We are developing a multiplex antibody-based assay for allergen potency. In previous work, the overall allergenicity of German cockroach (GCr) allergen extracts did not correlate with content of any known specific allergens. We have developed 14 avian scFv antibodies to GCr. In this study we begin to identify their target proteins. Targets of GCr-specific antibodies 2A1, 6A2 and 6A3, isolated by direct immunoprecipitation, were analyzed by SDS-PAGE and mass spectrometry (MS). The cDNA sequences of the target proteins of 2A1 and 6A2 were expressed in E. coli and purified using affinity chromatography. Allergenicity of these expressed proteins was determined by screening a GCr-allergic human serum pool (S1-Cr) for presence of specific IgE antibodies. Thermal stability of these proteins was determined by antibody binding following 1 min incubations at 40-80°C. The target proteins of antibodies 2A1 and 6A2 have an apparent mass of ∼33 kDa and ∼78 kDa respectively. BLAST analyses of peptide sequences obtained from MS indicate the following closest matches: 2A1: Per a 3 homologue (isoform 2, GU086323); 6A2: Bla g 7 (AF260897); 6A3: vitellogenin (AJ0005115). Targets of the other 11 antibodies remain unidentified. ELISA using S1-Cr indicates the presence of specific IgE for Per a 3 homologue and Bla g 7. Per a 3 homologue appears to be the only heat stable protein in GCr extract. We have identified 3 specific target allergens in GCr: vitellogenin, Bla g 7, and a heat stable Per a 3 homologue.

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.

RATIONALE: To improve the safety and efficacy of allergen immunotherapy, better methods are needed to measure the potency and composition of allergen extracts. The purpose of this study is to produce GCr-specific scFv antibodies from a chicken antibody cDNA library, and use these antibodies to analyze GCr extracts.METHODS: An avian antibody library was generated as previously described (Finlay, et al., Clin Exp Allergy. 2005; 35:1040-8). Unique GCr-specific antibodies were identified by sequencing. The phagemids of selected clones were transformed in E. coli, induced with IPTG and the soluble scFvs were purified by metal chelate affinity chromatography. The purified antibodies were used to analyze a GCr extract (E6CG) by ELISA and immunoblot. E6CG is a GCr internal standard extract with an estimated protein content of 20 g/L.RESULTS: The selected unique antibodies were purified and 32 kDa bands were analyzed using SDS-PAGE. 14 high affinity antibodies recognized specific proteins of varying molecular masses in immunoblot. Protein bands recognized by the expressed antibodies migrated at 17, 32, 50, 70, 76, and 102 kDa. Direct ELISA was used to define differential response to different doses of extracts. EC50s calculated from dose response curves ranged from 10−4 to 7 x 10−2 g/L of E6CG.CONCLUSION: Chicken scFv libraries proved to be a useful tool in preparation of unique scFv antibodies. We used these antibodies for analyses and identification of proteins in highly complex GCr extracts. RATIONALE: To improve the safety and efficacy of allergen immunotherapy, better methods are needed to measure the potency and composition of allergen extracts. The purpose of this study is to produce GCr-specific scFv antibodies from a chicken antibody cDNA library, and use these antibodies to analyze GCr extracts. METHODS: An avian antibody library was generated as previously described (Finlay, et al., Clin Exp Allergy. 2005; 35:1040-8). Unique GCr-specific antibodies were identified by sequencing. The phagemids of selected clones were transformed in E. coli, induced with IPTG and the soluble scFvs were purified by metal chelate affinity chromatography. The purified antibodies were used to analyze a GCr extract (E6CG) by ELISA and immunoblot. E6CG is a GCr internal standard extract with an estimated protein content of 20 g/L. RESULTS: The selected unique antibodies were purified and 32 kDa bands were analyzed using SDS-PAGE. 14 high affinity antibodies recognized specific proteins of varying molecular masses in immunoblot. Protein bands recognized by the expressed antibodies migrated at 17, 32, 50, 70, 76, and 102 kDa. Direct ELISA was used to define differential response to different doses of extracts. EC50s calculated from dose response curves ranged from 10−4 to 7 x 10−2 g/L of E6CG. CONCLUSION: Chicken scFv libraries proved to be a useful tool in preparation of unique scFv antibodies. We used these antibodies for analyses and identification of proteins in highly complex GCr extracts.

RATIONALE: German cockroach (GCr) has been reported to cause asthma-related disease in the US. Seven GCr allergens have been reported. We have developed single chain variable fragment antibodies (scFv) to GCr for a novel multiplex assay of GCr extract potency. To qualify these antibodies, we have examined target allergens by immunoprecipitation. METHODS: 14 unique scFv antibodies selected by phage display and panning were purified by affinity chromatography. Two of these, 2A1 and 6A2, were coupled to agarose beads, and incubated with GCr extract. The eluted proteins were analyzed by SDS-PAGE, N-terminal sequencing and mass spectrometry (MS). In addition, the gene sequence for the likely 2A1 target was obtained (from FTC), and the protein was expressed in E. coli and used for screening of GCr-allergic human serum pools for presence of specific IgE. RESULTS: The 2A1 target has an apparent mass of 78 kDa, and the N-terminal sequence is ATIPADKVFL. Sequence BLAST searches, using the N-terminal sequence and internal sequences obtained from MS, indicated the closest match to isoform 2 of a putative Per a 3 homologue (ACY40651). Direct ELISA and dot blot data using expressed recombinant Per a 3 homologue indicated the presence of allergen-specific IgE in screened pools. The 6A2 target has an apparent mass of 33 kDa, and the peptide sequences obtained from mass spectrometry suggest that it is Bla g 7. CONCLUSIONS: We have identified two GCr antigens, one of which appears to be a recently reported Per a 3 homologue, by immunoprecipitation using scFv.