Enzymatic chemiluminescence immunodetection method and reagent kit for human troponin I

Abstract

The invention belongs to the technical field of immunoassay analysis and particularly provides an enzymatic chemiluminescence immunodetection method for a human troponin I. The method comprises the steps of forming solid phase-antibody-antigen-enzyme labelled secondary antibody immune sandwich complexes and detecting the immune sandwich complexes with the method of chemiluminescence. The invention also provides an enzymatic chemiluminescence immunodetection reagent kit for the human troponin I. The detection method and the detection reagent kit are suitable for detecting and analyzing the human troponin I and have the advantages of large detection range, high sensitivity and high specificity.

Description

technical field

[0001] The invention belongs to the technical field of immunoassay and analysis, and specifically provides a human troponin I enzymatic chemiluminescence immunoassay method and kit, which are suitable for enzymatic chemiluminescence detection and analysis of human troponin I. Background technique

[0002] Human troponin I (cTnI) is the master regulatory protein of muscle and is directly involved in calcium-controlled muscle contraction. It accounts for 5% of myofibrillar protein. Calcium is released from the sarcoplasmic reticulum, binds to troponin and changes its structure, and the inhibited actin and myosin interaction begins, and muscle contraction occurs. If the calcium is removed, the troponin returns to its original state and the muscle relaxes. Cardiac troponin consists of three subunits: troponin T (TnT), troponin I (TnI) and troponin C (TnC). Cardiomyocyte damage is inevitable in patients with various coronary artery diseases. The clinical manif...

Examples

Embodiment 1

[0045] Example 1 Preparation of horseradish peroxidase (HRP)-labeled antibody

[0046] Concentrate the anti-human troponin I monoclonal antibody to a concentration of 2 mg / mL. Use the modified sodium iodate method for labeling, the specific steps are:

[0047] (1) Dissolve 5mg of HRP (purchased from Sigma, USA) in 0.5ml of double-distilled water, and add newly prepared 0.06Mol / L NaIO 4 0.5ml aqueous solution, mix well, set at 4°C for 30min;

[0048] (2) Add 0.5ml of 0.16Mol / L ethylene glycol aqueous solution and let stand at room temperature for 30min;

[0049] (3) Add 2.5ml of an aqueous solution containing 5mg of purified anti-human troponin I monoclonal antibody (clone number 9701), mix well, put it into a dialysis bag, and slowly add 0.05Mol / L pH9.5 carbonate buffer Stir and dialyze for 6h (or overnight) to combine;

[0050] (4) Take out the solution in the dialysis bag and add NaBH 4 Solution (5mg / ml) 0.2ml, mix well, set at 4°C, 2h;

[0051] (5) Slowly add an equal...

Embodiment 2

[0053] Example 2 Adsorption immobilization of coated antibody

[0054] Dilute anti-human troponin I monoclonal antibody (clone number 9707) to 2.5 μg / ml with 0.02M, pH7.4 phosphate buffer (PBS solution), add polystyrene microwell plate, 100 μL / well, 4 overnight at ℃, washed three times with washing solution, blocked with 1% bovine serum albumin 300 μL / well at room temperature for 2 hours, and then patted dry for later use.

[0055] The washing liquid preparation method is: sodium chloride (NaCl) 2g, sodium dihydrogen phosphate (NaH 2 P0 4 .12H 2 0) 2.9g, dipotassium hydrogen phosphate (K 2 HP0 4 ) 0.2g, potassium chloride (KCl) 0.2g, Triton X-1000.5mL, Tween-200.5mL, Bronidox-L 5g, make 1000mL with purified water, and use 0.2μm Filter and store at room temperature.

Embodiment 3

[0056] Example 3 Affinity immobilization of coated antibodies

[0057] Dilute avidin with 0.02M, PH7.4 phosphate buffer solution to 1.0 μg / ml, add to a polystyrene microwell plate, 100 μL / well, overnight at 4°C, wash with washing solution (the preparation method is the same as in Example 2) Three times, add biotin-labeled anti-human troponin I antibody (clone number 9707) (for biotin-labeling method, refer to Thermofisher’s Instructions for use of Sulfo-NHS-Biotin, product number is 21217, ), concentration 1.0 μg / ml, 100 μL / well, overnight at 4°C, washed three times with washing solution, blocked with 1% bovine serum albumin 300 μL / well for 2 hours at room temperature Pat dry and set aside.

technical field

[0001] The invention belongs to the technical field of immunoassay and analysis, and specifically provides a human troponin I enzymatic chemiluminescence immunoassay method and kit, which are suitable for enzymatic chemiluminescence detection and analysis of human troponin I. Background technique

[0002] Human troponin I (cTnI) is the master regulatory protein of muscle and is directly involved in calcium-controlled muscle contraction. It accounts for 5% of myofibrillar protein. Calcium is released from the sarcoplasmic reticulum, binds to troponin and changes its structure, and the inhibited actin and myosin interaction begins, and muscle contraction occurs. If the calcium is removed, the troponin returns to its original state and the muscle relaxes. Cardiac troponin consists of three subunits: troponin T (TnT), troponin I (TnI) and troponin C (TnC). Cardiomyocyte damage is inevitable in patients with various coronary artery diseases. The clinical manif...

Examples

Embodiment 1

[0045] Example 1 Preparation of horseradish peroxidase (HRP)-labeled antibody

[0046] Concentrate the anti-human troponin I monoclonal antibody to a concentration of 2 mg / mL. Use the modified sodium iodate method for labeling, the specific steps are:

[0047] (1) Dissolve 5mg of HRP (purchased from Sigma, USA) in 0.5ml of double-distilled water, and add newly prepared 0.06Mol / L NaIO 4 0.5ml aqueous solution, mix well, set at 4°C for 30min;

[0048] (2) Add 0.5ml of 0.16Mol / L ethylene glycol aqueous solution and let stand at room temperature for 30min;

[0049] (3) Add 2.5ml of an aqueous solution containing 5mg of purified anti-human troponin I monoclonal antibody (clone number 9701), mix well, put it into a dialysis bag, and slowly add 0.05Mol / L pH9.5 carbonate buffer Stir and dialyze for 6h (or overnight) to combine;

[0050] (4) Take out the solution in the dialysis bag and add NaBH 4 Solution (5mg / ml) 0.2ml, mix well, set at 4°C, 2h;

[0051] (5) Slowly add an equal...

Embodiment 2

[0053] Example 2 Adsorption immobilization of coated antibody

[0054] Dilute anti-human troponin I monoclonal antibody (clone number 9707) to 2.5 μg / ml with 0.02M, pH7.4 phosphate buffer (PBS solution), add polystyrene microwell plate, 100 μL / well, 4 overnight at ℃, washed three times with washing solution, blocked with 1% bovine serum albumin 300 μL / well at room temperature for 2 hours, and then patted dry for later use.

[0055] The washing liquid preparation method is: sodium chloride (NaCl) 2g, sodium dihydrogen phosphate (NaH 2 P0 4 .12H 2 0) 2.9g, dipotassium hydrogen phosphate (K 2 HP0 4 ) 0.2g, potassium chloride (KCl) 0.2g, Triton X-1000.5mL, Tween-200.5mL, Bronidox-L 5g, make 1000mL with purified water, and use 0.2μm Filter and store at room temperature.

Embodiment 3

[0056] Example 3 Affinity immobilization of coated antibodies

[0057] Dilute avidin with 0.02M, PH7.4 phosphate buffer solution to 1.0 μg / ml, add to a polystyrene microwell plate, 100 μL / well, overnight at 4°C, wash with washing solution (the preparation method is the same as in Example 2) Three times, add biotin-labeled anti-human troponin I antibody (clone number 9707) (for biotin-labeling method, refer to Thermofisher’s Instructions for use of Sulfo-NHS-Biotin, product number is 21217, ), concentration 1.0 μg / ml, 100 μL / well, overnight at 4°C, washed three times with washing solution, blocked with 1% bovine serum albumin 300 μL / well for 2 hours at room temperature Pat dry and set aside.

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