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.
Reagent Preparation
Make a 1:100 dilution of the concentrated Detect Antibody solution with Assay Buffer (1x) in a clean plastic tube as needed. The diluted Detect Antibody should be used within 30 minutes after dilution. Streptavidin-HRP Mix well prior to making dilutions. Make a 1:100 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1x) in a clean plastic tube as needed. The diluted Streptavidin-HRP should be used within 30 minutes after dilution.
Assay Procedure
1.Add 300 μL Washing Buffer (1x) per well, and allow it for about 30 seconds before aspiration.
2.Add 50 μL of Assay Buffer (1x) to each well.
3.Add 50 μL of Standard or sample to each well.
4.Add 50 μL of Detect Antibody to each well.
5.Seal the plate with an adhesive film. Incubate at room temperature (18 to 25 °C) for 2 hours on a microplate shaker set at 100 rpm.
6.Aspirate each well and wash by filling each well with 300 μL Washing Buffer (1x), repeat five times for a total six washes.
7.Add 100 μL of Streptavidin-HRP to each well.
8.Seal the plate with a fresh adhesive film. Incubate at room temperature (18 to 25 °C) for 45 minutes on a microplate shaker set at 100 rpm.
9.Repeat aspiration/wash as in step 8.
10.Add 100 μL of Substrate Solution to each well. Incubate for 10 - 30 minutes at room temperature. Protect from light.
11.Add 100 μL of Stop Solution to each well.
12.Measure the optical density value within 30 minutes by microplate 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).
Note
Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2 to 8 °C).
Restrictions
For Research Use only
Datasheet