Human red blood cell monoclonal antibody and its use

Abstract

The application discloses a human red blood cell monoclonal antibody and application thereof, and relates to the field of antibodies. The antibody disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody provides an important raw material source for detection of human red blood cells, and has good affinity or activity.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to a human erythrocyte monoclonal antibody and its applications. Background Technology

[0002] Lateral chromatography is a rapid, simple, and cost-effective immunoassay method widely used in point-of-care diagnosis of various diseases. Whole blood samples provide information closer to the physiological state and do not require complex sample processing procedures. This simplifies the process, saves testing time, and avoids errors and information loss that may be introduced during sample pretreatment. Therefore, whole blood has unique advantages in certain testing scenarios. For example, in the diagnosis of acute infectious diseases, whole blood testing can directly detect pathogens or pathogen antigens in the blood, such as bacteria, viruses, or parasites, significantly shortening testing time and providing more comprehensive clinical information, helping doctors make rapid diagnostic and treatment decisions.

[0003] While whole blood samples offer convenience for testing, their complex composition and physical properties can interfere with the chromatography process and the accurate detection of analytes, leading to false negative or false positive results. The main interference mechanisms of whole blood samples include: 1) Physical interference: The large number of red blood cells in whole blood can physically block the micropores of the chromatographic membrane during the chromatography process, severely slowing down or even completely preventing the forward flow of liquid and affecting the test results; 2) Background staining interference: After red blood cells rupture (hemolysis), they release hemoglobin. During the chromatography process, these hemoglobins will non-specifically adsorb onto various areas of the membrane, resulting in a light red background in the detection area, which will severely reduce the signal-to-noise ratio of the test results; 3) Interference from blood cell antigens: If the target substance to be detected is an antigen present on the red blood cell membrane (such as cardiac markers or viral antigens), then the red blood cells themselves will become a huge source of interference, either capturing too many markers or preventing the normal release and binding of antigens, affecting the detection reactivity; 4) The effect of hematocrit (HCT): Hematocrit refers to the percentage of red blood cells in the whole blood volume. The hematocrit varies greatly among different individuals (such as anemic patients, newborns, and residents of high altitudes). The design of test strips is usually optimized based on standard hematocrit, so abnormal hematocrit can become a potential factor for detection interference.

[0004] Currently, commercially available chromatographic test strips for whole blood sample testing address red blood cell interference using the following methods: 1) embedding a filter membrane (such as a glass fiber membrane) in the sample pad to physically trap red blood cells, allowing plasma to permeate for chromatography. This method is widely used but carries risks of filter clogging, decreased chromatographic efficiency, and sample loss; 2) adding a hemolysin to the test strip or accompanying buffer to rapidly lyse red blood cells and dilute their contents, reducing cell clumping. However, this method is less effective at eliminating background staining caused by hemoglobin release, potentially interfering with the interpretation of the detection signal; 3) coating the sample pad with human erythrocyte antibodies. When the whole blood sample flows through the sample pad, the human erythrocyte antibodies specifically bind to the antigens on the surface of red blood cells, forming a complex that filters out whole red blood cells. This method efficiently intercepts red blood cells, avoiding interference with the detection display. Comparatively, the third method, achieving red blood cell filtration through specific immune binding, is more accurate and reliable. Therefore, developing specific human erythrocyte monoclonal antibodies for whole blood testing is of great significance and necessity for improving detection performance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a monoclonal antibody targeting human erythrocytes, a detection kit, and their applications. The monoclonal antibody of this invention exhibits high binding activity and affinity for human erythrocytes. When applied to immunochromatographic test strips, the specific binding of the antigen and antibody enables the interception and filtering of erythrocytes in the sample, effectively improving detection accuracy. This invention is specifically achieved through the following technical solutions:

[0006] The first aspect of this invention provides a human erythrocyte monoclonal antibody, the antibody comprising:

[0007] The amino acid sequence is shown in the heavy chain complementarity-determining region 1 of SEQ ID No:1;

[0008] The amino acid sequence is shown in the heavy chain complementarity-determining region 2 of SEQ ID No:2;

[0009] The amino acid sequence is shown in the heavy chain complementarity-determining region 3 of SEQ ID No:3;

[0010] The amino acid sequence is shown in light chain complementarity-determining region 1 as indicated in SEQ ID No:4;

[0011] The amino acid sequence is shown in light chain complementarity-determining region 2 as indicated in SEQ ID No:5;

[0012] The amino acid sequence is shown in light chain complementarity-determining region 3 as shown in SEQ ID No:6;

[0013] The complementarity-determining region of the antibody is defined by the Kabat system.

[0014] Furthermore, the antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, and sc(Fv)2 fragment.

[0015] A second aspect of the present invention provides an antibody conjugate comprising, as described above, a human erythrocyte monoclonal antibody; the antibody conjugate is formed by conjugating the antibody with a label or a solid-phase carrier.

[0016] Furthermore, the label is selected from at least one of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, nanoparticle labeling, or radioactive labeling; the solid support is selected from at least one of microspheres, plates, or membranes.

[0017] A third aspect of the present invention provides a human erythrocyte detection reagent, the reagent comprising antibodies or conjugates thereof as described above.

[0018] A fourth aspect of the present invention provides a human erythrocyte detection reagent, the reagent comprising a capture reagent and a detection reagent, each comprising a human erythrocyte monoclonal antibody or conjugate thereof with a different sequence, wherein the human erythrocyte monoclonal antibody or conjugate thereof is selected from the antibodies or conjugates thereof as described above.

[0019] The present invention provides, in five aspects, the use of antibodies or their conjugates as described above in the preparation of products for detecting human erythrocytes.

[0020] The sixth aspect of the present invention provides a method for detecting human red blood cells for non-diagnostic purposes, the method comprising: forming an immune complex with an antibody or conjugate thereof as described above and an antigen in a sample.

[0021] The beneficial effects of this application are as follows: The anti-erythrocyte monoclonal antibody of this application can specifically recognize specific antigenic epitopes on the surface of erythrocytes, making it suitable for intercepting erythrocytes in whole blood samples. It captures erythrocytes through a specific binding reaction between the antigen and antibody, avoiding interference with subsequent detection and improving the accuracy of test results. Furthermore, because the antibody of this application has high specificity in binding to human erythrocytes, it can also be directly used in human erythrocyte detection reagents, effectively reducing cross-reactivity and improving the accuracy and sensitivity of erythrocyte detection, providing a more comprehensive tool for the research and diagnosis of erythrocyte-related diseases. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the formulation or unit dose practice or testing of this document, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting. Where specific conditions are not specified in the examples, they are performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are conventional products that are commercially available.

[0024] The first objective of this invention is to provide a monoclonal antibody against human erythrocytes, the antibody comprising: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID No:1-3; and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID No:4-6.

[0025] In this invention, "CDR," "CDRs," or "complementarity-determining region" refers to a highly variable region of the heavy and light chains of an immunoglobulin, specifically a region containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In a specific embodiment of this invention, CDRs refer to the highly variable region of the heavy and light chains of the antibody.

[0026] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0027] The methods for defining CDRs are well-known in the art and include: the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As described herein, the “Kabat definition” refers to the definition system described by Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). For the “Chothia definition,” see Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any of the above schemes but will still overlap at least partially with the CDR region defined by Kabat, although they may shorten or lengthen them based on predictions or experimental results for specific residues or residue groups.

[0028] In this invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by the Kabat system.

[0029] In this invention, the antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the following: Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, and sc(Fv)2 fragment.

[0030] A second objective of the present invention is to provide an antibody conjugate comprising, as described above, a human erythrocyte monoclonal antibody; the antibody conjugate being composed of the antibody conjugated to a label or a solid-phase carrier.

[0031] In this invention, the antibody conjugate is composed of an antibody coupled to a label or a solid-phase support. The label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, nanoparticle labeling, and radioactive labeling; the solid-phase support is selected from microspheres, plates, or membranes.

[0032] In optional embodiments, the aforementioned markers refer to substances that possess properties such as luminescence, color development, and radioactivity that can be directly observed with the naked eye or detected by instruments. These properties enable qualitative or quantitative detection of the corresponding target analytes. Examples include, but are not limited to, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

[0033] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0034] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar dyes, Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar dyes) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar dyes).

[0035] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0036] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0037] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0038] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0039] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0040] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0041] In an optional embodiment, the colloidal metal is colloidal gold.

[0042] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0043] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0044] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0045] The amino acid sequence of the variable region of the heavy chain of the antibody described in this invention is shown in SEQ ID NO.7, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.8.

[0046] In this invention, the "frame region" or "FR region" includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0047] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3.

[0048] -LCDR3-LFR4.

[0049] In an optional embodiment, the antibody further comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0050] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the antibody for human erythrocytes provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame region.

[0051] In an optional embodiment, the antibody or its antigen-binding fragment further includes a constant region.

[0052] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0053] In an optional implementation, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0054] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0055] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0056] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0057] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0058] In an optional implementation, the species source of the constant region is mice.

[0059] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region.

[0060] A third objective of this invention is to provide a detection reagent for human red blood cells, the reagent comprising the aforementioned antibody.

[0061] In optional embodiments, the human erythrocyte monoclonal antibody of the present invention is used to specifically capture and intercept erythrocyte interference in a sample. For example, the antibody of the present invention can be coated into the sample pad of an immunochromatographic assay reagent. When a whole blood sample flows through the sample pad, the erythrocytes are captured into the sample pad through the specific reaction between the antibody and the antigen, thereby intercepting the erythrocytes and preventing them from affecting subsequent detection. Alternatively, the antibody of the present invention can be conjugated to magnetic beads. By premixing the whole blood sample with the magnetic beads conjugated with the antibody of the present invention, interfering erythrocytes in the sample are specifically captured, and then separated by magnetic separation technology to eliminate erythrocyte interference in the whole blood sample, thereby reducing cross-reaction and improving the accuracy and sensitivity of the detection.

[0062] A fourth objective of this invention is to provide a human erythrocyte detection reagent, the reagent comprising a capture reagent and a detection reagent, each comprising a human erythrocyte monoclonal antibody or conjugate thereof with a different sequence, wherein the human erythrocyte monoclonal antibody or conjugate thereof is selected from the antibodies or conjugates thereof as described above.

[0063] In optional embodiments, the above kit can be used for detection methods involving the specific binding properties of human erythrocytes and their antibodies, such as immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and latex immunoturbidimetry. For example, in fluorescence immunochromatography, the antigen in the sample binds to the fluorescently labeled antibody on the binding pad, and the solution undergoes chromatography due to the siphon effect of the absorbent pad, moving towards the absorbent pad. When the complex moves to the detection line, it binds to the coated antibody on the T line, forming a "sandwich" type complex and accumulating on the T line. In chemiluminescence immunoassay, the antigen in the sample binds to the antibody coated on the magnetic beads, and after washing, it binds to the enzyme-labeled antibody (usually horseradish peroxidase HRP or alkaline phosphatase AP) to form a "solid-phase antibody-antigen-enzyme-labeled antibody" sandwich complex. It is understood that the antibody here does not specifically refer to a particular sequence of the present invention. Theoretically, any antibody that forms a "double-antibody sandwich" immune complex is acceptable; therefore, the antibody used as the capture antibody and the detection antibody is not theoretically specific.

[0064] A fifth object of the present invention is to provide the use of antibodies or their conjugates in the preparation of products for detecting human erythrocytes, and in the use of antibodies for blood cell capture in chromatographic whole blood programs.

[0065] A sixth objective of the present invention is to provide a method for detecting human red blood cells for non-diagnostic purposes, the method comprising: forming an immune complex with an antibody or conjugate thereof as described above and an antigen in a sample.

[0066] The technical solution of the present invention will be further described below through some specific embodiments.

[0067] Example 1: Preparation of monoclonal antibodies against human erythrocytes

[0068] (1) Mouse immunization and antibody detection

[0069] Twenty 6-8 week old SPF-grade female BALB / c mice (Hunan Silek Jingda Experimental Animal Co., Ltd.) were selected. Mixed red blood cells from various animals were used to immunize each 6-8 week old female SPF-grade BALB / c mouse via intraperitoneal injection of 100 μL of human red blood cells. Two weeks after the initial immunization, a second intraperitoneal injection of 100 μL of red blood cells was administered to each mouse. Three weeks later, blood was collected via tail vein, and the supernatant was collected by centrifugation and serum titer was determined by ELISA. Immunization was repeated every two weeks, and serum titer was measured. After two immunizations, the serum titer, after a million-fold dilution, reached a level higher than 1.5. A serum titer of 10 was selected for screening. 6 The spleens of the mice were used for cell fusion.

[0070] (2) Hybridoma antibody screening

[0071] In the antibody screening stage, red blood cells were first diluted 5-fold and added to 96-well plates at 100 μL / well. Hybridoma cell supernatant was added to perform an agglutination reaction. The hybridoma cell supernatant with good agglutination effect was then cultured on a larger scale. 100 μL of cell supernatant was then diluted 2-fold and added to 96-well plates at 100 μL / well. Whole blood samples from human blood types A, B, O, and AB were selected, and red blood cells were separated and added to 96-well plates at 50 μL / well. The agglutination reaction was observed. Then, an antibody that showed agglutination reaction with all four blood types and had high sensitivity was selected.

[0072] (3) Production and purification of monoclonal antibodies

[0073] Thirty 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL of paraffin oil to suppress the immune response. One week after injection, the mice were divided into 10 groups (3 mice / group), and 0.5 ml of the aforementioned human erythrocyte monoclonal antibody hybridoma cells (approximately 1 × 10⁻⁶ cells) were injected intraperitoneally into each group. 6 Quantity. Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and affinity purification with protein G to obtain the target antibody.

[0074] Example 2: Antibody gene sequence cloning and performance testing

[0075] 1. Identification of monoclonal antibody subtypes and cloning of gene sequences

[0076] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the heavy and light chain isotypes of the aforementioned human erythrocyte monoclonal antibodies, following the manufacturer's instructions. The specific procedures were as follows:

[0077] a. Dilute the capture antibody to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20.

[0078] b. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 min. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lambda-HRP) 1:3000 with diluent.

[0079] After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing three more times, add chromogenic buffer and incubate for approximately 5 minutes (depending on the reaction strength). Then, add 2 M sulfuric acid to terminate the reaction and read the OD450 absorbance. Based on the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells in good growth condition were collected, and total RNA was obtained from the hybridoma cells using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the Takara SMARTer RACE instruction manual, and the full-length sequence of the target antibody was amplified.

[0080] 2. Whole blood leakage detection

[0081] (1) Preparation of test strips

[0082] a. Preparation of the sample pad to be tested

[0083] To evaluate the function of the aforementioned antibody, the antibody was diluted to 0.2 mg / ml, and 1.75 ml was evenly sprayed onto a glass cellulose membrane, placed in a vacuum drying oven and dried for 2 hours, and then cut for later use.

[0084] b. Preparation of control sample pads

[0085] Take 1.75ml of ultrapure water and spray it evenly onto the glass cellulose membrane. Place it in a vacuum drying oven and dry for 2 hours. Cut it for later use.

[0086] c. The above-mentioned test sample pad and control sample pad are overlapped and cut with the backing and absorbent membrane respectively, and assembled into test strips and control test strips.

[0087] (2) Sample preparation

[0088] Twenty-six whole blood samples of four types (A, B, O, and AB) were collected for separation and preprocessing, and hematocrit (HCT) was adjusted. The range of high HCT values ​​was 52±2%, and the range of low HCT values ​​was 40-50%. The sample information is shown in the table below.

[0089] Table 1 Sample Information

[0090] Blood type HCT value % (high) Blood type HCT value % (low) AB 51.1 AB 47.6 AB 52.8 AB 43.9 AB 50.2 AB 46.2 B 51.5 B 48.6 B 53.5 B 47.5 B 51.1 A 44 B 51.4 A 47 A 51.2 A 40.1 A 52.9 A 46.7 O 51.5 A 46.1 O 51.7 O 45.6 O 52.4 O 40.2 / / O 41.1 / / O 46.5

[0091] (3) Performance evaluation

[0092] 100 μL of whole blood sample was added to the sample pad of the test strip, the test strip accelerated at 37℃ for 7 days, and the control test strip, respectively. After reacting for 15 min, the presence of obvious red blood cell exudation was observed in the window of each reagent card. The test results are shown in the table below.

[0093] Table 2. Erythrocyte exudation status

[0094] Batch number Control test paper Paper to be tested To be tested: 37°C for 7 days HCT value 52 ± 2% number of cases of blood leakage 2 / 12 0 / 12 2 / 12 HCT value 40-50% number of cases of blood leakage 0 / 14 0 / 14 0 / 14

[0095] The data above shows that the antibody of this invention can effectively intercept red blood cells in practical chromatography detection applications.

[0096] The partial amino acid sequence of the antibody of this invention is shown in the table below (defined by the Kabat system).

[0097] Table 3 Amino acid sequence listing

[0098] SEQ ID NO. Amino acid sequence HCDR1 SEQ ID NO. 1 NYGVN HCDR2 SEQ ID NO. 2 WINTNTGEPTYAEEFKG HCDR3 SEQ ID NO. 3 GDFRYFDY LCDR1 SEQ ID NO. 4 RSSQSLVHTNGKTYLH LCDR2 SEQ ID NO. 5 RVSNRFS LCDR3 SEQ ID NO. 6 SQSTHVPWT Heavy chain variable region SEQ ID NO. 7 QIQLVQSGPELMKPGETVKISCKASGYIFTNYGVNWVKQAPGRGLKWMGWINTNTGEPTYAEEFKGRFAFSLDTSATTAYLQINDLKNEDTATYFCARGDFRYFDYWGQGTTLTVSA Light chain variable region SEQ ID NO. 8 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHTNGKTYLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSESGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLDIK Heavy chain SEQ ID NO. 9 QIQLVQSGPELMKPGETVKISCKASGYIFTNYGVNWVKQAPGRGLKWMGWINTNTGEPTYAEEFKGRFAFSLDTSATTAYLQINDLKNEDTATYFCARGDFRYFDYWGQGTTLTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light chain SEQ ID NO. 10 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHTNGKTYLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSESGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLDIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC All data, reagents, and procedures described herein should be understood as illustrative rather than restrictive. Although the invention has been described in conjunction with the specific embodiments described above, many modifications and other variations will be apparent to those skilled in the art. All such modifications and other variations also fall within the scope of the invention.

Claims

1. A monoclonal antibody to human red blood cells, characterized in that, The antibody comprises: a heavy chain complementarity determining region 1 having an amino acid sequence as set forth in SEQ ID No: 1; a heavy chain complementarity determining region 2 having an amino acid sequence as set forth in SEQ ID No: 2; a heavy chain complementarity determining region 3 having an amino acid sequence as set forth in SEQ ID No: 3; a light chain complementarity determining region 1 having an amino acid sequence as set forth in SEQ ID No: 4; a light chain complementarity determining region 2 having an amino acid sequence as set forth in SEQ ID No: 5; a light chain complementarity determining region 3 having an amino acid sequence as set forth in SEQ ID No: 6; The complementarity determining regions are defined by the Kabat system.

2. The antibody of claim 1, wherein The antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

3. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody according to any one of claims 1-2; the antibody conjugate is formed by conjugation of the antibody with a label or a solid-phase carrier.

4. The antibody conjugate of claim 3, wherein, The label is selected from at least one of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a nanoparticle label or a radioactive label; the solid-phase carrier is selected from at least one of a microsphere, a plate or a membrane.

5. A test reagent for human red blood cells, characterized in that, The reagent comprises the antibody according to any one of claims 1-2 or the antibody conjugate according to any one of claims 3-4.

6. A test reagent for human red blood cells, characterized in that, The reagent comprises a capture reagent and a detection reagent, each of which comprises a human red blood cell monoclonal antibody or an antibody conjugate thereof having a different sequence, the human red blood cell monoclonal antibody being selected from the antibody according to any one of claims 1-2, and the human red blood cell monoclonal antibody conjugate being selected from the antibody conjugate according to any one of claims 3-4.

7. Use of the antibody according to any one of claims 1-2 or the antibody conjugate according to any one of claims 3-4 in the preparation of a reagent for detecting a human red blood cell product.