ELISA for human CCL2/MCP-1

R&D Systems ELISA for human CCL2/MCP-1

Judith Sunzenauer (last update 11.6.2013)



  • Bring all reagents and samples to room temperature before use.
  • Run duplicates.
  • Prepare Wash Buffer: Add 20ml of Wash Buffer Concentrate to 480ml a.dest (1:25 Dilution)
  • Discard Calibrator Diluent RD5L Concentrate (for urine/cell culture spernate only!!)
  • Reconstitute CCL2 Standard: add 5ml Calibrator RD6Q to vial of CXCL12 Standard (concentration of stock: 2000pg/ml). Allow the standard to sit for a minimum of 15min prior to making dilutions.
  • Dilute Plasma samples: 1:4 with Calibrator Diluent RD6Q
  • Produce the dilution series for CCL2 Standardcurve:

Prepare 8 prelabeled (#1-8) polypropylene tubes and fill each tube with 500µl of Calibrator Diluent RD6Q. Transfer 500µl Standard from the original vial to tube #1, mix thoroughly and transfer 500µl from tube #1 to #2,….. continue until tube #7. Fill tube #8 with Calibrator Diluent RD6Q.

Assay Procedure:

  • Add 50µl of Assay Diluent RD1-83 to each well.
  • Add 200µl of Standard Dilution #1-8 or sample per well.
  • Seal the Plate and incubate 2h at room temperature.
  • Aspirate each well.
  • Wash 3 times with 400µl Wash Buffer.
  • After the last wash, remove any remaining liquid and invert the plate and blot it against clean paper towels.
  • Add 200µl of CCL2/MCP-1 Conjugate to each well.
  • Seal and incubate for 2h at RT.

Prepare Substrate Solution: mix color Reagents A and B in equal volumes within 15min of use. Protect from light!

  • Wash 3 times with 400µl Wash Buffer.
  • Add 200µl of Substrate Solution to each well.
  • Incubate for 30min at RT and protect from light.
  • Add 50µl Stop Solution to each well. Gently tap the plate to ensure mixing.
  • Determine the OD at 450nm within 30min. Set wavelength correction to 540nm or 570nm.

Calculation of Results:

  • If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
  • Concentrations of the Standard Dilutions:
Std. # CCL2 pg/ml
1 1000
2 500
3 250
4 125
5 62.5
6 31.2
7 15.6
8 0


Assay Mean in EDTA Plasma(pg/ml) Range (pg/ml)
CCL2 153 1,7-2.000

Handling Photometer + Software:

Make sure the laser is warmed up prior to scanning for 15min


CCL2 Human Immunoassay

R&D Systems