Bispecific Antibody Production for Allergy Research

Recombinant Phl p 2 and Phl p 5 (major timothy grass pollen allergens) were obtained from Biomay AG (Vienna, Austria). Recombinant human ICAM-1 was purchased from R&D Systems (Minneapolis, MN, USA). Allergen-specific rabbit IgG antibodies were obtained by immunization of rabbits with purified recombinant allergens (Phl p 2, Phl p 5) using complete and incomplete Freund adjuvant (CFA, IFA), respectively (Charles River, Kislegg, Germany). All antibodies used for ELISA, flow cytometry and immunofluorescence staining are summarized in Table S2. Sera were obtained from patients suffering from grass pollen allergy according to case history and IgE serology after informed consent was obtained. Serum samples were analyzed in an anonymized manner with permission from the Ethics Committee of the Medical University of Vienna (EK1641/2014). Human Phl p 2- and Phl p 5-specific IgG₁ were expressed in Chinese hamster ovary (CHO)-K1 cells [23 (link),24 (link)] and purified via Protein G affinity chromatography (Thermo Fisher Scientific, Waltham, MA, USA), and their concentrations were determined by measuring the UV light absorption at 280 nm based on the assumption that the absorbance value for 1 mg/mL human IgG is 1.3. The purified Phl p 5-specific IgG₁ antibody was conjugated with Lightning-Link streptavidin (Innova Biosciences, Cambridge, UK) and termed αPhl p 5-IgG*Strep. Human ICAM-1-specific mouse IgG₁ antibody labeled with biotin (clone 15.2) was purchased from LifeSpan BioSciences (Seattle, WA, USA) and termed αICAM-1-IgG*Bio. To form bispecific antibody conjugates, termed αPhl p 5/αICAM-1, αPhl p 5-IgG*Strep and αICAM-1-IgG*Bio (1:0.25 ratio) were co-incubated for at least 1.5 h (h) at room temperature and overnight (o.n.) at 4 °C.