Related application of modified albumin as sealing agent in immunodetection

Abstract

The invention discloses related application of modified albumin as a sealing agent in immunodetection, and relates to the field of biological detection. According to the present invention, the chemically modified BSA with the charged surface is adopted as the blocking protein in the immunodetection, such that the surface affinity of the blocking agent on the solid phase carrier is improved, the problem that the existing blocking agent is easily replaced is avoided or reduced, and the non-specific adsorption during the immunodetection process is reduced so as to effectively improve the sensitivity of the immunodetection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and more particularly, to the related application of modified albumin as a blocking agent in immunoassay. Background Art

[0002] In the field of immunoassay, using a solid-phase carrier to achieve B / F separation is a common method. For example, in colloidal gold lateral flow immunoassay, nitrocellulose membrane is used as a carrier; in ELISA (enzyme-linked immunosorbent assay), enzyme-labeled plates are used as a carrier; in CLEIA (chemiluminescent immunoassay), magnetic microparticles are used as a carrier, and so on. Taking CLEIA as an example, during the immunoassay process, an antibody-antigen reaction occurs between the coated antibody on the magnetic microparticles and the analyte. Through magnetic separation, the immune complex can be separated from the sample to be detected. Then, a labeled antibody is added to react with it, and a signal is generated by the label to achieve quantitative or qualitative analysis of the substance to be measured. The sensitivity of various immunoassay methods using solid-phase carriers is affected by the background signal generated by the non-specific binding between the labeled antibody and the solid-phase carrier. In this case, even if the analyte does not exist in the sample, a positive measurement value may still be shown, resulting in a deviation from the true value.

[0003] To address the problem of non-specific binding between the labeled antibody and the solid-phase carrier, using more stringent post-treatment conditions, such as optimizing the working solution concentration of the labeled antibody and magnetic beads, adjusting the pH of the working solution, etc., may reduce the background signal generated by non-specific adsorption to a certain extent. However, these solutions need to be balanced in terms of detection performance. For example, introducing a stronger washing procedure may lead to signal loss, and changing the concentration of the labeled or coated antibody may result in insufficient antigen capture, thereby reducing the detection sensitivity.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide the related application of charge-modified albumin as a blocking agent in immunoassay.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, an embodiment of the present invention provides the application of modified albumin in the preparation of a blocking agent for immunoassay, wherein the modified albumin includes any one or more of positively charged albumin and negatively charged albumin. Among them, positively charged albumin is used in combination with a negatively charged solid-phase carrier, and negatively charged albumin is used in combination with a positively charged solid-phase carrier.

[0008] In a second aspect, an embodiment of the present invention provides an immunoassay method, which includes: performing B / F separation using the solid-phase carrier blocked with the modified albumin described in the foregoing embodiment.

[0009] In a third aspect, an embodiment of the present invention provides a reagent, a kit or a blocking agent for immunoassay, which includes: the modified albumin described in the aforementioned embodiment.

[0010] The present invention has the following beneficial effects:

[0011] The present invention adopts BSA with a surface charge after chemical modification as a blocking protein in immunoassay, thereby improving the surface affinity of the blocking agent for the solid phase carrier, avoiding or reducing the problem that the existing blocking agent is easily replaced, reducing nonspecific adsorption during the immunoassay, and thus effectively improving the sensitivity of the immunoassay. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0013] Blocking the nonspecific adsorption sites on the surface of magnetic beads by adding blocking agents is one of the solutions to reduce background. Blocking agents are a type of protein or polymer that can be adsorbed on the surface of solid phase carriers, which can coat the surface of hydrophobic particles, thereby reducing the occurrence of nonspecific adsorption. There are many types of blocking agents, among which bovine serum albumin (BSA) is the most widely used, but for some high-sensitivity detection projects, BSA still cannot meet the actual needs of the project. Due to the high surface free energy of the solid phase carrier, when exposed to a solution containing protein, it will quickly adsorb one or more layers of protein to form a "protein corona". Studies have found that the number and type of proteins on the "protein corona" are not fixed, and high-affinity proteins in the solution will gradually replace low-affinity proteins. This phenomenon is called the "Vorman effect". When using proteins such as BSA to block the nonspecific adsorption sites of magnetic beads, it is still possible that they will be replaced by labeled antibodies during the immunoassay process, resulting in nonspecific adsorption signals.

[0014] In this regard, the inventors of the present application have discovered and verified through a series of studies that using positively charged albumin or negatively charged albumin as the blocking protein in the blocking agent can enhance its surface affinity for the solid phase carrier, thereby achieving the effect of reducing non-specific adsorption. Different charge modifications of the blocking protein can be applied to different solid phase carrier systems. For example, after carboxyl or Tosyl magnetic beads are coated with antibodies, the exposed parts of their surfaces carry negative charges due to residual functional groups, especially when the pH of the immunoassay reaction system is relatively high. Therefore, positively charged modified BSA can achieve effective blocking. Similarly, for the surfaces of other positively charged solid phase carriers, negatively charged modified BSA will exhibit better blocking effects.

[0015] Specific technical solutions

[0016] On the one hand, the embodiments of the present invention provide the application of modified albumin in the preparation of a blocking agent for immunoassay, and the modified albumin includes any one or more of positively charged albumin and negatively charged albumin.

[0017] In some embodiments, the positive charge modification includes amination or onium ion formation.

[0018] In some embodiments, the amination includes primary amination, secondary amination or tertiary amination.

[0019] In some embodiments, the positively charged albumin is aminated albumin.

[0020] The present invention places no special limitation on the modification methods of positively charged and negatively charged albumin. The main inventive concept of the present invention is to use positively / negatively charged albumin as the blocking agent in immunoassay, and the specific modification methods can be implemented based on conventional modification methods.

[0021] In some embodiments, the preparation method of the aminated albumin includes: reacting albumin with an amination reagent to obtain the aminated albumin.

[0022] In some embodiments, the amination reagent includes any one or more of ethylenediamine, propylenediamine and 1,2,3-triaminoguanidine.

[0023] In some embodiments, the ratio of albumin to the amination reagent is: 1 g: 1 - 8 g.

[0024] In some embodiments, for every 1 g of albumin, the amount of the amination reagent added is any one or the range between any two of 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL and 10 mL.

[0025] In some embodiments, before mixing and reacting albumin with an amination reagent, the method for preparing the aminated albumin further includes: dissolving albumin in buffer 1 and adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0026] In some embodiments, the ratio of the albumin to the buffer 1 is: for every 1 g of albumin, 150 - 300 mL of buffer 1 is added.

[0027] In some embodiments, for every 1 g of albumin, the addition amount of buffer 1 is any one or the range between any two of 150, 160, 180, 200, 220, 240, 260, 280, 300 mL.

[0028] In some embodiments, the pH of the buffer 1 is 7 - 8; specifically, the pH can be any one or the range between any two of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.

[0029] In some embodiments, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is: for every 1 g of albumin, 50 - 200 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added.

[0030] In some embodiments, for every 1 g of albumin, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is any one or the range between any two of 50, 60, 80, 100, 120, 140, 160, 180, 200 mg.

[0031] In some embodiments, the reaction conditions of the albumin and the amination reagent include: 10 - 30 °C, 1 - 3 h. Specifically, the reaction temperature can be any one or the range between any two of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 °C. Specifically, the reaction time can be any one or the range between any two of 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3 h.

[0032] In some embodiments, the negatively charged modification includes carboxylation, sulfonation, sulfation, phosphorylation, or phosphonation.

[0033] In some embodiments, the negatively charged albumin is carboxylated albumin.

[0034] In some embodiments, the method for preparing the carboxylated albumin includes: reacting albumin with a carboxylating reagent to obtain carboxylated albumin.

[0035] In some embodiments, the carboxylation reagent includes any one or more of succinic anhydride, itaconic anhydride, and maleic anhydride.

[0036] In some embodiments, the ratio of albumin to the carboxylation reagent is: 1 g : 80 - 200 mg.

[0037] In some embodiments, for every 1 g of albumin, the addition amount of the carboxylation reagent is in the range of any one or between any two of 80, 100, 120, 140, 160, 180, 200 mg.

[0038] In some embodiments, before reacting albumin with the carboxylation reagent, the preparation method of the carboxylated albumin further includes: dissolving albumin in buffer 2.

[0039] In some embodiments, the ratio of albumin to buffer 2 is: for every 1 g of albumin, 150 - 300 mL of buffer 2 is added.

[0040] In some embodiments, for every 1 g of albumin, the addition amount of buffer 2 is in the range of any one or between any two of 150, 160, 180, 200, 220, 240, 260, 280, 300 mL.

[0041] In some embodiments, the pH of buffer 2 is 7 - 8. The pH can specifically be any one or in the range between any two of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.

[0042] In some embodiments, the reaction conditions of albumin and the carboxylation reagent are: 10 - 30 °C, 1 - 3 h. The reaction temperature can specifically be any one or in the range between any two of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 °C. The reaction time can specifically be any one or in the range between any two of 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3 h.

[0043] In some embodiments, the albumin includes BSA or HSA.

[0044] In some embodiments, the immunoassay is a heterogeneous immunoassay.

[0045] "Heterogeneous immunoassay", also known as heterogeneous immunoassay, refers to the need to separate unbound labels from the reaction system before signal detection. Otherwise, regardless of the concentration of the analyte, the signal levels generated are the same. Heterogeneous immunoassays usually rely on coating reaction substances such as antigens and antibodies on solid-phase substances (such as reaction plates, reaction micro-wells, or magnetic beads, etc.), separating unbound labeled antigens or antibodies from the reaction system through a washing step, and then checking the amount of antigen-antibody complexes through various different signal reporting systems. Since separation is a crucial step in the immunoassay process, many heterogeneous separation techniques have gradually emerged during the development of immunoassay technologies, such as centrifugation, filtration, solid-phase plates, magnetic particle separation using magnetic fields, etc.

[0046] In some embodiments, the heterogeneous immunoassay includes any one or more of: enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, electrochemiluminescence assay, radioimmunoassay (RIA), immunoradiometric assay (IRMA), and fluorescence immunoassay.

[0047] In some embodiments, the immunoassay includes a B / F separation step. "B" in "B / F" is the bound label (the complex of labeled antigen and specific antibody), and "F" is the free label.

[0048] In some embodiments, the blocking agent is used to block the solid-phase carrier.

[0049] In some embodiments, the solid-phase carrier includes at least one of a tube, a plate, a membrane, and microspheres.

[0050] In some embodiments, the solid-phase carrier includes magnetic beads.

[0051] In some embodiments, the solid-phase carrier and the modified albumin carry opposite charges.

[0052] On the other hand, the embodiments of the present invention also provide an immunoassay method, which includes: performing B / F separation using the solid-phase carrier blocked with the modified albumin described in any of the foregoing embodiments; the immunoassay method is not for the purpose of diagnosing or treating diseases.

[0053] In some embodiments, the solid-phase carrier and the modified albumin carry opposite charges.

[0054] In some embodiments, the steps of the detection method may include: coating an antigen or an antibody on the solid-phase carrier, or obtaining a solid-phase carrier coated with an antigen or an antibody; adding an analyte and related reagents (including a label that binds to the analyte) to the solid-phase carrier for mixing reaction; performing B / F separation after blocking the solid-phase carrier with the modified albumin; and performing signal detection.

[0055] In some embodiments, when performing blocking, the weight ratio of the modified albumin to the solid-phase carrier is 20:1 to 10:1.

[0056] In some embodiments, when using the modified albumin to block the solid-phase carrier, the modified albumin serves as the blocking protein in the blocking agent.

[0057] In some embodiments, the mass fraction of the blocking protein in the blocking agent is 0.1% to 5%.

[0058] In some embodiments, the pH of the blocking agent is 7 to 8. Specifically, the pH can be any one of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8, or the range between any two of them.

[0059] In some embodiments, the pH of the blocking agent is 7.2 to 7.6.

[0060] In some embodiments, the blocking agent further includes: a buffer solution.

[0061] In some embodiments, the blocking agent further includes Tween-20.

[0062] In some embodiments, the mass fraction of Tween-20 in the blocking agent is 0.1% to 5%.

[0063] In some embodiments, the mass fraction of Tween-20 in the blocking agent can be any one of 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or the range between any two of them.

[0064] In some embodiments, there are many detection situations that are not for the diagnosis or treatment of diseases. For example, when the analyte has nothing to do with the diagnosis or treatment of diseases, or the test sample is an environmental sample or an artificially made sample, the detection method is not directly for the diagnosis or treatment of diseases.

[0065] On the other hand, the embodiments of the present invention also provide a reagent, kit, or blocking agent for immunoassay, which includes: the modified albumin described in any of the foregoing embodiments.

[0066] In some embodiments, the reagent, kit, or blocking agent further includes a solid-phase carrier.

[0067] In some embodiments, the solid-phase carrier includes at least one of a tube, a plate, a membrane, and microspheres.

[0068] In some embodiments, the solid-phase carrier includes magnetic beads.

[0069] In some embodiments, the solid-phase carrier and the modified albumin carry opposite charges.

[0070] In some embodiments, the material of the solid-phase carrier is one or more of polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, and agarose gel.

[0071] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0072] Example 1

[0073] A kind of aminoated BSA, and its synthesis method is as follows:

[0074] Dissolve 1 g of BSA in 200 mL of PBS (pH 7.4), add 100 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), then add 6.675 mL of pure ethylenediamine solution, stir at room temperature for 2 hours, and remove unreacted small molecule impurities by dialysis.

[0075] Example 2

[0076] A kind of aminoated BSA, and its synthesis method is as follows:

[0077] Dissolve 1 g of BSA in 200 mL of PBS (pH 7.4), add 120 mg of succinic anhydride, stir at room temperature for 2 hours, and remove small molecule impurities by dialysis.

[0078] Test Example 1

[0079] Respectively prepare the corresponding blocking agents with aminoated BSA (Example 1), carboxylated BSA (Example 2), and conventional BSA (control BSA). The formula of the blocking agent is as follows: The blocking agent buffer is PBS (pH 7.4). In the blocking agent, the mass concentration of the protein is 1%, and the mass concentration of Tween-20 is 0.5%.

[0080] Taking the TP (syphilis antibody) magnetic particle chemiluminescence project as an example for verification, after the magnetic beads are coated with antibodies, add the corresponding blocking agent and incubate for 24 h. It is found from the test results of serum samples with different values (Table 1) that the carboxylated BSA blocking agent can significantly improve the sensitivity of low-value detection, and its P / N is increased by about 40%. This is mainly because the carboxylated BSA has a higher affinity for the surface of the magnetic beads in this system, can effectively block the hydrophobic sites, and reduce their aggregation.

[0081] Table 1 Detection results of the TP project

[0082]

[0083] Test Example 2

[0084] The three kinds of proteins, namely aminoated BSA (Example 1), carboxylated BSA (Example 2), and conventional BSA, were respectively formulated into corresponding blocking agents, and their formulations were as follows: The buffer of the blocking agent was HEPES (pH 8.0), the mass concentration of the protein in the blocking agent was 1%, and the mass concentration of Tween-20 was 0.5%.

[0085] Taking the hs-CTnI magnetic particle chemiluminescence project as an example for verification, after the magnetic beads were coated with antibodies, the corresponding blocking agent was added and incubated for 24 h, and the test results of serum samples with different values were found (Table 2).

[0086] Table 2 Detection Results of hs-CTnI Project

[0087]

[0088] It can be seen from the results that the blocking effect of conventional BSA on the hs-CTnI project is very poor, and its relatively high background leads to a significant decrease in the sensitivity of the project. This is mainly because the pH of the test buffer for this project is relatively high, and conventional BSA carries more negative charges in this buffer, resulting in poor affinity with the negatively charged magnetic bead surface. However, aminoated BSA can well overcome this problem. It can be seen from the test results that the sensitivity of the aminoated BSA blocking agent is about 2.5 times higher than that of the conventional BSA blocking agent.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a modified albumin in the preparation of a blocking agent for immunoassay, characterized in that, The modified albumin includes any one or more of positively charged albumin and negatively charged albumin.

2. The application according to claim 1, wherein The positive charge modification includes amination or onium ion formation, and the negative charge modification includes carboxylation, sulfonation, sulfation, phosphorylation or phosphonation; Optionally, the amination includes primary amination, secondary amination or tertiary amination.

3. The application according to claim 1, wherein The positively charged albumin is aminated albumin.

4. The application according to claim 1, characterized in that, The negatively charged albumin is carboxylated albumin.

5. The application according to claim 1, characterized in that The albumin includes BSA or HSA.

6. The application according to any one of claims 1 to 5, characterized in that, The immunoassay is a heterogeneous immunoassay; Optionally, the immunoassay includes a B / F separation step.

7. The application according to any one of claims 1 to 5, characterized in that, The blocking agent is used to block the solid phase carrier; Optionally, the solid phase carrier includes at least one of a tube, a plate, a membrane and microspheres; Optionally, the solid phase carrier includes magnetic beads; Optionally, the solid phase carrier and the modified albumin carry opposite charges.

8. An immunoassay method, characterized in that, It includes: performing B / F separation using a solid phase carrier blocked with the modified albumin according to any one of claims 1-7; Optionally, the solid phase carrier and the modified albumin carry opposite charges.

9. A reagent or kit for immunoassay, characterized in that, It includes: The modified albumin according to any one of claims 1 to 7.

10. The reagent or kit according to claim 9, characterized in that, The reagent or kit further includes a solid phase carrier; Optionally, the solid phase carrier includes at least one of a tube, a plate, a membrane and microspheres; Optionally, the solid phase carrier includes magnetic beads; Optionally, the solid phase carrier and the modified albumin carry opposite charges.

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