plasmonic ELISA for the detection of anti-leishmania sp. IgG antibodies

Author(s)

Marilene Oliveira dos Santos Maciel

• Document type: Master's Dissertation
• Press: Araçatuba. 2019-06-12.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária. Araçatuba
• Defense date: 2019-06-10
• Advisor: Valéria Marçal Felix Lima

Abstract

Dogs have been the target of control of Visceral Leishmaniasis (VL) in humans, as they are potential reservoirs of Leishmania infantum and play a key role in the epidemiological chain of the disease. Therefore, the diagnosis of Canine Leishmaniasis (CanL) in Brazil has been a challenge for endemic control organs, since they have limitations on sensitivity and specificity in endemic areas. In this perspective the present research aimed to develop and validate an indirect plasmonic ELISA rK28 (pELISA) for the diagnosis of CanL. For the development of pELISA, different concentrations of hydrogen peroxide, gold ions, biotinylated anti-dog IgG antibody and serum were tested in order to establish ideal values to each parameter. For the validation of the assay, 170 dog serum samples from endemic area to CanL and 26 healthy dog samples from an area nonendemic to the disease were tested by pELISA and compared with indirect ELISA rk28 and the imunocromatografic test (Dual Path Platform, TR_DPP®) using as gold standard assay the real-time PCR in blood samples and/or subconjunctival swab. The assay was standardized with the concentrations of 250 μM hydrogen peroxide, 0.30 mM gold ions, and dilution of the streptavidin-catalase conjugate of 1/50. The TR_DPP®, indirect ELISA rK28 and pELISA presented sensitivity of 79.0%, 89.5% and 94.7% and specificity of 90.1%, 91.4% and 100%, respectively. The highest predictive positive (100%), negative (99.3%) and accuracy (99.4%) values were observed in pELISA. Kappa coefficient between pELISA with real-time PCR showed excellent agreement (0.970), differently of indirect ELISA rK28 and TR_DPP®, which showed good agreement (0.645 and 0.551 respectively). The results revealed that the pELISA improved sensitivity and presented higher specificity compared to official method recommended by the Ministry of Health in Brazil and may increasing the practicality of diagnosis in resource-constrained countries, because it does not require sophisticated instruments to read, suggesting that this method can be used as an additional tool for the diagnosis of CanL in these areas. (AU)

• FAPESP's process: 17/11016-6 - Plasmonic ELISA for the ultra-sensitive detection of anti-Leishmania sp. IgG antibodies
• Grantee: Marilene Oliveira dos Santos Maciel
• Support Opportunities: Scholarships in Brazil - Master