Anti-5T4 [B3F1]

Invented at Cancer Research UK Manchester Institute

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Summary of the IgG subclasses of five m5T4 specific monoclonal antibodies (mAb, made in 5T4KO mice) recognizing distinct epitopes in the proximal and distal 5T4 extracellular LRR containing domains.
(Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365)

Titration of mAb activity in m5T4 specific ELISA. Anti-m5T4 monoclonal antibodies were titrated by doubling dilution between 0 and 0.1 mg/ml by sandwich ELISA.in 96-well ELISA plates pre-coated at 4C overnight with 1 mg/ml m5T4-pIgFc in borate buffer (100 mM boric acid 150 mM NaCl; pH 8.5). Antibody binding was detected using goat anti-mouse IgG conjugated to HRP. The plates were developed using tetramethyl benzidine, the colour reaction was stopped with 1 M sulphuric acid and the absorbance read at wavelength 450–650 nM.
(Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365)

Titration of mAb cell surface labeling of B16m5T4 tumor cells by flow cytometry. Cells were suspended in FACS buffer (PBS, 0.2% bovine serum albumin, 0.1% sodium azide) and labelled with the appropriate concentration of anti-m5T4 monoclonal antibodies diluted in FACS buffer for 30 mins on ice, washed with FACS buffer and labelled with rabbit anti-mouse IgG conjugated to FITC. 10,000 events were acquired and the data obtained was analysed using WinMIDI (version 2.8) software.
(Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365)

The chemotactic migration exhibited by differentiating WT-ES cells towards CXCL12 was abolished in the presence of the m5T4 specific mAb B1C3 (10 mg) but not in presence of mAb P1C9 or P1H10 (10 mg) or an irrelevant control antibody (10 mg). MAbs B3F1 and B5C9 (10 mg) reduced the chemotactic response. (2 = no CXCL12, + = 10ng CXCL12). Chemotaxis was assessed using transwell chambers.
(Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365)

MAb dose response of inhibition of chemotaxis towards CXCL12 in differentiating WT-ES cells.
(Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365)

Western blot analysis of mAb probing against recombinant m5T4-pIg showing recognition of m5T4 by all mAbs except B1C3.
(Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365)

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Catalogue Number	152711
Applications	ELISA FACS IF Fn WB
Antigen/Gene or Protein Targets	5T4 oncofetal glycoprotein
Synonyms	Trophoblast Glycoprotein; 5T4 Oncofetal Trophoblast Glycoprotein; Wnt-Activated Inhibitory Factor 1; 5T4 Oncotrophoblast Glycoprotein; 5T4 Oncofetal Antigen; WAIF1; M6P1; 5T4; 5T4AG
Reactivity	Mouse
Relevance	Monoclonal antibody directed against 5T4, with use as a prognostic aid of tumours. Background and Research Application The murine 5T4 monoclonal antibody, clone B3F1, identifies the human 5T4 oncofoetal antigen, also known as trophoblast glycoprotein. 5T4 is a highly glycosylated protein expressed by trophoblast and a few specialized adult epithelia and is an antagonist of the Wnt/ β-catenin signalling pathway. Up-regulation of 5T4 expression in some cancers is associated with poor clinical outcome; overexpression of human 5T4 cDNA in epithelial cells can alter their morphology and motility, supporting a role for such functions in cancer and development. 5T4 has limited expression in normal tissues, but is widespread in tumours, thus its expression is used as a prognostic aid. 5T4 is typically found in colorectal, ovarian and gastric tumours. 5T4 is a potential target for T cells, in antibody-directed immunotherapy.
Host	Mouse
Immunogen UniProt ID	Q9Z0L0
Subclass	IgG2a
Molecular Weight (kDa)	46.5
Recommended Growing Conditions	DMEM supplemented with heat inactivated 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin solution and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air.
Strain	C57BL/6
Notes	Production Details Purified using multi-step affinity chromatography with protein A. Storage Conditions Store at -20 degrees frozen. Avoid repeated freeze/thaw cycles. Points of Interest A study found that 5T4 was present in 85% of a study of 72 colorectal carcinomas and in 81% of a study of 27 gastric carcinomas. Clone B3F1 inhibits the chemotactic migration towards CXCL12 exhibited by differentiating WT-ES cells (to a lesser extent than clone B1C3). IC50 for the maximally inhibitory mAb B3F1 was 2.2 µg±0.8. Epitope: distal LRR domain of m5T4 Concentration 1mg/ml as standard
Research Area	Cancer, Stem Cell Biology

References

There are 7 reference entries for this reagent.

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References: 7 entries

Castro et al. 2012. Cancer Immunol Immunother. 61(7):1005-18. PMID: 22127365.

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Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365.

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CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic cells.

Europe PMC ID: 20376365

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Ward et al. 2003. J Cell Sci. 116(Pt 22):4533-42. PMID: 14576347.

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The 5T4 oncofoetal antigen is an early differentiation marker of mouse ES cells and its absence is a useful means to assess pluripotency.

Europe PMC ID: 14576347

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Woods et al. 2002. Biochem J. 366(Pt 1):353-65. PMID: 12003637.

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Characterization of the murine 5T4 oncofoetal antigen: a target for immunotherapy in cancer.

Europe PMC ID: 12003637

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References: 7 entries

Castro et al. 2012. Cancer Immunol Immunother. 61(7):1005-18. PMID: 22127365.

View

Southgate et al. 2010. PLoS One. 5(4):e9982. PMID: 20376365.

View

CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic cells.

View

Ward et al. 2003. J Cell Sci. 116(Pt 22):4533-42. PMID: 14576347.

View

The 5T4 oncofoetal antigen is an early differentiation marker of mouse ES cells and its absence is a useful means to assess pluripotency.

View

Woods et al. 2002. Biochem J. 366(Pt 1):353-65. PMID: 12003637.

View

Characterization of the murine 5T4 oncofoetal antigen: a target for immunotherapy in cancer.

View

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Invented at

	Cancer Research UK Manchester Institute
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Anti-5T4 [B3F1]

Invented at Cancer Research UK Manchester Institute

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References

References: 7 entries

References: 7 entries

Inventor Information

Invented at

Cancer Research UK Manchester Institute

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