Confining liquid capable of effectively reducing adsorption effect of enzyme-linked immunosorbent assay (ELISA) enzyme-free plate

Abstract

The invention discloses a confining liquid capable of effectively reducing the adsorption effect of an ELISA (enzyme-linked immunosorbent assay) enzyme-free plate. The confining liquid is a phosphate Tween buffer solution containing 2% of bovine serum albumin, the phosphate Tween buffer solution contains 0.05% of Tween-20, and the concentration of phosphate in the phosphate Tween buffer solution is 10 mM; % is the mass percentage content.

Description

Technical Field

[0001] The invention belongs to the technical field of antigen coating, and in particular relates to a blocking solution which can effectively reduce the adsorption of ELISA enzyme-immunoassay plates. Background Art

[0002] Enzyme immunosorbent assay (ELISA) is a commonly used technique in immunodiagnosis, and is used to detect trace substances in liquid specimens. Its basic method is to adsorb known antigens or antibodies on the surface of a solid phase carrier (polystyrene microplate), allow the enzyme-labeled antigen-antibody reaction to proceed on the solid phase surface, and use a washing method to wash away the free components in the liquid phase. Commonly used ELISA methods include the double antibody sandwich method and the indirect method. The former is used to detect macromolecular antigens, and the latter is used to determine specific antibodies. The antigen or antibody is connected to a certain enzyme to form an enzyme-labeled antigen or antibody, which retains both its immune activity and the activity of the enzyme. During the determination, the specimen to be tested (the antibody or antigen to be tested) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid phase carrier in different steps. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by a washing method, and finally the amount of enzyme bound to the solid phase carrier is in a certain ratio to the amount of the substance to be tested in the specimen. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the substance in the specimen, so qualitative or quantitative analysis can be conducted based on the depth of the color reaction. Since the enzyme has a high catalytic frequency, the reaction effect can be greatly amplified, thereby achieving a high sensitivity of the determination method.

[0003] Antigens used to coat solid phase carriers can be divided into three categories according to their sources: natural antigens, recombinant antigens, and synthetic polypeptide antigens. Natural antigens can be obtained from animal tissues, microbial cultures, etc., and must be extracted and purified before they can be used for coating. Coating the antigen reaction plate is the most important part of the entire experiment. The process of fixing the antigen or antibody is called coating. In other words, coating is the process of binding the antigen or antibody to the surface of the solid phase carrier. Proteins and polystyrene solid phase carriers are bound by physical adsorption, relying on the interaction between the hydrophobic groups on the protein molecular structure and the hydrophobic groups on the surface of the solid phase carrier. This physical adsorption is non-specific and is affected by the molecular weight, isoelectric point, concentration, etc. of the protein. The adsorption capacity of the carrier for different proteins is different. Large molecular proteins usually contain more hydrophobic groups than small molecular proteins, so they are easier to adsorb to the surface of the solid phase carrier. IgG has a strong adsorption force on solid phases such as polystyrene. Its connection mostly occurs on the Fc segment, and the antibody binding site is exposed to the outside. Therefore, the coating of antibodies generally adopts the direct adsorption method. Most protein antigens can also be coated in a similar way to antibodies. When the antigenic determinant exists in or is adjacent to the hydrophobic region, the direct adsorption of the antigen to the solid phase carrier may prevent the antigenic determinant from being fully exposed. In this case, the direct coating effect is not good, and the indirect capture coating method can be used, that is, the specific antibody against the antigen is pre-coated first, and then the antigen is solidified through the antigen-antibody reaction. The antigen indirectly bound to the solid phase is away from the carrier surface, and its antigenic determinant is also fully exposed. The purity of the indirectly coated antigen coated on the solid phase is greatly improved by the affinity chromatography of the solid phase antibody. Therefore, the capture coating method can also be used for antigens containing more impurities. The specificity and sensitivity of the test are improved, and the repeatability is also good. Another advantage of indirect coating is that the amount of antigen used is small, which is only 1 / 10 or even 1 / 100 of that of direct coating. Non-protein antigens that are not easily adsorbed on polystyrene carriers can be coated in a special way. Its preparation process is to first adsorb the antigen or antibody on the surface of the solid phase carrier (reaction plate), or it can be indirectly coated with an avidin-biotin system, that is, the carrier is first coated with avidin, and then the biotinylated antibody is added. This coating method is uniform and firm, and has been expanded to the quantitative determination of various antigenic substances. It is blocked with a blocking solution, dried and stored.

[0004] Currently, the commonly used blocking solution is mainly composed of EDTA, skim milk powder, gelatin, casein, and sugar. The prepared antigen-coated plate is sealed and stored in an environment of 2-8°C. It should be restored to room temperature before use to extend the service life of the coated plate. However, conventional blocking solutions and diluents have no effect on the detection of Aβ. It is necessary to invent a blocking solution and diluent that can effectively reduce the specific adsorption of Aβ.

[0005] Amyloid β (Aβ or Abeta) is a family of peptides formed by cleavage of the amyloid precursor protein (APP) by β- and γ-secretases and is thought to be involved in the pathogenesis of Alzheimer's disease. The presence of β-amyloid plaques, extracellular deposits of the protein, in the brain is a major hallmark of Alzheimer's disease. These plaques are formed by the spontaneous aggregation of monomeric Aβ into soluble Aβ oligomers, which aggregate together to form insoluble fibrils. Most Aβ peptides are 40 residues long (Aβ 1–40), with a small fraction being 42 residues (Aβ 1–42). Aβ 1-42 is generally considered to be more neurotoxic because the two extra amino acids in Aβ 1-42 make it more likely to misfold and aggregate. There is evidence that elevated plasma levels of Aβ 1-42 are associated with Alzheimer's disease.

[0006] There are too many formulas of blocking solution in the ELISA method and they are not targeted for the detected Aβ 42, so it is necessary to find a blocking solution that can increase the sensitivity of Aβ 42 in urine and reduce nonspecific binding. Antibodies and protein antigens generally use carbonate buffer at pH 9.6 as diluent, and phosphate buffer at pH 7.2 and Tris-HCL buffer at pH 7-8 are also used as diluents. Usually, after adding the coating solution to the ELISA plate wells, it is placed in a refrigerator at 4-8℃ overnight, and it is considered to have the same coating effect if it is kept warm at 37℃ for 2 hours. Blocking is the process of re-coating with a high concentration of irrelevant protein solution after coating. The concentration used for antigen or antibody coating is relatively low, and there are still unoccupied gaps on the surface of the solid phase carrier after absorption. Blocking is to allow a large amount of irrelevant proteins to fill these gaps, thereby excluding the re-adsorption of interfering substances in the subsequent steps of ELISA. The blocking procedure is similar to coating. The most commonly used blocking agent is 0.05%-0.5% bovine serum albumin, and 10% calf serum or 1% gelatin is also used as a blocking agent. 5% skim milk powder is also a good blocking agent. Its biggest feature is that it is cheap and can be used in high concentrations. High-quality instant edible low-fat milk powder can be used directly as a blocking agent, but because the composition of milk powder is complex and the carrier after blocking is not easy to preserve for a long time, it is rarely used in experiments. Summary of the invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provide a blocking solution which can effectively reduce the adsorption of ELISA enzyme-immunoassay plates.

[0008] In order to achieve the above object, the technical solution provided by the present invention is: The blocking solution is a phosphate-Tween buffer containing 2% bovine serum albumin, the phosphate-Tween buffer contains 0.05% Tween-20, and the concentration of phosphate in the phosphate-Tween buffer is 10 mM; the % is the mass percentage content.

[0009] Preferably, the phosphate Tween buffer further contains 0.018% sodium dihydrogen phosphate, 0.12% disodium hydrogen phosphate, and 0.818% sodium chloride; the % is the mass percentage content.

[0010] Preferably, the pH value of the phosphate-Tween buffer is 7.4.

[0011] Preferably, the blocking solution further contains 5% trehalose and 0.5% PVP; the % is the mass percentage.

[0012] The blocking solution that effectively reduces the adsorption of ELISA plates can be used to prepare a reagent that reduces the non-specific binding of Fc segments on cell surfaces.

[0013] The present invention will be further described below: The principle of Tween-20 in the blocking solution is that Tween-20 in the blocking solution is a non-ionic surfactant. Since there are more hydrophilic groups in the polysorbate molecule, namely polyoxyethylene, it has strong hydrophilicity. It blocks all unbound sites without replacing the target protein, binding to the epitope of the target protein, or cross-reacting with antibodies or detection reagents.

[0014] BSA (bovine serum albumin) in the blocking solution is an enzyme stabilizer to prevent enzyme decomposition and nonspecific adsorption. It is best to prepare it for immediate use. Even if the prepared solution is stored in a -20°C environment, it will cause the results to be high after a period of time. BSA is a globulin with a molecular weight of about 66.5kDa. It contains a large number of amino and carboxyl residues on the surface. The spatial structure of BSA can change with temperature, pH, and salt concentration. It is a flexible molecule. As a common blocking protein, BSA can bind to hydrophilic surfaces, hydrophobic surfaces, and activated covalently bound surfaces, such as activated carboxyl, amino, epoxy, tosyl, etc., to inhibit nonspecific adsorption, and is an ideal solid phase blocking agent. The surface of BSA contains a large number of carboxyl and amino groups, which can be used for binding to the activated groups on the surface to block excess sites. Studies have found that BSA is more compactly adsorbed on the solid surface in phosphate solution, and the structure formed is more stable. This is because phosphate binds to glycine and arginine on BSA, which can eliminate structural electrostatic repulsion. Therefore, BSA has a more stable structure in phosphate buffer and has a higher binding efficiency on the solid surface.

[0015] Trehalose in the blocking solution is the most stable type of natural disaccharide. Because it is not reducing, it has very good stability to heat and acid and alkali, and effectively protects the structure of biological molecules from being destroyed. Its protection mechanism is generally believed to be that the part of the body containing trehalose strongly binds water molecules, shares bound water with membrane lipids, or trehalose itself replaces membrane bound water, thereby preventing the denaturation of biological membranes and membrane proteins.

[0016] Tween-20 is a nonionic surfactant that blocks all unbound sites. Trehalose has very good stability and effectively protects the structure of biological molecules from being destroyed. Stably dispersed BSA can bind to antigens more uniformly and stably, exerting the antioxidant function of BSA.

[0017] When the blocking solution of the present invention is used to coat the antigen, the antibody is coated with 0.05 mol / L carbonate buffer (PH9.6) at 37°C for 2 hours, and then blocked with the above blocking solution to prepare an antigen-coated plate. The antibody is Aβ 42 antibody, and the Aβ 42 is Aβ 42 in urine. The coated antigen is a cell monoclonal antibody, and the detection antibody is an antibody labeled with biotin; the concentration of the coated antigen is 1 μg / mL, and the concentration of the detection antibody is 1:500. The blocking solution is 300 μL per well to ensure that the inner wall of the microplate is fully covered by the blocking solution to reduce the situation of incomplete blocking.

[0018] The blocking solution of the present invention can effectively reduce the non-specific binding of the Fc segment on the cell surface, has good blocking effect, high sensitivity, and is stable and durable when stored at 4° C., thereby reducing the influence of variables in the experiment. DETAILED DESCRIPTION Example 1

[0019] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0020] Wash the plate 4 times with PBST (Phosphate Tween Buffer) (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly tap dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a 4℃ refrigerator.

[0021] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0022] Wash the plate 4 times with PBST (1 min each time), repeat the liquid removal, plate tapping and adsorption operations after each wash. Add 300µL of PBS+2%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a 4℃ refrigerator.

[0023] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 10mM / L PBS buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0024] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a 4℃ refrigerator.

[0025] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 10mM / L PBS buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0026] Wash the plate 4 times with PBST (1 min each time), repeat the liquid removal, plate tapping and adsorption operations after each wash. Add 300µL of PBS+2%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a 4℃ refrigerator. Example 2

[0027] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0028] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a 4℃ refrigerator.

[0029] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0030] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+5%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a refrigerator at 4℃.

[0031] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0032] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+0.1%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a refrigerator at 4℃. Example 3

[0033] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0034] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA+5%trehalose (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a refrigerator at 4℃.

[0035] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0036] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA+2%trehalose (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a refrigerator at 4℃. Example 4

[0037] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0038] Wash the plate 4 times with PBST (1 min each time), repeat the liquid removal, plate tapping and adsorption operations after each wash. Add PBST+2%BSA+5%trehalose+0.5%PVP(10mM PBST PH 7.5) 300µL to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours after sealing. You can directly pat dry and put it in a 37℃ incubator for drying for 30 minutes, cover it with a sealing film, and store it in a refrigerator at 4℃.

[0039] Take Aβ42 antibody (dissolve the antibody into 1mg / mL), add it into 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain the final concentration of solution of 10 µg / mL, 2 µg / mL, 0.4µg / mL, 0.08µg / mL, 0.016µg / mL, 0.0032µg / mL, 0.0064µg / mL, 0µg / mL. Dispense into microplate at 100 µL / well, cover the ELISA plate with film, seal it and incubate it in 37℃ incubator for 2 hours.

[0040] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA+5%trehalose+2%PVP(10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a refrigerator at 4℃. Example 5

[0041] Take the antigen, add it to 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain a final concentration of 1 µg / mL. Dispense into microplates at 100 µL / well, cover the ELISA plate with film, seal it, and incubate it in a 37℃ incubator for 2 hours.

[0042] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA+0.5%PVP+5%trehalose (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a refrigerator at 4℃.

[0043] Add samples, dilute in two-fold series and add samples.

[0044] A, positive sample a, 100 μL per well.

[0045] B, positive sample b, 100 μL per well.

[0046] C, positive sample c, 100 μL per well.

[0047] D, positive sample d, 100 μL per well.

[0048] E, positive sample e, 100 μL per well.

[0049] F, positive sample f, 100 μL per well.

[0050] G, positive sample g, 100 μL per well.

[0051] H, positive sample h, 100 μL per well.

[0052] A, Aβ42 10 µg / mL, 100 μL per well.

[0053] B, Aβ42 2 µg / mL, 100 μL per well.

[0054] C, Aβ42 0.4 µg / mL, 100 μL per well.

[0055] D. Aβ42 0.08 µg / mL, 100 μL per well.

[0056] E, Aβ42 0.016µg / mL, 100μL per well.

[0057] F. Dilution solution, 100 μL per well.

[0058] G. Negative sample, 100 μL per well.

[0059] H, blank, 100 μL per well. Example 6

[0060] Take the antigen, add it to 0.05mol / L CB (sodium bicarbonate, sodium carbonate) buffer, mix well, and obtain a final concentration of 1 µg / mL. Dispense into microplates at 100 µL / well, cover the ELISA plate with film, seal it, and incubate it in a 37℃ incubator for 2 hours.

[0061] Wash the plate 4 times with PBST (1 min each time), repeat the operation of shaking, tapping and adsorption after each wash. Add 300µL of PBST+2%BSA (10mM PBST PH 7.5) to seal, cover the ELISA plate with film, and incubate in an incubator at 37℃ for 2 hours. You can directly pat dry and put it in a 37℃ incubator to dry for 30 minutes, cover it with a sealing film, and store it in a 4℃ refrigerator.

[0062] Add samples, dilute in two-fold series and add samples.

[0063] A, positive sample a, 100 μL per well.

[0064] B, positive sample b, 100 μL per well.

[0065] C, positive sample c, 100 μL per well.

[0066] D, positive sample d, 100 μL per well.

[0067] E, positive sample e, 100 μL per well.

[0068] F, positive sample f, 100 μL per well.

[0069] G, positive sample g, 100 μL per well.

[0070] H, positive sample h, 100 μL per well.

[0071] A, Aβ42 10 µg / mL, 100 μL per well.

[0072] B, Aβ42 2 µg / mL, 100 μL per well.

[0073] C, Aβ42 0.4 µg / mL, 100 μL per well.

[0074] D. Aβ42 0.08 µg / mL, 100 μL per well.

[0075] E, Aβ42 0.016µg / mL, 100μL per well.

[0076] F. Dilution solution, 100 μL per well.

[0077] G. Negative sample, 100 μL per well.

[0078] H, blank, 100 μL per well.

[0079] For the following dilutions, select the same dilution as the blocking solution to dilute Aβ42 to ensure that the experimental results are comparable.

[0080] Add the sample and dilute the biotin-labeled antibody (500-fold dilution) at 37°C for 1 hour.

[0081] Wash the plate 4 times with PBST and pat dry.

[0082] The antibody labeled with streptavidin was diluted with diluent (2000-fold dilution) and reacted at 37°C for 15 minutes.

[0083] Wash the plate 4 times with PBST and pat dry.

[0084] For color development, add 100 µL of color development solution, shake, and react for 10 minutes in the dark.

[0085] Stop solution: add 50 µL sulfuric acid to each well to stop the reaction.

[0086] Detection: Use a microplate reader to detect the OD value at 450nm within ten minutes and make a judgment based on the following result judgment criteria.

[0087] The test is established when the OD 450nm value of the positive control well is ≥ 0.3 and the OD value of the negative control well is < 0.1, otherwise it is invalid. When the test is established, the critical value (CΜT OFF) is calculated as: critical value = 0.26 + the average OD value of the negative control well (the average OD value of the negative control well is calculated as 0.03 if it is lower than 0.03). Result judgment: Samples with OD values ​​≥ critical value (CΜT OFF) were considered positive.

[0088] Samples with OD values ​​≤ critical value (CΜT OFF) were negative.

[0089] Test results (Table 1 to Table 6) Table 1 is the experimental results of Example 1, determining the coating solution and the basic diluent; Table 2 is the ELISA experimental results of Example 2, determining the BSA blocking solution concentration; Table 3 is the ELISA experimental results of Example 3, determining the trehalose concentration of the blocking solution; Table 4 is the ELISA experimental results of Example 4, determining the PVP concentration of the blocking solution; Table 5 is the ELISA experimental results of Example 5, the second blocking solution; Table 6 is the ELISA experimental results of Example 6, the first blocking solution.

[0090] Experiments were conducted and the results showed that the CB-coated plate was more specific than the PBS-coated plate, and the blocking solution 2% BSA+PBST had a strong blocking ability, which could block exposed binding sites and reduce nonspecific adsorption.

[0091] Table 1. Results of the experiments to determine the coating solution and basic dilution solution From the results in Table 1, it can be seen that CB coating has higher sensitivity than PBS when the blocking solution is the same but the coating solution is different. And the blank control shows that the CB coating blocking solution is 2% BSA + PBST, which is twice as high as the coated plate. Therefore, CB coating is selected, and 2% BSA + PBST is used as the blocking solution. Table 2. ELISA results for determining BSA blocking solution concentration CB coating blocking solution 5% BSA + PBST Blocking solution: 5% BSA + PBST CB coating blocking solution 2% BSA + PBST Blocking solution: 2% BSA + PBST CB coating blocking solution 0.1% BSA + PBST Blocking solution: 0.1% BSA + PBST Aβ42 10 µg / mL 0.1915 0.0944 0.4379 0.1319 0.5406 0.4508 Aβ42 2 µg / mL 0.1544 0.0930 0.2723 0.0836 0.2886 0.0983 Aβ42 0.4 µg / mL 0.1387 0.0785 0.2108 0.0797 0.2458 0.0742 Aβ42 0.08 µg / mL 0.1063 0.0794 0.1922 0.0711 0.2386 0.0978 Aβ42 0.016µg / mL 0.0999 0.0677 0.1502 0.0686 0.2055 0.0695 Aβ42 0.0032 µg / mL 0.0812 0.0636 0.1328 0.0621 0.1830 0.0824 Aβ42 0.0064 µg / mL 0.0649 0.0639 0.1112 0.0564 0.1637 0.0799 2%BSA+PBST 0.1300 0.0710 0.1496 0.0753 0.2289 0.0753 From the results in Table 2, we can see that 5% BSA blocking can effectively remove non-specificity, but at the same time the sensitivity decreases, which also leads to little difference between the coated plate and the blank plate; 2% BSA has a poorer effect on removing non-specificity than 5% BSA, but the sensitivity and signal-to-noise ratio are increased; 0.5% BSA blocking effect is not obvious. In summary, 2% BSA was selected as the blocking solution component.

[0093] Table 3. ELISA results for determining the trehalose concentration in the blocking solution From the results in Table 3, the OD value gradually decreased with the concentration of Aβ42 and was twice as correlated with the OD value of the blank plate. The blocking solution formula was selected as 5% trehalose, 2% BSA, and PBST.

[0094] Table 4. ELISA results for determining PVP concentration in blocking solution From the results in Table 4, the OD value gradually decreased with the concentration of Aβ42 and was twice as correlated with the OD value of the blank plate. 0.5% PVP had a better blocking effect than 2% PVP. The blocking solution formula was 0.5% PVP, 5% trehalose, 2% BSA, and PBST. Table 5. ELISA results of the second blocking solution From the results in Table 5, it can be seen that Aβ 42 in urine can be detected, and the sample detection OD value is twice that of the blank. The second dilution effectively improves the signal value, but at the same time the blank background will also increase.

[0095] Table 6. ELISA results of the first blocking solution From the results in Table 6, it can be seen that Aβ 42 in urine can be detected, and the sample detection OD value is twice that of the blank, and the blank OD value is controlled at 0.1 or below, which can effectively reduce nonspecificity and improve sensitivity.

[0096] The blocking effect of the blocking solution was compared, and it was found that the blocking effect of BSA at the same concentration was better than that of PVP. The blocking effect of the blank plate was better than that of 2% BSA concentration than that of 0.5% BSA.

[0097] Comparing the two commonly used coating antigen diluents CB and PBS in the existing ELISA technology, the experimental results show that the CB antigen coating diluent is more effective than PBS. By comparing different antigen diluents and blocking solutions, CB was finally selected as the antigen diluent. In this experimental result, the blocking solution 2% BSA + PBST has a more obvious blocking effect than 2% BSA + PBS, and the reason is Tween-20. Tween-20 is a non-ionic surfactant used as a blocking solution. Ionic detergents do not destroy the structure of proteins and can reduce the damage to the original interactions between proteins.

[0098] ELISA test results of different blocking solutions coated on the same plate. Further tests on the selected antigen coating diluent and blocking solution found that the overall effect of antigen diluent CB and blocking solution 2% BSA + PBST was more obvious than that of antigen diluent PBS and blocking solution 2% BSA + PBS. This can be well reflected from the data of the blank plate. Antigen diluent CB and blocking solution 2% BSA + PBST can effectively reduce nonspecific binding, enhance specific binding, and improve sensitivity.

[0099] It can effectively block proteins and other biological molecules from binding to the surface of the solid medium, does not affect the effectiveness of the reagents coated on the surface of the solid medium, does not cross-react with other active ingredients of the reagents in the reaction system, and can act as a stabilizer for solid surface coupling molecules.

[0100] Aβ 42 in urine can be detected, and the OD value of the sample detection is twice that of the blank, which proves that the first blocking solution of the present invention can effectively reduce the generation of nonspecificity and increase sensitivity.

Claims

1. A blocking solution that effectively reduces the adsorption of ELISA plates, characterized in that: The blocking solution is a phosphate-Tween buffer containing 2% bovine serum albumin, the phosphate-Tween buffer contains 0.05% Tween-20, and the concentration of phosphate in the phosphate-Tween buffer is 10 mM; the % is the mass percentage content.

2. The blocking solution for effectively reducing the adsorption of ELISA plates according to claim 1, characterized in that: The phosphate Tween buffer also contains 0.018% sodium dihydrogen phosphate, 0.12% disodium hydrogen phosphate, and 0.818% sodium chloride; the % is the mass percentage content.

3. The blocking solution for effectively reducing the adsorption of ELISA plates according to claim 2, characterized in that: The pH value of phosphate-Tween buffer is 7.

4.

4. The blocking solution for effectively reducing the adsorption of ELISA plates according to any one of claims 1 to 3, characterized in that: The blocking solution also contains 5% trehalose and 0.5% PVP; the % is the mass percentage content.

5. Use of the blocking solution for effectively reducing the adsorption of ELISA plates as claimed in any one of claims 1 to 3 in the preparation of a reagent for reducing the non-specific binding of Fc segments on cell surfaces.

6. Use of the blocking solution for effectively reducing the adsorption of ELISA plates as claimed in claim 4 in the preparation of a reagent for reducing the non-specific binding of Fc segments on cell surfaces.