IDENTIFICATION OF EPITOPES WITHIN BETA-LACTOGLOBULIN RECOGNIZED BY POLYCLONAL ANTIBODIES USING PHAGE DISPLAY AND PEPSCAN

Two different epitope mapping techniques were used to identify linear epitopes recognised by polyclonal IgG antibodies from rabbits immunise d with bovine beta lactoglobulin (BLG), which is generally regarded as a major allergen in milk. The first, PEPSCAN, was used to investigate the binding of several rabbit polyclonal antisera to sequential overl apping peptides (12-mers) across the sequence of BLG. Each peptide was synthesized on a different polypropylene PIN, and a standard ELISA pr ocedure was used to locate which of these peptides bound the antibodie s under investigation. Comparisons of PEPSCANs for antisera from six d ifferent rabbits showed that each rabbit recognized a similar set of e pitopes within BLG. PEPSCAN analysis also showed that polyclonal antib odies from the mouse recognize a set of epitopes similar to those reco gnized by the rabbit. The second epitope mapping technique is known as phage display and utilizes libraries of randomized short peptides fus ed to the coat proteins of filamentous phage as a source of epitopes f or analysis. A gene VIII phage display library was used in this study with constrained nonapeptides, which were screened for epitopes recogn ized by affinity purified rabbit anti-BLG IgG. Immobilised rabbit anti -BLG IgG was screened in two separate experiments, each consisting of three rounds of panning. For each separate experiment, a sensitive pha ge ELISA was used to screen several hundred single phage clones for bi nding to anti-BLG IgG immobilised on microtiter plates. As a result, a number of positive phage were identified from the two separate screen s of the library (19 different peptides were isolated, which resembled four different regions of BLG). The identified sequences were found t o constitute a subset of the linear epitopes recognized by the PEPSCAN technique. The coordinates of the crystal structure of BLG were used to display mapped epitopes on its structure. This study has permitted detailed mapping of the major linear antigenic regions within BLG reco gnised by IgG antibodies from immunised rabbits and mice. (C) 1998 Els evier Science B.V. All rights reserved.