Monoclonal Antibodies Against LipL32, The Major Outer Membrane Protein of PathogenicLeptospira: Production, Characterization, and Testing in Diagnostic Applications

Abstract

Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids.

Insights into Leptospirosis, a Neglected Disease

“…Outer membrane proteins like rLipL32 [Fernandes et al, 2007;Flannery et al, 2001] , rLipL41 [Flannery et al, 2001;Mariya et al, 2006] and immunoglobulin (Ig)-like Lig proteins (Croda et al, 2007;Srimanote et al, 2008) have been used as antigens in ELISA.…”

A Universal Vaccine against Leptospirosis: Are We Going in the Right Direction?

“…The localization of LipL32 is still unresolved; there is experimental data for both surface (56–59) and subsurface locations (21). Leptospiral genomes encode OMPs such as LptD, BamA-like, TonB-dependent receptors, and several other porins that play crucial roles in bacterial survival and potential role in pathogenicity.…”

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

“…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.…”

“…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.…”

“…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].…”