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PIPE-cloned human IgE and IgG4 antibodies: New tools for investigating cow's milk allergy and tolerance Abbreviations AIT allergen immunotherapy BAT basophil activation test BLG beta-lactoglobulin CM cow's milk CMA cow's milk allergy D1 name of engineered antibodies due to their published, variable regions ELISA enzyme-linked immunosorbent assay FA-AIT food allergen immunotherapy IL interleukin ISAC Immuno-Solid phase Allergy Chip OIT oral immunotherapy PBMC peripheral blood mononuclear cells PIPE cloning polymerase incomplete primer extension cloning RT room temperature SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis TBS Tris-buffered saline TBS-T TBS-Tween UHT milk ultra-high temperature processed milk Cow's milk (CM) allergy (CMA) is defined as an immune-mediated adverse response to CM proteins. Allergic children had significantly higher levels of IgE ( I P i < .01), but no differences in the IgG4 levels were seen between the groups (Figure S2), in accordance with previous studies.8 Our data suggest that the PIPE-cloned antibodies may be useful for a precise quantification of allergen-specific IgE and IgG4. [Extracted from the article]

Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG 1 , and IgG 4 sharing the same variable region against the major birch pollen allergen Bet v 1 using Polymerase Incomplete Primer Extension (PIPE) cloning. We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG 1 , and IgG 4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.