An immunoassay method for effectively reducing steric hindrance by directionally binding to the Fc site of an antibody

Abstract

The present invention discloses an immunoassay method for effectively reducing steric hindrance by directionally binding to the Fc site of an antibody. The method comprises the following steps: (1) mixing N-(5-amino-1-carboxypentyl)iminodiacetic acid with a nitrilotriacetic acid and nickel ions to form a chelating ligand; (2) mixing the chelating ligand with an Fc receptor carrying a His tag. The advantages of the present invention are that by directionally binding to the Fc site of the antibody, the Fab region of the antibody is fully exposed, thereby effectively reducing the steric hindrance between the antibody and the binding site, and significantly improving the sensitivity and linearity of the traditional sandwich immunoassay method.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological detection. Specifically, it relates to an immunological detection method that effectively reduces steric hindrance by directionally binding to the Fc site of an antibody. Background Art

[0002] NTA-Ni-His tag:

[0003] In 1987, Hochuli et al. invented the improved metal chelating ligand nitrilotriacetic acid (NTA). Along with the rapid development of modern molecular biology and recombinant protein technology, the NTA ligand has found important applications in purifying histidine-tagged proteins.

[0004] As is well known, the widely used metal ion is Ni 2+ , and there are a total of six ligand-binding sites around it. IDA is a tridentate ligand, that is, it can bind to three ligand-binding sites around Ni 2+ . Among the remaining three ligand-binding sites, two are used to bind histidine residues in the histidine-tagged protein, and the other is used for coordination binding with H2O. Therefore, the binding ability of IDA to Ni 2+ is relatively weak, resulting in the easy detachment of Ni 2+ bound to the medium, causing the performance of the medium to be unstable. While NTA is a tetradentate ligand, that is, NTA can bind to four ligand-binding sites around Ni 2+ , and the remaining two ligand-binding sites are used to bind histidine residues in the histidine-tagged protein.

[0005] Fc receptor protein:

[0006] Fc receptors refer to a series of receptors on the cell membrane surface that can bind to the Fc fragment of Ig. In immunology, cells with Fc receptors generally include B cells, killer cells, and macrophages. Antibodies are special proteins produced by immune cells that can bind to antigens such as microorganisms, toxins, or allergens. When the Fc receptors on immune cells bind to the antibody and its attached antigen, phagocytosis can be triggered, which will consume the antigen coated with the antibody.

[0007] Based on different types of antibodies, there will be different specific antigens. This also means that there are different Fc receptors, and each Fc receptor can bind to an antibody in its Fc region. Antibodies are also called immunoglobulins, abbreviated as Ig, and the most common type in the blood is IgG. Fc receptors are mainly divided into three categories, including Fc-γ receptors, Fc-α receptors, and Fc-ε receptors. Each type of Fc receptor contains several subtypes based on genetic homology. For example, Fc-γ receptors can bind to the Fc fragment of IgG, Fc-α receptors can bind to the Fc fragment of IgA, and Fc-ε receptors can bind to the Fc fragment of IgE.

[0008] Traditional double antibody sandwich method:

[0009] The double antibody sandwich method was first used in enzyme-linked immunosorbent assay (ELISA). In the traditional double antibody sandwich method, the antiserum containing the known antibody is adsorbed in the wells of a microtiter plate. After washing once, the antigen to be detected is added. If the known antibody and the antigen to be detected are specific, they will bind to each other. Then, an enzyme-linked antibody that reacts specifically with the antigen to be detected is added, so that one unit of antigen binds to two units of antibody simultaneously to form a "sandwich"; finally, the substrate of the enzyme is added, and the content of the antigen to be detected is judged according to the depth of the color of the colored enzymatic hydrolysis product produced. The double antibody sandwich method can be applied in various technical platforms, such as time-resolved technology platform, chemiluminescence technology platform, etc. As long as one unit of antigen binds to two units of antibody simultaneously to form a "sandwich", it is an application based on the double antibody sandwich method. However, in the traditional double antibody sandwich method, the antibody is bound by physical adsorption or chemical coupling. The antibody is adsorbed or bound randomly to the bottom of the microplate, and the Fab segment of the antibody is affected by steric hindrance and cannot fully expose the specific site, thus affecting the detection sensitivity and linearity. Summary of the Invention

[0010] The object of the present invention is to provide an immunoassay method that effectively reduces steric hindrance by specifically binding to the Fc site. This method is based on the double antibody sandwich detection technology. By specifically binding to the Fc site of the antibody, the Fab region of the antibody is fully exposed, thereby effectively reducing the steric hindrance between the antibody and the binding site, and significantly improving the detection sensitivity and linearity of the traditional double antibody sandwich method. For the comparison between the traditional immunoassay technology based on the double antibody sandwich method and the immunoassay technology based on the double antibody sandwich method of the present invention, see Figure 1 .

[0011] To overcome the above-mentioned shortcomings and deficiencies of the traditional double antibody sandwich method, the present invention chemically couples NTA to a polystyrene microplate. NTA and the Fc receptor with a His tag coordinate and bind Ni ions together. The Fc receptor specifically binds to the Fc fragment of the corresponding antibody to form a stable polystyrene microplate - NTA - Ni - His - Fc receptor - antibody structure ( Figure 2 ).

[0012] The technical solution of the present invention is as follows:

[0013] One of the technical solutions provided by the present invention is: an immunoassay method, which includes the following steps:

[0014] (1) Mix N-(5-amino-1-carboxypentyl)iminodiacetic acid with a nitrilotriacetic acid (NTA) group and nickel ions to form a chelating ligand;

[0015] (2) Mix the chelating ligand with an Fc receptor bearing a histidine (His) tag.

[0016] In some preferred embodiments, step (1) as described in one of the technical solutions includes the following steps:

[0017] 1-1) Spread the surfactant solution evenly in a microplate, and let it stand overnight at 2-8 °C for coating; after pouring off the surfactant solution, air-dry at room temperature. Dissolve 1% N-hydroxysuccinimide (NHS) and 0.1% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) with (10 mM - 50 mM, preferably 20 mM; pH 5.8 - 6.5, preferably 6.1) 2-(N-morpholino)ethanesulfonic acid buffer (MES), add it to the microplate, and shake well at room temperature for 10 min; add N-(5-amino-1-carboxypentyl)iminodiacetic acid, react at 37 °C for 30 min, and wash 3 times with deionized water; add 30 - 50 mM primary amine (such as hydroxylamine, ethanolamine, glycine) to block for 1 h, and wash 3 times with deionized water;

[0018] 1-2) Add nickel ion salt and mix well, shake well at room temperature for 60 min, and wash 3 times with deionized water.

[0019] In some preferred embodiments, step (2) as described in one of the technical solutions includes the following steps:

[0020] 2-1) Add the 6×His-Fc receptor solution (concentration 0.005% - 0.05%, preferably 0.02%, in 0.1 M PBS, pH 7.4 matrix) to the above microplate, react for 1 - 3 h, and wash 5 times with 0.1 M PBS buffer at pH 7.4; add the antibody against the analyte, react for 1 h, and wash 5 times with 0.1 M PBS buffer at pH 7.4; the mass percentage concentration of the antibody against the analyte is preferably 0.005% - 0.05%; more preferably 0.02%;

[0021] 2-2) Add a 5% - 30% (preferably 10%) glycerol solution (containing 0.05% preservative, either sodium azide or thimerosal), let it stand for 2 h to block, then drain and air-dry in a cool place for standby.

[0022] In some preferred examples, the microplate is a microplate made of polystyrene material.

[0023] In some preferred examples, in step (1) as described in one of the technical solutions, the surfactant has a characteristic structure A polyacrylic acid surfactant that can form a carboxyl layer on the polystyrene surface; preferably Eudragit L100-55, polyacrylic acid, polystyrene-block polyacrylic acid, or poly(styrene) block-poly(acrylic acid); and / or, the mass percentage concentration of the surfactant is 1-10%, preferably 2%.

[0024] As a preferred embodiment, the mass percentage concentration of N-(5-amino-1-carboxypentyl)iminodiacetic acid described in step (1) as described in one of the technical solutions is 0.1-2%, preferably 0.8%.

[0025] In some embodiments, the nickel ion salt described in step (1) as described in one of the technical solutions is a divalent nickel ion salt; preferably nickel acetate; more preferably 0.1M nickel acetate.

[0026] In some preferred embodiments, the 6×His-Fc receptor described in step (2) as described in one of the technical solutions is a recombinant Fc receptor with 6 histidines at the end.

[0027] In a preferred embodiment, the 6×His-Fc receptor is the 6×His-FcγR1 receptor.

[0028] In some embodiments, the immunoassay method as described in one of the technical solutions further includes the following steps:

[0029] (3) Dilute the sample to be tested 10 times with 0.1M PBS, add it to a microplate, and incubate at room temperature for 0.5 h;

[0030] (4) Wash, add the labeled antibody solution, and incubate at room temperature for 0.5 h;

[0031] (5) Wash, add the substrate (ELISA enzyme-linked immunosorbent assay), or the excitation solution (chemiluminescence method), or the enhancement solution (time-resolved immunofluorescence method), incubate, and read the value.

[0032] The positive and progressive effects of the present invention are as follows:

[0033] By specifically binding to the Fc site of the antibody, the present invention fully exposes the Fab region of the antibody, thereby effectively reducing the steric hindrance between the antibody and the binding site, and significantly improving the sensitivity and linearity of the detection. Brief Description of the Drawings

[0034] Figure 1 : Schematic diagram of the comparison between the traditional immunoassay technology based on the double antibody sandwich method and the immunoassay technology based on the double antibody sandwich method of the present invention;

[0035] The traditional sandwich immunoassay based on two antibodies binds antibodies through physical adsorption or chemical coupling. The antibodies are randomly adsorbed or bound to the bottom of the microplate. The Fab segments of the antibodies are affected by steric hindrance and cannot fully expose the specific sites (left); by the method of the present invention, the Fc fragments of the antibodies are directionally bound, the steric hindrance effect is eliminated, and the Fab ends are fully exposed, thereby improving the sensitivity and linearity of immunoassay (right).

[0036] Figure 2 : Schematic diagram of the binding principle of polystyrene microplate - NTA - Ni - His - Fc receptor - antibody;

[0037] NTA is chemically coupled to the polystyrene microplate. NTA and the His - tagged Fc receptor coordinate and bind Ni ions together. The Fc receptor specifically binds to the Fc fragments of the corresponding type of antibody to form a stable polystyrene microplate - NTA - Ni - His - Fc receptor - antibody structure.

[0038] Figure 3 : Linear relationship between the concentration of cyclosporine - BSA protein complex and the fluorescence signal value (Series 1 in the figure is the average value of duplicate wells of the fluorescence signal of the directional Fc - end sandwich immunoassay of the present invention, and Series 2 is the average value of duplicate wells of the fluorescence signal of the traditional sandwich immunoassay).

[0039] Figure 4 : Schematic diagram of the detection principle of the application of polystyrene microplate - NTA - Ni - His - Fc receptor - antibody in the time - resolved immunocompetitive assay of cyclosporine.

[0040] Figure 5 : Passing and Bablok regression curve graph.

[0041] Figure 6 : Bland - Altman bias graph (% bias).

[0042] Figure 7 : Bland - Altman bias graph (difference). Detailed implementation manners

[0043] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

[0044] The reagents and raw materials used in the present invention are all commercially available.

[0045] Example 1

[0046] When screening the concentration of cyclosporine-BSA protein complex, a comparative experiment was conducted between the directional Fc-terminal double antibody sandwich method and the traditional double antibody sandwich method:

[0047] Control microplate: The traditional physical adsorption coating method was used, and the method is as follows:

[0048] (1) Add 200 μL of anti-cyclosporine A antibody solution (concentration: 1 μg / mL, in 0.1 M PBS, pH 7.4 matrix) to each well and incubate overnight at 2 - 8°C.

[0049] (2) After centrifuging to dryness, immediately add 10% glycerol solution for blocking (250 μL per well) (containing 0.05% sodium azide as a preservative), let it stand for 2 h for blocking, then centrifuge to dryness and air-dry in a cool place for later use.

[0050] Example microplate: A 96-well plate using the method of the present invention for directionally binding to the Fc site of anti-cyclosporine A antibody for coating.

[0051] The coating method is the same as above.

[0052] The steps for coating the anti-cyclosporine A antibody on the microplate are as follows:

[0053] (1) Spread 2% Eudragit L100-55 solution evenly in the wells (polystyrene material), 200 μL per well, and incubate at 2 - 8°C overnight for coating;

[0054] (2) After discarding the surfactant solution, air-dry at room temperature. Dissolve 1% NHS (N-hydroxysuccinimide) and 0.1% EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) in 20 mM / L MES (pH 6.1), add 200 μL to each well, and shake well at room temperature for 10 min;

[0055] (3) Add 0.8% N-(5-amino-1-carboxypentyl)iminodiacetic acid, react at 37°C for 30 min, wash 3 times with deionized water; add 50 mM glycine (250 μL per well) for blocking for 1 h, and wash 3 times with deionized water;

[0056] (4) Add 0.1 M nickel acetate (200 μL per well), shake well at room temperature for 60 min, and wash 3 times with deionized water;

[0057] (5) Prepare 6×His-FcγR1 at a concentration of 0.02% (0.1 M PBS, pH 7.4), add 200 μL to each well, react at room temperature for 2 h, and wash 5 times with 0.1 M PBS, pH 7.4;

[0058] (6) Add 200 μL of anti-cyclosporine A antibody solution (concentration 0.02%, in 0.1 M PBS, pH 7.4 matrix) to each well, react at room temperature for 1 h, and wash 5 times with 0.1 M PBS, pH 7.4;

[0059] (7) Finally, block with 10% glycerol solution (250 μL per well) (containing 0.05% sodium azide as preservative), let it stand for 2 h, drain, and air-dry in a cool place for later use.

[0060] Use physiological saline as the test sample, and the remaining detection steps are the same for both reagents.

[0061] The concentrations of the cyclosporine-BSA protein complex and the fluorescence signal values are shown in Table 1.

[0062] Table 1: Concentrations of the cyclosporine-BSA protein complex and fluorescence signal values

[0063]

[0064] The linear relationship between the concentrations of the cyclosporine-BSA protein complex and the fluorescence signal values is as Figure 3 shown.

[0065] The results show that: a well-coated plate used in the present invention gives a curve with good linearity, while the traditional double-antibody sandwich method starts to level off after the protein complex concentration reaches 400 ng / mL and cannot continue to expand linearly, and the fluorescence signal at 1200 ng / mL has started to weaken. This result indicates that the directed Fc-end double-antibody sandwich method can have stronger fluorescence signals and better linearity compared to the traditional double-antibody sandwich method. The coating method of the present invention, which is different from the traditional double-antibody sandwich method, can direct the Fc-end, making the Fab-end of the antibody fully exposed, thereby achieving the purpose of enhancing sensitivity and linearity.

[0066] Example 2

[0067] A coating method for effectively reducing steric hindrance by directing the binding of antibodies to the Fc site is used in a kit for detecting the immunosuppressant cyclosporine A by time-resolved immunoassay:

[0068] Preparation of the main reagent:

[0069] Microplate: A 96-well plate using the coating method of the present invention for directing the binding of anti-cyclosporine A antibody to the Fc site.

[0070] Analysis buffer 1: A 0.05 mol / L Tris-hydrochloric acid buffer solution (pH 7.4) containing 0.1% bovine serum albumin, 2 mg / mL EDTA-2Na, 0.05% Tween-20, 0.9% sodium chloride, and 0.05% sodium azide.

[0071] Cyclosporine - BSA Protein Complex (100×): The main component is cyclosporine (carboxylated derivative) chemically conjugated to BSA protein via NHS / EDC. Concentration: 100 μg / mL; stored in 1 mol / L phosphate buffer solution (pH 6.8) containing 0.1% sodium azide and 1% Tween - 20.

[0072] Concentrated Wash Solution (20×): 0.2 mol / L Tris - HCl buffer solution (pH 7.4) containing 18% NaCl, 1% Tween - 20, and 0.1% sodium azide.

[0073] Analysis Buffer 2: 0.05 mol / L Tris - HCl buffer solution (pH 7.4) containing 0.1% bovine serum albumin, 0.05% sodium azide, 1 mg / mL EDTA, 0.01% Tween - 20, and 0.9% sodium chloride.

[0074] Anti - Cyclosporine Antibody - Eu (100×): Commercial anti - cyclosporine monoclonal antibody is labeled with DTTA - Eu and purified by chromatography; stored in 0.05 mol / L Tris - HCl buffer solution (pH 7.5) containing 0.2% bovine serum albumin, 0.1% sodium azide, and 0.9% sodium chloride.

[0075] Enhancement Solution: 0.1 mol / L potassium hydrogen phthalate buffer solution (pH 3.6) containing 15 μM / L β - naphthoyltrifluoroacetone, 60 μM / L tri - n - octylphosphine oxide, and 0.1% Triton X - 100.

[0076] Sample Pretreatment Reagent:

[0077] Both the whole blood lysing agent and whole blood sedimenting agent used are from the ABBOTT Cyclosporine Assay Kit (chemiluminescent microparticle immunoassay) ARCHITECT Cyclosporine Reagent Kit.

[0078] Calibrator:

[0079] Cyclosporine Calibrators A - F: After repeatedly freezing and thawing anticoagulated whole blood, preservatives are added, filtered, and prepared into a calibrator matrix; accurately weigh the national standard substance of cyclosporine, dissolve it in methanol solution to prepare a calibrator stock solution, calculate the dilution ratio, add it to the calibrator matrix according to the ratio, mix well to obtain the calibrator stock solution, and then serially dilute it with the calibrator matrix to each concentration calibrator.

[0080] Table 2: Concentrations of Cyclosporine Calibrators A - F

[0081]

[0082] Coat a microplate with anti-cyclosporine A antibody as follows:

[0083] (1) Spread 2% Eudragit L100-55 solution evenly in the wells (made of polystyrene), 200 μL per well, and let it stand for coating overnight at 2-8 °C;

[0084] (2) After discarding the surfactant solution, air-dry at room temperature. Dissolve 1% NHS (N-hydroxysuccinimide) and 0.1% EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) in 20 mM / L MES (pH 6.1), add 200 μL per well, and shake well at room temperature for 10 min;

[0085] (3) Add 0.8% N-(5-amino-1-carboxypentyl)iminodiacetic acid, react at 37 °C for 30 min, and wash 3 times with deionized water; Add 50 mM glycine (250 μL per well) for blocking for 1 h, and wash 3 times with deionized water;

[0086] (4) Add 0.1 M nickel acetate (200 μL per well), shake well at room temperature for 60 min, and wash 3 times with deionized water.

[0087] (5) Prepare 6×His-FcγR1 at a concentration of 0.02% (0.1 M PBS, pH 7.4), add 200 μL per well into the wells, react at room temperature for 2 h, and wash 5 times with 0.1 M PBS, pH 7.4.

[0088] (6) Add 200 μL of anti-cyclosporine A antibody solution (concentration: 1 μg / mL, in 0.1 M PBS, pH 7.4 matrix) per well, react at room temperature for 1 h, and wash 5 times with 0.1 M PBS, pH 7.4.

[0089] (7) Finally, block with 10% glycerol solution (250 μL per well) (containing 0.05% sodium azide as preservative), let it stand for blocking for 2 h, then drain, and air-dry in a cool place for standby.

[0090] Measurement steps:

[0091] 1. Reagent preparation:

[0092] (1) Dilute the concentrated washing solution (20×) with distilled water at a volume ratio of 1:20 before use and add it to the washing solution bottle of the plate washer.

[0093] (2) Equilibrate the analysis buffer, test samples, calibrators, and the required number of microplate strips at room temperature (18-25 °C).

[0094] (3) Within 1 hour before use, dilute the cyclosporine-BSA protein complex (100×) 100-fold with Analytical Buffer 2 according to the required dosage.

[0095] (4) Within 1 hour before use, dilute the anti-cyclosporine antibody-Eu (100×) 100-fold with Analytical Buffer 2 according to the required dosage.

[0096] 2. Sample pretreatment:

[0097] (1) Vortex the cyclosporine calibrator and samples thoroughly, and accurately pipette 100 μL into centrifuge tubes with corresponding numbers.

[0098] (Note: When pipetting, there should be no air bubbles in the sample solution to ensure the accuracy of sample aspiration, and the pipette tip must be replaced after each aspiration.)

[0099] (2) Pipette 50 μL of whole blood lysing agent into each centrifuge tube, and then pipette 200 μL of whole blood sedimenting agent into each centrifuge tube. Immediately vortex at a sufficient speed for 30 seconds to ensure thorough mixing of each sample.

[0100] (3) Place the vortexed centrifuge tubes into a centrifuge and centrifuge at 9000 rpm for 5 minutes. The clear and transparent supernatant obtained is the sample to be tested.

[0101] 3. Sample detection:

[0102] (1) Sequentially add 100 μL of Analytical Buffer 1, 30 μL of the extracted sample to be tested, and then add 50 μL of the diluted cyclosporine-BSA protein complex into the microplate wells. Oscillate on a plate shaker at a slow speed (about 700 rmp) at room temperature for 60 minutes.

[0103] (2) Place the microplate reaction strip into a plate washer and wash 2 times (the washing liquid volume per well each time is not less than 350 μL). If there is a small amount of residual liquid in the wells after washing, pat the microplate reaction strip dry on a clean and dust-free absorbent paper.

[0104] (3) Add 150 μL of the diluted anti-cyclosporine antibody-Eu to each well (discard the solution aspirated by the pipette tip for the first time), and oscillate at a slow speed at room temperature for 20 minutes.

[0105] (4) Place the microplate reaction strip into a plate washer and wash 6 times (the washing liquid volume per well each time is not less than 350 μL). If there is a small amount of residual liquid in the wells after washing, pat the microplate reaction strip dry on a clean and dust-free absorbent paper.

[0106] (5) Use a clean pipette tip to aspirate the enhancement liquid and add 150 μL to each well (discard the solution aspirated by the pipette tip for the first time), and oscillate at a slow speed at room temperature for 5 minutes.

[0107] (6) Detect the fluorescence value on a time-resolved fluorescence immunoassay analyzer (complete the measurement within 30 minutes).

[0108] In the examples, a double-antibody sandwich competitive immunoassay method was used to detect the content of cyclosporine drugs. The principle is as follows: After the sample pretreatment reagent extracts the small-molecule cyclosporine drug (hapten) in the sample, it competes with the chemically conjugated cyclosporine-protein complex (antigen) for binding to the antibody in the well. The more small-molecule cyclosporine drugs there are in the sample, the more binding sites for anti-cyclosporine monoclonal antibody, and the fewer binding sites available for the cyclosporine-protein complex. The sites bound to the small-molecule cyclosporine drug cannot form a sandwich structure, while the sites bound to the cyclosporine-protein complex can still bind to the monoclonal antibody labeled with europium (Eu) to form a double-antibody sandwich structure. After enhanced dissociation, the fluorescence signal is detected by a time-resolved fluorescence immunoassay analyzer. The fluorescence signal intensity is negatively correlated with the concentration of cyclosporine drugs in the sample, thus achieving the detection purpose (see the schematic diagram in Figure 4 ).

[0109] Using the kit prepared in the examples, a clinical sample comparison experiment was carried out with the Abbott CMIA cyclosporine assay kit. The calibration data is shown in Table 3.

[0110] Table 3: Calibration data for comparing clinical samples between the kit prepared in the examples and Abbott CMIA

[0111]

[0112] Note: For the fluorescence values shown in Table 3, the left column is the calibration data using the Abbott CMIA cyclosporine assay kit, and the right column is the calibration data using the kit prepared in the examples.

[0113] The control kit is the Abbott CMIA cyclosporine assay kit (microparticle chemiluminescence method). 130 clinical specimens were simultaneously detected, and the comparison data is shown in Table 4:

[0114] Table 4: Comparison data for detecting 130 clinical specimens using the Abbott CMIA cyclosporine assay kit (microparticle chemiluminescence method)

[0115]

[0116]

[0117]

[0118]

[0119] The Passing and Bablok regression curve is shown in Figure 5, the Bland-Altman bias plot (% bias) is shown in Figure 6 , the Bland-Altman bias plot (difference) is shown in Figure 7 .

[0120] Based on the above results, the double-antibody sandwich detection method of the present invention that effectively reduces steric hindrance by directed binding to the Fc site of the antibody can be used in a variety of technical platforms, such as enzyme-linked immunosorbent assay technology, chemiluminescence technology, or time-resolved immunocompetition technology for determining small molecule drugs like the cyclosporine assay kit used in the examples. The immunodetection method provided by the present invention that effectively reduces steric hindrance by directed binding to the Fc site of the antibody significantly improves the detection sensitivity of the traditional double-antibody sandwich method and reduces steric hindrance, thereby improving the utilization efficiency of the antibody.

Claims

1. An immunoassay method, characterized in that, It includes the following steps: (1) Mix N-(5-amino-1-carboxypentyl)iminodiacetic acid with nickel ions in the presence of nitrilotriacetic acid to form a chelating ligand; the mass percentage concentration of the N-(5-amino-1-carboxypentyl)iminodiacetic acid is 0.1-2%; (2) Mix the chelating ligand with the His-tagged Fc receptor; Step (1) includes the following steps: 1-1) Dissolve N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in 2-(N-morpholino)ethanesulfonic acid buffer, add to a microwell reaction plate coated with a surfactant and mix well, add N-(5-amino-1-carboxypentyl)iminodiacetic acid and mix well, wash with deionized water; add primary amine blocking, wash with deionized water; 1-2) Add nickel ion salt and mix well, wash with deionized water; The surfactant is Eudragit L100-55, polyacrylic acid, polystyrene-block-polyacrylic acid or polystyrene-block-polyacrylic acid; and, the mass percentage concentration of the surfactant is 1-10%; Step (2) includes the following steps: 2-1) Add the 6×His-Fc receptor solution to the microwell reaction plate, react for 1-3 h, wash with PBS buffer; add the antibody against the analyte, react for 1 h, wash with PBS buffer; the mass percentage concentration of the antibody against the analyte is 0.005%-0.05%; 2-2) Add glycerol solution, let stand for blocking, air dry for standby.

2. The immunoassay method according to claim 1, characterized in that, The immunoassay method meets one or more of the following conditions: The mass percentage concentration of N-(5-amino-1-carboxypentyl)iminodiacetic acid is 0.8%; The mass percentage concentration of the antibody against the analyte is 0.02%; and, The mass percentage concentration of the surfactant is 2%.

3. The immunoassay method according to claim 1 or 2, characterized in that, The microwell reaction plate is a microwell reaction plate made of polystyrene material.

4. The immunoassay method according to claim 1 or 2, characterized in that, The nickel ion salt in step (1) is a divalent nickel ion salt.

5. The immunoassay method according to claim 4, characterized in that, The nickel ion salt in step (1) is nickel acetate.

6. The immunoassay method according to claim 5, characterized in that, The nickel ion salt in step (1) is 0.1M nickel acetate.

7. The immunoassay method according to claim 1, characterized in that, The 6×His-Fc receptor in step (2) is a recombinant Fc receptor with 6 histidines at the end.

8. The immunoassay method according to claim 7, characterized in that, The 6×His-Fc receptor is the 6×His-FcγR1 receptor.

9. The immunoassay method according to claim 1, characterized in that, The immunoassay method further includes the following steps: (3) Dilute the sample to be detected with PBS, add to the microwell reaction plate, and incubate; (4) Add the labeled antibody solution and incubate; (5) Add the substrate or excitation solution or enhancement solution, incubate and read the value.

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