Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof

Abstract

The invention relates to a free thyroxine nanometer magnetic particle chemiluminescence assay kit and a preparation method thereof and a detection method thereof. The free thyroxine nanometer magnetic particle chemiluminescence assay kit comprises solutions containing fluorescein-labeled free thyroid antibodies, suspension liquid of magnetic particles covered with fluorescein-resistance antibodies and solutions containing alkaline-phosphatase-labeled free thyroxine antigens, wherein the alkaline-phosphatase-labeled free thyroxine antigens are formed by the connection of alkaline phosphatase and the free thyroxine antigens through crosslinking agent suberic acid succinimide ester. The free thyroxine nanometer magnetic particle chemiluminescence assay kit and the preparation method thereof and the detection method thereof have the advantage of enabling the free thyroxine to be quantificationally detected on the condition of lower cost, higher accuracy and higher precision.

Description

Technical field

[0001] The invention belongs to the field of biological technology, and specifically relates to a free thyroxine nano-magnetic particle chemiluminescence determination kit combining immunomagnetic particle separation technology and nano-magnetic particle chemiluminescence determination technology and a preparation method thereof. The invention also particularly relates to the preparation method of the kit. Background technique

[0002] Thyroxine, or 3,5,3',5'-tetraiodothyronine (T4), is a thyroid hormone with a molecular weight of 777. There are two forms of T4 in the cycle: bound state and free state. Under normal circumstances, there is a dynamic balance between the two forms. In circulation, 75% of T4 is bound to thyroid binding globulin (TBG), 15% is bound to prealbumin, and 10% is bound to albumin. Free T4 (FT4) only accounts for 0.03%, but it plays a real physiological role. When pregnancy, hepatitis, increased congenital TBG, and taking estrogen (oral co...

Examples

Embodiment 1

[0037] Example 1 Preparation of the first reagent

[0038] (1) Materials and equipment: anti-FT4 monoclonal antibody stored in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC), sodium bicarbonate and other reagents should reach the chemical Pure; G-25 gel purification column was purchased from GE.

[0039] (2) Preparation steps:

[0040] ① Prepare 0.5 mg / mL FITC solution with 0.1~0.2mol / L carbonate buffer with pH 9.0~10.0;

[0041] ②Add the FITC solution prepared in step ① to the antibody solution according to the ratio of FT4 antibody to FITC molecule ratio of 1:20, mix well, and let stand at room temperature for 12 hours to react to generate FT4 antibody-FITC conjugate;

[0042] ③The reaction solution after step ② is separated by a G-25 gel column to remove unreacted FITC to obtain a solution containing FT4 antibody-FITC conjugate (ie FITC-labeled FT4 antibody);

[0043] ④Dilute the solution containing FT4 antibody-FITC conjugate obtained i...

Embodiment 2

[0044] Example 2 Preparation of the second reagent

[0045] (1) Materials and equipment: FT4 antigen (solid powder, purity more than 95%); alkaline phosphatase stored in phosphate buffer (ALP solution, ALP purity is about 99%, specific activity is about 1500 U / mg, and the concentration is 10mg / mL); coupling agent DSS was purchased from THERMO, chemical reagents such as TRIS should be chemically pure; G-25 gel purification column is a product of GE.

[0046] (2) Preparation steps:

[0047] ①Take 1 mg of FT4 antigen, add DMSO to dissolve the antigen to a concentration of 20-50 mg / mL, add DSS 0.5 mg, react at room temperature for 2 hours, dilute the reaction solution 1:10 with DMSO, and store at 2-8°C for later use;

[0048] ②Take 1mg of ALP solution and use 0.1M pH9.5 NaHCO 3 Buffer dilute the ALP solution to 1mg / mL, add the FT4-DMSO solution prepared in step ① to the diluted ALP buffer for the ligation reaction, add the FT4-DMSO solution volume to 1 / 20 of the volume of the ALP buffer, ...

Embodiment 3

[0050] Example 3 Preparation of magnetic separation reagent

[0051] (1) Materials and instruments:

[0052] Magnetic particle suspension: magnetic particle content 5wt%, magnetic particle contains carboxyl (COOH) active group, carboxyl content per gram (g) magnetic particle (dry weight) is not less than 0.4 millimoles (mmol), with super paramagnetism, The diameter is between 0.5-2μm

[0053] Anti-FITC antibody: It can be a polyclonal antibody or a monoclonal antibody, with a purity of more than 90wt%, and a dilution titer of more than 1:1 million;

[0054] 2-Morpholine ethanesulfonic acid (MES), carbodiimide (EDC), TRIS and other reagents should be chemically pure.

[0055] (2) Preparation steps:

[0056] ① Take a suspension of 100 mg of magnetic particles, magnetically separate the supernatant, and resuspend in 10 mL of 0.05 mol / L, pH 4.5-5 MES buffer;

[0057] ②Add 2~4mg of anti-FITC antibody and suspend at room temperature for 30~60min;

[0058] ③Add 0.5~1mL of a freshly prepared 10mg...

Technical field

[0001] The invention belongs to the field of biological technology, and specifically relates to a free thyroxine nano-magnetic particle chemiluminescence determination kit combining immunomagnetic particle separation technology and nano-magnetic particle chemiluminescence determination technology and a preparation method thereof. The invention also particularly relates to the preparation method of the kit. Background technique

[0002] Thyroxine, or 3,5,3',5'-tetraiodothyronine (T4), is a thyroid hormone with a molecular weight of 777. There are two forms of T4 in the cycle: bound state and free state. Under normal circumstances, there is a dynamic balance between the two forms. In circulation, 75% of T4 is bound to thyroid binding globulin (TBG), 15% is bound to prealbumin, and 10% is bound to albumin. Free T4 (FT4) only accounts for 0.03%, but it plays a real physiological role. When pregnancy, hepatitis, increased congenital TBG, and taking estrogen (oral co...

Examples

Embodiment 1

[0037] Example 1 Preparation of the first reagent

[0038] (1) Materials and equipment: anti-FT4 monoclonal antibody stored in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC), sodium bicarbonate and other reagents should reach the chemical Pure; G-25 gel purification column was purchased from GE.

[0039] (2) Preparation steps:

[0040] ① Prepare 0.5 mg / mL FITC solution with 0.1~0.2mol / L carbonate buffer with pH 9.0~10.0;

[0041] ②Add the FITC solution prepared in step ① to the antibody solution according to the ratio of FT4 antibody to FITC molecule ratio of 1:20, mix well, and let stand at room temperature for 12 hours to react to generate FT4 antibody-FITC conjugate;

[0042] ③The reaction solution after step ② is separated by a G-25 gel column to remove unreacted FITC to obtain a solution containing FT4 antibody-FITC conjugate (ie FITC-labeled FT4 antibody);

[0043] ④Dilute the solution containing FT4 antibody-FITC conjugate obtained i...

Embodiment 2

[0044] Example 2 Preparation of the second reagent

[0045] (1) Materials and equipment: FT4 antigen (solid powder, purity more than 95%); alkaline phosphatase stored in phosphate buffer (ALP solution, ALP purity is about 99%, specific activity is about 1500 U / mg, and the concentration is 10mg / mL); coupling agent DSS was purchased from THERMO, chemical reagents such as TRIS should be chemically pure; G-25 gel purification column is a product of GE.

[0046] (2) Preparation steps:

[0047] ①Take 1 mg of FT4 antigen, add DMSO to dissolve the antigen to a concentration of 20-50 mg / mL, add DSS 0.5 mg, react at room temperature for 2 hours, dilute the reaction solution 1:10 with DMSO, and store at 2-8°C for later use;

[0048] ②Take 1mg of ALP solution and use 0.1M pH9.5 NaHCO 3 Buffer dilute the ALP solution to 1mg / mL, add the FT4-DMSO solution prepared in step ① to the diluted ALP buffer for the ligation reaction, add the FT4-DMSO solution volume to 1 / 20 of the volume of the ALP buffer, ...

Embodiment 3

[0050] Example 3 Preparation of magnetic separation reagent

[0051] (1) Materials and instruments:

[0052] Magnetic particle suspension: magnetic particle content 5wt%, magnetic particle contains carboxyl (COOH) active group, carboxyl content per gram (g) magnetic particle (dry weight) is not less than 0.4 millimoles (mmol), with super paramagnetism, The diameter is between 0.5-2μm

[0053] Anti-FITC antibody: It can be a polyclonal antibody or a monoclonal antibody, with a purity of more than 90wt%, and a dilution titer of more than 1:1 million;

[0054] 2-Morpholine ethanesulfonic acid (MES), carbodiimide (EDC), TRIS and other reagents should be chemically pure.

[0055] (2) Preparation steps:

[0056] ① Take a suspension of 100 mg of magnetic particles, magnetically separate the supernatant, and resuspend in 10 mL of 0.05 mol / L, pH 4.5-5 MES buffer;

[0057] ②Add 2~4mg of anti-FITC antibody and suspend at room temperature for 30~60min;

[0058] ③Add 0.5~1mL of a freshly prepared 10mg...

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