Generation and purification of monoclonal antibodies against Der f 2, a major allergen from Dermatophagoides farinae

Abstract

Monoclonal antibodies (mAbs) are needed for the quantitation of environmental allergens for precise diagnosis and immunotherapy. In this study, we produced and purified monoclonal antibodies against Der f 2, one of the major allergens of the house dust mite Dermatophagoides farina, in order to develop an assay for the detection of this allergen. BALB/c mice were immunized four times with the protein Der f 2 together with an adjuvant after which splenocytes were collected and fused with SP2/0 (myeloma cells) in the presence of polyethylene glycol (PEG). The fused cells were selected in the presence of Hypoxanthine-Aminopterin-Thymidine (HAT) and then Hypoxanthine-Thymidine (HT) medium. Positive cells were screened with ELISA and subcloned by limited dilution at least three times to achieve stable mAb-producing clones. Four stable mAb-producing clones were obtained. One clone with IgG1 isotype and another with IgG2b isotype were chosen to produce large amounts of mAb by inoculation of the cells into the abdominal cavity of mice. Ascites were collected and the mAbs were purified using protein A affinity chromatography. Testing of the ascites by ELISA showed the titration of IgG1 and IgG2b to be higher than 1/10⁶ dilution. The specificity of both antibodies was confirmed by immunoblotting. Thus, we produced two mAb clones against Der f 2 that can be used to create a precise quantitative method to identify allergen components in dust samples and facilitate further study in Der f 2 component-resolved diagnosis (CRD).