Antibody for detecting RhD blood group antigen and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of immune diagnosis, and in particular to an antibody for detecting RhD blood group antigens and an application thereof. Background Art

[0002] Blood group antigens are molecular structures present on the surface of red blood cells. Based on their expression, humans are divided into various blood group systems, such as the ABO and Rh systems. 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 has the strongest antigenicity. Therefore, the Rh blood group system most commonly detects RhD antigen.

[0004] The Rh blood type system generally lacks natural antibodies, so the first blood transfusion may not produce adverse reactions. However, after receiving Rh-positive blood, an Rh-negative recipient may develop anti-Rh antibodies. If a subsequent transfusion of Rh-positive blood is given, these antibodies will attack the incoming Rh-positive blood, resulting in a hemolytic transfusion reaction, destroying red blood cells and causing illness or death. If an Rh-negative mother is pregnant with 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 and enter the fetal circulation, causing the fetal red blood cells to agglutinate and destroy them. This can lead to severe anemia in the fetus or even death.

[0005] RhD blood type antigen detection and matching are crucial in medical applications such as blood transfusion, organ transplantation, and blood typing. Currently, the most commonly used blood typing methods include the slide method, test tube method, microcolumn gel method, and solid-phase method. Most hospitals use the slide method, which involves placing a drop of anti-D antibody on a glass slide and then adding the red blood cells to be tested. If the red blood cells contain RhD antigens on their surface, they will agglutinate with the anti-D antibodies, indicating RhD-positive blood. If they do not agglutinate, the blood is RhD-negative.

[0006] The above detection method requires antibodies against RhD blood type antigens. The current anti-RhD blood type antigen antibodies have poor low-temperature stability. They are prone to aggregation or denaturation when stored for a long time at 2-8°C, resulting in a decrease in potency. This forces the addition of high-concentration stabilizers (such as sucrose, trehalose) or reliance on frozen storage, which increases costs and transportation difficulties. Summary of the Invention

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

[0008] To achieve the above objectives, according to a first aspect of the present invention, an antibody for detecting an RhD blood group antigen is provided, wherein the antibody comprises three complementarity determining regions having a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 15 and three complementarity determining regions having a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 18, wherein two consecutive arginine residues in the complementarity determining regions are selectively or independently mutated to uncharged amino acids.

[0009] To achieve the above objectives, according to a second aspect of the present invention, an antibody for detecting an RhD blood group antigen is provided, wherein the antibody comprises 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 an RhD blood group antigen is provided, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein 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: 18; and wherein two consecutive arginine residues in the light chain variable region are either mutated or both are independently mutated to uncharged amino acids.

[0011] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody for detecting an RhD blood group antigen is provided, wherein 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.

[0012] In order to achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0013] To achieve the above object, according to a sixth aspect of the present invention, a kit for detecting RhD blood group antigens is provided, wherein the kit comprises the above-mentioned antibody or antibody conjugate.

[0014] In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a method for detecting RhD blood group antigens, comprising: a) contacting the antibody, antibody conjugate, or kit with a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of RhD blood group antigens in the test sample.

[0015] To achieve the above objectives, according to an eighth aspect of the present invention, there is provided use of the above antibodies, antibody conjugates, or kits in preparing products for detecting RhD blood group antigens or preparing products for identifying blood types. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is an example of a visual observation result of "4+"; Figure 2 This is an example of a visual observation result of "3+"; Figure 3 This is an example of a visual observation result of "2+"; Figure 4 This is an example of a visual observation result of "1+"; Figure 5 This is an example of a “negative” result from naked eye observation; Figure 6 Visual inspection appearance results for stability assessment. DETAILED DESCRIPTION

[0018] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0019] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0020] In the present invention, the term "antibody" is used in the broadest sense and includes full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, and antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. Antibody antigen-binding fragments are molecules comprising the CDRs of an antibody that lack some of the amino acids present in the full-length chain but are still capable of specific binding 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 diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), and minimal antibody recognition units. Antigen-binding fragments of these antibodies are capable of binding to the same antigen as the parent antibody.

[0021] Antigen-binding fragments of antibodies generally possess the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that antigen-binding fragments of these antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain these antigen-binding fragments. Antigen-binding fragments can also be obtained through recombinant genetic techniques known to those skilled in the art, or through synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0022] Herein, 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. The heavy chain constant region is located at the C-terminus of the heavy chain of an antibody molecule. Each heavy chain constant region comprises, from N-terminus to C-terminus, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional). The amino acid sequences and structures of the heavy chain constant regions vary between different antibody types (e.g., IgG, IgA, IgM, etc.), but all share relatively conserved structural features that enable the heavy chain constant region to perform its biological functions. The heavy chain constant regions, 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 the division and sequence identification results of constant region segments may not be completely consistent in different bioinformatics databases or software. 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 region segments that can be identified and divided by those skilled in the art using common knowledge and conventional methods all fall within the scope of protection of the present invention.

[0023] 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, and the starting or ending 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 a sequence of the corresponding segment that is longer or shorter than the reference sequence.

[0024] As used herein, the term "hinge region" refers to the polypeptide connecting the CH1 and CH2 domains in the heavy chain constant region of an antibody. This region is rich in proline and therefore easily stretches and bends, for example, containing at least one proline. The hinge region is typically dimeric, consisting of two polypeptides with the same amino acid sequence. The specific amino acid sequence is not limited and is within the scope of protection of this application. Hinge regions from different species and subclasses are well known.

[0025] Herein, 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 of different species are well known.

[0026] As used herein, the term "IgM tail peptide" refers to a short peptide sequence located at the C-terminus of the CH4 region of an IgM antibody. The specific amino acid sequence is not limited and is within the scope of protection of this application. The IgM tail peptide contains cysteine, which is involved in polymer formation, and can also bind to the J chain to further stabilize the polymer structure.

[0027] In the present invention, the terms "include" and "comprising" are open expressions, that is, they include the contents specified in the present invention, but do not exclude other aspects of the contents.

[0028] In the present invention, the term "optionally" generally means that the subsequently described event or circumstance may but need not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0029] In a first aspect, an embodiment of the present invention provides an antibody for detecting an RHD blood group antigen, wherein the antibody comprises three complementarity determining regions having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 15 and three complementarity determining regions having a light chain variable region with the amino acid sequence shown in SEQ ID NO: 18, wherein two consecutive arginine (Arginine, R, Arg) residues in the complementarity determining regions are selectively mutated or both are independently mutated to uncharged amino acids.

[0030] In an optional embodiment, the uncharged amino acid is selected from glycine (Glycine, G, Gly), alanine (Alanine, Ala), serine (Serine, S, Ser) and threonine (Threonine, T, Thr).

[0031] In an optional embodiment, two consecutive arginine residues in the complementarity determining region are selectively mutated, and the first of the two arginine residues is mutated to glycine.

[0032] In an optional embodiment, two consecutive arginine residues in the complementarity determining region are selectively mutated, and the second arginine residue of the two arginine residues is mutated to serine.

[0033] The mutation of two consecutive arginine residues in the complementary determining region to uncharged amino acids is preferred to mutation to charged amino acids. The charged amino acids are selected from glutamic acid, aspartic acid, lysine, or histidine.

[0034] In an optional embodiment, the aforementioned antibody comprises three complementarity determining regions of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15 and three complementarity determining regions of a light chain variable region having the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 25.

[0035] Furthermore, the antibody has improved RHD blood group antigen binding titer, affinity, and / or specificity.

[0036] It should be understood that the leucine residues and isoleucine residues in the above-mentioned complementarity determining regions can be optionally replaced with each other, and all fall within the scope of protection of the present invention.

[0037] In an optional embodiment, the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems or a combination of multiple systems.

[0038] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0039] In the present invention, the heavy chain complementarity determining region is represented by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2, and HCDR3; the light chain complementarity determining region is represented by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2, and LCDR3. In the present invention, the term "antibody pair" refers to the use of multiple antibodies in pairs, which can be two antibodies, three antibodies, four antibodies, or more, but at least two antibodies.

[0040] The precise amino acid sequence boundaries of the CDRs can be determined by numbering using a number of 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).

[0041] The boundaries of CDRs may vary depending on the definition method. For example, the Kabat definition is based on structural alignment, while the Chothia definition 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 through software (such as Abysis). Table 1 below exemplifies the positions of CDRs determined by the Kabat, Chothia, IMGT, AbM and Contact definitions. There are other CDR definition methods that may not strictly follow one of the above schemes and may shorten or extend them based on predictions or experimental results of specific residues or residue groups. Therefore, other CDRs not limited to those in Table 1 also fall within the scope of protection of the present disclosure.

[0042] Table 1: CDR Definition 1

[0043] 1 All CDR definitions in Table 1 are numbered according to the Chothia numbering scheme (see below), with heavy chain amino acid numbers represented by "H+numbers" and light chain amino acid numbers represented by "L+numbers." One of ordinary skill in the art can unambiguously assign this Chothia numbering scheme to any variable region sequence.

[0044] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems or a combination of multiple systems.

[0045] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0046] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0047] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

[0048] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0049] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0050] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0051] In a second aspect, an embodiment of the present invention provides an antibody for detecting RHD blood group antigens, wherein the antibody comprises the following complementary 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 as EINHSGSTTYNPSLKS (SEQ ID NO: 2); HCDR3 comprising or consisting of the amino acid sequence shown as 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, which comprises or consists of the amino acid sequence shown in GKNNRPS (SEQ ID NO: 5); LCDR3 comprises or consists of the amino acid sequence shown by NSRDSSGNHRI (SEQ ID NO: 6).

[0052] It should be understood that the leucine residues and isoleucine residues in the complementarity determining regions can be optionally replaced with each other, and all fall within the scope of protection of the present invention.

[0053] According to an embodiment of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0054] The antibodies of the present invention also include a framework region. In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding the CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0055] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0056] In an optional embodiment, the antibody of the first aspect or the second aspect further comprises the framework regions shown by HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0057] In an optional embodiment, the above-mentioned HFR1 includes the sequence shown in SEQ ID NO:7 or an amino acid sequence with at least 80% identity thereto; the above-mentioned HFR2 includes the sequence shown in SEQ ID NO:8 or an amino acid sequence with at least 80% identity thereto; the above-mentioned HFR3 includes the sequence shown in SEQ ID NO:9 or an amino acid sequence with at least 80% identity thereto; the above-mentioned HFR4 includes the sequence shown in SEQ ID NO:10 or an amino acid sequence with at least 80% identity thereto; the above-mentioned LFR1 includes the sequence shown in SEQ ID NO:11 or an amino acid sequence with at least 80% identity thereto; the above-mentioned LFR2 includes the sequence shown in SEQ ID NO:12 or an amino acid sequence with at least 80% identity thereto; the above-mentioned LFR3 includes the sequence shown in SEQ ID NO:13 or an amino acid sequence with at least 80% identity thereto; and the above-mentioned LFR4 includes the sequence shown in SEQ ID NO:14 or an amino acid sequence with at least 80% identity thereto.

[0058] As used herein, the term "percent identity" refers to the degree to which amino acids in two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Amino acid sequence identity comparisons can be performed using various methods known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, which are well known in the art.

[0059] In other embodiments, the amino acid sequences of the framework regions of the antibodies provided herein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions described above.

[0060] In a third aspect, an embodiment of the present invention provides an antibody for detecting an RHD blood group antigen, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO: 15, the amino acid sequence of the light chain variable region being shown in SEQ ID NO: 18, and two consecutive arginine residues in the light chain variable region being selectively mutated or both being independently mutated into uncharged amino acids.

[0061] In an alternative embodiment, the uncharged amino acid is selected from glycine, alanine, serine and threonine.

[0062] In an optional embodiment, one of two consecutive arginine residues in the light chain variable region is mutated, and the first of the two arginine residues is mutated to glycine.

[0063] In an optional embodiment, two consecutive arginine residues in the light chain variable region are selectively mutated, and the second arginine residue of the two arginine residues is mutated to serine.

[0064] 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.

[0065] It should be understood that the leucine residues and isoleucine residues in the variable region can be optionally replaced with each other, and all fall within the scope of protection of the present invention.

[0066] In an optional embodiment, the antibody described in the first, second, and third aspects above further comprises a constant region.

[0067] In an alternative embodiment, the above-mentioned constant region includes a heavy chain constant region and / or a light chain constant region.

[0068] In an optional embodiment, the species origin of the above-mentioned constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0069] In an optional embodiment, the heavy chain constant region of the above-mentioned 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 a κ-type or λ-type light chain constant region; the λ-type light chain constant region can be selected from λ1, λ2, λ3, and λ4 subtypes.

[0070] In an optional embodiment, 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.

[0071] In an optional embodiment, the constant region of the above-mentioned 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 that is at least 80% identical to each of the constant regions.

[0072] It should be noted that, in some embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region.

[0073] In a fourth aspect, an embodiment of the present invention provides an antibody for detecting an RHD blood group antigen, wherein 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, the amino acid sequence of the light chain is shown in SEQ ID NO: 20, and two consecutive arginine residues in the variable region of the light chain are selectively mutated or both are independently mutated to uncharged amino acids.

[0074] In an alternative embodiment, the uncharged amino acid is selected from glycine, alanine, serine and threonine.

[0075] In an optional embodiment, two consecutive arginine residues in the variable region of the light chain are selectively mutated, and the first of the two arginine residues is mutated to glycine.

[0076] In an alternative embodiment, two consecutive arginine residues in the variable region of the light chain are selectively mutated, and the second arginine residue of the two arginine residues is mutated to serine.

[0077] 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.

[0078] 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 replaced with each other, and all fall within the scope of protection of the present invention.

[0079] In a fifth aspect, an embodiment of the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the antibody according to the first aspect, the second aspect, the third aspect or the fourth aspect.

[0080] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0081] In an optional embodiment, the above-mentioned antibody conjugate further comprises a marker or purification tag coupled to the antibody.

[0082] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0083] In an optional embodiment, the above-mentioned label is selected from but not limited to metal ions, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, electron-dense labels, adamantane and nanoparticle labels.

[0084] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0085] In an optional embodiment, the fluorescent dye is selected from but not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0086] In an optional embodiment, the enzyme is selected from but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphate glucose deoxidase.

[0087] In an optional embodiment, the radioactive isotope is selected from but not limited to 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 Such as 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.

[0088] In an optional embodiment, the chemiluminescent reagent is selected from but not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lophan and its derivatives, and peroxalate and its derivatives.

[0089] In an optional embodiment, the above-mentioned marker is an acridinium ester.

[0090] In an optional embodiment, the nanoparticle marker is selected from but not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0091] In an optional embodiment, the colloid is selected from but not limited to colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres and latex.

[0092] In an optional embodiment, the colloidal metal is selected from but not limited to colloidal gold, colloidal silver and colloidal selenium.

[0093] In an optional embodiment, the labeled antibody is labeled by binding to a labeling substance.

[0094] It should be noted that after the antibody is labeled with a marker to become a labeled antibody, it neither affects the characteristics of the antibody immune response nor changes the activity of the marker itself.

[0095] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.

[0096] In an optional embodiment, the solid phase carrier is selected from microspheres, latex particles, microfluidic chips, magnetic beads, microplates or nitrocellulose membranes.

[0097] In an optional embodiment, the solid phase carrier is a magnetic bead.

[0098] In an optional embodiment, the above-mentioned antibody conjugate further includes a binding partner conjugated to the antibody.

[0099] In an alternative embodiment, the binding partner comprises biotin / avidin or a biotin derivative / avidin derivative.

[0100] In the present invention, the term "binding partners" refers to a pair of molecules that can bind to each other through non-covalent interactions. This binding is usually specific and can be temporary or form a more stable complex.

[0101] In the present invention, the binding partner can be bound to the label or solid phase through various binding forms such as physical adsorption, electrostatic adsorption or covalent binding. Such binding / conjugation methods are application methods known to those skilled in the art.

[0102] In a sixth aspect, an embodiment of the present invention provides a kit for detecting RHD blood group antigens, wherein the kit comprises the above-mentioned antibody or antibody conjugate.

[0103] It should be noted that the meaning of the kit in this application can be considered equivalent to the meaning of the reagent.

[0104] In an optional embodiment, the kit further comprises at least one of a sample pretreatment reagent (such as a sample purification and enrichment reagent, a lysis solution, etc.), a cleaning solution (such as water, etc.), a buffer solution (such as PBS or Tris, etc.), a stabilizing solution, and a colorimetric reagent for the signal substance.

[0105] The antibody conjugates or antibodies described in certain embodiments or examples of the present invention are capable of binding to RHD blood group antigens. Therefore, kits containing such antibody conjugates or antibodies are capable of effectively performing qualitative or quantitative detection of RHD blood group antigens. The kits provided herein can be used, for example, in immunoagglutination, immunochromatography, immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of RHD blood group antigens and antibodies thereto. As previously described, the antibodies of the present invention exhibit improved RHD blood group antigen binding potency, affinity, stability, and / or specificity. Therefore, kits containing such antibodies exhibit improved detection sensitivity and specificity and / or reduce missed detections.

[0106] In some embodiments, the antibodies of the present invention may be monoclonal antibodies or polyclonal antibodies.

[0107] In alternative embodiments, the antibody conjugates or antibodies of the present invention can be prepared using methods known in the art.

[0108] In an alternative embodiment, the antibodies of the present invention can be prepared by immunogen immunization and / or chemical synthesis.

[0109] In alternative embodiments, the effects of the antibody pairs or antibodies of the invention, such as binding activity and / or cross-reactivity, can be tested using any appropriate in vitro assays, cell-based assays, in vivo assays, animal models, and the like.

[0110] In alternative embodiments, the assay can include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, and the like.

[0111] In alternative embodiments, the kits of the invention include reagents suitable for performing an immunoassay.

[0112] In alternative embodiments, the kit of the present invention can be used to perform immunoassays, such as ELISA, indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), and other non-enzyme-linked antibody binding tests or methods.

[0113] In a seventh aspect, an embodiment of the present invention provides a method for detecting RHD blood group antigens, comprising: a) contacting the antibody, antibody conjugate or reagent with a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of RHD blood group antigens in the test sample.

[0114] In an optional embodiment, the method is a slide method, a test tube method, a microcolumn gel method, or a solid phase method.

[0115] To achieve the above objectives, according to an eighth aspect of the present invention, there is provided use of the above antibodies, antibody conjugates or reagents in detecting RHD blood group antigens or preparing products for detecting RHD blood group antigens or preparing products for identifying blood types.

[0116] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0117] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Colligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0118] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0119] Based on extensive and innovative research on RHD blood type antibodies, the inventors discovered that RHD blood type antibodies without the mutations described in this application are prone to precipitation during storage at low temperatures (e.g., 2-8°C). Through charge analysis and structural modeling of the antibodies, they discovered that two concentrated arginine residues in the antibody light chain sequence may contribute to excessive local positive charge. Consequently, they designed a mutant RHD blood type antibody. The preparation, activity characterization, and performance testing of the antibodies are demonstrated in the Examples.

[0120] Example 1. Preparation of Antibodies In this example, 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. Primer synthesis and gene sequencing were performed by Invitrogen. The cell line secreting wild-type RhD antibody was a pre-existing cell line in our company and was revived for future use.

[0121] 1. Amplification of Antibody Genes 1) Wild-type antibodies mRNA was extracted from a cell line secreting the parental Anti-RhD monoclonal antibody. DNA products were obtained by RT-PCR. This product was then inserted into the pMD-18T vector after A-addition using rTaq DNA polymerase and transformed into DH5α competent cells. After colonies grew, heavy and light chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing. The resulting gene sequences were analyzed against the Kabat antibody database and analyzed using VNTI 11.5 software to confirm that the heavy and light chain primer pairs correctly amplified the antibody variable region genes.

[0122] Restriction endonuclease cleavage sites were introduced into the eukaryotic expression vector, hereinafter referred to as the 3.4A expression vector. Based on the above antibody variable region gene sequencing results, gene-specific primers for the light chain variable region and heavy chain variable region of the antibody were designed, with restriction sites and protective bases at both ends, respectively. The variable region and constant region were connected by Overlap PCR amplification method, thereby amplifying the complete light chain gene and heavy chain gene of the wild-type antibody.

[0123] 2) Chimeric antibodies Upstream and downstream primers were designed for the CH1-hinge region (ASTKGPSVFPL-EPKSC) of human IgG1 antibodies, the CH2-tail peptide (PVIAELPPKVSV-MSDTAGTCY) of human IgM antibodies, and the heavy chain variable region, respectively. These primers, each with restriction endonuclease sites and protected bases at both ends, were used to generate the complete heavy chain gene of the chimeric antibody by overlap PCR. The complete light chain gene of the chimeric antibody is identical to that of the wild-type antibody.

[0124] 3) Mutant antibodies Mutation points were designed for the light chain of the chimeric antibody, and primers containing the desired mutation points were generated. These primers were flanked by restriction endonuclease sites and protective bases. Overlap PCR was then performed to obtain the complete light chain gene for the mutant antibody. The complete heavy chain gene for the mutant antibody was the same as that for the chimeric antibody.

[0125] 2. Construction of recombinant antibody expression plasmid The heavy chain and light chain genes of the above antibodies and the 3.4A vector were digested with restriction endonucleases. After the gene fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were respectively connected to the 3.4A expression vector to obtain recombinant expression plasmids for the heavy chain and light chain, respectively.

[0126] 3. Recombinant Antibody Production Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3~5 × 10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 3.4×10 6 cells / ml, wash the cells, and re-dissolve them with culture medium. At the same time, use the culture medium to prepare plasmid DNA and transfection reagent diluents respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well, and let it stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well, take samples and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a speed of 120 rpm and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The supernatant of the centrifugation was affinity purified using a protein A affinity chromatography column to obtain purified antibodies. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table: Table 2: Antibody sequences

[0127] Example 2: HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography) Purity Identification Take 20 μg of purified antibody and determine the sample purity by SEC according to the conditions in Table 3 below.

[0128] Table 3: Chromatographic conditions for HPLC-SEC

[0129] The yield and purity are shown in Table 4.

[0130] Table 4: Yield and purity

[0131] The results showed that the expression level, assembly efficiency and purity of the mutant antibody were better than those of the wild-type antibody and chimeric antibody.

[0132] Example 3: Antibody performance testing 1. Anti-RhD blood group antigen antibody titer detection Antibody dilution: All antibodies were diluted to 0.3 mg / ml with PBS. Based on 0.3 mg / ml, the antibodies were diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, 512-fold, and 1024-fold to serve as RhD blood group antibody working solution.

[0133] Take a 5 ml test tube and add 50 μL of RhD red blood cell suspension.

[0134] Add 100 μl of the RhD blood group antibody working solution diluted in step 1) and mix immediately.

[0135] Centrifuge at 900 g for 15 s in an Eppendorf centrifuge.

[0136] 5) After removing the test tube, gently tap the bottom 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 the mutant and chimeric antibodies are superior to those of the wild-type antibody. The mutant antibody can effectively bind to the RhD blood group antigen at low concentrations.

[0137] Table 5: Anti-RhD blood group antigen antibody titer

[0138] Table 6: Reaction result evaluation criteria (this evaluation criteria is applicable to the result evaluation in all examples in this application)

[0139] Results: Using 4+ as the standard, the antibody titer was calculated using the following formula: 1 / ((original antibody concentration / 0.3) × dilution factor); The wild-type antibody titer is: 1:64; The titer of chimeric antibody is: 1:256; Mutant 1 titer: 1:256; The titer of mutant 2 is: 1:256.

[0140] 2. Stability assessment 2.1 Visually inspect the appearance. Pour the antibody solution into a transparent vial and incubate at 4°C overnight. Remove the vial, quickly invert it upside down three times, and place it in the corresponding tube rack. Visually inspect the appearance of the antibody solution and take photos to record the actual appearance.

[0141] See Figure 6From the appearance, there are obvious precipitations of wild-type antibodies and chimeric antibodies, while the mutant antibody state is clear.

[0142] 2.2 Turbidity detection: 200 μl of antibody solution was added to a 96-well plate and incubated at low temperature for 2 hours. The plate was quickly taken out and shaken on a shaker for 3 minutes. The absorbance was then measured at 350 nm and 550 nm using a BioTek Epoch.

[0143] Table 10: Turbidity test results

[0144] From the results in Table 10, it can be seen that the mutant antibody can improve the clarity of the antibody at low temperature.

[0145] Some of the amino acid sequences involved in this application are shown in Table 11:

[0146] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be 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 three complementarity determining regions having a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 15 and three complementarity determining regions having a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 18, wherein two consecutive arginine residues in the complementarity determining regions are either mutated or both are independently mutated to uncharged amino acids; Optionally, the uncharged amino acid is selected from glycine, alanine, serine and threonine; Optionally, the complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems or a combination of multiple systems.

2. An antibody for detecting RhD blood group antigen, characterized in that The antibody comprises 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 as EINHSGSTTYNPSLKS (SEQ ID NO: 2); HCDR3 comprising or consisting of the amino acid sequence shown as 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, which comprises or consists of the amino acid sequence shown in GKNNRPS (SEQ ID NO: 5); LCDR3 comprises or consists of the amino acid sequence shown by NSRDSSGNHRI (SEQ ID NO: 6).

3. The antibody according to claim 1 or 2, characterized in that The antibody further comprises the framework regions represented by HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, the HFR1 comprises the sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises the sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises the sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises the sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises the sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises the sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and The LFR4 comprises the sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. 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: 18; two consecutive arginine residues in the light chain variable region are mutated one by one or both independently to uncharged amino acids; Optionally, the uncharged amino acid is selected from glycine, alanine, serine and threonine.

5. The antibody according to any one of claims 1 to 4, characterized in that The antibody further comprises a constant region; Optionally, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one 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 a κ-type or λ-type light chain constant region; Optionally, 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; Optionally, the constant region of the antibody comprises: The heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 16; and the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 19; or an amino acid sequence that is at least 80% identical to each of the constant regions.

6. 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 is shown in SEQ ID NO: 17, the amino acid sequence of the light chain is shown in SEQ ID NO: 20, and two consecutive arginine residues in the variable region of the light chain are either mutated or both are independently mutated to uncharged amino acids; Optionally, the uncharged amino acid is selected from glycine, alanine, serine and threonine.

7. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody according to any one of claims 1 to 6; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a marker or purification tag coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

8. A kit for detecting RhD blood group antigen, characterized in that: The kit comprises the antibody according to any one of claims 1 to 6 or the antibody conjugate according to claim 7.

9. A method for detecting RhD blood group antigen, characterized in that: include: a) contacting the antibody according to any one of claims 1 to 6, the antibody conjugate according to claim 7, or the kit according to claim 8 with a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of RhD blood group antigens in the test sample.

10. Use of the antibody according to any one of claims 1 to 6, the antibody conjugate according to claim 7, or the kit according to claim 8 in the preparation of a product for detecting RhD blood group antigens or a product for identifying blood types.

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