Spot enzyme-linked immunosorbent assay materials and methods
(Take the quick diagnosis of FMD as an example)
Dot ELISA (DELISA) is an immunoassay method established by replacing the polystyrene micro-reaction plate commonly used in immunoenzyme solid-phase carrier method with cellulose membrane. DELISA not only retains the advantages of conventional ELISA, but also makes up for the insufficiency of antigen or antibody coating on the carrier, so it has high sensitivity, strong specificity, less sample used, less material, no special equipment, The results are easy to judge and easy to store for a long time. Therefore, since its inception, this method has been widely used in the detection of antigens and antibodies and the selection of hybridoma cells with its unique advantages.
The basic principle of DELISA is basically the same as that of conventional ELISA and immunoenzyme staining. That is, the antigen or antibody is first adsorbed on the surface of the cellulose film (such as nitrocellulose membrane, NC), and maintains its immunological activity. A series of immune reactions of enzyme markers to form an enzyme-labeled antigen-antibody complex. With the participation of the substrate, the enzyme on the conjugate catalyzes the substrate to hydrolyze and oxidize it to another colored substance, calming the antigen-antibody complex The spot where the substance is adsorbed exhibits color spots visible to the naked eye. The result of the test can be judged by the presence or absence of color spots and the depth of color. DELISA commonly used detection methods are direct method, indirect method, double antibody sandwich method, double sandwich method and competition method.
1 Material
(1) Carrier: nitrocellulose membrane 022 ～ 045μm, Schleiche & Schucll or domestic; mixed nitrocellulose membrane 022μm, produced by Shanghai Pharmaceutical Industry Research Institute.
(2) Insulation solution and washing solution: The incubation solution contains 01% BSA, 005% Tween20 PBS (001mol / L, pH 74); the washing solution is 005% Tween20 PBS.
(3) Blocking agent: PBS containing 1% BSA or 4% egg white or 3% white gelatin.
(4) Foot-and-mouth disease monoclonal antibodies: Foot-and-mouth disease virus type A monoclonal antibody (AFMcAb) and type O polyclonal antibody (OFPcAb, prepared by immunizing BALB / c mice with type O standard virus), provided by the Veterinary Research Institute of Xinjiang Academy of Animal Science.
(5) A, O, C, ZB standard antigens for foot-and-mouth disease: provided by Lanzhou Veterinary Research Institute.
(6) Freeze-dried horseradish peroxidase labeled rabbit anti-BALB / c mouse antibody: produced by the Institute of Biological Products of the Ministry of Health.
(7) Substrate solution: 3,3 diaminobenzidine 50mg, 005mol / L, pH value 76 TrisHCl
Buffer 100ml, dissolve and filter, store at 4 ° C protected from light. Before use, press 003% to add H2O2.
(8) Preparation of the tested antigen: blisters can be directly applied. The blister skin was washed with physiological saline, dried, weighed, shredded, ground with neutral glass sand, and made into a 1: 5 ~ 1:10 suspension with physiological saline, soaked at room temperature for 2h, after shaking, 1500 ~ Centrifuge at 2 000 r / min for 10 minutes, and the supernatant can be used for spotting.
2 Method
DELISA indirect procedure
(1) Indentation: Cut a piece of nitrocellulose membrane or mixed cellulose membrane according to the number of samples. Drill holes with a diameter of 3 ～ 5mm
Device (agar plate punch), in order to apply marks on the smooth surface of the membrane in order, as the spot. Soak the membrane in distilled water. After completely soaking, take it out and dry it at room temperature.
(2) Spotting: Use a micropipette to draw 1 ～ 2μl (or use glass rod or capillary to dip a drop) of the tested antigen solution
Inside the circle, dry naturally. In order to increase the amount of antigen adsorption, the second spotting can be performed after the spotting is dried. At the same time as negative and positive antigen control.
(3) Blocking: Place the membrane in a plate or a small pool, add a blocking agent, and soak at 37 ℃ for 30 to 60 minutes. Remove for a while, rinse twice with lotion, and dry.
(4) Reaction with AFMcAb or OFCcAb: AFMcAb or OFCcAb is diluted appropriately with a warming solution, added to the reaction tank, and covered with a membrane at 37 ° C for 2h.
(5) Washing: Rinse the membrane 3 times with washing solution, 3 min each time, and dry.
(6) Reaction with enzyme conjugate: freeze-dried horseradish peroxidase-labeled rabbit anti-BALB / c mouse antibody was diluted 1:80 with a warming solution and covered with a membrane at 37 ° C for 1 h.
(7) Washing: 3 times, the method is the same as above.
(8) Color development: immerse the membrane in the substrate solution and stain for 5 to 15 minutes in the dark.
(9) Terminate the reaction: rinse the filter membrane with distilled water and dry it.
3 Result judgment
Under normal conditions of yin and yang controls, brown spots appear in the membrane circle as positive and colorless as negative. After the film is dried, it can be stored for a long time.
When using DELISA indirect method, double antibody sandwich method and other detection methods, if the membrane is coated with a known standard antigen or antibody instead of the sample to be tested, the above "diagnostic membrane" can be pressed for convenient operation Cut into small pieces and place them in the small holes of the microplate. In the microwell, a series of antigen-antibody reactions and enzymatic reactions occur between the "diagnostic membrane" and the corresponding test sample and diagnostic solution. In the test, the amount of the test sample and the diagnostic solution are 50 μl per well, and the other reaction conditions and operation methods are the same as the conventional ELISA.
4 matters needing attention
1 The sampling volume is not easy to be too large, so as not to overflow the indentation circle, causing the mixing of various samples. If you want to increase the adsorption amount of the sample, you can perform the second sampling after one drying.
2 After the membrane is coated, it must be sealed tightly to prevent non-specific adsorption of antigens or antibodies, and to prevent the background staining of the membrane from being too dark.
3 When judging the result, it should be compared with the negative and positive control spots repeatedly to try to overcome the error caused by naked eye observation.
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