After verifying the compatibility of HCP ELISA with the sample matrix and finding the minimum required dilution (MRD) of the sample, the next step is to validate the precision of the ELISA method and complete the entire process of ELISA methodological validation required by regulations.

What are precision and repeatability?

Precision refers to the degree of consistency between multiple test results, usually evaluated by the repeatability of the experiment and expressed as the coefficient of variation (% CV).

Calculation formula:% CV=(standard deviation/mean concentration) × 100%

Through this formula, two types of precision can be evaluated:

Intra batch precision: measures the repeatability between different wells within a single experiment. The strict intra batch CV indicates that the sample reading is not affected by the sampling location or operating procedure.

Inter batch precision: measures the repeatability between multiple independent experiments. Ensuring that the inter batch CV is within an acceptable range (usually<15-20%, depending on regulatory requirements) is a key indicator to demonstrate that this method can consistently and stably produce reliable results.

Regulatory requirements

ICH Q2(R1)

Method: Conduct at least 9 measurements at 3 concentration levels, covering the analysis range of the measurement (repeat 3 times at 3 concentrations)

Standard: Not specified.

US FDA

Method: Conduct at least 5 measurements at 3 concentration levels, covering the analysis range of the measurement (repeat 5 times at 3 concentrations)

Standard: The CV% of precision at each concentration level should be within ± 20%, and the CV% of LLOQ should not exceed 25%.

EMA

Method: Conduct at least 5 measurements at 4 concentrations (within LLOQ, three times LLOQ, 30-50% of standard curve concentration, at least 75% of ULL, repeat 5 times at 4 concentrations).

Standard: The intra - and inter batch precision of each concentration level quality control should not exceed 15%, and the CV% of LLOQ should not exceed 20%.

Common problem troubleshooting: Causes of high CV values

If the CV value of the experimental results is higher than the expected range in the reagent kit instructions, it is likely that there is a problem with the operating process or equipment. We recommend that you follow the following steps to troubleshoot and optimize the experiment.

1. Board washing operation

Problem cause:

Excessive and vigorous washing of the plate is a common cause of abnormally high CV values, including the use of automatic washing machines, forceful tapping, and other operations, as it may lead to non-specific dissociation of antigen antibody complexes, resulting in excessively high CV values.

Optimization suggestions:

To achieve optimal repeatability, we strongly recommend the bottle washing and plate washing method, which can ensure that the cleaning time for each hole is basically consistent by adding washing solution in alternating directions. For detailed instructions on washing the board, please refer to the following text.

2. Enzyme linked immunosorbent assay (ELISA) performance

Problem cause:

When there is a significant fluctuation in the detection value in the low absorbance range (such as OD value<0.1), it may indicate a potential problem with the enzyme-linked immunosorbent assay (ELISA). Malfunctions of light sources, monochromators, or filters may result in intermittent signal abnormalities or noise, which are typically not identifiable through conventional calibration procedures, but can have a significant impact on the sensitivity of detection as well as the limit of quantification (LOQ) and limit of detection (LOD). This type of problem often manifests as a standard deviation of 0.020 or lower in OD readings between pores in blank pores or pores containing only non adsorbent liquids.

Optimization suggestions:

For HRP-TMB colorimetric ELISA, it is recommended to use dual wavelength readings (such as detection wavelength of 450nm and reference wavelength of 650nm). By using the dual wavelength reading method, non-specific signals (such as background OD value at 650nm) can be subtracted from the signal value at the detection wavelength (such as 450nm), thereby improving the accuracy and reliability of the detection results. This method can minimize the inter hole differences caused by stains or plastic defects, thereby improving precision and accuracy.

(Note: The OD value of Flat noodles with good quality at the reference wavelength (such as 650nm) is usually very low (about 0.040 on average), and the standard deviation of OD value between holes is very small (about 0.002). If the actual detected OD value is much higher than 0.040 and the standard deviation is greater than 0.004, it indicates that there may be a problem with the enzyme-linked immunosorbent assay (ELISA) reader. ）

3. Reagent contamination

Problem cause:

Cygnus' ELISA kit has a sensitivity of up to pg/mL to ng/mL. Potential high concentrations of analytes in laboratory environments (such as HCP or BSA in culture media and upstream process samples, with concentrations up to mg/mL) can easily contaminate key components in the kit. If the detection results show significant deviations in the absorbance values of individual wells in the well plate, and the deviation data values of the same kit are similar after repetition, it may be due to contamination of the wells, standards, or enzyme-linked antibodies in the well plate.

Optimization suggestions:

Perform ELISA experiments as far away as possible from the upstream sample processing area, avoid cross use of pipettes, clean the operating table and instrument, and use suction heads with filter cartridges as much as possible.

4. Analysts and instruments

Problem cause:

Improper operation, uncalibrated pipettes, or use of suction tips with substandard quality may all have adverse effects on the precision of the test results.

Optimization suggestions:

Inviting another analyst to conduct parallel testing using different equipment in an independent laboratory may help you determine whether the reason for poor precision is due to inexperienced operators or issues with conventional laboratory tools, such as uncalibrated pipettes or poor quality suction tips.

The successful completion of precision and repeatability validation of ELISA methods is the cornerstone of ensuring data reliability and driving project progress. Cygnus provides corresponding QS (Qualification Summary) files for each detection kit. If you have any requirements, please directly contact Cygnus China's general agent, XMJ. If you encounter any problems while using the Cygnus HCP ELISA kit, our technical support team will be dedicated to providing you with solutions and suggestions.

Cygnus Technologies has been dedicated to providing products and analytical methods for the biotechnology and biopharmaceutical industries for over 25 years, with the aim of accelerating the research and development phase and improving product quality. The biotechnology residue assay kit developed and produced by Cygnus is used to detect specific impurities in over 50 different expression systems. Cygnus, as an expert in high-sensitivity analytical techniques for immune testing in biotechnology applications, has been widely recognized by regulatory agencies such as FDA, NMPA, EMA, and over 95% of biopharmaceutical companies worldwide for its products and services.

XMJ as the exclusive distributor of Cygnus in China, has established long-term and stable cooperative relationships with many well-known pharmaceutical companies and CRO/CMO enterprises in China. Over the years, XMJ's products and services have helped many companies accelerate the R&D stage, improve drug quality, purity, and safety, accelerate the optimization of R&D processes, reduce product launch time, and lower QC costs. If you are interested in the above products, please feel free to call the customer service hotline of XMJ at 400-050-4006 or visit the website m.rmb365.cn for more information.