Production of a Chaetomium globosum enolase monoclonal antibody

Abstract

Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species.

FIG. 1.

Sequence alignment of putative and characterized enolase derived from Chaetomium and other closely related fungal species. The full-length sequence of Chaetomium globosum enolase was identified by translation of the cDNA sequence and is identified as cDNA. Alignment was performed using Clustal Omega. Database accession numbers identify putative or enolase sequences. CHAGB, putative uncharacterized protein Chaetomium globosum; THITE, putative uncharacterized protein Thielavia terrestris; NEUCR, enolase Neurospora crassa; ASPFU, enolase Aspergillus fumigatus. Highlighted yellow represents the epitope of MAb 1C7 and homology between CHAGB, THITE, and NEUCR. Highlighted red boxes represent two amino acid substitutions in the ASPFU enolase sequence and correspond to glutamic acid (E) for glutamine (Q) and asparagine (N) for aspartic acid (D). Symbols correspond to (*) positions that have a single, fully conserved residue; (:) indicates conservation between groups of strongly similar properties; (.) indicates conservation between groups of weakly similar properties. The sequence analysis was performed on February 21, 2014.

FIG. 2.

Cloning strategy for recombinant enolase in pASK-IBA6 vector and expression in Escherichia coli (A). Ethidium bromide-stained agarose gel electrophoresis image of enolase genes amplified with primers CgEno_F1 and CgEno_R1 from panel A using PCR. PCR was performed on four separate cDNA preparations (lanes 1–4) and also a genomic DNA preparation. Note the slightly faster migration pattern of cDNA amplicons compared to genomic DNA. bp=DNA standard with length in base pairs (bp) indicated to the left (B). Characterization of purified recombinant C. globosum enolase using SDS-PAGE. Lane 1, MW markers; lane 2, Strep-Tactin purified recombinant C. globosum enolase fraction (C).

FIG. 3.

Western blot analysis of IgG₁ isotype MAb 1C7 clone reactivity to recombinant C. globosum enolase. Lanes 2–6 represent multiple MAb 1C7 clones that were selected and purified following the limiting dilution steps. Lane kDa, MW markers; lane 1, 3% SMPBST; lane 2, 1C7-4D4-1D4; lane 3, 1C7-3E3-1C4; lane 4, 1C7-3E3-1D4; lane 5, 1C7-3D4-1C4; lane 6, 1C7-3D4-1D4; lane 7, rCgEno murine polyclonal antibody.

FIG. 4.

Cross-reactivity analysis of IgG₁ isotype MAb 1C7 reactivity to enolase derived from Chaetomium species and A. fumigatus spore extracts. Lane kDa, MW markers; lane 1, C. globosum (UAMH 9683); lane 2, C. globosum (UAMH 841); lane 3, C. indicum (UAMH 7031); lane 4, C. atrobrunneum (ATCC 64497); lane 5, A. fumigatus (ATCC 13073); lane 6, rCgEno.

FIG. 5.

Epitope mapping of MAb 1C7 reactivity. Each spot represents a decapeptide of rCgEno sequence. Decapeptides were sequential with an overlap of two amino acids. In addition to the rCgEno sequence, the N-terminal purification Strep-tag II and the Factor Xa cleavage site are included.