Phospho-ZAP-70 (Tyr319) & Total ZAP-70 Cellular Assay Kits

Phospho-ZAP-70 (Tyr319) for monitoring T-lymphocyte activation

This cell-based assay is designed to monitor the modulation of phosphorylated ZAP-70 (Tyr319) which is a readout of T cell activation.

ZAP-70 is a cytoplasmic protein tyrosine kinase expressed in T and NK cells, and which plays a critical role in the events involved in initiating T-cell responses by the antigen receptor. Following TCR engagement, Zap-70 is rapidly and transiently phosphorylated on several tyrosine residues through autophosphorylation and transphosphorylation by the tyrosine kinase Lck. Increased Zap-70 kinase activity mediates downstream signaling events such as the linker for the activation of T cells (LAT) and the 76 kDa SH2-domain-containing leukocyte protein (SLP-76), which are subsequently phosphorylated by active ZAP-70. The consequences of these early signaling events eventually lead to T-cell activation, proliferation, and differentiation.

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total ZAP-70 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-Zap-70 antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with Zap-70, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

Assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-ZAP-70 can be quantitatively detected using the HTRF phospho-ZAP-70 (Tyr319) and total ZAP-70 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

Simplified pathway

Function and regulation of ZAP-70

• TCR engagement promotes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytosolic side of the TCR/CD3 complex by lymphocyte protein tyrosine kinase (Lck).

• Zap-70 is recruited to the TCR/CD3 complex where it becomes phosphorylated and activated, promoting recruitment and phosphorylation of downstream adaptor or scaffold proteins.

Product Performance

1. HTRF assay compared to Western Blot using phospho & total ZAP-70 cellular assays on human Jurkat cells

Jurkat cells were grown in a T175cm2 flask for 2 days in RPMI culture medium supplemented by 10% FBS, at 37°C in 5% CO2 atmosphere. 4.5 ml of cell suspension (13 x 106 cells / mL) were stimulated with anti-CD3 antibody (20 µg / mL final concentration during 2.5min). After stimulation, cells were lysed at RT for 30 min by adding 2.25 mL of 4X supplemented lysis buffer. Neat lysate was then serially diluted in the same supplemented lysis buffer. 16 µL of each dilution were analyzed in parallel by HTRF or by Western Blot.

2. Cell density optimisation for Phospho ZAP-70 (Tyr319)

This assay was performed using the two-plate assay protocol. Jurkat cells were plated in a 96-well culture plate at 200K cells or 400K cells and 25 µL / well, in complete RPMI culture medium with 10% FBS. Cell stimulation was done by anti-CD3 Ab at the final concentration of 20 µg / mL for 2.5 min. Cells lysis was performed using 10 µL of 4X supplemented lysis buffer. 16 µL of each cell lysate were transferred to the detection plate for analysis using HTRF Phospho ZAP-70 (Tyr319) assays.

3. Kinetics of cell stimulation

This assay was performed using the two-plate assay protocol. Jurkat cells were plated in a 96-well culture plate at 400 K cells, using 25 µL / well in complete RPMI culture medium with 10% FBS. Cell stimulation was performed using the anti-CD3 Ab at the final concentration of 20 µg / mL for different stimulation times (min). After stimulation, cells were lysed with 10 µL of 4X supplemented lysis buffer. 16 µl of each cell lysate type were analyzed by HTRF Phospho ZAP-70 (Tyr319) and Total ZAP-70 assays.

4. Monitoring of the immune checkpoint inhibitor PD-L1 activity

As Phospho ZAP-70 (Tyr319) is a readout of T cell activation, it was used to monitor the inhibitory effect of PD-L1 recombinant protein on T cell activation.
This assay was performed using the two-plate assay protocol.
400 000 Jurkat cells were plated in 25 µL of complete RPMI culture medium with 10% FBS in a 96-well culture plate. Cells were then pre-incubated 24 hours with different concentrations of PD-L1 recombinant protein. After incubation, the cells were stimulated for 2.5 min with 20 µg / mL anti-CD3 Ab (final concentration) and cell lysis was done directly by adding 10 µL of 4X supplemented lysis buffer, followed by a RT 30 min incubation. 16µL of each experimental sample were analyzed using Phospho ZAP-70 (Tyr319) and Total ZAP-70 assays.

Each Phospho ZAP-70 result was then normalized with the Total ZAP-70 amount and plotted on a graph.

A slight but significant inhibitory effect of PD-L1 recombinant protein was recorded with the Phospho ZAP-70 (Tyr319) assay at the final concentration of 40 µg / mL.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
ZAP-70 phospho-Y319 kit - 500 tests64ZAPPEG
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ZAP-70 phospho-Y319 kit - 10,000 tests64ZAPPEH
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ZAP-70 total kit - 500 tests64ZATPEG
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ZAP-70 total kit - 10,000 tests64ZATPEH
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Companion products

DescriptionCat. noProduct insertMSDS
ZAP-70 phospho-Y319 kit - control lysate64ZAPTDA
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ZAP-70 total kit - control lysate64ZATTDA
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Literature