Human PD-1 Quantikine ELISA Kit

Catalog #: DPD10
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QC289: Quantikine Immunoassay Control Set 1078 for human PD-1

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Procedure

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Summary Precision Recovery Linearity Data Examples Product Datasheets Preparation and Storage Background Related Research Areas

Human PD-1 Quantikine ELISA Kit Summary

Assay Type

Solid Phase Sandwich ELISA

Format

96-well strip plate

Assay Length

4.5 hours

Sample Type & Volume Required Per Well

Cell Culture Supernates (50 uL), Cell Lysates (50 uL), Serum (50 uL), EDTA Plasma (50 uL), Heparin Plasma (50 uL), Urine (50 uL)

Sensitivity

3.27 pg/mL

Assay Range

15.6 - 1,000 pg/mL (Cell Culture Supernates, Cell Lysates, Serum, EDTA Plasma, Heparin Plasma, Urine)

Specificity

Natural and recombinant human PD-1.

Cross-reactivity

< 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.

Interference

No significant interference observed with available related molecules.

Product Summary

The Quantikine™ Human Programmed Death-1 (PD-1) Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human PD-1 in cell culture supernates, cell lysates, serum, plasma, and urine. It contains HEK293-expressed recombinant human PD-1 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human PD-1

showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human PD-1.

Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision.

Cell Culture Supernates, Cell Lysates, Serum, EDTA Plasma, Heparin Plasma, Urine

	Intra-Assay Precision			Inter-Assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	125	319	655	131	304	626
Standard Deviation	3.49	5.6	10.8	7.36	14.6	25.8
CV%	2.6	1.8	1.6	5.6	4.8	4.1

Recovery

The recovery of human PD-1 spiked to levels throughout the range of the assay was evaluated.

Sample Type	Average % Recovery	Range %
Cell Culture Media (n=4)	100	97-103
Cell Lysis Buffer (n=1)	96	94-97
EDTA Plasma (n=4)	86	81-93
Heparin Plasma (n=4)	85	75-93
Serum (n=4)	88	77-98
Urine (n=4)	89	83-97

Linearity

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human PD-1 were serially diluted with calibrator diluent to produce samples with values within the dynamic range of the assay.

Scientific Data

N/A

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Human PD-1 ELISA Standard Curve

ELISA

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Human PD-1 Quantikine Recovery Competitor Comparison PD-1 is spiked at three known concentrations throughout the range of the assay and run to measure response of the spiked sample matrix. Serum recovery is 95% compared to 136% for the top competitor. EDTA Plasma recovery is 87% compared to 157% for the top competitor. Culture media recovery is 94% compared to 58% for the top competitor. In spike and recovery experiments, natural samples are spiked with the recombinant target analyte of interest to identify interference caused by sample matrices.

Product Datasheets

Product Datasheet

COA

SDS

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: PD-1

Programmed Death-1 (PD-1), also known as Programmed cell death protein and CD279, is an extensively studied immune checkpoint inhibitory receptor. Given PD-1's role in peripheral tolerance, it is not surprising that increased PD-1 expression is a mechanism for immune escape, which is permissive for cancer growth and metastasis (1,2). PD‐1 is encoded by the PDCD1 gene (3). The PD-1 glycoprotein is a monomeric 50-55kDa type 1 transmembrane protein that belongs to the immunoglobulin (Ig) superfamily (4). PD-1 expression and induction have been well studied (3,5,6). It is expressed in CD4⁺ and CD8⁺ T cells as well as B cells, macrophages, some dendritic cell subsets and NK cells. PD-1 is induced by T cell receptor (TCR) signaling as well as interleukin 2 (IL-2), IL-7 and type 1 interferons. A variety of reagents are used to experimentally induce PD-1 expression, including, phorbol 12-myristate 13 acetate, ionomycin, concanavalin A, CD3/CD28 antibodies, or most notably lymphocytic choriomeningitis virus in vivo (6).

PD-1 expression is also regulated post-translationally (2). The PD-1 extracellular domain is glycosylated at asparagine (N) residues N49 and N72 while in the endoplasmic reticulum. PD-1 subsequently transits to the golgi apparatus where it is fucosylated at the same sites by the core fucosylase FUT8. This post translational modification represents a novel therapeutic target as T cells have a more robust anticancer response due to reduced surface expression of de-fucosylated PD-1. PD-1 is polyubiquinated at lysine (K) residue K48 by the E3 ligase F-Box 38 (FBX028) (2,7) which results in PD-1 degradation via the proteosome.

PD-1 serves as the receptor for programmed death ligand 1 (PD-L1/CD274/B7-H1) and PD-L2 (CD273/B7-DC) (8,9). The interaction of the extracellular domains of PD-1 and PD-L1 causes a confirmational change that results in the phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and the immunoreceptor tyrosine-based switch motif (ITSM) by Src family kinases (4). Src homology 2 containing tyrosine phosphatase 2 (SHP-2) and SHP-1 are recruited in order to attenuate T cell activating signals via dephosphorylation of downstream signaling cascades.

Long Name:

Programmed Death-1

Entrez Gene IDs:

5133 (Human); 18566 (Mouse); 301626 (Rat); 100533201 (Porcine); 486213 (Canine); 102123659 (Cynomolgus Monkey)

Alternate Names:

CD279 antigen; CD279; hPD-1; PD1; PD-1; PD1hPD-l; PDCD1; programmed cell death 1; programmed cell death protein 1; Protein PD-1; SLEB2

Related Research Areas

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

Prepare all reagents, standard dilutions, and samples as directed in the product insert.
Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

Add 50 µL of Assay Diluent to each well.

50 µL Assay Diluent

Add 50 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours on a horizontal orbital microplate shaker.
Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.

50 µL Standard, Control, or Sample

Add 200 µL of Human PD-1 Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.
Aspirate and wash 4 times.

200 µL Conjugate

Add 200 µL Substrate Solution to each well. Incubate for 30 minutes on the benchtop. Protect from light.

200 µL Substrate Solution

Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.

50 µL Stop Solution

Citation for Human PD-1 Quantikine ELISA Kit

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1