ELISA (enzyme-linked immuno sorbent assay) kit composition and kit for detecting advanced oxidized protein products

Abstract

The invention belongs to the technical field of biology, and particularly relates to an ELISA (enzyme-linked immuno sorbent assay) kit composition and kit for detecting advanced oxidized protein products. The kit composition is prepared from the following raw materials: 20 to 50 mM of Tris-HCl, 100 to 200 mM of NaCl, 0.5 to 2.0 percent (w / v) of fetal calf serum, 0.1 to 0.5 percent (w / v) of sodium caseinate, 1 to 5 mM of disodium ethylene diamine tetraacetate, 1 to 5 percent (w / v) of trehalose, 0.05 to 0.2 percent (w / v) of polyvinylpyrrolidone and 0.02 to 0.05 percent (v / v) of ProCin 300. The kit composition provided by the invention is applied to the kit, so that the kit has better accuracy, sensitivity and stability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an ELISA kit composition and kit for detecting late-stage oxidized protein products. Background Technology

[0002] Late oxidative protein products (AOPPs) are bistyrosine-containing protein cross-links formed from proteins (especially albumin) during myeloperoxidase-mediated oxidation. Elevated AOPP levels are closely associated with the severity and prognosis of end-stage renal disease, diabetic complications, inflammatory diseases, and cardiovascular diseases. Enzyme-linked immunosorbent assay (ELISA) has become the mainstream technique for detecting AOPPs due to its high throughput, relatively simple operation, and good specificity. Currently, most commercially available AOPP ELISA kits employ a double-antibody sandwich or competitive assay principle; however, most suffer from insufficient sensitivity and specificity, poor long-term stability, and weak anti-interference capabilities, limiting the accuracy and wide applicability of the assay.

[0003] For example, patent CN116375855A discloses a monoclonal antibody that specifically recognizes AOPPs and its applications. The inventors obtained a hybridoma cell line secreting the monoclonal antibody AP-4C5 (accession number CCTCCNO.: C202232) through experimental screening. The monoclonal antibody AP-4C5 can specifically recognize the oxidized groups in AOPPs without reacting with natural proteins. A sandwich ELISA system was established using AP-4C5 as the coating antibody and HRP-AP-4C5 as the detection antibody, which can detect AOPP-HSA and AOPP-FB at concentrations of 0-20 μg / ml and can quantitatively detect AOPPs in human serum. A multi-monoclonal antibody sandwich ELISA system was established using commercially available anti-human albumin (HSA) polyclonal antibody as the coating antibody and HRP-AP-4C5 as the detection antibody, which can detect AOPPs at concentrations of 0-40 μg / ml and can quantitatively detect AOPPs in the plasma of healthy individuals and patients. However, the long-term stability of this invention still needs improvement.

[0004] For example, the literature "Study on the Molecular Mechanism of Late Oxidation Protein Products Inducing Chondroitin Inflammatory Response," Southern Medical University, 2019.000600. This study elucidates the effects of late oxidation protein products on the expression of interleukins and tumor necrosis factor. However, the research on kits related to late oxidation protein products in this study is inadequate.

[0005] Therefore, there is an urgent need to develop an ELISA kit composition for detecting late-stage oxidized protein products that has high sensitivity and specificity, good long-term stability, and high detection accuracy. Summary of the Invention

[0006] The purpose of this invention is to provide an ELISA kit composition and kit for detecting late-stage oxidized protein products, and related technologies, to solve the technical problems of insufficient sensitivity and specificity, poor long-term stability, and low accuracy of existing ELISA kits for late-stage oxidized protein products, or combinations thereof.

[0007] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0008] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0009] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0010] In a first aspect, the present invention provides an ELISA kit composition for detecting late-stage oxidized protein products, comprising the following raw materials: 20-50mM Tris-HCl, 100-200mM NaCl, 0.5%-2.0% w / v fetal bovine serum, 0.1%-0.5% w / v sodium caseinate, 1-5mM disodium EDTA, 1%-5% w / v trehalose, 0.05%-0.2% w / v polyvinylpyrrolidone, and 0.02%-0.05% v / v ProClin 300.

[0011] Preferably, the kit composition comprises the following raw materials: 30 mM Tris-HCl, 150 mM NaCl, 1.0% w / v fetal bovine serum, 0.25% w / v sodium caseinate, 2 mM disodium EDTA, 3% w / v trehalose, 0.1% w / v polyvinylpyrrolidone, and 0.03% w / v ProClin300.

[0012] Preferably, the pH of the kit composition is 5.5-6.5, more preferably 6.0.

[0013] Secondly, the present invention also provides a method for preparing the above-mentioned reagent kit composition, comprising the following steps: mixing Tris-HCl, NaCl, fetal bovine serum, sodium caseinate, disodium ethylenediaminetetraacetate, trehalose and polyvinylpyrrolidone in proportion, then adding ProClin 300 and mixing, adjusting the volume, and filtering to obtain the kit composition.

[0014] Preferably, the filtration is performed using a 0.22μm filter membrane under positive pressure.

[0015] Preferably, the volume adjustment is performed to a volume of 1000 mL.

[0016] Thirdly, the present invention also provides the application of the above-described kit composition in the preparation of an ELISA kit for detecting late-stage oxidized protein products.

[0017] Preferably, the kit composition is used to improve the accuracy, sensitivity, and stability of the kit.

[0018] Fourthly, the present invention also provides an ELISA kit for detecting late-stage oxidized protein products.

[0019] Preferably, the kit comprises: the above-described kit composition, quality control materials, and calibrators.

[0020] Preferably, the calibrator is prepared by using late-stage oxidized protein products as raw materials for the calibrator, dissolving them in the kit composition, and mixing thoroughly.

[0021] Preferably, the method for preparing the quality control product is as follows: using late-stage oxidized protein products as raw materials for the quality control product, dissolving them in the kit composition, and mixing thoroughly to obtain the product.

[0022] The present invention has the following beneficial effects: The reagent kit composition provided by this invention improves the accuracy, sensitivity, and stability of the kit through the interaction between its components. Specifically, fetal bovine serum and sodium caseinate work synergistically to occupy binding sites for interfering substances, jointly enhancing the stability and accuracy of the kit; polyvinylpyrrolidone increases solution viscosity, reduces surface adsorption, and stabilizes the reaction interface. Detailed Implementation

[0023] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0024] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0025] The AOPPs detection kit used in this invention was purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml024017.

[0026] The fetal bovine serum used in this invention was purchased from Merck Life Sciences, Inc., catalog number F8687.

[0027] The sodium caseinate used in this invention was purchased from Maclean Biotechnology Co., Ltd., product number C6334.

[0028] The ProClin 300 used in this invention was purchased from McLean Biotechnology Co., Ltd., product number P675347.

[0029] Example 1: An ELISA kit composition for detecting late-stage oxidized protein products. Composed of the following raw materials: 30 mM Tris-HCl, 150 mM NaCl, 1.0% w / v fetal bovine serum, 0.25% w / v sodium caseinate, 2 mM disodium EDTA, 3% w / v trehalose, 0.1% w / v polyvinylpyrrolidone and 0.03% w / v ProClin300, balance purified water, pH 6.0.

[0030] Example 2: An ELISA kit composition for detecting late-stage oxidized protein products. Composed of the following raw materials: 20 mM Tris-HCl, 100 mM NaCl, 0.5% w / v fetal bovine serum, 0.1% w / v sodium caseinate, 1 mM disodium EDTA, 1% w / v trehalose, 0.05% w / v polyvinylpyrrolidone and 0.02% w / v ProClin300, balance purified water, pH 6.5.

[0031] Example 3: An ELISA kit composition for detecting late-stage oxidized protein products. Composed of the following raw materials: 50 mM Tris-HCl, 200 mM NaCl, 2.0% w / v fetal bovine serum, 0.5% w / v sodium caseinate, 5 mM disodium EDTA, 5% w / v trehalose, 0.2% w / v polyvinylpyrrolidone and 0.05% w / v ProClin300, balance purified water, pH 5.5.

[0032] The above Examples 1-3 were prepared according to the following method: Mix Tris-HCl, NaCl, fetal bovine serum, sodium caseinate, disodium EDTA, trehalose, and polyvinylpyrrolidone according to the specified ratio, then add ProClin 300 and mix well. Make up to 1000 mL and filter through a 0.22 μm filter membrane to obtain the final product.

[0033] Comparative Example 1 The difference from Example 1 is that 0.5% w / v sodium caseinate was replaced with 0.5% w / v fetal bovine serum, and it was prepared as follows: Tris-HCl, NaCl, fetal bovine serum, disodium EDTA, trehalose and polyvinylpyrrolidone were mixed in the specified proportions, ProClin 300 was added and mixed, and the volume was adjusted to 1000 mL. The mixture was then filtered through a 0.22 μm filter membrane to obtain the final product.

[0034] Comparative Example 2 The difference from Example 1 is that 1.0% w / v fetal bovine serum was replaced with 1.0% w / v sodium caseinate, and the preparation was carried out as follows: Tris-HCl, NaCl, sodium caseinate, disodium EDTA, trehalose and polyvinylpyrrolidone were mixed in the specified proportions, ProClin 300 was added and mixed, and the volume was adjusted to 1000 mL. The mixture was then filtered through a 0.22 μm filter membrane to obtain the final product.

[0035] Comparative Example 3 The difference from Example 1 is that polyvinylpyrrolidone is replaced with polyethylene glycol 4000, and it is prepared according to the following method: Tris-HCl, NaCl, fetal bovine serum, sodium caseinate, disodium EDTA, trehalose and polyethylene glycol 4000 are mixed in the specified proportions, ProClin 300 is added and mixed, the volume is adjusted to 1000 mL, and filtered through a 0.22 μm filter membrane to obtain the final product.

[0036] Comparative Example 4 The difference from Example 1 is that trehalose is replaced with sucrose, and it is prepared as follows: Tris-HCl, NaCl, fetal bovine serum, sodium caseinate, disodium EDTA, sucrose and polyvinylpyrrolidone are mixed in the specified proportions, ProClin 300 is added and mixed, the volume is adjusted to 1000 mL, and the mixture is filtered through a 0.22 μm filter membrane to obtain the final product.

[0037] Comparative Example 5 The difference from Example 1 is that the proportions of each component are different, specifically: 10mM Tris-HCl, 250mM NaCl, 2.5% w / v fetal bovine serum, 0.05% w / v sodium caseinate, 0.5mM disodium EDTA, 8% w / v trehalose, 0.25% w / v polyvinylpyrrolidone, and 0.03% w / v ProClin 300.

[0038] Detection example The sample diluent in the AOPPs detection kit (purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.) was replaced with the kit compositions prepared in Examples 1-3 and Comparative Examples 1-5 above, and the methodological indicators were evaluated as follows: (1) Accuracy: The recovery rate should be between 85% and 115%; (2) Blank detection limit: should not be higher than 16.53 pg / mL; (3) Linear correlation coefficient: Within the specified linear interval (16.53-10000 pg / mL): calculate the linear correlation coefficient (r), generally r≥0.990; (4) Precision: Repeat the test on a sample 10 times and calculate the coefficient of variation (CV). Generally, CV ≤ 8%, the smaller the CV, the better the precision. (5) Inter-batch variation: Different batches of reagents are used to test the same sample. The inter-batch relative range (R) is calculated. Generally, R ≤ 10.0%. The smaller the R, the smaller the inter-batch variation.

[0039] (6) Long-term stability: The reagents were stored at 2-8℃ for 28 months, and their accuracy, limit of detection, linear correlation coefficient, inter-batch difference and precision were evaluated every 3 months. The results are shown in Tables 1-6.

[0040] Table 1 Performance data for day 0

[0041] Table 2 Long-term stability of accuracy

[0042] Table 3 Long-term stability of the limit of detection

[0043] Table 4 Long-term stability of linear correlation coefficients

[0044] Table 5. Long-term stability of batch differences

[0045] Table 6 Long-term stability of precision

[0046] As can be seen from Table 1, the reagent composition prepared in the embodiments of the present invention, when applied to the kit, gives the kit better accuracy and sensitivity.

[0047] As can be seen from the comparison between Table 1 and Tables 2-6, the reagent composition prepared in the embodiments of the present invention, when applied to the kit, gives the kit better long-term stability.

[0048] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An ELISA kit composition for detecting late-stage oxidized protein products, characterized in that, Including the following raw materials: 20-50mM Tris-HCl, 100-200mM NaCl, 0.5%-2.0% w / v fetal bovine serum, 0.1%-0.5% w / v sodium caseinate, 1-5mM disodium EDTA, 1%-5% w / v trehalose, 0.05%-0.2% w / v polyvinylpyrrolidone, and 0.02%-0.05% v / v ProClin 300.

2. The reagent kit composition according to claim 1, characterized in that, Including the following raw materials: 30 mM Tris-HCl, 150 mM NaCl, 1.0% w / v fetal bovine serum, 0.25% w / v sodium caseinate, 2 mM disodium EDTA, 3% w / v trehalose, 0.1% w / v polyvinylpyrrolidone, and 0.03% w / v ProClin 300.

3. The reagent kit composition according to claim 1, characterized in that, The pH of the kit composition is 5.5-6.

5.

4. A method for preparing the reagent kit composition according to any one of claims 1-3, characterized in that, Includes the following steps: Mix Tris-HCl, NaCl, fetal bovine serum, sodium caseinate, disodium EDTA, trehalose, and polyvinylpyrrolidone in the specified proportions, then add ProClin 300 and mix well. Make up to a final volume and filter to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The filtration process uses a 0.22μm filter membrane.

6. Use of the kit composition according to any one of claims 1-3 in the preparation of an ELISA kit for detecting late-stage oxidized protein products.

7. An ELISA kit for detecting late-stage oxidized protein products, characterized in that, Includes the kit composition according to any one of claims 1-3.

8. The ELISA kit according to claim 7, characterized in that, The kit also includes quality control materials and calibrators.

9. The ELISA kit according to claim 7, characterized in that, The method for preparing the calibrator is as follows: using late-stage oxidized protein products as raw materials for the calibrator, dissolving them in the kit composition according to any one of claims 1-3, and mixing thoroughly to obtain the calibrator.

10. The ELISA kit according to claim 7, characterized in that, The method for preparing the quality control product is as follows: using late-stage oxidized protein products as raw materials for the quality control product, dissolving them in the kit composition according to any one of claims 1-3, and mixing thoroughly to obtain the product.

Images