Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor

[0028] Example 1

[0029] 1. Preparation and characterization of the secondary antibody probe

[0030] (1) Preparation of magnetic nanoparticles

[0031] Preparation of magnetic nanoparticles: For specific preparation methods, please refer to the invention patent application with publication number CN 101302361A.

[0032] Characterization of magnetic nanoparticles: X-ray fluorescence spectroscopy (XRF) was used to analyze Fe 3 O 4 / ZrO 2 For characterization, Zr-K appeared β (17.8keV), Zr-K α (15.8keV), Zr-L β (2.1ke), Zr-L α (2.0keV) peak and Fe-K β (7.1KeV), Fe-K α (6.4keV) peak, indicating the presence of Zr and Fe elements in the magnetic particles.

[0033] (2) Preparation and characterization of antibody-loaded nanospheres

[0034] Preparation of antibody-loaded nanospheres: Disperse 10 mg of magnetic nanoparticles in 5 mL pH 7.0 phosphate buffer, add 1 mg horseradish peroxide-labeled alpha-fetoprotein secondary antibody (HRP-anti-AFP), and stir for 6 hours , Magnetic separation un

[0058] Example 2 (Comparative Experiment 1)

[0059] 1. Experimental materials

[0060] 1. Antigen and antibody: AFP antigen standard, alpha-fetoprotein monoclonal primary antibody (anti-AFP), horseradish peroxidase-labeled alpha-fetoprotein polyclonal antibody (HRP-anti-AFP) and AFP ELISA kit All purchased Zhengzhou Bosai Biotechnology Co., Ltd.

[0061] 2. Amperometric immunosensor: prepared according to the method described in Example 1.

[0062] 3. Secondary antibody probe

[0063] 3.1. Sample: The secondary antibody probe prepared by the method described in Example 1.

[0064] 3.2. Reference substance

[0065] Reference substance 1:

[0066] (1) Preparation of magnetic nanoparticles

[0067] Preparation of magnetic nanoparticles: For specific preparation methods, please refer to the invention patent application with publication number CN 101302361A.

[0068] (2) Preparation and characterization of antibody-loaded nanospheres

[0069] Preparation of antibody-loaded nanospheres: Disperse 10

[0086] Example 3

[0087] 1. Preparation and characterization of the secondary antibody probe

[0088] (1) Preparation and characterization of magnetic nanoparticles

[0089] Same as in Example 1 step 1.

[0090] (2) Preparation and characterization of antibody-loaded nanospheres

[0091] Preparation of antibody-loaded nanospheres: Disperse 10 mg of magnetic nanoparticles in 5 mL of pH 7.0 phosphate buffer, and add 1 mg of horseradish peroxidase-labeled HIV p24 antibody (HRP-anti-HIV p24). After stirring for 6 hours, a magnetic field was applied to separate the unbound antibody to obtain antibody-loaded nanospheres.

[0092] (3) Preparation of nanospheres

[0093] Same as Example 1 step 3 shown.

[0094] (4) Preparation of secondary antibody probe

[0095] Same as Example 1 step 4 shown.

[0096] 2. Using secondary antibody probe to detect antigen concentration

[0097] (1) Preparation of amperometric immunosensor

[0098] (a) Graphene / chitosan (GS / CS) is modified into a film on the surface