• Pre-coated 96-Well Microplate
• Standard
• Standard Diluent Buffer
• Wash Buffer
• Detection Reagent A
• Detection Reagent B
• Diluent A
• Diluent B
• TMB Substrate
• Stop Solution
• Plate Sealer

• 37°C incubator
• Multi and single channel pipettes and sterile pipette tips
• Squirt bottle or automated microplate washer
• 1.5 ml tubes
• Distilled water
• Absorbent filter papers
• 100 ml and 1 liter graduated cylinders
• Microplate reader (wavelength: 450 nm)
• ELISA Shaker

• 1) Set standard, test samples and control wells.

• 2) Aliquot 100 µl of diluted standard into the standard wells.

• 3) Aliquot 100 µl of Standard Diluent buffer into control (zero) well.

• 4) Aliquot 100 µl of diluted samples into the sample wells. Incubate for 1 hr at 37 °C.

• 5) Aliquot 100 µl of Detection Reagent A to each well. Incubate for 1 hr at 37 °C.

• 6) Wash 3 times.

• 7) Aliquot 100 µl of Detection Reagent B to each well. Incubate for 90 mins at 37 °C.

• 8) Wash 5 times.

• 9) Aliquot 90 µl of TMB Substrate to each well. Incubate for 10-20 mins at 37 °C.

• 10) Aliquot 50 µl of Stop Solution.

• 11) Measure the OD at 450 nm.

Intra-assay Precision (Precision within an assay): 3 samples with low, medium and high levels of Tumor Necrosis Factor Alpha (TNFA) were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, medium and high levels of Tumor Necrosis Factor Alpha (TNFA) were tested on 3 different plates, 8 replicates in each plate.

CV (%) = (Standard Deviation / mean) × 100

Intra-Assay: CV<10%

Inter-Assay: CV<10%