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Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay.

Sato M, Kojima K, Sakuma C, Murakami M, Tamada Y, Kitani H - Sci Rep (2014)

Bottom Line: Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications.To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates.These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

View Article: PubMed Central - PubMed

Affiliation: 1] Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan [2].

ABSTRACT
Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated "affinity" silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

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ScFv-conjugated silk film efficiently detects the target protein.Specific binding was quantified by ELISA using 96-well plates coated with silk film derived from W1, S01, or C03 strains. (a) The indicated amount of GST or GST-WASP15 was added to each silk film-coated well. (b) A mouse macrophage RAW264.7 cell line was lysed. The lysate was diluted by half (1/2 dil.), one-quarter (1/4 dil.), or not diluted (1/1 dil.) and added to each silk film-coated well or control well coated with silk film made from silk solution after autoclaving. (c) The diluted or non-diluted cell lysate was added to each silk film-coated well which 3 months have passed since it was made. Values are mean ± standard error of the mean from three independent experiments. *P < 0.05; ** P < 0.01; ***P < 0.001; n.s., not significant; n.d., not detectable.
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f3: ScFv-conjugated silk film efficiently detects the target protein.Specific binding was quantified by ELISA using 96-well plates coated with silk film derived from W1, S01, or C03 strains. (a) The indicated amount of GST or GST-WASP15 was added to each silk film-coated well. (b) A mouse macrophage RAW264.7 cell line was lysed. The lysate was diluted by half (1/2 dil.), one-quarter (1/4 dil.), or not diluted (1/1 dil.) and added to each silk film-coated well or control well coated with silk film made from silk solution after autoclaving. (c) The diluted or non-diluted cell lysate was added to each silk film-coated well which 3 months have passed since it was made. Values are mean ± standard error of the mean from three independent experiments. *P < 0.05; ** P < 0.01; ***P < 0.001; n.s., not significant; n.d., not detectable.

Mentions: To test the specific affinity of silk film derived from anti-WASP-scFv-conjugated S01 silk solution to the target protein, we performed ELISA using wild-type or transgenic silk film-coated 96-well plates. The intensity of absorbance of the S01 silk-film coated wells exponentially increased as the concentration of the target protein (GST-WASP15) increased, whereas W1 and C03 silk film-coated wells did not (Fig. 3a). These results suggest that anti-WASP-scFv fused with FibL preserves its specific affinity to the target protein in the silk-film format.


Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay.

Sato M, Kojima K, Sakuma C, Murakami M, Tamada Y, Kitani H - Sci Rep (2014)

ScFv-conjugated silk film efficiently detects the target protein.Specific binding was quantified by ELISA using 96-well plates coated with silk film derived from W1, S01, or C03 strains. (a) The indicated amount of GST or GST-WASP15 was added to each silk film-coated well. (b) A mouse macrophage RAW264.7 cell line was lysed. The lysate was diluted by half (1/2 dil.), one-quarter (1/4 dil.), or not diluted (1/1 dil.) and added to each silk film-coated well or control well coated with silk film made from silk solution after autoclaving. (c) The diluted or non-diluted cell lysate was added to each silk film-coated well which 3 months have passed since it was made. Values are mean ± standard error of the mean from three independent experiments. *P < 0.05; ** P < 0.01; ***P < 0.001; n.s., not significant; n.d., not detectable.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921627&req=5

f3: ScFv-conjugated silk film efficiently detects the target protein.Specific binding was quantified by ELISA using 96-well plates coated with silk film derived from W1, S01, or C03 strains. (a) The indicated amount of GST or GST-WASP15 was added to each silk film-coated well. (b) A mouse macrophage RAW264.7 cell line was lysed. The lysate was diluted by half (1/2 dil.), one-quarter (1/4 dil.), or not diluted (1/1 dil.) and added to each silk film-coated well or control well coated with silk film made from silk solution after autoclaving. (c) The diluted or non-diluted cell lysate was added to each silk film-coated well which 3 months have passed since it was made. Values are mean ± standard error of the mean from three independent experiments. *P < 0.05; ** P < 0.01; ***P < 0.001; n.s., not significant; n.d., not detectable.
Mentions: To test the specific affinity of silk film derived from anti-WASP-scFv-conjugated S01 silk solution to the target protein, we performed ELISA using wild-type or transgenic silk film-coated 96-well plates. The intensity of absorbance of the S01 silk-film coated wells exponentially increased as the concentration of the target protein (GST-WASP15) increased, whereas W1 and C03 silk film-coated wells did not (Fig. 3a). These results suggest that anti-WASP-scFv fused with FibL preserves its specific affinity to the target protein in the silk-film format.

Bottom Line: Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications.To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates.These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

View Article: PubMed Central - PubMed

Affiliation: 1] Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan [2].

ABSTRACT
Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated "affinity" silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

Show MeSH
Related in: MedlinePlus