Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

Yan, Weiwei; Saleem, Muhammad Hassan; McDonough, Patrick L.; McDonough, Sean P.; Divers, Thomas J.; Chang, Yung-Fu

doi:10.1128/cvi.00245-13

Cited by 13 publications

(29 citation statements)

References 41 publications

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“…The DNA insert of each clone was verified by DNA sequencing, and the recombinant plasmid was then transformed into E. coli BL21(DE3) (Stratagene, Santa Clara, CA) for expression. Protein expression and purification were performed as previously described (11). The concentration of purified protein was then determined using the Bradford method, and the protein was finally used for ELISA (11).…”

Section: Methodsmentioning

confidence: 99%

“…Protein expression and purification were performed as previously described (11). The concentration of purified protein was then determined using the Bradford method, and the protein was finally used for ELISA (11).…”

Section: Methodsmentioning

confidence: 99%

“…The MAT was used as the reference method to determine serum titers, using live L. interrogans as antigen, as previously described (11,13).…”

Section: Methodsmentioning

confidence: 99%

“…The performance of the ELISA was evaluated using the MAT as the reference method (gold standard) (11). First, we compared the ELISA results for the individual recombinant proteins to the results of the MAT.…”

Section: Methodsmentioning

confidence: 99%

“…Commercial enzyme-linked immunosorbent assay (ELISA) kits using antigens derived from a nonpathogenic Leptospira strain (e.g., Leptospira biflexa serovar Patoc) have generally been found to have lower sensitivities than that of the MAT, because the ELISA antigens do not detect all infecting serovars (9,10). From a previous study, we found that rLigACon can be a useful antigen for indirect ELISA (11). In this study, we evaluated 3 other recombinant antigens, rLipL21, rLoa22, and rLipL32, as well as rLigACon4-8, in an attempt to improve the specificity and sensitivity of the indirect ELISA for equine leptospirosis.…”

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confidence: 99%

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Serodiagnosis of Equine Leptospirosis by Enzyme-Linked Immunosorbent Assay Using Four Recombinant Protein Markers

Yan

McDonough

et al. 2014

Clin Vaccine Immunol

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Leptospirosis, caused by Leptospira spp., is one of the most common zoonotic diseases in the world. We tested four recombinant proteins of Leptospira interrogans, namely, rLipL21, rLoa22, rLipL32, and rLigACon4-8, to evaluate their potential for use as antigens for the diagnosis of equine leptospirosis. We employed equine sera (n ‫؍‬ 130) that were microscopic agglutination test (MAT) negative and sera (n ‫؍‬ 176) that were MAT positive for the 5 serovars that most commonly cause equine leptospirosis. The sensitivity and specificity of ELISA compared to MAT were 82.39% and 86.15%, respectively, for LigACon4-8, 77.84% and 92.31%, respectively, for Loa22, 77.84% and 86.15%, respectively, for LipL32, and 84.66% and 83.85%, respectively, for LipL21. When one of the two antigens was test positive, the sensitivity and specificity of ELISA were 93.75% and 78.46%, respectively, for rLigACon4-8 and LipL32, 93.18% and 76.15%, respectively, for rLigACon4-8 and LipL21, 89.77% and 80.77%, respectively, for rLigACon4-8 and Loa22, 91.48% and 78.46%, respectively, for LipL21 and Loa22, 93.75% and 76.92%, respectively, for LipL21 and LipL32, and 90.34% and 80.77%, respectively, for Loa22 and LipL32. In conclusion, we have developed an indirect ELISA utilizing rLigACon4-8, rLoa22, rLipL32, and rLipL21 as diagnostic antigens for equine leptospirosis. The use of four antigens in the ELISA was found to be sensitive and specific, the assay was easy to perform, and the results concurred with the results of the standard Leptospira MAT.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The MAT was used as the reference method to determine serum titers, using live L. interrogans as antigen, as previously described (11,13).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Serodiagnosis of Equine Leptospirosis by Enzyme-Linked Immunosorbent Assay Using Four Recombinant Protein Markers

Yan

McDonough

et al. 2014

Clin Vaccine Immunol

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Moving towards the immunodiagnosis of staphylococcal intramammary infections

Fabres-Klein

Aguilar

Silva

et al. 2014

Eur J Clin Microbiol Infect Dis

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Bovine mastitis is the primary disease of dairy cattle that has a great impact on the dairy industry. It is estimated that worldwide economic losses due to mastitis range between US$82 and US$131 per cow/year. A fast and efficient diagnosis of the disease remains a major bottleneck that directly influences the speed with which treatment decisions and management are undertaken. Microbiological culture remains the gold standard in the identification of bacteria that cause mastitis, but the method has inherent limitations, such as a delay in obtaining results and cost, and requires special care during the collection and processing of the sample. For this reason, multiple groups have devoted efforts to develop alternative methods that, preferably, can be easily accomplished in the field. The specificity of the antigen-antibody reaction has enabled the emergence of major diagnostic methods used in clinical practice, such as immunoassays, which have significant advantages in terms of speed, sensitivity, specificity, and portability. Commercially, immunodiagnostics have been used in the detection of various diseases in cattle. However, in several cases, only a presumptive diagnosis can be made, which requires confirmation using culture-based methods. This review discusses the immunological-based assays developed since the 1990s for the detection of Staphylococcus aureus, which is considered the primary pathogen of contagious bovine mastitis. Although no ideal antigens ensure the accurate performance of tests and the costs need to be reduced to allow for good market competitiveness, immunoassays, particularly lateral flow immunoassay and immunoagglutination, have emerged as promising tests to be used in the field.

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Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis

Nagalingam

Thirumalesh

Kalleshamurthy

et al. 2015

Trop Anim Health Prod

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This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

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Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

Cited by 13 publications

References 41 publications

Serodiagnosis of Equine Leptospirosis by Enzyme-Linked Immunosorbent Assay Using Four Recombinant Protein Markers

Serodiagnosis of Equine Leptospirosis by Enzyme-Linked Immunosorbent Assay Using Four Recombinant Protein Markers

Moving towards the immunodiagnosis of staphylococcal intramammary infections

Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis

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