Abstract:Antibodies have long been the main approach used for localizing proteins of interest by light microscopy. In the past 5 yr or so, and with the advent of superresolution microscopy, the diversity of tools for imaging has rapidly expanded. One main area of expansion has been in the area of nanobodies, small single-chain antibodies from camelids or sharks. The other has been the use of artificial scaffold proteins, including Affimers. The small size of nanobodies and Affimers compared with the traditional antibod… Show more
“…These findings indicated that CEA binding Affimers have better sensitivity and specificity than the corresponding antibody. This could be due to their being physically smaller than antibodies (~ 3 nm for Affimer vs. ~ 14 nm for IgG) facilitating better penetration into fixed cells 14 , 30 . Figure 2 Cell binding and selectivity of anti-CEA Affimers on LoVo cells in comparison to monoclonal antibody.…”
Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.
“…These findings indicated that CEA binding Affimers have better sensitivity and specificity than the corresponding antibody. This could be due to their being physically smaller than antibodies (~ 3 nm for Affimer vs. ~ 14 nm for IgG) facilitating better penetration into fixed cells 14 , 30 . Figure 2 Cell binding and selectivity of anti-CEA Affimers on LoVo cells in comparison to monoclonal antibody.…”
Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.
“…These small proteins are ~10kDa in size, and can be specifically dye-labelled using a unique cysteine placed at either the N-or the Cterminus. This places the dye molecule close to the target protein [3], reducing 'linkage error'. The small size of the Affimer additionally improves its ability to penetrate dense structures over antibodies [2].…”
This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This version may be subject to change during the production process.
“…Rather large size and multivalence of antibodies, however, limit their application as labeling agents in imaging approaches. Over the past years, the popularity of antigen-binding fragments of antibodies and single-chain nanobodies derived from camelids or shark antibodies grew vastly ( Beghein and Gettemans, 2017 ; Carrington et al., 2019 ; Leslie, 2018 ). Both types of molecules are much smaller than full-length antibodies, yet possess similar binding properties to their target proteins ( Harmsen and De Haard, 2007 ; Sahl et al., 2017 ).…”
Summary
Advanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs). However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organization of green fluorescent protein (GFP) tagged proteins after labeling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently labeled nanobodies with the GFP-labeled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the FPs, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labeling for studying dynamic and quantitative protein organization in the plasma membrane of living cells using advanced imaging techniques.
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