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
Distilled or deionized water
Microtiter Plate Reader software capable of measuring at 450 nm
Dishwasher - Automatic or Manual (Dispenser)
Calibrate adjustable precision pipettes and glass or plastic tubes to dilute solutions
Assay Time
Less than 4 hours
Reagent Preparation
1. Collect samples in pyrogen/endotoxin-free tubes.
2. If the sample cannot be tested immediately after collection, freeze the sample. Freeze samples to avoid multiple freeze-thaw cycles. Thaw completely and mix well (do not vortex) prior to analysis.
3. Avoid using hemolytic or blood lipid serum. If a large amount of particulate matter is present in the sample, centrifuge or filter the sample prior to analysis.
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
1. Read the absorbance at 450nm. Read the plate within 3 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
16-1690 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.
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.
Datasheet