Monitoring method of antibody coupled latex microspheres and application thereof
A technology of antibody coupling and latex, which is applied in the field of immunoassay medical detection, can solve the problems of affecting detection results and low coupling efficiency
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Embodiment 1
[0051] Example 1: Preparation of antibody-latex microsphere coupling complex
[0052] (1) Prepare 0.02-0.05mol / L MES buffer solution, adjust the pH to 6.00-6.50 with 0.05mol / L sodium hydroxide solution, and mix thoroughly;
[0053] (2) Weigh 0.2383g of HEPES solid and dissolve it in 100ml of 0.1M, pH 3.5 sodium citrate aqueous solution;
[0054] (3) Take a certain amount of adiponectin (NADP) monoclonal antibody NA1, dilute to 2 mg / ml with the buffer in (2), and set aside;
[0055] (4) Take 2ml of latex microspheres with a particle size of 123nm and a solid content of 5%, dilute to 100ml with the solution in (1), stir and mix, and shake at a constant temperature of 37°C for 30 minutes;
[0056] (5) The activator needs to be prepared and used immediately. Weigh 0.2490g of EDC solid, make it into 1mg / ml with purified water, and then add it to the reaction solution of (4), place on a shaker, and shake at a constant temperature of 37°C for 30 minutes;
[0057] (6) Take 0.5ml of ...
Embodiment 2
[0059] Example 2: Monitoring of antibody-latex microsphere coupling process
[0060] (1) After adding the diluted antibody solution obtained in (3) in step (7) of Example 1, sample 200ul at different time points of the reaction, and the sampling time points are 10min, 20min, 30min, 40min, 50min, 60min. Under the conditions of 16000rpm and 15min, centrifuge each sample with a centrifuge, remove the supernatant and resuspend the centrifuged product with purified water, and detect the fluorescence signal value at each sampling time point with a UV spectrophotometer, and the UV detection wavelength is 565nm.
Embodiment 3
[0061] Example 3: Monitoring of antibody-latex microsphere coupling process under different pH MES buffer conditions
[0062] Select 0.05mol / L MES buffer solution with different pH (pH is 6.00, 6.10, 6.20, 6.30, 6.40, 6.50 respectively), and prepare the antibody-latex microsphere coupling complex according to the method of Example 1, according to Example 2 The method was used to measure the fluorescence signal value of the resuspension solution at 10min, 20min, 30min, 40min, 50min, and 60min, so as to evaluate the coupling efficiency.
[0063] The experimental results (Table 1) show that changing the pH of the MES buffer will directly affect the coupling efficiency; but no matter how much the pH of the MES buffer is, within 60 minutes after the start of the reaction, the fluorescence signal value increases with the reaction time increases, indicating that the coupling efficiency is proportional to the reaction time.
[0064] According to Table 1, when the reaction is 10min an...
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