Specification
Sensitivity: 0.188 ng/mL (The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero).
Detection Range: 0.313–20 ng/mL
Specificity: This kit recognizes Biotin. No significant cross-reactivity or interference was observed.
Repeatability: Coefficient of variation is <10%.
Test principle
This kit uses Competitive-method. The microplate provided in this kit has been pre-coated with Biotin. During the reaction, Biotin in the sample or standard competes with a fixed amount of Biotin on the solid phase supporter for sites on the Avidin-HRP specific to Biotin. Excess Avidin-HRP and unbound sample or standard are washed from the plate. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by adding Stop Solution and the color change can be measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Biotin in samples can be calculated by comparing the OD of the samples with the standard curve.
Calculation of results
Average the duplicate readings for each
standard and samples. Plot a four-parameter logistic curve on log-log graph
paper, with standard concentration on the x-axis and OD values on the y-axis.
If samples have been diluted, the
concentration calculated from the standard curve must be multiplied by the
dilution factor. If the OD of the sample surpasses the lower limit of the
standard curve, you should re-test it after appropriate dilution. The actual
concentration is the calculated concentration multiplied dilution factor.
SUMMARY
1. Add 50
μL standard or sample to each well.
2. Immediately add 50 μL Avidin-HRP to each well.
3. Incubate for 30 min at 37℃
4. Aspirate and wash
5 times
5. Add 90 μL
Substrate Reagent. Incubate 15 min at 37℃
6. Add
50 μL Stop Solution. Read at 450 nm immediately.
7. Calculation
of results
Kit
components & Storage
The unopened kit can be stored at 4℃ for 1 month. If the kit is not
used within 1 month, store the items separately according to the following
conditions since the kit is received.
|
Item
|
Specifications
|
Storage
|
|
Micro ELISA
Plate (Dismountable)
|
8×12 or 8×6 *
|
-20℃, 12 months
|
|
Reference
Standard
|
2/1 vial
|
|
Concentrated
Avidin-HRP
|
1 vial,
120 μL/60 μL *
|
-20℃(shading
light), 12 months
|
|
Reference
Standard & Sample Diluent
|
1 vial, 20 mL/12
mL *
|
4℃, 12 months
|
|
HRP Conjugate
Diluent
|
1 vial, 14 mL
|
|
Concentrated
Wash Buffer (25×)
|
1 vial, 30 mL
|
|
Substrate
Reagent
|
1 vial, 10 mL
|
4℃(shading
light)
|
|
Stop Solution
|
1 vial, 10 mL
|
4℃
|
|
Plate Sealer
|
5/3pieces *
|
|
|
Product Description
|
1 copy
|
|
Certificate of Analysis
|
1 copy
|
Note: All reagent bottle caps must be tightened to
prevent evaporation and microbial pollution.
The volume of reagents in partial shipments is a little more than the
volume marked on the label, please use in measuring instead of directly
pouring.
Other supplies required
Microplate
reader with 450nm wavelength filter
High-precision
transferpettor, EP tubes and disposable pipette tips
37°C Incubator, Deionized
or distilled water
Absorbent
paper
