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: Wash the plate twice before adding the standard and sample.
• Step 2: Add 50ul standard or sample into each well. Immediately add 50ul biotin-labeled antibody working solution into each well(The tip can't be reused after touching the liquid.), gently shake the plate for 1min, seal closely and statically incubate for 45 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 into each well, 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	2.269	2.384	2.315
12.5	1.615	1.681	1.648
25	1.34	1.394	1.367
50	1.027	1.069	1.048
100	0.819	0.853	0.836
200	0.589	0.613	0.601
400	0.395	0.411	0.403
800	0.241	0.251	0.246

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

Dilute the sample with a certain amount of AMSH 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.