Complementing antibody profiles: Assessing antibody function on antigen microarrays

Abstract

Antibody effector functions other than neutralization depend on interactions with soluble and cellular components of the immune system. Antigen recognition is usually oligoclonal, with the different clones of antibodies belonging to different classes, subclasses, glycoforms and having different affinities and epitope specificities. Thus, composition of immune complexes determines biological effects mainly via interactions with FcR and complement proteins. Antibodies are capable of triggering any of the three pathways of complement activation and antigen recognition of complex antigens often results in the activation of more than one pathway. These events can be tracked in a multiplex format using antigen microarrays, where complement products bind to elements of the microarray. By controlling cation concentrations and detecting various complement components (C1q, C4, C3) contribution of the different pathways can be identified. Parallel measurement of antibodies and complement proteins provides a novel way of looking at interactions between antigen and antibodies. We propose the use of immune complex signatures, composite depictions of antibody and complement content of immune complexes characterizing healthy and diseased populations. Normalized interquartile ranges of antibody binding (IgM, IgG) and complement deposition (C4, C3) are projected onto radar charts to produce patterns that can distinguish normal and altered immune responses.The comprehensive interaction studies of serum antibodies and complement with arrays of antigens can generate profiles for a better understanding of disease mechanism.1 Antibodies, complement and arrayed antigenThe complement system participates in wide ranging biological phenomena, starting from regulation of organogenesis and regeneration through mediation of inflammation to promoting opsonic phagocytosis to modulation of adaptive immunity. This variability is partly due to the fact that the system has about 30 components and also due to the presence of a variety of receptors on different cell types. While complement activation usually implies the triggering of one of the pathways which then proceeds to the formation of the membrane attack complex, it is important to note that the cascade of enzymatic cleavage events can be blocked at several points by natural inhibitory and regulatory molecules, producing a different outcome. Antibodies are usually associated with classical pathway, where antigen-bound IgM or IgG provides binding sites for C1q, leading to activation of the associated serine proteases C1r and C1s. However, it is important to note that not only classical but also the lectin and alternative pathways can Otherwise, epitope density on the microarray can be controlled by adjusting concentration of the antigen solution used for array printing. At a given epitope density antibodies with higher affinity will be more potent complement activators as the time spent in antigen-bound form will be longer. Therefore, on part of the antigen, quality and epitope densit...

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