Antibody and method for identifying collagen triple-helix structure and application

Abstract

The invention provides an antibody and a method for identifying a collagen triple helix structure and application, and belongs to the technical field of antibodies, the antibody can specifically identify collagen with a high-grade structure, and a novel technical method for identifying the collagen structure is provided for the industry. The invention also provides a method for screening and identifying the antibody of the collagen triple-helix structure. The method comprises the steps of antigen selection, immune method selection, antibody screening and preparation, and specific recognition of the antibody on the collagen triple-helix structure.

Description

Technical Field

[0001] The invention belongs to the technical field of antibodies, and in particular relates to a method for screening and identifying antibodies with triple helical structures of collagen. Background Art

[0002] As one of the most abundant proteins in the body, collagen plays an important role in maintaining the structure and function of skin, bones, tendons and other tissues. The triple helix structure of collagen is the basis of its biological activity, and its specific identification and detection are of great significance for the diagnosis and treatment of collagen-related diseases. Therefore, the development of antibodies that can accurately identify the triple helix structure of collagen has become a hot topic in current research.

[0003] Currently, there are many antibodies that recognize collagen on the market, but they are only based on sequence recognition (such as recognition of linear sequences) rather than recognition of triple helix structures. In addition, due to the lack of unified standards and methods, the specificity and accuracy of these antibodies vary, which seriously affects the quality of collagen-related research and applications. Summary of the invention

[0004] Based on this, the present invention provides antibodies for identifying the triple helix structure of collagen, and also provides antibody screening and preparation processes, which are of great significance for improving antibody quality and promoting the development of collagen-related industries.

[0005] The present invention provides an antibody for identifying a triple helix structure of collagen, wherein the antibody for identifying a triple helix structure of collagen comprises:

[0006] (i) the complementarity determining region (HCDR) of the heavy chain variable region,

[0007] The amino acid sequence of its HCDR1 is shown in SEQ ID NO: 1, 9, 17 or 25,

[0008] The amino acid sequence of its HCDR2 is shown in SEQ ID NO: 2, 10, 18 or 26,

[0009] The amino acid sequence of its HCDR3 is shown in SEQ ID NO: 3, 11, 19 or 27; and

[0010] (ii) the complementarity determining region (LCDR) of the light chain variable region,

[0011] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, 12, 20 or 28,

[0012] The amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, 13, 21 or 29,

[0013] The amino acid sequence of its LCDR3 is shown in SEQ ID NO:6, 14, 22 or 30.

[0014] Preferably, the HCDR and LCDR of the antibody are selected from one of the following:

[0015]

[0016]

[0017] Preferably, the heavy chain variable region (VH) of the antibody has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23 or SEQ ID NO:31; and the light chain variable region (VL) has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24 or SEQ ID NO:32.

[0018] Use of the antibody or antigen-binding fragment of the present invention in any of the following:

[0019] i) preparing a detection kit;

[0020] ii) preparing antibody drugs;

[0021] iii) Preparation of antibody-drug conjugates.

[0022] In one embodiment, antibody conjugates, multispecific antibodies (preferably bispecific antibodies), fusion proteins or pharmaceutical compositions can be used to prepare drugs for prolonging the lifespan of animals, especially dogs, and the main component comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0023] Preferably, the antibody conjugate further comprises a coupling portion coupled to the antibody or antigen-binding fragment thereof, wherein the coupling portion is selected from a purification tag, a cytotoxic agent, a detectable marker, a radioactive isotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol, etc.

[0024] Preferably, the multispecific antibody further comprises antibodies or antigen-binding fragments against other antigens and / or other antigen epitopes.

[0025] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0026] Preferably, the pharmaceutical composition is used alone or in combination with one or more drugs.

[0027] The present invention also provides a method for screening and identifying antibodies for the collagen triple helix structure, comprising the following steps:

[0028] i) Selecting antigens: Collagen samples with triple helical structure characteristics identified by circular dichroism are used as antigens;

[0029] ii) Select the immunization scheme: select mice as the immunized animals, and immunize with antigens and adjuvants;

[0030] iii) Screening and preparation of antibodies: Screening and preparation of antibodies using hybridoma technology or single B cell technology;

[0031] iv) Identification:

[0032] ① The ELISA test antibody can recognize collagen with a triple helical structure identified by circular dichroism, but does not recognize collagen with a non-triple helical structure identified by circular dichroism;

[0033] ②WB identified that the antibody's recognition epitope was a conformational epitope.

[0034] Preferably, the collagen sample described in step i) has a thermal stability greater than 37° C. and can be maintained for more than 3 months in an in vitro accelerated stability test.

[0035] Preferably, the collagen sample in step i) is coupled to one or more of BSA, KLH and OVA.

[0036] Preferably, the mice in step ii) are female BALB / c mice of 6-8 weeks old.

[0037] Preferably, the adjuvant in step ii) is a water-soluble agent; more preferably, the water-soluble agent is QuickAntibody-Mouse5W.

[0038] Preferably, flow sorting is used when screening antibodies in step iii).

[0039] Preferably, the antibody in step iv) can recognize triple-helical collagen identified by circular dichroism and the ELISA value is more than 10 times that of non-triple-helical collagen identified by circular dichroism.

[0040] Preferably, the antibody in step iv) can recognize the triple helical structure of different types of collagen.

[0041] Circular dichroism

[0042] The structural characterization of collagen is carried out by circular dichroism (CD) spectroscopy, which is commonly used in the art. CD is a spectroscopic method used to determine the structure of compounds with chiral structures that can produce differential absorption of left-right optical rotation, and is mainly used to determine the asymmetry of molecular structures. Generally, biomacromolecules contain chiral groups and structures, so CD is often used to measure and observe changes in the structure and conformation of biomacromolecules. The CD characteristics of the triple helix structure of collagen generally have a positive absorption peak near 220-230nm and a negative absorption peak near 190-200nm. The position of the absorption peak will shift with the change of amino acid sequence and length.

[0043] Antibody titer

[0044] The antibody titer is a test indicator to measure the antibody level in serum. It refers to the physical state of the antibody and its retention time in the body. The amount of reaction with the antigen indicates its immune effect. The antibody titer is usually expressed in the form of "1:X" (X represents the maximum dilution multiple at which the antibody can be detected). The antibody titer in normal human serum is generally less than 1:64.

[0045] Peptide coupling

[0046] Peptides are usually too short to be immunogenic. When preparing collagen, it can be coupled to carriers such as MAP, KLH, OVA, BSA and THY to enhance immunogenicity. In addition, multiple coupling methods can also be used.

[0047] Hybridoma technology

[0048] Hybridoma technology is based on cell fusion technology, which fuses sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproduction ability into hybridoma cells. After hybridoma cells produce antibodies, they are then subjected to limiting dilution to finally obtain monoclonal antibodies.

[0049] By using flow cytometry to sort single antigen-specific B cells, it is possible to effectively obtain and express antibody sequences targeting the antigen to obtain recombinant monoclonal antibodies.

[0050] Antibody positive and negative judgment

[0051] In qualitative immunoassays, the basis for determining the positive or negative results is the CUT OFF value. Determining the appropriate CUT OFF value (positive judgment value) is important for accurately determining the results. The CUT OFF value should be determined to minimize the incidence of false negative and false positive results obtained based on it.

[0052] The test kit result is judged as positive when S (sample measurement value) / N (average measurement value of negative pair) ≥ 2.1, based on the fact that 2.1 times of the negative reference serum is used as the CUT OFF value.

[0053] Antibody affinity

[0054] Antibody affinity refers to the dissociation equilibrium constant between the antibody and the target antigen. 50 EC refers to the half effective concentration, which is used to measure the affinity of the antibody. 50 The value indicates the concentration of the antibody required to achieve its effect under specific experimental conditions. 50 It indicates that the antibody has a higher affinity for the target substance.

[0055] Sequence identity

[0056] "Identity" as used herein refers to the relationship between the sequences of two or more proteins or polypeptide molecules, such as the percentage of identical residues between amino acids determined by alignment and comparison of the sequences, and can be calculated based on the size of the smallest molecule to be compared. The default gap weight provided by the program is used to perform optimal alignment.

[0057] "Conservative amino acid substitution" means that an amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophilicity). In general, conservative amino acid substitutions will not substantially change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other in conservative substitutions, the percentage sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. Therefore, sequences resulting from such changes having a sequence identity greater than 90% are considered to still fall within the scope of the present invention. Specifically, the latter sequence has a sequence homology of 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) with the sequences SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31 or SEQ ID NO:39, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32 or SEQ ID NO:40 listed in the application.

[0058] Complementarity Determining Region (CDR)

[0059] Herein, "complementarity determining region" or "CDR region" or "CDR" are used interchangeably, and the three CDRs of the heavy chain are referred to as CDR-H1, CDR-H2 and CDR-H3, and the three CDRs of the light chain are referred to as CDR-L1, CDR-L2 and CDR-L3.

[0060] The most commonly used coding rules for the CDR are provided by Kabat EA et al. In addition, IMGT (Lefranc, 2003), Chothia (Al-Lazikani, 1997) and the like all provide CDR coding rules, which are well known to those skilled in the art. The CDR of the antibody in the present application can be determined by those skilled in the art according to any scheme in the art.

[0061] It should be noted that the boundaries of the CDRs of the same antibody obtained based on different definition methods may be different, that is, the CDR sequences of the variable regions of the same antibody obtained under different definition methods are different. Therefore, when the complementary determining region sequence of the latter antibody contains the sequence of the CDR described in this application, but the antibody whose claimed CDR boundaries are different from the specific CDR boundaries defined in this application is only due to the use of different CDR boundary definition methods, it still falls within the scope of protection of this application.

[0062] In a specific embodiment of the present invention, the CDR is defined according to the IMGT numbering system, and the number and position of the CDR amino acid residues in the VL region and VH region of the antibody or antigen-binding fragment are determined by the IMGT numbering rules.

[0063] Reagent test kit

[0064] The kit of the present invention comprises a reagent for detecting the presence and / or level of triple-helical collagen in a sample of a subject, wherein the reagent comprises an antibody or an antigen-binding fragment that specifically binds to triple-helical collagen, preferably, the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof.

[0065] Further, the kit further comprises a detection agent, which is selected from a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a fluorescein-type label, umbelliferone, lissamine, cyanine, Texas Red, a paramagnetic label, a radioactive label, biotin, streptavidin / biotin, avidin / biotin, a hapten, digoxigenin, a metal complex, a metal, an enzyme, colloidal gold or a combination thereof.

[0066] Further, the detection method or principle is selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology (Simoa), flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot, protein chip, positron emission tomography and / or single photon emission computed tomography. Preferably, the kit includes reagents, materials, containers and / or devices required for the detection selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology, flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot and / or protein chip. Preferably, the enzyme-linked immunosorbent assay is selected from direct enzyme-linked immunosorbent assay, indirect enzyme-linked immunosorbent assay, direct sandwich enzyme-linked immunosorbent assay and indirect sandwich enzyme-linked immunosorbent analysis.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] The method of the present invention is suitable for preparing all antibodies that need to recognize spatial structural epitopes, especially triple helical epitopes of collagen. The immunogen selection, immune experimental scheme, etc. provided therein ensure the specificity and accuracy of the antibody, guide the research and development of antibodies that specifically recognize and bind to collagen trimers, and provide a new technical method for identifying the triple helical structure of collagen in the fields of medical treatment, cosmetology and tissue engineering. The present invention also provides multiple antibodies that specifically recognize and bind to the triple helical structure of collagen, which can be used to detect the expression level of triple helical collagen in organisms and assist in the diagnosis of related diseases; it can provide important tools for studying the biological functions of triple helical collagen in organisms, the mechanism of disease occurrence, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the circular dichroism spectroscopy identification result of triple-helical collagen and non-triple-helical collagen.

[0070] Figure 2 The fused hybridoma cells are observed under a microscope.

[0071] Figure 3 This is the color reaction result of the positive wells for ELISA detection of hybridoma cell monoclonal.

[0072] Figure 4 The results of flow cytometry analysis were obtained using antibodies that specifically recognized triple-helical collagen.

[0073] Figure 5These are the SDS-PAGE results for the expression and purification of antibodies 2B10, 3H9, 5G12, and 8H11.

[0074] Figure 6 Western Blot results showing the specific binding of antibodies 2B10, 3H9, 5G12, and 8H11 to triple-helical collagen.

[0075] Figure 7 These are the affinity (EC50) test results for antibodies 2B10, 3H9, 5G12, and 8H11.

[0076] Figure 8 These are the subtype identification results of antibodies 2B10, 3H9, 5G12, and 8H11. DETAILED DESCRIPTION

[0077] The present invention does not specifically limit the preparation method of the recombinant vector, and the conventional preparation method of the recombinant vector in the art can be used. In the present invention, the gene can be synthesized by a biotechnology company. The present invention does not specifically limit the separation and purification method, and the conventional protein separation and purification method in the art can be used; the preferred technical scheme is described in the examples.

[0078] In the embodiments of the present invention, the triple helical structure collagen sample used has the same amino acid sequence as the non-triple helical structure collagen sample, but is expressed and purified by eukaryotic cells, and circular dichroism spectra identify it as having a triple helical structure; while it is expressed and purified by prokaryotic cells, and circular dichroism spectra identify it as not having a triple helical structure.

[0079] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0080] Example 1 Circular dichroism identification of triple helical collagen samples and non-triple helical collagen

[0081] The recombinant human collagen (antigen 1) expressed and purified by mammalian cells and the recombinant human collagen (antigen 2) expressed and purified by E. coli were dissolved in 20mM PB buffer (PH7.4) to a concentration of 0.5mg / ml. The sample concentration was further diluted to 0.01mg / ml and the volume was 3ml. The sample to be tested was transferred to the 10mm×10mm sample cell of the circular dichroism spectrometer. The scanning wavelength range was set to 190nm-260nm, the scanning speed was 100nm / min, the scanning temperature was room temperature, and the CD spectra were the average of three scans. The results are shown in Figure 1As shown, antigen 1 has a maximum characteristic positive peak at 220-230nm and a negative peak at 190-200nm, which indicates that the collagen sample has a triple helix structure; antigen 2 has no CD characteristic peak, which indicates that the collagen sample has no triple helix structure.

[0082] Example 2 Preparation and screening of antibodies that can recognize triple-helical collagen using hybridoma technology

[0083] 2.1 Mouse immunization process

[0084] BALB / c mice aged 6-8 weeks were selected for immunization by hind leg intramuscular injection. The antigen was diluted with physiological saline to prepare a 50 μl volume with a total amount of 20 μg of antigen 1 (endotoxin-free), which was thoroughly mixed with 50 μl of adjuvant at a ratio of 1:1.

[0085] Before the initial immunization, blood was collected and serum titer was tested as a negative control for ELISA. Mice were immunized by intramuscular injection in the hind legs of the calf, with 100 μl injected into each mouse. Blood was collected before immunization, and two, three, and four immunizations were performed on days 21, 35, and 56, and one booster immunization was performed in the same manner. Blood was collected before shock immunization and serum titer was tested. Shock immunization was performed when the titer was >10,000. On day 70 (3 days before fusion), 20 μg of antigen 1 (endotoxin-free) was intraperitoneally immunized for shock immunization, and fusion or flow sorting was performed on the third day after shock immunization (day 73).

[0086] 2.2ELISA detection of serum titer of immune mice

[0087] The serum of mice at different immunization days was centrifuged and stored in a refrigerator at -20°C. The collagen with triple helical structure described in Example 1 was used as antigen 1.

[0088] For coating, select appropriate coating antibodies or antigens with high purity and adsorb them at pH 9.0 to 9.6, usually overnight at 2-8°C.

[0089] Buffer preparation:

[0090] Washing buffer PBST: PBS + 0.05% Tween-20

[0091] Blocking buffer: PBS + 2% BSA.

[0092] The specific steps of the test are as follows:

[0093] 1) Dilute antigen 1 with coating buffer, add the diluted antigen to the ELISA plate, usually 100 μL per well, and incubate overnight (12-18 hours) at 2-8°C.

[0094] 2) Blocking: Fill the well plate with blocking buffer (2% BSA solution) to block the surface not occupied by the antigen and incubate in a 37°C incubator for 2 hours.

[0095] 3) Washing: After removing the blocking solution, wash the wells with a washing buffer (such as PBS containing 0.05% Tween 20) to remove unbound substances.

[0096] 4) Adding Samples and Standards: Dilute mouse serum in blocking buffer at a multiple of 1000, 2000, 4000, 8000, 16000, 32000, and 64000, then add to the microplate, usually 100 μL per well, and incubate at 37°C for 2 hours.

[0097] 5) Wash again: After the serum incubation is completed, wash the plate again to remove unbound proteins.

[0098] 6) Substrate development: Add substrate solution (TMB) and incubate until the color develops to sufficient intensity, usually within 15-30 minutes.

[0099] 7) Stop reaction and read: Stop the enzyme reaction with stop solution and measure the absorbance on a microplate reader.

[0100] Table 1 ELISA test mouse serum titer

[0101]

[0102]

[0103] 2.3 Hybridoma technology screening positive monoclones that recognize triple helical collagen

[0104] Resuscitate and passage myeloma cells SP2 / 0 in advance, and replace with fresh culture medium one day before the experiment. Take SP2 / 0 with a viability of >95%, discard the supernatant, blow and mix with DMEM culture medium to obtain a cell suspension, transfer the cell suspension to a 50ml centrifuge tube, centrifuge at room temperature, 800rpm, for 10min, discard the supernatant, repeat the operation, and wash the cell pellet 3 times. Add DMEM culture medium to the cell pellet, gently blow and mix to obtain a cell suspension, count and record the cells, and place at 4℃ for later use.

[0105] The mice were killed by dislocating the neck after removing the eyeballs and taking whole blood, and then soaked in 75% alcohol for 5 minutes for disinfection. The mice were taken out and placed on a sterile operating table, with the left ventral side facing up. The skin was cut open, and long dark spleen tissue was visible. The peritoneum was lifted on the lower side of the spleen, and the spleen was exposed after cutting. The spleen was lifted with forceps, and the connective tissue under the spleen was separated with scissors to remove the spleen. DMEM culture medium was added to the culture dish, and the spleen was carefully and gently ground into the culture dish with a 5ml syringe piston. After grinding, a 70-mesh disposable cell screen was placed on top of a 50mL centrifuge tube. A disposable pipette was used to transfer the ground spleen cell suspension to the cell screen, and DMEM culture medium was added to rinse the culture dish and screen to obtain a spleen cell suspension. The culture dish and screen were centrifuged at room temperature for 10 minutes, and the supernatant was discarded. The precipitate was added with DMEM culture medium, resuspended and blown, centrifuged, and the supernatant was discarded. The washing was repeated 3 times. The precipitate was added with DMEM culture medium, resuspended and blown to mix, the cells were counted and recorded, and placed at 4℃ for use.

[0106] Prepare unimmunized 6-8 week old BALB / c mice, kill them, and take spleen cells as feeder layer cells according to the above method. Prepare 96-well plates and lay feeder layer cells one day before the experiment. Peritoneal macrophages from unimmunized 6-8 week old BALB / c mice can also be used as feeder layer cells.

[0107] Take out SP2 / 0 and immune spleen cells, gently blow and mix them evenly, mix them in a 50ml centrifuge tube at a ratio of spleen cells: SP2 / 0 = 1:5-1:10, and centrifuge them at room temperature at 800rpm for 10min. Discard the supernatant, break up the cell pellet, and place the cell pellet in a 37℃ water bath for 5-10min. The entire process of cell fusion is in a 37℃ water bath. Place 50% PEG and cell pellet in a 37℃ water bath purified water (beaker), gently shake the cell pellet while adding 1ml of 50% PEG dropwise, and then add DMEM culture medium dropwise to the pellet. After gently inverting and mixing, let it stand in a 37℃ incubator for 5min, centrifuge at room temperature at 800rpm for 10min, and discard the supernatant. Resuspend the pellet with complete culture medium (DMEM+20% FBS+1% P / S) and gently blow and mix it into a cell suspension. The spleen cells were added to a 96-well plate covered with feeder cells using a spray gun and cultured in a 5% CO2, 37°C constant temperature incubator.

[0108] On the second day, add 5×HAT screening medium, on the third day, change the medium and add 2×HAT, and then change the medium every other day and add 1×HAT. On the third to fifth day, the fused hybridoma cells can be observed under a microscope ( Figure 2 ) and calculate the fusion rate of hybridoma cells. On the 10th day, the medium was replaced with 1xHT screening medium.

[0109] 2.4ELISA detection of hybridoma cell secreted antibody supernatant

[0110] The specific operation steps are the same as 2.2. 450 Wells with absorbance values ​​greater than 1.0 are marked and subsequently flow cytometry single-cell sorting is performed into a 96-well culture plate with feeder cells to select monoclonal positive wells. After 10 days of culture, an ELISA test is performed, and the positive wells are expanded and cultured again and sorted until the ELISA test results are almost all positive, which proves that a monoclonal antibody that specifically recognizes triple-helical collagen has been obtained ( Figure 3 ). The test results are shown in Table 2.

[0111] Table 2 ELISA test monoclonal positive wells

[0112] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.574 0.282 0.368 0.443 0.422 0.416 0.472 0.458 0.531 0.498 0.472 0.280 B 0.605 0.514 0.516 0.544 0.520 0.639 0.528 0.533 0.526 0.530 0.530 0.417 C 0.427 0.514 0.541 0.523 0.505 0.550 0.507 0.525 0.510 0.498 0.511 0.505 D 0.300 0.530 0.523 0.517 0.516 0.515 0.505 0.525 0.501 0.500 0.525 0.520 E 0.416 0.509 0.509 0.522 0.513 0.509 0.495 0.503 0.509 0.490 0.525 0.511 F 0.580 0.540 0.525 0.507 0.523 0.515 0.531 0.498 0.503 0.486 0.529 0.516 G 0.368 0.519 0.567 0.530 0.530 0.516 0.507 0.533 0.537 0.511 0.533 0.490 H 0.520 0.332 0.354 0.312 0.322 0.321 0.369 0.337 0.297 0.317 0.283 0.233

[0113] Example 3 Preparation and screening of antibodies that can recognize triple-helical collagen using single B cell technology

[0114] 3.1 Fluorescent labeling of triple-helical collagen

[0115] Dissolve the protein to be labeled in PBS buffer to a concentration of 2 mg / ml. Dissolve the fluorescent dye in DMSO to make a stock solution with a concentration of 1 mg / mL. Dilute the stock solution to the appropriate labeling concentration before use. Mix the protein solution and the fluorescent dye solution so that the molar ratio of protein to fluorescent dye is 1:10 to 1:50. Add an appropriate amount of PBS buffer to the reaction system to make the total volume about 100 μL. Incubate the reaction system at room temperature or 37°C for 1-2 hours to promote the labeling reaction. After the labeling reaction is completed, add an appropriate amount of stop solution (such as glycine or boric acid buffer) to terminate the reaction. In order to remove unbound fluorescent dye, dialysis is used to purify the labeled protein. Store the purified labeled protein in PBS buffer and store it at 4°C away from light.

[0116] 3.2 Flow cytometry to obtain antibodies that specifically recognize triple-helical collagen

[0117] The spleen cells of the immunized mice in Example 2 were washed with DMEM medium and centrifuged at room temperature at 1000 rpm for 5 min. The cell pellet was resuspended with 10-20 ml of D-PBS + 2% FBS, washed 1-2 times, and centrifuged at room temperature at 1000 rpm for 5 min. The cell pellet was resuspended with D-PBS + 2% FBS, and 5 ml of red blood cell lysis buffer was added, mixed, allowed to stand for 1-2 min, and centrifuged at 4°C at 1000 rpm for 5 min. The cell pellet was resuspended with D-PBS + 2% FBS and the cells were counted. 5×10 6 Cells were stained and sorted by flow cytometry, 1×10 6Each tube, 1 ml / tube, add Fc block first, let stand at 4℃ for 15min, centrifuge at 4℃ 1000rpm for 5min. Discard the supernatant, add D-PBS + 2% FBS to resuspend the cell pellet, add flow cytometry antibodies accordingly, let stand at 4℃ for 15-20min, centrifuge at 4℃ 350×g for 5min. Add 7AAD before loading, and flow sort single B cells into 96-well plate.

[0118] Table 3 Flow cytometry fluorescent dyes and corresponding marker molecules

[0119]

[0120]

[0121] The sorting results are as follows Figure 4 As shown, first circle the lymphocyte population according to FSC and SSC, then use 7AAD to distinguish between live and dead cells, and then circle the B cells according to the marker molecules of T cells and B cells. The B cell subtypes that secrete antibodies include IgG, IgM, IgD, etc. Circle the B cells that secrete IgG subtype antibodies, and finally, according to the specific recognition method of antigen and antibody, circle the single-positive and double-positive cell populations that can bind to the antigen, and sort the single B cells into a 96-well cell culture plate containing cell lysate, and then perform molecular cloning to obtain the antibody sequence.

[0122] Example 4 Molecular cloning to obtain antibody sequences

[0123] Hybridoma cells / flow cytometry sorted single B cells were lysed, RNA was extracted, and reverse transcribed into cDNA. The specific operation steps refer to the instructions of FastPure Cell / Tissue Total RNAIsolation Kit V2 (RC112-01) of Novagen and the reverse transcription kit of AG. Constructed into the pcDNA3.1 vector containing the constant region of the heavy and light chains of the antibody, transformed into DH5α, plate resistance screening, bacterial liquid PCR identified as positive clones, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared in the IMGT database to obtain the variable region sequences of antibodies 2B10, 3H9, 5G12, 8H11 and the corresponding antibody families.

[0124] The heavy chain variable region (VH) and light chain variable region (VL) of the above antibodies are shown in Table 4.

[0125] Table 4

[0126] Antibody ID / variable region Amino acid sequence (SEQ ID NO.) 2B10-VH 7 2B10-VL 8 3H9-VH 15 3H9-VL 16 5G12-VH 23 5G12-VL 24 8H11-VH 31 8H11-VL 32

[0127] Example 5 Antibody Expression and Purification

[0128] The strain with correct sequencing was expanded and cultured, and the plasmid was extracted using the Tiangen Endotoxin-Free Plasmid Extraction Kit (DP117-TA). For specific operation steps, please refer to the corresponding instructions.

[0129] One day before transfection, culture 293F cells at a density of 1-1.5×10 6 cells / ml, and the cell viability is greater than 98%. Take 1mg of the recombinant plasmid and add it to 10ml of Opti-MEM medium, then add 2ml of PEI transfection reagent, mix well and let stand for 15min, add the transfection mixture to 1L of 293F cells, observe and count the number of live cells and cell viability on a daily basis, and collect samples for purification when the cell viability drops below 60% when the cells express to the 5th to 7th day. Centrifuge at 8000rpm for 20min, collect the supernatant, and filter. Load the Protein A filler into the chromatography column, pass 10 column volumes of ultrapure water, and wash the residual ethanol. After washing, balance the column with 10 column volumes of PBS, add the collected supernatant to the chromatography column, load the sample 3 to 5 times, wash with PBS, elute with glycine solution (pH3.0, 0.1mol / L), and add an appropriate volume of Tris-HCl (pH8.0, 1mol / L) to the eluent to neutralize until the solution is neutral. The purified samples were subjected to SDS-PAGE electrophoresis to verify the molecular weight and purity of the protein. The protein samples were mixed with protein loading buffer (-DTT, +DTT), placed in a metal bath, boiled at 95°C for 10 minutes, and then loaded on the gel. The protein concentration was determined by the BCA method to estimate the yield of the target protein obtained after affinity purification.

[0130] The results are as follows Figure 5 As shown, antibodies 2B10, 3H9, 5G12, and 8H11 were successfully expressed and purified.

[0131] Example 6 ELISA detection of monoclonal antibodies to distinguish triple helical and non-triple helical collagen

[0132] The specific operation steps were the same as 2.2. Antigen 1 and antigen 2 were coated separately, and three duplicate wells were repeated. Different monoclonal antibodies 2B10, 3H9, 5G12, and 8H11 were added and incubated in sequence. Finally, the color was developed and the ELISA was used to read the values. Monoclonal antibodies that can distinguish between triple helical and non-triple helical collagens were successfully screened and obtained. The ELISA detection values ​​are shown in Table 5.

[0133] Table 5

[0134]

[0135] Example 7 Western Blot Detection of Recognition Epitopes of Recombinant Antibodies

[0136] Western Blot (WB) experiments can identify whether antibodies are targeting conformational epitopes or linear epitopes, which mainly depends on the treatment of proteins during the experiment and the characteristics of antibodies. In WB experiments, proteins are heat-denatured before electrophoresis. This step causes the protein to unfold into a linear polypeptide chain by destroying the hydrogen bonds in the secondary and tertiary structures of the protein. This treatment method mainly affects the spatial conformation of the protein, exposing linear epitopes that may have been hidden in the native state. Linear epitope antibodies recognize epitopes composed of continuous amino acid residues in the protein sequence. Since proteins unfold into linear polypeptide chains after heat denaturation, linear epitopes are not affected by denaturation, and both natural and denatured proteins contain them. Conformational epitope antibodies recognize epitopes composed of amino acid residues that are close but not continuous in space. Conformational epitopes depend on the three-dimensional structure of the protein, and heat denaturation will destroy this structure, causing the conformational epitope to disappear. Due to the denaturation of proteins in WB experiments, antibodies are mainly used to identify linear epitopes. This is because the denatured protein unfolds into a linear polypeptide chain, exposing linear epitopes that may have been hidden in the native state and are thus recognized by antibodies.

[0137] Add 5× protein loading buffer (containing DTT) to antigen 1, mix well, and place in a metal bath at 95℃ for 10min. Take the sample after the metal bath and add it to the loading hole of the protein precast gel, run SDS-PAGE electrophoresis at 160V for 32min. Soak the cut 0.45μm PVDF membrane in methanol solution, and after 1min, transfer the PVDF membrane to the transfer solution with tweezers. Place the SDS-PAGE gel and PVDF membrane together in two layers of filter paper, remove bubbles, and run at 400mA for 30min. After the transfer, the PVDF membrane was blocked with 5% skim milk powder for 2 h, washed with TBST three times for 10 min each time, and monoclonal antibodies 2B10, 3H9, 5G12, and 8H11 were added in sequence, incubated on a shaker for 1 h, washed with TBST three times for 10 min each time, and then goat anti-mouse HRP-labeled antibody (1:10000 dilution) was added, incubated on a shaker for 1 h, washed with TBST three times for 10 min each time, and 1 ml of developer was added for development.

[0138] The results are as follows Figure 6 As shown, there is no target protein band in the WB results, which indicates that the monoclonal antibodies 2B10, 3H9, 5G12, and 8H11 recognize conformational epitopes, which can indirectly reflect the binding of the antibodies to collagen with a triple-helical spatial structure.

[0139] Example 8 Indirect ELISA to detect EC of recombinant antibodies and triple helical collagen 50 value

[0140] One day in advance, 2 μg / ml triple helical collagen was coated in a 96-well ELISA plate at 4°C. The next day, the coating solution was discarded, 2% BSA, 300 μL / well solution was added for blocking, 37°C for 60 min, PBST was used to wash the plate 4 times, the purified recombinant antibody was gradiently diluted with PBS to the corresponding concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.0156 μg / ml and added to the blocked ELISA plate, 100 μL / well, incubated at 37°C for 60 min, enzyme-labeled secondary antibody was diluted 1:20000 times, added to the ELISA plate, incubated at 37°C for 60 min, TMB color development was performed for 20 min, color development was stopped, and the absorbance at 450 and 650 nm was measured. Adding sample diluent was used as a negative control.

[0141] The results are as follows Figure 7 As shown, the EC of antibody 2B10 50 The value is 1.492 μg / ml, and the EC of antibody 3H9 50 The value is 0.032 μg / ml, and the EC of antibody 5G12 50 The value is 0.0834 μg / ml, and the EC of antibody 8H11 50 The value is 1.224μg / ml.

[0142] Example 9 Antibody subtype detection

[0143] Remove the Mouse monoclonal antibody subtype identification kit (proteintech) from 4℃ and equilibrate at room temperature for 30 minutes. Prepare the corresponding reagents, 1×PBST, enzyme-labeled plates and 1×goat anti-mouse IgM+IgG-HRP, dilute the antibody to 200ng / mL with 1×PBST, and add it to the sample wells of the enzyme-labeled plates, 50uL / well. No incubation is required, add 1×goat anti-mouse IgM+IgG-HRP to the sample wells, 50uL / well. Mix gently on the mixer, or tap the sides of the plate rack gently for 1min. Cover with the sealing film and incubate at room temperature for 1h. Discard the liquid in the well, wash the plate 3 times with 1×PBST, and pat dry on absorbent paper. Add the prepared color development solution (A solution: B solution = 1:100, that is, 10uA solution is mixed with 1mL B solution, and use it immediately after mixing.) to the wells, 100uL / well. Color development at room temperature in the dark for 10-20min. Add stop solution to each well, 100uL / well. Interpretation of results: The results can be judged by naked eyes by comparing with the figure below, or by reading OD on a microplate reader 450 The darkest well or the one with the highest OD value corresponds to the corresponding subtype. Each sample should have one positive well in AF (i.e., the sample heavy chain type) and one positive well in G and H (i.e., the sample light chain type).

[0144] like Figure 8As shown, the subtypes of antibodies 2B10, 3H9, and 5G12 were identified as IgG1 and Kappa, and the subtype of 8H11 was identified as IgG1 and Lambda.

Claims

1. An antibody for identifying the triple helix structure of collagen, characterized in that: Include: (i) the complementarity determining region (HCDR) of the heavy chain variable region, The amino acid sequence of its HCDR1 is shown in SEQ ID NO: 1, 9, 17 or 25, The amino acid sequence of its HCDR2 is shown in SEQ ID NO: 2, 10, 18 or 26, The amino acid sequence of its HCDR3 is shown in SEQ ID NO: 3, 11, 19 or 27; and (ii) the complementarity determining region (LCDR) of the light chain variable region, The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, 12, 20 or 28, The amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, 13, 21 or 29, The amino acid sequence of its LCDR3 is shown in SEQ ID NO:6, 14, 22 or 30.

2. The antibody according to claim 1, characterized in that Comprising a complementarity determining region (HCDR) of a heavy chain variable region and a complementarity determining region (LCDR) of a light chain variable region, wherein the HCDR and LCDR of the antibody are selected from one of the following:

3. The antibody according to claim 1, characterized in that: The heavy chain variable region (VH) has more than 90% identity with the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23 or SEQ ID NO:31; and the light chain variable region (VL) has more than 90% identity with the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24 or SEQ ID NO:

32.

4. Use of the antibody according to any one of claims 1 to 3 in any of the following: i) preparing a detection kit; ii) preparing antibody drugs; iii) Preparation of antibody-drug conjugates.

5. A method for screening and identifying antibodies for the triple helix structure of collagen, characterized in that: The steps include: i) Selecting antigens: Collagen samples with triple helical structure characteristics identified by circular dichroism are used as antigens; ii) Select the immunization scheme: select mice as the immunized animals, and immunize with antigens and adjuvants; iii) Screening and preparation of antibodies: Screening and preparation of antibodies using hybridoma technology or single B cell technology; iv) Identification: ① The ELISA test antibody can recognize collagen with a triple helical structure identified by circular dichroism, but does not recognize collagen with a non-triple helical structure identified by circular dichroism; ②WB identified that the antibody's recognition epitope was a conformational epitope.

6. The method according to claim 5, characterized in that The collagen sample described in step i) has a thermal stability greater than 37° C. and can be maintained for more than 3 months in an in vitro accelerated stability test.

7. The method according to claim 5, characterized in that The collagen sample described in step i) is coupled with a carrier protein.

8. The method according to claim 5, characterized in that The mice in step ii) are female BALB / c mice of 6-8 weeks old.

9. The method according to claim 5, characterized in that The adjuvant in step ii) is a water-soluble agent.

10. The method according to claim 5, characterized in that The ELISA measurement value of the triple-helical collagen identified by circular dichroism in step iv) is more than 10 times that of the non-triple-helical collagen identified by circular dichroism.

Images