Components
Assay plate (12 × 8 coated Microwells)
Standard (freeze dried)
Biotin-antibody (100 × concentrate)
HRP-avidin (100 × concentrate)
Biotin-antibody Diluent
HRP-avidin Diluent
Sample Diluent
Wash Buffer (25 × concentrate)
TMB Substrate
Stop Solution
Adhesive Strip (for 96 wells)
Instruction manual
Material not included
Microplate reader capable of measuring absorbance at 450nm, with the correction wavelength set at 540nm or 570nm.
An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.
Squirt bottle, manifold dispenser or automated microplate washer.
Absorbent paper for blotting the microtiter plate.
100mL and 500mL graduated cylinders.
Deionized or distilled water.
Pipettes and pipette tips.
Test tubes for dilution.
Reagent Preparation
Biotin-antibody (1×) - Centrifuge the vial before opening.
HRP-avidin (1×) - Centrifuge the vial before opening.
Wash Buffer (1×) - If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved.
Standard - Centrifuge the standard vial at 6000-10000rpm for 30s.
Assay Procedure
1.Add 100µL of standard or sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37°C.
2.Remove the liquid of each well, don't wash.
3.Add 100µL of Biotin-antibody (1×) to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37°C.
4.Aspirate each well and wash, repeating the process two times for a total of three washes.After the last wash, remove any remaining wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5.Add 100µL of HRP-avidin (1×) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
6.Repeat the aspiration/wash process for five times as in step 6.
7.Add 90µL of TMB Substrate to each well. Incubate for 20 minutes at 37°C. Protect from light.
8.Add 50µL of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
9.Determine the optical density of each well within 5 minutes using a microplate reader set to 450nm.
Calculation of Results
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit.
Assay Precision
Intra-assay precision (precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess precision.
Inter-assay precision (precision between assays): Three samples of known concentration were tested in twenty assays to assess precision.
Intra-assay: CV% less than 8%
Inter-assay: CV% less than 10%
Precaution of Use
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.
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
May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.
Note
May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.
Restrictions
For Research Use only
