Electrophoresis flowing type ELISA method based on polyelectrolyte multilayer (PEM) substrate
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A flow-type, electrophoretic technology, used in instruments, measuring devices, scientific instruments, etc., to achieve the effect of shortening the reaction time
Inactive Publication Date: 2015-12-16
BEIJING UNIV OF CHEM TECH
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[0003] The purpose of the present invention is to solve the problem of low efficiency of sample detection in the ELISA system and further solve the problem of low sensitivity to provide a method for...
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Embodiment 1
[0020] CA Porous Membrane Substrate Electrophoresis ELISA
[0021] 1) Configuration of the solution:
[0022] a) Configuration of 50mM Tris-HCl 0.15MNaCl solution: Dissolve 1.15175g Tris-base solid in 125mL ultrapure water, take 0.84mL HCl and dissolve it in 100mL ultrapure water to obtain 0.1M HCl solution, after dissolving, add the HCl solution dropwise to Tris- Adjust the pH of the base solution to 7.4, set the volume to 250 mL, weigh 2.208 g of NaCl solid and add it to Tris-HCl.
[0023] b) Configuration of pH=7.4 concentration of 0.15M phosphate buffer (PB): weigh 10.74gNa 2 HPO 4 12H 2 Add O solid into 200mL ultrapure water, weigh 4.68gNaH 2 PO 4 2H 2 O solid was added into 200mL ultrapure water, and NaH 2 PO 4 solution added to Na 2 HPO 4 In the solution, adjust pH=7.4.
[0024] c) 0.05% Tween200.03MPB configuration: Take 250 μL Tween-20 and add it to 500 mL 0.03MPB buffer.
[0025] d) 2MH 2 SO 4 Configuration: take 8mL ultrapure water and add 1mL concentr...
Embodiment 2
[0032] 1. Preparation of PEMs modified CA porous membrane
[0033] 1) Prepare PDDA and PSS solution. Weigh 2mg each of PDDA and PSS solids, and dissolve them in 10mL of 50mM Tris-HCl0.15MNaClpH=7.4 solution to obtain PDDA and PSS concentrations of 0.2mg / mL.
[0034]
[0035] 2) Preparation of PEMs modified CA porous membrane. The CA membrane soaked in ultrapure water was put into PDDA solution, soaked for 2min at room temperature, and then washed with ultrapure water for 30s to remove the unadsorbed polyelectrolyte solution. Then put it into the PSS solution, soak it at room temperature for 2min, and then wash it with ultrapure water for 30s. The above PDDA and PSS are alternately performed to prepare 7 layers of positively charged self-assembled multilayer PEMs ((PDDA / PSS) 3 PDDA). 2. PEMs-CA porous membrane substrate electrophoresis ELISA
[0036] 1) with embodiment 1 step 1)
[0037] 2) Same as step 2) of Example 1, PEMs-CA replaces CA.
[0038] 3) Same as Step 3) ...
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Abstract
The invention discloses an electrophoresis flowing type ELISA method based on a PEM substrate, belonging to the field of biomaterials. The method comprises the following steps: allowing a first antibody to be adsorbed on the surface of a porous membrane and then carrying out blocking agent protein adsorption; carrying out antigen-first antibody reaction under the driving of electrophoresis; carrying out second antibody-antigen reaction and carrying out color development and quantification on the enzyme-labeled second antibody, i.e., after the antigen-first antibody reaction under the driving of electrophoresis, placing the porous membrane substrate having undergone electrophoresis in a second antibody solution, carrying out a reaction, then carrying out cleaning, placing the obtained porous membrane substrate in a substrate solution of second antibody identification enzyme and carrying out color development; and carrying out quantitative analysis on antigen by detecting absorbance. Preferably, an electropositive PDDA/PSS modified cellulose acetate membrane PEMs-CA is used. According to the invention, through modification of PEMs, non-specific adsorption is reduced; electrophoresis driving force enables antigen molecules to be locally concentrated around the first antibody in a short period of time; so rapid and sensitive detection is realized.
Description
technical field [0001] The invention belongs to the field of biological materials, in particular to the preparation of polyelectrolyte multilayers (PEMs) and the method of enzyme-linked immunosorbent assay (ELISA) driven by electrophoresis. Background technique [0002] In recent years, since the establishment of immunological methods, immunosensors, as emerging biosensors, have been favored for their high specificity, sensitivity and stability in identifying substances. ELISA is a small amount of protein detection and quantitative test method, widely used in food, industry, environmental monitoring, and clinical medicine and other fields. There are several problems in the static incubation reaction of ELISA on the conventional polystyrene substrate (polystyrene: PS) substrate: (1) the primary antibody on PS is easily denatured, and the site that reacts with the antigen is not easy to be exposed on the substrate; (2) The blocking effect is not good, which leads to the non-s...
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