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
96-well ELISA plate reader (microplate spectrophotometer)
ELISA plate washer
(Optional) Microplate shaker
Pipettes and pipettors
Refrigerator
96-well plate
Assay Time
Less than 24 hours
Reagent Preparation
1. Dilute the Coating Antibody 1:100 in carbonate-bicarbonate buffer.
2. Add diluted Coating Antibody to each well.
3. Aspirate Coating Antibody solution and add Blocking Buffer to each well.
4. Aspirate Blocking Buffer and proceed to assay or allow to dry overnight at room temperature.
Assay Procedure
1. For the standard curve, add the standard to the corresponding hole. For sample, add diluted sample to the well.
2. Cover well and incubate at room temperature for 2-4 hours or gently shake at 4°C overnight.
3. Discard the solution and wash it 3-5 times with 1X wash buffer. Use a multi-channel pipette or automatic cleaner to fill each hole with a cleaning buffer. Complete liquid removal at each step is essential for good performance. After the last wash, remove any remaining wash buffer by suction or decanting. Turn the board upside down and drain with a clean paper towel.
4. Add the prepared biotin conjugates into each well.
5. Incubate gently at room temperature for 1-2 hour.
6. Discard the solution. Repeat the washing in Step 3.
7. Add the prepared Streptavidin-HRP solution to each well.
8. Incubate gently at room temperature, shaking for 30-50 minutes.
9. Discard the solution. Repeat the washing in Step 3.
10. Add TMB substrate to each well. The substrate will begin to turn blue.
11. Incubate at room temperature, dark, for 30-45 minutes, shaking gently.
12. Add termination solution to each well. Tap lightly on the sides of the boards to blend. The solution in the hole changes from blue to yellow.
Calculation of Results
Generate the standard curve by plotting the average absorbance obtained for each Standard concentration on the vertical (Y) axis vs. the corresponding bovine IL-4 concentration (pg/mL) on the horizontal (X) axis. Calculate results using graph paper or curve-fitting statistical software. Determine the bovine IL-4 amount in each sample by interpolating from the absorbance value (Y-axis) to bovine IL-4 concentration (X-axis) using the standard curve.
Assay Precision
15.6-1000 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 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.
