Using Human Single Chain Variable Fragment (scfv) Antibodies for Identification of Potential Allergens of Neurospora Crassa

Neurospora crassa has been used as a platform for rapid and cost-effective vaccine production (Allgaier et al. Biologicals 2009; 37:128). The purpose of this study is to screen Neursopora crassa extract for the existence of potential allergens using human scFv antibodies. A highly complex human scFv phage library (Creative Bio-labs) was panned and screened against spent soytone-based Neurospora medium. Affinity-purified soluble scFv antibodies were screened against spent medium. Antibody-binding Neurospora proteins were identified by electrophoresis and immunoblotting and, when possible, isolated using protein-L based immunoprecipitation. One such target protein was excised from a Coomassie-stained gel and identified using laser capture mass spectrometry. Out of 65 positive clones four unique clones were identified by sequencing. All of the Neurospora-specific antibodies were affinity purified. All 4 of the antibodies recognized a specific band of about 70 kDa molecular mass by immunoblot analysis. SDS-PAGE of immunoprecipitated product also revealed protein running at the level of ∼70 kDa molecular weight. Mass spectrometric analysis performed on the protein band was consistent with glucoamylase I precursor of Neurospora crassa (XP_956966). The glucoamylase shares 54% identity and 65% similarity with glucoamylase of Aspergillus niger (Q870G8). Aspergillus-derived glucoamylase has been associated with occupational allergies (Quirce et al. Ann Allergy Asthma Immunol 2002; 89:197). Human scFv antibodies were successfully used for identification of a potential allergen, glucoamylase I precursor, in Neurospora crassa extract.