A method for detecting antibodies by capture and uses thereof

Abstract

The application relates to a method for detecting antibodies by capture method, and application, which completes detection in the form of anti-Ig antibody-detecting antibody-modified antigen. The modified antigen is obtained by modifying an antigen by betaines, oxidized tertiary amines or polyethylene glycols, and is applied to immune detection, so that the detection sensitivity is improved, and the detection background is reduced.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a method and application for detecting antibodies using a capture method. Background Technology

[0002] Immunoassay utilizes the specific reaction between antigens and antibodies for detection, employing isotopes, enzymes, chemiluminescent substances, etc., to display the detection signal. It is commonly used to detect trace substances such as proteins and hormones. Immunodiagnostics plays a vital role in clinical diagnosis. Among these methods, the capture assay is a frequently used approach for antibody detection. This method involves linking anti-Ig antibodies to a solid-phase carrier to form a solid-phase anti-Ig antibody. Then, a sample and a specific antigen-label are added, ultimately forming a complex of "anti-Ig antibody - test Ig antibody - specific antigen - label." The label signal is then detected using specific techniques to arrive at the final result. The capture assay is the preferred method for many immunoassay antibody detection methods. Summary of the Invention

[0003] This invention provides a detection method based on the capture method, which reduces detection background and improves detection sensitivity.

[0004] A capture method for detecting antibodies, wherein the method completes the detection by forming an anti-Ig antibody-analyte antibody-modified antigen, wherein the modified antigen includes an antigen and a modifier linked to the antigen, and the modifier is a betaine, an oxidized tertiary amine, or a polyethylene glycol;

[0005] The method includes:

[0006] Anti-Ig antibodies linked to a solid-phase support are used to capture the target antibody in the sample.

[0007] The antibody to be tested binds to the modified antigen;

[0008] The modified antigen binds directly and / or indirectly to the detection marker;

[0009] The detection marker is detected.

[0010] The present invention also provides a modified antigen, comprising an antigen and a modifier linked to the antigen, wherein the modifier is a betaine, a tertiary amine oxide, or a polyethylene glycol.

[0011] The present invention also provides the use of the modified antigens described above in the preparation of kits for detecting antibodies.

[0012] The present invention also provides a kit comprising an anti-Ig antibody, a modified antigen as described above, and a detection marker. Attached Figure Description

[0013] Figure 1A schematic diagram illustrating the principle of an immune detection method according to an embodiment of the present invention. Detailed Implementation

[0014] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0016] An embodiment of the present invention provides a capture method for detecting antibodies, wherein the method completes the detection in the form of anti-Ig antibody-analyte antibody-modified antigen, wherein the modified antigen includes an antigen and a modifier linked to the antigen, and the modifier is a betaine, an oxidized tertiary amine, or a polyethylene glycol;

[0017] The method includes:

[0018] Anti-Ig antibodies linked to a solid-phase support are used to capture the target antibody in the sample.

[0019] The antibody to be tested binds to the modified antigen;

[0020] The modified antigen binds directly and / or indirectly to the detection marker;

[0021] The detection marker is detected.

[0022] In some implementations, the modified antigen is directly bound to the detection marker, for example, the modified antigen is directly labeled with the detection marker, such as acridine ester.

[0023] In some embodiments, the modified antigen binds indirectly to the detection marker. In some embodiments, the modified antigen binds indirectly to the detection marker via a first bridging molecule and a second bridging molecule; and / or the modified antigen binds indirectly to the detection marker via an antibody against the antigen. In some embodiments, the first bridging molecule is biotin, a carbohydrate, or a tag protein, and the second bridging molecule is avidin, anti-biotin antibody, lectin, or an antibody capable of specifically binding to the tag protein. It is understood that the specific types of the first and second bridging molecules are not limited thereto; the first and second bridging molecules constitute a pair of bridging molecules, and common bridging molecules with affinity properties, such as receptor-ligand, antibody-antigen, etc., can be selected as needed. In some embodiments, the first bridging molecule is linked to the antigen via protein fusion; for example, a tag protein (such as GST) is fused to the antigen. In some embodiments, the first bridging molecule is linked to the antigen via chemical means (such as covalent bonds, hydrogen bonds, etc.).

[0024] In some embodiments, within a reaction system, the modified antigen directly binds to the detection marker and indirectly binds to the detection marker; for example, within a reaction system, the modified antigen directly binds to acridine ester, and the modified antigen indirectly binds to acridine ester.

[0025] In some embodiments, within a reaction system, the modified antigen indirectly binds to the detection marker simultaneously using two or more modes; for example, in one reaction system, the modified antigen indirectly binds to acridine ester via biotin-avidin, and the modified antigen indirectly binds to acridine ester via a tag protein-antibody; for example, in one reaction system, the modified antigen indirectly binds to acridine ester via biotin-avidin, and the modified antigen indirectly binds to acridine ester via an antibody against the antigen; for example, in one reaction system, the modified antigen indirectly binds to acridine ester via a tag protein-antibody, and the modified antigen indirectly binds to acridine ester via an antibody against the antigen; for example, in one reaction system, three or more indirect binding modes are used simultaneously to detect the marker, the modified antigen indirectly binds to acridine ester via a tag protein-antibody, the modified antigen indirectly binds to acridine ester via an antibody against the antigen, and the modified antigen indirectly binds to acridine ester via biotin-avidin.

[0026] In some embodiments, the above method further includes a washing step after the anti-Ig antibody linked to the solid-phase carrier contacts the sample. It is understood that the specific sample addition pattern can be selected as needed. For example, the anti-Ig antibody linked to the solid-phase carrier and the modified antigen can be added to the sample first, followed by washing, and then the addition of a second bridging molecule linked to a detection marker and an antibody against the antigen linked to the detection marker; or the anti-Ig antibody linked to the solid-phase carrier can be added to the sample first, followed by washing, and then the modified antigen, the second bridging molecule linked to the detection marker, and the antibody against the antigen linked to the detection marker, etc., can be added. In some embodiments, washing is performed before detecting the detection marker. It is understood that the above method is a specific example of the application of modified antigens in the capture method, and it can also be applied to indirect methods or other forms of immunoassay methods.

[0027] In this invention, contact refers to the process by which the sample comes into contact with and is mixed with the anti-Ig antibody attached to the solid-phase carrier, thereby causing any possible components therein to undergo an immune response.

[0028] In some implementations, the detection markers are metal particles, fluorescent markers, chromophore markers, electron-dense markers, chemiluminescent markers, radioactive markers, or enzyme markers, but are not limited to these. It is understood that the corresponding detection method can be selected as needed, depending on the detection marker; for example, fluorescent markers can be detected using fluorescence detection methods, and enzyme markers can be detected using substrate reactions, etc.

[0029] In some implementations, the detection markers are rhodamine, fluorescein, quantum dots, digoxigenin-labeled probes, radioisotopes, radioactive contrast agents, ultrasound contrast agents, photosensitizers, fluorescent microspheres, colloidal gold, acrid esters, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, sugar oxidase, glucose oxidase, galactose oxidase, or glucose-6-phosphate dehydrogenase.

[0030] In some embodiments, the modifier is a betaine, a tertiary amine oxide, or a polyethylene glycol. The betaine includes, for example, sulfobetaines, carboxybetaines, and phosphate betaines. It is understood that derivatives of the above substances can also function effectively as substitutes. These electrically neutral modifiers can reduce the high background of proteins used in immunoassays by improving protein solubility. The polyethylene glycol can be, for example, polyethylene glycol or polyethylene glycol fatty acid esters. Preferably, the polyethylene glycol is linear polyethylene glycol and / or branched polyethylene glycol with a molecular weight of 100–3000.

[0031] In some implementations, the anti-Ig antibody is an anti-human IgM antibody, an anti-human IgG antibody, etc., which can be adjusted according to the type of antibody to be tested. Furthermore, the anti-human IgM antibody can be an anti-μ chain antibody, etc.

[0032] An embodiment of the present invention provides a modified antigen, which includes an antigen and a modifier respectively linked to the antigen. The modifier is a betaine, a tertiary amine oxide, or a polyethylene glycol.

[0033] In some implementations, the antigen is a recombinant antigen or a natural antigen. Optionally, the antigen may be Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus antigen, EBV herpesvirus antigen, parvovirus antigen, or varicella-zoster virus antigen. It is understood that the specific type of antigen is not limited to these and may be adjusted according to the type of antibody to be tested.

[0034] An antigen conjugate according to an embodiment of the present invention includes the modified antigen described above and a second bridging molecule connected to a detection marker, wherein the second bridging molecule is connected to a first bridging molecule; or includes the modified antigen described above and a solid-phase carrier, wherein the solid-phase carrier is connected to the antigen.

[0035] In some implementations, the solid support is magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips, but is not limited to these; other conventional solid supports may be selected as needed.

[0036] The application of the modified antigen or antigen conjugate as described above in the preparation of a kit for detecting antibodies, according to one embodiment of the present invention.

[0037] One embodiment of the kit of the present invention includes an anti-Ig antibody and a modified antigen or an antigen conjugate as described above.

[0038] In some implementations, the kit further includes one or more of a solid-phase carrier, a first bridging molecule, a second bridging molecule, an antibody against the antigen, and a detection marker.

[0039] The present invention will be further described in detail below with reference to specific embodiments.

[0040] Example 1: Detection of Toxoplasma gondii (TOX) IgM

[0041] 1. IgM capture antibody magnetic bead coating

[0042] Mix 10 mg of Tosyl magnetic beads (0.05 μm to 3 μm in diameter) solution, remove the supernatant by magnetic separation, wash three times with 0.02 M PB buffer, resuspend, add 0.5 mg of coating antibody, mix well, and incubate at 37 °C for 2–4 h under continuous rotation. Remove the supernatant by magnetic separation, wash three times with 0.02 M PB buffer, block with 20% lysine, remove the supernatant by magnetic separation, resuspend the magnetic beads in stock solution, the concentration of antibody coating on the magnetic beads is 5%, collect and store at 2–8 °C for subsequent experiments.

[0043] 2. TOX antigen modification and biotinylation

[0044] Activation of PEG(n)-COOH and Bio-COOH groups

[0045] The carboxyl groups were activated in DMSO solvent at a molecular ratio of n(-COOH):n(EDC):n(NHS) = 1:1:3 for 30 min at room temperature. The activated modified groups and activated biotin groups were mixed with TOX antigen in the designed ratio and reacted at room temperature for 2 h. The reaction was terminated with 1 M lysine and reacted for another 30 min at room temperature. The mixture was then packed into a desalting column and purified by elution with 20 mM PBS (pH 7.4). The modified antigen was collected.

[0046] 3. Streptavidin AE labeling

[0047] The SA concentration was adjusted to 1 mg / mL using 0.1 M boric acid (pH 5.5) + 0.02% Tw20 buffer. A 4 mM acridine ester was prepared using DMSO. The mixture was then added according to a molecular ratio of n(SA):n(AE) = 1:2. After mixing, the mixture was reacted in the dark for half an hour. The mixture was then packed into a desalting column and eluted with 0.01 M boric acid (pH 5.5) for purification. The SA-acrididine ester conjugate was collected.

[0048] 4. TOX antibody AE labeling

[0049] The concentration of TOX antibody was adjusted to 2 mg / mL with 20 mM PB (pH 7.4) buffer. 4 mM acridine ester was prepared with DMSO. Then, the materials were added according to the molecular ratio of n(antibody):n(AE) = 1:5. The mixture was mixed and reacted in the dark for half an hour. After that, it was packed into a desalting column and eluted with 20 mM PB (pH 7.4) to purify the antibody-acrididine ester conjugate.

[0050] 5. Testing

[0051] (1) Dilute the sample with sample processing solution at a ratio of 1:100. Then take 100 μL of the diluted sample and 50 μL of 0.1 mg / mL capture antibody magnetic bead working solution, mix well, and react at 37 °C for 15 min. Adsorb the magnetic beads with a magnetic adsorption plate and remove the supernatant. Then add PBST washing solution to wash the magnetic beads, adsorb them with the magnetic plate, remove the supernatant, and repeat this operation 4 times.

[0052] (2) Add 50 μL of 2 μg / mL TOXAg-Bio (modified or unmodified) working solution and 100 μL of 250 ng / mL SA-AE (with or without 50 ng / mL TOX antibody AE labeling), mix well, and react at 37℃ for 15 min. Adsorb the magnetic beads using a magnetic adsorption plate and remove the supernatant. Then add PBST washing buffer to wash the magnetic beads, adsorb them using the magnetic plate, remove the supernatant, and repeat this operation 4 times.

[0053] (3) Add 100 μL of luminescent excitation solution A and 100 μL of luminescent excitation solution B, and collect the luminescent signal using a luminometer.

[0054] Table 1

[0055]

[0056]

[0057] Note: Mode A: MP-Ab1+IgM / Ag-AE (i.e., forming a complex of anti-Ig antibody-detection antibody-antigen-detection marker)

[0058] Mode B: MP-Ab1+IgM / Ag+Ab2-AE (i.e., forming a complex of anti-Ig antibody-detection antibody-antigen-antibody against antigen-detection marker)

[0059] Mode C: MP-Ab1+IgM / Ag-Bio+SA-AE (i.e., forming a complex of anti-Ig antibody-detection antibody-antigen-first bridging molecule-second bridging molecule-detection marker)

[0060] Mode D: MP-Ab1+IgM / Ag(PEG)-Bio+SA-AE (i.e., forming a complex of anti-Ig antibody-analyte antibody-modified antigen-first bridging molecule-second bridging molecule-detection marker)

[0061] E mode: MP-Ab1+IgM / Ag(PEG)-Bio+SA-AE+Ab2-AE (i.e., forming a complex of anti-Ig antibody-analyte antibody-modified antigen-(first bridging molecule-second bridging molecule-detection marker) / (antibody against antigen-detection marker)).

[0062] SD: Test blank

[0063] (MP-Ab1: Magnetic bead anti-μ chain antibody; IgM: Antibody to be tested; Ag(PEG)-Bio: Biotinylated PEGylated TORCH antigen; SA-AE: Streptavidin AE (acridoid ester) marker; Ab2-AE: TORCH antigen antibody AE (acridoid ester) marker)

[0064] Based on the results in Table 1, we can conclude that:

[0065] (1) Comparison of detection modes A and B: Under detection mode B, the P / N value of the positive and negative serum detection signals increased, indicating that the positive and negative serums had a certain degree of differentiation, which shows that the detection sensitivity can be improved by using antibody indirect labeling.

[0066] (2) Comparison of detection modes B and C: The P / N value of positive and negative samples is higher in mode C than in mode B, indicating that the sensitivity of mode C is higher than that of mode B. SA / Bio mode can improve detection sensitivity.

[0067] (3) Comparison of C and D detection modes: The P / N value of positive and negative samples is higher in mode D than in mode C, indicating that proper PEG modification of the antigen can improve detection sensitivity.

[0068] (4) Comparison of D and E detection modes: The P / N value of positive and negative samples is higher in mode E than in mode D, indicating that using two labeling modes, such as Ab2-AE and SA-AE, can improve detection sensitivity.

[0069] Example 2: Rv IgM Detection

[0070] Rv IgM detection was performed according to the method in Example 1, and the results are shown in Table 2.

[0071] Table 2

[0072]

[0073] Note: Mode A: MP-Ab1+IgM / Ag-AE (i.e., forming a complex of anti-Ig antibody-detection antibody-antigen-detection marker)

[0074] Mode B: MP-Ab1+IgM / Ag+Ab2-AE (i.e., forming a complex of anti-Ig antibody-detection antibody-antigen-antibody against antigen-detection marker)

[0075] Mode C: MP-Ab1+IgM / Ag-Bio+SA-AE (i.e., forming a complex of anti-Ig antibody-detection antibody-antigen-first bridging molecule-second bridging molecule-detection marker)

[0076] Mode D: MP-Ab1+IgM / Ag(PEG)-Bio+SA-AE (i.e., forming a complex of anti-Ig antibody-analyte antibody-modified antigen-first bridging molecule-second bridging molecule-detection marker)

[0077] E mode: MP-Ab1+IgM / Ag(PEG)-Bio+SA-AE+Ab2-AE (i.e., forming a complex of anti-Ig antibody-analyte antibody-modified antigen-(first bridging molecule-second bridging molecule-detection marker) / (antibody against antigen-detection marker)).

[0078] SD: Test blank

[0079] (MP-Ab1: Magnetic bead anti-μ chain antibody; IgM: Antibody to be tested; Ag(PEG)-Bio: Biotinylated PEGylated TORCH antigen; SA-AE: Streptavidin AE (acridoid ester) marker; Ab2-AE: TORCH antigen antibody AE (acridoid ester) marker)

[0080] Based on the results in Table 3, we can conclude that:

[0081] (1) Comparison of detection modes A and B: Under detection mode B, the P / N value of positive and negative serum detection signals increases, indicating that the detection sensitivity can be improved by using antibody indirect labeling.

[0082] (2) Comparison of detection modes B and C: The P / N value of positive and negative samples is higher in mode C than in mode B, indicating that the sensitivity of mode C is higher than that of mode B. SA / Bio mode can improve detection sensitivity.

[0083] (3) Comparison of C and D detection modes: The P / N value of positive and negative samples is higher in mode D than in mode C, indicating that proper PEG modification of the antigen can improve detection sensitivity.

[0084] (4) Comparison of D and E test mode data: The P / N value of positive and negative samples is higher in mode E than in mode D, indicating that using two labeling modes, such as Ab2-AE and SA-AE, can improve detection sensitivity.

[0085] Example 3: Sulfobetaine Modification

[0086] N-propanesulfonyl betaine was activated by carboxylation and modified with antigen according to the method in Example 1, and then Rv IgM was detected. The results showed that MP-Ab1+IgM / Ag(NDSB)-Bio+SA-AE+Ab2-AE in E mode can reduce the detection background and improve the detection sensitivity.

[0087] N-propanesulfonate betaine was activated by carboxylation and modified with antigen according to the method in Example 1, and then Rv IgG was detected. The results showed that MP-Ab1+IgG / Ag(NDSB)-Bio+SA-AE+Ab2-AE in E mode can reduce the detection background and improve the detection sensitivity.

[0088] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A capture method for detecting antibodies for non-diagnostic purposes, characterized in that, The method completes the detection by forming an anti-Ig antibody-detector antibody-modified antigen, wherein the modified antigen includes an antigen and a modifier linked to the antigen, and the modifier is a betaine, an oxidized tertiary amine, or a polyethylene glycol. The method includes: Anti-Ig antibodies linked to a solid-phase support are used to capture the target antibody in the sample. The antibody to be tested binds to the modified antigen; The modified antigen binds directly and / or indirectly to the detection marker; The detection marker is detected; the modified antigen binds indirectly to the detection marker through a first bridging molecule and a second bridging molecule; The modified antigen binds indirectly to the detection marker via an antibody targeting the antigen.

2. The method according to claim 1, characterized in that, The first bridging molecule is biotin, carbohydrate, or a tag protein, and the second bridging molecule is avidin, anti-biotin antibody, lectin, or an antibody that can specifically bind to the tag protein.

3. The method according to any one of claims 1 to 2, characterized in that, The method also includes a washing step after the anti-Ig antibody attached to the solid-phase carrier comes into contact with the sample.

4. The method according to any one of claims 1 to 2, characterized in that, The detection markers are metal particles, fluorescent markers, chromophore markers, electron-dense markers, chemiluminescent markers, radioactive markers, or enzyme markers.

5. The method according to any one of claims 1 to 2, characterized in that, The betaines include sulfobetaines, carboxybetaines, and phosphate betaines.

6. The method according to any one of claims 1 to 2, characterized in that, The modifier includes polyethylene glycol or polyethylene glycol fatty acid esters.

7. The method according to any one of claims 1 to 2, characterized in that, The anti-Ig antibody is an anti-human IgM antibody or an anti-human IgG antibody, for example, it can be an anti-μ chain antibody.

8. A modified antigen, characterized in that, The modified antigen is the modified antigen according to claim 1, comprising an antigen and a modifier linked to the antigen, wherein the modifier is a betaine, a tertiary amine oxide, or a polyethylene glycol; the modified antigen indirectly binds to the detection marker through a first bridging molecule and a second bridging molecule; The modified antigen binds indirectly to the detection marker via an antibody targeting the antigen.

9. The modified antigen according to claim 8, characterized in that, The betaines include sulfobetaines, carboxybetaines, and phosphate betaines.

10. The modified antigen according to claim 8, characterized in that, The modifier is polyethylene glycol or polyethylene glycol fatty acid ester.

11. The use of the modified antigen of claim 8 in the preparation of a kit for detecting antibodies.

12. A reagent kit, characterized in that, Includes the anti-Ig antibody, modified antigen, and detection marker as described in any one of claims 1-7.

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