Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof
A technology of nano-magnetic particles and chemiluminescence, which is applied in chemiluminescence/bioluminescence, analysis and measurement devices through chemical reactions of materials, and can solve problems such as poor process stability, limited detection effect, and cumbersome preparation process , to achieve cost advantages, simple and time-saving operation, and high sensitivity
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Embodiment 1
[0053] Example 1 Preparation of the first reagent
[0054] (1) Materials and equipment: Carbohydrate-related antigen CA19-9 (CA19-9) monoclonal antibody stored in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC) ), sodium carbonate and other reagents should be chemically pure; G-25 gel purification column is purchased from GE.
[0055] (2) Preparation steps:
[0056] ① Prepare 0.5mg / mL FITC solution with 0.1~0.2mol / L carbonate buffer with pH 9.0~10.0;
[0057] ②According to the ratio of CA19-9 antibody to FITC molecule ratio of 1:20, add the FITC solution prepared in step ① to the antibody solution, mix well, and let stand at room temperature for 12 hours to react to generate CA19-9 antibody-FITC conjugate;
[0058] ③ Separate the reaction solution after step ② through a G-25 gel column to remove unreacted FITC, and obtain a solution containing CA19-9 antibody-FITC conjugate (ie FITC-labeled CA19-9 antibody);
[0059] ④Dilute the solution con...
Embodiment 2
[0060] Example 2 Preparation of the second reagent
[0061] (1) Materials and equipment: CA19-9 monoclonal antibody stored in phosphate buffer (purity over 95wt%, concentration of 2mg / mL); alkaline phosphatase stored in phosphate buffer (ALP solution, ALP purity is About 99%, the specific activity is about 1500U / mg, the concentration is 10mg / mL); the cross-linking agent SMCC, 2-IT is purchased from THERMO, the chemical reagents such as TRIS should be chemically pure; the G-25 gel purification column is GE company's product.
[0062] (2) Preparation steps:
[0063] ①Take 1mg of CA19-9 antibody, add 2-4μL of 10mg / mL coupling agent 2-IT solution, leave it at room temperature for 20min, add 10μL of 0.1mol / L glycine solution, leave it at room temperature for 5min, use G-25 gel Desalt the column, collect the activated CA19-9 antibody, store it at 2-8°C for later use;
[0064] ②Take 1.5mg of ALP solution, add 10-20μL of 5mg / mL SMCC solution, let stand at room temperature for 30min, remove ...
Embodiment 3
[0067] Example 3 Preparation of magnetic separation reagent
[0068] (1) Materials and instruments:
[0069] Magnetic particle suspension: magnetic particle content 5wt%, magnetic particle contains carboxyl group (COOH) active group, carboxyl group 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.
[0070] 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;
[0071] 2-Morpholine ethanesulfonic acid (MES), carbodiimide (EDC), TRIS and other reagents should be chemically pure.
[0072] (2) Preparation steps:
[0073] 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;
[0074] ②Add 2~4mg of anti-FITC antibody and suspend at room temperature for 30~60min;
[0075] ③Add 0.5~1mL of a freshly ...
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