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Biotin Quantitative Determination ELISA Kit

SKU: orb1946751

Description

Biotin Quantitative Determination ELISA Kit

Research Area

Protein Biochemistry

Images & Validation

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Application Notes
This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with Biotin protein. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Biotin. Next,Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Biotin in the samples is then determined by comparing the OD of the samples to the standard curve.

Key Properties

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ReactivityAll
Sample TypesSerum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.
Assay TypeCompetitive
Assay Time0.75H
Range7.81-500 pg/mL
Sensitivity5.35 pg/mL

Procedure & Performance

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Assay Principle
This kit is based on a competitive ELISA detection method. The microtiter plate is pre-coated with target antigen. During the assay, the target antigen in the sample or standard competes with the immobilized target for binding sites on the biotinylated detection antibody specific to the target antigen. After incubation, excess conjugate and unbound sample or standard are removed by washing. HRP–streptavidin (SABC) is then added to each well and incubated, followed by the addition of TMB substrate solution. The enzymatic reaction is terminated by adding an acidic stop solution, and the color change is measured at 450 nm using a microplate reader. The concentration of the target antigen in the samples is determined by comparison with the standard curve and is inversely proportional to the OD450 value.
Kit Components
1. ELISA Microplate
2. Standards
3. Detection Antibody
4. HRP-Streptavidin Conjugate
5. TMB Substrate
6. Dilution buffers
7. Stop Solution
8. Wash Buffer
9. Plate Sealers
10. Manual
Reagent Preparation
1. Wash Buffer: Prepare the 1X Wash Buffer using distilled water according to the manual.
2. Standard: Perform gradient dilution according to the instructions in the manual.
3. Other Concentrated Reagents: Dilute the concentrated reagents using the Dilution Buffers provided in the kit to 1 X working solutions as instructed in the manual. Always use a clean pipette tip for each different solution.
Assay Procedure
This procedure is for reference only.

After the kit equilibrates to room temperature, add standard or sample to the wells and incubate.
Aspirate and wash the plate for three times.
Immediately add Biotinylated Detection Antibody Working Solution to each well and incubate.
Aspirate and wash the plate for three times
Add Streptavidin-HRP Working Solution to each well and incubate.
Add TMB Substrate Reagent to each well and incubate.
Add Stop Reagent to each well, read the plate at OD 450 nm immediately, and calculate the results.
Materials Required
1. Microplate readers
2. Centrifuge
3. Incubator
4. Automated plate washer
5. Single-channel or multi-channel high-precision pipettes
6. Disposable pipette tips
7. Sterile tubes
8. Eppendorf tubes
9. Absorbent paper
10. Loading slots
Precision
Intra-assay Precision (Precision within an assay): CV% ≤ 10%
Intra-assay precision was evaluated by testing multiple replicates of samples within the same plate.

Inter-assay Precision (precision between assays): CV% ≤ 10%
Inter-assay precision was evaluated by testing samples across different plates.
Calculation of Results
1. Average the duplicate readings for each Standard, Control, and Sample, and subtract the mean optical density of the zero Standard.
2. Construct a standard curve by plotting the target concentration on the y-axis against absorbance on the x-axis and draw a curve through the data points.
3. Determine the sample concentration by substituting the OD450 value into the standard curve. For diluted samples, multiply the calculated value by the corresponding dilution factor.
Curve Fitting Softwares
1. Curve Expert
2. Thermo SkanIt RE
3. SciDAVis
4. LabPlot
5. ……

Storage & Handling

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StorageRefer to the Storage Guidelines in the Manual
Expiration Date6 months from date of receipt.
DisclaimerFor research use only

Alternative Names

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Vitamin H, Coenzyme R, VB7, Vitamin B7, Biopeiderm

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Quality Guarantee

Quality Guarantee

Explore bioreagents carefree to elevate your research. All our products are rigorously tested for performance. If a product does not perform as described on its datasheet, our scientific support team will provide expert troubleshooting, a prompt replacement, or a refund. For full details, please see our Terms & Conditions and Buying Guide. Contact us at [email protected].

Quick Database Links

Gene Symbol
Biotin

Protocol Information

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48 T
$ 310.00
96 T
$ 430.00
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