1. Microplate reader (wavelength: 450nm)
2. 37°C incubator (CO2 incubator for cell culture is not recommenced.)
3. Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
4. Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
5. Sterile tubes and Eppendorf tubes with disposable tips
6. Absorbent paper and loading slot
7. Deionized or distilled water

• Step 1: Add 100ul standard or sample into each well, seal the plate and statically incubate for 90 minutes at 37°C.
• Washing: Wash the plate twice without immersing.
• Step 2: Add 100ul biotin-antibody working solution, seal the plate and statically incubate for 60 minutes at 37°C.
• Washing: Wash the plate three times and immerse for 1min each time.
• Step 3: Add 100ul HRP-Streptavidin Conjugate (SABC) working solution, seal the plate and statically incubate for 30 minutes at 37°C.
• Washing: Wash the plate five times and immerse for 1min each time.
• Step 4: Add 90ul TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
• Step 5: Add 50ul stop solution. Read at 450nm immediately and calculate.

This product is detected by QC department and meets performance required in the manual. (Laboratory Humidity: 20%-60%; Temperature: 18°C -25°C; Equilibrate TMB substrate to 37°C before staining. After adding into the ELISA wells, incubate for 15min at 37°C in dark.)

Due to different assay environments and operations, assay data below and standard curve are provided for reference. Experimenters should establish standard curve according to their own assay.

STD.(pg/ml)	OD-1	OD-2	Average
0	0.086	0.091	0.088
15.625	0.134	0.141	0.137
31.25	0.182	0.192	0.186
62.5	0.308	0.323	0.314
125	0.514	0.54	0.524
250	0.823	0.865	0.84
500	1.372	1.442	1.4
1000	2.052	2.157	2.094

Add a certain amount of Hmgb1 into the sample. Calculate the recovery by comparing the measured value with the expected amount of Hmgb1 in the sample.

Dilute the sample with a certain amount of Hmgb1 at 1:2, 1:4 and 1:8 to get the recovery range.

Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.

Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.

Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.