Development of ELISA Assays for Measurement of Can f 1 and Can f 3 in Dog Allergen Extracts

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RationaleDog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3.MethodsMouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts.ResultsScreening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays.ConclusionssELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts. RationaleDog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. Dog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. MethodsMouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. Mouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. ResultsScreening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. Screening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. ConclusionssELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts. sELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts.