Liquid phase protein chip for combined detection of five colorectal cancer markers, and preparation method thereof
A colorectal cancer and joint detection technology, applied in measuring devices, biological testing, material inspection products, etc., can solve the problems of inaccurate detection data, poor pertinence, slow detection speed, etc., achieve high sensitivity, reduce costs, The effect of saving inspection time
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specific Embodiment 1
[0038] Preparation of monoclonal antibodies to M2-PK, K-ras, APC, AKT and PI3K.
[0039] 1. Preparation of immune antigen
[0040] Prepare gene recombination M2-PK, K-ras, APC, AKT and PI3K protein through the engineering strain of a kind of Escherichia coli carrying M2-PK, K-ras, APC, AKT and PI3K protein gene; -PK, K-ras, APC, AKT and PI3K proteins were injected into different mice for immunization.
[0041] 2. Preparation and screening of hybridoma cells
[0042] Select the serum antibody titer to reach 1×10 6 Mice were injected with M intraperitoneally 3 days before fusion 2 - PK, K-ras, APC, AKT and PI3K antigens 100 μg. The mouse spleen was obtained in a sterile manner, and the splenocyte suspension was mixed with the mouse myeloma cell line NS-1 in the logarithmic growth phase at a ratio of 10:1, and 45% polyethylene glycol (PEG ) to facilitate integration. Specifically, polyethylene glycol (PEG) is added according to the following steps. In a water bath at 37°C,...
specific Embodiment 2
[0047] Preparation of nano-gold-coated quantum-encoded microspheres and M 2 - Conjugation of PK monoclonal antibody, K-ras monoclonal antibody, APC monoclonal antibody, AKT monoclonal antibody and PI3K monoclonal antibody.
[0048] 1. Preparation of gold nanoparticles
[0049] The glass container in the present invention is pickled and rinsed with distilled water, and then siliconized. The method of siliconization treatment is to soak for several minutes with 5% dichlorosilane in chloroform solution, rinse with distilled water and dry for later use. The preparation method and process of gold nanoparticles are as follows: the HAuC 14 First prepare a 0.01% aqueous solution, take 100mL and heat it to boiling; accurately add 10mL of 1% trisodium citrate (Na3C6H5O7 2H2O) aqueous solution under stirring, continue heating and boiling for 15min, at this time, a light yellow chloroauric acid aqueous solution can be observed After sodium citrate is added, it turns gray quickly, then ...
specific Embodiment 3
[0056] The microspheres coated with gold nanoparticles and labeled with quantum dots were coupled with monoclonal antibodies to M2-PK, K-ras, APC, AKT and PI3K.
[0057] Here, the M2-PK monoclonal antibody is taken as an example for illustration, and the coupling of other monoclonal antibodies is the same as that of the M2-PK monoclonal antibody, and will not be repeated here.
[0058] The concentration of the M2-PK monoclonal antibody in the test tube is 50nmol / L, and the phosphate buffer solution of pH=6.0 is diluted to 30 μg / mL under electromagnetic stirring, Tween-80 is added to 1% (V / V), and then Add 0.01g quantum dot coded microspheres coated with gold nanoparticle layer, continue to stir for 10 minutes, then add 5% BSA solution (1%) in the test tube to seal the unreacted site, after the sealing is completed, the The microspheres were centrifuged and washed with PBS buffer, and the washing was repeated 3 times. After calculation, the density of the probe is about 0.01 μ...
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