, either 3-23 or VH26, rearranged either directly to JH6b1 or through DN1 to JH4b1, resulting in an extremely short CDR3 region (six amino acids, codons 95 to 102) with a conserved glycine-tyrosine-glycine motif (codons 95 to 97) (4, 22, 39). Antibodies with these characteristics are called canonical with respect to the HibCP antibody response as proposed by Pinchuk et al. (39), using the terminology for Ig gene combinations dominating certain antibody responses in mice. The canonical light chain expresses an idiotope (HibId-1) recognized by the monoclonal antibody LuC9 (31). Judged by expression of this idiotope, the canonical antibody has been detected in 85% of postvaccination sera constituting on average 60% of the HibCP-specific IgG (31). In comparison to noncanonical antibodies, the canonical antibody is generally of higher avidity, shows higher levels of in vitro bactericidal activity, and is more protective in infant rats (30, 36). A structural analysis may therefore improve our understanding of natural and vaccination-induced resistance to Hib disease. Furthermore, the antibody response to HibCP may be a model of more general relevance for human antibody Arimoclomol maleate responses to antigens with a limited number of epitopes. MATERIALS AND METHODS Sources of Ig sequences for antigen-binding fragment (Fab)-encoding constructs. A set of canonical heavy (clone ToPG438) and light (clone ToP218) chains was selected among published plasmid clones of reverse-transcribed and PCR-amplified Ig mRNA (6, 22). The mRNA was derived from purified HibCP-specific antibody-secreting cells (AbSC) present in the circulation of a healthy adult male (22 years of age) 9 days after vaccination with a single dose of a HibCP-tetanus toxoid (TT) conjugate (ActHib; Pasteur Mrieux Serum et Vaccines, Lyon, France). Arimoclomol maleate The A18b germ line sequence was obtained from a published plasmid clone (A18b clone 002) derived from PCR-amplified genomic DNA (25). The IGVH 3-23 germ line sequence was obtained from a plasmid clone (To2317) from PCR-amplified DNA, and the JH6b1 germ line sequence was obtained from the clone ToPG335 (22). Arimoclomol maleate PCRs for the construction of Fab-expressing vectors. All PCRs were performed in a final volume of 50 l containing 1 PFU reaction buffer, 0.2 mM deoxynucleoside triphosphate, 0.078 U of polymerase (Stratagene, La Jolla, Calif.), and 0.55 U of polymerase (Life Technologies, Paisley, United Kingdom) mixed with 0.55 U of Taq-Start antibody (Clontech Laboratories, Palo Alto, Calif.) and 5 pmol of gene-specific primer pairs. After an initial denaturation for 4 min at 94C, 20 to 30 PCR cycles, consisting of 30 s at 94C, 1 min at 55C, 1.5 min at 72C, and a final 10-min step at 72C, were performed. Cloning of Fab-encoding constructs. The cloning procedures used for Fab-encoding constructs, described below briefly, were previously described in detail (22). (i) Cloning of the VH domain. One hundred nanograms of the plasmid ToPG438 was used as a template for a 20-cycle PCR amplification of the VH domain sequence. Gene-specific primers were placed in framework region 1 (FR1) and FR4 and contained an Taqpolymerases with anti-antibody for 20 cycles as described above. The resulting full-length kappa light chain PCR.