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Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis.

Sato R, Obonai T, Tsumura R, Tsumoto K, Koga Y, Yasunaga M, Matsumura Y - Cancer Sci. (2014)

Bottom Line: Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer.The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells.This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Therapeutics, Research Center for Innovative Oncology, National Cancer Center Hospital East, Chiba, Japan; Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.

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Related in: MedlinePlus

Purification of anti-mTF scFv. Anti-mTF scFv was produced in an Escherichia coli system and purified by gel-filtration chromatography. (a) Western blotting of anti-mTF scFv with anti-His-tag antibody. lane1: size marker; lane2: soluble form of anti-mTF scFv; lane3: solubilized anti-mTF scFv in an inclusion body. Arrow indicates 28 kDa band. (b) Size-exclusion chromatography following nickel affinity chromatography of the soluble anti-mTF scFv. There were monomers (arrow) and dimers of anti-mTF scFv. (c) SDS-PAGE. CBB staining of purified anti-mTF scFv. Lane 1: size marker; lane 2: anti-mTF scFv. Arrow indicates 28 kDa.
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fig02: Purification of anti-mTF scFv. Anti-mTF scFv was produced in an Escherichia coli system and purified by gel-filtration chromatography. (a) Western blotting of anti-mTF scFv with anti-His-tag antibody. lane1: size marker; lane2: soluble form of anti-mTF scFv; lane3: solubilized anti-mTF scFv in an inclusion body. Arrow indicates 28 kDa band. (b) Size-exclusion chromatography following nickel affinity chromatography of the soluble anti-mTF scFv. There were monomers (arrow) and dimers of anti-mTF scFv. (c) SDS-PAGE. CBB staining of purified anti-mTF scFv. Lane 1: size marker; lane 2: anti-mTF scFv. Arrow indicates 28 kDa.

Mentions: We determined the sequences of the VH and VL regions of our anti-mTF monoclonal antibody. The specificity was validated using IGBLAST, according to the method described in a previous report.(17,18) The construction of anti-mTF scFv is shown in Figure 1. Western blot analysis showed that the anti-mTF scFv was expressed in a soluble form in the supernatant of the cell lysate and in an insoluble form in the inclusion bodies (Fig. 2a). The anti-mTF scFv with a 6-His tag was purified using affinity chromatography and size-exclusion chromatography. The results of size-exclusion chromatography indicated that there were monomers and dimers of the anti-mTF scFv in the supernatant (Fig. 2b). Finally, the monomer scFv, which represented the single-chain protein as judged by visualization of a single band of 28 kDa on SDS-PAGE, was purified and used for this study (Fig. 2c).


Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis.

Sato R, Obonai T, Tsumura R, Tsumoto K, Koga Y, Yasunaga M, Matsumura Y - Cancer Sci. (2014)

Purification of anti-mTF scFv. Anti-mTF scFv was produced in an Escherichia coli system and purified by gel-filtration chromatography. (a) Western blotting of anti-mTF scFv with anti-His-tag antibody. lane1: size marker; lane2: soluble form of anti-mTF scFv; lane3: solubilized anti-mTF scFv in an inclusion body. Arrow indicates 28 kDa band. (b) Size-exclusion chromatography following nickel affinity chromatography of the soluble anti-mTF scFv. There were monomers (arrow) and dimers of anti-mTF scFv. (c) SDS-PAGE. CBB staining of purified anti-mTF scFv. Lane 1: size marker; lane 2: anti-mTF scFv. Arrow indicates 28 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317963&req=5

fig02: Purification of anti-mTF scFv. Anti-mTF scFv was produced in an Escherichia coli system and purified by gel-filtration chromatography. (a) Western blotting of anti-mTF scFv with anti-His-tag antibody. lane1: size marker; lane2: soluble form of anti-mTF scFv; lane3: solubilized anti-mTF scFv in an inclusion body. Arrow indicates 28 kDa band. (b) Size-exclusion chromatography following nickel affinity chromatography of the soluble anti-mTF scFv. There were monomers (arrow) and dimers of anti-mTF scFv. (c) SDS-PAGE. CBB staining of purified anti-mTF scFv. Lane 1: size marker; lane 2: anti-mTF scFv. Arrow indicates 28 kDa.
Mentions: We determined the sequences of the VH and VL regions of our anti-mTF monoclonal antibody. The specificity was validated using IGBLAST, according to the method described in a previous report.(17,18) The construction of anti-mTF scFv is shown in Figure 1. Western blot analysis showed that the anti-mTF scFv was expressed in a soluble form in the supernatant of the cell lysate and in an insoluble form in the inclusion bodies (Fig. 2a). The anti-mTF scFv with a 6-His tag was purified using affinity chromatography and size-exclusion chromatography. The results of size-exclusion chromatography indicated that there were monomers and dimers of the anti-mTF scFv in the supernatant (Fig. 2b). Finally, the monomer scFv, which represented the single-chain protein as judged by visualization of a single band of 28 kDa on SDS-PAGE, was purified and used for this study (Fig. 2c).

Bottom Line: Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer.The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells.This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Therapeutics, Research Center for Innovative Oncology, National Cancer Center Hospital East, Chiba, Japan; Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.

Show MeSH
Related in: MedlinePlus