PD-1 Pathway Recombinant Protein Collection

Biotinylated PD-1 Pathway Proteins

ELISA is an assay technique routinely used to characterize protein-protein interaction. It’s often used in initial high-throughput screening (HTS) of compound library. ACROBiosystems has developed a range of pre-biotinylated PD-1 pathway proteins. Biotinylated proteins can serve as two-way antibodies for both capture and detection in ELISA assays. They demonstrate higher specificity and detection sensitivity than traditional antibodies.

	Product Description	Cat. No.	Species	Structure
Pair 1	Human PD-L1 / B7-H1 Protein, Fc Tag, low endotoxin	PD1-H5258
Pair 2	Mouse PD-L1 / B7-H1 Protein, Fc Tag	PD1-M5251
	Biotinylated Mouse PD-1 / PDCD1, Fc Tag, Avi Tag (AviTagTM)	PD1-M82F4
Pair 3	Human PD-1 / PDCD1 Protein, Fc Tag (HPLC-verified), low endotoxin	PD1-H5257
	Biotinylated Human PD-L1 / B7-H1, His Tag & Fc Tag, Avi Tag (AviTagTM)	PD1-H82F3
Pair 4	Mouse PD-1 / PDCD1 Protein, Fc Tag	PD1-M5259
	Biotinylated Mouse PD-L1 / B7-H1, Fc Tag, Avi Tag (AviTagTM)	PD1-M82F5

To further simplify your research, we’ve developed a PD-1[Biotin]: PD-L1 Inhibitor Screening Kit (Cat. No. EP-101). This assay employs a simple colorimetric ELISA platform, which measures the binding between immobilized human PD-L1 and in-house developed biotinylated PD-1 protein. This product is uniquely suitable for rapid and high-throughput screening of putative PD-1 and PD-L1 inhibitors.

Fig. 2 Immobilized PD-L1 at 2 μg/mL (100 μL/well) can bind biotinylated human PD-1 with a linear range of 0.038 - 0.6 μg/mL when detected by Streptavidin-HRP.

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Fig. 3 Inhibition of PD-1-PD-L1 binding by an anti-PD-1 neutralizing antibody is measured by the PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair (Cat. No. EP-101).

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Biotinylated proteins can be used along with fluorophore-tagged SA to detect/isolate cells expressing particular surface markers. In a US patent titled "ANTI-PD-1 ANTIBODIES AND METHODS OF USE THEREOF" (US patent 20160159905), the authors (from Pfizer Inc.) used ACROBiosystems' biotinylated PD-L1 (Cat. No. PD1-H82F3) and biotinylated PD-L2 (Cat. No. PD2-H82F6) to compete with candidate antibodies in a binding assay against PD-1 expressing cells. This assay provides measurement for the binding activity of the corresponding antibodies.

Biopanning has gradually become the mainstream approach for antibody development. The AviTag^TM biotinylated proteins can be used with SA-coated magnetic beads/surface in biopanning. It’s a much better choice than non-biotinylated, or chemically biotinylated proteins because it offers higher coating density and uniformed antigen presentation.

Please note that the Fc tag is generally undesired for these experiments because it generates false positive results. We have developed AviTag^TM PD-1 pathway proteins with only His tag attached. These products are recommended specifically for biopanning.

Molecule	Cat. No.	Species	Structure	Purity	Features
PD-1	PD1-H82E4
PD-L1	PD1-H82E5
PD-L2	PD2-H82E8

Cell based assay verified proteins

As a professional supplier of pharmaceutical companies, we have established a cell based assay platform to verify the activity of the target protein, which provide cell based experiment data support for drug screening.

ACROBiosystems used human PD-1 overexpressed 293 cells made in-house in flow cytometry assay and cell based ELISA, and the results showed good performance of PD-L1 (Cat. No. PD1-H5258) and PD-L2 (Cat. No. PD2-H5251) binding to PD-1.

Fig. 4 Flow Cytometry assay shows that recombinant human PD-L1 (Cat. No. PD1-H5258) can bind to 293 cell overexpressing human PD-1.The concentration of PD-L1 used is 10ug/mL

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Fig. 5 Immobilized cell surface PD-1 (5x104 of cells per well) can bind Human PD-L2 Protein, Fc Tag (Cat. No. PD2-H5251) with an EC50 of 0.018 μg/mL (Routinely tested).

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Fig. 7 Immobilized cell surface PD-1 (5x10⁴ of cells per well) can bind Human PD-L1, Fc Tag (Cat. No. PD1-H5258) with an EC50 of 0.029 μg/mL.

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Fig. 8 Immobilized cell surface PD-1 (5x10⁴ of cells per well) can bind Human PD-L2, Fc Tag (Cat. No. PD2-H5251) with an EC50 of 0.018 μg/mL.

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References

• Ishida Y., et al., 1992, EMBO J. 11 (11): 3887–95.

• Blank C., et al., 2007, Cancer Immunol. Immunother. 56 (5): 739–45.

• Agata Y., et al., 1996, Int. Immunol. 8 (5): 765–72.

• Freeman GJ., et al., 2000, J. Exp. Med. 192 (7): 1027–34.

• Latchman Y., et al., 2001, Nat. Immunol. 2 (3): 261–8.

• Yamazaki T., et al., 2002, J. Immunol. 169 (10): 5538–45.

• Dong H., et al., 1999, Nat. Med. 5:1365-1369.

• Freeman G.J., et al., 2000, J. Exp. Med. 192:1027-1034.

• Nishimura H., et al., 2001, Trends in Immunology 22:265.

• Tseng S.-Y., et al., 2001, J. Exp. Med. 193:839-846.

• Latchman Y., et al., 2001, Nat. Immunol. 2:261-268.

• He X.-H., et al., 2004, Acta Biochim. Biophys. Sin. 36:284-289.

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