MycologyA quantitative enzyme-linked immunoassay (ELISA) to approximate complement-fixing antibody titers in serum from patients with coccidioidomycosis☆
Introduction
Coccidioidomycosis (CM), also known at San Joaquin Valley fever, is both a regionally (McCotter et al., n.d.; Wilson et al., 2019) and nationally (Benedict et al., 2018) important systemic fungal infection, often confused with other respiratory infections, cancer, or rheumatologic conditions (Gabe et al., 2017; Manning et al., 2018; Shahab et al., 2017; Stockamp and Thompson 3rd., 2016). Because the clinical manifestations of CM overlap with such a diverse range of other illnesses, diagnosis typically requires specific laboratory testing. While direct microscopic detection of spherules in clinical specimens (Saubolle et al., 2007), growth of the fungus in culture, PCR detection of Coccidioides-specific DNA sequence (Saubolle et al., 2018), or measurement of coccidioidal antigen (Durkin et al., 2009; Kassis et al., 2015) are all clinically available approaches, diagnosis of CM is most frequently accomplished by detecting specific anti-coccidioidal antibodies in serum. Of the several serologic tests commercially performed for this purpose, the assay for anti-coccidioidal complement-fixing (CF) antibodies and the immunodiffusion method designed to measure the antibodies directed against the same antigen (Huppert et al., 1967; Wieden et al., 1983) are the only tests whose quantitation has prognostic value (Pappagianis and Zimmer, 1990), and for this reason, they have remained in clinical use for over the past 60 years (Smith et al., 1956).
Despite its long history, the CF antibody assay has several limitations for all but the most specialized laboratories. Several biologics reagents (tanned sheep red blood cells, fresh serum complement, heat-sensitive coccidioidal antigen preparations) are required for the procedure, each of which is supplied by different suppliers, potentially introducing a source of inter-laboratory variability. The test itself is complex and labor-intensive. Day-to-day agreement among replicates is not always achieved, and for this reason, some laboratories repeat a prior specimen contemporaneously with a current one to determine more directly any difference in titer between the two (Pappagianis and Zimmer, 1990). Recommended procedures are not uniform (Huppert et al., 1970), there is no information available regarding differences obtained between different reference laboratories, and a national performance testing program has not been established. A method which avoided these limitations would be a useful alternative.
There is broad agreement that the protein within complex coccidioidal extracts that reacts with CF antibodies is the 427 amino acid product of the chitinase gene, CTS1 (Yang et al., 1996; Yu et al., 2005; Zimmermann et al., 1996a), and its crystal structure has been determined (Hollis et al., 2000). The recombinant expression product of CTS1 (rCTS1) has been considered as a reagent for an enzyme-linked immunoassay (ELISA) to measure CF antibodies, found to have potential, but was not pursued further (Johnson et al., 1996). Truncations rCTS1(aa20–111) and rCTS1(aa280–427) were not reactive with sera from patients with CM, and rCTS1(aa20–310) eliminated cross-reactivity that was seen with rCTS1(aa20–427) in sera from patients with histoplasmosis and blastomycosis (Yang et al., 1997a). Little has been done further with these observations reported over 20 years ago, perhaps in large part because coccidioidomycosis is an orphan disease for which relatively small commercial incentive exists to improve its diagnosis and management.
In this report, we detail additional work with recombinant products of truncations of rCTS1. We have restricted further the domain immunoreactive with antibodies in serum from patients with CM and characterized the epitope(s) responsible for antibody binding. Also, we describe our assay results with reference to a standard curve, permitting expression of antibody binding as an antibody concentration instead of titers. These results support a recombinant truncation of CTS1 as a useful reagent for the eventual transition of quantitative CF antibody assays to an ELISA platform.
Section snippets
Human sera
A serum library used for these studies was composed of remnant specimens tested for anti-coccidioidal antibodies and quantitative immunodiffusion results were from at the Southern Arizona Veterans Health Care System Medical Center. All specimens were de-identified and the University of Arizona Institutional Review Board has determined that their use was not human experimentation. For some studies, a serum pool was created from portions of 50 separate sera. The calculated geometric mean CF titer
Anti-coccidioidal antibody binding is restricted to rCTS1(aa111–310), but not to smaller rCTS truncations
Since prior work (Yang et al., 1997a) had demonstrated that rCTS truncations between amino acids 1 and 110 and beyond amino acid 310 had no binding to antibodies in patients with CM, our initial study focused on the rCTS1(aa111–310) and compared it to rCTS1(aa20–310). As shown in Fig. 1, the shorter truncation (27.4 kDa) demonstrates at least as much binding as to the previously published rCTS1(aa20–310) (34.7 kDa). Further, adding increasing concentrations of rCTS1(aa111–310) to antisera used
Discussion
In this report, we constrained the earlier localization of Coccidioides-specific antigenic determinants of rCTS1 from rCTS1(aa20–310) (Yang et al., 1997b) to rCTS1(aa111–310). Our absorption studies where we mixed rCTS1(aa111–310) with CF-positive serum virtually eliminated any binding to rCTS1(aa20–310) on the nitrocellulose membrane, indicating that rCTS1(aa111–310) accounted for all of the binding by rCTS1(aa20–310). However, none of four smaller, overlapping truncations of this regions
Funding
This research was funded in part by the National Institutes of Health, grant 5-R01-AI132140.
Authors’ roles
T Peng: Investigation, Methodology, Writing – review and editing.
Y Zong: Investigation, Writing – review and editing
MDL Johnson: Investigation, Methodology, Resources, Visualization, Supervision, Writing – review and editing.
SV Menghani: Investigation, Writing – review and editing
ML Lewis: Investigation
JN Galgiani: Conceptualization, Data curation, Methodology, Project administration, Resources, Supervision, Writing – original draft, review and editing.
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Disclosures: MDL Johnson and JN Galgiani are inventors on a patent application in connection with the results shown in this report.