Allele Researchers Engineer Modified Nanoantibodies to Increase Sensitivity in Biochemical Assays

Researchers at Allele have published new work demonstrating a novel application for nanoantibodies (nAbs) in direct signal amplification. nAbs have distinguishable qualities that set them apart from their traditional IgG counterparts, including significantly smaller size, better stability, and excellent specificity. However, because of their small size, there are no suitable secondary antibodies for traditional assays like immunohistochemistry, immunofluorescence, and other biochemical assays that require an enhanced signal.

The researchers engineered a modified nAb, termed “nAb Plus,” to directly amplify nAb signal detection through the addition of a small scaffolding protein containing numerous reporter binding sites. nAb Plus bypasses the need for secondary antibodies or additional amplification steps, streamlining biochemical assays and decreasing costs of reagents. The authors demonstrate the use of nAb Plus using immunohistochemistry, an assay typically requiring one or more signal amplification steps. However, nAb Plus could also be incorporated in any biochemical assay needing signal enhancement.

Abstract: Revealing the spatial arrangement of molecules within a tissue through immunohistochemistry (IHC) is an invaluable tool in biomedical research and clinical diagnostics. Choosing both the appropriate antibody and amplification system is paramount to the pathologic interpretation of the tissue at hand. The use of single domain VHH nanoantibodies (nAbs) promise more robust and consistent results in IHC, but are rarely used as an alternative to conventional immunoglobulin G (IgG) antibodies. nAbs are originally obtained from llamas and are the smallest antigen-binding fragments available. To determine whether the unique biophysical properties of nAbs give them an advantage in IHC, we first compared a basic fibroblast growth factor nAb to polyclonal IgG antibodies using tissue isolated from pancreatic adenocarcinoma. The nAb was extremely effective in antigen signal detection and allowed for a more streamlined and reproducible protocol. Furthermore, because nAbs are expressed in Escherichia coli from a single gene, they are quite amenable to genetic engineering. As such, we then covalently bound a highly biotinylated amplifier protein to basic fibroblast growth factor and p16 nAbs (termed nAb Plus), resulting in improved IHC sensitivity. The use of a biotinylated nAb Plus not only achieved local, covalent signal amplification, but also eliminated the need for a secondary antibody and subsequent amplification steps. These results highlight nAbs as valuable alternatives to conventional IgG antibodies, decreasing overall processing time and costs of reagents while increasing sensitivity and reproducibility across individual IHC assays.

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