Antioxidant low density lipoprotein monoclonal antibodies and uses thereof

Abstract

The application discloses an oxidized low-density lipoprotein monoclonal antibody and application thereof, and belongs to the technical field of antibodies. The oxidized low-density lipoprotein monoclonal antibody provided by the application has high specificity, high affinity and high sensitivity. An immunoturbidimetric detection kit prepared based on the oxidized low-density lipoprotein monoclonal antibody can quantitatively detect oxidized low-density lipoprotein in peripheral blood samples, and has a good diagnosis effect on cardiovascular diseases caused by atherosclerosis (AS).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to antioxidant low-density lipoprotein monoclonal antibodies and their applications. Background Technology

[0002] Low-density lipoprotein (LDL) is a lipoprotein particle that carries cholesterol into peripheral tissue cells. Oxidized low-density lipoprotein (Ox-LDL) is formed when these particles are oxidized and modified. Ox-LDL is a crucial factor in the development and progression of atherosclerosis (AS). Serum Ox-LDL expression is significantly elevated in hypertensive AS patients, and its expression level is positively correlated with CIMT (carotid intima-media thickness) in hypertensive patients, thus contributing to cardiovascular disease. Therefore, monitoring and controlling serum Ox-LDL levels in hypertensive patients can effectively improve AS and reduce the risk of cardiovascular disease.

[0003] The main method for conventional Ox-LDL detection is enzyme-linked immunosorbent assay (ELISA) using different antibodies. This method is relatively cumbersome and time-consuming, and is not suitable for continuous and rapid detection. Therefore, a simpler method is needed to detect oxidized low-density lipoprotein. Summary of the Invention

[0004] To address the above issues, this invention provides an antioxidant low-density lipoprotein monoclonal antibody and its applications. By adding a second specific antibody to the traditional monoclonal antibody model, the antigen is simultaneously bound by both antibodies through dual-site recognition, which allows dispersed small complexes to crosslink into larger aggregates, significantly improving the intensity and stability of turbidity signals.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention uses low-density lipoprotein as a raw material and oxidizes it in vitro with copper sulfate to obtain oxidized low-density lipoprotein.

[0007] This invention uses oxidized low-density lipoprotein (ODL) as an immunogen to immunize New Zealand white rabbits, and screens to obtain cell clones 3E7 and 4B12 that can secrete antibodies against ODL. The nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region are obtained by RT-PCR amplification. These nucleotide sequences are cloned into a eukaryotic expression vector, and CHO-K1 suspension cells are transiently transfected using a transfection method. The supernatant is collected after culture, and the supernatant is purified using Protein A to obtain antibodies with a purity greater than 90%.

[0008] The antibody 3E7 includes a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 2; the second antibody is antibody 4B12, which includes a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 4.

[0009] The nucleotide sequence of the heavy chain variable region of antibody 3E7 is shown in SEQ ID No: 5, and the nucleotide sequence of the light chain variable region of antibody 3E7 is shown in SEQ ID No: 6.

[0010] The nucleotide sequence of the heavy chain variable region of antibody 4B12 is shown in SEQ ID No: 7, and the nucleotide sequence of the light chain variable region of antibody 4B12 is shown in SEQ ID No: 8.

[0011] An immunoconjugate comprising an antibody portion and a coupling portion coupled to the antibody portion, the antibody portion comprising the antioxidant low-density lipoprotein antibody of claim 1, and the coupling portion being selected from polypropylene latex microspheres.

[0012] An oxidized low-density lipoprotein (ODL) detection kit comprises a first antibody and a second antibody; the first antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; the second antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4.

[0013] The kit is based on latex-enhanced immunoturbidimetric assay for detecting oxidized low-density lipoprotein (ODL) and includes reagents R1, R2, R3, and calibrators. Reagent R1 is a latex microsphere solution conjugated with anti-Ox-LDL antibody 3E7; reagent R2 is a latex microsphere solution conjugated with anti-Ox-LDL antibody 4B12; reagent R3 is a reaction buffer; and the calibrators and quality control samples consist of different concentrations of Ox-LDL.

[0014] The beneficial effects of this invention are as follows: This invention utilizes dual-site recognition to allow antigens to bind simultaneously to two antibodies, significantly enhancing the intensity and stability of turbidity signals and enabling more accurate detection and quantification. This technology requires less sample volume, greatly reducing the burden on patients; it offers rapid detection, shortening diagnostic waiting times; it is simple and easy to operate, lowering the technical requirements for operators; its economical and practical features make it widely cost-effective; and its excellent specificity ensures the accuracy of test results. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of SDS-PAGE analysis of antibodies 3E7 and 4B12 in Example 3 of the present invention; Figure 2 This is the calibration curve of the antioxidant low-density lipoprotein detection kit in this invention. Detailed Implementation

[0016] The present invention will be further illustrated below with reference to specific embodiments. These examples are merely illustrative and not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0017] Example 1 Preparation of oxidized low-density lipoprotein Low-density lipoprotein (LDL) was dialyzed against PBS buffer at 4°C for 12 hours to remove antioxidants or salts from the original storage solution. Protein concentration was then determined using the BCA method. The dialyzed LDL was then mixed with PBS containing CuSO4 to allow CuSO4 to be absorbed. 2+ The final concentration was 50 μM, and the final concentration of low-density lipoprotein was 1 mg / mL. After incubation at 37°C in the dark for 12 hours, the reaction was terminated by adding EDTA to a final concentration of 1 mM. Dialysis was then continued at 4°C with PBS buffer for 12 hours to completely remove Cu. 2+ Finally, store in PBS (pH 7.4) containing 0.01 μM EDTA at 2–8 °C, protected from light.

[0018] Example 2 Preparation of antioxidant low-density lipoprotein antibodies New Zealand white rabbits were immunized with the oxidized low-density lipoprotein prepared in Example 1. For the initial immunization, oxidized low-density lipoprotein was mixed with Freund's complete adjuvant in an equal ratio, and three New Zealand white rabbits were subcutaneously injected with an immunization dose of 500 μg per rabbit. Subsequently, every 14 days, oxidized low-density lipoprotein was mixed with Freund's incomplete adjuvant in an equal ratio for the second and third subcutaneous immunizations. Ten days after the third immunization, blood was collected from the marginal ear vein, and the serum was obtained by centrifugation. The serum was serially diluted, and the serum before immunization was used as a negative control. The serum titer was measured and is shown in Table 1.

[0019] Table 1: Serum titer detection of New Zealand White rabbits

[0020] New Zealand white rabbit No. 3, which had the highest serum titer, was given a booster immunization. Three days later, blood was collected from the marginal ear vein under aseptic conditions to prepare a single-cell suspension of lymphocytes. Positive cells were sorted by flow cytometry and cultured in 96 culture plates for 7-10 days. The presence of specific antibodies in the cell supernatant was detected by indirect ELISA. Two wells with high positive OD values ​​were selected, namely 3E7 and 4B12.

[0021] Example 3 Preparation, purification, and titer detection of recombinant antibodies against antioxidant low-density lipoprotein Total RNA was extracted from the two positive cells in Example 2 and reverse transcribed to obtain cDNA. The nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were obtained by PCR amplification.

[0022] The above-mentioned heavy and light chain nucleotide sequences were cloned into the constructed rabbit antibody expression vector, and CHO-K1 suspension cells were transfected using transient transfection. After 7 days of culture, the supernatant was collected. The collected cell supernatant was centrifuged at 12000 rpm for 10 min and then purified by Protein A affinity chromatography. The antibody was eluted with 0.1 M glycine-hydrochloric acid solution (pH 2.7) and neutralized with 1 M pH 9.0 Tris buffer. The purified antibody was dialyzed overnight with PBS solution, and the antibody purity was detected by SDS-PAGE. The results showed that both antibody heavy chains had bands at 50 KD, and the antibody light chains had bands at 25 KD. The purity of both was greater than 90% according to grayscale analysis. Figure 1 .

[0023] Oxidized low-density lipoprotein was diluted to 1 μg / ml with CBS, coated overnight at 4°C, and then blocked with 10% skim milk powder for 2 h. The purified antibodies were serially diluted to 100 μL per well and incubated at 37°C for 1 h. After washing, goat anti-rabbit IgG-HRP was added, and the plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and after washing, TMB was added for color development. The OD450 value was read after stopping the reaction with stop solution. The titers of monoclonal antibodies 3E7 and 4B12 were 8 × 10⁻⁶. 6 and 4×10 7 .

[0024] Example 4 Sequencing of monoclonal antibodies Sequencing of the variable regions of the heavy and light chains of two monoclonal antibodies, 3E7 and 4B12, was performed. Sequencing analysis yielded the amino acid sequences of the variable regions of the heavy and light chains of both antibodies. Example 5 Oxidized Low-Density Lipoprotein Assay Kit The Oxidized Low-Density Lipoprotein (OLDL) Assay Kit includes: Reagent R1, Reagent R2, Reagent R3, and a series of OLDL calibrators and quality control products.

[0025] (1) Reagent R1: Polypropylene latex microspheres coupled with the primary antibody; (2) Reagent R2: Polypropylene latex microspheres coupled with a second antibody; (3) Reagent R3: 0.01 M PBS, pH 7.4, containing 0.1% BSA, 0.05% Tween-20, and 0.02% p300; (4) Calibrators and quality control samples: Oxidized low-density lipoprotein was prepared into calibrators with concentrations of 0 U / L, 5 U / L, 10 U / L, 20 U / L, 50 U / L, and 100 U / L using diluent. A high-concentration oxidized low-density lipoprotein sample (100 U / L) was diluted with diluent to 10 U / L and 50 U / L, which were used as low-value and high-value quality control samples, respectively.

[0026] Example 6 reagent kit accuracy Using the test kit described in Example 5, one sample each of the high-concentration (50±10 U / L) and low-concentration (10±2 U / L) oxidized low-density lipoprotein (ODL) corporate reference samples were tested, with three repeated tests. The test results are shown in Table 2. Based on the relative deviation of the test results, the relative deviation of the three test results for the two reference samples was within 5%, indicating that the test kit has good accuracy.

[0027] Table 2. Accuracy results of the oxidized low-density lipoprotein detection kit

[0028] Example 7 linear range of the reagent kit The expected linear range of the oxidized low-density lipoprotein (ODL) assay kit in Example 5 is 5.0–100 U / L. One high-value sample (120 U / L) and one low-value sample (2 U / L) were selected near the upper and lower limits of this range. These samples were diluted with calibrator diluent at different ratios to prepare five different concentration levels (5 U / L, 10 U / L, 20 U / L, 50 U / L, and 100 U / L). The ODL assay kit from Example 5 was used to detect these samples. The results are shown in Table 3. After curve fitting and linear analysis, the linear regression equation for the assay kit is: y = 1.0476x - 0.5726, R0. 2 =0.9998>0.995( Figure 2 The results indicate that the expected linear range is met; the relative deviation between the measured value and the theoretical value for each concentration sample is no more than 10%, which is acceptable.

[0029] Table 3. Detection results of the linear range of the oxidized low-density lipoprotein assay kit.

[0030] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antioxidant low-density lipoprotein antibody, characterized in that, The antibody comprises a first antibody and a second antibody. The first antibody is antibody 3E7, which includes a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:

2. The second antibody is antibody 4B12, which includes a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:

4.

2. The nucleic acid encoding the antioxidant low-density lipoprotein antibody of claim 1, characterized in that, The nucleotide sequence of the heavy chain variable region of antibody 3E7 is shown in SEQ ID No: 5, and the nucleotide sequence of the light chain variable region of antibody 3E7 is shown in SEQ ID No: 6; the nucleotide sequence of the heavy chain variable region of antibody 4B12 is shown in SEQ ID No: 7, and the nucleotide sequence of the light chain variable region of antibody 4B12 is shown in SEQ ID No:

8.

3. An expression vector comprising the nucleic acid of claim 2.

4. A transgenic cell line comprising the nucleic acid of claim 2 or the expression vector of claim 3.

5. An immunoconjugate, characterized in that, It comprises an antibody portion and a coupling portion conjugated to the antibody portion, the antibody portion comprising the antioxidant low-density lipoprotein antibody of claim 1, and the coupling portion being selected from polypropylene latex microspheres.

6. A kit for detecting oxidized low-density lipoprotein, characterized in that, The mixture comprises a first antibody and a second antibody; the first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2; the second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO:

4.

7. The oxidized low-density lipoprotein detection kit according to claim 6, characterized in that, The kit is based on latex-enhanced immunoturbidimetry for the detection of oxidized low-density lipoprotein.

8. The oxidized low-density lipoprotein detection kit according to claim 6, characterized in that, It includes the first antibody and the second antibody used; the first antibody is a binding antibody, and the second antibody is an antibody used by the Overseas Chinese Federation.

9. The oxidized low-density lipoprotein detection kit according to claim 6, characterized in that, The kit includes polypropylene latex microspheres R1 coated with the first antibody, polypropylene latex microspheres R2 coated with the second antibody, buffer reagent R3, and a series of oxidized low-density lipoprotein calibrators and quality control products; the buffer reagent R3 is 0.01 M PBS, pH 7.4, containing 0.1% BSA, 0.05% Tween-20 and 0.02% p300 by volume.

10. The use of the first antibody or the second antibody as described in claim 1 in the preparation of an oxidized low-density lipoprotein detection reagent or kit.

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