Monoclonal antibody for alpha1-antitrypsin detection and application thereof

Abstract

The invention provides a monoclonal antibody for detecting alpha1-antitrypsin and application of the monoclonal antibody. The monoclonal antibody is any one of a monoclonal antibody A1 P39-5 and a monoclonal antibody A1 P40-6 or a combination of the monoclonal antibody A1 P39-5 and the monoclonal antibody A1 P40-6. The Serpin A1 antibody prepared by the invention has extremely high affinity, so that the detection sensitivity of a detection kit based on the antibody to Serpin A1 in serum reaches a pg / mL level which is far higher than a microgram / mL level of a conventional immunoturbidimetry. Meanwhile, the detection linear range is wide, a high-concentration sample does not need to be diluted for multiple times, and the operation error is reduced.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a monoclonal antibody for the detection of α1-antitrypsin and its application. Background Technology

[0002] α1-Anttrypsin (AAT), also known as Serpin A1, is an acute-phase reactive glycoprotein mainly synthesized and secreted into the blood by the liver. It belongs to the serine protease inhibitor superfamily. Serpin A1 mainly functions to inhibit neutrophil elastase (NE) in vivo and is an important protective factor for maintaining tissue homeostasis, especially the structural integrity of lung tissue.

[0003] During infection or inflammatory responses, neutrophils are recruited in large numbers to the lesion site and release elastase to degrade pathogen-associated proteins and damaged tissue components. However, when elastase activity is excessive or unregulated, it can nonspecifically degrade elastin and other extracellular matrix components abundant in the alveolar walls, leading to lung tissue structural damage. Serpin A1 forms a 1:1 stoichiometric covalent complex with NE through its reaction center loop (located at Met358-Ser359), irreversibly inactivating elastase and thus limiting the damage to the body's own tissue caused by the inflammatory response.

[0004] The Serpin A1 encoding gene exhibits significant genetic polymorphism, with some mutations closely associated with the development of hereditary α1-antitrypsin deficiency (AATD). The most common pathogenic mutation is the Z-type mutation, which causes Serpin A1 protein to misfold and form aggregates within hepatocytes, hindering its effective secretion into the bloodstream. This abnormality can lead to dual pathological consequences: firstly, the abnormally aggregated Serpin A1 polymers within hepatocytes are cytotoxic, causing hepatocyte damage and potentially progressing to neonatal hepatitis, childhood or adult cirrhosis, and even hepatocellular carcinoma; secondly, a significant decrease in functional Serpin A1 levels in the blood leads to uncontrolled elastase activity in neutrophils in the lungs, continuously damaging alveolar structure and ultimately causing progressive emphysema. Exogenous stimuli such as smoking can further reduce the inhibitory activity of Serpin A1 through oxidative modification, thereby accelerating the progression of lung function impairment.

[0005] Therefore, α1-antitrypsin deficiency is typically characterized by a significant decrease in serum Serpin A1 protein levels or functional impairment, and its clinical diagnosis and disease monitoring are highly dependent on the accurate determination of serum Serpin A1 protein concentration. Currently, the main screening method for serum Serpin A1 protein is immunoturbidimetry. However, this method has limited sensitivity and a high detection limit, which limits its application in detecting low-abundance Serpin A1 samples, early disease screening, and research, making it difficult to meet the demand for highly sensitive and specific detection.

[0006] Therefore, there is an urgent need for a more sensitive, more specific, and applicable method for the detection of Serpin A1 protein suitable for scientific research and clinical applications, so as to achieve accurate determination of Serpin A1 content in serum, thereby providing a reliable technical means for the early diagnosis, efficacy evaluation and mechanism research of related diseases. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a monoclonal antibody for α1-antitrypsin detection and its application.

[0008] This invention provides a monoclonal antibody for the detection of α1-antitrypsin, wherein the monoclonal antibody is any one of monoclonal antibody A1 P39-5 and monoclonal antibody A1 P40-6 or a combination thereof; The amino acid sequence of the heavy chain variable region of A1 P39-5 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region of A1 P39-5 is shown in SEQ ID NO.3; The amino acid sequence of the heavy chain variable region of A1 P40-6 is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of A1 P40-6 is shown in SEQ ID NO.5.

[0009] The present invention also provides a nucleic acid encoding the monoclonal antibody described herein.

[0010] The present invention also provides biological materials containing the said nucleic acid.

[0011] In some embodiments, the biomaterial is an expression cassette, vector, transposon, or host cell.

[0012] The present invention also provides a complex or conjugate comprising the monoclonal antibody.

[0013] The present invention also provides a detection reagent comprising the aforementioned monoclonal antibody.

[0014] The present invention also provides the application of the detection reagent in the in vitro quantitative or qualitative detection of α1-antitrypsin in biological samples.

[0015] In some embodiments, the biological sample is any one or more of serum, plasma, whole blood, urine, sputum, bronchoalveolar lavage fluid, nasal secretions, cell culture supernatant, or tissue homogenate.

[0016] The present invention also provides an α1-antitrypsin ELISA detection kit, comprising: (1) A solid-phase carrier coated with a primary antibody; (2) A second antibody labeled with a detection marker; The first antibody is A1 P39-5, and the second antibody is A1 P40-6; or the first antibody is A1 P40-6, and the second antibody is A1 P39-5; The amino acid sequence of the heavy chain variable region of A1 P39-5 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region of A1 P39-5 is shown in SEQ ID NO.3; The amino acid sequence of the heavy chain variable region of A1 P40-6 is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of A1 P40-6 is shown in SEQ ID NO.5.

[0017] In some embodiments, the detection marker is horseradish peroxidase, alkaline phosphatase, or a fluorescent marker.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) High sensitivity: The Serpin A1 antibody prepared in this invention has extremely high affinity, enabling detection kits based on this antibody (such as chemiluminescence assay) to achieve a detection sensitivity of pg / mL for Serpin A1 in serum, which is much higher than the μg / mL level of conventional immunoturbidimetric assays. At the same time, it has a wide linear detection range, eliminating the need for multiple dilutions of high-concentration samples and reducing operational errors.

[0019] (2) High specificity: The antibody of the present invention screens for specific conformational epitopes or unique sites of Serpin A1, and has no cross-reaction with other serine protease inhibitors in serum (such as α1-antitrypsin, antithrombin III, etc.). It can also effectively avoid interference from Serpin A1 degradation products, greatly reducing the false positive and false negative rates of detection and ensuring the accuracy of the results.

[0020] (3) The optimized buffer system and blocking formulation of the kit effectively resist the effects of common endogenous interfering substances such as lipemia, jaundice, and hemolysis. Compared with the traditional immunoturbidimetric method, the kit of this invention is effective in handling complex samples. Attached Figure Description To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results of Serpin A1 protein expression and function identification in Example 1 of this invention are shown in Figure 1. A represents the SDS-page result, and B represents the verification result that the purified Serpin A1 protein can bind to the commercial NE monoclonal antibody.

[0022] Figure 2 The serum titer of BALB / c mice after the third immunization in Example 2 of this invention.

[0023] Figure 3 This is a growth trend diagram of Serpin A1 hybridoma in Example 2 of the present invention.

[0024] Figure 4 This is the result of Serpin A1 hybridoma monoclonal ELISA detection in Example 2 of the present invention.

[0025] Figure 5 The results of SDS-page analysis of the Serpin A1 hybridoma monoclonal antibody in Example 2 of this invention are shown.

[0026] Figure 6 This is the result of the competitive ELISA of Serpin A1 hybridoma monoclonal antibody in Example 2 of the present invention.

[0027] Figure 7 This is a schematic diagram of the principle of the Serpin A1 ELISA detection kit in Example 3 of the present invention.

[0028] Figure 8 Example 3 of this invention compares the standard curves of laboratory and commercial Serpin A1 ELISA kits.

[0029] Figure 9 This is the specificity verification result of the Serpin A1 antibody in Example 4 of the present invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] Example 1: Construction of a eukaryotic expression system for SerpinA1 protein and its functional identification (1) Plasmid construction and transformation: The Serpin A1 protein encoded by the human Serpin A1 gene (NCBI gene number 5265) was sent to Guangzhou Ruibo Biotechnology Co., Ltd. for synthesis. The synthesized protein sequence is shown in SEQ ID NO.1. After synthesis, it was inserted into the eukaryotic expression vector pcDNA3.1 for protein expression.

[0032] (2) Transfection and expression: PEI transfection reagent was used to transfect expi-293F cells. After 5 days, the supernatant was collected and the expression was detected by WB.

[0033] (3) Protein purification: After confirming expression, the NE protein with the His tag was purified using a Ni-NTA affinity column, and the purity of the purified SerpinA1 protein was confirmed using SDS-PAGE.

[0034] (4) Functional identification: The binding affinity between SerpinA1 and the specific antibody (commercial) was detected using Western blotting (WB), such as... Figure 1 As shown. Figure 1 SDS-PAGE results for sample A showed successful expression of SerpinA1 protein, with a size of 55 kDa, consistent with the theoretical size. The purified SerpinA1 protein was loaded onto the sample, and a commercially available SerpinA1 monoclonal antibody was used as the primary antibody. Figure 1 The exposure results of B showed that the purified SerpinA1 protein could bind to the commercial NE monoclonal antibody, indicating that the purified SerpinA1 protein has biological activity.

[0035] Example 2: Construction of an efficient SerpinA1-specific murine monoclonal antibody hybridoma screening platform (1) Immunization method for B-cell donor mice: 100 μg / mouse of Freund's complete adjuvant emulsified antigen was injected subcutaneously in the back; 2 weeks later, blood was collected from the cheek to detect antibody titers. At the same time, 200 μg / mouse of Freund's incomplete adjuvant emulsified antigen was injected subcutaneously in the back; 2 weeks later, blood was collected from the cheek to detect antibody titers. One month after the second immunization, Balb / c mice with high antibody titers were selected and injected intraperitoneally with approximately 200 μg / mouse of antigen for 3 consecutive days, followed by cell fusion.

[0036] (2) Cell fusion method: Mice with the highest immunogenic titer were selected, their spleens were isolated, and splenocytes were obtained. After electrofusion, the cells were mixed with HAT medium and added to 96-well plates at 200 μL / well. On day 8 post-fusion, the medium was replaced with HT medium at 120 μL / well. On day 10, the growth of monoclonal cells was observed under a microscope, and the supernatant was collected for ELISA detection. Cells from positive wells were digested and sorted by flow cytometry at 1 cell / well on the same day. After another 7 days, monoclonal growth was observed, and positive wells were digested and expanded into 6-well plates for further culture via ELISA. Cells from positive wells were digested and injected intraperitoneally into BALB / c mice. Seven days later, the ascites fluid was purified to obtain a monoclonal antibody specifically binding to Serpin A1 protein.

[0037] Serpin A1 protein was used as an immunogen for ELISA coating. After the third immunization of mice, whole blood was collected from the tail of the mice and centrifuged at 5000 rpm for 20 minutes at room temperature to obtain serum. This serum was used as the primary antibody for ELISA experiments.

[0038] The results are as follows Figure 2 As shown, Serpin A1 protein was successfully used for hybridoma immunization in BALB / c mice, and the serum antibody titer in mice reached 10. 7 .

[0039] Spleen cells from mice immunized with Serpin A1 were fused with SP2 / 0 myeloma cells, as follows: Figure 3 As shown, the hybridoma cells showed good growth trends on the second, third, fourth, sixth and eighth days after fusion, and obvious hybridoma clusters could be seen.

[0040] Serpin A1 protein was used as an immunogen for ELISA coating, and the supernatant of cultured hybridoma cells was used as the primary antibody for ELISA experiments. Figure 4 As shown, the image displays positive single clones in a 96-well plate during the screening of Serpin A1 hybridoma single clones. The darker the color in the image, the stronger the binding affinity of its supernatant to NE.

[0041] The eight most potent Serpin A1 hybridoma monoclonal antibodies were purified using protein A agarose gel beads and then subjected to SDS-PAGE. Figure 5 As shown, all monoclonal antibody heavy chains are at 50 kDa and light chains are at 25 kDa, the antibody size is correct, and the bands are clear.

[0042] Example 3: Screening and functional identification of antibody pairs for ELISA detection kits according to Figure 6The obtained highly specific Serpin A1 monoclonal antibodies were paired into antibody pairs, with one monoclonal antibody serving as the capture antibody and the other as the detection antibody. Competitive ELISA experiments were then performed to screen for antibody pairs with non-overlapping epitopes.

[0043] First, coat the well with Serpin A1 protein overnight (0.2 μg / well). The next day, block with 300 μL / well of 1% BSA, add 20 μg / mL of the competing antibody (each antibody needs to be added, 50 μL / well), incubate at room temperature for 2 hours, add 4 times the concentration of biotin-labeled antibody at room temperature for 2 hours, add streptavidin-HRP and incubate at room temperature for 1 hour, then develop the color with chromogenic solution.

[0044] The amino acid sequences of the following chains are shown in SEQ ID NO. 2: A1 P39-5H chain V region; K chain V region: A1 P40-6 H chain V region; K chain V region: A1 P42-6 H chain V region; K chain V region: A1 P41-6 H chain V region; K chain V region: A1 P42-6 H chain V region; K chain V region: A1 P41-6 H chain V region; K chain V region: A1 P41-6 H chain V region; K chain V region: A1 P38-3 H chain V region; L chain V region: A1 P41-1 H chain V region; K ... The amino acid sequence of the V region of the A1 P38-2 H chain is shown in SEQ ID NO.15; the amino acid sequence of the V region of the K chain is shown in SEQ ID NO.16; and the amino acid sequence of the V region of the K chain is shown in SEQ ID NO.17.

[0045] according to Figure 6 The competitive ELISA results show the percentage of two antibodies that do not compete for epitopes. The higher the value, the greater the probability that the antibody pair will not compete for the surface of the Serpin A1 protein. Therefore, antibodies that do not compete for epitopes were selected and paired to prepare ELISA detection kits. It was found that the antibodies A1 P39-5 and A1 P40-6 had low detection limits and high specificity in the prepared ELISA detection kits.

[0046] according to Figure 7The method involves first coating the well with the capture antibody A1 P39-5 (0.2 μg / well) overnight. The next day, the well is blocked with 300 μL / well of 1% BSA, followed by the addition of the detection sample or standards at different concentrations of NE protein. After incubation at room temperature for 2 hours, the biotin-labeled A1 P40-6 detection antibody (1.5 μg / well) is added, and incubation is repeated for another 2 hours at room temperature. Finally, streptavidin-HRP is added, and the well is incubated at room temperature for one hour before color development. This antibody pair of A1 P39-5 and A1 P40-6 is used to prepare an ELISA kit. The results of the kit are then compared with those of a commercially available kit from a well-known international brand, both detecting the same SerpinA1 immunogen.

[0047] When using A1 P39-5 as the capture antibody and A1 P40-6 as the detection antibody to create a detection kit, the standard curve and sensitivity for detecting Serpin A1 protein are as follows: Figure 8 As shown, its sensitivity reaches 100 pg / mL. A comparative experiment was conducted between this detection kit and a commercially available kit from a well-known international brand. When detecting the same Serpin A1 immunogen, the Serpin A1 ELISA kit of this invention showed a lower detection limit and higher sensitivity.

[0048] Example 4: Specificity verification test of Serpin A1 antibody The specificity of the monoclonal antibodies A1 P39-5 and A1 P40-6 obtained by screening in this invention against α1 antitrypsin (Serpin A1) was verified, and their cross-reaction with α1-antichymotrypsin (also known as Serpin A3), which belongs to the same family of serine protease inhibitors and has high amino acid homology, was evaluated.

[0049] Serpin A3 protein (0.2 μg / well) was coated overnight. The next day, the cells were blocked with 300 μL / well of 1% BSA. Then, 10 μg / mL and 50 μL / well of A1 P39-5 and A1 P40-6 monoclonal antibodies were added. After incubation at 37°C for 1 hour, goat anti-mouse-HRP secondary antibody was added. After incubation at 37°C for 1 hour, the cells were developed using a colorimetric solution.

[0050] Experimental results are as follows Figure 9 As shown, Figure 9 As shown in Figure A, both A1 P39-5 and A1 P40-6 produced strong signals when coated with the Serpin A1 antigen, indicating that both monoclonal antibodies could bind specifically to the Serpin A1 protein with high affinity. Figure 9As shown in Figure B, when the coating antigen was Serpin A3, the absorbance values ​​of both antibodies were close to the background level. Even with increased antibody concentration, no significant signal was observed, and no saturation binding curve was formed. This indicates that A1 P39-5 and A1 P40-6 exhibit almost no cross-recognition with Serpin A3 and have extremely low non-specific binding levels.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0052] sequence list SEQ ID NO.1: EDPQGD AAQKTDTSHH DQDHPTFNKI TPNLAEFAFS LYRQLAHQSN STNIFFSPVSIATAFAMLSL GTKADTHDEI LEGLNFNLTE IPEAQIHEGF QELLRTLNQP DSQLQLTTGN GLFLSEGLKLVDKFLEDVKK LYHSEAFTVN FGDTEEAKKQ INDYVEKGTQ GKIVDLVKEL DRDTVFALVN YIFFKGKWERPFEVKDTEEE DFHVDQVTTV KVPMMKRLGM FNIQHCKKLS SWVLLMKYLG NATAIFFLPD EGKLQHLENELTHDIITKFL ENEDRRSASL HLPKLSITGT YDLKSVLGQL GITKVFSNGA DLSGVTEEAP LKLSKAVHKAVLTIDEKGTE AAGAMFLEAI PMSIPPEVKF NKPFVFLMIE QNTKSPLFMG KVVNPTQK SEQ ID NO.2: RLRATGLNSGFVFMELLSCTSLCTIILEGLNQDLPDCLIYPSDKETHYYPKFLDAILTVDPSSFTAYMQLISLTSEDSAVFYCAR SEQ ID NO.3: MLAVCSLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRVNRLVDGVP.SRFSGSGSGQDFSLDITSLEYEDMGFYYCLHFDEF SEQ ID NO.4: IDVEVLSRGLISFSACFWRHINDIYLPWVKQRPDQGLEWIGRIDPANGNTKYDPKFQ.GKGTMTVDTCSKTAYLQLFSLTSEDTAVYYCA SEQ ID NO.5: MTCCSFFSRCEGHTPDRASSSVTYMNWYHQKPGSSPKPWIYDTSNLASGVP.ARFSGSGSGTSYSLKSAEWRLNMLPLIT SEQ ID NO.6: RYRSRGVLRLSCAASGFTFTKFAMSWVRLAPGKGLEWVSAISGTGGNTYYADSVKGRFTISRDNSKNTLSLQMNSLRAEDTAVYYCAK SEQ ID NO.7: SFSGREHRHCKSSQSLLYTSNNANYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAIYYCQQYWSTP SEQ ID NO.8: SSTGTSVPARAGVVFPLSLYMSSLGPPGSREGLEWVSVIRATTHKEYYADSVKGRFSISRDDSQDTVFMQLSSLKVEDTAVYYCVR SEQ ID NO.9: CIVERVTITCRASQSINNYLNWFRQRTGIAPKFLIYAASTLQSGVPSRFSGSGSGTYFTLTISSLQPEDFATYYCQQSYSA SEQ ID NO.10: KIVSAWGVLRLSCAASGFTFSNYVMSWVRQAPGKGLEWVSAVLGSGSNTYYTESVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCAK SEQ ID NO.11: QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLTIYDVSKRPSGVPDRFSGSKSGNTASLTIFGLQAEDEADYYCCSYAGSY SEQ ID NO.12: SPYVVRVLTLSCVASELTFTSYWMTWVRQAPGKGLEYVADIKHDGSEKNYVDSAKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYC SEQ ID NO.13: DVSGESTINCKSSHSLNSNNKNYLAWYQRKPGQPPKLFIYWASTRESGVPERFSGSGSGTDFTLTIDNLQAEDVAAYYCQQYHTG SEQ ID NO.14: NFTAWQVLRLSCAASGFTFHDYVMYWVRQVPGRGLEWVSGISCNGGTTVYADSVKGRFTVSRDNAKNSLYLQMNSLRPEDTAFYYCAK SEQ ID NO.15: RWRLLRAARRNKRRRTAGQGKAQHTFRPPLFIFSVSFLLGVPPPVFGGGASGTDFPLTTLSLPPEYFLNLLLPPY SEQ ID NO.16: LFLMMLEVLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEQ ID NO.17: RTARESPFTLPGQSRSYLVAGLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSY。

Claims

1. A monoclonal antibody for the detection of α1-antitrypsin, characterized in that, The monoclonal antibody is any one or a combination of monoclonal antibody A1 P39-5 and monoclonal antibody A1 P40-6. The amino acid sequence of the heavy chain variable region of A1 P39-5 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region of A1 P39-5 is shown in SEQ ID NO.3; The amino acid sequence of the heavy chain variable region of A1 P40-6 is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of A1 P40-6 is shown in SEQ ID NO.

5.

2. A nucleic acid encoding the monoclonal antibody as described in claim 1.

3. Biological material containing the nucleic acid of claim 2.

4. The biomaterial according to claim 3, characterized in that, The biological material is an expression cassette, vector, transposon, or host cell.

5. A complex or coupling compound, characterized in that, It includes the monoclonal antibody as described in claim 1.

6. A detection reagent, characterized in that, Includes the monoclonal antibody as described in claim 1.

7. The use of the detection reagent according to claim 6 in the in vitro quantitative or qualitative detection of α1-antitrypsin in biological samples.

8. The application according to claim 7, characterized in that, The biological sample is any one or more of the following: serum, plasma, whole blood, urine, sputum, bronchoalveolar lavage fluid, nasal secretions, cell culture supernatant, or tissue homogenate.

9. An α1-antitrypsin ELISA detection kit, characterized in that, include: (1) A solid-phase carrier coated with a primary antibody; (2) A second antibody labeled with a detection marker; The first antibody is A1 P39-5, and the second antibody is A1 P40-6; or the first antibody is A1 P40-6, and the second antibody is A1 P39-5; The amino acid sequence of the heavy chain variable region of A1 P39-5 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region of A1 P39-5 is shown in SEQ ID NO.3; The amino acid sequence of the heavy chain variable region of A1 P40-6 is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of A1 P40-6 is shown in SEQ ID NO.

5.

10. The α1-antitrypsin ELISA detection kit according to claim 9, characterized in that, The detection marker is horseradish peroxidase, alkaline phosphatase, or a fluorescent marker.

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