Experimental Procedure

1. Remove number of strips desired,and allow to acclimate to room temperature.The unused strips and desiccant should be placed back into the sealed aluminum foil bag and stored at 2-8℃.

2. Set aside blank wells (if measuring at dual-wavelength,the blank wells can be ignored)

3. Add standards or samples to their corresponding wells (100μL for each well).Please remember that the 0ng/mL well should be 100μL of Standard Diluent.Seal the wells/plate with the adhesive tape strip,and incubate at 37℃ for 90min.

4. Prepare required quantity of Biotinylated Antibody 30min in advance.

5. Wash ELISA plate 2 times

6. Add prepared Biotinylated Antibody to each well (100μL per well).Seal reaction wells with adhesive tape strip,and incubate at 37℃ for 60min.

7. Prepare required quantity of Enzyme Conjugate 30min in advance.

8. Wash ELISA plate 3 times.

9. Add prepared Enzyme Conjugate to each well other than the blank wells (100μl for each).Seal the wells with the adhesive tape strip,and incubate at 37℃ for 30min.

10. Wash ELISA plate 5 times.

11. Add 100μL of the prepared Color Reagent to individual wells (also into blank well),incubate protected from light at 37℃.When the coloration of the highest standards become darker,and the color gradient appears,the incubation can be stopped.The chromogenic reaction should be controlled to within 30 min.

12. Add 100μL Color Reagent C to each individual well (also into blank well).Mix well.Read OD（450nm）within 10 min.

Take the concentration values of standards as X- and OD readings as the Y-coordinates.Use a smooth line to connect each coordinate point of the standard values.The concentration of samples can be found by inputting the sample OD values into the line equation for the standard curve.It is recommended to employ professional curve software (e.g.curve expert 1.3) to analyze and compute the results.

Note

1. The re-dissolved standard cannot be stored again once prepared,so please do not attempt to re-freeze it once it has been reconstituted.

2. Due to shaking/inversion during transport,centrifuging of the tubes/bottles of the kit might be necessary to consolidate the material contained within.Tubes should be shaken manually or centrifuged for 1 min at 1000rpm to pool all material to the bottom.

3. Concentrated washing buffer might crystallize slightly.Use a water bath to help the dissolution during diluting process.The crystals must be totally dissolved when preparing the washing buffer.

4. The prepared standard is intended to only be a single-use aliquot,so please do not try to re-use the standard that has already been tested.Please use the second vial provided if you run the assay again.

5. Only use reagents/components that came directly with this kit.Do not mix batches/lots from other orders of this kit,or from different kits.

6. Ensure the reagents are well mixed.For the reagents in the microplate,adequate mixing is particularly important for accurate test results.It is recommended to employ a micro-oscillator (at the lowest frequency).If a micro-oscillator is not available,please slightly shake the microplate manually for 1 min,in a circular motion in order to make sure the wells are sufficiently mixed.

7. Please ensure the kit has been brought to room temperature prior to beginning the assay.

8. Standards are always recommended to be tested in duplicate or triplicate.

9. Place the unused microplate strips into the foil bag at 2-8℃ for storage (if you intend to use the strips within a relatively short timeframe).

10. The chromogen reagent is sensitive to light,therefore please avoid exposing to light.

11. Kits that have passed their expiration date should not be used.

12. When using dual-wavelength,the wavelengths should be set at 450nm and 630nm.

13. All the samples,washing buffer and wastes should be treated as biowaste.Color Reagent C is 1M sulfuric acid,so please pay close attention to safety when it is used.

14. Sample addition should always be done via pipette or similar instrument.Calibrate the instrument prior to running the assay in order to avoid experimental errors.Please add samples to the wells quickly,as it is recommended to control the sample addition time to less than ~5 minutes.You might want to consider multi-pronged pipettes if this helps with the loading time.

15. Do not re-use the adhesive strips.Cut them to size if you only use part of the plate at a time.Always discard after use.

16. New standard curves should be made for every new run of the assay.If the observed concentrations of test samples are too high (OD value of the sample is higher than that of standard well maximum concentration),dilute by a certain factor,and correct for said factor in the end calculations.

17. Samples containing NaN3 cannot be tested due to NaN3 inhibiting the activities of horseradish peroxidase (HRP).

18. When washing plate via plate washer,the volume of buffer injected into each well should be slightly more than 350μl.Make sure the sampling head is not jammed or blocked.Also,if washing by hand,please take care when using an absorbent material to remove excess water – make sure this absorbent material wasn’t used to clean any of the other reagents to prevent contamination.

19. After the coloration reaction termination by Color Reagent C,please read OD within 10 minutes.

20. If duplicate wells were performed,the mean value of the wells should be used.

21. Hemolyzed samples may cause false positive results,so we consider these samples to be incompatible with this kit.

22. During the assay,please try to control the humidity to ~60%.

23. We recommend regularly checking the thermostat and calibration in order to confirm the incubation temperature remains at a stable 37℃.