Components
Pre-coated 96 well plate
Standard
Assay Diluent concentrate
Biotinylated Detection Antibody
SAV-HRP
Wash Buffer
Chromogen
Stop Solution
Adhesive Plate Covers
Material not included
Microplate reader capable of reading at a wavelength of 450 nm
Variable volume Microdrop Pipettes
Adjustable multi-channel pipette
Reagent reservoir
Bottle or dishwashing systems
Distilled or deionized water
Serological pipettes
Disposable straw Tips
Sample matrix free of endogenous interferon
Assay Time
Less than 4 hours
Reagent Preparation
1. Prepare Wash Buffer
Allow wash buffer concentrate to reach room temperature and mix to redissolve any precipitated salts. Dilute Wash Buffer Concentrate into deionized or distilled water. Store the concentrate and 1X wash buffer in the refrigerator.
2. Prepare the thinner
The test diluent should be diluted with deionized or distilled water before use.
3. Preparation of Biotin Conjugation
Spin the biotin conjugate briefly before use. Gently stir up and down with a pipette. Collect samples in pyrogen/endotoxin-free tubes. If samples cannot be tested immediately after collection, freeze samples. Freeze samples to avoid multiple freeze-thaw cycles.
4. Pre-dilute samples
For different types of samples, different dilution solutions should be used for dilution.
5. Prepare 1X Streptavidin-HRP Solution
Quickly swirl Streptavidin-HRP before use and mix gently up and down with a pipette as a precipitate may form during storage. Do not store the diluted solution for future use.
6. Dilution Standards
Dilute standards using glass or plastic tubes. Simply spin off a vial of lyophilized standards.
Assay Procedure
1. Determine the number of microbars required for the number of samples required for testing and the appropriate number of holes required for running blanks and standards. Measurements should be repeated for each sample, standard sample, blank sample, and optional control sample. Remove the excess microstrip from the holder and store in an aluminum foil bag sealed with a desiccant provided from 2°C to 8°C.
2. Wash the microporous bar twice with washing buffer for each well, and thoroughly suck out the microporous contents between two washes. Allow the wash buffer to sit in the hole for about 10-15 seconds before suction. Be careful not to scratch the surface of the micropores. After the last washing step, empty the holes and tap the microstrip on an absorbant pad or paper towel to remove excess Wash buffer.
3. Standard dilution on microplate: Add calibration dilution to all standard holes in duplicate. Standard for preparation of pipette, in duplicate, in two separate Wells. The contents of the two Wells were mixed by repeated pumping and ejection and transferred to the other two Wells separately. Be careful not to scratch the inner surface of the micropores. Continue this process 6-8 times to create two rows of standard diluents. Discard the contents of the last microhole.
4. Add the calibration diluent in duplicate to the blank well.
5. Add sample thinner to sample well.
6. Add each sample in duplicate to the sample well.
7. Prepare biotin conjugates according to the manual.
8. Add biotin-Conjugate to all Wells.
9. Cover with a sticky film and incubate on a microplate oscillator at room temperature (18°C to 25°C) for 2-3 hours.
10. Prepare Streptavidin-HRP according to the operation manual.
11. Remove the film and empty the hole. Clean the microstrip 6-8 times according to the operation manual.
12. Add diluted Streptavidin-HRP to all the well, including the blank well.
13. Cover with a sticky film and incubate on a microplate oscillator at room temperature (18°C to 25°C) for 1-2 hours.
14. Remove film and empty holes. Clean the microstrip 6-8 times according to the operation manual.
15. Move TMB substrate solution into all Wells.
16. Incubate the micropores at room temperature (18°C to 25°C) for 10-30 minutes. Avoid direct exposure to the light. Color development on the plate should be monitored and the substrate reaction stopped before positive Wells are no longer properly recorded. The ideal chromogenic time period must be determined separately for each assay.
17. Stop the enzyme reaction by rapidly draining the stop solution into each well. It is important to stop the solution distributing quickly and evenly throughout the micropore to completely inactivate the enzyme. The results must be read immediately after adding the termination solution, or within 1 hour if the microstrip is stored at 2°C to 8°C in dark.
18. Read the absorbance of each micropore on a spectrophotometer, using 450 nm as the main wavelength (620 nm as the reference wavelength is optional; 610 nm to 650 nm is acceptable). Use blank holes to blank the reader according to the manufacturer's instructions. Determine absorbance of sample and standard.
Calculation of Results
1. Read the absorbance at 450nm. Read the plate within 2 hours of adding the stop solution.
2. Generate a standard curve using curve fitting software.
3. Read the concentrations of unknown samples and controls from the standard curve. The sample dilution is corrected by an appropriate factor, resulting in a number of values (s) for the sample.
Assay Precision
15.6-1000 pg/mL
Format
Sandwich ELISA Kit
Precaution of Use
All products are supplied for research and laboratory use only.
Handling Advice
All blood components and biological materials should be treated with precautions as potentially hazardous. Strictly follow the management principles of the Centers for Disease Control and Prevention, Occupational Safety and Health Administration for handling and disposing of infectious agents.
Storage
The ELISA Kits are shipped at 2 to 8°C. Upon receipt, store the kits at 2 to 8°C in dark.
Expiry Date
Stability If properly stored, all components are stable for up to 12 months. For expiry dates for the entire kit, see the kit label. The expiration date of each ingredient is shown on the bottle label. The expiration date of a kit ingredient is guaranteed only if the ingredient is properly stored and, in the event of repeated use of an component, the reagent will not be contaminated by the first treatment.
Note
Each production lot of this ELISA kit is quality tested to meet criteria such as sensitivity, specificity, precision and lot-to-lot consistency. See the manual for more information on validation.
