Comparative immunoenzymatic analysis of IgG and IgM peptides from LipL32 protein for the development of novel Leptospirosis immunological diagnostic tests

Introduction:Leptospirosis is a worldwide zoonosis with high morbimortality, caused by pathogenic spirochetes of the genus Leptospira.The development and validation of ELISA-based assays may improve access to diagnosis and support early detection.Structural analysis of the LipL32 protein revealed putative protein-protein interaction sites, suggesting its involvement in host-pathogen interaction.These findings reinforce the need for effective diagnostic strategies using suitable technologies to provide faster and more accurate detection of this difficult-to-diagnose disease.Objectives: Analyze comparatively the IgG and IgM antibody responses using two ELISA formats -NeutrAvidin ELISA and indirect ELISA-employing biotinylated synthetic peptides derived from the LipL32 protein of Leptospira as antigens for the development of novel immunological diagnostic tests for leptospirosis. Methodology:In-house indirect and NeutrAvidin ELISAs were performed using 96-well plates sensitized with 1 g of biotinylated peptides.For indirect ELISA, plates were incubated overnight at 4 C and blocked with 2.5% BSA, while NeutrAvidin plates were incubated for 2 h at room temperature.Patient sera and negative control were diluted 1:100 for IgG and 1:50 for IgM, followed by goat antihuman IgG (1:30,000) or IgM (1:15,000) HRP-conjugated.Reactions were developed with TMB, stopped with H 2 SO 4 , and read at 450 nm.Plates were washed with PBS-T between steps, and incubations were performed for 1 h at 37 C for indirect ELISA and for 30 min at room temperature for NeutrAvidin ELISA.Statistical analyses were performed using one-way ANOVA and Tukey's test (P<0.05). Results:The results allowed the identification and characterization of LipL32 epitopes.IgG and IgM responses against synthetic peptides showed distinct reactivity patterns across different phases of infection, with most peptides exhibiting significantly higher absorbance in leptospirosis-positive sera compared to negative control (P<0.05 to <0.001).The LipL32/10G peptide showed significant reactivity in all infection phases but displayed moderate cross-reactivity with syphilis sera.The LipL32/5M peptide showed statistical difference in leptospirosis-positive sera with greater sensitivity in the indirect ELISA and marked cross-reactivity with syphilis, especially in the NeutrAvidin assay. Conclusion:These findings demonstrate the diagnostic potential of LipL32 peptides at different stages of infection and support their application in the development of novel immunological assays, including biplex diagnostic approaches for leptospirosis/syphilis.