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 color intensity of the chromogenic substrate. The color reaction usually takes place at 37°C for about 10 minutes, after which a stop solution is added to terminate the reaction. The absorbance is then measured using a microplate reader. Since the optical path of each well is perpendicular to the reader, it's essential to keep the bottom of each well clean and ensure accurate volumes of both the substrate and stop solution are used, as any variation can affect the final readings. Below are some common issues encountered during ELISA experiments and solutions to address them.
All test wells remain colorless. This may be due to issues with the enzyme conjugate or the substrate, such as failure, leakage, or incorrect addition. A common approach to identify the cause is to mix the enzyme conjugate with the substrate and observe if color develops. If it does, the issue might be with the positive control or an incorrect substrate addition. If no color appears, try using a new enzyme conjugate and reagent to see if the problem persists. If the color doesn’t develop, it could indicate that the substrate has failed. In cases where the stop solution was mistakenly added, washing the plate and repeating the development step may help.
If all wells show color, this is often due to residual unbound enzyme conjugate from improper washing or an extended incubation time. Washing is a critical step in ELISA, and it should strictly follow the protocol. If necessary, you can extend the soaking time or increase the number of washes by 1-2 times. Failure to do so may lead to false positives (or false negatives in competitive methods).
When quality control wells are colorless, it may indicate a decrease in enzyme conjugate activity, assuming the substrate and controls were correctly used. Quality control samples are essential for determining the success of the test. Even if the positive control shows color, a significant change in absorbance may suggest the need for repeating the batch to avoid missing weak positive results.
The substrate in domestic ELISA kits is typically a mixture of two components: a hydrogen peroxide solution and either TMB or OPD. These are unstable and can develop color if mishandled. If the solution changes color upon mixing or even after a single drop, it’s likely degraded or contaminated and should be discarded immediately.
**Common Problems in ELISA Experiments**
| Description | Causes | Abnormal Results | Countermeasures |
|-------------|--------|------------------|-----------------|
| All wells are colorless | Expired reagents, mixed kit components, or misaddition | No color in positive control | Check reagent expiration, lot numbers, and confirm correct kit components. Avoid mixing different kits or batches. |
| Substrate or developer leakage | Improper handling or pipetting | Color not developed | Follow manual steps carefully. Ensure liquid levels are consistent after adding reagents. |
| Contaminated plates | Residual enzyme conjugate | False positives | Wash plates thoroughly and follow washing instructions. |
| Stop solution mistaken for diluent | Incorrect labeling or use | Inaccurate reading | Always double-check labels and use distilled water for preparation. |
| Weak color or low sensitivity | Expired reagents, improper storage | Low signal | Check reagent expiry and storage conditions. Store kits as instructed. |
| Uneven reagent temperature | Reagents stored at wrong temperature | Inconsistent results | Allow reagents and samples to equilibrate to room temperature before use. |
| Inaccurate pipetting | Poor technique or dirty tips | Inconsistent readings | Calibrate pipettes and ensure proper tip fitting. |
| Inactive enzyme | Contaminated buffer or poor washing | No color | Use buffers with inhibitors like sodium azide and ensure clean containers. |
| Incubation time/temperature not met | Incorrect settings | Incomplete reaction | Maintain 37–38°C and follow incubation time guidelines. Avoid opening the incubator. |
| Over-washing or incorrect dilution | Too many washes or wrong concentration | Low signal | Dilute washing solution as instructed and reduce washing force. |
| Insufficient or improperly added developer | Wrong order or speed | Incomplete reaction | Add A first, then B, and mix thoroughly. |
| Poor quality distilled water | Contaminated water | Inconsistent results | Test distilled water for purity and use only when confirmed safe. |
| Weak color in controls | Sample lacks strong positives | Unclear results | Retest or add preservatives like sodium azide to prevent degradation. |
| Sample degradation | Repeated freezing/thawing | Inconsistent results | Store samples properly and avoid multiple freeze-thaw cycles. |
| Instrument misconfiguration | Incorrect filter or settings | Inaccurate readings | Reset microplate reader and verify filter compatibility. |
Proper storage of the ELISA kit and strict adherence to operating procedures are essential to avoid common experimental errors. Following these guidelines will help ensure accurate and reliable results in your ELISA experiments.
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