AlphaLISA Human and Mouse CXCL12 Detection Kit, 500 assay points

Principle of the AlphaLISA CXCL12 assay

The AlphaLISA CXCL12 assay is based on an AlphaLISA sandwich immunoassay involving a biotinylated anti-CXCL12 antibody bound to Streptavidin-coated AlphaLISA Donor beads and an anti-CXCL12 antibody conjugated to AlphaLISA Acceptor beads. In the presence of the CXCL12 the beads come into proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer within the Acceptor beads, resulting in emission with λmax at 615 nm. The intensity of the signal is directly proportional to the concentration of CXCL12 present in the sample.

Protocol of the AlphaLISA human and mouse CXCL12 assay

The AlphaLISA CXCL12 assay can be run in a 96- or 384-well detection plate (50 µL final). As described here, samples or standards are dispensed directly into the assay plate for the detection of CXCL12 by AlphaLISA reagents. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Human and mouse CXCL12 assay details

Intra-assay precision table

Each of the 3 samples were measured 24 times, and the % CV was calculated for each sample. Samples were mouse recombinant CXCL12.

Inter-assay precision table

Each of the samples were measured in 3 independent experiments (3 days), and the % CV was calculated for each sample. Samples were mouse recombinant CXCL12.

Spike and recovery table

Known concentrations of standard (exogenous analyte) were spiked into human plasma samples containing the endogenous analyte. Non-spiked and spiked samples were measured in the assay.

Dilutional linearity table

A sample of mouse recombinant CXC12 was serially diluted. Concentrations of CXCL12 in neat and diluted samples were determined.

Specificity table

Cross reactivity were assessed using recombinant proteins and tested at 10,000 pg/mL. The assay is human CXCL12 isoform α specific as isoforms β and γ were not detected. The assay is also compatible with mouse CXCL12.

Validation of AlphaLISA CXCL12 detection kit on human cell supernatants

BJ human fibroblast cells were plated in a 96-well culture plate at 200k and 400k cells/well in MEM medium with 10% FBS for 24H at 37°C. Then, 5 µL of supernatants were transferred into an AlphaPlate-384 to measure the concentration of secreted CXCL12. The standard curve was prepared in MEM medium + 10% FBS. All other assay components were diluted in AlphaLISA BSA Assay Buffer and added into the wells using the kit protocol. AlphaLISA counts were measured and the CXCL12 concentration in each supernatant sample was interpolated from the standard curve.

Validation of AlphaLISA CXCL12 detection kit on human cell supernatants

NIH-3T3 mouse fibroblast cells were plated in a 96-well culture plate at 200k and 400k cells/well in DMEM medium with 10% FBS for 24H at 37°C. Then, 5 µL of supernatants were transferred into an AlphaPlate-384 to measure the concentration of secreted CXCL12. The standard curve was prepared in DMEM medium + 10% FBS. All other assay components were diluted in AlphaLISA BSA Assay Buffer and added into the wells using the kit protocol. AlphaLISA counts were measured and the CXCL12 concentration in each supernatant sample was interpolated from the standard curve.

Validation of AlphaLISA CXCL12 detection kit on human plasma

Normal platelet poor plasma samples from healthy volunteers were tested for endogenous level of CXCL12.

5 µL of plasma was transferred into an AlphaPlate-384 to measure the concentration of endogenous CXCL12. The standard curve was prepared in FBS. All other assay components were diluted in AlphaLISA BSA Assay buffer and added into the wells using the kit protocol. AlphaLISA counts were measured and the CXCL12 concentration in each supernatant sample was interpolated from the standard curve.