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 IgG3 format, for improved compatibility with existing reagents, assays and techniques.

Application Notes

The original mouse IgG3 antibody was generated and its specificity confirmed by binding to gonococcal LOS in a solid-phase RIA. A biotin-labeled version of the antibody was used in an inhibition assay, and the antibody was also coupled to agarose as an immunopurificant to remove anti-mouse IgG antibodies from human sera. The epitope recognized by the antibody was characterized through immunoblotting and inhibition assays with treated (modified) gonococcal samples. It was found that the antibody bound both native and modified LOS, indicating recognition of a specific structure on the oligosaccharide portion of the LOS. Further specificity evaluation was conducted using enzyme-linked immunosorbent assay (ELISA) and immunodot blot assays, which confirmed the antibody's specificity for gonococcal LOS. To assess potential cross-reactivity with human blood group antigens, three approaches were employed: inhibition ELISA, hemagglutination (HA) assay, and competitive ELISA. In all cases, the antibody was specific to gonococcal LOS. Testing against a panel of gonococcal strains revealed that the antibody bound to 13 out of 20 strains, demonstrating broad recognition across different isolates, though not universally. In bactericidal assays, the antibody effectively killed prototypic gonococcal strains (24-1, WG, and 71H). Additionally, it opsonized gonococcal bacteria, promoting phagocytosis by polymorphonuclear leukocytes (PMNL), as demonstrated in an opsonophagocytosis assay (Gulati et al., 1996; PMID: 8940213). The antibody was also used to identify and characterize peptides that bind to it through biopanning, and it was employed in various assays, including Western blotting (WB), flow cytometry (FC), and inhibition ELISA. It was used to characterize synthetic peptides and assess antibody responses against LOS in animal models (Ngampasutadol et al., 2006; PMID: 16125281). In passive immunization experiments, the antibody was administered to mice before infection, demonstrating its ability to alter the course of gonococcal infection by inhibiting bacterial growth. It was effective in vitro at killing gonococcal strains and promoting phagocytosis by human PMNL, and it provided some protection against infection in vivo when mice were challenged with gonococcal strains expressing the targeted epitope (Gulati et al., 2013; PMID: 24009500). A humanized version of this antibody was developed to enhance its therapeutic potential in humans. The humanized antibody retained the bactericidal activity of the original mouse antibody and was able to activate complement-mediated killing, as determined via complement-dependent bactericidal assays. The antibody reduced bacterial load in infected mice, with better infection clearance compared to control antibody-treated mice. It also exhibited strong cross-species activity, maintaining efficacy in human complement systems while minimizing the risk of immune reactions associated with murine antibodies (US20210188952A1).