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

PLoS Negl Trop Dis. 2022 May 12;16(5):e0009805. doi: 10.1371/journal.pntd.0009805. eCollection 2022 May.


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. 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.

Methods: 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. Next, we evaluated the agreement between Protein A-ELISA and Anti-IgG ELISA in an expanded sample set (n = 487).

Results: Optimal 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).

Conclusions: Altogether, 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.

MeSH terms

  • Animals
  • Dogs
  • Enzyme-Linked Immunosorbent Assay / methods
  • Humans
  • Immunoglobulin G
  • Plague* / diagnosis
  • Plague* / veterinary
  • Rabbits
  • Reproducibility of Results
  • Rodentia
  • Sensitivity and Specificity
  • Staphylococcal Protein A
  • Zika Virus Infection*
  • Zika Virus*


  • Immunoglobulin G
  • Staphylococcal Protein A

Grant support

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grants #422612/2016–2 and 400785/2019-6) awarded to AMPA and CRSR, respectively. CCX received scholarships from the Brazilian funding agencies: Fundação de Amparo ao Estado de Pernambuco (FACEPE; grant # IBPG-1050-2.02/14) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES - Finance Code 001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.