Components
Plate, Standard, Diluent
Material not included
Microplate reader capable of measuring absorbance at 450 nm, with correction wavelength set at 570 nm or 630 nm.
Pipettes and pipette tips.
50 μL to 300 μL adjustable multichannel micropipette with disposable tips.
Multichannel micropipette reservoir.
Beakers, flasks, cylinders necessary for preparation of reagents.
Deionized or distilled water. Polypropylene test tubes for dilution.
Assay Procedure
1.Add the Standard working solution to the first two columns: Each concentration of the solution is added in duplicate, to one well each, side by side (100 μL for each well).Cover the plate with the sealer provided in the kit. Incubate for 90 min at 37 °C.
2.Remove the liquid out of each well, do not wash. Immediately add 100 μL of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently mix up. Incubate for 1 hour at 37 °C.
3.Aspirate or decant the solution from each well, add 350 μL of wash buffer to each well. Soak for 1~2 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times.
4.Add 100 μL of HRP Conjugate working solution to each well. Incubate for 30 min at 37 °C.
5.Aspirate or decant the solution from each well, repeat the wash process for five times as conducted in step 3.
6.Add 90 μL of Substrate Reagent to each well. Cover with a new plate sealer. Incubate for about 15 min at 37 °C. Protect the plate from light.
7.Add 50 μL of Stop Solution to each well.
8.Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.
Calculation of Results
Average the duplicate optical density readings for each standards and sample, then subtract the average optical density value of the zero standard. Standard Concentration as horizontal axis, optical density (OD) Value as the vertical axis, regressing the data and create a standard curve using computer software.
Assay Precision
Intra-assay precision (precision within an assay): Three serum-based and buffer-based samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay precision (precision between assays): Three serum-based and buffer-based samples of known concentration were tested in six separate assays to assess inter-assay precision.
Precaution of Use
This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice
The kit should not be used beyond the expiration date on the kit label.
Do not mix or substitute reagents with those from other lots or sources.
If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.
Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time/temperature and kit age can cause variation in binding.
This assay is designed to eliminate interference by soluble receptors, binding proteins and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Storage Comment
Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2 to 8 °C).
Opened/Reconstituted Reagents: Up to 1 month at 2 - 8 °C.
Note
Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2 to 8 °C).
Opened/Reconstituted Reagents: Up to 1 month at 2 - 8 °C.
Restrictions
For Research Use only