Development of an Antigen-independent Affinity Assay to study the Binding of IgG to FcgammaR

University essay from Lunds universitet/Kemiska institutionen

Author: Anette Mårtensson; [2012]

Keywords: Proteinvetenskap; Chemistry;

Abstract: Fc gamma receptors (FcγRs) are membrane-bound receptors which bind the Fc fragment of antigen-bound IgGs. The binding generates cell signaling and subsequently an immunological response. In this way FcγRs are important links between the humoral and cellular parts of the immune system. Three families of FcγRs have been identified in human, FcγRI, FcγRII and FcγRIII. FcγRI has high affinity for IgG and is the only receptor capable of binding IgG in monomeric form. The other receptors have low to intermediate affinity for IgG and can only bind immune complexes. The receptors vary in IgG subclass specificity and also differ in the cellular response they give rise to upon IgG binding. The total immune response is a balance between activating and inhibiting signals. The use of therapeutic antibodies for treatment of human diseases has grown immensely since the introduction in the 80s. Second and third generation antibodies are now entering the market. Much research is conducted on optimizing IgG regarding FcγR binding since this interaction to large extent dictates the cellular immunological response of IgG. An appropriate and reliable screening method to measure and compare the affinities of FcγRs for IgG in vitro would be of great assistance in the evaluation of therapeutic antibodies. In this work two such assays were developed, one based on ELISA and one on flow cytometry, both suitable for screening of approximately 24 IgGs. A number of human and murine FcγRs were expressed in soluble and cell-bound form and the apparent affinities of these for different IgGs were assayed using antigen-independent immune complexes. Receptor-specific IgG binding could be detected with both assay set-ups and discrimination between IgGs with different apparent affinity could be made.

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