SignalKine™ Sandwich ELISA Protocol

This ELISA was developed for the detection and quantitation of Cytokines 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 20X wash buffer in dH₂O.

SignalKine™ Assay Diluent A01:

Buffered solution supplied as a 5X solution. 10 µl of Assay Diluent 1 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 (EDTA), plasma (heparin), or serum samples.

hTNF-α 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.

hTNF-α Standard Preparation

Add 0.1 ml dH₂O to reconstitute lyophilized TNF-α recombinant protein standard (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 serum/plasma samples, protein standard is diluted with Serum Diluent, (provided in kit). For samples in cell culture media, protein standard is diluted in fresh cell culture media corresponding to sample media. (Not supplied)

A standard range of 10,000, 2,500, 625, 156.3, 39.0, 9.8, 2.4, and 0 pg/ml is set up. This can be achieved by diluting the protein standard (100,000 pg/ml) 1:10, which yields 10,000 pg/ml. From the 10,000 pg/ml solution, serial four-fold dilutions are performed to achieve the other standard concentrations.

We recommend diluting 50 µl of the 100,000 pg/ml protein standard into 450 µl of appropriate Serum Diluent or cell culture medium to yield the 10,000 pg/ml standard. Serial four-fold dilution can be achieved by setting up six tubes with 300 µl of the appropriate diluent and then serially transferring 100 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, serum, and EDTA, citrate and heparin plasma 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 contamination, clotted or hemolyzed samples and interpret these results judiciously.

Absorbance values falling within the standard curve range can be determined directly from the standard curve. Sample absorbance values higher than the 10,000 pg/ml 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 Assay Diluent to each well.
3. Add 40 µl of hTNF-α Protein Standards and sample 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 Biotinylated hTNF-α Detection Antibody per well. Recover 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. Recover with plate sealer and incubate for 30 min at room temperature.
8. Wash plates 4 times with 1X Wash Buffer.
9. Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution and Stable Peroxide Buffer.
10. Add 50 μl of the Working Solution to each well.

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 at right are hTNF-α Standard Curves, one diluted in Serum Diluent and the second diluted 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.