DEVELOPMENT OF AN ELISA ASSAY FOR DETECTING ANTI-HISTOPLASMA CAPSULATUM ANTIBODIES USING RECOMBINANT DNA TECHNOLOGY

CABEZA M.; LOAIZA-OLIVA M.; ABUD J.E.; GAMARRA, S. ; RODRIGUEZ, H.A.; GARCÍA-EFFRON G.

Igauzy Falls

Simposio; INFOCUS (The Latin American Forum of Fungal Infections in Clinical Practice); 2023

Local Organizing Committee Infocus LATAM

Objective:In Argentina, one of the diagnostic methods for histoplasmosis involves a technique that uses a completeHistoplasma capsulatum antigen (named histoplasmin) for the detection of antibodies through DoubleImmunodiffusion. This technique is easy to perform but has the drawback of using an antigen obtained manuallyfrom live cultures, making the reagent difficult to standardize. The objective of this work was to produce an H.capsulatum antigen (derived from M antigen) using recombinant DNA technology and to implement it in an ELISAassay to evaluate the presence of antibodies in plasma samples from patients suspected of having a non-identifiedmycosis.Materials and Methods:A structural model of the M protein was constructed using the homology modeling program MODELLER. Thegenerated information was used to define the most antigenic regions using the epitope prediction software ElliPro.The target sequence was amplified by PCR using genomic DNA as a template. The nucleotide sequences were firstligated into a vector for conservation and sequencing (pGEM-T Easy) and subsequently into an inducible expressionvector, which generates a hexahistidine tag fusion. Expression was then induced, and the crude extract was obtainedby sonication. From this, the protein of interest was purified using immobilized metal affinity chromatography(IMAC). Antigenicity was assessed by Dot Blot using anti-histoplasmin serum obtained from rabbits and sera fromconfirmed patients. An indirect ELISA using the recombinant antigen for plate sensitization was designed andoptimized. A secondary anti-human IgG antibody conjugated to peroxidase was used, and a TMB/hydrogenperoxide solution was employed for detection. Finally, this technique was used to detect the presence of anti-histoplasma antibodies in 270 plasma samples from suspected mycosis patients.Results:The constructed structural model allowed for the definition of the antigenic portion of interest in the M antigen. PCRsuccessfully amplified this sequence, and there was good expression of the protein in E. coli. The protein waseffectively purified in a single step using IMAC. The recombinant protein exhibited antigenicity when assessed byDot Blot. Finally, the developed ELISA successfully detected the presence of anti-M antibodies in 25 out of the 270screened plasma samples.Conclusion:The developed ELISA method is capable of detecting the presence of anti-M antibodies in patient´s plasma samples,particularly useful for retrospective analysis of biological banks with a large number of samples. The clinicalsignificance of the presence of anti-M antibodies is being evaluated in consideration of other available sample data.