Precautions for the use of antithrombin III assay kit

The use of the antithrombin III assay kit must strictly follow the following precautions to ensure the accuracy of the test results and operational safety:

1. Preparation before operation

Only for in vitro diagnostic use

The kit is only used for quantitative detection of antithrombin III (AT-III) activity in plasma samples in the laboratory, and is prohibited from being used for other purposes.

Carefully read the instructions

Before operation, it is necessary to fully read the reagent kit instructions, familiarize oneself with the experimental process, reagent composition, and storage conditions, and avoid result deviation caused by improper operation.

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Preparation of reagents and equipment

Reagent balance: Reagents taken out from refrigerated environments (2 ℃~8 ℃) should be equilibrated at room temperature (18 ℃~25 ℃) for 15-30 minutes to avoid sudden temperature changes affecting reagent performance.

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Equipment calibration: Ensure that the enzyme-linked immunosorbent assay (ELISA) reader, washing machine, and other equipment have been calibrated, and the sampler needs to be regularly maintained to avoid cross contamination.

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Self provided items: Prepare high-precision sample dispensers, gun heads, distilled water, and other auxiliary tools according to the requirements of the reagent kit.

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2. Experimental Environment Requirements

Avoid harsh environments

The experiment should be conducted in a clean, non corrosive environment (such as sodium hypochlorite, acid alkali, ether) and dust to prevent contamination of reagents or samples.

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Temperature and humidity control

It is recommended to maintain the experimental environment temperature at 18 ℃~25 ℃ and humidity at 30%~70% to avoid extreme conditions affecting reaction stability.

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III. Sample Processing Standards

Sample Collection and Preservation

Serum Samples: Use test tubes free of pyrogens and endotoxins to avoid cell irritation. After collection, centrifuge at 3000 rpm for 10 minutes to separate serum and red blood cells.

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Plasma samples: anticoagulation with EDTA, citrate or heparin, centrifugation at 3000 rpm for 30 minutes to obtain supernatant.

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Other samples: Cell supernatant needs to be centrifuged at 3000 rpm for 10 minutes to remove particles;

; The homogenized tissue needs to be crushed with physiological saline and centrifuged to obtain the supernatant. Storage conditions: If the sample is not tested immediately, it should be packaged and frozen at -20 ℃ to avoid repeated freezing and thawing. When thawing, it should be evenly and fully thawed at room temperature.

Sample dilution and addition

Dilution operation: The sample to be tested needs to be diluted with sample diluent in proportion (such as 5-fold dilution). When adding the sample, add it to the bottom of the enzyme-linked immunosorbent assay plate well to avoid touching the well wall. Gently shake and mix well.

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High concentration sample processing: If the OD value of the sample exceeds the highest concentration well of the standard, it is necessary to dilute it with a sample diluent by a certain factor (n times) and then retest. The final result should be multiplied by the total dilution factor (x n x 5).

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IV. Reagent Usage Guidelines

Validity and Storage of Reagents

Validity: Reagents should be used within their validity period. Unopened reagent kits should be stored at 2 ° C to 8 ° C, and their validity period is usually 6 to 24 months (refer to the instructions for details).

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Storage after opening: The remaining reagents should be sealed in a timely manner and stored at 2 ° C to 8 ° C to avoid moisture or contamination.

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Reagent reconstitution and dilution

Standard reconstitution: Freeze dried standards need to be diluted with standard diluent according to the instructions, allowed to stand at room temperature for 10 minutes, and repeatedly reversed to aid dissolution, then diluted proportionally to the desired concentration.

. Dilution of washing solution: Concentrated washing solution may precipitate crystals. When diluting, it can be dissolved by heating in a water bath and diluted with distilled water in proportion (such as 30 times) for later use.

Substrate stored away from light: The substrate solution should be stored away from light. Before use, use a sterilized pipette to aspirate the required volume. Discard the remaining substrate and do not pour it back into the original bottle.

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5. Key points of experimental operation

Processing of wrapping strips

After opening the wrapping strip, the remaining part should be sealed in a sealed bag to prevent moisture from affecting the experimental results.

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Sample addition and mixing

Micro pipette nozzle: Do not mix to avoid cross contamination.

. Mixing of samples in the well: Shake and mix evenly to ensure no bubbles are present, otherwise it may affect the absorbance detection.

Washing steps

Thoroughly wash: The washing solution should be filled into each well, but should not be used too vigorously to avoid the formation of bubbles.

. After each wash, shake off the liquid in the hole and finally pat off any remaining liquid in the hole.

Washing machine usage: It is recommended to use a washing machine. If manually washing, it needs to be repeated more than 5 times to ensure thorough washing.

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Reaction condition control

Incubation time and temperature: Strictly control the reaction time and temperature for each step according to the instructions (such as incubating at 37 ℃ for 30 minutes), and the operation should be compact to avoid temperature fluctuations affecting the results.

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Light avoidance reaction: After adding the luminescent substrate, it is necessary to avoid light at room temperature, as strong light may affect the measurement results.

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VI. Safety and Waste Management

Personal Protection

When handling reagents and samples, disposable gloves should be worn, and hands should be washed thoroughly after operation to avoid direct contact with skin or mucous membranes.

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Waste disposal

All samples and used reagent kits should be considered as potential infectious substances, and should be disposed of harmlessly according to local government and national regulations, such as high-pressure sterilization or handed over to professional institutions for disposal.

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7. Special case handling

Crystallization of concentrated washing solution

If crystallization occurs in concentrated washing solution, it can be dissolved by heating in a water bath, and dilution does not affect the experimental results.

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Water sample substances in the wells of the enzyme-linked immunosorbent assay (ELISA) plate

There may be a small amount of water sample substances in the newly opened wells of the ELISA plate, which is a normal phenomenon and will not affect the experimental results.

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Handling of Abnormal Results

If the experimental results are abnormal (such as OD values exceeding the standard curve range), it is necessary to check the sample dilution factor, reagent expiration date, and operating procedures, and if necessary, re experiment.

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