A detection kit based on beta-trace protein monoclonal antibody and application thereof

Abstract

The application belongs to the technical field of biomedicine, and discloses a detection kit based on a beta-trail protein monoclonal antibody and application thereof. The application provides two strains of monoclonal antibodies which specifically recognize different antigen epitopes of the beta-trail protein, have high affinity and good specificity, and can be used for detection of the beta-trail protein. The application also provides a beta-trail protein detection kit with good performance, which has a wide linear range, high precision, good repeatability and strong applicability. The detection kit prepared by using the monoclonal antibody can be applied to kidney disease screening detection, disease monitoring, prognosis evaluation and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a detection kit based on a monoclonal antibody against β-trace protein and its application. Background Technology

[0002] In clinical practice, the most commonly used endogenous substances for assessing glomerular filtration rate (GFR) are serum creatinine (Scr) and endogenous creatinine clearance (Ccr). Serum creatinine (Scr) is affected by factors such as age and muscle mass, and its level only increases in moderate to severe renal impairment. Therefore, it cannot reflect changes in the condition of patients with near-normal glomerular filtration rates. Endogenous creatinine clearance (Ccr) is a more sensitive indicator of GFR decline than Scr, but measuring Ccr requires accurate 24-hour urine collection, leading to poor patient compliance. Furthermore, in patients with renal insufficiency, increased compensatory tubular secretion of creatinine may result in an overestimation of Ccr. In recent years, some new serum biomarkers, such as cysteine ​​protease inhibitor C (cystatin C, CysC) and beta trace protein (BTP), have been discovered as indicators of glomerular filtration rate.

[0003] Beta-trace protein (BTP) is a glycosylated, bifunctional monomeric protein, also known as lipocalin-type prostaglandin D synthase (L-PGDS). It contains 168 amino acids and has a molecular weight between 23,000 and 29,000. It catalyzes the synthesis of prostaglandin D2 and the transport of lipophilic substances. BTP has been found in various human body fluids and tissue fluids, and its content in the body is not affected by factors such as sex, age, or nutrition. Almost all of it is excreted through the kidneys. Serum BTP levels are more sensitive in assessing kidney damage in hemodialysis patients, monitoring acute kidney injury in kidney transplant patients, evaluating kidney injury in elderly diabetic patients, and early prediction of hypertensive disorders of pregnancy (HDCP), reflecting an early decline in glomerular filtration rate (GFR). The urinary BTP excretion rate can predict the progression of proteinuria and kidney damage, reflecting renal tubular dysfunction, making it an ideal marker of kidney injury.

[0004] β-Traces Protein (BTP) detection mainly employs ELISA and immunoturbidimetry, which are performed on specific protein analyzers. However, these methods are expensive and lack standardization. Traditional immunoturbidimetry uses a single antibody model for baseline turbidity detection, and its sensitivity and stability require improvement.

[0005] In view of the shortcomings of the prior art, the present invention provides a β-trace protein detection kit and its preparation method. Based on the traditional single antibody mode, a second specific antibody is added. Through dual-site recognition, the antigen is simultaneously bound by the two antibodies, which can cross-link the dispersed small complexes into larger aggregates, significantly improving the intensity and stability of the turbidity signal, and making the instrument more accurate in detection and quantification. Summary of the Invention

[0006] This invention provides a detection kit based on β-trace protein monoclonal antibody and its application, in order to solve the problems of low sensitivity, narrow linear range and poor accuracy of existing β-trace protein detection antibodies, and improve the performance of β-trace protein antibody raw materials and β-trace protein antigen detection reagent products.

[0007] A first aspect of the present invention provides a β-trace protein antibody or an antigen-binding fragment thereof, comprising:

[0008] The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; or, The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4.

[0009] Optionally, the antibody or its antigen-binding fragment may recognize and / or specifically bind to epitopes of β-trace proteins as shown in SEQ ID NO: 5 or SEQ ID NO: 6.

[0010] Optionally, the antibody is a monoclonal antibody; the antigen-binding fragment of the antibody is selected from F(ab)2, Fab, or scFv.

[0011] A second aspect of the present invention provides an immunoconjugate comprising an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the aforementioned β-trace protein antibody or its antigen-binding fragment, and the coupling portion is selected from polypropylene latex microspheres.

[0012] A third aspect of the present invention provides a β-trace protein detection kit comprising a first antibody and a second antibody; the first antibody comprising 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 comprising 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.

[0013] Optionally, the kit is based on latex-enhanced immunoturbidimetry for the detection of β-trace proteins.

[0014] Optionally, the kit includes a first antibody and a second antibody; the first antibody, as a binding antibody, binds to the antigenic epitope of the β-trace protein to complete preliminary antigen recognition; the second antibody, as a bridging antibody, binds to another antigenic epitope of the β-trace protein, bridging and cross-linking the small complexes of "antigen-first antibody" to form a multimeric immune complex, which significantly increases the particle size, significantly improves the turbidity, and enhances the detection sensitivity.

[0015] Optionally, the kit includes polypropylene latex microspheres R1 coated with the first antibody, polypropylene latex microspheres R2 coated with the second antibody, buffer R3, and a series of calibrators and quality control products for β-trace protein antigens.

[0016] In a fourth aspect, the present invention provides the use of the above-described β-trace protein antibody or its antigen-binding fragment in the preparation of a diagnostic reagent or kit for kidney injury.

[0017] The beneficial effects of this invention are as follows: This invention, through immunization with recombinant β-trace protein antigen and screening of monoclonal antibodies, obtained β-trace protein monoclonal antibodies with high affinity and good specificity that recognize different antigen sites. Furthermore, through appropriate reagent preparation, a β-trace protein detection kit with excellent performance was obtained.

[0018] The β-trace protein detection kit of the present invention has high accuracy, high sensitivity, wide linear range, and high correlation; the reagent has high precision, good repeatability, and strong applicability. Attached Figure Description

[0019] Figure 1 This is an SDS-PAGE gel image of the expression and purification of β-trace protein in Example 1 of this invention; Figure 2 This is an SDS-PAGE gel image of the monoclonal antibody in Example 3 of this invention; Figure 3 This is the detection result analysis curve of the linear range of the β-trace protein kit in Example 8 of the present invention. Detailed Implementation

[0020] 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.

[0021] Example 1: Recombinant expression of β-trace protein Based on the β-trace protein sequence information (NP_000945.3) published by the National Center for Biotechnology Information (NCBI), its full-length sequence was cloned into the pCDNA3.1 eukaryotic expression vector. The resulting plasmid was transfected into HEK293 cells using PEI transfection reagent and cultured for 7 days in a constant temperature shaker at 37°C, 8% CO2, and 120 rpm. The cell supernatant after expression was centrifuged at 4°C, 12000 rpm for 10 min, filtered through a 0.45 μm filter membrane, and purified by nickel affinity chromatography to obtain the recombinant β-trace protein, approximately 23-29 kDa. Figure 1 ).

[0022] Example 2: Preparation of hybridoma cell lines for β-trace protein monoclonal antibodies The recombinant β-trace protein prepared in Example 1 was used as an immunogen to immunize BALB / c mice. For the initial immunization, the antigen and Freund's complete adjuvant were mixed in an equal proportion and injected subcutaneously, with each mouse receiving 50 μg, and three mice were immunized. Every 14 days, the antigen and Freund's incomplete adjuvant were mixed in an equal proportion and injected subcutaneously for the second and third immunizations, with each mouse receiving 30 μg. Ten days after the third immunization, blood was collected from the tail vein, centrifuged, and the serum was serially diluted. The serum before immunization was used as a negative control, and the serum titer was measured, as shown in Table 1.

[0023] Table 1. Serum titers of mouse immune cells

[0024] Three days after a booster immunization of mice with the highest serum titer, spleen cells were harvested under aseptic conditions. SP2 / 0 and spleen cells were mixed in a centrifuge tube and centrifuged to remove the supernatant. 1 ml of pre-warmed PEG was slowly added dropwise to a 50 ml centrifuge tube and incubated for 1 min. Then, pre-warmed DMEM medium was added to a final volume of 35 mL. The mixture was incubated at 42°C for 15 min, followed by centrifugation at 800 rpm for 10 min, and the supernatant was discarded. 100 mL of pre-warmed HAT medium was added, and the mixture was gently pipetted to mix. The mixture was then transferred to 200 μL of feeder cells in each well of a 96-well plate and incubated statically in a CO2 incubator. Seven days later, the supernatant of the fusion cells was analyzed using an indirect ELISA method. Positive hybridomas were screened for clones, yielding six hybridoma cell lines secreting specific anti-β-trace protein monoclonal antibodies, named 2G8, 2F6, 3E4, 4A7, 4B6, and 5H2. The antibody subtype results showed that all were IgG1.

[0025] Example 3: Preparation, purification, and titer detection of β-trace protein monoclonal antibodies Liquid paraffin was injected intraperitoneally into mice. 7-10 days later, the hybridoma cell lines obtained in Example 2 were inoculated into the peritoneum of the mice. After the mice's abdomens expanded, ascites fluid was collected. The collected ascites fluid was centrifuged at 8000 rpm / min for 10 min, and the supernatant was collected. 10-20 times the volume of Protein A equilibration buffer was added to the supernatant for Protein A affinity chromatography purification. The antibody was eluted with 0.1 M glycine-hydrochloric acid solution (pH 3.0) and neutralized with 1 M Tris buffer (pH 8.8). The purified antibody was dialyzed against PBS, and the antibody purity was detected by SDS-PAGE. The results showed that all six antibody strains had a heavy chain band at 50 KD and a light chain band at 25 KD. The purity of all strains was greater than 95% according to grayscale analysis. Figure 2 .

[0026] β-traces protein was diluted to 2 μg / ml with CBS, coated overnight at 4°C, and then blocked with 10% skim milk powder for 2 h. The purified antibody was serially diluted, 100 μl per well, and incubated at 37°C for 1 h. After washing, goat anti-mouse IgG-HRP was added, and the plate was incubated at 37°C for 1 h. 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 2G8, 2F6, 3E4, 4A7, 4B6, and 5H2 were 1x10⁻⁶. 6 1x10 7 1x10 7 1x10 8 1x10 6 and 1x10 7 .

[0027] Example 4: Screening of monoclonal antibodies against β-trace proteins that recognize different antigen binding sites Monoclonal antibodies recognizing different antigen-binding sites of β-trace proteins were screened using ELISA pairing. First, purified monoclonal antibodies 2G8, 2F6, 3E4, 4A7, 4B6, and 5H2 were used as capture and detection antibodies, respectively, for antibody pairing verification. Microplates were coated with 2 μg / ml of antibody and incubated overnight at 4°C. After blocking with 10% skim milk powder at 37°C for 2 h, the plates were washed, and 100 μl of different concentrations (0-100 ng / ml) of β-trace protein were added. The plates were incubated at 37°C for 60 min, washed again, and then 100 μl of HRP-labeled 2G8, 2F6, 3E4, 4A7, 4B6, and 5H2 at a concentration of 0.1 μg / ml were added and incubated at 37°C for 30 min. After washing, TMB substrate was added, and the reaction was carried out for 15 min. The reaction was terminated with stop solution, and the absorbance (OD450) was measured using a microplate reader. The pairing results are shown in Table 2. One pair of paired antibodies was screened.

[0028] Table 2: Monoclonal antibody pairing results for β-trace protein

[0029] Example 5: Sequencing and Epitope Analysis of Monoclonal Antibodies (1) Antibody Sequencing: The variable regions of the light and heavy chains of the two monoclonal antibody hybridoma cell lines 2F6 and 5H2 were sequenced. After sequencing analysis, the amino acid sequences of the heavy and light chain variable regions of the first antibody 2F6 and the second antibody 5H2 were obtained. The amino acid sequence of the heavy chain variable region of 2F6 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of 5H2 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. Both antibody subtypes are mouse antibody IgG1.

[0030] (2) Antibody binding epitope analysis: The binding epitopes of the two monoclonal antibodies 2F6 and 5H2 were analyzed. The binding epitopes of both antibodies were linear epitopes, and the relevant information is shown in Table 4. Among them, the binding epitope of monoclonal antibody 2F6 is amino acids 89 to 102 of the full-length β-trace protein, and the amino acid sequence is shown in SEQ ID NO: 5, binding epitope aa89-aa102; the binding epitope of monoclonal antibody 5H2 is amino acids 29 to 42 of the full-length β-trace protein, and the amino acid sequence is shown in SEQ ID NO: 6, binding epitope aa89-aa102.

[0031] Example 6: Establishment of a β-trace protein detection kit based on latex-enhanced immunoturbidimetry A latex reagent was prepared by covalently coupling the aforementioned β-trace protein antibody to carboxylated latex microspheres. A reaction buffer containing buffer, stabilizer, and turbidity enhancer was used. Key parameters such as antibody coating amount, reagent ratio, reaction pH, and temperature were optimized. After performance verification including calibrator values, precision, accuracy, linearity, and anti-interference, a β-trace protein quantitative detection kit compatible with fully automated biochemical analyzers was finally established. This kit includes reagents R1, R2, buffer reagent R3, calibrators, and quality control samples.

[0032] (1) Reagent R1: Polypropylene latex microspheres coupled with the primary antibody; (2) Reagent R2: Polypropylene latex microspheres coupled with a second antibody; (3) Buffer reagent R3: 0.01 M PBS, pH 7.4, containing 0.1% BSA, 0.05% Tween-20 and 0.02% p300 by volume; (4) Calibrators and quality control samples: The recombinant β-trace protein antigen was prepared into calibrators with concentrations of 0 μg / mL, 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL using the calibrator diluent. A high-concentration β-trace protein sample (100 μg / mL) was diluted with the calibrator diluent to 1 μg / mL and 10 μg / mL, which were used as low-value and high-value quality control samples, respectively.

[0033] Example 7: Accuracy of the Reagent Kit Using the detection kit described in Example 6, one sample each of the high-concentration (10±1 μg / mL) and low-concentration (1±0.1 μg / mL) β-trace protein enterprise reference samples were tested, with three repeated tests. The results are shown in Table 3. 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 kit has good accuracy.

[0034] Table 3. Accuracy results of the β-trace protein detection kit

[0035] Example 8: Linear Range of the Kit The expected linear range of the β-trace protein detection kit in Example 6 is 0.1-20 μg / mL. Two samples were selected, one high-value sample (21.5 μg / mL) and one low-value sample (0.15 μg / mL), each near the upper and lower limits of this range. These samples were diluted at different ratios using zero-value β-trace protein calibrator diluent to prepare five different concentration levels (0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL). The β-trace protein detection kit from Example 6 was used to detect these samples. The detection results are shown in Table 4. After curve fitting and linear analysis, the linear regression equation of the detection kit is: y = 1.0519x - 0.1227, R0. 2 =0.9997>0.995 (e.g.) Figure 3 As shown in the figure, the preliminary judgment is that it meets the expected linear range requirements; the relative deviation between each measured value and the theoretical value of each concentration sample is no more than 10%, and the expected linear range is acceptable.

[0036] Table 4. Detection results of the linear range of the β-trace protein detection kit.

[0037] 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. A β-trace protein antibody, characterized in that, The antibody is a monoclonal antibody, comprising a first antibody and a second antibody. The first antibody has 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 has 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.

2. The antibody according to claim 1, characterized in that, The antigen-binding fragment of the antibody is selected from F(ab)2, Fab, or scFv, and can recognize and / or specifically bind to epitopes of β-trace proteins as shown in SEQ ID NO: 5 or SEQ ID NO:

6.

3. A nucleic acid encoding the β-trace protein antibody or its antigen-binding fragment as described in claim 1 or 2.

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

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

6. An immunoconjugate, characterized in that, It comprises an antibody portion and a coupling portion conjugated to the antibody portion, wherein the antibody portion comprises the β-trace protein antibody of claim 1 or its antigen-binding fragment, and the coupling portion is a polypropylene latex microsphere.

7. A β-trace protein detection kit, characterized in that, The antibody comprises a first antibody and a second antibody; the first antibody is a binding antibody and the second antibody is a bridging 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.

8. The β-trace protein detection kit according to claim 7, characterized in that, The kit is based on latex-enhanced immunoturbidimetric assay for the detection of β-trace protein.

9. The β-trace protein detection kit according to claim 7, 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 β-trace protein 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.

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