Development of Monoclonal Antibody-Based Immunofluorescence Assay for Detection of Leptospires in Urine

Abstract

Leptospirosis is a common  life-threatening zoonotic  disease. The disease  is often underdiagnosed, especially  in the acute phase, due  to variations  in presenting  symptoms,  some of which may be mistaken for other diseases. The low sensitivity  gold standard culture  isolation is a  microscopic  agglutination test  (MAT).  In this  study,  a  hybridoma secreting  monoclonal antibody (MAb)  broadly  specific  to 24  Leptospira serovars including serovars Australis, Autumnalis, Hallum, Bangkok,  Bataviae, Bratislava,  Canicola,  Cynopteri, Djasiman,  Grippoty­ phosa, Hardjo,  Hebdomadis, Icterohaemorrhaegiae, Louisiana,  Pomona,  Pyrogenes, Ranarum, Saigon,  Sarmin,  Sejroe,  Shermani, Tarassovi,  Wolffi, and  Patoc of the  Leptospira genus was produced. The MAb, named immunoglobulin M kappa (IgMK), reacted  to secretory  and whole cell antigens  of  Leptospira spp.,  but  not with  the following  8 bacterial  genera:  Aeromonas hydrophila, Escherichia coli, Klebsiella  pneumoniae, Proteus  spp.,  Pseudomonas aeruginosa, Salmonella spp.,  Shigella  spp.,  and  Streptococcus spp.  A MAb-based   immunofluorescence assay (MAb-IFA) subsequently developed reacted to all of the 24 known serovars of Leptospira, but not with the 8 aforementioned bacterial  genera. A minimum  of 1.5 leptospires/uL or at least

30 leptospires in 20 uL of sample volume is required  for  Leptospira identification by Mab-IFA. The  MAb-IFA  is a sensitive and  low-cost antigen  detection  assay  that  can  be performed in a routine  clinical laboratory. Early and accurate diagnosis of leptospirosis will facilitate  improved care and  better  patient outcomes.