Introduction to Antithrombin III Assay Kit

Introduction to Antithrombin III Assay Kit

I. Product Definition and Application

Antithrombin III Assay Kit is an in vitro diagnostic reagent used for quantitative detection of antithrombin III (AT-III) activity in plasma. AT-III is one of the most important anticoagulant substances in the human body, which maintains coagulation balance by inhibiting the serine protease activity of thrombin and active coagulation factors (such as IX, X, XI, XII). Its function accounts for about 70% of the total activity of the anticoagulant system. This kit assists in evaluating coagulation status, screening for congenital AT-III deficiency, reducing the risk of postoperative venous thrombosis and pulmonary embolism, and monitoring the effectiveness of anticoagulant therapy by detecting AT-III activity.

II. Detection Principle

The reagent kit mainly adopts the following two technical principles:

Chromogenic substrate method: thrombin catalyzed cleavage to synthesize substrates (such as H-D-HHT-L-Ala-L-Arg-pNA · 2AcOH), releasing p-nitroaniline (PNA), and monitoring the absorbance change at 405nm wavelength.

. The absorbance value is inversely proportional to AT-III activity, and the concentration of AT-III in the sample is calculated using a standard curve. Enzyme linked immunosorbent assay (ELISA): Using a double antibody one-step sandwich method, the sample, standard, and HRP labeled detection antibody are added in sequence. After incubation and washing, the substrate TMB is used for color development. The color depth is positively correlated with the AT-III antibody content in the sample, and the concentration is calculated by measuring the absorbance (OD value) at 450nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader.

III. Product Composition

The reagent kit usually contains the following core components:

Thrombin reagent: such as 25nCat freeze-dried bovine thrombin, used to catalyze substrate reactions.

. Thrombin substrates, such as H-D-HHT-L-Ala-L-Arg-pNA · 2AcOH, are used as reaction chromogenic substances. Dilution buffer: containing Tris HCl, NaCl, NaEDTA, heparin sodium and other components, used to adjust the pH and ionic strength of the reaction system. Other auxiliary reagents, such as stop buffer, chromogenic reagent, washing buffer, etc., are used to control the reaction process or clean non-specific binding substances.

IV. Specifications and Models

Different manufacturers produce reagent kits with different specifications. Common combinations include:

Thrombin reagents: 4 × 2mL, 6 × 5mL, 6 × 15mL, etc.

. Thrombin substrates: 4 × 2mL, 3 × 3mL, 6 × 3mL, etc. Dilution buffer: 4 × 5mL, 1 × 30mL, 1 × 100mL, etc. Users need to choose appropriate specifications based on experimental needs and instrument compatibility.

Fifth, Precautions for Use

Operating Environment: Avoid using in environments containing corrosive vapors and dust such as sodium hypochlorite, acid alkali, or ether to prevent reagent deterioration.

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Sample processing:

Serum sample: After being left at room temperature for 2 hours or overnight at 4 ℃, centrifuge at 1000g for 20 minutes and collect the supernatant.

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Plasma samples: Anticoagulate with EDTA or heparin, centrifuge at 2-8 ℃ and 1000g for 20 minutes within 30 minutes after collection.

. All samples should avoid repeated freezing and thawing, and hemolytic specimens should not be tested.

Experimental procedure:

After opening the wrapping strip, the remaining part needs to be sealed and stored to prevent moisture.

. Micro pipette tips should not be mixed to prevent cross contamination. The sample inside the hole should be shaken and mixed evenly to avoid the presence of bubbles.

The washing should be thorough, and the washing solution should be filled into each hole but avoid excessive force. It is recommended to use a washing machine.

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Safety precautions: Wear disposable gloves when handling reagents and samples, and wash hands thoroughly after operation.

. All waste must be treated as potentially infectious substances and comply with local regulations. Storage conditions: The reagent should be stored at 2-8 ℃ to avoid freezing. After opening, any remaining reagents should be promptly sealed and used within their expiration date.

Reaction control: After adding the luminescent substrate, the reaction should be carried out at room temperature in the dark, as strong light may affect the results.

. The reaction time and temperature for each step should be strictly controlled, and the operation should be compact.

Sixth, Clinical Significance

AT-III activity testing is an important indicator for evaluating coagulation function, and its clinical applications include:

Screening for thrombophilia: Congenital AT-III deficiency is one of the risk factors for hereditary thrombophilia, and early detection can reduce the risk of thrombotic events.

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Disease diagnosis: Secondary AT-III deficiency is common in liver and gallbladder diseases, disseminated intravascular coagulation (DIC), and heparin treatment. A decrease in AT-III levels has diagnostic value.

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Treatment monitoring: AT-III activity testing can guide dosage adjustment and efficacy evaluation in antithrombin replacement therapy or heparin anticoagulant therapy.

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