Antibody combinations and their use in the manufacture of detection products for detecting tps

Abstract

This invention relates to the field of biological detection, and more particularly to antibody combinations and their application in the preparation of detection products for TPS. The monoclonal antibodies provided in this invention are named 7#, 13#, and 16#. This invention also discloses the preparation method and application of these antibodies. This invention uses prokaryotically recombinantly expressed CK18 as an immunogen to immunize mice. Sixteen anti-TPS monoclonal antibodies were obtained through screening and purification using hybridoma technology. The obtained antibodies were labeled with horseradish peroxidase. Epitope identification was performed on the synthesized peptides, and cross-pairing of different epitope antibodies was used to select one antibody suitable for coating and two antibodies suitable for labeling. These were then applied to detect TPS in human serum. The double-antibody sandwich detection method established using the above antibodies can be applied to a magnetic microparticle chemiluminescence platform, and has the advantages of high sensitivity, simple operation, and short detection time. It can provide key materials for the subsequent development of commercial kits for TPS detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to antibody combinations and their application in the preparation of detection products for detecting TPS. Background Technology

[0002] Tissue-specific peptide antigen (TPS) is a soluble polypeptide fragment derived from cytokeratin 18 (CK18), primarily released into the bloodstream by actively proliferating epithelial cells. It exhibits characteristic elevations in the serum of patients with various epithelial-derived tumors. Due to its close correlation with cell proliferation status, TPS has been widely used for adjunctive monitoring, efficacy evaluation, and prognostic assessment of malignant tumors such as breast cancer, lung cancer, prostate cancer, ovarian cancer, and bladder cancer.

[0003] Currently, the concentration of TPS in serum is mainly detected clinically using immunological methods, with the sandwich assay system based on monoclonal antibody M3 being a representative example. This antibody specifically recognizes phosphorylated epitopes on the CK18 molecule, exhibiting good sensitivity and repeatability. Multiple studies and clinical practices have shown that TPS detection results are well correlated with changes in tumor burden and demonstrate practical value in dynamically monitoring treatment response. Furthermore, TPS is often used in combination with other tumor markers (such as CA15-3, CEA, and CA125), further enhancing its effectiveness in comprehensive treatment strategies.

[0004] In recent years, with the development of antibody engineering technology, high-affinity and high-specificity paired antibodies against TPS have emerged, providing a technological foundation for establishing a more stable and accurate quantitative detection system. Commercially available reagent kits are widely used in clinical laboratories, and their detection results show good consistency with clinical course, validating the reliability of TPS as a functional tumor marker. Based on this, developing novel TPS detection reagents with independent intellectual property rights and performance comparable to or even superior to existing products is of positive significance for improving the independent innovation capability and clinical adaptability of my country's in vitro diagnostic reagents. Summary of the Invention

[0005] In view of this, the present invention provides an antibody combination and its application in the preparation of a detection product for TPS. The invention first immunizes Balb / c mice with TPS as an immunogen, then uses hybridoma technology to screen and purify 16 monoclonal antibodies against tissue polypeptide-specific antigens. These antibodies are then labeled with horseradish peroxidase (HRP). Through site identification and cross-pairing, one coated antibody and two labeled antibodies are selected and applied to detect TPS in human serum. The detection method established using these paired antibodies has the advantages of simple operation and high correlation with the values ​​of the imported reagent "Tissue Polypeptide-Specific Antigen Detection Kit - Enzyme-Linked Immunosorbent Assay (TPSELISA)" (IDLBiotech AB), providing key materials for the subsequent development of commercial kits for the detection of tissue polypeptide-specific antigens.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a coated antibody comprising: a heavy chain variable region and a light chain variable region, wherein: (1) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:7, SEQ ID No:8, and SEQ ID No:9, respectively; and (2) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:10, SEQ ID No:11, and SEQ ID No:12, respectively; and / or (3) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (1) or (2), and which has the same function as the amino acid sequence described in (1) or (2); or (4) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (1), (2) or (3).

[0007] In some embodiments of the present invention, the coated antibody includes: (5) The heavy chain variable region has the amino acid sequence shown in SEQ ID No:1; and (6) The light chain variable region has an amino acid sequence as shown in SEQ ID No:2; and / or (7) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (5) or (6), and which has the same function as the amino acid sequence described in (5) or (6); or (8) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (5), (6) or (7).

[0008] The present invention also provides a labeled antibody comprising: a heavy chain variable region and a light chain variable region, wherein: (9) The heavy chain variable region is composed of CDR1 region, CDR2 region and CDR3 region; The CDR1 region has the amino acid sequence shown in SEQ ID No:13; the sequence of the CDR2 region is ISYSX1SX2, wherein: X1 is selected from D or G; X2 is selected from I or K; the sequence of the CDR3 region is AX3X4X5YX6X7X8X9FX. 10 Y; where: X3 is selected from E or S; X4 is selected from G or I; X5 is selected from Y or D; X6 is selected from R or A; X7 is selected from Y or E; X8 is selected from S or G; X9 is selected from W or Y; X 10 Choose from A or D; and (10) The light chain variable region is composed of CDR1 region, CDR2 region and CDR3 region; The CDR1 region has the amino acid sequence shown in SEQ ID NO:18; the CDR2 region has the amino acid sequence shown in SEQ ID NO:19; and the sequence of the CDR3 region is: QQWSSX 11 PPT; where: X 11 Selected from S or N; and / or (11) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (9) or (10), and which has the same function as the amino acid sequence described in (9) or (10); or (12) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (9), (10) or (11).

[0009] In some embodiments of the present invention, the labeled antibody includes: labeled antibody 1 and / or labeled antibody 2; The labeled antibody 1 includes: (13) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:13, SEQ ID No:14, and SEQ ID No:16, respectively; and (14) The CDR1, CDR2 and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:18, SEQ ID No:19 and SEQ ID No:20, respectively; and / or (15) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (13) or (14), and which has the same function as the amino acid sequence described in (13) or (14); or (16) An amino acid sequence having more than 90% identity with the amino acid sequence described in (13), (14) or (15); and / or The labeled antibody 2 includes: (17) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:13, SEQ ID No:15, and SEQ ID No:17, respectively; and (18) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:18, SEQ ID No:19, and SEQ ID No:21, respectively; and / or (19) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (17) or (18), and which has the same function as the amino acid sequence described in (17) or (18); or (20) An amino acid sequence that is more than 90% identical to the amino acid sequence described in (17), (18) or (19).

[0010] In some embodiments of the present invention, the labeled antibody includes: (20) The heavy chain variable region has an amino acid sequence as shown in SEQ ID No:3 or SEQ ID NO:5; and (21) The light chain variable region has an amino acid sequence as shown in SEQ ID No:4 or SEQ ID NO:6; and / or (22) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (20) or (21), and which has the same function as the amino acid sequence described in (20) or (21); or (23) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (20), (21) or (22).

[0011] The present invention also provides modified antibodies, including: modified antibody 1 or modified antibody 2; The modified antibody 1 comprises: the above-mentioned coated antibody and the labeling molecule 1; The modified antibody 2 comprises: the above-mentioned labeled antibody and labeled molecule 2; The marker molecule 1 and the marker molecule 2 may be the same or different.

[0012] In some embodiments of the present invention, the labeling molecule 1 and the labeling molecule 2 in the modified antibody include one or more of the following: fluorescent dye, enzyme, chemiluminescent substance, radioactive isotope and quantum dot.

[0013] In some embodiments of the present invention, the marker molecule 1 and the marker molecule 2 in the modified antibody include streptomycin and / or biotin.

[0014] In some embodiments of the present invention, the modified antibody 2 comprises: the labeled antibody 1 and the labeled molecule 2; or

[0015] The above-mentioned labeled antibody 2 and labeled molecule 2.

[0016] The present invention also provides an antibody combination, comprising: a first antibody and a second antibody; The first antibody is selected from the above-mentioned coated antibody or the modified antibody 1 among the above-mentioned modified antibodies; The second antibody is selected from the labeled antibody or the modified antibody 2 described above.

[0017] This invention also provides biological materials, including any of the following: (24) A nucleic acid molecule encoding the above-coated antibody or the above-labeled antibody or the above-modified antibody or the above-combination of antibodies; (25) An expression vector containing nucleic acid molecules as described in (24); (26) Transformation and / or transfer into host cells containing the expression vector described in (25); (27) A mixture obtained by culturing host cells as described in (26).

[0018] In some embodiments of the present invention, in the above-described biological material, the nucleic acid molecule encoding the antibody-coated heavy chain variable region has: a nucleotide sequence as shown in SEQ ID NO:22; and / or

[0019] The nucleic acid molecule encoding the light chain variable region of the coated antibody has: a nucleotide sequence as shown in SEQ ID NO:23; and / or

[0020] The nucleic acid molecule encoding the heavy chain variable region of the labeled antibody 1 has: a nucleotide sequence as shown in SEQ ID NO:24; and / or

[0021] The nucleic acid molecule encoding the light chain variable region of the labeled antibody 1 has: a nucleotide sequence as shown in SEQ ID NO:25; and / or

[0022] The nucleic acid molecule encoding the heavy chain variable region of the labeled antibody 2 has: a nucleotide sequence as shown in SEQ ID NO:26; and / or

[0023] The nucleic acid molecule encoding the light chain variable region of the labeled antibody 2 has the nucleotide sequence shown in SEQ ID NO:27.

[0024] This invention also provides the application of any of the following in the preparation of TPS detection products: (28) The above-mentioned coating antibody; and / or (29) The above-mentioned labeled antibodies; and / or (30) The above-mentioned modified antibodies; and / or (31) The above antibody combination; and / or (32) The above-mentioned biological materials.

[0025] This invention also provides detection reagents and / or detection kits, including any of the following: (33) The above-mentioned coating antibody; and / or (34) The above-mentioned labeled antibodies; and / or (35) The above-mentioned modified antibodies; and / or (36) The above antibody combination; and / or (37) The above-mentioned biological materials.

[0026] The beneficial effects of this invention include: This invention uses prokaryotically recombinant CK18 as an immunogen to immunize Balb / c mice. Sixteen monoclonal antibodies against TPS were obtained through hybridoma technology screening and purification. These antibodies were then labeled with horseradish peroxidase (HRP). Epitope identification was performed using synthesized peptides, and cross-pairing of different epitope antibodies was used to select one antibody suitable for coating and two antibodies suitable for labeling. These were then applied to the detection of TPS in human serum. The dual-antibody sandwich detection method established using these antibodies can be applied to a magnetic microparticle chemiluminescence platform, exhibiting advantages such as high sensitivity, simple operation, and short detection time. This method can provide key materials for the subsequent development of commercial kits for TPS detection. Detailed Implementation

[0027] This invention discloses antibody combinations and their application in the preparation of detection products for detecting TPS.

[0028] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0029] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0030] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0031] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0032] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0033] The purpose of this invention is to provide a method for preparing antibodies against tissue polypeptide-specific antigens and their applications, in order to solve the problems existing in the prior art. The antibodies prepared by this method can be used to detect tissue polypeptide-specific antigens in human serum, providing key materials for the subsequent development of commercial kits for detecting tissue polypeptide-specific antigens.

[0034] This invention discloses three mouse monoclonal antibodies against tissue polypeptide-specific antigen (TPS), namely 7#, 13#, and 16#. 7# has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:2; 13# has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:4; and 16# has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:5 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:6.

[0035] This invention provides a method for preparing the aforementioned anti-TPS monoclonal antibody, comprising the following steps: Step 1: After emulsifying the recombinant CK18 antigen with an equal volume of adjuvant, mice were immunized three times. Spleen cells were collected and hybridized with myeloma cells. Step 2: Using traditional hybridoma technology, monoclonal antibody hybridoma cells are obtained through three rounds of screening; Step 3: Inject monoclonal antibody hybridoma cell lines into the peritoneal cavity of Balb / c mice, and collect ascites fluid 7 days later; Step 4: Purify the ascites fluid using the octanoic acid-ammonium sulfate method to obtain monoclonal antibodies.

[0036] The present invention also provides an application of the aforementioned anti-TPS antibody in the detection of TPS in human serum.

[0037] The sequence involved in this invention: SEQ ID NO: 1 7# heavy chain variable region: IEVQLEESGPGLVKPSQSLSLTCTVTGYSVTSDYAWNWIRQFPGNKLEWMGYIGYSGSISYNPSLKSRISITRDTSKNQFFLQLNSVTTGDSATYCAEGYYRYSWFAYWGQGTLVTVSA.

[0038] SEQ ID NO:2 7# light chain variable region: DIVMSQFPSSLAVSVGEKVTVSCKSSQSLLYSNNQKNYLAWYQQKPGQSPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCHQYYTYPYTFGGGTKLEIK.

[0039] SEQ ID NO: 3 13# heavy chain variable region: SDVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSDSINYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAEGYYRYSWFAYWGQGTLVTVSA.

[0040] SEQ ID NO:4 13# light chain variable region: QIVLTQSPAIMSASPGEKVTMTCSASSSVRSMNWYQQKSGTSPKRWVYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSSPPTFGAGTKLELK.

[0041] SEQ ID NO:5 16# heavy chain variable region: SDVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSKSYNPSLKSRFSITRDTSKNQFFLQLNSVTTEDTATYYCASIDYAEGYFDYWGQGTTLTVSS.

[0042] SEQ ID NO:6 16# light chain variable region: QIVLTQSPAIMSASPGEKVTMTCSASSSVRSMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEADDAATYYCQQWSSNPPTFGGGTKLEIK.

[0043]

[0044] Nucleotide sequence:

[0045] In Examples 1 to 3 of this invention, all raw materials and reagents used can be purchased from the market.

[0046] The present invention will be further illustrated below with reference to the embodiments: Example 1: Screening and preparation of anti-TPS antibodies 1.1 Animal Immunization Using CK18 recombinant antigen as the immunogen, 6-8 week old Balb / c mice were immunized subcutaneously at a dose of 100 μg / mouse, for a total of 3 immunizations, with each immunization spaced 2 weeks apart. Cell fusion was performed 5 days after the last immunization, and a booster immunization was performed 3 days before fusion at the same dose (100 μg / mouse).

[0047] 1.2 Screening of positive hybridoma cells

[0048] Hybridoma cells were fused using conventional cell fusion methods. Seven days after fusion, hybridoma cells were screened using indirect ELISA. The optimal antigen coating concentration and serum dilution were determined using a matrix method to establish an ELISA screening system for monoclonal antibodies.

[0049] The specific steps are as follows: 1) Using CK18 recombinant antigen as the coating antigen, dilute with coating buffer to 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL and 0.125 μg / mL respectively, add 50 μL / well to ELISA plates, and coat at 4℃ for 12~16 h; discard the coating buffer and wash twice with PBST.

[0050] 2) Add blocking solution containing 1% Casein, 150 μL per well, and seal at 37°C for 2 h; discard the blocking solution and dry at 37°C.

[0051] 3) Dilute negative serum and positive serum at 1:100, 1:200, 1:400, 1:800 and 1:1600 respectively, add 100 μL to each well, react at 37℃ for 1 h, and wash 5 times with PBST.

[0052] 4) Add HRP-labeled goat anti-mouse secondary antibody, 50 μL / well, react at 37℃ for 1 h, and wash 5 times with PBST.

[0053] 5) Add 100 μL of TMB substrate chromogenic solution to each well. After developing the color at room temperature in the dark for 10 min, add 50 μL of stop solution (2 mol / L H2SO4) to each well and read the OD value at 450 nm using a microplate reader.

[0054] 6) Result determination: The optimal working conditions are defined as the antigen coating concentration and serum dilution corresponding to the positive well OD value closest to 1.0, the negative well OD value < 0.1 and the P / N value ≥ 2.1.

[0055] For culture wells that initially showed positive results, limiting dilution was used for cloning purification. After each cloning, the cells were cultured for 7 days, and the cell culture supernatant was tested using a CK18 antigen-coated plate. Wells with positive results and good cell monoclonal activity were selected for further limiting dilution cloning until all cloned cells showed 100% positive results.

[0056] 1.3 Production and Purification of Monoclonal Antibodies

[0057] Balb / c mice were intraperitoneally injected with 0.5 mL of sterile paraffin oil per mouse, followed by intraperitoneal injection of anti-TPS hybridoma cells (10⁻⁶) 7 days later. 6 (each animal). Ascites fluid was collected after 7 days, and the monoclonal antibody in the ascites fluid was purified by the caprylic acid-ammonium sulfate method. After the protein concentration was measured, it was stored at -20℃.

[0058] 1.4 Identification of Monoclonal Antibody Subtypes

[0059] The Beijing Yiqiao Antibody Subtype Identification Kit was used to analyze the subtypes of 16 monoclonal antibodies. The results showed that all of them were of the IgG type and the light chain was the κ chain.

[0060] 1.5 Monoclonal Antibody Titer Detection

[0061] The TPS antigen was diluted to 1 μg / mL with 0.05 mmol / L, pH 9.6 CB buffer, and 50 μL was added to each well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. After washing twice with PBST, 100 μL of 1% Casein blocking buffer was added to each well, and the plate was blocked at 37°C for 2 h. The purified monoclonal antibodies (all at 5 mg / mL) were serially diluted with CB buffer (initial dilution 1:8000, followed by 1:16000, 1:32000, 1:64000, etc.). 50 μL of each diluted antibody solution was added to each well of the antigen-coated plate, with CB buffer used as a negative control. The plate was incubated at 37°C for 30 min, washed five times with PBST, and dried. 100 μL of 1:4000 diluted HRP-labeled goat anti-mouse IgG (SIGMA) was added to each well, and the plate was incubated at 37°C for 30 min. Wash 5 times with PBST and blot dry. Add 100 μL of ordinary enzyme-labeled substrate solution per well, and incubate at room temperature in the dark for 10 min. Stop the reaction by adding 50 μL of 0.1 mol / L H2SO4 per well. Measure the absorbance (OD value) at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Table 1 below.

[0062] Table 1 Absorbance at different dilution factors

[0063]

[0064] As shown in Table 1, the average OD value of the negative control was 0.059, and the titers of all 16 monoclonal antibodies showed high levels.

[0065] 1.6 Site Identification of Monoclonal Antibodies

[0066] To identify antibody sites, a series of peptides were synthesized based on antigenic epitopes predicted by different software. Peptide 1 corresponded to the M3 epitope; peptide 2 was the specific C-terminus of the fragment generated after CK18 cleavage. These peptides were conjugated with bovine serum albumin and coated onto enzyme immunoassay plates for reactivity verification. Results showed that the 16 antibodies could be divided into three categories based on their reactivity with different conjugated peptides: Category I (2#, 3#, 9#, 12#, 13#, 16#) recognized only the peptide 1 epitope; Category II (7#, 11#) recognized only the peptide 2 epitope; and Category III showed non-specific reactions or no reaction with either peptide. Based on the peptide reactivity results, Category I antibodies were paired with Category II antibodies, and antibodies 2#, 3#, 9#, 12#, 13#, and 16# were paired with antibodies 7# and 11#, respectively. The peptide reactivity test results are shown in Table 2 below.

[0067] Table 2

[0068]

[0069] All of the above antibodies can be HRP conjugated using the sodium periodate method. The specific implementation steps are the commonly used procedures in the industry and will not be described in detail here.

[0070] Example 2: Application of anti-TPS antibody in the detection of human tissue peptide-specific antigens

[0071] 2.1 Double Antibody Sandwich Method for TPS Detection

[0072] (1) Preparation of antibody-coated plates: Dilute the first two types of antibodies with sodium carbonate buffer to a final concentration of 5 μg / mL. Add 100 μL / well to a 96-well microplate and coat overnight at 4°C. After washing twice with PBST, add 1% Casein blocking buffer (100 μL / well) and block at 37°C for 2 h.

[0073] (2) Dilute TPS to 1000 ng / mL, and then dilute it to 4 concentrations in a 4-fold gradient. Record the concentration of the diluent as 0. Add 100 μL of each concentration of antigen to the antibody-coated plate per well, incubate at 37°C for 0.5 h, wash the plate 5 times with PBST washing buffer, and pat dry with absorbent paper.

[0074] (3) According to the site classification, add HRP-labeled antibodies (1:1000 dilution) at different sites from the coated antibody, 100 μL per well, incubate at 37℃ for 0.5 h, wash 5 times with PBST, add luminescent substrate, and read the luminescence value on the luminescence analyzer.

[0075] Based on the paired detection results, antibody #7, when paired with labeled antibodies #13 and #16, showed good sensitivity and overall good correlation. Some paired detection data are as follows: Table 3

[0076]

[0077] 2.2 Magnetic microparticle coating

[0078] The antibody was conjugated with magnetic beads. The magnetic beads used were carboxyl-modified on their surface. The carboxyl activation step and the antibody-magnetic bead conjugation step were performed in two separate steps. 0.45 mg of antibody was conjugated with 3 mg of magnetic beads, and the volume was adjusted to 3 mL using a protein-containing preservation solution after conjugation. 2.3 Clinical Sample Testing Twelve tissue polypeptide-specific antigen-positive samples were collected from a hospital and tested using the double-antibody sandwich method of this invention. The two paired antibodies were used to detect the hospital clinical samples on a magnetic microparticle platform. The overall correlation between the paired detection signal values ​​of the 7# coated and 13# and 16# labeled antibodies and the detection values ​​of "TPS ELISA" (IDLBiotechAB) reached more than 0.95.

[0079] Table 4

[0080]

[0081] Example 3: Monoclonal Antibody Sequencing

[0082] The following primers were synthesized based on the constant region sequence of the antibody gene: LF5′-GACATTGTGATGACCCAGTCTCCT-3′ (as shown in SEQ ID NO:28); LR5′-TGGACACTGTTGGGGCCGCATCGGCCCT-3′ (as shown in SEQ ID NO:29); HF5′-CAGGTGCAGCTGCAGGAGTCAGGA-3′ (as shown in SEQ ID NO:30); HR5′-GATAGACAGATGGGGGTGTCGTTTTGGC-3′ (as shown in SEQ ID NO:31); Hybridoma cells #7, #13, and #16 were extracted using Trizol Reagent reagent (3 × 10⁶ cells each). 6 The total RNA (number of cells) was reverse transcribed into cDNA.

[0083] The heavy chain variable region (VH) of the monoclonal antibody was amplified by PCR using HF and HR primers; the light chain variable region (VL) was amplified using LF and LR primers. All PCR reactions were performed using a hot-start program with the following conditions: 95℃ for 5 min; 95℃ for 15 s, 55℃ for 45 s, 72℃ for 30 s, for a total of 30 cycles; followed by a 72℃ extension for 7 min.

[0084] After separation of PCR products by 1% agarose gel electrophoresis, the target fragment was recovered and purified. The purified fragment was cloned into the pMD18-T vector, transformed into E. coli DH5α competent cells, and screened on LB agar plates containing the appropriate antibiotics. White single colonies were picked and inoculated into LB liquid medium containing ampicillin for expansion culture.

[0085] After screening positive clones, recombinant plasmids were extracted using the QIAGEN plasmid extraction kit and sequenced to finally determine the heavy and light chain variable region sequences of each monoclonal antibody.

[0086] Mouse monoclonal antibody 7# has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:2.

[0087] Mouse monoclonal antibody 13# has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:4.

[0088] Mouse monoclonal antibody 16# has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:5 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:6.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A coated antibody, characterized in that, include: Heavy chain variable region and light chain variable region, wherein: (1) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:7, SEQ ID No:8, and SEQ ID No:9, respectively; and (2) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:10, SEQ ID No:11, and SEQ ID No:12, respectively; and / or (3) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (1) or (2), and which has the same function as the amino acid sequence described in (1) or (2); or (4) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (1), (2) or (3).

2. The coated antibody as described in claim 1, characterized in that, include: (5) The heavy chain variable region has an amino acid sequence as shown in SEQ ID No:1; and (6) The light chain variable region has an amino acid sequence as shown in SEQ ID No:2; and / or (7) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (5) or (6), and which has the same function as the amino acid sequence described in (5) or (6); or (8) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (5), (6) or (7).

3. A labeled antibody, characterized in that, include: Heavy chain variable region and light chain variable region, wherein: (9) The heavy chain variable region is composed of CDR1 region, CDR2 region and CDR3 region; The CDR1 region has the amino acid sequence shown in SEQ ID No:13; the sequence of the CDR2 region is ISYSX1SX2, wherein: X1 is selected from D or G; X2 is selected from I or K; the sequence of the CDR3 region is AX3X4X5YX6X7X8X9FX. 10 Y; where: X3 is selected from E or S; X4 is selected from G or I; X5 is selected from Y or D; X6 is selected from R or A; X7 is selected from Y or E; X8 is selected from S or G; X9 is selected from W or Y; X 10 Choose from A or D; and (10) The light chain variable region is composed of CDR1 region, CDR2 region and CDR3 region; The CDR1 region has the amino acid sequence shown in SEQ ID NO:18; the CDR2 region has the amino acid sequence shown in SEQ ID NO:19; and the sequence of the CDR3 region is: QQWSSX 11 PPT; where: X 11 Selected from S or N; and / or (11) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (9) or (10), and which has the same function as the amino acid sequence described in (9) or (10); or (12) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (9), (10) or (11).

4. The labeled antibody as described in claim 3, characterized in that, include: Labeled antibody 1 and / or labeled antibody 2; The labeled antibody 1 includes: (13) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:13, SEQ ID No:14, and SEQ ID No:16, respectively; and (14) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:18, SEQ ID No:19, and SEQ ID No:20, respectively; and / or (15) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (13) or (14), and which has the same function as the amino acid sequence described in (13) or (14); or (16) An amino acid sequence having more than 90% identity with the amino acid sequence described in (13), (14) or (15); and / or The labeled antibody 2 includes: (17) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:13, SEQ ID No:15, and SEQ ID No:17, respectively; and (18) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:18, SEQ ID No:19, and SEQ ID No:21, respectively; and / or (19) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (17) or (18), and which has the same function as the amino acid sequence described in (17) or (18); or (20) An amino acid sequence that is more than 90% identical to the amino acid sequence described in (17), (18) or (19).

5. The labeled antibody as described in claim 3 or 4, characterized in that, include: (20) The heavy chain variable region has an amino acid sequence as shown in SEQ ID No:3 or SEQ ID NO:5; and (21) The light chain variable region has an amino acid sequence as shown in SEQ ID No:4 or SEQ ID NO:6; and / or (22) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (20) or (21), and which has the same function as the amino acid sequence described in (20) or (21); or (23) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (20), (21) or (22).

6. A modified antibody, characterized in that, include: Modifying antibody 1 or modifying antibody 2; The modified antibody 1 comprises: the coated antibody and the labeling molecule 1 as described in claim 1 or 2; The modified antibody 2 comprises: the labeled antibody and the labeled molecule 2 as described in any one of claims 3 to 5; The marker molecule 1 and the marker molecule 2 may be the same or different.

7. An antibody combination, characterized in that, include: First antibody and second antibody; The first antibody is selected from the coated antibody as described in claim 1 or 2 or the modified antibody 1 as described in claim 6; The second antibody is selected from the labeled antibody as described in any one of claims 3 to 5 or the modified antibody 2 as described in claim 6.

8. A biomaterial, characterized in that, include: Any of the following items: (24) A nucleic acid molecule encoding the coated antibody as described in claim 1 or 2, the labeled antibody as described in any one of claims 3 to 5, the modified antibody as described in claim 6, or the antibody combination as described in claim 7; (25) An expression vector containing nucleic acid molecules as described in (24); (26) Transformation and / or transfer into host cells containing the expression vector described in (25); (27) A mixture obtained by culturing host cells as described in (26).

9. Any of the following applications in the preparation of TPS testing products: (28) The coated antibody as described in claim 1 or 2; and / or (29) The labeled antibody as described in any one of claims 3 to 5; and / or (30) The modified antibody as described in claim 6; and / or (31) The antibody combination as described in claim 7; and / or (32) The biomaterial as described in claim 8.

10. Detection reagents and / or detection kits, characterized in that, Includes any of the following: (33) The coated antibody as described in claim 1 or 2; and / or (34) The labeled antibody as described in any one of claims 3 to 5; and / or (35) The modified antibody as described in claim 6; and / or (36) The antibody combination as described in claim 7; and / or (37) The biomaterial as described in claim 8.

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