Additional Text

Purification

Purified

Antibody Clonality

Recombinant Monoclonal

Storage Note

Store at 4⁰C for up to 3 months. For longer storage, aliquot and store at -20⁰C.

Short Description

This chimeric human antibody was made using the variable domain sequences of the original Mouse IgG2a format, for improved compatibility with existing reagents, assays and techniques.

Application Notes

The original antibody (mouse IgG2a) was generated and characterized for binding specificity by Western blotting. It demonstrated bactericidal activity against Neisseria meningitidis strains MC58, M2934, and BZ83, with titers ranging from 256 to 8,192, and was successfully applied in both WB and FACS assays on strains MC58, M2934, BZ83, M4030, F6124, and BZ133. Bactericidal activity was restricted to a subset of variant 1 strains, underscoring its dependence on specific sequence conservation. Site-directed mutagenesis of recombinant GNA1870 variant 1 revealed that substituting arginine 204 with histidine abolished antibody recognition in WB, confirming Arg204 as a critical residue for binding (Giuliani et al., 2005; PMID: 15664958). Further WB analysis evaluated binding to fHbp from 10 diverse N. meningitidis strains (MC58, M4030, M01-240149, F6124, LPN17592, M01-240345, NZ98/254, M2197, M6190, and M2937). The antibody recognized fHbp in 6 strains-MC58, M4030, M01-240149, F6124, LPN17592, and M01-240345-and showed strong bactericidal activity only against MC58, suggesting strain-specific recognition driven by sequence variability. FACS confirmed surface binding to the same 6 strains, consistent with WB results. Epitope mapping by NMR using a Fab fragment revealed that the epitope does not overlap with the factor H (fH) binding site, permitting fH binding to meningococcal cells even in the presence of the antibody (Scarselli et al., 2009; PMID: 19100746). Cooperative bactericidal activity was observed when this antibody was combined with JAR 5 against strain H44/76, achieving a BC50 of <1 µg/ml. The spatial separation of its epitope at R204 and JAR 5's at G121 (~16 Å) supports previous findings on optimal epitope spacing for synergy (Beernink et al., 2008; PMID: 18591239). SPR analysis showed the antibody has a high association rate (Ka = 1.2 µM-¹s-¹) and very low dissociation rate when binding immobilized fHbp, indicating strong and stable interaction, though Kd could not be reliably determined. The antibody does not compete with JAR 3 or JAR 5 for fHbp binding and binds live N. meningitidis strain H44/76 at concentrations ranging from 0.8 to 40 µg/ml, with binding equivalent to that of JAR 3 and JAR 5. Its binding is unaffected by physiological concentrations of human fH (~90 µg/ml). Despite robust binding, the antibody does not inhibit fH-fHbp interaction, as shown by ELISA and FACS-even at high concentrations. It activates the classical complement pathway via C1q-mediated C4b deposition but induces only limited C3b deposition, indicating weak amplification via the alternative pathway. Unlike fH-blocking antibodies such as JAR 3 and JAR 5, this antibody fails to prevent fH binding and lacks bactericidal activity in normal human serum (BC50 >100 µg/ml). In contrast, it exhibits potent bactericidal activity in fH-depleted complement (BC50 = 1.25 µg/ml), which is abolished upon reconstitution with fH (BC50 >100 µg/ml), confirming that fH interferen