ELISA is one of the most widely used techniques in immunological detection and diagnostic research. The principle and procedure of ELISA are well documented, and results are typically interpreted based on the intensity of color development from the chromogenic substrate. The color reaction usually takes about 10 minutes at 37°C, after which a stop solution is added to terminate the reaction. The absorbance is then measured using a microplate reader. It's important to ensure that the bottom of each well is clean during testing, as any residue or incorrect volume of reagent can affect the final reading.
If all test wells remain colorless, it could indicate issues such as faulty enzyme conjugate, missing or incorrect substrate, or problems with the positive control. To troubleshoot, mix the enzyme conjugate with the substrate and observe if color develops. If it does, the issue may be with the positive control or substrate addition. If no color appears, try using a new batch of enzyme conjugate and substrate to confirm the problem. If the stop solution was added prematurely, washing the plate and repeating the development step may help.
Another common issue is when all wells develop color, which often happens due to an unclean plate or excessive incubation time. Proper washing is essential in ELISA, and following the manufacturer’s instructions carefully can prevent false positives (or false negatives in competitive assays). If the quality control wells are colorless, this might indicate reduced enzyme activity, not necessarily a problem with the substrate or controls. In such cases, repeating the test with a new batch is recommended to avoid missing weak positive results.
The substrate in many domestic ELISA kits consists of two components, A and B, typically hydrogen peroxide and TMB or OPD solutions. These are unstable and can produce color if mishandled. If the solution changes color upon mixing or after a drop, it should be discarded immediately.
Here is a summary of common ELISA issues and their solutions:
| **Description** | **Causes** | **Countermeasures** |
|------------------|------------|---------------------|
| All wells are colorless | Expired reagents, mixed kit components, or missed additions | Check reagent expiration dates and storage conditions; do not mix different kits or batches |
| Positive control fails to develop color | Substrate leakage, incorrect dilution, or improper handling | Ensure proper pipetting and reagent preparation; double-check labels and volumes |
| Weak color or low sensitivity | Poor reagent quality, incorrect incubation time or temperature | Verify reagent integrity, follow incubation guidelines strictly |
| Plate contamination | Unwashed plates or improper washing | Increase wash cycles and extend soaking time |
| Color development too fast or too slow | Incorrect developer volume or timing | Use accurate pipettes and follow precise timing steps |
| Instrument settings incorrect | Mismatched filters or wrong wavelengths | Reset the microplate reader and verify filter compatibility |
Proper storage of the ELISA kit is crucial. Always store reagents according to the manufacturer’s instructions. Following the correct procedures for each step can significantly reduce errors and improve the reliability of your results. By paying attention to detail and maintaining good lab practices, you can ensure more consistent and accurate ELISA outcomes.
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