Validation Report #029766

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Chemokine (C-C Motif) Ligand 2 (CCL2) (ABIN365052) has been independently validated for Enzyme-linked immunosorbent assay by the Science Exchange network of experts.

The Science Exchange network is used to independently validate research reagents, protocols, and results to ensure accuracy and reproducibility.

Validation number: #029766
Validated on: 07/18/14

Summary

Antigen

Chemokine (C-C Motif) Ligand 2 (CCL2)

Catalog number
Supplier

Cusabio

Supplier catalog number
Lot number

Z02184065

Method validated
Laboratory
Validation number
Positive Control

Human serum - expression is ~16 pg/mL

Negative Control

Goat serum (non-reactive species)

Notes

Target protein was detected in the positive control sample and not in the negative control sample as expected.

Independent Results

Full Methods

ELISA kit

  • Antigen: Chemokine (C-C Motif) Ligand 2 (CCL2)
  • Catalog number: ABIN365052
  • Supplier: Cusabio
  • Supplier catalog number: csb-e04655h
  • Lot number: Z02184065

Controls

  • Positive control: Human serum (Sigma Aldrich, Cat# H6914-20ML, Lot# SLBK2170V)
  • Negative control: Goat serum (Sigma Aldrich, Cat# G9023-10ML, Lot# SLBH2670V)

Protocol

  1. All reagents were brought up to room temperature for 30 minutes prior to use. The 1x Wash Buffer was prepared by adding 20 mL of 25x Wash Buffer Concentrate to 480 mL of distilled/deionized water and mixing thoroughly.
  2. The vial of Standard was reconstituted with 1 mL of Sample Diluent, mixed, and allowed to sit for 15 minutes with gentle agitation.
  3. The standard curve was prepared by creating a 2-fold dilution series of seven standards (including the original undiluted vial) using Sample Diluent. Sample Diluent alone served as the 0 pg/mL standard.
  4. The assay plate was removed from the foil pouch and 100 μL of each standard and sample were added to the appropriate wells, in triplicate. The plate was covered with the adhesive strip provided and incubated for 2 hours at 37 °C.
  5. Approximately 10 minutes before the incubation ended, a 1x Biotin-antibody solution was prepared by diluting 60 μL of 100x Biotin-antibody into 5940 μL of Biotin-antibody Diluent.
  6. The liquid from each well was removed.
  7. 100 μL of 1x Biotin-antibody solution was added to each well, and the plate was covered with a new adhesive strip, and incubated for 1 hour at 37 °C.
  8. Approximately 10 minutes before the incubation ended, a 1x HRP-avidin solution was prepared by diluting 60 μL of 100x HRP-avidin into 5940 μL of HRP-avidin Diluent.
  9. Each well was aspirated and washed, repeating the process two times for a total of three washes. Each well was washed by filling each well with 1x Wash Buffer and letting it stand for 2 minutes. After the last wash, remaining Wash Buffer was removed and the plate was inverted and blotted against clean, absorbent paper towels.
  10. 100 μL of 1x HRP-avidin solution was added to each well, the plate was covered with a new adhesive strip, and incubated for 1 hour at 37 °C.
  11. The aspiration/wash procedure from Step 9 was repeated for an additional 5 washes.
  12. 90 μL of TMB Substrate was added to each well. The plate was protected from light and incubated for 15-30 minutes at 37 °C, with periodic checking to prevent overdevelopment.
  13. 50 μL of Stop Solution was added to each well and mixed thoroughly. The optical density (OD) of each well was measured within 5 minutes using a microplate reader set to 450 nm.
  14. A standard curve was generated by plotting the OD value for each standard on the y-axis against the concentration on the x-axis using Excel. A line of best fit through the points on the graph was used to generate the equation x = (y-0.1865) / 0.0019.
  15. The equation was used to calculate CCL2 concentrations of the samples based on their average OD values.

Experimental Notes

At high concentrations the readout of the assay is non-linear.

Figures

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Figure 1: Human CCL2 full standard curve graph.
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Figure 2: Human CCL2 partial standard curve graph (linear range).
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Figure 2: Table of absorbance readings (OD 450 nm) for standard curve, positive (human serum) and negative (goat serum) control samples. Value for Average Reading is derived from the average of three readings (OD 450nm). An equation x = (y-0.1865) / 0.0019 was generated from the partial standard curve (in the linear range) and used to calculate CCL2 concentrations shown in the Table.
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