Antibody Identification for Antigen Detection in Formalin-Fixed Paraffin-Embedded Tissue Using Phage Display and Naïve Libraries

Abstract

Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.

pH-Responsive Upconversion Mesoporous Silica Nanospheres for Combined Multimodal Diagnostic Imaging and Targeted Photodynamic and Photothermal Cancer Therapy

“…Tumors and vital organs (heart, kidneys, liver, lungs, and spleen) were isolated, fixed in 10% formalin, embedded in paraffin, and sectioned into 4 μm slices using a Leica RM2235 microtome. The tissue sections were then dewaxed on microscope slides and stained with hematoxylin and eosin (H&E) using standard procedures. , For immunohistochemistry (IHC) analysis, samples were prepared according to a published protocol . Briefly, tissue sections were treated with a heat-induced epitope retrieval solution (10 mM sodium citrate buffer, pH 6.0) for antigen recovery, blocked with 8% BSA, incubated overnight at 4 °C with the cleaved caspase-3 primary antibody (Asp175, 1:800), and then subjected to sequential incubations (45 min at room temperature) in biotinylated secondary antibody and streptavidin-horseradish peroxidase (Abcam).…”

Progress on Phage Display Technology: Tailoring Antibodies for Cancer Immunotherapy

“…The cells can be presented adhered to a surface or in solution ( Figure 2 ), and there is a wide range of methods for conducting the selection. Cells can be attached to a surface, forming a monolayer that will later receive the phage library [ 66 , 94 , 95 ], or it is still possible to use a whole biological tissue, which preserves cellular interactions and the natural presentation of antigens on the cell surface, which can be altered in the cell isolation process [ 96 ]. Dorfmueller et al [ 63 ] isolated antibodies against corneal endothelial surface antigens.…”

Assessing Tissue Fixation Time and Quality with Label-free Mid Infrared Spectroscopy and Machine Learning

“…How thoroughly a tissue is fixed has a significant impact on antigen detection via modern immunohistochemistry (IHC) since most antibodies do not recognize native unfixed structures. 2–4 The level of tissue fixation significantly impacts detected protein expression, which drives medical opinion on diagnosis, prognosis, and treatment. 5–11 …”