A new multi-species Protein A-ELISA assay for plague diagnosis in humans and other mammal hosts

Abstract

Background: The 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. Conventional IgG-ELISA is limited by the need of specific conjugates for multiple plague hosts. Methods: Thus, we 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. Results: Optimal conditions were found with 250ng/well of F1 and 1:500 serum dilution. Protein A-ELISA showed high repeatability and reproducibility. The positive/negative OD ratios were higher in Protein A-ELISA and there was no significant cross-reaction with other pathogenic yersiniae. The overall sensitivity/specificity, area under the curve and Kappa rates for Protein A-ELISA were 93.9/98.9%; 0.993 and 0.938, respectively. Similar results were observed in each species separately. There was a strong agreement between Protein A and IgG assays (kappa=0.973) in independent analysis (n=487). Conclusions: Altogether, the Protein A-ELISA showed high performance when compared both to HA and IgG-ELISA, with a polyvalent single protocol that requires reduced amounts of antigen and can be employed to any plague hosts.