As the gold standard for bacterial endotoxin detection, the accuracy of TAL/LAL Reagent test results is directly related to the safety assessment of pharmaceuticals, medical devices, and other products. Since endotoxin detection is highly sensitive to factors such as the operating environment, reagent performance, and sample characteristics, deviations in any link may lead to distorted results. Therefore, establishing a scientific result verification system and judging the accuracy of test results from multiple dimensions is a core link in ensuring test quality.
I. Laying the Foundation for Accuracy through Reagent Performance Verification
The quality of TAL/LAL Reagent itself is a prerequisite for reliable test results, which needs to be verified from three aspects: sensitivity, stability, and specificity:
(I) Sensitivity Recheck
Each batch of TAL/LAL Reagent must undergo sensitivity verification using standard endotoxin (CSE/RSE) before use. In accordance with pharmacopoeial requirements, standard endotoxin is diluted to three concentrations: 0.5λ, 1λ, and 2λ of the reagent's labeled sensitivity (λ). Each concentration is set with 3 replicate tubes, and a negative control is also established. If the gel formation rate at the 1λ concentration is ≥90%, the gel formation rate at the 0.5λ concentration is ≤10%, and no gel formation occurs in the negative control, it indicates that the reagent's sensitivity meets the labeled value and can be used for detection; otherwise, if the results deviate from this range, the reagent needs to be recalibrated or replaced.
(II) Stability Monitoring
The activity of TAL/LAL Reagent is easily affected by storage conditions, so its stability needs to be verified through comparative experiments. Divide the same batch of reagents into two groups: one group is stored under specified conditions (refrigerated at 2-8°C), and the other group is subjected to accelerated destruction treatment (such as placing at 37°C for 24 hours). Then, test their reactivity to standard endotoxin respectively. If the sensitivity of the accelerated treatment group decreases by more than 50% compared with the normal group, it indicates that the reagent's stability is insufficient, which may lead to low test results, and the reagent needs to be discarded.
(III) Specificity Verification
The G factor in TAL/LAL Reagent may react with 1,3-β-D-glucan to produce false positives, so it is necessary to exclude interference through specific experiments. Add 1,3-β-D-glucan (concentration ≥100pg/mL) to the reagent. If no gel formation occurs or there is no significant change in the photometric signal, it indicates that the reagent has good specificity; if a positive reaction occurs, it is necessary to select recombinant TAL/LAL Reagent with G factor removed (such as rFC reagent) for re-testing.
II. Eliminating Systematic Errors through Experimental Environment and Operation Control
Environmental pollutants and non-standard operations are common causes of distorted results, which need to be checked through strict quality control measures:
(I) Environmental Cleanliness Verification
Regularly test the endotoxin contamination level in the experimental environment, which can adopt the "blank control method": add pyrogen-free water to TAL/LAL Reagent. If the blank control shows a positive result, it indicates that there is endotoxin contamination in the operating environment (such as a clean bench, operating table) or experimental instruments. At this time, it is necessary to re-sterilize the instruments by dry heat at 250°C for 30 minutes and disinfect the environment until the blank control result is negative.
(II) Operational Normativity Check
Focus on key operational links such as pipetting accuracy and incubation conditions:
Pipetting error: Use a calibrated pipette and verify the pipetting volume by the gravimetric method (for example, when pipetting 100μL of liquid, the weight should be between 99-101mg) to ensure the accurate ratio of reagent to sample;
Incubation conditions: Use a calibrated constant temperature water bath or incubator, and monitor the temperature (37±1°C) and time (60±2 minutes for the gel method) in real-time to avoid abnormal enzyme activity due to temperature fluctuations.
III. Verifying the Effectiveness of the Detection System through a Control System
Establishing a multi-level control is a core means to judge the accuracy of results, including positive control, negative control, and sample interference control:
(I) Positive Control (PC)
Add standard endotoxin of known concentration (usually 2λ) to TAL/LAL Reagent. If the expected positive result does not appear (such as no gel formation or the photometric signal does not reach the threshold), it indicates that the reagent is invalid or there is an error in the operation (such as improper incubation temperature), and the experiment needs to be repeated.
(II) Negative Control (NC)
Use pyrogen-free water instead of the sample for detection. If a positive result appears, it indicates the presence of exogenous endotoxin contamination (such as reagent contamination, operational cross-contamination), the test result is invalid, and it is necessary to check the pollution source and retry.
(III) Sample Interference Control (PPC)
Add standard endotoxin (concentration 2λ) to the sample and calculate the recovery rate (recovery rate = measured value / theoretical value × 100%). If the recovery rate is within the range of 50%-200%, it indicates that the sample has no obvious interference and the test result is reliable; if the recovery rate is outside this range, it is necessary to eliminate the interference through dilution, pH adjustment, etc., and then re-test. For example, for samples containing high concentrations of EDTA, 0.05M Ca²⁺ can be added to neutralize the interference, and then verify whether the recovery rate meets the standard.
IV. Ensuring Result Consistency through Methodological Comparison and Data Traceability
(I) Comparison of Different Detection Methods
For key samples, two methods with different principles can be used for comparison (such as the gel method and the kinetic turbidimetric method). If the deviation between the test results of the two methods is ≤50%, it indicates that the results have good consistency; if the deviation is too large, it is necessary to analyze the reasons (such as sample matrix interference, reagent sensitivity difference) and take the method with qualified recovery rate verification as the standard.
(II) Data Recording and Traceability
Complete records of key parameters in the detection process, including reagent batch, standard endotoxin concentration, incubation conditions, control results, etc., to form traceable experimental records. At the same time, regularly participate in inter-laboratory comparisons (such as proficiency testing programs). If the test results are within the acceptable range (such as Z value ≤2), it indicates that the laboratory's detection system is stable and the accuracy of the results has been externally verified.
V. Judgment on the Accuracy of Test Results for Special Samples
For complex matrix samples (such as blood products, biological agents), the following points need to be paid extra attention:
Turbidity interference: If the sample itself has turbidity (such as liposome injection), it is necessary to remove particles by centrifugation (10,000×g, 15 minutes) or filtration (0.22μm filter membrane), and then compare the test results before and after treatment to ensure that turbidity does not affect signal reading;
pH impact: If the sample pH is <6.0 or >8.0, it is necessary to adjust it to 6.5-7.5 with pyrogen-free acid-base solution, and then detect the recovery rate before and after adjustment to avoid enzyme activity inhibition caused by abnormal pH.
To judge the accuracy of TAL/LAL Reagent test results, it is necessary to build a verification system from multiple dimensions such as reagent performance, operational specifications, control verification, and method comparison. Only when the reagent sensitivity meets the standard, the control system is effective, the sample has no interference, and the operation process is traceable, the test result is reliable. In actual operation, it is necessary to combine sample characteristics and pharmacopoeial requirements, flexibly use various verification methods, and timely check the causes of abnormal results to provide a scientific basis for product quality control.
