Abstract
The Feline Immunodeficiency Virus (FIV) is a lentivirus that affects cats worldwide. The detection of the virus’ 24 Kda capsid protein (p24) antibody is the most common way to diagnose FIV infections. In Brazil, the most used commercial assay for FIV diagnosis is a rapid ELISA test based on the lateral flow principle, with high sensitivity and specificity (SNAP®test). However, the high cost of this imported assay undermines not only the diagnosis of this infection but also its epidemiology. The objective of this study was to optimize and validate an indirect ELISA using the protein p24 recombinant antigen (r-p24) with national technology and maintaining sensitivity and specificity comparable to those of available commercial rapid ELISA tests. Twenty-six reference cats’ sera samples (13 negative and 13 positives for FIV), previously analyzed by PCR and the SNAP® test, were used as a reference to optimize the ELISA. Serum samples of 226 cats from private owners and from shelters of the metropolitan area of Rio de Janeiro were used to validate the assay. The sensitivity and specificity of the r-p24 ELISA were 95.4% and 99.4%, respectively. When compared to the SNAP® Combo Test, the accuracy of r-p24 ELISA was 99%, with a Kappa index of 0.96. Our results indicate that the national technology-based ELISA r-p24 has sensibility and specificity values that are comparable to rapid ELISA kits. This test is therefore recommendable, using national technology for FIV routine diagnosis, research, and epidemiological studies.
Highlights
The Feline Immunodeficiency Virus (FIV) is one of the main pathogens of domestic cats (Felis catus) and worldwide spread (Lutz, 1990)
The ELISA optimization experiments pointed that the best difference between the OD450 of the positive and negative sera reference samples were obtained with assays using microplates coated with r-p24 antigen at 100 ng/100 μL, sera dilutions at 1:100 in 0.05% PBST and 10% of skimmed milk powder; and rabbit anti-cat IgG conjugate diluted at 1:8000 in 0.05% PBST and 1% BSA (Figure 1)
Reference negative sera samples resulted in an average OD450 of 0.144, whilst positive samples resulted in an average OD450 of 1.385
Summary
The Feline Immunodeficiency Virus (FIV) is one of the main pathogens of domestic cats (Felis catus) and worldwide spread (Lutz, 1990). The Brazilian Association of Pet Products (Abinpet) reported that the country has the second largest concentration of domestic cats of the world. The Abinpet estimated an annual growth rate of the Brazilian feline population by 8%. It will reach about 40 million animals in 2020 (Associação Brasileira de Produtos para Animais de Estimação, 2014). Once the clinical signs of FIV infected animals are nonspecific and similar to other diseases, laboratory diagnosis is critical for the identification and subsequent treatment (Hosie et al, 2009; European Advisory Board on Cat Diseases, 2012). The European Advisory Board on Cat Diseases (ABCD) proposes the identification and segregation of infected cats as effective measures to minimize the risk of virus transmission to other cats, to prevent opportunistic fatal infections or lymphomas (Hosie et al, 2009)
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