Antibodies detecting the rhod blood group antigen and use thereof

Abstract

The application discloses an antibody for detecting RhD blood group antigen and application thereof, and relates to the technical field of immunodiagnosis. The antibody for detecting RhD blood group antigen provided by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, and the antibody provides an important raw material source for the detection of RhD blood group antigen and has improved low-temperature stability.

Description

Technical Field

[0001] This invention relates to the field of immunodiagnostic technology, and more specifically, to an antibody for detecting RhD blood group antigen and its application. Background Technology

[0002] Blood group antigens are molecular structures present on the surface of red blood cells. Based on their different expression patterns, humans are divided into several different blood group systems, such as the ABO blood group system and the Rh blood group system. Blood group antibodies are immune proteins that target specific blood group antigens and possess antibody activity corresponding to those antigens.

[0003] In the Rh blood group system, there are five common antigens: D, C, E, c, and e. Among them, D antigen is the most potent. Therefore, the RhD antigen is the most frequently tested antigen in the Rh blood group system.

[0004] The Rh blood group system generally does not have natural antibodies, so adverse reactions may not occur during the first transfusion. However, Rh-negative recipients who receive Rh-positive blood may develop immune anti-Rh antibodies. If they receive Rh-positive blood again, these antibodies will attack the external Rh-positive blood, causing a hemolytic transfusion reaction that destroys red blood cells and can lead to illness or death. If an Rh-negative mother is carrying an Rh-positive fetus, and the Rh factor on the fetal red blood cells enters the mother's bloodstream for some reason (such as bleeding caused by placental abruption), the mother will produce anti-agglutinins (anti-Rh antibodies). These anti-agglutinins then cross the placenta into the fetal circulation, causing the fetal red blood cells to agglutinate and be destroyed. This can lead to severe anemia in the fetus, or even death.

[0005] RhD blood group antigen testing and matching are crucial in medical applications such as blood transfusions, organ transplantation, and blood typing. Currently, commonly used blood typing methods include: slide method, test tube method, microcolumn gel method, and solid-phase method. Hospitals mostly use the slide method, which involves placing a drop of anti-D antibody on a glass slide, then adding the red blood cells to be tested. If the red blood cells contain RhD antigens, they will agglutinate with the anti-D antibody, indicating RhD positive blood; if they do not agglutinate, it indicates RhD negative blood.

[0006] The above detection methods require antibodies against RhD blood group antigen. Current antibodies against RhD blood group antigen have poor low-temperature stability. They are prone to aggregation or denaturation when stored at 2-8°C for a long time, resulting in a decrease in potency. This forces the addition of high-concentration stabilizers (such as sucrose and trehalose) or reliance on cryopreservation, increasing costs and transportation difficulties. Summary of the Invention

[0007] This application provides an antibody that provides an important source of raw materials for the detection of RhD blood group antigens, and has improved low-temperature stability, making it easy to store and transport.

[0008] To achieve the above objective, according to a first aspect of the present invention, an antibody for detecting RhD blood group antigen is provided, the antibody comprising three complementarity-determining regions having a heavy chain variable region as shown in SEQ ID NO:15 and three complementarity-determining regions having a light chain variable region as shown in SEQ ID NO:18, wherein two consecutive arginine residues in the complementarity-determining regions are mutated, either one or both independently, to an uncharged amino acid.

[0009] To achieve the above objectives, according to a second aspect of the present invention, an antibody for detecting RhD blood group antigen is provided, the aforementioned antibody comprising the following complementarity-determining regions: HCDR1, comprising or consisting of the amino acid sequence shown in GYFWT (SEQ ID NO:1); HCDR2, comprising or consisting of the amino acid sequence shown in EINHSGSTTYNPSLKS (SEQ ID NO:2); HCDR3, comprising or consisting of the amino acid sequence shown in GFSWGGYNYGFAIDY (SEQ ID NO:3); LCDR1, comprising or consisting of the amino acid sequence shown in QGDSLX1X2YYGS (SEQ ID NO:4), wherein X1 and X2 are each independently selected from glycine, alanine, serine, and threonine; LCDR2, comprising or consisting of the amino acid sequence shown in GKNNRPS (SEQ ID NO:5); and LCDR3, comprising or consisting of the amino acid sequence shown in NSRDSSGNHRI (SEQ ID NO:6).

[0010] To achieve the above objectives, according to a third aspect of the present invention, an antibody for detecting RhD blood group antigen is provided, the antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region being as shown in SEQ ID NO:18; wherein one or both consecutive arginine residues in the light chain variable region are mutated to an uncharged amino acid.

[0011] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody for detecting RhD blood group antigen is provided, the antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:17 and the amino acid sequence of the light chain is shown in SEQ ID NO:20.

[0012] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibody described above.

[0013] To achieve the above objectives, according to a sixth aspect of the present invention, a kit for detecting RhD blood group antigen is provided, the kit comprising the aforementioned antibody or antibody-drug conjugate.

[0014] To achieve the above objectives, according to a seventh aspect of the present invention, a method for detecting RhD blood group antigens is provided, comprising:

[0015] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody, antibody conjugate, or kit is contacted with the sample to be tested to form an immune complex; and

[0016] b) Detect the presence of the immune complex, the presence of which indicates the presence of RhD blood group antigen in the test sample.

[0017] To achieve the above objectives, according to an eighth aspect of the present invention, the use of the above-described antibody, antibody-drug conjugate, or kit in the preparation of products for detecting RhD blood group antigens or in the preparation of products for identifying blood types is provided. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Example image for visual observation of a result of "4+";

[0020] Figure 2 Example image for visual observation of a result of "3+";

[0021] Figure 3 Example image for visual observation showing a result of "2+";

[0022] Figure 4 Example image for visual observation showing a result of "1+";

[0023] Figure 5 Example image of a visual observation showing a "negative" result;

[0024] Figure 6 Visual inspection results for stability assessment. Detailed Implementation

[0025] 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0026] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0027] In this invention, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. An antigen-binding fragment of an antibody is a substance containing an antibody CDR that lacks some amino acids present in the full-length chain but can still specifically bind to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), and the smallest antibody recognition unit. The antigen-binding fragments of the aforementioned antibodies can bind to the same antigen as the parent antibody.

[0028] Antigen-binding fragments of antibodies typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the antigen-binding fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments. Antigen-binding fragments can also be obtained using recombinant genetic techniques known to those skilled in the art or synthesized using, for example, automated peptide synthesizers, such as those sold by Applied BioSystems.

[0029] In this paper, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) are all components of the heavy chain constant region, which is located at the C-terminus of the heavy chain of the antibody molecule. Each heavy chain constant region, from the N-terminus to the C-terminus, includes the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional). Different types of antibodies (such as IgG, IgA, IgM, etc.) have different amino acid sequences and structures in their heavy chain constant regions, but they all have relatively conserved structural features. These conserved structures enable the heavy chain constant region to perform its biological function. The heavy chain constant region, CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) of different species and subclasses are well known in the art, and their amino acid sequences can be determined based on bioinformatics databases, such as the IMGT database (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). It should be understood that different bioinformatics databases or software may not have completely consistent results in the division and sequence identification of constant regions. However, those skilled in the art have a general and unified understanding of the concept, division and sequence identification of constant regions and their segments. Therefore, the constant regions that those skilled in the art can identify and divide using common knowledge and ordinary methods are all within the scope of protection of this invention.

[0030] For example, the amino acid sequence of the corresponding segment (such as the IgM CH2 region) divided by the IMGT database can be used as the reference sequence. The start or end position of the reference sequence can be moved forward by several amino acid residues (i.e., moved to the IgM CH1 region) or backward by several amino acid residues (i.e. moved to the IgM CH3 region) to obtain the corresponding segment sequence which is longer or shorter than the reference sequence.

[0031] In this document, the term "hinge region" refers to a polypeptide that links the CH1 and CH2 domains within the constant region of the heavy chain of an antibody. This region is rich in proline, thus allowing for stretching and bending, and typically contains at least one proline (P). Hinge regions are usually dimers, consisting of two polypeptides with the same amino acid sequence. Specific amino acid sequences are not limited and are all within the scope of protection of this application. Hinge regions of different species and subclasses are well known.

[0032] In this paper, the term "tail peptide" refers to a short peptide sequence of about a dozen amino acid residues at the end of the CH3 or CH4 region of an antibody. Tail peptides from different species are well known.

[0033] In this document, the term "IgM tail peptide" refers to a short peptide sequence at the end of the CH4 region of an IgM antibody, located at the C-terminus of the CH4 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. The IgM tail peptide contains cysteine ​​residues that participate in polymer formation and can also bind to the J chain to further stabilize the multimeric structure.

[0034] In this invention, the terms "comprising" and "including" are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0035] In this invention, the term "optionally" generally means that the event or condition described below may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] In a first aspect, embodiments of the present invention provide an antibody for detecting RHD blood group antigens. The antibody comprises three complementary determinant regions having a heavy chain variable region as shown in SEQ ID NO:15 and three complementary determinant regions having a light chain variable region as shown in SEQ ID NO:18. In the complementary determinant regions, one or both of two consecutive arginine (R, Arg) residues are mutated to uncharged amino acids.

[0037] In an optional embodiment, the above-mentioned uncharged amino acid is selected from glycine (G, Gly), alanine (A, Ala), serine (S, Ser), and threonine (T, Thr).

[0038] In an optional implementation, one of two consecutive arginine residues in the complementarity-determining region is mutated, with the first arginine residue being mutated to glycine.

[0039] In an optional implementation, one of the two consecutive arginine residues in the complementarity-determining region is mutated, and the second arginine residue is mutated to serine.

[0040] The mutation of two consecutive arginine residues in the complementarity-determining region to an uncharged amino acid is preferred over a mutation to a charged amino acid. The charged amino acid is selected from glutamic acid, aspartic acid, lysine, or histidine.

[0041] In an optional embodiment, the aforementioned antibody comprises three complementary determining regions having the heavy chain variable region shown in amino acid sequence SEQ ID NO:15 and three complementary determining regions having the light chain variable region shown in amino acid sequence SEQ ID NO:24 or SEQ ID NO:25.

[0042] Furthermore, the antibody has improved RHD blood group antigen binding titer, affinity, and / or specificity.

[0043] It should be understood that the leucine residues and isoleucine residues in the complementarity-determining region described above can be optionally substituted for each other, and both are within the scope of protection of this invention.

[0044] In an optional implementation, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0045] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions 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 specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0046] In this invention, the heavy chain complementarity-determining region (CDR) is denoted by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is denoted by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2, and LCDR3. In this invention, the term "antibody pair" refers to the use of multiple antibodies paired together, which can be two, three, four, or more antibodies, but at least two antibodies.

[0047] The precise amino acid sequence boundaries of a CDR can be determined by numbering using many well-known definitions, including Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol.262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27 (1):55-77 (“IMGT” numbering scheme).

[0048] The boundaries of CDRs may vary depending on the definition method. For example, the Kabat definition method is based on structural alignment, while the Chothia definition method is based on structural information. These two schemes place certain insertions and deletions in different positions, resulting in different numbering. CDRs under different definition methods can be obtained using software (such as Abysis). Table 1 below exemplarily lists the positions of CDRs determined by the Kabat, Chothia, IMGT, AbM, and Contact definition methods. Other CDR definition methods may not strictly follow one of the above schemes and may shorten or lengthen them based on predictions or experimental results of specific residues or residue groups. Therefore, other CDRs not limited to those in Table 1 are also within the scope of this disclosure.

[0049] Table 1: CDR Definition 1

[0050]

[0051] 1 The CDRs defined in Table 1 are numbered according to the Chothia numbering scheme (see below). Amino acids on the heavy chain are numbered with "H + number," and amino acids on the light chain are numbered with "L + number." Those skilled in the art can readily map this Chothia numbering scheme to any variable region sequence.

[0052] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0053] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0054] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0055] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0056] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0057] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0058] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0059] Secondly, embodiments of the present invention provide an antibody for detecting RHD blood group antigens, wherein the antibody includes the following complementarity-determining region:

[0060] HCDR1, which contains, or is composed of, the amino acid sequence shown in GYFWT (SEQ ID NO:1);

[0061] HCDR2, which contains, or is composed of, the amino acid sequence shown in EINHSGSTTYNPSLKS (SEQ ID NO:2);

[0062] HCDR3, which contains, or is composed of, the amino acid sequence shown in GFSWGGYNYGFAIDY (SEQ ID NO:3);

[0063] LCDR1, comprising or consisting of the amino acid sequence shown in QGDSLX1X2YYGS (SEQ ID NO:4), wherein X1 and X2 are each independently selected from glycine, alanine, serine and threonine;

[0064] LCDR2, which contains, or is composed of, the amino acid sequence shown in GKNNRPS (SEQ ID NO:5);

[0065] LCDR3 contains, or is composed of, the amino acid sequence shown in NSRDSSGNHRI (SEQ ID NO:6).

[0066] It should be understood that the leucine residues and isoleucine residues in the complementarity-determining region can be optionally substituted for each other, and both are within the scope of protection of this invention.

[0067] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0068] The antibody of the present invention also includes a frame region. In the present invention, the "frame region" or "FR" region includes a heavy chain frame region and a light chain frame region, which refers to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR. The heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including LFR1, LFR2, LFR3 and LFR4 frame regions.

[0069] 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-LCDR3-LFR4.

[0070] In an optional embodiment, the antibody described in the first and second aspects further comprises the framework regions shown by HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0071] In an optional embodiment, HFR1 comprises the sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it; HFR2 comprises the sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; HFR3 comprises the sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it; HFR4 comprises the sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it; LFR1 comprises the sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it; LFR2 comprises the sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it; LFR3 comprises the sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it; and LFR4 comprises the sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity with it.

[0072] In this invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0073] In other embodiments, the amino acid sequences of each frame region of the antibody 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 regions described above.

[0074] Thirdly, embodiments of the present invention provide an antibody for detecting RHD blood group antigens. The aforementioned antibody includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:18. Two consecutive arginine residues in the light chain variable region are mutated, either one or both are mutated independently, to uncharged amino acids.

[0075] In an optional embodiment, the uncharged amino acid is selected from glycine, alanine, serine, and threonine.

[0076] In an optional embodiment, one of two consecutive arginine residues in the light chain variable region is mutated, with the first arginine residue being mutated to glycine.

[0077] In an optional embodiment, one of two consecutive arginine residues in the light chain variable region is mutated, and the second arginine residue is mutated to serine.

[0078] In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24 or SEQ ID NO:25.

[0079] It should be understood that the leucine residues and isoleucine residues in the variable region can be optionally substituted for each other, and both are within the scope of protection of this invention.

[0080] In an optional implementation, the antibody described in the first, second, and third aspects above further includes a constant region.

[0081] In an optional implementation, the aforementioned constant region includes a heavy chain constant region and / or a light chain constant region.

[0082] In an optional implementation, the species source of the aforementioned constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0083] In optional embodiments, the heavy chain constant region of the antibody is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and / or; the light chain constant region is selected from the κ-type or λ-type light chain constant region; the λ-type light chain constant region can be selected from the λ1, λ2, λ3, and λ4 subtypes.

[0084] In an optional embodiment, the aforementioned heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM, and / or the tail peptide of IgM.

[0085] In an optional embodiment, the constant region of the antibody includes: a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:16; and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:19; or an amino acid sequence having at least 80% identity with each of the constant regions.

[0086] It should be noted that, in some 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.

[0087] Fourthly, embodiments of the present invention provide an antibody for detecting RHD blood group antigens. The antibody comprises a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID NO:17, and the amino acid sequence of the light chain is shown in SEQ ID NO:20. In the variable region of the light chain, one of two consecutive arginine residues is mutated or both are mutated independently to uncharged amino acids.

[0088] In an optional embodiment, the uncharged amino acid is selected from glycine, alanine, serine, and threonine.

[0089] In an optional embodiment, one of two consecutive arginine residues in the variable region of the light chain is mutated, with the first arginine residue being mutated to glycine.

[0090] In an optional embodiment, one of two consecutive arginine residues in the variable region of the light chain is mutated, and the second arginine residue is mutated to serine.

[0091] In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO:17, and the amino acid sequence of the light chain is shown in SEQ ID NO:21 or SEQ ID NO:22.

[0092] It should be understood that the leucine residues and isoleucine residues in the variable region of the heavy chain or the variable region of the light chain can be optionally substituted for each other, and both are within the scope of protection of this invention.

[0093] Fifthly, embodiments of the present invention provide an antibody conjugate, the antibody conjugate comprising the antibodies described in the first, second, third, or fourth aspects.

[0094] In an optional administration method, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody.

[0095] In optional administration methods, the antibody conjugate may further include a marker or purification tag conjugated to the antibody.

[0096] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0097] In optional embodiments, the markers are selected from, but not limited to, metal ions, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, electron-dense markers, adamantane, and nanoparticle markers.

[0098] 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.

[0099] In optional embodiments, the fluorescent dyes are selected from, but 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).

[0100] In optional embodiments, the enzymes are selected from, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0101] In optional embodiments, the radioactive isotopes mentioned above are selected from, but not limited to, those selected from, and others. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0102] In optional embodiments, the chemiluminescent reagent is selected from, but 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.

[0103] In an optional embodiment, the marker is acridine ester.

[0104] In optional embodiments, the above-mentioned nanoparticle markers are selected from, but not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0105] In optional embodiments, the colloid is selected from, but not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0106] In an optional embodiment, the colloidal metal is selected from, but not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0107] In an optional implementation, the labeled antibody is labeled by binding the coupler to the labeler.

[0108] It should be noted that when an antibody is labeled with a marker to become a labeled antibody, it does not affect the characteristics of the antibody's immune response, nor does it change the activity of the marker itself.

[0109] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0110] In optional embodiments, the solid support is selected from microspheres, latex particles, microfluidic chips, magnetic beads, microporous plates, or nitrocellulose membranes.

[0111] In an optional embodiment, the solid support is a magnetic bead.

[0112] In an optional embodiment, the antibody conjugate further includes a binding partner conjugated to the antibody.

[0113] In an optional embodiment, the binding coupler includes biotin / avidin or biotin derivative / avidin derivative protein.

[0114] In this invention, the term "binding pair" refers to a pair of molecules capable of binding together through non-covalent interactions. This binding is typically specific and can be temporary or form a more stable complex.

[0115] In this invention, the binding of the aforementioned binding couple to the marker or solid phase can take various forms, such as physical adsorption, electrostatic adsorption, or covalent bonding. Such binding / combining methods are known to those skilled in the art.

[0116] Sixthly, embodiments of the present invention provide a kit for detecting RHD blood group antigens, the aforementioned kit comprising the above-mentioned antibody or antibody-drug conjugate.

[0117] It should be noted that the meaning of "kit" in this application can be considered equivalent to the meaning of "reagent".

[0118] In an optional embodiment, the kit may further include at least one of the following: sample pretreatment reagents (such as sample purification and enrichment reagents, lysis buffers, etc.), washing solutions (such as water), buffers (such as PBS or Tris), stabilizing solutions, and chromogenic reagents for signal substances.

[0119] The antibody conjugates or antibodies in some specific embodiments or examples of this invention can bind to RHD blood group antigens. Therefore, kits containing said antibody conjugates or antibodies can effectively perform qualitative or quantitative detection of RHD blood group antigens. The kits provided by this invention can be used, for example, for detections involving the specific binding properties of RHD blood group antigens and their antibodies, such as immunoagglutination, immunochromatography, immunoblotting, and immunoprecipitation. As mentioned above, the antibodies of this invention have improved RHD blood group antigen binding titer, affinity, stability, and / or specificity. Therefore, kits containing said antibodies have improved detection sensitivity, specificity, and / or reduced false negatives.

[0120] In some embodiments, the antibody of the present invention may be a monoclonal antibody or a polyclonal antibody.

[0121] In optional embodiments, the antibody conjugates or antibodies of the present invention can be prepared using methods known in the art.

[0122] In an optional embodiment, the antibody of the present invention can be prepared by immunogen immunization and / or chemical synthesis.

[0123] In alternative embodiments, any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc., can be used to detect the effects of the antibody pair or antibody, such as binding activity and / or cross-reactivity.

[0124] In optional embodiments, the assay may include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, etc.

[0125] In an optional embodiment, the kit of the present invention includes reagents suitable for performing immunoassays.

[0126] In optional embodiments, the kit of the present invention can be used for immunoassays, such as ELISA, indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), and other non-enzyme-linked antibody binding assays or methods.

[0127] Seventhly, embodiments of the present invention provide a method for detecting RHD blood group antigens, comprising:

[0128] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody, antibody conjugate, or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0129] b) Detect the presence of the immune complex, the presence of which indicates the presence of RHD blood group antigen in the test sample.

[0130] In optional embodiments, the method is a slide method, a test tube method, a microcolumn gel method, or a solid-phase method.

[0131] To achieve the above objectives, according to an eighth aspect of the present invention, the use of the above-described antibody, antibody-drug conjugate, or reagent in detecting RHD blood group antigens or in preparing products for detecting RHD blood group antigens or in preparing products for identifying blood types is provided.

[0132] 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 practice or testing of formulations or unit doses herein, 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 in nature.

[0133] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. "Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JEColigan et al., eds., 1991), each of which is explicitly incorporated herein by reference.

[0134] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0135] Based on long-term inventive research on RHD blood group antibodies, the inventors discovered that RHD blood group antibodies without the mutations described in this application are prone to precipitation during low-temperature storage (e.g., 2-8°C). Through charge analysis and structural simulation of the antibodies, it was found that two concentrated arginine residues in the antibody's light chain sequence may cause excessively high local positive charges. Therefore, a mutant RHD blood group antibody was designed. The preparation, activity identification, and performance testing of the antibody are illustrated in the examples.

[0136] Example 1: Preparation of Antibody

[0137] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. The BD SMART™ RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by Invitrogen. The cell line secreting wild-type RhD antibody was an existing cell line from our company, which was revived for later use.

[0138] 1. Amplification of antibody genes

[0139] 1) Wild-type antibodies

[0140] mRNA was extracted from cell lines secreting parental Anti-RhD monoclonal antibodies, and DNA products were obtained by RT-PCR. These products were then inserted into the pMD-18T vector after an A-addition reaction using rTaq DNA polymerase. The vector was transformed into DH5α competent cells, and after colony growth, four clones of each heavy and light chain genes were collected and sent to a gene sequencing company for sequencing. The resulting gene sequences were analyzed in the Kabat antibody database, and VNTI 11.5 software was used to confirm that the antibody variable region genes amplified by both heavy and light chain primer pairs were correct.

[0141] Restriction endonuclease sites were introduced into the eukaryotic expression vector, hereinafter referred to as the 3.4A expression vector. Based on the sequencing results of the antibody variable region gene, specific primers for the light chain variable region and heavy chain variable region genes of the antibody were designed, with restriction sites and protective bases at both ends, respectively. The variable region and constant region were connected by the overlap PCR amplification method, thereby amplifying the complete light chain gene and heavy chain gene of the wild-type antibody.

[0142] 2) Chimeric antibodies

[0143] Primers were designed for the CH1 hinge region (ASTKGPSVFPL~EPKSC) of the human IgG1 antibody, the CH2 tail peptide (PVIAELPPKVSV~MSDTAGTCY) of the human IgM antibody, and the heavy chain variable region, respectively. Each primer had a restriction endonuclease cleavage site and a protective base at both ends. Overlap PCR was performed to obtain the complete heavy chain gene of the chimeric antibody. The complete light chain gene of the chimeric antibody is the same as that of the wild-type antibody.

[0144] 3) Mutant antibodies

[0145] Mutation sites were designed on the light chain of the chimeric antibody to generate primers containing the desired mutation sites, with restriction endonuclease cleavage sites and protective bases at both ends. Overlap PCR was then performed to obtain the complete light chain gene of the mutant antibody. The complete heavy chain gene of the mutant antibody is the same as that of the chimeric antibody.

[0146] 2. Construction of recombinant antibody expression plasmid

[0147] The heavy and light chain genes of the above antibodies and the 3.4A vector were digested with restriction endonucleases. After purification and recovery of the gene fragments and vectors, the heavy chain gene and light chain gene were respectively ligated into the 3.4A expression vector to obtain recombinant expression plasmids of heavy chain and light chain, respectively.

[0148] 3. Recombinant antibody production

[0149] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3-5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 3.4 × 10⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C (120 rpm) with 8% CO2 for 13 days. After 13 days, the samples were centrifuged. The supernatant was purified using a protein A affinity chromatography column to obtain the purified antibody. The heavy chain (H) and light chain (L) sequences of the above antibody are shown in the table below.

[0150] Table 2: Antibody Sequences

[0151]

[0152] Example 2: Purity identification by HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography)

[0153] Take 20 μg of purified antibody and determine the SEC purity of the sample according to the conditions in Table 3 below.

[0154] Table 3: Chromatographic conditions for HPLC-SEC

[0155]

[0156] The yield and purity are shown in Table 4.

[0157] Table 4: Yield and Purity

[0158]

[0159] The results showed that the expression level, assembly efficiency, and purity of the mutant antibody were superior to those of the wild-type antibody and the chimeric antibody.

[0160] Example 3: Antibody Performance Testing

[0161] 1. Anti-RhD blood group antigen antibody titer detection

[0162] Antibody dilution: All antibodies were diluted with PBS to 0.3 mg / ml. Based on the 0.3 mg / ml level, the antibodies were diluted 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 times to prepare working solutions for RhD blood group antibodies.

[0163] Take a 5 ml test tube and add 50 μL of RhD blood type red blood cell suspension.

[0164] Add 100 μl of the diluted RhD blood group antibody working solution from step 1) and mix immediately.

[0165] Centrifuge at 900g for 15 seconds using an Eppendorf centrifuge.

[0166] 5) After removing the test tube, gently tap the bottom of the tube with your finger and observe the results under good light. The results are shown in the table below. The results show that the titers of mutant and chimeric antibodies are superior to those of wild-type antibodies. Mutant antibodies can effectively bind to RhD blood group antigens at low concentrations.

[0167] Table 5: Anti-RhD blood group antigen antibody titers

[0168]

[0169] Table 6: Evaluation Criteria for Reaction Results (These evaluation criteria apply to the evaluation of results in all embodiments of this application)

[0170]

[0171] Results: Antibody titer was calculated using 4+ as the standard, calculated as: 1 / ((original antibody concentration / 0.3) × dilution factor).

[0172] The wild-type antibody titer was 1:64.

[0173] The titer of the chimeric antibody was 1:256.

[0174] The titer of mutant type 1 is 1:256;

[0175] The titer of mutant type 2 is 1:256.

[0176] 2. Stability assessment

[0177] 2.1 Visual inspection of appearance: Pour the antibody solution into a transparent vial and place it at 4°C overnight; take out the vial and quickly invert it 3 times, place it in the corresponding tube rack, visually inspect the appearance of the antibody solution, and record the actual appearance by taking a picture.

[0178] See Figure 6 From the appearance, wild-type antibodies and chimeric antibodies show obvious precipitation, while mutant antibodies are clear.

[0179] 2.2 Turbidity detection: 200 μl of antibody solution was added to a 96-well plate and incubated at low temperature for 2 hours. After being quickly removed, the plate was shaken on a shaker for 3 minutes. The absorbance was then measured at 350 nm and 550 nm using BioTek Epoch.

[0180] Table 10: Turbidity Detection Results

[0181]

[0182] As shown in Table 10, mutant antibodies can improve the clarity of antibodies at low temperatures.

[0183] The partial amino acid sequences involved in this application are shown in Table 11:

[0184]

[0185] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody for detecting RhD blood group antigen, characterized in that, The antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 are, in order, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:15, and LCDR1, LCDR2, and LCDR3 are, in order, the light chain variable regions LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:24 or 25. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody for detecting RhD blood group antigen, characterized in that, The antibodies include the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: HCDR1 has the amino acid sequence shown in GYFWT (SEQ ID NO:1); HCDR2 has the amino acid sequence shown in EINHSGSTTYNPSLKS (SEQ ID NO:2); HCDR3 has the amino acid sequence shown in SEQ ID NO:3; LCDR1 is the amino acid sequence shown in QGDSLX1X2YYGS, wherein X1 is G and X2 is R, or X1 is R and X2 is S; LCDR2 has the amino acid sequence shown in GKNNRPS (SEQ ID NO:5); LCDR3 has the amino acid sequence shown in NSRDSSGNHRI (SEQ ID NO:6); HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

3. The antibody according to claim 1 or 2, characterized in that, The antibody also includes the framework regions shown in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

4. The antibody according to claim 3, characterized in that, The amino acid sequence of HFR1 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:7; The amino acid sequence of HFR2 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:8; The amino acid sequence of HFR3 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:9; The amino acid sequence of HFR4 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:10; The amino acid sequence of LFR1 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:11; The amino acid sequence of LFR2 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:12; The amino acid sequence of LFR3 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:13; and The amino acid sequence of LFR4 is an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO:

14.

5. An antibody for detecting RhD blood group antigen, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24 or 25.

6. The antibody according to claim 1, 2, or 5, characterized in that, The antibody also includes a constant region.

7. The antibody according to claim 6, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

8. The antibody according to claim 6, characterized in that, The constant region includes the heavy chain constant region and the light chain constant region.

9. The antibody according to claim 8, characterized in that, The heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

10. The antibody according to claim 8, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM, and / or the tail peptide of IgM.

11. The antibody according to claim 8, characterized in that, The constant region of the antibody includes: The heavy chain constant region having at least 80% identity with the sequence shown in SEQ ID NO:16; and the light chain constant region having at least 80% identity with the sequence shown in SEQ ID NO:

19.

12. An antibody for detecting RhD blood group antigen, characterized in that, The antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO:17, and the amino acid sequence of the light chain being as shown in SEQ ID NO:21 or 22.

13. An antibody conjugate, characterized in that, The antibody conjugate comprises: the antibody according to any one of claims 1 to 12; and at least one of biotin, a label, a purification tag, and a solid-phase carrier conjugated to the antibody.

14. The antibody conjugate according to claim 13, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

15. A kit for detecting RhD blood group antigen, characterized in that, The kit comprises the antibody as described in any one of claims 1 to 12 or the antibody conjugate as described in claim 13 or 14.

16. A method for detecting RhD blood group antigen, characterized in that, include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the antibody of any one of claims 1 to 12, the antibody conjugate of claim 13 or 14, or the kit of claim 15 is brought into contact with the sample to be tested to form an immune complex. and b) Detect the presence of the immune complex, the presence of which indicates the presence of the RhD blood group antigen in the test sample; The method described is not intended for the diagnosis or treatment of disease.

17. Use of the antibody of any one of claims 1 to 12, the antibody conjugate of claim 13 or 14, or the kit of claim 15 in the preparation of products for detecting RhD blood group antigens or for the preparation of products for identifying blood types.

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