Components
Capture Antibody
Detection Antibody
Standard
IOCyto Detect™10X Coating Buffer
IOCyto Detect™5X ELISA/ELISPOT Diluent
Enzyme
Substrate
Certificate of Analysis
96 Well Plate
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 24 hours
Reagent Preparation
1. Coated buffer (1X)
Dilute PBS (10X) at 1:10 in deionized water.
2. Capture antibody
Antibodies (250X) were captured in the coated buffer (1X) with 1:250 dilution.
3. 5X ELISA/ELISPOT dilution
Dilute a concentrate (5X) of 1:5 in deionized water.
4. Standard
The protein standard was reconstructed by adding distillate water. The redissolved volume is indicated on the label of the standard bottle. Let the standard redissolve for 10-30 minutes. Swirl or mix gently to ensure complete uniform dissolution.
5. Detect antibody
Detection of antibodies in 1:250 diluted ELISA/ELISPOT solution (250X).
6. Enzyme
Dilute HRP concentrate with 1:100 in ELISA/ELISPOT diluted solution (100X).
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
Plates were read at 450 nm. If there is wavelength subtraction, subtract 570nm from the 450nm value and analyze the data.
Assay Precision
4-500 pg/mL
Format
Uncoated 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.
Some components may need storage at -20°C.
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.
