Goat Atrial Natriuretic Peptide (ANP) ELISA Kit
This kit is designed for research use only and not intended for diagnostic or therapeutic purposes.
Principle of Operation
The Goat Atrial Natriuretic Peptide (ANP) ELISA Kit utilizes a double-antibody sandwich immunoassay to measure ANP levels in biological samples. The process involves coating a microplate with goat ANP-specific antibodies, followed by the addition of the sample containing ANP. An HRP-labeled antibody is then introduced, forming an antigen-antibody-enzyme complex. After washing, TMB substrate is added, resulting in a color change that correlates with the concentration of ANP in the sample. The reaction is stopped using a stop solution, and absorbance is measured at 450 nm to determine the concentration via a standard curve.
Kit Composition
- 30× Washing Solution – 20ml × 1 bottle
- Stop Solution – 6ml × 1 bottle
- Enzyme Standard Reagent – 6ml × 1 bottle
- Standard (640 pg/ml) – 0.5ml × 1 bottle
- Enzyme-labeled Plate – 12 wells × 8 strips
- Standard Dilutions – 1.5ml × 1 bottle
- Sample Diluent – 6ml × 1 bottle
- Instructions – 1 copy
- Reagent A – 6ml × 1 bottle
- Color Developer B – 6ml × 1 bottle
- Sealing Film – 2 sheets
- Sealed Bag – 1
Sample Requirements
It is recommended to process samples as soon as possible after collection. If storage is necessary, keep samples at -20°C and avoid repeated freeze-thaw cycles. Avoid using samples containing NaN3, as it may inhibit HRP activity.
Procedure
- Standard Dilution: Prepare serial dilutions of the standard according to the provided instructions.
- Loading: Add 50 μl of standard and 40 μl of sample diluent to each well, then add 10 μl of the sample. Ensure proper mixing without touching the well walls.
- Incubation: Seal the plate and incubate at 37°C for 30 minutes.
- Washing: Wash the plate five times with diluted washing solution and dry thoroughly.
- Add Enzyme: Introduce 50 μl of enzyme-labeled reagent to each well except blank controls.
- Second Incubation: Repeat the incubation step at 37°C for 30 minutes.
- Color Development: Add 50 μl of TMB developer to each well and incubate for 15 minutes at 37°C.
- Stop Reaction: Add 50 μl of stop solution to terminate the reaction and observe color change.
- Measurement: Read the absorbance at 450 nm within 15 minutes of adding the stop solution.
Calculation
Plot a standard curve using the OD values of standards against their concentrations. Use this curve to determine the sample concentration, adjusting for any dilution factor applied during the procedure.
Precautions
- Allow the kit to reach room temperature before use and store unused components properly.
- Diluted washing solution may crystallize; gently warm if needed.
- Use a pipette for accurate measurements and maintain consistent timing.
- Always include a standard curve and consider sample dilution if OD values exceed the range.
- Do not reuse sealing films to prevent cross-contamination.
- Keep substrates away from light and follow all safety protocols.
- Dispose of all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of discrepancies, the English manual takes precedence.
Storage and Shelf Life
Store the kit at 2–8°C. The shelf life is 6 months from the date of manufacture.
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