CloneID: S69-4
Antigen Long Description: The original antibody was generated by immunizing BALB/c mice with a synthetic neoglycoconjugate antigen — Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA (Kdo4-BSA).
Origin Pub PMID: 16282606
Buffer Composition: PBS with 0.02% Proclin 300.
Available Custom Conjugation Options: AP, HRP, Fluorescein, APC, PE, Biotin Type A, Biotin Type B, Streptavidin, FluoroProbes 647H, Atto488, APC/Cy7, PE/Cy7
Specificity Statement: This antibody is specific for Kdo4, a Chlamydophila psittaci Lipopolysaccharide (LPS) tetrasaccharide (3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo)) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. This antibody specifically binds to the LPS of Chlamydophila psittaci but not to LPS from Chlamydophila pneumoniae or Chlamydia trachomatis. Furthermore, it binds exclusively to the LPS and O-deacylated LPS of Chlamydophila psittaci.
Application Notes (Clone): This antibody demonstrated high affinity and specificity for structures containing the branched Kdo4 tetrasaccharide or the 2→4/2→4Kdo3 trisaccharide, enabling differentiation of Chlamydophila psittaci from other chlamydial species. Furthermore, it shows potential for detecting individual molecular lipopolysaccharide (LPS) species and studying the enzymology of Kdo transferases. Its binding affinity to neoglycoconjugate antigens was assessed using ELISA checkerboard titrations. It displayed comparable affinity for 2→4/2→4Kdo3-BSA and Kdo4-BSA and weak binding was observed with 2→4Kdo2-BSA, 2→8Kdo2-BSA, and 2→8/2→4Kdo3-BSA, but only at high antibody and antigen concentrations. This antibody was also tested against BSA glycoconjugates containing 4'-monophosphoryl derivatives of deacylated C. psittaci LPS. It demonstrated similar reactivity with 2→4/2→4Kdo3-GlcNAc2-4P-BSA and Kdo4-GlcNAc2-4P-BSA. Further experiments confirmed its binding to isolated LPS and whole bacteria. Inhibition assays using free oligosaccharides as inhibitors in ELISA demonstrated that the antibody achieved 50% inhibition with 2→4/2→4Kdo3-allyl, Kdo4-allyl, and Kdo4-GlcNAc2-P2 at concentrations of 4.4 µM, 1.7 µM, and 2.1 µM, respectively. An immunofluorescence (IF) assay on infected L929 and HL cells confirmed the antibody's recognition of a consistent epitope in C. psittaci inclusions, while remaining negative for Chlamydophila pneumoniae and Chlamydia trachomatis. Additional IF testing using various chlamydial strains confirmed the antibody's specificity, as all four tested C. psittaci strains displayed positive staining. Surface plasmon resonance (SPR) binding studies were conducted to determine the interaction between the original antibody format (mouse IgG1) with various ligands; the dissociation constants (Kd) for the immobilized antibody were measured for Kdo4-GlcNAc2-P2, 2→4/2→4Kdo3-GlcNAc2-P2, and 2→8/2→4Kdo3-GlcNAc2-P2, resulting in Kd values of 10 µM, 20 µM, and 200 µM, respectively. When the immobilized scFv version of the antibody was used, the binding constants at 150 mM NaCl were 10 µM, 100 µM, and 800 µM for Kdo4-GlcNAc2-P2, 2→4/2→4Kdo3-GlcNAc2-P2, and 2→8/2→4Kdo3-GlcNAc2-P2, respectively. The SPR studies revealed that this antibody exhibits a higher affinity for Kdo4-GlcNAc2-P2 compared to 2→4/2→4Kdo3-GlcNAc2-P2, and significantly reduced affinity for the Kdo-trisaccharide containing 2→8-linked terminal Kdo (Müller-Loennies et al., 2006; PMID: 16282606).
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