Evaluation of a multi-species Protein A-ELISA assay for plague serologic diagnosis in humans and other mammal hosts

BackgroundThe Hemagglutination assay (HA) is widely used in plague diagnosis, however, it has a subjective interpretation and demands high amounts of antigen and other immunobiological supplies. On the other hand, the conventional Anti-IgG ELISA is limited by the need of specific conjugates for multiple plague hosts, which leaves a gap for new diagnostic methods able to cover both the diagnosis of human cases and the epidemiological surveillance of multiple sentinel species. MethodsWe developed an ELISA Protein A-peroxidase method to detect anti-F1 antibodies across several species, including humans. To determine the cut-off and performance rates, HA results from 288 samples (81 rabbits, 64 humans, 66 rodents and 77 dogs) were used as reference. Next, we evaluated the agreement between Protein A-ELISA and Anti-IgG ELISA in an expanded sample set (n = 487). ResultsOptimal conditions were found with 250ng/well of F1 and 1:500 serum dilution. Protein A-ELISA showed high repeatability and reproducibility. We observed good correlation rates between the Protein A and IgG ELISAs optical densities and a higher positive/negative OD ratio for the Protein A-ELISA method. The overall sensitivity, specificity and area under the curve for Protein A-ELISA were 94%, 99% and 0.99, respectively. Similar results were observed for each species separately. In the analysis of the expanded sample set, there was a strong agreement between Protein A and IgG assays (kappa = 0.97). Furthermore, there was no cross-reaction with other common infectious diseases, such as dengue, Zika, Chagas disease, tuberculosis (humans) and ehrlichiosis, anaplasmosis and leishmaniasis (dogs). ConclusionsAltogether, the Protein A-ELISA showed high performance when compared both to HA and Anti-IgG ELISA, with a polyvalent single protocol that requires reduced amounts of antigen and can be employed to any plague hosts. Author summaryHere, we developed and evaluated an ELISA diagnostic test based on the Protein A-peroxidase conjugate that allows the test to be used for plague laboratorial diagnosis not only in humans, but also in a wide range of mammalian species. This particularity is specifically important for plague epidemiological surveillance, given that Yersinia pestis, the causative agent of plague, have a long list of animal reservoirs across distinct ecosystems. Briefly, we first evaluated the best reaction parameters, such as antigen concentration, serum and protein A-conjugate dilutions. Next, we used serum samples from humans, dogs, rodents and rabbits (n = 288) with known results for plague serology by a conventional method, to evaluate the performance of the new Protein A-ELISA test. We observed a good performance of the novel Protein A-ELISA test, with high sensitivity and specificity rates. Evaluation of the coefficient of variation revealed that the test measurements suffer little variation, and therefore, has high repeatability and reproducibility. Next, by evaluating 487 samples, we observed a high degree of concordance between the Protein A-ELISA with a conventional IgG-based ELISA. Furthermore, this test showed no significant cross-reaction with other common infectious diseases. Altogether, the Protein A-ELISA showed high performance when compared both to HA and Anti-IgG ELISA, with a single protocol that requires reduced amounts of antigen and can be employed to several plague hosts.