Monoclonal antibody capable of quantitatively detecting arrdc4 in exosomes and application thereof

Abstract

This invention relates to the field of biomedicine and discloses a monoclonal antibody capable of quantitatively detecting ARRDC4 in exosomes and its applications. This invention provides a novel monoclonal antibody (A-1) capable of quantitatively detecting ARRDC4 in exosomes. This antibody exhibits high specificity, high affinity, high accuracy, and high reproducibility for ARRDC4. Therefore, this antibody can be used to prepare kits for detecting ARRDC4 in exosomes, filling a technological gap in exosomal ARRDC4 detection, establishing a method for tumor diagnosis and prognostic assessment based on exosomal ARRDC4, and providing a new biomarker for liquid biopsy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a monoclonal antibody capable of quantitatively detecting ARRDC4 in exosomes and its applications. Background Technology

[0002] Arrestins are a class of intracellular molecules that play a crucial role in regulating signal transduction of G protein-coupled receptors. In recent years, increasing research has shown that proteins containing the arrestin-associated domain (ARRDC) are closely related to tumorigenesis and development. ARRDC4 (arrestin-associated domain protein 4), as an important member of the α-arrestin family, participates in the biogenesis of exosomes and related microvesicles. ARRDC4-mediated microvesicles (ARMMs) are rich in markers such as CD63 and CD81. Through the ARMM pathway, ARRDC4 participates in the transmission of metabolic regulatory signals (such as insulin signaling) and the diffusion of antiviral signals. Multiple studies in recent years have reported that ARRDC4 is associated with the development of various tumors. In colorectal cancer, breast cancer, and other malignant tumors, ARRDC4 is expressed at low levels, and its expression level is positively correlated with patient survival prognosis, suggesting that ARRDC4 may be a potential tumor suppressor. In prostate cancer, ARRDC4 gene polymorphism is significantly associated with tumor invasiveness, further supporting the important role of ARRDC4 in tumorigenesis and development. Mechanistic studies have shown that ARRDC4 can inhibit the overactivation of pro-cancer signaling pathways by regulating ubiquitination and endocytosis of receptor proteins such as EGFR. Downregulation or loss of function of ARRDC4 can lead to tumor cell proliferation, migration and enhanced drug resistance.

[0003] Exosomes are small vesicles secreted by cells that carry various bioactive molecules, including proteins, lipids, and RNA (such as miRNAs and mRNAs), transmitting signals between cells and regulating cellular behavior. Exosomes secreted by tumor-initiating cells have multiple regulatory effects on other tumor cells. For example, exosomes can activate the migration and invasiveness of other tumor cells by transmitting specific signaling molecules. miRNAs and proteins carried in tumor-initiating cell exosomes can enhance the extracellular matrix metastasis (EMT) and metastatic ability of tumor cells by regulating extracellular matrix degradation and cytoskeleton remodeling. Tumor-initiating cell exosomes can deliver some drug resistance-related molecules to non-initiating cell subsets within the tumor. These molecules may help tumor cells resist chemotherapy, radiotherapy, and other treatments by altering drug metabolism pathways or activating anti-apoptotic signals, leading to drug resistance or treatment failure. In addition, exosomes secreted by tumor-initiating cells can also promote tumor cell survival and proliferation, immune evasion, and regulate and modify the tumor microenvironment (TME). It is noteworthy that ARRDC4, as a key regulatory molecule of exosomes, not only participates in exosome biogenesis, but can also be encapsulated or anchored on the surface of exosomes, and transmitted between cells along with exosomes, thereby diffusing tumor suppressor signals to recipient cells. Therefore, the level of ARRDC4 in exosomes may more accurately reflect the biological state of tumor cells and the dynamic changes in their signaling networks.

[0004] Therefore, ARRDC4 in exosomes holds promise as a biomarker for tumor drug response, providing new reference for the clinical diagnosis, treatment monitoring, and prognostic assessment of related diseases. However, current high-specificity and high-sensitivity detection tools for ARRDC4 in exosomes are still incomplete, limiting its application in clinical diagnosis. Therefore, developing a monoclonal antibody that can specifically recognize the ARRDC4 protein in exosomes has significant clinical application value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a monoclonal antibody capable of quantitatively detecting ARRDC4 in exosomes and its applications. This invention provides a novel monoclonal antibody (A-1) capable of quantitatively detecting ARRDC4 in exosomes. This antibody exhibits high specificity, high affinity, high accuracy, and high reproducibility for ARRDC4. Therefore, this antibody can be used to prepare kits for detecting ARRDC4 in exosomes, filling a technological gap in exosomal ARRDC4 detection. It can be used to establish methods for tumor diagnosis and prognostic assessment based on exosomal ARRDC4, providing a new biomarker for liquid biopsy.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides a monoclonal antibody or its antigen-binding fragment for quantitative detection of ARRDC4 in exosomes, named A-1. The amino acid sequence of the monoclonal antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region; wherein: the light chain variable region includes CDR1, CDR2 and CDR3, whose amino acid sequences are, in order, positions 24-34, 50-56 and 89-97 of SEQ ID NO. 1; the heavy chain variable region includes CDR1, CDR2 and CDR3, whose amino acid sequences are, in order, positions 31-35, 50-65 and 98-106 of SEQ ID NO. 2.

[0007] Furthermore, the light chain variable region also includes framework regions FR1, FR2, FR3 and FR4, and their amino acid sequences are, in order, positions 1-23, 35-49, 57-88 and 98-107 of SEQ ID NO. 1; the heavy chain variable region also includes framework regions FR1, FR2, FR3 and FR4, and their amino acid sequences are, in order, positions 1-30, 36-49, 66-97 and 107-117 of SEQ ID NO. 2.

[0008] Furthermore, the light chain variable region includes the amino acid sequence shown in SEQ ID NO.1: QIVLTQSPALMSASPGEKVTMTCSASASVNYVYWYQQKPGSSPKAWIYLTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQHWSSNPLTFGAGTKLELK; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.2: QVQLQQPGSELVRPGASVNLSCKASGYTFTSYWMHWVKQRHGQGLEWIGNIYPGSGNTNYDEKFKRKAILTVDTSSSTAYMHLNSLTSEDSAVYYCARWLTGTWNFDVWGAGTTVTVSS.

[0009] Furthermore, the amino acid sequence of the monoclonal antibody or its antigen-binding fragment further includes a heavy chain constant region and a light chain constant region; wherein: the heavy chain constant region is IgG, IgM, or IgA; more preferably, the IgG is IgG1, IgG2, IgG3, or IgG4. The light chain constant region is a lambda(λ) constant region or a kappa(κ) constant region.

[0010] Preferably, the monoclonal antibody or its antigen-binding fragment includes any one of Fab, Fv, scFv, F(ab')2, linear antibody, and single-domain antibody.

[0011] Preferably, the monoclonal antibody or its antigen-binding fragment is mouse, human, chimeric, or humanized.

[0012] Most preferably, the light chain of the monoclonal antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.3; the heavy chain contains the amino acid sequence shown in SEQ ID NO.4.

[0013] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or its antigen-binding fragment thereof, comprising nucleotide sequences SEQ ID NO.5 and SEQ ID NO.6 that respectively encode amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0014] Most preferably, the polynucleotide comprises nucleotide sequences SEQ ID NO.7 and SEQ ID NO.8 that respectively encode amino acid sequences as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0015] Thirdly, the present invention provides the application of the above-mentioned monoclonal antibody or its antigen-binding fragment in the preparation of a kit for quantitative detection of ARRDC4 in exosomes.

[0016] Furthermore, the ARRDC4 is ARRDC4 in exosomes from serum, body fluids, or cell culture supernatants.

[0017] Fourthly, the present invention provides the use of the above-mentioned monoclonal antibody or its antigen-binding fragment in the preparation of a kit for the diagnosis or treatment of diseases associated with ARRDC4 in exosomes for monitoring or prognostic assessment.

[0018] Preferably, the diseases associated with ARRDC4 in exosomes are colorectal cancer and breast cancer.

[0019] Fifth, the present invention provides a chemiluminescent detection kit for quantitative detection of ARRDC4 in exosomes, comprising: a CD63+CD81 antibody coupled with magnetic beads for capturing CD63+CD81 positive exosomes; and the monoclonal antibody or its antigen-binding fragment thereof coupled with acridine ester as a detection antibody.

[0020] More preferably, the chemiluminescence detection kit comprises the following components: Reagent 1: 0.1~0.5 mg / mL affinity magnetic beads, with 1.0-10.0 ug / mL of A-1 antibody bound to the surface, 10-50 mM Tris-HCl or 20-100 mM phosphate buffer or 10-100 mM MME buffer (pH = 7.2~7.4), 0.10-1.00% BSA, 0-0.05% Tween 20, 0.05-0.1% PC300.

[0021] Reagent 2: 0.05–0.50 μg / mL acridinium ester (NSP-SA-NHS or NSP-DMAE-NHS) labeled A-1 antibody, 20–100 mM phosphate buffer or 10–100 mM MES buffer (pH = 6.0–7.4), 0.10–1.00% BSA, 0.05–0.1% PC300, 0–0.05% Tween 20 Diluents: 20mM Tris-HCl or 10-100mM phosphate buffer, 2.0%-5.0% newborn calf serum or 0.1-1.0% BSA, 0-0.05% Tween 20.

[0022] Compared with the prior art, the beneficial effects of the present invention are: (1) The amino acid sequence of the monoclonal antibody (A-1) provided by the present invention is different from that reported in existing literature or patents. It is a novel antibody that can detect ARRDC4 in exosomes. This antibody has the characteristics of high specificity, high affinity, high accuracy and high reproducibility for ARRDC4.

[0023] (2) The monoclonal antibody (A-1) provided by the present invention has the characteristics of high specificity, high affinity, high accuracy and high repeatability for ARRDC4, and can quantitatively detect ARRDC4 in exosomes.

[0024] (3) The monoclonal antibody (A-1) provided by the present invention can be used to prepare a kit for detecting ARRDC4 in exosomes, filling the technical gap in the detection of exosome ARRDC4. It can be used to establish a tumor diagnosis and prognostic assessment method based on exosome ARRDC4, providing a new biomarker for liquid biopsy. Attached Figure Description

[0025] Figure 1 This is a graph showing the SDS-PAGE purity analysis of the prokaryotic ARRDC4 protein. Figure 2 Image showing the initial screening of antibody-positive clones of exosomal ARRDC4; Figure 3 A selection diagram for optimal positive clones of exosomal ARRDC4; Figure 4SDS-PAGE purity analysis of exosomal ARRDC4 antibody (A-1); Figure 5 This is an identification diagram showing the specific binding of A-1 antibody to ARRDC4; Figure 6 This is the standard curve for the exosome chemiluminescence reagent kit. Detailed Implementation

[0026] First, a monoclonal antibody or its antigen-binding fragment for quantitative detection of ARRDC4 in exosomes, named A-1, has an amino acid sequence comprising a light chain variable region and a heavy chain variable region; wherein: the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region sequentially include positions 24-34, 50-56, and 89-97 of SEQ ID NO.1; the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region sequentially include positions 31-35, 50-65, and 98-106 of SEQ ID NO.2.

[0027] Furthermore, the light chain variable region also includes framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 sequentially include positions 1-23, 35-49, 57-88, and 98-107 of SEQ ID NO. 1; the heavy chain variable region also includes framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 sequentially include positions 1-30, 36-49, 66-97, and 107-117 of SEQ ID NO. 2.

[0028] Furthermore, the light chain variable region includes the amino acid sequence shown in SEQ ID NO.1: QIVLTQSPALMSASPGEKVTMTCSASASVNYVYWYQQKPGSSPKAWIYLTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQHWSSNPLTFGAGTKLELK; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.2: QVQLQQPGSELVRPGASVNLSCKASGYTFTSYWMHWVKQRHGQGLEWIGNIYPGSGNTNYDEKFKRKAILTVDTSSSTAYMHLNSLTSEDSAVYYCARWLTGTWNFDVWGAGTTVTVSS.

[0029] Furthermore, the amino acid sequence of the monoclonal antibody or its antigen-binding fragment further includes a heavy chain constant region and a light chain constant region; wherein: the heavy chain constant region is IgG, IgM, or IgA; more preferably, the IgG is IgG1, IgG2, IgG3, or IgG4. The light chain constant region is a lambda(λ) constant region or a kappa(κ) constant region.

[0030] Preferably, the monoclonal antibody or its antigen-binding fragment includes any one of Fab, Fv, scFv, F(ab')2, linear antibody, and single-domain antibody.

[0031] Most preferably, the monoclonal antibody or its antigen-binding fragment comprises, in its light chain, the amino acid sequence shown in SEQ ID NO. 3 (QIVLTQSPALMSASPGEKVTMTCSASASVNYVYWYQQKPGSSPKAWIYLTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQHWSSNPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC); and in its heavy chain, the sequence shown in SEQ ID NO. 3. The amino acid sequence shown in NO.4 (QVQLQQPGSELVRPGASVNLSCKASGYTFTSYWMHWVKQRHGQGLEWIGNIYPGSGNTNYDEKFKRKAILTVDTSSSTAYMHLNSLTSEDSAVYYCARWLTGT WNFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).

[0032] Preferably, the monoclonal antibody or its antigen-binding fragment is mouse, human, chimeric, or humanized.

[0033] Second, the present invention provides a polynucleotide encoding the monoclonal antibody or its antigen-binding fragment, comprising nucleotide sequences SEQ ID NO. 5 (caaattgttctcacccagtctccagcactcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagcgcaagtgtaaattatgtgtactggtaccagcagaagccaggatcctcccccaaagcctggatttatctcacatccaacctggcttctggagtccctggtcgcttcagtggcagtgggtctgggacctcttattctctaacaatcagtagcatggaggctgaagatgctgccacttattactgccagcactggagtagtaatccgctcacgttcggtgctggggacctcttattctctaacaatcagtagcatggaggctgaagatgctgccacttattactgccagcactggagtagtaatccgctcacgttcggtgctgggaccaagctggagctgaaa) and SEQ ID NO. NO.6 (caggtccaactgcagcagcagcctgggtctgagctggtgaggcctggagcttcagtgaacctgtcctgcaaggcttctggctacacattcaccagctactggatgcactgggtgaagcagaggcatggacaaggccttgagtggattggaaatatttatcctggtagtggtaatactaac tacgatgagaagttcaagaggaaggccatactgaccgtagacacatcctccagcacagcctacatgcacctcaacagcctgacatctgaggactctgcggtctattactgtgcgagatggctaactgggacatggaacttcgatgtctggggcgcagggaccacggtcaccgtctcctca).

[0034]

[0035] Third, a chemiluminescent assay kit for quantitative detection of ARRDC4 in exosomes, comprising: a CD63+CD81 antibody coupled with magnetic beads for capturing CD63+CD81 positive exosomes; and the monoclonal antibody or its antigen-binding fragment coupled with acridine ester as the detection antibody.

[0036] Preferably, the chemiluminescence detection kit comprises the following components: Reagent 1: 0.1~0.5 mg / mL affinity magnetic beads, with 1.0-10.0 ug / mL A-1 antibody bound to the surface, 10-50 mM Tris-HCl or 20-100 mM phosphate buffer or 10-100 mM MES buffer (pH = 7.2~7.4), 0.10-1.00% BSA, 0-0.05% Tween 20, 0.05-0.1% PC300.

[0037] Reagent 2: 0.05–0.50 μg / mL acridinium ester (NSP-SA-NHS or NSP-DMAE-NHS) labeled A-1 antibody, 20–100 mM phosphate buffer or 10–100 mM MES buffer (pH = 6.0–7.4), 0.10–1.00% BSA, 0.05–0.1% PC300, 0–0.05% Tween 20 Diluents: 20mM Tris-HCl or 10-100mM phosphate buffer, 2.0%-5.0% newborn calf serum or 0.1-1.0% BSA, 0-0.05% Tween 20.

[0038] A method for preparing a monoclonal antibody for quantitative detection of ARRDC4 in exosomes, comprising the following steps: Immunization of animals: Balb / c mice were injected with ARRDC4 protein; each mouse was injected with 100 μL (100 μg / mL), for a total of 5 mice. Each mouse was immunized 3-5 times, with each immunization spaced 2 weeks apart.

[0039] ELISA titer test: Blood was collected from the tail vein of mice and centrifuged to obtain plasma. The titer of the glycocholic acid-antibody complex in mouse plasma was tested by ELISA, and mice with the highest titer were selected.

[0040] Mice were sacrificed by cervical dislocation to obtain their spleens. The spleens were washed with antibiotics and then with PBS until they turned grayish-white. After centrifugation and washing, a spleen cell suspension was obtained. SP2 / 0 cells and spleen cells were mixed at a ratio of 1:10 and then seeded into 96-well plates. The cell supernatant was collected after 8-10 days.

[0041] Positive clones of ARRDC4 were screened by ELISA and expanded cultured until hybridoma cell lines with monoclonal antibodies were obtained.

[0042] Hybridoma cells were injected intraperitoneally into mice to obtain ascites. The ascites was purified using a Protein A column to obtain monoclonal antibody A-1, which was then analyzed by SDS-PAGE and its A280 concentration was determined.

[0043] Example 1: Preparation of exosomal ARRDC4 antibody (A-1) Antigen preparation: The gene encoding the target protein is inserted into a plasmid using molecular cloning technology. This plasmid carries a His-tag sequence. The constructed expression vector is then transformed into competent *E. coli* cells. After culturing the bacteria to a suitable density in culture medium, an inducer is added to initiate large-scale expression of the target protein. The expressed bacterial cells are collected, and the cell walls and membranes are broken by sonication to release the internal proteins, generating inclusion bodies. The precipitate is then subjected to renaturation treatment. Using the tag on the target protein (e.g., His-tag), high-purity one-step purification is performed using a nickel column. Finally, the purity and molecular weight (e.g., [missing information]) are identified by SDS-PAGE electrophoresis. Figure 1 (As shown).

[0044] Immunization of animals: Balb / c mice were injected with exosomal ARRDC4 antigen; each mouse was injected with 100 μL of 100 μg / mL exosomal ARRDC4 antigen, for a total of 5 mice. Each mouse was immunized 5 times, with an interval of 2 weeks between each immunization.

[0045] ELISA titer assay: Blood was collected from the tail vein of mice and centrifuged to obtain plasma. The titer of exosomal ARRDC4 antigen in mouse plasma was tested by ELISA, and mice with the highest titer were selected.

[0046] Mice were sacrificed by cervical dislocation to obtain their spleens. The spleens were washed with antibiotics and then with PBS until they turned grayish-white. After centrifugation and washing, a spleen cell suspension was obtained. SP2 / 0 cells and spleen cells were mixed at a ratio of 1:10 and then seeded into 96-well plates. The cell supernatant was collected after 8 days.

[0047] Positive clones of exosomal ARRDC4 were screened using ELISA: the initial screening measured the reaction between cell supernatant and ARRDC4 protein. Results are as follows: Figure 2As shown, the cells corresponding to the blue-labeled wells are positive clones. These positive clones were cultured in a large-scale manner until hybridoma cell lines that stably secrete ARRDC4 monoclonal antibodies were obtained. The screening steps are as follows: Dilute humanized CD81 antibody to 1 μg / mL with CBS buffer, add 100 μL to each well of the microplate, and coat overnight at 4°C; add 250 μL of TBST washing buffer to each well, let stand for 1 min, pat dry, and repeat the washing 4 times; then add 10% newborn calf serum-TBST blocking buffer to each well and incubate at 37°C for 1 h; dilute exosome ARRDC4 to 1 μg / mL, add 100 μL to each well, and incubate at 37°C for 1 h; repeat the above washing steps; dilute GAM (goat anti-mouse IgG) with 5% newborn calf serum-TBST at a ratio of 1:6000, add 100 μL to each well, and incubate at 37°C for 30 min; repeat the washing steps again; add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well sequentially, react at room temperature in the dark for 5 min, and finally add 50 μL of stop solution. The OD values ​​of each well were measured using a microplate reader under dual wavelengths (primary wavelength 450 nm, secondary wavelength 630 nm, optical path 1.0). The final screening results are as follows: Figure 3 As shown, among A-1 to A-22, the supernatant of A-1 cells showed the highest reaction amplitude and the strongest titer. Therefore, A-1 cell line was selected as the hybridoma cell line for secreting the target monoclonal antibody.

[0048] Hybridoma cell lines secreting monoclonal antibodies, obtained through the above screening process, were cultured, and total RNA was extracted from the cells using standard biological methods. Using the total RNA as a template, cDNA was synthesized via reverse transcription using the PrimeScript™ 1st Strand cDNA Synthesis Kit (TAKARA). The cDNA was then amplified using primers for the antibody's constant region. After separation by agarose gel electrophoresis, the DNA fragments were purified and recovered to obtain the amino acid sequence of the antibody's variable region.

[0049] The target gene fragment, generated by splicing the sequenced monoclonal antibody variable region with the mouse IgG1 constant region, was cloned into the pcDNA3.4 expression vector to prepare a transfection-grade expression plasmid. Cells were cultured in serum-free medium (Thermo Fisher Scientific), seeded in shake flasks, and incubated on a shaker at 37°C and 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and ExpiFectamine™ 293 transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 18 hours of transfection, ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2 were added. After 6 days of cell culture, the supernatant was collected for purification, and the purity of the finally purified monoclonal antibody A-1 was analyzed by SDS-PAGE. Figure 4 ) and A280 concentration determination.

[0050] Its light chain amino acid sequence is shown in SEQ ID NO.3: QIVLTQSPALMSASPGEKVTMTCSASASVNYVYWYQQKPGSSPKAWIYLTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQHWSSNPLTFGAGTKLELKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0051] The amino acid sequence of its heavy chain is shown in SEQ ID NO.4: QVQLQQPGSELVRPGASVNLSCKASGYTFTSYWMHWVKQRHGQGLEWIGNIYPGSGNTNYDEKFKRKAILTVDTSSSTAYMHLNSLTSEDSAVYYCARWLTGTWNFDVWGAG TTVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0052] Example 2: Specificity test of A-1 antibody 1) Dilute ARRDC1, ARRDC2, ARRDC3, and ARRDC4 to a concentration of 1 μg / mL with CBS at pH 9.6, add 100 μL to each well, and coat overnight at 4°C; 2) Add 250 μL of TBST washing solution to each well, let stand for 1 min, then pat dry. Repeat 4 times. 3) Fill the wells with 10% newborn calf serum TBST and incubate at 37°C for 1 hour; 4) Dilute the A-1 antibody to a concentration of 1 μg / mL, and set up blank wells. Add 100 μL to each well and incubate at 37°C for 1 h. 5) Repeat step 2) of washing; 6) Dilute GAM with 5% newborn calf serum TBST at a ratio of 1:6000, add 100 μL to each well, and incubate at 37°C for 30 min; 7) Repeat step 2) of washing; 8) Add 50 μL of colorimetric solution A and 50 μL of colorimetric solution B to each well, react at room temperature for 5 min, then add 50 μL of stop solution. Set the microplate reader to a main wavelength of 450 nm, a secondary wavelength of 630 nm, and a path length of 1.0, and detect the OD value.

[0053] 9) Results are as follows Figure 5As shown, the A-1 antibody reacts only with ARRDC4 and shows no cross-reactivity with ARRDC1, ARRDC2, or ARRDC3, confirming that it can specifically recognize ARRDC4.

[0054] Example 3: Affinity test of A-1 antibody 1) Dilute exosome ARRDC4 with CBS at pH=9.6 at 1:200 and 1:400 respectively, add 100 μL to each well, and coat overnight at 4℃; Add 250 μL of TBST washing solution to each well, let stand for 1 min, then pat dry. Repeat 4 times. The enzyme-labeled wells were filled with 10% newborn calf serum TBST and blocked, and incubated at 37°C for 1 hour. 4) Dilute the A-1 antibody 1000 times and perform 14 serial dilutions, add 100 μL to each well, and incubate at 37°C for 1 h; Repeat step 2) of washing; GAM was diluted with 5% newborn calf serum TBST at a ratio of 1:6000. 100 μL was added to each well and incubated at 37°C for 30 min. Repeat step 2) of washing; 8) Add 50 μL of colorimetric solution A and 50 μL of colorimetric solution B to each well, react at room temperature for 5 min, then add 50 μL of stop solution. Set the microplate reader to a main wavelength of 450 nm, a secondary wavelength of 630 nm, and a path length of 1.0, and detect the OD value.

[0055] 9) The results are shown in Table 1. The affinity of antibody A-1 for ARRDC4 is 0.355 nM. Table 1: Results of A-1 antibody affinity assay Example 4: Application of exosomal ARRDC4 antibody in acridinium ester chemiluminescence reagent kit Reagent kit formulation: Reagent 1: 0.2 mg / mL affinity magnetic beads with surface-bound A-1 antibody, 50 mM phosphate buffer (pH = 7.4), 0.5% BSA, 0.05% Tween 20, 0.1% PC300.

[0056] Reagent 2: 0.05–0.50 μg / mL acridinium ester (NSP-SA-NHS) labeled A-1 antibody, 20 mM MES buffer (pH = 6.0–7.4), 0.5% BSA, 0.1% PC300, 0.05% Tween 20 Diluent: 20mM phosphate buffer, 0.5% BSA, 0.05% Tween 20.

[0057] Standard curve: Confirm that the Shine i1910 device is operating normally; set the detection parameters according to Table 2; calibrate the standard with concentration gradients of 0.00, 0.05, 2.00, 5.00, 10.00, and 20.00 ng / mL using the instrument, and obtain the standard curve using four-parameter fitting as shown below. Figure 6 As shown.

[0058] Table 2: Detection parameters of the exosome ARRD4 kit Repeatability: Within the same experimental batch, using the same kit and the same instrument (Shine i1910), three concentrations were set: high (19.5 ng / mL), median (10.5 ng / mL), and low (1.5 ng / mL). Each concentration was tested 20 times. The mean, standard deviation (SD), and coefficient of variation (CV = SD / Mean × 100%) of the 20 test results were calculated. The CVs for the high, median, and low concentration samples were [values ​​missing], meeting the testing requirements (kit CV ≤ 10%). The results are shown in Table 3.

[0059] Table 3: Repeatability of Chemiluminescence Reagent Kit Accuracy: Exosome ARRDC4 was diluted to 190, 100, and 10 ng / mL, respectively, and mixed with artificial serum at a ratio of 1:9 to form the experimental group. An equal volume of the diluent was mixed with artificial serum at a ratio of 1:9 to form the control group. The above kit was used to detect the experimental and control groups, and the recovery rate was calculated. The results are shown in Table 4. The recovery rates of the experimental group samples with theoretical values ​​of 19, 10, and 1 ng / mL were 102.07%, 102.83%, and 101.00%, respectively, with an average recovery rate of 101.97%, all of which met the detection requirements (the kit requires a recovery rate range of 80%~120%).

[0060] Table 4: Accuracy of Chemiluminescence Reagent Kits Example 5: Evaluation of the effect of surfactants on the exosome ARRDC4 chemiluminescence reagent kit Three common surfactants (Tween 20, RIPA, and Triton X-100) were selected, and three different concentrations of each were set (Sample 1: 10.29 ng / mL; Sample 1: 4.89 ng / mL; Sample 1: 1.02 ng / mL). The concentration of exosome ARRDC4 in each sample was measured, with each sample repeated three times and the average value taken. The influence of surfactants on the detection was evaluated based on the magnitude of the deviation (<5% was negligible, >15% was severe interference). The results are shown in Table 5: Tween 20 concentrations less than 0.05% had no effect on the detection of exosome ARRDC4, while RIPA and Triton X-100 caused severe interference. Therefore, special attention should be paid to the influence of surfactants when detecting exosome ARRDC4. It is recommended not to add surfactants, or to add only a small amount of Tween-20 (concentration ≤0.05%) to the kit.

[0061] Table 5: Effect of surfactants on the exosome ARRDC4 chemiluminescence reagent kit In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody or its antigen-binding fragment for quantitative detection of ARRDC4 in exosomes, characterized in that: Its amino acid sequence includes a light chain variable region and a heavy chain variable region; wherein: The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region include positions 24-34, 50-56 and 89-97 of SEQ ID NO.1, respectively; The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region include positions 31-35, 50-65 and 98-106 of SEQ ID NO.2, respectively.

2. The monoclonal antibody or its antigen-binding fragment as described in claim 1, characterized in that: The light chain variable region further includes framework regions FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 include positions 1-23, 35-49, 57-88 and 98-107 of SEQ ID NO.1, respectively. The heavy chain variable region further includes framework regions FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 include positions 1-30, 36-49, 66-97 and 107-117 of SEQ ID NO.2, respectively.

3. The monoclonal antibody or its antigen-binding fragment as described in claim 2, characterized in that: The light chain variable region includes the amino acid sequence shown in SEQ ID NO.1; The heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO.

2.

4. The monoclonal antibody or its antigen-binding fragment as described in claim 3, characterized in that: Its amino acid sequence also includes heavy chain constant regions and light chain constant regions; among which: The heavy chain constant region is IgG, IgM, and IgA; The light chain constant region is either the lambda(λ) constant region or the kappa(κ) constant region.

5. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, characterized in that: The light chain contains the amino acid sequence shown in SEQ ID NO.3; The heavy chain contains an amino acid sequence as shown in SEQ ID NO.

4.

6. A polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-5, characterized in that: It contains nucleotide sequences SEQ ID NO.5 and SEQ ID NO.6 that can encode amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

7. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-5 in the preparation of a kit for the quantitative detection of ARRDC4 in exosomes.

8. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-5 in the preparation of a kit for the diagnosis or treatment of diseases associated with ARRDC4 in exosomes for monitoring or prognostic assessment.

9. The application as described in claim 8, characterized in that: The diseases associated with ARRDC4 in exosomes are colorectal cancer and breast cancer.

10. A chemiluminescent assay kit for quantitative detection of ARRDC4 in exosomes, characterized in that: include: CD63+CD81 antibody used to capture CD63+CD81 positive exosomes with coupled magnetic beads; As a detection antibody, a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-5 is used to conjugate acridine ester.

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