How to Run Single Molecule Counting (SMC^®) Assays

Read on to learn how to run a Single Molecule Counting (SMC^®) assay from start to finish with this step-by-step guide, using the SMC^® Human IL-6 High Sensitivity Immunoassay Kit as an example. You can also watch short videos to guide you through the steps from reconstituting the standard, preparing the standard curve, preparing and dispensing beads and detection antibody, and elution and neutralization steps.

• How to Reconstitute the Standard
• How to Prepare the Standard Curve
• How to Prepare and Dispense Beads
• How to Prepare and Dispense the Detection Antibody
• How to Perform Elution and Neutralization Steps
• Materials

How to Reconstitute the Standard

Below describes the steps involved in reconstituting the standard for the SMC^® IL-6 assay.

• Remove the foil and seal and loosen the rubber seal
• Be careful not to remove the seal from the vial
• Reconstitute the IL-6 lyophilized standard in 250 µL of ultra-pure water
• Invert the vial several times to mix
• Allow the vial to sit for 5-10 minutes

You can also watch a short video outlining these steps.

How to Prepare the Standard Curve

Below outlines the steps in preparing the standard curve for the SMC^® IL-6 assay.

Dispensing of the Standard Diluent

• In a 12-channel reservoir, add 1,000 µL of Standard Diluent to wells 2 through 5
• Then, add 500 µL of Standard Diluent to wells 6 through 12

Preparation of Serial Dilutions for Standard Curve

• Refer to the standard value assignment on the Certificate of Analysis for the starting concentration of the IL-6 Standard vial
• Dilute the IL-6 Standard stock down to 50 pg/mL in at least a 1 mL final volume
• Transfer 1 mL of the 50 pg/mL standard stock solution into well 1 of the 12-channel reservoir
• Transfer 500 µL from well 1 into well 2, mixing thoroughly
• Continue serial dilutions from well 2 stopping at well 11, mixing thoroughly and consistently each time
• Mix well 12 even though you do not add anything to it

Dispensing of the Samples and Standard Curve Into the Assay Plate

• Pipette 75 µL of the appropriate Standard to the corresponding wells on the assay plate
• Pipette 75 µL of Sample to the appropriate replicate wells in the assay plate

You can also view each of these steps in action in this video.

How to Prepare and Dispense Beads

Below are the steps in preparing and dispensing the beads for the SMC^® IL-6 assay.

Preparation of the Capture Beads

• Mix IL-6 Capture Antibody Coated Beads on a rotisserie-style mixer until all beads are resuspended (recommended time is at least 30 minutes)
• Then, add the entire vial of Coated Beads to 11.0 mL of Assay Buffer
• Rinse the bead vial with 0.55 mL of Assay Buffer and ensure that all beads have been transferred
• Mix by gentle inversion
• Dispense the beads into a reservoir in preparation for dispensing into the assay plate

Transfer of the Capture Coated Beads with the Assay Plate

• Pipette 100 µL per well of the diluted coated beads into the assay plate
• Resuspend the beads between each dispense using the pipet

Capture Incubation

• Seal the assay plate with a clear adhesive plate seal
• Apply pressure to the seal to prevent leaking and cross-contamination
• Incubate for 2 hours at 25 °C on microplate incubator/shaker
  • For the recommended Boekel Scientific Jitterbug™ Heated Microplate Shaker (VWR Cat. No. 35821-065):
    • Put the plate on the recommended Jitterbug™ Heated Microplate Shaker
    • Click onto “Mix 5”
    • Agitation will be done at 25 °C for 2 hours

See each step performed in this short video.

How to Prepare and Dispense the Detection Antibody

Below shows the steps in preparing and dispensing the detection antibody for the SMC^® IL-6 assay.

• For the BioTek^® 405™TSUVS Plate Washer (Cat. No. 95-0004-05):
  • Put the plate on the manifold
  • Select wash program “Post Capture Wash”
  • Click on “Run”

How to Prepare the Detection Antibody

• Prepare 1X detection antibody: add 250 µL of 20S detection antibody into 4,750 µL of Assay Buffer
• Agitate the dilution by inversion
• Filter the diluted detection antibody using a syringe with a 0.2 µm filter into a reservoir

Dispense the Detection Antibody into the Assay Plate

• After removal of the plate from the plate washer, dispense 20 µL per well of Detection Antibody
• Firmly seal the assay plate with a clear adhesive plate seal
• Incubate for 1 hour at 25 °C on microplate incubator/shaker
  • For the recommended Boekel Scientific Jitterbug™ Heated Microplate Shaker (VWR Cat. No. 35821-065):
    • Put the plate on the recommended Jitterbug™ Heated Microplate Shaker
    • Click onto “Mix 5”
    • Agitation will be done at 25 °C for 1 hour

Watch this short video to see how to prepare and dispense the detection antibody.

How to Perform Elution and Neutralization Steps

Below shows the steps in preparing and dispensing the detection antibody for the SMC^® IL-6 assay.

Detection Wash 1

• BioTek^® 405™TSUVS Plate Washer (Cat. No. 95-0004-05):
  • Put the plate on the manifold
  • Select wash program “4 Cycle Pre-Transfer”
  • Click on “Run”

After Detection Wash Incubation

• For the recommended Boekel Scientific Jitterbug™ Heated Microplate Shaker (VWR Cat. No. 35821-065):
  • After the 4CyPrTr wash, put the plate on the recommended Jitterbug™ Heated Microplate Shaker
  • Click onto “Mix 3”
  • Agitation will be done at 25 °C for 1-5 minutes

Detection Wash 2

• BioTek^® 405™TSUVS Plate Washer (Cat. No. 95-0004-05):
  • Put the plate back on the manifold
  • Select wash program “Final aspiration”
  • Click on “Run”

Dispense of Buffer B

• After removal from the plate washer, dispense 11 µL per well of Buffer B
• Seal the assay plate with a clear adhesive plate seal

Elution Incubation

• For the recommended Boekel Scientific Jitterbug™ Heated Microplate Shaker (VWR Cat. No. 35821-065):
  • Put the plate on the recommended Jitterbug™ Heated Microplate Shaker
  • Click onto “Mix 5”
  • Agitation will be done at 25 °C for 10 minutes

Dispense of Buffer D

• Add 11 µL per well of Buffer D into a fresh 96-well assay plate
• Seal the assay plate with a clear adhesive plate seal

Dispense of the Eluate in the Second 96-Well Plate

• Put the assay plate on the SMC^® magnet
• Wait for 2 minutes at room temperature to allow the beads to settle and form a tight pellet
• A bead pellet will form which looks like a red dot on the edge of the wells
• Transfer 10 µL of the eluate from the assay plate by aspirating directly from the V-bottom of the plate into the fresh 96-well plate previously filled in with Buffer D
• Avoid touching the beads pellet
• Change the tip for every transfer

Last Shaking Step

• For the recommended Boekel Scientific Jitterbug™ Heated Microplate Shaker (VWR Cat. No. 35821-065):
  • Put the SMC^® 384-well read plate on the recommended Jitterbug™ Heated Microplate Shaker with its shoe
  • Click onto “Mix 5”
  • Agitation will be done for 2 minutes
  • Centrifuge the plate for 1 minute (1,100 x g)

Dispense of the Neutralized Eluate in the 384-Well Plate

• Review the process of using quadrants on a 384-well plate
• Transfer 20 µL of the neutralized eluate per well to the corresponding wells of the SMCxPRO^® reading plate

Before Plate Reading

• Seal the reading plate with a clear adhesive plate seal
• Centrifuge the plate for 1 minute at room temperature, at approximately 1,100 x g
• Remove the plate sealer, inspect the reading plate wells, and remove bubbles if they are present

Plate Reading

• Seal the plate with an aluminum foil seal
  • Be strong with the sealing
  • Take the shoe off the plate
• Put the plate into the SMCxPRO^® instrument for reading

View this video for key elution and neutralization steps in the SMC^® protocol.

For Research Use Only. Not For Use In Diagnostic Procedures.