TECHNOLOGY

Standard Curve FAQ

Q1: Why are the standards in ELISA kits made into lyophilized powder instead of pre-diluted liquid standard solutions?

A: The lyophilization process (freeze-drying) dehydrates and freezes protein solutions at low temperatures, which can maximize the preservation of biological activity and extend the shelf life. Lyophilized standards are easier to transport and store at room temperature, avoiding the protein degradation or microbial contamination that may occur in liquid form.

Q2: How long can the prepared standards be stored? Can I use it after 3 days?

A: To make the standard curves, the standards solutions should be freshly prepared and used promptly. The maximum concentration (e.g., 200 pg/mL in the example in this article) of the standards solutions can be stored at 4°C for 7 days or -20°C for 1 month. Please do not freeze-thaw repeatedly. Due to the low content of other concentrations (except the maximum concentration), the analyte will be degradated significantly within 24 hours, so it must be freshly prepared and used immediately.

Q3: What should I do if there are a small amount of undissolved substances after dissolving the standard?

A: Appropriately extend the standing time or perform gentle vortex oscillation again. If the substance remains undissolved, it may indicate that the standard has been inactivated or denatured. It is recommended to contact the supplier for consultation.

Q4: Why does the OD value of the standard curve not matching that in the datasheet? Does this mean the experiment failed?

A: Due to differences in operator habits, experimental conditions, plate washing techniques, and chromogenic termination time (which cannot be completely consistent and accurate), there will be certain differences in the standard curve each time. In practice, as long as the OD value of the maximum concentration in the standard curve is between 1.0 and 3.5, and there are obvious differences in the OD values of all concentration points, the standard curve can be regarded as valid.

Q5: Is it necessary to generate a new standard curve every time? Can I use the standard curve provided in the datasheet or the one created in the previous experiment?

A: Yes, a new standard curve must be created every time. This is because the standard curve is an indicator of whether the current ELISA experiment is successful. It is impossible to determine whether the current experiment is successful if you don’t create the standard curve. It will also cause the false positive or false negative results in sample concentration determination.

Q6: What should I do if the expected concentration of the sample is very high? Or I’m not sure whether the diluted sample concentration will exceed the range of the standard curve or not?

A: The specific dilution factor can be determined through pre-experiments based on literature data, experimental experience, etc., or you can consult the technical support.

Q7: What causes the standard curve have no concentration gradient?

A: The absence of a concentration gradient usually indicates the contamination of samples or standards, incorrect dilution, excessively high conjugate antibody concentration, or prolonged chromogenic time. It is necessary to check the operation process, avoid the contamination and strictly follow the datasheet for dilution.

Q8: What causes a low R² value of the standard curve?

A: A low R² value indicates poor fitting of the standard curve, which may be caused by uneven distribution of data or large experimental errors. You can try to increase the number of concentration gradient, or reduce the operational errors.

Q9: What should I do if the absorbance value of the maximum concentration on the standard curve is too high?

A: It maybe the over-chromogenic reaction, or non-specific signals caused by single-wavelength detection. It is suggested to Control the chromogenic time, and use dual-wavelength detection to reduce the impact of non-specific signals.

Q10: What should I do if the absorbance value of the maximum concentration on the standard curve is too low?

A: It maybe the incomplete dissolution, repeated freeze-thaw cycles, or improper incubation conditions of the standard substances. Please try to avoid above all situations and operate strictly according to the requirements of the datasheet. In addition, chromogenic time and temperature are also crucial, please ensure the appropriate chromogenic time and temperature, or you can try to extend the chromogenic time if necessary.

Standards processing in ELISA experiments is a sophisticated process that combines scientific principles and operational techniques. Any detail may become a key factor that affect the results. Only strictly follow the operating specifications and fully understand the principles, an ideal standard curve will be created, reliable and accurate experimental results will be obtained finally.

Details determine success or failure; precision is the top priority.