Recombinant antibodies against ngal and their preparation and use

Abstract

The application discloses an anti-NGAL recombinant antibody and a preparation and application thereof, and belongs to the technical field of antibodies. The anti-NGAL recombinant antibody is prepared by taking a recombinant NGAL protein antigen (21th-198th amino acid of human NGAL protein (P80188)) as an immunogen, and is obtained through B cell screening, heavy and light chain vector construction, expression and multiple rounds of screening and detection. The anti-NGAL recombinant antibody can specifically recognize NGAL protein and can be used for diagnosing diseases such as kidney diseases.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to anti-NGAL recombinant antibodies and their preparation and application. Background Technology

[0002] Neutrophil gelatinase associated lipocalin (NGAL, also known as LCN2, Lipocalin-2, 24p3, MSFI) is a member of the apolipoprotein family. Under physiological conditions, NGAL is expressed at low levels in neutrophils, kidneys, colon, and lung epithelial cells. When the kidneys experience ischemic or nephrotoxic damage, NGAL expression in renal tubular epithelial cells increases dramatically. NGAL protects renal tissue from inflammatory cell damage by inducing apoptosis in neutrophils infiltrating the renal tubular interstitium. It is a marker of renal structural damage, systemic inflammation, and oxidative stress.

[0003] Currently, the clinical applications of NGAL mainly include: (1) an early marker of acute kidney injury (AKI). Under normal circumstances, the concentration range of NGAL in urine is 0.7 to 9.6 ng / mL, and the concentration range of NGAL in plasma is 3 to 106 ng / mL. When AKI occurs, the concentration of NGAL in blood and urine usually rises rapidly, with the most significant increase at 2 hours (tens to hundreds of times higher than the critical value). Traditional indicators such as serum creatinine (sCr) and urinary enzymes often rise significantly after 24 to 72 hours, indicating that NAGL is an earlier and more sensitive indicator of AKI than serum creatinine; (2) predicting the progression of chronic kidney disease (CKD). In the late stages of chronic kidney diseases such as lupus nephritis, IgA nephropathy, glomerulonephritis, polycystic kidney disease, and diabetic nephropathy, tubulointerstitial damage occurs. NGAL, as an early marker of renal tubular injury, also plays an important role in the pathological process of CKD and can be used as an independent biological indicator for CKD risk assessment, disease staging, and hemodialysis adequacy; (3) assess the degree of renal damage in cardiorenal syndrome (CRS). The sensitivity and specificity of serum NGAL in diagnosing CRS are 100% and 86.7%, respectively. Close monitoring of NGAL levels is helpful for clinicians to correctly evaluate the degree of renal function damage in CRS and take timely intervention and treatment to reduce the occurrence of complications; (4) assist in judging the adequacy of renal dialysis. Studies have shown that the serum NGAL content of maintenance hemodialysis (MHD) patients is significantly higher than that of healthy controls, and the serum NGAL of MHD patients with adequate dialysis is significantly higher than that of patients with inadequate dialysis. Therefore, it is believed that NGAL level can better reflect the adequacy of dialysis.

[0004] NGAL (Non-Grade Acid Albumin) offers advantages such as early detection, high sensitivity and specificity, and ease of interpretation, playing a crucial role in the early diagnosis, prevention, treatment, and improvement of prognosis of AKI. Secondly, NGAL is also highly suitable as a biomarker for drug safety and efficacy testing; for example, urinary NGAL levels significantly decrease in patients with hypertensive nephropathy after taking ARB antihypertensive drugs. Furthermore, given NGAL's advantages in reflecting nephrotoxic damage, it can also be applied in new drug development, enabling early exclusion of unsafe drugs and early determination of drug efficacy, significantly reducing new drug development costs and ensuring the safety of research subjects.

[0005] Currently, immunological methods are commonly used in clinical practice to detect NGAL in serum (plasma) and urine. Therefore, developing antibodies with high specificity, high sensitivity, and good NGAL protein binding ability is of great significance. Summary of the Invention

[0006] 1. Purpose of the invention

[0007] One of the objectives of this application is to provide a recombinant NGAL protein antigen that has good immunomodulatory activity and specificity.

[0008] The second objective of this application is to provide an anti-NGAL recombinant antibody, which is prepared using recombinant NGAL protein antigen as an immunogen. It can specifically recognize NGAL protein and can be applied to the detection of NGAL protein molecules by ELISA, immunoblotting, immunohistochemistry, cell immunofluorescence, flow cytometry, etc., thereby improving the accuracy of NGAL protein detection.

[0009] 2. Technical Solution

[0010] To solve the above problems, the technical solution adopted in this application is as follows:

[0011] This application provides a recombinant NGAL protein antigen with the amino acid sequence shown in SEQ ID NO:1. This recombinant NGAL protein antigen contains only amino acids 21 to 198 of the human NGAL protein (P80188), which are specific dominant antigenic epitopes. This ensures that the anti-NGAL recombinant antibody prepared using it as an immunogen can specifically recognize the NGAL protein, thereby improving the accuracy of NGAL molecular detection.

[0012] This application also provides a method for preparing the above-mentioned recombinant NGAL protein antigen, which specifically includes the following steps:

[0013] S1, Construction of recombinant expression vector: The nucleic acid encoding the above recombinant NGAL protein antigen is cloned into the expression vector to obtain the recombinant expression vector;

[0014] S2, Eukaryotic expression system for recombinant protein expression: The obtained recombinant expression vector is transfected into mammalian cells, and the expression supernatant of the recombinant protein is obtained by culturing.

[0015] S3, Isolation and purification of recombinant NGAL protein antigen: The supernatant in S2 was isolated and purified to obtain the recombinant NGAL protein antigen.

[0016] This application also provides the application of the above-mentioned recombinant NGAL protein antigen in the preparation of anti-NGAL recombinant antibodies.

[0017] Furthermore, the above applications include:

[0018] (1) After immunizing New Zealand white rabbits with recombinant NGAL protein antigen and Freund's adjuvant, spleens were removed and B lymphocytes were isolated.

[0019] (2) Culture and screen antigen-specific B lymphocytes and lyse the cells to release the nucleic acids therein;

[0020] (3) The nucleic acid was reverse transcribed into mRNA, and the variable region sequence was obtained by PCR amplification using gene-specific primers for the antibody heavy chain variable region (VH) and light chain variable region (VL).

[0021] (4) Insert the variable region sequence into a vector plasmid containing the constant region (heavy chain, light chain) gene to obtain an anti-NGAL recombinant antibody expression plasmid;

[0022] (5) Select a eukaryotic expression system for expression, culture to obtain supernatant, and affinity purify to obtain recombinant anti-NGAL antibody.

[0023] This application also provides an anti-NGAL recombinant antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises one of the amino acid sequences shown in SEQ ID NO. 2, 4, 6, 8, and the light chain variable region comprises one of the amino acid sequences shown in SEQ ID NO. 3, 5, 7, 9.

[0024] Furthermore, the above-mentioned anti-NGAL recombinant antibody has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.2 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO.3.

[0025] Furthermore, the above-mentioned anti-NGAL recombinant antibody has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.4 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO.5;

[0026] Furthermore, the above-mentioned anti-NGAL recombinant antibody has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 6 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 7;

[0027] Furthermore, the above-mentioned anti-NGAL recombinant antibody has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.8 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO.9.

[0028] Furthermore, the aforementioned anti-NGAL recombinant antibody also includes a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:10 and the light chain constant region includes the amino acid sequence shown in SEQ ID NO:11.

[0029] This application also provides a nucleic acid that encodes the heavy chain variable region and / or light chain variable region of the above-mentioned anti-NGAL recombinant antibody.

[0030] Furthermore, the nucleic acid encoding the heavy chain variable region of the anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.12, which is used to encode the heavy chain variable region of the anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.2.

[0031] Furthermore, the nucleic acid encoding the heavy chain variable region of the anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.14, which encodes the heavy chain variable region of the anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.4.

[0032] Furthermore, the nucleic acid encoding the heavy chain variable region of the anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.16, which is used to encode the heavy chain variable region of the anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.6.

[0033] Furthermore, the nucleic acid encoding the heavy chain variable region of the anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.18, which encodes the heavy chain variable region of the anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.8.

[0034] Furthermore, the nucleic acid encoding the light chain variable region of the above-mentioned anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.13, which is used to encode the light chain variable region of the above-mentioned anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.3.

[0035] Furthermore, the nucleic acid encoding the light chain variable region of the above-mentioned anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.15, which is used to encode the light chain variable region of the above-mentioned anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.5.

[0036] Furthermore, the nucleic acid encoding the light chain variable region of the anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.17, which is used to encode the light chain variable region of the anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.7.

[0037] Furthermore, the nucleic acid encoding the light chain variable region of the above-mentioned anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.19, which is used to encode the light chain variable region of the above-mentioned anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.9.

[0038] Furthermore, the aforementioned nucleic acid also encodes the heavy chain constant region and / or light chain constant region of the aforementioned anti-NGAL recombinant antibody.

[0039] Furthermore, the nucleic acid encoding the heavy chain constant region of the anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.20, which encodes the heavy chain constant region of the anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.10.

[0040] Furthermore, the nucleic acid encoding the light chain constant region of the anti-NGAL recombinant antibody includes the nucleotide sequence shown in SEQ ID NO.21, which is used to encode the light chain constant region of the anti-NGAL recombinant antibody, including the amino acid sequence shown in SEQ ID NO.11.

[0041] This application also provides a recombinant expression vector containing the aforementioned nucleic acid.

[0042] This application also provides a recombinant expression cell, which includes the above-described recombinant expression vector or the above-described nucleic acid.

[0043] This application also provides the application of the above-mentioned nucleic acid in the preparation of recombinant anti-NGAL antibodies.

[0044] This application also provides the application of the above-mentioned recombinant expression vector in the preparation of anti-NGAL recombinant antibodies.

[0045] This application also provides the application of the above-mentioned recombinant expression cells in the preparation of anti-NGAL recombinant antibodies.

[0046] This application also provides a method for preparing an anti-NGAL recombinant antibody, the method comprising: transfecting cells with the above-mentioned recombinant expression vector to obtain recombinant expression cells; culturing the transfected recombinant expression cells; collecting the supernatant and purifying it to obtain an anti-NGAL recombinant antibody.

[0047] This application also provides the application of the above-mentioned anti-NGAL recombinant antibody, or the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant expression cell, or the above-mentioned method for preparing the anti-NGAL recombinant antibody in the detection of NGAL protein molecules, or in the preparation of products for detecting NGAL protein molecules.

[0048] Furthermore, the above-mentioned methods for detecting NGAL proteins include any one or more of ELISA, Western blotting, immunohistochemistry, immunofluorescence, and flow cytometry.

[0049] Furthermore, the aforementioned products for detecting NGAL protein molecules include any one or more of the following: kits, antibody conjugates, antibody chips, etc.

[0050] This application also provides a kit for detecting NGAL protein molecules, the kit comprising the above-mentioned anti-NGAL recombinant antibody, with the anti-NGAL recombinant antibody as the primary antibody.

[0051] Furthermore, the above-mentioned kit is a flow cytometry detection kit, which includes the above-mentioned anti-NGAL recombinant antibody and also includes flow cytometry detection reagents.

[0052] Furthermore, the above-mentioned kit is an immunohistochemical detection kit, comprising: the above-mentioned anti-NGAL recombinant antibody, and immunohistochemical detection reagents.

[0053] 3. Beneficial effects

[0054] Compared with the prior art, the advantages of this application are as follows:

[0055] (1) The recombinant NGAL protein antigen provided in this application has good immunogenicity and high purity, which ensures that the anti-NGAL recombinant antibody prepared with it as an immunogen can only specifically recognize NGAL protein molecules, thus improving the accuracy of NGAL protein molecule detection.

[0056] (2) The anti-NGAL recombinant antibody provided in this application can specifically recognize the NGAL protein and can be used to detect NGAL molecules. In particular, combining any two of them and using them in pairs improves the detection sensitivity and accuracy of NGAL molecules.

[0057] (3) The recombinant anti-NGAL antibody provided in this application can recognize natural NGAL protein in human serum, exhibiting good specificity and affinity. It can be used not only for chemiluminescence detection but also specifically recognize human NGAL-positive cells. When used in immunoassays, especially immunohistochemical detection, it provides clean background staining without non-specific staining, demonstrating high reliability and strong anti-interference ability in NGAL protein detection. Furthermore, it boasts high analytical sensitivity and specificity, effectively avoiding false positives and false negatives. It is suitable for high-accuracy and high-precision detection of NGAL protein in various pathological samples. This recombinant antibody can be used in the preparation of cells or conjugates, diagnostic reagents or kits, drug development, and other fields, possessing significant application value in basic research and clinical pathological testing. Attached Figure Description

[0058] Figure 1 This is an SDS-PAGE result of the purified recombinant NGAL protein antigen expressed in mammalian cells. Lane 1 is the protein molecule marker, and lane 2 is the purified recombinant NGAL protein antigen.

[0059] Figure 2 This is an ELISA test to detect the efficacy and specificity of recombinant NGAL protein antigen immunization in rabbits. Among them, K1306, K1307 and K1308 are serums obtained from three different rabbits, and NC refers to the serum of unimmunized blank rabbits.

[0060] Figure 3 This is an electrophoresis image of the antibody heavy and light chain variable regions obtained by 1% agarose gel electrophoresis, where: the middle band is the DNA marker band, the left lane is the heavy chain band, and the right lane is the light chain band.

[0061] Figure 4 This is an SDS-PAGE result image of anti-NGAL recombinant antibody. Samples marked 1-4 are non-reducing antibody samples, and samples marked 5-8 are reducing antibody samples.

[0062] Figure 5 This is an ELISA assay showing the binding of the anti-NGAL recombinant antibody to the commercially available NGAL recombinant protein (Hytest, Cat.#8NL2).

[0063] Figure 6 This is an immunoblot assay showing the binding of the anti-NGAL recombinant antibody to the NGAL molecule. 1 is MCF-7 whole-cell lysis buffer (which does not express the NGAL molecule and can be used as negative cell material), 2 is COLO 205 whole-cell lysis buffer, 3 is SW480 whole-cell lysis buffer, and 4 is A431 whole-cell lysis buffer.

[0064] Figure 7This is a graph showing the results of cellular immunofluorescence detection of the specific binding of anti-NGAL recombinant antibody to positive tumor cells.

[0065] Figure 8 This is an image showing the results of immunohistochemical (IHC) staining. Detailed Implementation

[0066] The present application will be further described below with reference to specific embodiments.

[0067] It should be noted that terms such as "upper", "lower", "left", "right", and "middle" used in this specification are only for clarity of description and are not intended to limit the scope of implementation. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of this application.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0069] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0070] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0071] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0072] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0073] As used in this article, antibodies, recombinant antibodies, and recombinant monoclonal antibodies are interchangeable and are globulins that can specifically bind to corresponding antigens (epitopes).

[0074] Example 1

[0075] This embodiment provides the expression and purification of recombinant NGAL protein antigen.

[0076] In this embodiment, NGAL protein was used as the target antigen. Referring to the amino acid sequence of the human NGAL protein (P80188) published on NCBI, online software such as UniProt and Epitome were used to analyze the NGAL protein structure, linear site prediction, and antigen-antibody interaction residues. Amino acids 21 to 198 of the NGAL protein were selected as the specific dominant antigenic epitopes, and a Gly4Ser and a 10×His tag were added to the C-terminus to facilitate expression and purification. The specific sequence is as follows:

[0077] QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIK SYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGGGSHHHHHHHHHH(SEQ ID NO: 1).

[0078] Transfect the mammalian cell expression plasmid containing the above sequence into a cell density of 3 × 10⁶ cells. 6 Cells were cultured in 100 mL of Expi293F cells for 5 days. The cell supernatant was collected, purified using a nickel column to obtain the corresponding specific recombinant NGAL protein antigen, and analyzed by SDS-PAGE. The results are as follows: Figure 1 As shown, the size of the purified specific recombinant NGAL protein antigen is between 22.2kD and 24kD, which is consistent with the prediction results of the online software, and the antibody purity can reach 95%.

[0079] Example 2

[0080] This embodiment provides the screening, preparation, purification, and validation of anti-NGAL recombinant antibodies.

[0081] Recombinant anti-NGAL antibodies were obtained by immunizing New Zealand white rabbits with the recombinant NGAL protein antigen purified in Example 1 and Freund's adjuvant. Specifically, this included:

[0082] (1) Animal Immunization

[0083] Using the recombinant NGAL protein antigen purified in Example 1 as an immunogen, three New Zealand rabbits (K1306, K1307, and K1308) were immunized, each receiving 600 μg of recombinant NGAL protein antigen via a multi-site dorsal immunization protocol. Specifically: for the first immunization, 1 mL of complete Freund's adjuvant was mixed with 600 μg of recombinant NGAL protein antigen and emulsified; for the second immunization 3 weeks later, 1 mL of incomplete Freund's adjuvant was mixed with 600 μg of recombinant NGAL protein antigen and emulsified, again via a multi-site dorsal immunization protocol; for the third immunization 5 weeks later, 300 μg of recombinant NGAL protein antigen was mixed with the same volume of 0.01 M PBS (pH 7.4), again via a multi-site dorsal immunization protocol; and for the last 7 weeks, the same immunization procedure (i.e., 300 μg of recombinant NGAL protein antigen mixed with the same volume of 0.01 M PBS (pH 7.4)) was repeated. Seven days after the last immunization, blood was collected from the marginal ear vein of the rabbits and the rabbit antiserum was obtained by centrifugation at 4000 rpm for 20 min.

[0084] The immunogenicity of recombinant NGAL protein antigen was evaluated by detecting the antibody titer in rabbit antiserum using an indirect ELISA method. The specific experimental steps were as follows: Recombinant NGAL protein antigen was diluted with carbonate buffer (50 mM, pH 9.6), and 100 μL / well was coated onto the microplate and incubated overnight at 4°C. The next day, the plate was blocked with 1% BSA at 37°C for 1 hour. Rabbit antiserum was serially diluted as the primary antibody and incubated at 37°C for 1 hour. Then, 1:5000 dilution of goat anti-rabbit secondary antibody (Hangzhou Startech Biotechnology, S0B4002) was added, and the plate was incubated at 37°C for 1 hour. Finally, chromogenic reagent was added, and the absorbance at OD450 was read using a microplate reader.

[0085] ELISA test results as follows Figure 2 As shown, the rabbit antiserum titer against NGAL protein is not less than 1:64000, indicating that the recombinant NGAL protein antigen has good immunogenicity.

[0086] (2) Splenic cell separation

[0087] Perform aseptic procedures in a biosafety cabinet, remove a 10cm cell culture dish, and add 20mL of basal culture medium (RPMI 1640 + 1% Pen Strep) (RPMI 1640: GIBCO, 4873966; Pen Strep: Absin, abs9244-100mL) for later use. Using sterile scissors and forceps, remove excess connective tissue and adipose tissue from the freshly removed spleen. Wash the spleen 10 times with basal culture medium (RPMI 1640 + 1% Pen Strep), and then place it on a 10cm cell culture dish containing a 100-mesh cell strainer. Using a 10mL disposable syringe, draw 10mL of basal culture medium and inject it into the spleen at several points, slowly flushing out the spleen cells. Then, use the syringe plunger to grind and crush the spleen tissue as thoroughly as possible until the entire spleen tissue is nearly white. The ground cells are filtered through a cell sieve into the culture medium. The culture medium containing the cells is transferred to a sterile 50mL centrifuge tube using a pipette. The tube is centrifuged at 400g for 5 minutes at room temperature. The supernatant is discarded, and the cells are retained. Five volumes of erythrocyte lysis buffer (Absin, ABS9101-500 mL) are added, and lysis is performed at room temperature for 5 minutes. After timing, 30mL of basal culture medium is added to terminate the reaction. The tube is centrifuged at 400g for 5 minutes at room temperature, and the supernatant is discarded. 40mL of room-temperature basal culture medium is added, and the cell pellet is resuspended by pipetting. The tube is centrifuged at 400g for 5 minutes at room temperature, and the supernatant is discarded. 20mL of room-temperature basal culture medium (RPMI 1640 + 1% Pen Strep) is added, and the cell pellet is resuspended by pipetting. Cell counting is performed, and the cell density is adjusted to 102. 5 / mL for later use.

[0088] (3) Obtaining NGAL-specific B lymphocytes

[0089] Following the instructions for using immunomagnetic beads (Cytiva, 30152105010150), NGAL protein was conjugated to the magnetic beads. The mixture of NGAL protein-conjugated magnetic beads and isolated B lymphocytes was incubated at room temperature for 50 minutes and then placed in a magnetic rack. After 5 minutes, all the magnetic beads sank to the bottom. The supernatant was discarded, and sterile PBS was added to wash the cells. The washing process was repeated 3 times. The cells obtained at the end were NGAL-specific B lymphocytes.

[0090] The isolated B lymphocytes were diluted several times and placed in 96-well cell culture plates. RPMI 1640 medium containing 10% fetal bovine serum (Hyclone, SH30406.05) and 2 μg / mL IL2 (Noveprotein, CO13) was added, and the plates were cultured at 37°C and 5% CO2 for 7 days. The supernatant was collected for indirect ELISA identification of antibodies. The detection method was the same as the ELISA method for rabbit antiserum in step (1).

[0091] (4) Cloning of the gene encoding the rabbit monoclonal antibody and construction of the plasmid

[0092] Specific B lymphocytes that tested positive were lysed, and total RNA was obtained using a TurboCapture 96mRNA Plate (QIAGEN, 72251) kit. cDNA was obtained using a reverse transcription kit (TonkBio, TD002-B), and the reverse transcription product was used as a template for PCR. Primers were designed to amplify the antibody-encoded heavy chain variable region (VH) and light chain variable region (VL) sequences. The reaction program was as follows: VH: 95℃ 5 min, 95℃ 30 s, 70℃ 30 s, 72℃ 1 min, 72℃ 10 min for 35 cycles; VL: 95℃ 5 min, 95℃ 30 s, 55℃ 30 s, 72℃ 1 min, 72℃ 10 min for 35 cycles. The amplification products were detected by 1% agarose gel electrophoresis. Figure 3 Then, a single target band was selected for gel recovery. The nucleic acid sequences of the forward and reverse primers for VL and VH are shown below:

[0093] VL-Primer-F: 5'-GCTCGTGATGACCCAGACTCCA-3' (SEQ ID NO. 22),

[0094] VL-Primer-R: 5'-CCACCTCGGTCCCTCCG-3' (SEQ ID NO. 23);

[0095] VH-Primer-F: 5'-CCTGGTCGCTGTGCTCAAAGGTGTCCAG-3' (SEQ ID NO. 24);

[0096] VH-Primer-R: 5'-CGTTGGTCAGTGTGCCGCTA-3' (SEQ ID NO. 25).

[0097] The target band recovered from the gel and a mammalian cell expression vector containing the constant region gene were transformed into TOP10 competent cells (Shanghai Weidi Biotechnology Co., Ltd., DF1010) via homologous recombination. The cells were cultured at 37°C for 12 hours. Single clones were picked and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using Snapgene software to screen for VH and VL sequences that match the characteristics of rabbit-derived antibody sequences.

[0098] The VH and VL sequences for screening rabbit monoclonal antibodies that specifically bind to the NGAL protein are as follows:

[0099] Recombinant antibody 1 (H14L73):

[0100] The amino acid sequence of the heavy chain variable region (H14L73VH) of the H14L73 recombinant antibody is as follows:

[0101] VLKGVQCQSVEESGGRLVTPGTPLTLTCTISGFSLSSYYMSWVRQAPGKGLEWIGIIYASGRTYYANWAKGRFTISKTSSTTVTLRMTSLTAADTATYFCARYGGGIDTRLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGY (SEQ ID NO: 2),

[0102] The amino acid sequence of the light chain variable region (H14L73VL) of the H14L73 recombinant antibody is as follows:

[0103] AYDMTQTPASVEVAVGGTVTIKCQASESINSWLAWYQQKPGQPPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQQGYTGNYIENS(SEQ ID NO:3);

[0104] The nucleic acid sequence encoding the heavy chain variable region (H14L73VH-DNA) of the H14L73 recombinant antibody is as follows:

[0105] GTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAATCTCTGGATTCTCCCTCAGTAGCTACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTTATGCTAGTGGTAGGACATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGAGAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCCAGATATGGTGGTGGTATTGATACTCGGTTGGATCTCTGGGGCCAGGGCACTCTGGTTACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTAC(SEQ ID NO:12),

[0106] The nucleic acid sequence encoding the recombinant antibody light chain variable region H14L73 (H14L73VL-DNA) is:

[0107] GCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGAGCATTAATAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTAGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATTAGTGGCGTGCAGTGTGATGATGCTGCCACTTACTACTGTCAACAGGGTTATACTGGTAATTATATTGAGAATAGT(SEQ ID NO:13)。

[0108] Recombinant antibody 2 (H1L65):

[0109] The amino acid sequence of the recombinant antibody heavy chain variable region H1L65 (H1L65VH) is:

[0110] VLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSTNAVSWVRQAPGEGLEWIGVMQSGGTTDYASWTKGRFTISRTSTTVDLKITSPTTEDTATYSCARGGNRFDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGY(SEQ ID NO:4),

[0111] The amino acid sequence of the light chain variable region (H1L65VL) of the H1L65 recombinant antibody is as follows:

[0112] ALVMTQTPAPVSVAVGGTVTINCQASESVYNNNRLSWLQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGWKSYSHDYFAFGGGTEV (SEQ ID NO: 5).

[0113] The nucleic acid sequence encoding the heavy chain variable region (H1L65VH-DNA) of the H1L65 recombinant antibody is as follows:

[0114] GTGCTGAAGGGCGTGCAGTGCCAGAGCGTGGAGGAGAGCGGCGGCAGGCTGGTGACCCCCGGCACCCCCCTGACCCTGACCTGCACCGTGAGCGGCATCGACCTGAGCACCAACGCCGTGAGCTGGGTGAGGCAGCCCCGGCGAGGGCCTGGAGTGGATCGGCGTGATGCAGAGCGGCGGCACCACCGACTACGCCAGCTGGACCAAGGGCAGGTTCACCATCAGC AGGACCAGCACCACCGTGGACCTGAAGATCACCAGCCCCCACCGAGGACACCGCCACCTACAGCTGCGCCAGGGGCGGCAACAGGTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCCAGCCCAAGGCCCCCAGCGTGTTCCCCTTGGCCCCCTGCTGCGGCGACACCCCCAGCAGCACCGTGACCCTGGGCTGCCTGGTGAAGGGCTAC(SEQ ID NO:14),

[0115] The nucleic acid sequence encoding the light chain variable region (H1L65VL-DNA) of the H1L65 recombinant antibody is as follows:

[0116] GCCCTGGTGATGACCCAGACCCCCGCCCCCGTGAGCGTGGCCGTGGGCGGCACCGTGACCATCAACTGCCAGGCCAGCGAGAGCGTGTACAACAACAGGCTGAGCTGGCTGCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTACGCCAGCACC CTGGCCAGCGGCTGCCCAGCAGGTTCAAGGGCAGCGGCAGCGGCACCCAGTTCACCCTGACCATCAGCGGCGTGCAGTGCGACGACGCCGCCACCTACTACTGCGCCGGCTGGAAGAGCTACAGCCACGACTACTTCGCCTTCGGCGGCGGCACCGAGGTG(SEQ ID NO:15).

[0117] Recombinant antibody 3 (H267L31):

[0118] The amino acid sequence of the heavy chain variable region (H267L313VH) of the H267L313 recombinant antibody is as follows:

[0119] VLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSGYYMHWVRQAPGKGLEWIGIIYISGSPYYAHWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARGGYAGDSGYDYGTEYNLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL(SEQ ID NO:6),

[0120] The amino acid sequence of the light chain variable region (H267L313VL) of the H267L313 recombinant antibody is as follows:

[0121] AYDMTQTPASVEVAVGGTVTMKCQASQSISNYLNWYQQKPGQRPKLLIYRASTLASGVPSRVSGSGSGTQFTLTISGVECADAATYYCQQDYTSTNVDNIFGGGTEV (SEQ ID NO: 7).

[0122] The nucleic acid sequence encoding the heavy chain variable region (H267L313VH-DNA) of the H267L313 recombinant antibody is as follows:

[0123] GTGCTGAAGGGCGTGCAGTGCCAGAGCCTGGAGGAGAGCGGCGGCAGGCTGGTGACCCCCGGCACCCCCCTGACCCTGACCTGCACCGCCAGCGGCTTCAGCCTGAGCGGCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCTACATCAGCGGCAGCCCCTACTACGCCCACTGGGCCAAGGGCAGGTTCACCATCAGCAGGACCAGCACCACCGTGGACCTGAAGATCACCAGCCCCACCACCGAGGACACCGCCACCTACTTCTGCGCCAGGGGCGGCTACGCCGGCGACAGCGGCTACGACTACGGCACCGAGTACAACCTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCCAGCCCAAGGCCCCCAGCGTGTTCCCCCTGGCCCCCTGCTGCGGCGACACCCCCAGCAGCACCGTGACCCTGGGCTGCCTGGTGAAGGGCTACCTG(SEQ ID NO:16),

[0124] The nucleic acid sequence encoding the light chain variable region of the recombinant antibody H267L313 (H267L313 VL-DNA) is:

[0125] GCCTACGACATGACCCAGACCCCCGCCAGCGTGGAGGTGGCCGTGGGCGGCACCGTGACCATGAAGTGCCAGGCCAGCCAGAGCATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCCAGAGGCCCAAGCTGCTGATCTACAGGGCCAGCACCCTGGCCAGCGGCGTGCCCAGCAGGGTGAGCGGCAGCGGCAGCGGCACCCAGTTCACCCTGACCATCAGCGGCGTGGAGTGCGCCGACGCCGCCACCTACTACTGCCAGCAGGACTACACCAGCACCAACGTGGACAACATCTTCGGCGGCGGCACCGAGGTG(SEQ ID NO:17).

[0126] Recombinant antibody 4 (H129L202):

[0127] The amino acid sequence of the heavy chain variable region (H129L202VH) of the H129L202 recombinant antibody is as follows:

[0128] VLKGVQCQSVEESGGGLVKPGTSLTLTCKASGFSFSSGYWLYWVRQAPEKGLEWIGAIYADSGSTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARSLNTAITDDYYALFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL(SEQ ID NO:8);

[0129] The amino acid sequence of the light chain variable region (H129L202VL) of the H129L202 recombinant antibody is as follows:

[0130] AAVLTQTPSPVSAAVGGTVTIGCQSSQSIYNNRDLAWYQQKQGQPPKLLIYRASKLASGVPSRFSGSGSGTQFNLTISGVQCDDAATYYCLGGYDDDEDTVFGGGTEV (SEQ ID NO: 9).

[0131] The nucleic acid sequence encoding the heavy chain variable region (H129L202VH-DNA) of the H129L202 recombinant antibody is as follows:

[0132] GTGCTGAAGGGCGTGCAGTGCCAGAGCGTGGAGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCACCAGCCTGACCCTGACCTGCAAGGCCAGCGGCTTCAGCTTCAGCAGCGGCTACTGGCTGTACTGGGTGAGGCAGGCCCCCGAGAAGGGCCTGGAGTGGATCGGCGCCATCTACGCCGACAGCGGCAGCACCTACTACGCCAGCTGGGCCAAGGGCAGGTTCACCATCAGCAAGACCAGCAGCACCACCGTGACCCTGCAGATGACCAGCCTGACCGCCGCCGACACCGCCACCTACTTCTGCGCCAGGAGCCTGAACACCGCCATCACCGACGACTACTACGCCCTGTTCAACCTGTGGGGCCCCGGCACCCTGGTGACCGTGAGCAGCGGCCAGCCCAAGGCCCCCAGCGTGTTCCCCCTGGCCCCCTGCTGCGGCGACACCCCCAGCAGCACCGTGACCCTGGGCTGCCTGGTGAAGGGCTACCTG(SEQ ID NO:18),

[0133] The nucleic acid sequence encoding the light chain variable region of the H129L202 recombinant antibody (H129L202 VL-DNA) is:

[0134] GCCGCCGTGCTGACCCAGACCCCCAGCCCCGTGAGCGCCGCCGTGGGCGGCACCGTGACCATCGGCTGCCAGAGCAGCCAGAGCATCTACAACAACAGGGACCTGGCCTGGTACCAGCAGAAGCAGGGCCAGCCCCCCAAGCTGCTGATCTACAGGGCCAGCAAGCTGGCCAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCCAGTTCAACCTGACCATCAGCGGCGTGCAGTGCGACGACGCCGCCACCTACTACTGCCTGGGCGGCTACGACGACGACGAGGACACCGTGTTCGGCGGCGGCACCGAGGTG(SEQ ID NO:19).

[0135] The sequences of the heavy chain constant region (CH) and light chain constant region (CL) of the rabbit monoclonal antibody that specifically binds to the NGAL protein are as follows:

[0136] The amino acid sequence of the recombinant antibody heavy chain constant region (CH) is as follows:

[0137] SGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPI AHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:10),

[0138] The amino acid sequence of the light chain constant region (CL) of the above recombinant antibody is as follows:

[0139] FGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCK(SEQ ID NO:11);

[0140] The nucleic acid sequence encoding the recombinant antibody heavy chain constant region (CH-DNA) is as follows:

[0141] AGCGGCACACTGACCAACGGCGTGAGGACCTTTCCTAGCGTGAGACAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTGTCCGTGACAAGCAGCTCCCAGCCCGTGACATGCAACGTGGCCCACCCCGCCACAAACACAAAGGTGGATAAGACAGTGGCCCCTAGCACCTGCAGCAAGCCCACCTGTCCCCCTCCTGAGCTGCTGGGCGGCCCTTCTGTGTTTATCTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACATGCGTGGTGGTGGACGTGAGCCAGGACGATCCTGAGGTGCAGTTCACCTGGTACATCAATAACGAGCAGGTGAGAACAGCCAGACCTCCTCTGAGAGAGCAGCAGTTCAACTCCACCATCAGAGTGGTGAGCACCCTGCCCATCGCCCACCAGGACTGGCTGAGGGGCAAGGAGTTTAAGTGTAAGGTGCACAACAAGGCCCTGCCCGCCCCTATCGAGAAGACCATCAGCAAGGCCAGGGGCCAGCCCCTGGAGCCTAAGGTGTACACCATGGGCCCCCCTAGGGAGGAGCTGAGCTCCAGGTCCGTGAGCCTGACCTGCATGATCAATGGCTTCTACCCTTCCGACATCTCCGTGGAGTGGGAGAAGAACGGCAAGGCCGAGGATAACTACAAGACCACACCTGCCGTGCTGGACTCCGACGGCAGCTACTTCCTGTACAGCAAGCTGAGCGTGCCCACAAGCGAGTGGCAGAGGGGCGATGTGTTCACCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCCATCTCCAGGTCCCCCGGCAAG(SEQ ID NO:20);

[0142] The nucleic acid sequence encoding the recombinant antibody light chain constant region (CL-DNA) is:

[0143] TTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCT GGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAG(SEQID NO:21).

[0144] (5) Expression, purification and validation of recombinant anti-NGAL antibody

[0145] Expression vectors containing the complete encoding of the rabbit monoclonal antibody light chain gene and heavy chain gene were paired 1:1 and co-transfected into cells at a density of 3 × 10⁶ cells / year. 6 Cell supernatant was collected after culturing 100 mL of Expi 293F cells for 5 days. The antibody was purified by affinity chromatography using Protein A resin (Beyotime, P2019-50 ml). The purity and molecular weight of the anti-NGAL recombinant antibody were preliminarily identified using SDS-PAGE. 2 μg of antibody was added to an appropriate amount of loading buffer to make a total volume of 20 μL. Reducing and non-reducing SDS-PAGE samples were prepared simultaneously. A 10% SDS-PAGE gel was prepared, and samples were loaded. Electrophoresis was performed at 80 V for 30 min, followed by 120 V until clear bands were observed. The gel was removed after electrophoresis, stained with Coomassie brilliant blue, and after 15 min, the staining solution was removed. The gel was rinsed thoroughly with water and then destained with destaining solution until clear bands were observed. The results are as follows: Figure 4 As shown, lanes 1-4 contain purified antibodies H14L73 (RMB1019005), H1L65 (RMB1019006), H267L313 (RMB1019007), and H129L202 (RMB1019008), respectively.

[0146] Example 3

[0147] This embodiment provides the application of the anti-NGAL recombinant antibody in Example 2 in the detection of NGAL protein molecules. The detection methods include ELISA, Western blotting, immunofluorescence assay, immunohistochemistry, etc.

[0148] (1) ELISA analysis of the binding characteristics of anti-NGAL recombinant antibody

[0149] The purified antibody was specifically bound to the NGAL protein using an ELISA method. The detection method was the same as the rabbit antiserum ELISA method described in step 1 of Example 1, except that the antigen used in this ELISA was a commercially available recombinant NGAL protein (Hytest, Cat.#8NL2). The ELISA results are as follows: Figure 5 As shown, H14L73, H1L65, H267L313, and H129L202 all exhibited good binding effects with commercially available NGAL recombinant proteins.

[0150] (2) Immunoblotting analysis of the specificity of anti-NGAL recombinant antibodies

[0151] Cell lysates were prepared from well-cultured MCF-7, COLO 205, SW480, and A431 cells using SDS loading buffer. MCF7 cells, which do not express NGAL protein, served as a negative control. SDS-PAGE electrophoresis was performed using standard methods. The target protein gel region was transferred to a PVDF membrane (activated with methanol) and transferred at a constant current of 120 mA for 90 min. After transfer, the PVDF membrane was blocked in 5% skim milk powder on a shaker at room temperature for 1 h. The rabbit monoclonal antibody against NGAL protein (0.5 mg / mL) from Example 2 was diluted 1:1000 as the primary antibody and incubated on a shaker at room temperature for 2 h. The membrane was washed three times with TBST for 10 min each time. HRP-labeled goat anti-rabbit secondary antibody (1:10000) (Hangzhou Startech Biotechnology, S0B4002) was added and incubated on a shaker at room temperature for 1 h. The membrane was washed five times with TBST for 10 min each time. The ECL colorimetric solution is used to expose and develop the film for imaging analysis in a luminescent imaging system.

[0152] Immunoblotting results as follows Figure 6 As shown, the recombinant antibody H129L202 can specifically bind to the NGAL protein.

[0153] (3) Cell immunofluorescence assay to analyze the specific recognition of NGAL-positive tumor cells by anti-NGAL recombinant antibody.

[0154] After pre-seeding cell spreaders into 24-well cell culture plates, cells were seeded at a density of 1×10⁶ cells / well. 5A431 (NGAL-positive cell material) and MCF-7 (NGAL-negative cell material) cells were cultured at / mL for 12h, and the cell status was observed. After the cells were in good condition, the culture supernatant was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde (PFA) (Beyotime, P0099-500ml) at room temperature for 15min, washed three times with PBS, and blocked at 37℃ for 2h with 5% skim milk powder. After blocking, the cells were washed three times with PBS and stored at 4℃ for later use. The rabbit monoclonal antibody against NGAL protein (0.5 mg / mL) from Example 2 was diluted 1:50 to serve as the primary antibody. This was added to the prepared cell culture plate and incubated at 37°C for 60 min, followed by washing three times with TBST. Goat anti-rabbit fluorescent secondary antibody (Hangzhou Startech Biotechnology, S0B4004) was added under light-protected conditions, and the plate was incubated at 37°C for 45 min, followed by washing three times with TBST. DAPI (Beyotime, PO131-25 ml) was added under light-protected conditions to stain the nuclei. After incubation at room temperature for 5 min, the plate was washed three times with TBST. A mounting medium was then placed on a glass slide and placed over the mounting medium from one side. Fluorescence was observed under an inverted fluorescence microscope.

[0155] Cell immunofluorescence detection results as follows Figure 7 As shown, the recombinant antibody H129L202 can specifically bind to cells expressing NGAL protein, but does not bind to MCF-7 cells that do not express NGAL molecules.

[0156] (4) Immunohistochemical analysis of anti-NGAL recombinant antibody

[0157] Paraffin tissue microarrays containing human tonsil, kidney, pancreatic cancer, and breast cancer tissues were placed in a drying oven and baked at 65°C for approximately 1 hour for dewaxing and hydration. After dewaxing, the sections were removed and rinsed three times with distilled water. The sections were then placed in a staining box containing EDTA retrieval solution and placed in an autoclave for antigen retrieval under high temperature and pressure. After heating, the sections were allowed to cool naturally. They were then rinsed thoroughly with distilled water and washed three times with 1×PBS. The antigen-retrieved sections were placed in 3% H2O2 solution for 10 minutes, followed by three washes with 1×PBS. Non-specific antigens were blocked with 5% BSA + 10% sheep serum (prepared with PBST) at room temperature for 30 minutes. After blocking, the blocking solution was removed from the sections, diluted primary antibody was added, and the sections were incubated overnight at 4°C. The following morning, the sections were rinsed three times with 1×PBST, followed by the addition of secondary antibody and incubation at room temperature for 30 minutes, followed by three washes with 1×PBST. AB staining for 1-2 minutes, which can be observed with the naked eye until a brownish-yellow color appears. After staining, rinse directly with tap water to stop the staining process. Hematoxylin counterstaining for 2-5 minutes. After rinsing with distilled water, destain with PBS for 30 seconds to regain blue color, rinse with distilled water again, and then dehydrate the sections. After dehydration and drying with a series of alcohols, add 50-100 μL of neutral resin to each section, and then mount with a coverslip. After mounting, air dry the sections overnight, and observe under a microscope the next day and scan the sections with a tissue sectioner to obtain the immunohistochemical results.

[0158] Immunohistochemical test results as follows Figure 8 As shown, the recombinant antibody H129L202 exhibited NGAL molecule-specific positive staining signals in human tonsils, human kidneys, human pancreatic cancer, and human breast cancer tissues.

[0159] Example 4

[0160] This embodiment provides a screening method for detecting anti-NGAL recombinant antibody pairings in biological samples.

[0161] This embodiment provides a method for screening paired recombinant anti-NGAL antibodies to detect NGAL molecules in biological samples using a double-antibody sandwich ELISA method.

[0162] One of the anti-NGAL recombinant antibodies from Example 2 will be used as the capture antibody, and the other will be used as the labeling antibody (Biotin labeling). The capture antibody will be coated into the wells of the ELISA plate. First, a biological sample (patient serum) will be added. After incubation, the unbound antigen will be washed away. Then, the labeling antibody will be added. After incubation, the unbound labeling antibody will be washed away. Finally, the colorimetric solution will be added for color development.

[0163] The specific steps are as follows: Dilute the capture antibody to 2 μg / mL with coating buffer, add 50 μL / well to each well of the ELISA plate, and coat overnight at 4°C. Pat the ELISA plate dry, add 200 μL of blocking buffer (PBS containing 1% BSA) to each well, block at 37°C for 2 hours, and then pat dry. Add 50 μL of positive blood sample to each well (pre-dilute the blood sample to 100 ng / mL with 0.5% BSA / PBS). Incubate at 37°C for 1 hour, then wash the plate 3 times. Add 50 μL of biotinylated antibody to each well (pre-dilute the labeled antibody to 0.5 μg / mL with 0.5% BSA / PBS). Incubate at 37°C for 1 hour on a shaker, then wash the plate 3 times. Add 50 μL of SA-HRP to each well (pre-diluted 1:20000 with 0.5% BSA / PBS), incubate at 37°C for 1 hour on a shaker, then wash the plate 3 times. Add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 10 min. Then, add 100 μL of 4.9% H3PO4 to each well to terminate the reaction. Read the OD value at 450 nm using a microplate reader.

[0164] The results of the anti-NGAL recombinant antibody pairing screening are shown in Table 1.

[0165] Table 1. Statistical analysis of P / N values ​​of different anti-NGAL recombinant antibodies.

[0166]

[0167]

[0168] As shown in Table 1, a positive result cannot be detected when the P / N ratio of the same monoclonal antibody is less than 2.1; however, when two different monoclonal antibodies are used in combination, a positive result can be detected when the P / N ratio is greater than or equal to 2.1.

Claims

1. An anti-NGAL recombinant antibody, characterized in that, The recombinant antibody includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

9.

2. A nucleic acid, characterized in that, The nucleic acid encodes the heavy chain variable region and the light chain variable region of the anti-NGAL recombinant antibody according to claim 1.

3. A nucleic acid according to claim 2, characterized in that, The nucleic acid includes nucleic acids with nucleotide sequences as shown in SEQ ID NO. 18 and SEQ ID NO.

19.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in claim 2 or 3.

5. A recombinant expression cell, characterized in that, The recombinant expression cell comprises the recombinant expression vector of claim 4, or the nucleic acid of claim 2 or 3.

6. The use of the nucleic acid of claim 2 or 3, or the recombinant expression vector of claim 4, or the recombinant expression cell of claim 5 in the preparation of anti-NGAL recombinant antibody.

7. The method for preparing the anti-NGAL recombinant antibody according to claim 1, characterized in that, The method includes: transfecting cells with the recombinant expression vector of claim 4 to obtain recombinant expression cells; culturing the transfected recombinant expression cells; collecting the supernatant and purifying it to obtain anti-NGAL recombinant antibody.

8. The use of the anti-NGAL recombinant antibody of claim 1, or the nucleic acid of claim 2 or 3, or the recombinant expression vector of claim 4, or the recombinant expression cell of claim 5 in the preparation of a kit for detecting NGAL protein.

9. The application according to claim 8, characterized in that, The detection of NGAL protein includes any one or more of the following methods: ELISA, Western blotting, immunohistochemistry, immunofluorescence, and flow cytometry.

10. A kit for detecting NGAL protein, characterized in that, The kit includes the anti-NGAL recombinant antibody as described in claim 1.

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