DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis

Abstract

A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.

Production of monoclonal antibodies for immunoaffinity purification and quantitation of Blo t 1 allergen in mite and dust extracts

“…In this study, all mice immunized with Blo t 1 DNA with electroporation produced high titre specific antibodies recognizing both the recombinant and native forms of the allergen. We and others have previously shown that monoclonal and polyclonal antibodies derived from DNA‐immunized animals recognized the recombinant and the native forms of the protein encoded by the immunized DNA [20–25]. This study likewise supports the claim that electroporation enhances gene delivery in vivo resulting in increased expression and elevated immune responses [26, 27].…”

“…In the present study, the DNA‐immunized mice were boosted with recombinant yBlo t 1 resulting in the further increase of the Blo t 1‐specific antibody production (data not shown). Although we have previously shown that an exclusive DNA‐based immunization protocol is sufficient to induce a strong immune response in mice useful for fusion experiments [20], a protein boost might be important in order to obtain a wide spectrum of matured, class‐switched, and high affinity mAbs [21]. Furthermore, the type of immune response generated by DNA immunization is influenced by a wide array of factors including the nature of the antigen, the expression vector used, and the route of administration [28], thus we used protein boost to complement any potential deficiency generated by DNA immunization to ensure a successful hybridoma experiment.…”

“…An exclusive DNA-based immunization protocol [20] or in conjunction with protein boost [21] for the production of mAbs against HDM allergens has been reported by our laboratory previously. Here, we report the production, characterization and applications of Blo t 1 mAbs generated by DNA immunization with electroporation and protein boosts.…”

Anisakis simplex: from Obscure Infectious Worm to Inducer of Immune Hypersensitivity

“…They are paramyosin and tropomyosin proteins, respectively, and are similar to the paramyosins and tropomyosins of other species (reviewed by Weiler [279]). Ani s 2, for instance, is similar to paramyosin allergens from dust mites (Blo t 11 from the mite Blomia tropicalis) (222). The similarity in sequence of these highly conserved proteins is probably the cause of cross-reactivity in IgE binding to A. simplex proteins seen for German cockroaches, chironomids, and dust mites (96,132,279).…”

Expression of the Blomia tropicalis paramyosin Blo t 11 and its immunodominant peptide in insect cells

“…Exponential‐phase High Five cells were seeded at a density of 2×10 6 cells/ml and infected with the high‐titre P‐3 viral stock at an MOI (multiplicity of infection) of 5 or 10. Cultures were incubated for 72 h at 28 °C with shaking at 130 rev./min and the culture supernatant containing the secreted protein was harvested on day 3. mAbs (monoclonal antibodies) to Blo t 11, P2E7G1 and P2E7G10, which are able to detect Blo t 11 and fD equally well ([22] and J. D. A. Ramos, unpublished work), were produced and purified from mouse ascites [22], and coupled with CNBr‐activated Sepharose 4B (Amersham Biosciences, Uppsala, Sweden) beads that were then packed into 10 ml columns and washed with TBS (Tris‐buffered saline; 0.01 M Tris and 0.15 M NaCl, pH 7.5) as described previously [22]. The culture supernatants containing the secreted Blo t 11 and fD proteins were passed through the P2E7G10–Sepharose and P2E7G1–Sepharose columns respectively.…”