Development of monoclonal antibodies to recombinant terrelysin and characterization of expression in Aspergillus terreus

Abstract

Aspergillus terreus is an emerging pathogen that mostly affects immunocompromised patients, causing infections that are often difficult to manage therapeutically. Current diagnostic strategies are limited to the detection of fungal growth using radiological methods or biopsy, which often does not enable species-specific identification. There is thus a critical need for diagnostic techniques to enable early and specific identification of the causative agent. In this study, we describe monoclonal antibodies (mAbs) developed to a previously described recombinant form of the haemolysin terrelysin. Sixteen hybridomas of various IgG isotypes were generated to the recombinant protein, of which seven demonstrated reactivity to the native protein in hyphal extracts. Cross-reactivity analysis using hyphal extracts from 29 fungal species, including 12 Aspergillus species and five strains of A. terreus, showed that three mAbs (13G10, 15B5 and 10G4) were A. terreus-specific. Epitope analysis demonstrated mAbs 13G10 and 10G4 recognize the same epitope, PSNEFE, while mAb 15B5 recognizes the epitope LYEGQFHS. Time-course studies showed that terrelysin expression was highest during early hyphal growth and dramatically decreased after mycelial expansion. Immunolocalization studies demonstrated that terrelysin was not only localized within the cytoplasm of hyphae but appeared to be more abundant at the hyphal tip. These findings were confirmed in cultures grown at room temperature as well as at 37 6C. Additionally, terrelysin was detected in the supernatant of A. terreus cultures. These observations suggest that terrelysin may be a candidate biomarker for A. terreus infection.

Fungal hemolysins

“…Current knowledge suggests that hemolysins are pore-forming toxins that interact with specific ligands on the surface of various target cells [1]. Although extensively studied in various bacterial species [1,2], hemolysins have also been reported in fungi [3,4], plants [5], invertebrates [6– 8], and mammals (perforins) [9]. Bacterial hemolysins have been well characterized due to the role of these proteins in pathogenesis and, their structural details, mechanisms of hemolysis, ligand differences on target cells, and diagnostic potential have been described [2,10– 19].…”

Production of aChaetomium globosumEnolase Monoclonal Antibody

“…(42) Targeting a highly expressed protein such as enolase was hypothesized to result in improved immunoassay sensitivity compared to other fungal antigens, such as terrelysin that may only be expressed during a specific growth stage of a fungal species. (21) Although the production of the IgG 1 isotype MAb 1C7 is an exciting development for the detection of C. globosum and other members of Sordariomycetes, the results of these preliminary immunoassay studies demonstrated that MAb 1C7…”

“…Fusion of splenocytes with SP2/ 0-AG14 myeloma cells (ATCC# CRL-1581) was performed as previously described. (21,22) Hybridomas were selected by growing cells in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Rockville, MD) supplemented with 1 mM sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.292 mg/mL L-glutamine, 100 mM sodium hypoxanthine, 16 mM thymidine, 10% fetal calf serum (FCS, HyClone, Logan, UT), and 100 U/mL IL-6 (Boerhinger, Mannheim, Germany). DMEM was also supplemented with azaserine for selective propagation of hybridomas.…”

“…The MAb 15B5 served as an IgG 1 isotype control as it reacts with an epitope of the hemolytic protein terrelysin. (21) The membrane was washed in TBST three times for 5 min each and incubated with goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Promega) diluted 1:50000 in blocking buffer for 1 h at RT on a shaker. The membrane was washed thoroughly in TBST three times for 5 min each and developed with ECL Western blotting substrate (Promega) as per the manufacturer's instructions.…”

Monoclonal Antibody 1C7 Against Chaetomium globosum Enolase

“…Fusion of splenocytes with SP2/0-AG14 myeloma cells was performed as previously described, (1,2) and hybridoma cells were cultivated in Dulbecco's Modified Eagle Medium. The supernatant fluid from confluent hybridomas was diluted 1:2 (v/v) in PBS 0.05% Tween-20 containing 3% non-fat dry skim milk powder and tested using a recombinant C. globosum enolase indirect ELISA.…”