title: 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 1/10(6) 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).
authors.affiliation: The Second Affiliated Hospital of Guangzhou Medical University, The State Key Laboratory of Respiratory Disease, Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology.
authors.forename: Huifang
authors.initials: H
authors.lastname: Chen
authors.forename: Kejun
authors.initials: K
authors.lastname: Zhang
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.forename: Chenxia
authors.initials: C
authors.lastname: Xu
authors.forename: Zehong
authors.initials: Z
authors.lastname: Zou
authors.forename: Ailin
authors.initials: A
authors.lastname: Tao
mesh_terms: D000485:Allergens / Q000276:immunology*; D000630:Aminopterin; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D039741:Antigens, Dermatophagoides / Q000276:immunology*; D060829:Arthropod Proteins / Q000276:immunology*; D001201:Ascites / Q000276:immunology; D002459:Cell Fusion; D002460:Cell Line; D040001:Dermatophagoides farinae / Q000276:immunology*; D006825:Hybridomas; D007042:Hypoxanthines; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D039981:Pyroglyphidae / Q000276:immunology*; D013936:Thymidine
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451160:Dermatophagoides farinae antigen f 2; D007042:Hypoxanthines; D007074:Immunoglobulin G; D000630:Aminopterin; D013936:Thymidine