Monoclonal antibody combinations against human neutrophil gelatinase-associated lipocalin and uses thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a combination of monoclonal antibodies against human neutrophil gelatinase-associated lipocalin and application thereof. The combination is composed of monoclonal antibodies 2E7 and 1H6, the CDR amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody 2E7 are shown in SEQ ID NO. 1-6, and the CDR amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody 1H6 are shown in SEQ ID NO. 7-12. The antibody combination has high specificity and high affinity, can effectively capture and detect target antigens, and reduces the risk of non-specific binding and cross-reaction. In a colloidal gold immunochromatographic detection platform, the antibody combination shows excellent sensitivity and stability, can be used for preparing an in vitro detection kit for hNGAL, and provides a stable and reliable biological recognition tool for rapid and accurate detection of an acute kidney injury related index.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a combination of monoclonal antibodies against human neutrophil gelatinase-associated lipid transport proteins and their applications. Background Technology

[0002] Neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin-2 (LCN2), is a special granular component of neutrophils and a member of the human lipocalin family.

[0003] NGAL has a molecular weight of approximately 25 kDa. Mature NGAL proteins exist in vivo as monomers, homodimers, or heterodimers and bind to matrix metalloproteinase-9 (MMP-9). NGAL possesses a variety of biological functions. It exerts antibacterial effects by chelating bacterial siderophores, acts as a chemokine in inflammatory responses to stimulate neutrophil migration and adhesion, and participates in the differentiation and functional regulation of epithelial cells as a growth factor, closely related to tumorigenesis and development.

[0004] NGAL expression in the human body exhibits certain spatiotemporal and tissue-cell specificity. It is expressed at low levels in various tissues such as the kidneys, heart, liver, and lungs, but almost not in the brain and peripheral nerves, colon, uterus and ovaries, and thyroid cells. As a lipid transport protein, NGAL can bind small molecules and distribute throughout the body via blood circulation. Under normal circumstances, NGAL has a short half-life in blood and urine, approximately 2-4 hours. In the kidneys, NGAL is filtered through the glomeruli into the renal tubules and reabsorbed by the tubular epithelial cells. In acute kidney injury (AKI), due to impaired proximal tubular reabsorption function and upregulation of NGAL gene expression following tubular epithelial cell damage, urinary NGAL levels significantly increase within 2-6 hours of AKI onset, and this change precedes other traditional renal function markers such as cystatin C (Cys-C) and serum creatinine (sCr). Furthermore, the systemic inflammatory response triggered by AKI stimulates immune cells and extrarenal organs such as the liver and lungs to synthesize and release NGAL, leading to a rapid increase in plasma NGAL levels. Therefore, NGAL can reflect renal tubular damage earlier and more directly, and can be used for early diagnosis, risk stratification, and prognosis of acute kidney injury, making it an important early biomarker for AKI. In addition, NGAL also has important applications in the early diagnosis and progression monitoring of chronic kidney disease, cardiorenal syndrome, and diabetic nephropathy.

[0005] Currently, the main detection methods for NGAL include latex immunoturbidimetry, double-antibody sandwich immunochemiluminescence assay, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Among these, latex immunoturbidimetry offers advantages such as ease of operation, low cost, and automation; however, existing reagents suffer from insufficient sensitivity, poor stability, and susceptibility to interference from lipemia. While immunochemiluminescence assays offer high sensitivity and accuracy, they rely on expensive equipment and have stringent reagent storage requirements, limiting their application in primary healthcare institutions. ELISA and immunoblotting methods involve complex procedures, long detection cycles, and limited repeatability and standardization, thus being primarily used in research. In contrast, colloidal gold immunochromatography offers advantages such as ease of operation, rapid detection, and no need for complex equipment, making it suitable for point-of-care testing (POCT) scenarios and meeting the clinical needs for early rapid screening of AKI.

[0006] However, all of the aforementioned immunoassay methods rely on the specific binding reaction between antibodies and antigens, and the affinity, specificity, and stability of the antibody directly determine the performance of the assay. Furthermore, the supply of high-quality monoclonal antibodies against human NGAL is limited, with some relying on imports, resulting in high costs. Therefore, the demand for high-performance human NGAL assay reagents and antibody raw materials remains unmet. Summary of the Invention

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a combination of monoclonal antibodies against human neutrophil gelatinase-associated lipocalin and its application, which solves the technical problems of limited sources and high costs of high-quality monoclonal antibodies against human NGAL (neutrophil gelatinase-associated lipocalin), as well as the insufficient sensitivity, poor stability and dependence on expensive instruments of existing detection reagents, making it difficult to meet the needs of primary medical institutions and point-of-care testing.

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include: A monoclonal antibody ensemble against human neutrophil gelatinase-associated lipotransferase, the monoclonal antibody ensemble comprising monoclonal antibody 2E7 and monoclonal antibody 1H6; The heavy chain variable region of the monoclonal antibody 2E7 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3. The light chain variable region of the monoclonal antibody 2E7 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.6. The heavy chain variable region of the monoclonal antibody 1H6 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.9. The light chain variable region of the monoclonal antibody 1H6 includes three complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.12.

[0009] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO. 14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.16.

[0010] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.18.

[0011] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.20.

[0012] This application provides the use of the monoclonal antibody combination described herein in the preparation of a tool for recognizing human neutrophil gelatinase-associated lipid transport proteins.

[0013] In a further embodiment, the tool includes reagents, kits, test strips, and antibody chips; The test strips include colloidal gold test strips.

[0014] In a further embodiment, the colloidal gold test strip uses monoclonal antibody 2E7 as the capture antibody and monoclonal antibody 1H6 as the labeling antibody.

[0015] In a further embodiment, the colloidal gold test strip includes a nitrocellulose membrane, a gold-labeled conjugate pad, a sample pad, and an absorbent pad sequentially connected to a backing plate.

[0016] In a further embodiment, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 2E7, the control line is coated with goat anti-mouse IgG, and the gold-labeled binding pad is coated with monoclonal antibody 1H6.

[0017] The method for preparing the monoclonal antibody combination provided in this application is as follows: Step S1: Immunize female BALB / c mice with purified recombinant antigen; Step S2: Take all spleen cells from immunized mice and fuse them with SP2 / 0 myeloma cells in the logarithmic growth phase, screen and culture them to obtain hybridoma cells; Step S3: Expand the culture of the hybridoma cells obtained by screening, inject them into female BALB / c mice via intraperitoneal injection, collect the ascites fluid, and purify it to obtain monoclonal antibody 2E7 and monoclonal antibody 1H6. The recombinant antigen is human neutrophil gelatinase-associated lipid transport protein, the amino acid sequence of which is shown in SEQ ID NO. 22, and the nucleotide sequence encoding the human neutrophil gelatinase-associated lipid transport protein is shown in SEQ ID NO. 21.

[0018] Beneficial effects: This application provides a monoclonal antibody ensemble against human neutrophil gelatinase-associated lipocalin (hNGAL), consisting of monoclonal antibodies 2E7 and 1H6. This ensemble efficiently and specifically recognizes hNGAL. The complementarity-determining regions (CDRs) of the variable regions of both the heavy and light chains are clearly defined, as shown in SEQ ID NO. 1-12, ensuring high specificity and high affinity of the antibodies. This antibody ensemble can capture and detect the target antigen, reducing the risk of non-specific binding and cross-reactivity. The ensemble exhibits excellent sensitivity and stability in detection platforms such as colloidal gold immunochromatography, providing a stable and reliable biorecognition tool for the in vitro quantitative or qualitative detection of hNGAL, as well as for the development and preparation of reagents related to acute kidney injury (AKI). Attached Figure Description

[0019] Figure 1 The image shows the SDS-PAGE results of hNGAL protein purification. Figure 2 The image shows the identification results of the hNGAL recombinant protein. Figure 3 The image shows the identification results of the purified monoclonal antibody; Figure 4 This is a schematic diagram of colloidal gold assembly. Figure 5 This is a graph showing the specificity analysis results of the test strip. Figure 6 This is a graph showing the sensitivity test results of the test strip card; Figure 7 This is a graph showing the results of antibody binding identification.

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Detailed Implementation

[0021] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0022] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0023] This invention successfully prepared a monoclonal antibody that specifically recognizes human NGAL protein using hybridoma technology and elucidated its key sequence information. Based on this, an optimal antibody pair combination was screened, and a colloidal gold immunochromatographic test strip for human NGAL detection was successfully constructed. Verification showed that this antibody combination exhibited good detection sensitivity and specificity for human NGAL antigen, enabling rapid detection of human NGAL and providing reliable core raw materials and technical support for the development of related in vitro diagnostic products.

[0024] The detection or identification methods described in this application do not involve the diagnosis of diseases.

[0025] Example 1.

[0026] 1. Expression of recombinant hNGAL (human neutrophil gelatinase-associated lipotransferase) protein The hNGAL gene originates from NM_005564.5, and was synthesized by Qingke Biotechnology through codon optimization and cloned into the pET28a vector.

[0027] The nucleotide sequence of the hNGAL gene is shown in SEQ ID NO.21: .

[0028] The amino acid sequence is shown in SEQ ID NO.22: MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYW IRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG.

[0029] The synthesized recombinant plasmid pET28a-hNGAL failed to achieve effective expression after transformation into BL21(DE3) competent cells. Subsequently, it was re-cloned into the pCold-TF prokaryotic expression vector for expression purification. Using the recombinant plasmid pET28a-hNGAL as a template, the hNGAL gene was amplified, cloned, and ligated into the pCold-TF vector. BL21(DE3) competent cells were then transformed using standard methods and induced to express the gene. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Inoculate 1% of the total culture medium volume into LB medium (containing 100 μg / mL ampicillin), incubate at 37°C with shaking at 220 rpm for about 3 hours until the OD600 reaches 0.6-0.9, add IPTG to a final concentration of 0.1 mM, and induce at 16°C with shaking at 200 rpm for 16 hours before collecting the bacterial cells.

[0030] 2. Purification of hNGAL recombinant protein Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TM Purification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second intervals between sonications. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The 500 mM elution peak was collected. The purified protein was obtained via SDS-PAGE electrophoresis. Figure 1 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C.

[0031] Figure 1 In the diagram, M stands for protein marker, number 1 represents pre-induction of bacterial cells, number 2 represents lysed precipitate, number 3 represents lysed supernatant, number 4 represents sample flow through, number 5 represents 100mM imidazole elution buffer, and number 6 represents 500mM imidazole elution buffer (the molecular weight of recombinant hNGAL protein is 75.8kDa).

[0032] The results are as follows Figure 1 As shown, there is a distinct main band near the expected size position (between 70-95 kD), and after purification by Ni column affinity chromatography, high-purity hNGAL was obtained, which can be used for subsequent experiments.

[0033] 3. Indirect ELISA to identify the activity of hNGAL recombinant protein The purified recombinant hNGAL protein was coated onto an ELISA plate, and its reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was hNGAL mouse monoclonal antibody from Sinocare (catalog number: 10222-H08H), labeled Ab1. The recombinant protein was first coated into microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), at a concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20) and patted dry. The monoclonal antibody was diluted with PBS in gradients of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of each diluted antibody was added to each well of the antigen-coated microplate. A negative control (Ab2) was used as a separate mouse monoclonal antibody (Sino-US TREM-2 monoclonal antibody: 11084-MM08), diluted to the same concentration. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL / well of HRP-labeled goat anti-mouse IgG (5000-fold diluted with PBS) was added. The plates were incubated at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer (Beijing Meike Wande) was added, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction. The OD450 nm value was measured using a microplate reader. Results are as follows: Figure 2 . Figure 2 The purified recombinant hNGAL protein showed a significant positive reaction with the commercial hNGAL monoclonal antibody, and this antigen can be used for subsequent mouse immunization and antibody screening.

[0034] 4. Screening for monoclonal antibodies against hNGAL recombinant protein 4.1 Mouse Immunization Mice were immunized with high-purity hNGAL recombinant protein. Specifically, 6-week-old female BALB / c mice were immunized subcutaneously at multiple sites with a mixture of hNGAL recombinant protein and an equal volume of Freund's complete adjuvant (total volume 200 μL), at a dose of 20 μg / mouse. At weeks 4 and 8, booster immunizations were administered subcutaneously at multiple sites with a mixture of the same dose and an equal volume of Freund's incomplete adjuvant. At week 12, mice were immunized by direct injection of insulin (5 μg / mouse) into the spleen. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 20 μg of recombinant hNGAL protein via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0035] 4.2 Screening of hybridoma cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway up the bottom of the well, clones that reacted positively with the hNGAL recombinant protein were selected by indirect ELISA. Since the immunogen was a prokaryotically expressed, His-tagged recombinant protein, background components needed to be screened to identify specific cell lines targeting the hNGAL protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0036] 4.3 Screening of positive clones using indirect ELISA: To efficiently screen for hNGAL-specific monoclonal antibodies, both recombinant hNGAL protein expressed in *E. coli* and commercially available recombinant hNGAL protein (nearshore, catalog number DCH31, C-terminal 6×His tag, expressed in *E. coli*) were used for detection. Simultaneously, a recombinant protein with the same vector, pCold-TF-fFGF23 (patent number: 202511127219.2), was used for reverse screening to further improve the specificity of the screening antibodies. This combined screening effectively avoids obtaining non-specific antibodies that only recognize the tag sequence or impurities related to the expression system, thereby improving the recognition ability of the screening antibodies for hNGAL protein and the reliability of practical applications.

[0037] Specifically, this involves coating microplates with two different preparation sources of recombinant hNGAL protein and pCo. ld-TF fFGF23 antigen (coating buffer: carbonate buffer: sodium carbonate 1.59g, sodium bicarbonate 2.93g, diluted to 1L pure water, pH 9.6), coating concentration 1μg / mL, incubated overnight at 4℃. The next day, the coating buffer was discarded, and the cells were blocked with 3% sucrose + 2% BSA, 150 μL per well, incubated at 37℃ for 2 hours, then washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4), and blotted dry. 50 μL of cell culture supernatant was added, and a negative control was prepared using another unrelated mouse monoclonal antibody (Sino Biotech TREM-2 monoclonal antibody: 11084-MM08, code Ab2), diluted 1 μg / mL, and reacted at 37℃ for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, add 50 μL / well of TMB chromogenic buffer, and incubate at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewand, 1001SA) to stop the reaction. Measure the OD450nm value using a microplate reader. Select positive cell lines that react with hNGAL recombinant proteins from two sources but not with the control recombinant protein for subsequent experiments. The screening results are shown in Table 1.

[0038] Table 1: Screening results of monoclonal antibodies.

[0039]

[0040] After the selected hybridoma cell lines were expanded and cultured, 0.2 mL (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0041] 5. Purification and Identification of Monoclonal Antibodies Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 mL of the supernatant, add 4 mL of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8 °C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8 °C. Centrifuge at 12000 rpm for 30 minutes at 2–8 °C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl (pH 9) and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS (pH 7.4) for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0042] The purified antibody was diluted to 1 μg / mL, and another mouse monoclonal antibody (Sino Biotech TREM-2 monoclonal antibody: 11084-MM08, code Ab2) was used as a negative control at the same dilution. The antibody was detected by indirect ELISA for its interaction with recombinant hNGAL protein (pCold-TF-hNGAL), nearshore hNGAL antigen (hNGAL, nearshore), and irrelevant antigen pCold-TF-fFGF23 (fFGF23). Results are shown below. Figure 3 The selected monoclonal antibodies reacted with both self-produced and near-shore hNGAL recombinant proteins, but showed no significant reaction with the control antigen pCold-TF-fFGF23 recombinant protein, indicating that the obtained monoclonal antibodies have good specificity and high affinity.

[0043] 6. Preparation of test strips coated with different monoclonal antibodies against hNGAL: The 22 selected hNGAL monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1 mg / mL with 0.01 M PBS, pH 7.4) were sprayed horizontally in a linear pattern using a scribing instrument, with a spray volume of 0.8 μL / cm per line, forming the detection line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL with 0.01 M PBS (pH 7.4), were sprayed horizontally in a linear pattern at 6 mm intervals, forming the control line (C line).

[0044] 7. Colloidal gold pairing of hNGAL monoclonal antibodies Preparation of antibody-colloidal gold labeled complex.

[0045] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes, and the colloidal gold particles were allowed to stabilize before cooling to room temperature. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in gradients of 1 μL, 2 μL, 3 μL, 4 μL (optimal), 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. After mixing, 5 μg of the hNGAL monoclonal antibody to be labeled was added to each tube, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and incubation at room temperature was continued for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.05M Tris + 0.5% Triton x100 + 1.5% NaCl + 0.1% SDS, pH 8.0) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0046] 8. Screening of paired monoclonal antibodies Nitrocellulose membranes streaked with different monoclonal antibodies against hNGAL were paired with different colloidal gold-labeled monoclonal antibodies. hNGAL protein (nearshore, DCH31) was diluted to 20 ng / mL for detection, while the control antigen protein pCold-TF-fFGF23 was diluted to 20 ng / mL as a negative antigen for detection. Combinations showing deep staining for hNGAL protein without reacting with the control protein were screened. The screening process is shown in Table 2. Therefore, the optimal pairing for detecting recombinant hNGAL protein was determined to be 2E7 streaking and 1H6 gold labeling.

[0047] Table 2: Results of screening paired monoclonal antibodies using hNGAL recombinant protein.

[0048]

[0049] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0050] Table 2 shows the screening results using nearshore hNGAL recombinant protein diluted to a concentration of 20 ng / mL as a positive antigen. The detection results for pCold-TF-fFGF23 protein and blank dilution were negative and are not presented. The results showed that the 2E7 monoclonal antibody, applied with a membrane-stretching assay, combined with the 1H6 monoclonal antibody for labeling, produced the deepest staining of the hNGAL recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody anti-2E7 as the capture antibody and monoclonal antibody 1H6 as the labeling antibody can specifically recognize the hNGAL recombinant protein.

[0051] 9. Preparation and Assembly of Colloidal Gold Test Strips Preparation of gold-labeled pads: Using a 6mm×300mm glass fiber membrane, the prepared colloidal gold-labeled antibody was evenly dropped onto the glass fiber at a rate of 1200μL / strip, allowed to air dry naturally, and then dried at 37℃ for 2 hours for later use. See Figure 4 , Figure 4 This is a schematic diagram of the colloidal gold assembly. A 60mm × 300mm PVC backing plate is used for support, on which a sample pad, a gold pad (gold-labeled binding pad), an NC membrane, and an absorbent pad are attached. The nitrocellulose membrane is coated with two lines: a detection line (T line, monoclonal antibody 2E7 streaking) and a control line (C line, goat anti-mouse IgG). The gold-labeled binding pad is coated with monoclonal antibody 1H6 and dried at 37℃ for 12 hours. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with colloidal gold plastic casings, exposing the sample pad at the sample application well of the casing and the control and detection lines at the result observation wells. The colloidal gold test strip is now assembled.

[0052] 10. Specificity test of test strips Recombinant protein samples: pCold-TF-hNGAL recombinant protein (hNGAL), pCold-TF-fFGF23 recombinant protein (fFGF23, expressed by the applicant), hLCN1 recombinant protein (Sinochem, 11583-H08H), hCys-C recombinant protein (Nearshore, DCI55), pCold-TF-fNGAL (fNGAL, expressed by the applicant, see patent CN120399062B), and hemoglobin (Hb, supernatant from high-speed centrifugation after red blood cell lysis, and protein concentration was determined by ultra-micro spectrophotometer, the main protein of which is hemoglobin) were diluted to 1 μg / mL with sample diluent for detection. Add 80 μL of the diluted sample to the sample well of the test strip. Simultaneously, add another 80 μL of the diluent to a new test strip as a blank control. Determine the results within 20 minutes. If both the T and C lines show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid.

[0053] The sample dilution solution was 0.01M PBS + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4. Results are shown below. Figure 5 , Figure 5 The results of the specificity analysis of the test strip show that the test strip can specifically detect hNGAL recombinant protein, while it does not show color for fFGF23, hLCN1, hCys-C, fNGAL recombinant protein, hemoglobin, and blank dilution control, indicating that the test strip has good specificity.

[0054] 11. Sensitivity test of test strips The pCold-TF-hNGAL recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL before detection. Figure 6 The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 1 ng / mL, while the blank dilution, i.e. the sample dilution (0.01M PBS + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) (0 ng / mL), did not show color development, indicating that the limit of detection for hNGAL recombinant protein on the test strip is 1 ng / mL.

[0055] 12. Monoclonal antibody binding activity Based on the selection of paired antibodies using colloidal gold, the selected paired monoclonal antibodies and other murine unrelated monoclonal antibodies were serially diluted (concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.1 ng / mL, respectively) using the aforementioned indirect ELISA method to evaluate their binding activity with the pCold-TF-hNGAL recombinant protein. Other murine monoclonal antibodies (Sinosure TREM-2 monoclonal antibody: 11084-MM08) were used as negative controls (Ctrl) to exclude the influence of non-specific binding. Results are shown below. Figure 7 . Figure 7 In the text, “Ctrl” represents the negative control mouse monoclonal antibody. Monoclonal antibodies 2E7 and 1H6 still showed strong signals at concentrations as low as 10 ng / mL.

[0056] This invention utilizes hybridoma technology to screen and obtain monoclonal antibody pairs capable of specifically recognizing the hNGAL protein. These antibody pairs efficiently recognize the recombinant hNGAL protein, exhibiting good specificity and sensitivity. This invention applies these monoclonal antibody pairs to an immunoassay platform, constructing a rapid test strip based on colloidal gold immunochromatography. This test strip demonstrates high sensitivity to the recombinant hNGAL protein and exhibits no cross-reactivity with other proteins, making it suitable for rapid, on-site detection.

[0057] 13. Gene sequence of monoclonal antibodies Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0058] membrane-scraped monoclonal antibody 2E7 sequence Light chain variable region nucleotide sequence: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.18: gatgttgtgatgacccaacctccactctccctgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttttacacagtgatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaagctcctgatctacaaaagtt tccagccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcagagtacacatgttcctccgacgttcggtggaggcaccaagctggaaatcaaa.

[0059] Light chain variable region amino acid sequence: The amino acid sequence of the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO. 14: DVVMTQPPLSLPVSLGDQASISCRSSQSLLHSDGNTYLHWYLQKPGQSPKLLIYKVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPTFGGGTKLEIK.

[0060] Light chain CDR area annotation: The amino acid sequence of CDR-L1 in the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.4: CDR-L1: RSSQSLLHSDGNTYLH; The amino acid sequence of CDR-L2 in the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.5: CDR-L2: KVSSRFS; The amino acid sequence of CDR-L3 in the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO. 6: CDR-L3: SQSTHVPPT.

[0061] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.17: cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactttggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacaatggagaggcaacatatgctgatgac tttaagggacgatttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaagatcaggatataggtacgacgtcgttgcttattattactatactatggactactggggtcaaggaacctcagtcaccgtctcctca.

[0062] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO. 13: QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGWINTYNGEATYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSGYRYDVVAYYYYTMDYWGQGTSVTVSS.

[0063] Heavy chain CDR region annotation: The amino acid sequence of the CDR-H1 of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.1; CDR-H1: NFGMN; The amino acid sequence of the CDR-H2 of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.2; CDR-H2: WINTYNGEATYADDFKG; The amino acid sequence of CDR-H3 in the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.3; CDR-H3: SGYRYDVVAYYYYTMDY.

[0064] Gold monoclonal antibody 1H6 sequence Light chain variable region nucleotide sequence: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.20: gacattgtgttgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatgtcctgcagagccagtgaaagtgttgatagttctggcaacagtttcatgcactggtaccagcagaaaccaggacagccacccaaactcctcatctatcgtgcatc caacctagaatctggtatccctgccaggttcagtggcggtgggtctaggacagacttcaccctcaccattaatcccgtggaggctgatgatgttgcaacctattactgtcagcaaggtaatgaggatccattcacgttcggctcggggacaaagttggaagtaaaa.

[0065] Light chain variable region amino acid sequence: The amino acid sequence of the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.16: DIVLTQSPASLAVSLGQRATMSCRASESVDSSGNSMHWYQQKPGQPPKLLIYRASNLESGIPARFSGGGSRTDFTLTINPVEADDVATYYCQQGNEDPFTFGSGTKLEVK.

[0066] Light chain CDR area annotation: The amino acid sequence of CDR-L1 in the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.10: CDR-L1: RASESVDSSGNSFMH; The amino acid sequence of CDR-L2 in the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.11: CDR-L2: RASNLES; The amino acid sequence of CDR-L3 in the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.12: CDR-L3: QQGNEDPFT.

[0067] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO. 19: caggttactctgaaagagtctggccctgggatattgcagccctcccagaccctcagtctgacttgttctttctctgggttttcactgagcacttctggtatgggtgtgagctggattcgtcagccttcaggaaagggtctggagtggctggcacacatttactggggatgatgacaagcgctataa cccatccctgcagagccggctcacaatctccaaggatacctccagcaaccaggtattcctcaagatcaccagtgtggacactgcagatactgccacatactactgtgctcgatccccctataggttcgacgtgggagatgctatggactactggggtcaaggaacctcagtcaccgtctcctca.

[0068] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.15; QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLQSRLTISKDTSSNQVFLKITSVDTADTATYYCARSPYRFDVGDAMDYWGQGTSVTVSS.

[0069] Heavy chain CDR region annotation: The amino acid sequence of the CDR-H1 of the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.7: CDR-H1: TSGMGVS; The amino acid sequence of the CDR-H2 region of the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.8: CDR-H2; HIYWDDDKRYNPSLQS; The amino acid sequence of CDR-H3 in the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO. 9: CDR-H3: SPYRFDVGDAMDY.

[0070] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0071] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody combination against human neutrophil gelatinase-associated lipid transporter protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 2E7 and monoclonal antibody 1H6; The heavy chain variable region of the monoclonal antibody 2E7 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 2E7 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6. The heavy chain variable region of the monoclonal antibody 1H6 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

9. The light chain variable region of the monoclonal antibody 1H6 includes three complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody combination against human neutrophil gelatinase-associated lipid transporter protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.

14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.

16.

3. The monoclonal antibody combination against human neutrophil gelatinase-associated lipid transporter protein according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.

18.

4. The monoclonal antibody combination against human neutrophil gelatinase-associated lipid transporter protein according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1H6 is shown in SEQ ID NO.

20.

5. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for recognizing human neutrophil gelatinase-associated lipid transport protein.

6. The application according to claim 5, characterized in that, The tools include reagents, kits, test strips, and antibody chips; The test strips include colloidal gold test strips.

7. The application according to claim 6, characterized in that, The colloidal gold test strip uses monoclonal antibody 2E7 as the capture antibody and monoclonal antibody 1H6 as the labeling antibody.

8. The application according to claim 7, characterized in that, The colloidal gold test strip includes a nitrocellulose membrane, a gold-labeled conjugate pad, a sample pad, and an absorbent pad, which are sequentially connected to the back plate.

9. The application according to claim 8, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 2E7, the control line is coated with goat anti-mouse IgG, and the gold-labeled conjugate pad is coated with monoclonal antibody 1H6.

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