TECHNOLOGY

ELISA Procedures 

- Reconstitution and Dilution of Standards

In ELISA (Enzyme-Linked Immunosorbent Assay), the quality of the standard curve determines the reliability and accuracy of experimental results directly. An ideal standard curve not only requires a good linear relationship but also a sufficient dynamic range to accurately reflect the true concentration of the analyte in the sample. The preparation of the standard curve starts with the standards processing—reconstitution and dilution.

1. Reconstitution of Standards

Reagent Equilibration: Take the ELISA kit out of the 4°C refrigerator, and place the lyophilized standard powder and standard diluent at room temperature for approximately 20 minutes for Equilibration. It can avoid the incomplete dissolution at low temperatures and inaccurate concentration calculation caused by temperature differences.

Brief Centrifugation: Before opening the cap of the standard powder tube, please perform a brief centrifugation (e.g., 10,000 rpm for 1 minute) to sediment all powder that adhered to the tube wall or cap due to the transportation.

 Resuspended by adding buffer: Please add 1 mL of Reference Standard & Sample Diluent to standard tube, tighten the tube cap and keep stand for 10 minutes, then invert the tube several times to ensure the full dissolution, followed by gentle mixing to avoid the foaming. The concentration is the maximum concentration of the standard curve in ELISA kit (e.g., the detection range of the RE3186M Mouse IL-6 ELISA Kit is 3.12–200 pg/mL, so the first concentration is the maximal concentration is (200 pg/mL) of the standard curve).

Important Caution: Don’t blow it vigorously with micropipette before powder dissolved completely , to avoid  undissolved protein powder was taken away by the micropipette, which may cause the decrease in the actual concentration.

2. Gradient Dilution of Standards

The reconstituted standard solution needs to be diluted into a series of concentrations for curve plotting, and a 2-fold serial dilution is generally used. Please Take 7 clean 1.5 mL microcentrifuge tubes (EP tubes), label them from A to G in sequence, and add 250 μL of Standard & Sample Diluent to each tube (the volume can be adjusted according to the actual usage, e.g., 500 μL).

Gradient Dilution Operation (Taking 2-Fold Dilution as an Example): Taking the RE3186M kit with a standard concentration of 200 pg/mL as an example: the ELISA standard curve typically consists of 8 points, including 7 serial dilution concentrations and 1 diluent as the blank control. The following are schematic diagram and table for standard dilution:

Notes

• Thorough Mixing: After each gradient dilution is completed, it is necessary to pipette 10 times and vortex for 5 seconds to ensure thorough mixing before proceeding to the next step. This is the most crucial step to ensure the accuracy of the gradient.
• Brief Centrifugation:Before drawing liquid from the previous concentration tube, please briefly centrifuge the tube, it will move all small liquid drops from the tube’s cap and wall to the bottom, to ensures the accuracy of the drawn volume.

Standard Dilution Video (Reference Only)