SignalKine™ Sandwich ELISA Protocol

This ELISA was developed for the detection and quantification of cytokines, chemokines, and growth factors in cell culture media, serum, and plasma. Recombinant protein standards are run alongside samples. Concentration of target in samples can be determined from the standard curve. We recommend running replicates of standards and samples.

Reagent Preparation

All reagents should be brought to room temperature before use.

Wash Buffer

Prepare 1X wash buffer by diluting ELISA Wash Buffer (20X) in dH₂O.

SignalKine™ Assay Diluent A01:

Buffered solution supplied as a 5X solution. 10 µl of assay diluent is added to each well before addition of 40 µl of standard and samples.

SignalKine™ Sample Diluent S01

Diluent for diluting standards and samples when working with plasma (citrate), plasma (heparin), or serum samples.

SignalKine™ Sample Diluent S03

Diluent for diluting standards and samples when working with plasma (EDTA) samples.

hIL-2 Detection Rabbit mAb (Biotinylated)

Supplied lyophilized. Reconstitution with 60 µl of dH₂O yields a 100X Concentrated Stock solution. Incubate at room temperature for 15 min with occasional gentle mixing to fully reconstitute. Dilute 100X solution 1:100 in Detection Antibody Diluent (green solution). A 96 well plate will need 50 µl/well of 1X solution, so approximately 5 ml of reagent is needed. Dilute 50 µl of 100X stock into 5 ml of Detection Antibody Diluent or dilute as much as needed if not using full 96 well plate.

HRP-linked Streptavidin

Supplied lyophilized. Reconstitution with 60 µl of dH₂O yields a 100X concentrated stock solution. Incubate at room temperature for 15 min with occasional gentle mixing to fully reconstitute. Dilute 100X solution 1:100 in HRP Diluent (red solution). A 96 well plate requires 50 µl/well of 1X solution, so approximately 5 ml of reagent is needed. Dilute 50 µl of 100X stock into 5 ml of HRP Diluent or dilute as much as needed if not using full 96 well plate.

Chemiluminescent Detection Substrate

Working Solution is prepared by mixing equal parts of Luminol/Enhancer Solution and Stable Peroxide Buffer. 50 µl of Working Solution is added to each well. A plate-based luminometer is used to measure Relative Light Units (RLU) at 425 nM within 1–10 min following addition of substrate.

Optimal signal intensity is achieved when read within 10 min. Longer periods between addition of substrate and reading plate may result in decreased signal intensity.

Working Solution is stable for 24 hr at room temperature.

Human IL-2 Standard Preparation

Add 0.1 ml dH₂O to reconstitute Human IL-2 Standard (Lyophilized) (10,000 pg) to yield a 100,000 pg/ml solution. Let sit at room temperature for 15 min with occasional gentle mixing to fully reconstitute standard protein.

For detection in plasma (citrate), plasma (heparin), and serum, samples and protein standards are diluted with SignalKine™ Sample Diluent S01 (provided in kit). For plasma (EDTA), samples and protein standards are diluted in SignalKine™ Sample Diluent S03 (provided in kit). For cell culture media, samples and protein standards are diluted in fresh cell culture media corresponding to sample media (not supplied).

A standard range of 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0 pg/ml is set up. This can be achieved by diluting the protein standard, (100,000 pg/ml) 1:50, which yields 2000 pg/ml. From the 2000 pg/ml solution, serial two-fold dilutions are performed to achieve the other standard concentrations.

We recommend diluting 20 µl of the 100,000 pg/ml protein standard into 980 µl of either SignalKine™ Sample Diluent S01, SignalKine™ Sample Diluent S03 or cell culture medium to yield the 2000 pg/ml standard. Serial two-fold dilution can be achieved by setting up six tubes with 200 µl of the appropriate diluent and then serially transferring 200 µl from the preceding tube. Mix well before each transfer. Use the appropriate diluent as the 0 pg/ml standard.

Sample Preparation

This assay has been validated with cell culture medium, plasma (citrate), plasma (EDTA), plasma (heparin), and serum samples. After sample collection, store in single use aliquots at −20°C. Avoid multiple freeze/thawing.

Particulates in cell culture media should be removed by centrifugation. Note any contaminated, clotted, or hemolyzed samples and interpret these results judiciously.

Absorbance values falling within the hIL-2 standard curve range can be determined directly from the standard curve. Sample absorbance values higher than the 2000 pg/ml hIL-2 value should be diluted so that the absorbance values fall into standard curve range.

Procedure

1. Once microwell strips have reached room temperature, take out as many strips as needed and insert into strip holder. Unused microwell strips should be resealed and stored at 4°C.
2. Add 10 µl of 5X SignalKine™ Assay Diluent A01 to each well.
3. Add 40 µl of hIL-2 Standards and samples to the wells. Cover with plate sealer and incubate for 2 hr at room temperature.
4. Wash plates 4 times with 1X Wash Buffer.

Note: Maximum wash volume is 170 µl/well. These are low volume microwells and have a smaller volume capacity than full size microwells.

5. Add 50 µl of 1X hIL-2 Detection Rabbit mAb (Biotinylated) per well. Cover with plate sealer and incubate for 1 hr at room temperature.
6. Wash plates 4 times with 1X Wash Buffer.
7. Add 50 µl of 1X HRP-linked Streptavidin per well. Cover with plate sealer and incubate for 30 min at room temperature.
8. Wash plates 4 times with 1X Wash Buffer.
9. Add 50 µl of Chemiluminescent Detection Substrate Working Solution per well.
10. Use a plate-based luminometer to measure Relative Light Units (RLU) at 425 nM within 1–10 min following addition of the substrate. Optimal signal intensity is achieved when read within 10 min.

Calculation of Results

Average replicates for standards and samples.

Using a graphing/curve fitting software, plot a 4-parameter logistic curve fit. A standard curve can also be generated by plotting the standard concentration on the x-axis and the absorbance corresponding to the standard concentration on the y-axis. A best-fit curve is drawn through the points on the graph.

Sample concentrations are determined by interpolating from the standard curves. Account for sample dilution by multiplying determined concentration by dilution factor.

Typical Data

Represented here are IL-2 Standard Curves, one diluted in SignalKine™ Sample Diluent S01, a second diluted in SignalKine™ Sample Diluent S03 and a third in cell culture media (RPMI + 10% FBS). Although these are typical of the standard curves that will be generated using this kit, a new set of standards should be run for each new experiment.