Beyond the IFA: Revisiting the ELISA as a More Sensitive, Objective, and Quantitative Evaluation of Spotted Fever Group Rickettsia Exposure

Abstract

Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever group Rickettsia (SFGR) species. The immunofluorescence antibody assay (IFA) has been the gold standard for the serodiagnosis of rickettsial infections such as spotted fever rickettsiosis (SFR). However, the IFA is semi-quantitative and subjective, requiring a high level of expertise to interpret it correctly. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Rickettsia parkeri infection in the guinea pig. Our ELISA is an objective, quantitative, and high-throughput assay that shows greater sensitivity and resolution in observed titers than the IFA. We methodically optimized relevant parameters in sequence for optimal signal-to-noise ratio and low coefficient of variation% values. We used a guinea pig model as it is a part of our overall research efforts to understand the immunological and clinical response to SFGR species after tick transmission. Guinea pigs are a useful model to study SFR and show clinical signs of SFR, such as fever and eschars. We anticipate that this assay will be easily adapted to other hosts, including humans and other SFGR species.

Evaluating the Clinical and Immune Responses to Spotted Fever Rickettsioses in the Guinea Pig‐Tick‐Rickettsia System

“…R. parkeri (Alugubelly et al, 2021;Goddard, 2003;Levin et al, 2020;Philip, Lane, & Casper, 1981). One may also employ guinea pigs to assess the pathogenicity of other SFGR and evaluate putative nonpathogenic endosymbionts (Snellgrove et al, 2021).…”

“…The latter two agents cause a milder disease typically distinguishable from Rocky Mountain spotted fever, with one or more eschars—red, erythematous lesion with necrotic center on the skin—rather than a maculopapular rash. Of the three species, R. parkeri and R. rickettsii have been studied using the guinea pig model, and tick‐borne transmission has been extensively investigated for R. rickettsii and R. parkeri (Alugubelly et al., 2021 ; Goddard, 2003 ; Levin et al., 2020 ; Philip, Lane, & Casper, 1981 ). One may also employ guinea pigs to assess the pathogenicity of other SFGR and evaluate putative nonpathogenic endosymbionts (Snellgrove et al., 2021 ).…”

“…This protocol describes an enzyme‐linked immunosorbent assay (ELISA) for monitoring the immune response in guinea pigs to rickettsial infection (Alugubelly et al., 2021 ). The principal advantages of this assay over the immunofluorescence assay (IFA) are that it is quantitative rather than semi‐quantitative, more objective, and more suitable for high‐throughput studies or diagnostics.…”

“…When you monitor the antibody titer by ELISA, you should expect the following intra‐plate precision from replicates: CV% ≤6.2 ( x̄ = 3.3); high % deviation (% deviation of highest single OD from mean OD) ≤11.3 ( x̄ = 4.5); low % deviation (% deviation of lowest single OD from mean OD) ≤9.3 ( x̄ = 3.9), where OD = optical density. You should expect the following inter‐plate precision: CV% ≤12.8 ( x̄ = 8.8) (Alugubelly et al., 2021 ).…”

An Update on the Laboratory Diagnosis of Rickettsia spp. Infection

“…Typically, an OD ≥ 0.5 is used as a diagnostic cut-off; however, independent validation is lacking [ 35 ]. Recently, quantitative ELISA has been developed for SFG rickettsial infections, moving away from arbitrary OD cut-offs and instead calculating cut-offs directly from negative controls, with final results reported in titres [ 37 ]. Together, these advancements allow for more accurate and objective interpretation of results [ 37 ].…”