Development?of?gold?nanoparticle-based?lateral?flow?immunoassay?for?detection?of?pathogenic?leptospiral?antigen

Abstract

Leptospirosis, caused by pathogenic Leptospira spp., is a global re-emerging zoonosis and endemic disease in tropical and sub-tropical countries including Thailand. Point-of-care testing (POCT) for rapid diagnosis of leptospirosis is needed for prompt and appropriate treatment particularly in resource-poor settings. Lateral flow immunoassay (LFA)-based POCT devices are rapid, user friendly, cheap, have one-step analysis, and need no sophisticated equipment. LipL32 is a common target for diagnostic tests of leptospirosis because it is the major outer membrane protein (OMP) that is specific and highly conserved among pathogenic Leptospira. This study aimed to develop the gold nanoparticle (AuNP)-based LFA for detection of pathogenic leptospiral antigen. To develop the LFA, this study used two clones of anti-LipL32 mouse monoclonal antibodies (mAb3 and mAb82) and a rabbit anti-LipL32 polyclonal antibody (pAb) that showed specific binding to all 22 reference pathogenic Leptospira serovars found in Thailand and did not bind to 2 non-pathogenic serovars. These mAb3 and mAb82 bind different epitopes on LipL32 as demonstrated by the competitive inhibition-based enzyme-linked immunosorbent assay. The lowest limit of detection (LoD) was achieved by the LFA comprising anti-LipL32 mAb82-conjugated AuNPs on the conjugate pad and the anti-LipL32 pAb on the test line. Different sizes of AuNPs including 10, 20, 30 and 40 nm in diameter were successfully synthesized by seeded growth synthesis using citrate reduction. The LoD of 40-nm mAb82-conjugated AuNPs was 100- and 10-fold lower than that of 20-nm and 30-nm AuNPs, respectively, to detect sonicated L. interrogans serovar Pomona spiked in sera. In the present study, the lowest LoD obtained after various optimization of anti-LipL32 mAb82-conjugated AuNP-based LFA was 5 x 103 cells of whole cell lysates in sera. This AuNP-based LFA was evaluated with acute sera from patients with leptospirosis and unrelated diseases. Pretreatment of sera with 4.5% Tween 20 improved the sensitivity of the LFA from 6% (3 of 50) to 24% (12 of 50) to detect acute phase sera from known cases of leptospirosis. Negative detection was observed in all 30 (100%) pretreated and untreated sera of patients with unrelated diseases and healthy person resulting in 100% specificity of this LFA. The new strategies are required to improve the sensitivity of this potential anti-LipL32 mAb82 conjugated AuNP-based LFA for better diagnosis of acute phase leptospirosis in the future.