Monoclonal antibodies against the leptospiral immunoglobulin-like proteins A and B conserved regions

Abstract

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira that affects humans and a wide variety of animals. Recently the genomes of Leptospira interrogans, Leptospira borgpetersenii and Leptospira biflexa species were sequenced allowing the identification of new virulence factors involved in survival and pathogenesis of bacteria. LigA and LigB are surface-exposed bacterial adhesins whose expression is correlated with the virulence of Leptospira strains. In this study, we produced and characterized five monoclonal antibodies (MAbs) against a recombinant fragment of LigB (rLigBrep) with approximately 54kDa that comprise the portions of LigA and LigB (domains 2-7). The 5 MAbs obtained were of the IgG1 (2) and IgG2b (3) isotypes and their affinity constants for rLigBrep ranged from 7×10(7) M(-1) to 4×10(8) M(-1). The MAbs were able to react with the native antigen on the L. interrogans, L. borgpetersenii and Leptospira noguchii surfaces by indirect immunofluorescence, immunoblotting and immunoelectron microscopy. These results demonstrate that the MAbs anti-rLigBrep can be useful to complement genetic studies and to aid studies aiming understanding the role of Lig proteins in Leptospira pathogenesis and the development of Lig-based vaccines and improved diagnostic tests for leptospirosis.

Diagnostic Accuracy of Recombinant Immunoglobulin-like Protein A-Based IgM ELISA for the Early Diagnosis of Leptospirosis in the Philippines

“…borgpetersenii may explain the lower sensitivity. It is plausible that, although the LigB molecule has the same repeated immunoglobulin-like domains where antibodies bind to, the C-terminal 80 kDa domain of LigB might mask the antibody epitope regions [ 34 ].…”

Monoclonal Antibodies Recognizing the Surface Autolysin IspC of Listeria monocytogenes Serotype 4b: Epitope Localization, Kinetic Characterization, and Cross-Reaction Studies

“…High affinity is a requirement of diagnostic tests, such as immunomagnetic separation or flow cytometry [25]. The affinities of the MAbs from this study, in the nanomolar range, are within the biologically useful range for current diagnostic tests such as ELISA and M2773, M2775, M2781, M2792 and M2797 have a particularly high affinity compared to other MAbs produced using a similar methodology [26], [27], [28]. In a clinical setting, high-affinity MAbs have a greater neutralizing potential than low-affinity MAbs during passive immunization [29].…”

Phenotypic and Molecular Characterization of Leptospira interrogans Isolated from Canis familiaris in Southern Brazil

“…2). The mAbs used in this study were developed by our group [7,18] and are useful tools for phenotypic characterization of pathogenic Leptospira [10,19]. Canines are continuously exposed to risk factors since they have free access to contaminated environments [17] and consequently are considered as significant reservoirs for human infection in many tropical countries [20].…”

“…Aliquots of 10 lL were pipetted into the slide chambers and incubated at 30°C until dry. The slides were then blocked with 10 % fetal bovine serum (FBS) in PBS, washed twice with PBS, and coated for 1 h at 30°C with mAbs against LipL32 [7] and leptospiral immunoglobulin-like proteins [18] diluted in 10 % FBS in PBS. The slides were then washed twice with 10 % FBS in PBS, and a 1:100 dilution of goat anti-mouse fluorescein isothiocyanate (FITC) conjugate (Invitrogen, USA) was added and incubated for 1 h in a dark, humid chamber at 30°C.…”

“…To verify the virulence and pathogenicity of Leptospira isolate, IFI was performed using monoclonal antibodies (mAbs) against membrane-associated antigens, LipL32 and leptospiral immunoglobulin-like proteins [7,18]. Slide chambers (ICN Biomedicals Inc., CA, USA) were coated with a 0.01 % poly-L-lysine solution for 1 h at 30°C.…”