Recombinant Antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays in Dogs

Abstract

Animal leptospirosis is one of the most common zoonotic diseases in the United States and around the world. In a previous study, we applied four recombinant antigens, rLipL21, rLoa22, rLipL32 and rLigACon4-8 of Leptospira interrogans (L. interrogans) for the serological diagnosis of equine leptospirosis (Ye et al, Serodiagnosis of equine leptospirosis by ELISA using four recombinant protein markers, Clin. Vaccine. Immunol. 21:478–483). In this study, the same four recombinant antigens were evaluated for their potential to diagnose canine leptospirosis by ELISA. A total of 305 canine sera that were Leptospira microscopic agglutination test (MAT)-negative (n = 102) and MAT-positive (n = 203) to 5 serovars (Pomona, Grippotyphosa, Icterohaemorrhagiae, Canicola and Hardjo) were tested. When individual recombinant antigens were used, the sensitivity and specificity of ELISA were 97.5% and 84.3% for rLigACon4-8; 89.7% and 81.4% for rLoa22; 92.6% and 84.3% for rLipL32 and 99.5% and 84.3% for rLipL21, respectively compared to the MAT. The sensitivity and specificity of ELISA were, 92.6% and 91.2% for rLigACon4-8 and rLipL32, 97.5% and 84.3% for rLigACon4-8 and rLipL21, 89.7% and 87.3% for rLigACon4-8 and rLoa22, 89.7% and 87.3% to rLipL21 and rLoa22, 92.6% and 91.2% for rLipL21 and rLipL32 and 89.2% and 94.1% for rLoa22 and rLipL32 when one of the two antigens was test positive. The use of all four antigens in the ELISA assay was found to be sensitive and specific, easy to perform, and agreed with the results of the standard Leptospira Microscopic Agglutination test (MAT) for the diagnosis of canine leptospirosis.

Development and analytical validation of an enzyme-linked immunosorbent assay based on baculovirus recombinant LipL32 protein antigen for the accurate detection of canine leptospirosis

“…The emergence of leptospirosis world-wide resulted in an increased demand for safe, standardized and validated diagnostic methods for the diagnosis of leptospirosis (20) (14). The development of recombinant proteins is one approach that is suitable for the safe production of high quality ELISA reagents given that are easily expressed, produced and standardize with high degree of consistency between batches without the biosecurity risks associated extraction of leptospiral antigen from pure microbiological culture preparations (6, 20). Here, we developed an ELISA based on a baculovirus recombinant LipL32 protein with potential application as diagnostic antigen in an indirect ELISA system for detection of antibodies in dogs plasma samples.…”

“…Concordance rate experiments showed that the ELISALipL32 was capable to detect more (57/57) samples positive for antibodies to rLipL32 that the RTPCR (45/57) reference method. This can be explained because of the fact that after infection, Leptospira rapidly proliferates in the blood stream producing an acute phase that lasts for about 10 days when molecular diagnosis is capable to produce a positive result (20). On the other hand, antibodies can be detected in blood by the 6 th to 10th day post infection, which coincides with the clearance of Leptospira from the blood stream.…”

“…To overcome the limitations of MAT, several ELISA platforms for the diagnosis of leptospirosis has been developed based on the use of whole cell antigens for detection of antibodies (10, 16), or by the use of specific antibodies to detect circulating leptospiras in serum or plasma samples (5, 10). More recently, several ELISA platforms have been developed based on highly specific recombinant proteins of Leptospira for the diagnosis of leptospirosis in both humans (5, 6, 10), and different animal species such as bovine (2), equine (19), swine (9), and dogs (20), with similar or higher specificity and sensitivity levels than MAT. The use of the adhesion protein LipL32, a highly conserved immunodominant protein expressed only in pathogenic species of Leptospira (14), has been successfully adapted into different serological platforms for the diagnosis of leptospirosis in humans (6) and animals (2, 9, 20).…”

“…More recently, several ELISA platforms have been developed based on highly specific recombinant proteins of Leptospira for the diagnosis of leptospirosis in both humans (5, 6, 10), and different animal species such as bovine (2), equine (19), swine (9), and dogs (20), with similar or higher specificity and sensitivity levels than MAT. The use of the adhesion protein LipL32, a highly conserved immunodominant protein expressed only in pathogenic species of Leptospira (14), has been successfully adapted into different serological platforms for the diagnosis of leptospirosis in humans (6) and animals (2, 9, 20). In this study, we report the development and analytical validation of an ELISA system based on the LipL32 protein of Leptospira expressed in a baculovirus expression system.…”

Efficacy of the Rabbit Polyclonal Anti-leptospira Antibody against Homotype or Heterotype Leptospira Infection in Hamster

“…After incubation at 30°C for 2 hours, the antiserum was performed for agglutination using dark field microscopy. Final titers represent the reciprocal of the highest serum dilution showing at least 50% agglutination of antigen in the suspension [29]. …”