1994;93:852C861. models (20, 23, 38). Antibodies to the ends of the polysaccharide have not been explained. Antibodies to HibCP are predominated by molecules (mostly immunoglobulin G [IgG]) transporting a kappa light chain encoded from the variable (V) region VII A2 gene (Immunogenetics database [IMGT] nomenclature, IGKV 2D-29) rearranged to one of the becoming a member of (J) genes, J1, J2, or J3 (47). The VJ genes are only slightly mutated and have prolonged third complementarity-determining areas (CDR) (10 amino acids, codons 89 to 97) having a characteristic arginine in GS-9451 the place of VJ recombination (codon 95A; nomenclature relating to Kabat and colleagues [27]) (1, 3, 6, 31, 46). Two highly homologous alleles in the A2 locus, A2a and A2c, have been used. The related weighty chain is definitely encoded by one of the highly homologous weighty 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) having 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 mixtures dominating particular antibody reactions in mice. The canonical light chain expresses an idiotope (HibId-1) identified by the monoclonal antibody LuC9 (31). Judged by manifestation of this idiotope, the canonical antibody has been recognized in 85% of postvaccination sera constituting normally 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 Rabbit Polyclonal to TRPS1 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 GS-9451 of more general relevance for human being antibody reactions to antigens with a limited quantity of epitopes. MATERIALS AND METHODS Sources of Ig sequences for antigen-binding fragment (Fab)-encoding constructs. A set of canonical weighty (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 blood 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). The A18b germ collection sequence was from a published plasmid clone (A18b clone 002) derived from PCR-amplified genomic DNA (25). The IGVH 3-23 germ collection sequence was from a plasmid clone (To2317) from PCR-amplified DNA, and the JH6b1 germ collection sequence was from the clone ToPG335 (22). PCRs for the building of Fab-expressing vectors. All PCRs were performed in a final volume of 50 l comprising 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 Systems, 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 methods utilized for Fab-encoding constructs, explained below briefly, were previously explained in detail (22). (i) Cloning of the VH website. One hundred nanograms of the plasmid ToPG438 was used like a template for any 20-cycle PCR amplification of the VH website sequence. Gene-specific primers were placed in platform region 1 (FR1) and FR4 and contained an Taqpolymerases with anti-antibody for 20 cycles as explained above. The producing full-length kappa light chain PCR product was size purified, digested with Taqpolymerases with anti-Taq for 20 cycles. The producing full-length kappa light chain PCR products were size purified and digested with K100CP (1 mg/ml; kindly supplied by Uffe Skov S?rensen, Statens Seruminstitut, Copenhagen, Denmark) at 37C to demonstrate specificity. GS-9451 (iii) Cross-reactivity with additional polysaccharides. ELISA.