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
5 mL and 10 mL graduated pipettes
Adjustable single channel micropipette from 5 µL to 1000 µL with disposable tip
Adjustable multi-channel micropipette from 50 µL to 300 µL with disposable tip
Multichannel micropipette storage tank
Beakers, flasks and cylinders required for preparation of reagents
Washing liquid conveying device
Able to operate at 450 nm (620 nm as Optional reference wavelength)
Glass distilled water or deionized water
A statistical calculator with a linear regression program
Analysis.
Assay Time
Less than 4 hours
Reagent Preparation
1. The buffer concentrate should be brought to room temperature and diluted before starting the test procedure.
2. If crystals form in the buffer concentrate, heat gently until the crystals are completely dissolved. See the kit manual for the rest.
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.Calculate the mean absorbance values for each set of replicate standards and samples.
2. Create a standard curve by plotting the mean absorbance of each standard concentration on the ordinate and the human TNFα concentration on the abscissa.
3. To determine the circulating human TNFα concentration for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line onto the standard curve.
4. At the intersection, the vertical line is extended to the abscissa and the corresponding human TNFα concentration is read.
5. The sample has been diluted, so the concentration read from the standard curve must be multiplied by the dilution factor.
Assay Precision
0.31-20.0 pg/mL
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.
Datasheet
