In the field of in vitro diagnostics (IVD), Although the development of IVD test kits is fraught with difficulties and involves numerous complex links, from laboratory performance validation to performance optimization, every step is related to the quality and clinical application effectiveness of the kit. But through strict laboratory performance verification, multiple aspects of sensitivity and specificity are checked, and targeted performance optimization is carried out, including the optimization of biological raw materials and reagent formulas. Experimenters can improve the quality of the reagent kit to better meet clinical needs.

(Biological buffer Tris)

Room performance verification

1. Sensitivity evaluation

Determination of detection limit: By detecting a series of standard samples with gradually decreasing concentration gradients, the low target concentration that the reagent kit can reliably detect is determined, which is the detection limit. The determination method is to perform multiple tests on blank samples or low concentration samples, and calculate the average and standard deviation of the detection signal. Generally, the concentration or amount corresponding to the mean of the blank sample signal plus three times the standard deviation is used as the low detection limit. Usually, at least 10-20 repeated experiments are used to calculate the average and standard deviation of the concentration, ensuring the accuracy and reliability of the detection limit. The low detection limit reflects the ability of the reagent kit to detect low concentration samples.

2. Functional sensitivity: Functional sensitivity refers to the low analyte concentration that a detection system can reliably detect under specific precision requirements (usually expressed as coefficient of variation (CV)). It considers the repeatability and reliability of the detection results, which can better reflect the performance of the detection method in practical applications. The determination method is generally to perform multiple repeated tests on samples of different concentrations, calculate the precision (CV) of each concentration level, and use the low concentration at which the imprecision meets the specified requirements (such as CV ≤ 20%) as the functional sensitivity. When detecting the level of a certain hormone in the blood, functional sensitivity can inform doctors at what concentration the test results may have significant errors, which requires careful interpretation. It is important for clinical diagnosis to judge the reliability of the test results and determine the scope of application of the testing method.

3. Low concentration sample testing: In addition to determining the detection limit, extensive testing of low concentration samples close to the detection limit concentration is also required. Prepare multiple samples with concentrations slightly above the detection limit, test the number of samples that can correctly present positive results, and calculate the positive detection rate. Ideally, the positive detection rate should be above a specific standard (such as 80% -90%) to demonstrate the effectiveness of the reagent kit in detecting low concentration samples.

Specific evaluation

1. Cross reaction detection: Select interfering substances that are structurally similar to the target or may exist in actual detection scenarios, and prepare samples containing these substances for detection. If the detection line of the test kit does not show color (i.e. does not produce a positive signal) in these sample tests, it indicates that the test kit has good specificity and no cross reaction has occurred. It is usually required to test for possible interfering substances.

2. Clinical sample validation: Collect a large number of clinical samples, including positive samples diagnosed with the target disease and negative samples from individuals with similar or healthy conditions. For the cancer biomarker detection kit, collect at least 100 samples from patients diagnosed with a certain type of cancer through pathology, as well as samples from patients with other non cancer conditions and healthy individuals undergoing physical examinations. By testing these samples, the true positive rate (the proportion of actual positive samples detected as positive) and true negative rate (the proportion of actual negative samples detected as negative) are calculated to evaluate the specificity of the reagent kit in clinical practice.

performance optimization

1. Optimization of biological raw materials

Antibody/antigen screening: If the kit is based on the principle of immunochromatography, when the clinical results are not ideal, antibodies or antigens with higher affinity and specificity can be re screened. Prepare more monoclonal antibodies through phage display technology or hybridoma technology, and then perform affinity assays on these antibodies, selecting antibodies with lower affinity constants (KD values) to improve the sensitivity and specificity of the kit.

Improvement of nucleic acid probe design: For nucleic acid chromatography based reagent kits, optimize the design of nucleic acid probes. Adjust the length, base sequence, and modification method of the probe to improve its hybridization efficiency and specificity with the target nucleic acid. Add special modifying groups (such as fluorescent groups, biotin, etc.) to the 5 'or 3' end of the probe to enhance the detection signal or facilitate subsequent signal capture.

2. Optimization of buffer reagent formula

Involving point membrane solution, labeling solution, binding pad solution, sample pad solution, etc. Change the pH value, ionic strength, and composition of the buffer to optimize the activity of bioactive materials and markers. In immunochromatographic reagents, adjusting the pH value of the buffer from 7.4 to 7.2 may make the binding of antibodies and antigens more stable, thereby improving detection sensitivity. Determine the ideal buffer conditions by experimentally testing the effects of different buffer formulations on the performance of the reagent kit.

Product packaging

Hubei Xindesheng, as a manufacturer of IVD reagent raw materials, sells products such as biological buffering agents, color reagents and luminescent reagents, enzyme preparations, antigen antibodies, etc., all of which are of analytical grade with low impurities and interference, suitable for various detection kits. If you have any purchasing needs, please feel free to contact us!