Abstract
The production costs for monoclonal antibodies (MAbs) utilized in medical diagnostic kits are inevitably high because the MAbs are mostly obtained from hybridoma cell culture. Here, we report the development and validation of a novel affinity silk protein produced by transgenic silkworm technology as a possible alternative diagnostic tool for cancers. We generated a transgenic silkworm expressing a cDNA construct containing fibroin L-chain fused to a single-chain variable fragment (scFv) derived from a MAb against carcinoembryonic antigen (CEA). The transgenic cocoons were dissolved in aqueous lithium bromide solution, applied to 96-well plates, and analysed by enzyme-linked immunosorbent assay. The scFv-conjugated affinity silk protein specifically recognized CEA as well as the parental MAb. The binding activity was retained after several months of storage in coated plates or concentrated solution. Thus, the scFv-conjugated affinity silk protein provides a potentially useful alternative to conventional MAbs in medical diagnostic kits.
Highlights
Monoclonal antibodies (MAbs) are utilized in various medical applications, such as the diagnosis of infectious diseases or cancers, in either 96-well plates or designed devices
The levels of scFv-conjugated fibroin L-chain (FibL) expression were estimated to be approximately 10% of endogenous FibL by densitometric analysis, which is comparable to the average expression levels in the previous transgenic silk experiments[3,4,5,13]. These results demonstrate that anti-carcinoembryonic antigen (CEA)-011-scFv and 022-scFv constructs fused with FibL are sufficiently expressed as fusion proteins in silk fibres in the transgenic cocoon shells
W1 and S01 silk solution did not show a non-specific reaction in these assays regardless of the storage period. These results suggest that the binding activity of S13 silk solution containing anti-CEA-011-scFv-conjugated FibL can be stably preserved for at least 6 months when dissolved in 9 M lithium bromide (LiBr) solution and kept at 4 °C
Summary
Monoclonal antibodies (MAbs) are utilized in various medical applications, such as the diagnosis of infectious diseases or cancers, in either 96-well plates or designed devices. The transgenic cocoons were dissolved in aqueous lithium bromide (LiBr) solution, the silk solution was dialysed, concentrated, freeze-dried, and processed into powder This scFv-conjugated silk powder immunoprecipitated its target protein, WASP13. We processed cocoons expressing anti-WASP scFv-conjugated fibroin protein into a thin film by dissolving it in LiBr solution and drying it in 96-well plates to demonstrate the specific detection of WASP by enzyme-linked immunosorbent assay (ELISA)[14]. These results clearly suggested that scFv-conjugated affinity silk protein, in powder or film form, can recognize the target protein and provide useful alternatives for affinity purification or immunodetection of the antigen, respectively. Our present study demonstrates that scFv-conjugated affinity silk protein could be potentially used as an alternative to conventional affinity reagents for the diagnosis of human cancer
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