Modification and Application of Antibodies and Their Fragments for Immunostaining in Live Lymphatic Tissue Slices

The research presented in this thesis focuses on 3 major themes: 1.) the in-house modification of antibodies, 2.) the validation/testing of antibodies after modification, and 3.) the use of modified antibodies as an immunostaining reagent in live lymphatic tissue slices. For most research labs, the development of novel antibody-dependent methods or antibody-based research tools are often limited by the lack availability of commercial recombinant antibodies, which are best suited to efficiently generate fragments, bispecific, or otherwise modified antibodies. However, the in-house generation of recombinant antibodies is nontrivial. High costs, specialized equipment, and necessary expertise are barriers to in-house recombinant antibody production; thus, the average research lab will may choose to use monoclonal/polyclonal antibodies to make their desired modification by other, more cost effective, methods. Chapter 2 discusses the use of enzymatic digestion of antibodies, as it is more cost effective and approachable than recombinant production for small-scale in-house generation of fragments. The benefits, challenges, validation, and suggested optimization workflow for enzymatic production of antibody fragments are presented in detail. Chapter 3 discusses the application of microfluidics to address optimization and validation needs for another form of antibody modification – the labeling or conjugation of an antibody to another molecule or tag. When labeling, the number of labeled tags (labeling ratio) is often used to approximate the function/affinity of the antibody. Optimizations of these reactions can be time-consuming and costly; thus, there is a need for a rapid, cost-effective, and user-friendly (microfluidic) platform to test antibody labeling ratios. Chapter 4 presents one such application of antibodies labeled with fluorophores for immunohistochemical staining of lymph node slices for spatial determination of substructures and identification of cell surface markers in tissue samples. Chapter 5 presents a novel method to observe the release of cytokine signals from immune cells in live lymph tissue using a conjugated bispecific antibody “dual affinity reagent.” We discuss the production of the dual affinity reagent and propose several validation experiments for its use in observing spatially discrete cytokine release from lymph tissue in response to immune stimuli. We believe that studying live tissue spatial morphology changes and cytokine release in response to certain diseases may allow us in the future to obtain new unique insights into disease progression and may identify key pathways, events, or sub-structures to target for new therapies.