Two reference monoclonal antibodies against the meningococcal P1. the VR1 and VR2 domains, respectively. Both subtype regions of PorA are generally determined by an enzyme-linked immunosorbent assay (ELISA) (1) or a blotting assay (31, 33) with reference monoclonal antibodies (MAbs) directed against epitopes in VR1 or VR2; as a result, each PorA can bind two different subtype-specific MAbs. In addition, variations in VR1 and VR2 are analyzed by sequencing of genes (4, 8, 17C20, 23C26). In a earlier characterization of meningococcal isolates, the two reference MAbs against the common P1.15 subtype, MN3C5C (1) and 2-1-P1.15, did not show identical binding patterns (29). The epitope for MN3C5C offers previously been mapped to a 3-amino-acid sequence in VR2 (19), but that for 2-1-P1.15 has not been reported. Because those MAbs have been used for serological characterization of a number of large strain collections (1, 3, 9, 27), the aim of our study was Rabbit Polyclonal to STAT1 to elucidate the reason for their different specificities. (Parts of this work were offered at the Tenth International Pathogenic Conference, Baltimore, Md., 8 to 13 September 1996 ). For this purpose, whole-cell suspensions of 707 strains, isolated between 1987 and 1995 from individuals with meningococcal disease in Norway, were screened on dot blots with a panel of serotype- and subtype-specific MAbs as explained elsewhere (31). Strains that were positive RAD001 kinase inhibitor with MN3C5C and 2-1-P1.15 on dot blots were also immunoblotted with those MAbs following sodium dodecyl sulfate (SDS) gel electrophoresis of boiled RAD001 kinase inhibitor cell suspensions (31). PorA bands on the blots, along with the corresponding PorA bands in SDS gels, stained with Coomassie amazing blue, were scanned by densitometry (30). The rationale behind this analysis was that PorA epitope variants might be revealed by their weaker antibody binding after antigen denaturation. Isolates were also characterized by multilocus enzyme electrophoresis from the combination of alleles at 14 enzyme loci (7). Distinctive multilocus genotypes were designated as electrophoretic types (ETs). For DNA sequencing of the gene, chromosomal DNA was isolated from a loopful of cells, suspended in 400 l of TE buffer (10 mM Tris-HClC1 mM EDTA [pH 8.0]), essentially as described previously (10), except for a 2-h lysozyme treatment. One microliter of DNA, diluted 1:5, was amplified in a PCR assay (total volume, 50 l) with the primer pair 5-AAACTTACCGCCCTCGTA-3 and RAD001 kinase inhibitor 5-TTAGAATTTGTGGCGCAAACCGAC-3 (8). Sequencing of PCR products was performed as reported previously (8) or by automated sequencing using an ABI Prism 377 and the Big Dye Terminator Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems). The epitope for MAb 2-1-P1.15 was localized by reacting the MAb in an ELISA (22) with synthetic 25- to 29-mer peptides corresponding to loops 1 (VR1), 4 (VR2), and 5 of the subtype P1.19,15 PorA from reference strain H355 (18, 25). The peptides were used in the oxidized state and bound directly to the plate. Detailed epitope mapping was performed by the Geysen method with pins derivatized to allow cleavage of the completed peptides from the pins (15). Twenty-three overlapping decapeptides (each shifted along the sequence by 1 amino acid) that spanned all of VR1 from P1.19,15 PorA were prepared. A 4-amino-acid spacer (SGSG) was added N-terminally to each decapeptide, and the completed peptides were biotinylated at the N terminus before cleavage from the pins (15). The SGSG spacer served to raise the reactive peptides from the surface of the ELISA plate and allow for mobility and conformational freedom of the potentially reactive sequences. The biotinylated peptides were bound to ELISA plates previously coated with streptavidin (50 l of 50 g ml?1, dried overnight at 37C). After three washes, peptides diluted to 50 g ml?1 in phosphate-buffered saline were added, and the plates were incubated for 2 h at room temperature. RAD001 kinase inhibitor The MAb was diluted 1:1,000 and allowed to react with the peptides overnight at room temperature. Alkaline phosphatase-labelled anti-mouse immunoglobulin G (1 g ml?1) was used as the second antibody and incubated for 2 h at room temperature. The assay was completed and read as described previously (22). Dot blot analysis showed that 25 of the 707 patient strains expressed PorAs that reacted with both reference MAbs, MN3C5C and 2-1-P1.15, whereas 12 strains bound 2-1-P1.15 but not MN3C5C (Table ?(Table1).1). Five of the latter strains also expressed epitopes for the P1.1, P1.2, or P1.14 subtype-specific MAbs. All RAD001 kinase inhibitor but 1 of the 37 strains belonged to serogroup B, and all strains expressed a class 3 PorB.