An anti-idiotypic antibody for detecting and neutralizing anti-D antibody and its uses

Abstract

The present invention discloses an anti-idiotypic antibody for detecting and neutralizing anti-D antibody and its uses, including a heavy chain variable region and a light chain variable region. The above heavy chain variable region contains HCDR1, HCDR2 and HCDR3 sequences, wherein: the amino acid sequence of the above HCDR1 has the amino acid sequence shown in SEQ ID No.1; the amino acid sequence of the above HCDR2 is the amino acid sequence shown in SEQ ID No.2; the amino acid sequence of the above HCDR3 is the amino acid sequence shown in SEQ ID No.3. This anti-idiotypic antibody can specifically bind to anti-D antibody and can be applied to clinical blood group detection or the prevention or treatment of anti-D antibody-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an anti-idiotypic antibody for detecting and neutralizing anti-D antibody and its use. Background Art

[0002] The Rh blood group system is the most complex one among the human red blood cell blood group systems, and its importance is second only to the ABO blood group system. The D antigen has the strongest immunogenicity among all antigens of the Rh blood group system, which means it is most likely to cause an immune response in the body. Studies have found that Rh-negative individuals may produce anti-D when they are first transfused with more than 2 ml of Rh-positive red blood cells. In recent years, studies have shown that some D variant individuals with incomplete expression of the D antigen due to RHD gene mutation will also produce anti-D under the stimulation of the D antigen due to the deletion of D antigen epitopes; among the population, the relatively common weak D15 type, RHD-CE(2-9)-D fusion type, DVI-III type, etc., all have literature reports of producing anti-D. Anti-D is mainly an IgG-type immune antibody, which can be produced through immune pathways such as blood transfusion or pregnancy. Anti-D can cause acute or chronic hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN). Therefore, in clinical work, accurate detection of anti-D is the basic guarantee for blood transfusion safety and also the basis for guiding the prevention, diagnosis and treatment of Rh-HDFN.

[0003] At present, the method for detecting IgG-type anti-D is the indirect antiglobulin test. The principle is to use an anti-Fc second antibody as a mediator to cause agglutination of red blood cells bound with IgG antibodies. It has the disadvantages of cumbersome operation and being greatly affected by the technical level of operators. The concentration of anti-D is usually detected by the indirect antiglobulin test using the serial dilution method, expressed as titer or dilution ratio, and it is unable to accurately detect the amount of anti-D, also having the disadvantage of rough results.

[0004] Currently, the method used for detecting red blood cell group antibodies is the immuno - serological method, whose principle is to detect unknown antibodies with known antigens. Since it is currently impossible to obtain a single, purified red blood cell group antigen, only red blood cells carrying multiple antigens can be selected for antibody detection, and the specificity of unexpected antibodies is determined according to a certain reaction pattern. This common method is restricted by the technical operation level of laboratory personnel and methodology, and is more dependent on the panel red blood cells. Whether the antigen spectrum of the panel red blood cells is comprehensive and whether antigens fall off during storage both affect the accuracy of experimental results, threatening the safety and effectiveness of clinical blood transfusion. For patients who produce unexpected blood group antibodies, to eliminate the influence of unexpected antibodies, only blood with corresponding negative antigens can be taken during blood transfusion treatment. In emergency situations, due to this limitation, compatible blood may not be found, delaying the treatment of patients; for common Rh - negative pregnant women who produce anti - D, if the anti - D titer is high, the risk and severity of HDFN increase, often resulting in stillbirth and miscarriage in the early pregnancy. If the anti - D in the pregnant woman's body can be reduced and the high - risk period in the early pregnancy can be passed, it can create opportunities for treatment such as intrauterine blood transfusion in the middle and late pregnancy.

[0005] To solve the above problems, an "antigen" that can bind to the Fab region of anti - D is needed. Since the RhD protein is a transmembrane protein, in addition to transmembrane folding, it also requires interaction with Rh - related proteins to form a spatial conformation. If the protein is obtained by extraction from the red blood cell membrane, the quaternary structure of the RhD protein will be lost and the RhD antigenicity will also disappear; similarly, when synthesizing proteins through genetic engineering, only amino acid chains without RhD antigenicity can be obtained. Summary of the Invention

[0006] The object of the present invention is to provide an anti - idiotype antibody for detecting and neutralizing anti - D antibodies and its uses. This anti - idiotype antibody can specifically bind to anti - D antibodies and can be applied to clinical blood group detection, or for preventing or treating anti - D antibody - related diseases.

[0007] The present invention adopts the following technical scheme: An anti - idiotype antibody for detecting and neutralizing anti - D antibodies, including a heavy - chain variable region and a light - chain variable region. The above - mentioned heavy - chain variable region contains HCDR1, HCDR2, and HCDR3 sequences, where:

[0008] The amino acid sequence of the above - mentioned HCDR1 is the amino acid sequence shown in SEQ ID No.1;

[0009] The amino acid sequence of the above - mentioned HCDR2 is the amino acid sequence shown in SEQ ID No.2;

[0010] The amino acid sequence of the above - mentioned HCDR3 is the amino acid sequence shown in SEQ ID No.3.

[0011] Furthermore, the light chain variable region contains any one of the LCDR1, LCDR2, and LCDR3 sequences, where:

[0012] The amino acid sequence of the above-mentioned LCDR1 is the amino acid sequence shown in SEQ ID No. 4;

[0013] The amino acid sequence of the above-mentioned LCDR2 is the amino acid sequence shown in SEQ ID No. 5;

[0014] The amino acid sequence of the above-mentioned LCDR3 is the amino acid sequence shown in SEQ ID No. 6.

[0015] The above SEQ ID No. 5 is as follows: LVS.

[0016] Furthermore, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 7; the amino acid sequence of the above-mentioned light chain variable region is as shown in SEQ ID No. 8.

[0017] Furthermore, the anti-idiotypic antibody is a murine antibody.

[0018] Furthermore, the constant region of the heavy chain of the anti-idiotypic antibody is the heavy chain constant region of murine IgM, IgG1, IgG2a, IgG2b, or IgG3, and the constant region of the light chain of κ or λ type.

[0019] The present invention also discloses the use of the above-mentioned anti-idiotypic antibody for detecting and neutralizing anti-D antibody, the use of the anti-idiotypic antibody in the preparation of a kit for detecting the content of anti-D antibody in serum; or the use in the preparation of a kit for neutralizing anti-D antibody.

[0020] The present invention also discloses the use of the above-mentioned anti-idiotypic antibody for detecting and neutralizing anti-D antibody, its use in the preparation of drugs for anti-D antibody-related diseases.

[0021] The beneficial effects of the present invention are: the anti-idiotypic antibody has high affinity for anti-D antibody, can block the erythrocyte agglutination mediated by anti-D antibody, can be used to detect various blood group unexpected antibody mixed samples containing anti-D, and can also be used for the risk assessment and clinical diagnosis and treatment of Rh-HDFN, which has important application value for improving clinical blood transfusion safety and disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a test diagram for the preparation of human anti-D antibody and the preparation of immunogen by pepsin digestion; where: A is the SDS-PAGE electrophoresis diagram before and after protein purification of the crude product of the anti-D antibody red blood cell eluate; B is the SDS-PAGE electrophoresis diagram of the product before and after pepsin digestion of the anti-D antibody.

[0023] Figure 2 It is a graph for measuring the serum antibody titer of anti-D-F(ab’)2 protein immunized mice.

[0024] Figure 3 It is a graph for screening anti-idiotypic antibodies by enzyme-linked immunosorbent assay.

[0025] Figure 4 It is an SDS-PAGE electrophoresis graph of anti-idiotypic antibody; wherein: M is the prestained protein Marker; 1 represents the 153# anti-idiotypic antibody.

[0026] Figure 5 It is a fitting graph of anti-idiotypic antibody detected by Fortebio.

[0027] Figure 6 It is a graph of the neutralizing effect of anti-idiotypic antibody on the erythrocyte agglutination mediated by different anti-D antibodies; wherein: a is the neutralizing effect of anti-idiotypic antibody on the erythrocyte agglutination mediated by different anti-D antibodies, selecting Shanghai anti-D antibody and Millipore anti-D antibody; b is the neutralizing effect of anti-idiotypic antibody on the erythrocyte agglutination mediated by anti-D serum sample, selected from human anti-D antibody.

[0028] Figure 7 It is the identification of recombinant anti-idiotypic antibody; wherein A is the SDS-PAGE electrophoresis graph of anti-idiotypic antibody; B. The erythrocyte deagglutination experiment of recombinant anti-idiotypic antibody. Detailed implementation mode

[0029] The present invention will be described in detail below in conjunction with the accompanying drawings and specific implementation modes.

[0030] An anti-idiotypic antibody for detecting and neutralizing anti-D antibody and its use in the present invention. The term "antibody" refers to an immunoglobulin molecule containing four polypeptide chains, namely two heavy chains H and two light chains L interconnected by disulfide bonds, and its polymers, such as IgM. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. The heavy chain variable region is abbreviated as VH, and the heavy chain constant region is abbreviated as CH. The heavy chain constant region contains three domains, namely CH1, CH2 and CH3. Each light chain contains a light chain variable region and a light chain constant region. The light chain variable region is abbreviated as VL, and the light chain constant region is abbreviated as CL. The light chain constant region contains one domain CL1. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions, written as CDR, which are interspersed with conserved regions called framework regions FR. The framework region is written as FR. From the N-terminus to the C-terminus, the light and heavy chain variable domains both contain FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0031] The term "antigen-binding portion" of an antibody refers to the part or region of the intact antibody molecule responsible for binding the antigen. The antigen-binding portion can include the heavy chain variable region, the light chain variable region, or both. The antigen-binding portion of an antibody can be prepared from the intact antibody molecule using any suitable standard techniques, including proteolytic digestion or recombinant genetic engineering techniques, etc. Non-limiting examples of antigen-binding portions include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and minimal recognition units composed of amino acid residues mimicking the hypervariable regions of antibodies, such as isolated CDRs. The term "antigen-binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetra-bodies, and minibodies, etc. For example, the Fd fragment in the present invention refers to the antibody fragment composed of VH and CH1 domains; the Fv fragment is composed of VL and VH domains in a single arm of the antibody; the dAb fragment is composed of the VH domain.

[0032] Complementary determining regions are the regions in the variable region that have the greatest influence on the affinity and specificity of the antibody. CDRs usually include CDR1, CDR2, and CDR3; there are two common ways to define the CDR sequences of VH or VL, namely the Kabat definition and the Chothia definition. The CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the present invention, the Kabat definition is used to define the CDR sequences. In the present invention, CDR1, CDR2, and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2, and HCDR3, respectively; CDR1, CDR2, and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2, and LCDR3, respectively.

[0033] For the variable region sequence of a given antibody, the CDR region sequences in the variable region sequence can be analyzed in various ways, for example, it can be determined using the online software Abysis.

[0034] In the present invention, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope, for example, the ability of an antibody to bind to a specific antigen with an affinity at least twice as great as its affinity for a non-specific antigen. However, it should be understood that an antibody can specifically bind two or more antigens related to its sequence. For example, the antibody in the present invention can specifically bind to human anti-D antibody.

[0035] In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody constituting the population is identical except for possible naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided that they exhibit the desired biological activity.

[0036] In the present invention, the term "murine antibody" refers to any antibody in which all domain sequences are murine sequences. Such antibodies can be produced by hybridomas. The term "anti-D antibody-related disease" includes diseases and / or disorders associated with anti-D antibodies. Anti-D antibody-related diseases or disorders include one or more of hemolytic disease of the fetus and newborn.

[0037] In the present invention, the term "treatment" refers to any type of intervention or method performed on a subject or the administration of an active agent to the subject, wherein the aim is to reverse, alleviate, improve, inhibit or mitigate or prevent symptoms, complications, conditions or the progression, development, severity or recurrence associated with a disease. The term "prevention" refers to the administration to a subject not suffering from a disease to prevent the occurrence of the disease or, if present, to minimize its impact.

[0038] The present invention discloses an anti-idiotypic antibody for detecting and neutralizing anti-D antibodies. The anti-idiotypic antibody prepared against the antigenic determinant of the variable region can specifically bind to the Fab region of anti-D antibodies and can act as a mimic of the antigen, and is used for detecting the content of anti-D antibodies in serum; or for neutralizing anti-D antibodies. It comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises at least one of the HCDR1, HCDR2 and HCDR3 sequences, wherein:

[0039] The amino acid sequence of the HCDR1 is shown as SEQ ID No.1: GYSFTGYY.

[0040] The amino acid sequence of the HCDR2 is shown as SEQ ID No.2: VNPTNAGT.

[0041] The amino acid sequence of the HCDR3 is shown as SEQ ID No.3: ARDGYYVENYAMDY.

[0042] The light chain variable region comprises at least one of the LCDR1, LCDR2 and LCDR3 sequences, wherein:

[0043] The amino acid sequence of the LCDR1 is shown in SEQ ID No.4: QSLLDSGGKTY.

[0044] The amino acid sequence of the LCDR2 is shown in SEQ ID No.5: LVS.

[0045] The amino acid sequence of the LCDR3 is shown in SEQ ID No.6: WQGTHFPQT.

[0046] The anti-idiotypic antibody is a murine antibody, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.7:

[0047] EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYIHWVRQSHGKSLEWIGR VNPTNAGTFYNQKFKGKAILTVDKSSSTAYMELRSLTSEDSAVYYCARDGYYV ENYAMDYWGQGTSVTVSS. That is, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (121aa).

[0048] The amino acid sequence of the light chain variable region is shown in SEQ ID No.8: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSGGKTYLNWLFQRPGQSPKRLIY LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGT KLEIK. That is, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (112aa).

[0049] The binding portion of the anti-idiotypic antibody is selected from: F(ab’)2 fragment

[0050] The above anti-D antibody is a primate anti-D antibody; preferably, the primate anti-D antibody is selected from human anti-D antibody or monkey anti-D antibody.

[0051] The anti-idiotypic antibody is a murine antibody. The constant region of the heavy chain of the anti-idiotypic antibody is the heavy chain constant region of murine IgM, IgG1, IgG2a, IgG2b or IgG3, and the kappa or lambda light chain constant region.

[0052] As a specific embodiment, the present invention provides a murine anti-idiotypic antibody No. 153# for detecting and neutralizing anti-D antibody. 153# is an artificial number.

[0053] In the present invention, hybridoma cells were screened, and the antibodies secreted by them can bind to anti-D antibody proteins. In the present invention, the idiotype and subtype of the anti-idiotype antibody can be determined using antigens specific to specific antibody types and subtypes, and the mimic RhD antigen isotype can be measured using an ELISA assay.

[0054] An anti-idiotype antibody for detecting and neutralizing anti-D antibodies in the present invention further comprises a murine antibody constant region. The murine antibody constant region includes the heavy chain constant regions of murine IgM, IgG1, IgG2a, IgG2b or IgG3, and the light chain constant region of the κ or λ type. An anti-idiotype antibody for detecting and neutralizing anti-D antibodies is a primate anti-D antibody, and the primate anti-D antibody is selected from human anti-D antibodies or monkey anti-D antibodies.

[0055] The present invention also discloses a method for preparing an anti-idiotype antibody for detecting and neutralizing anti-D antibodies, comprising the following steps: culturing host cells under expression conditions to express an anti-idiotype monoclonal antibody of anti-D antibodies; separating and purifying the expressed anti-idiotype monoclonal antibody of anti-D antibodies. Based on the affinity purification of the anti-idiotype antibody of anti-D antibodies, such as non-denaturing gel purification, HPLC or RP-HPLC, purification on a protein A column, the anti-idiotype monoclonal antibody of anti-D antibodies is purified into a substantially homogeneous substance, for example, a single band on SDS-PAGE electrophoresis.

[0056] Example 1

[0057] Purification of anti-D antibodies: The crude product of the anti-D antibody eluate from red blood cells was affinity-purified with Protein A, and the anti-D antibody was obtained by elution. The harvested antibody molecules were identified by SDS-PAGE electrophoresis, and the molecular weight was as expected, as shown in Figure 1 Figure A. The anti-D antibody was ultrafiltered and replaced with a solvent into a 50 mM citric acid buffer at pH 3.0, digested with pepsin at 37°C for 2 h, and purified with Mabsure LX to remove the Fc fragment and intact undigested protein. The flow-through was collected and passed through a Capto L purification column to remove pepsin and other antibody fragments, and the F(ab')2 protein was obtained by elution. The harvested protein was identified by SDS-PAGE electrophoresis. As can be seen from Figure 1 Figure B, the reduced molecular weight of the target protein F(ab')2 segment was 25 kD, and the non-reduced molecular weight was about 100 kD, which was as expected and could be used for subsequent immunization experiments.

[0058] Example 2

[0059] Preparation of hybridomas of anti-idiotype antibodies:

[0060] I. Female Balb / c mice (Beijing Huafukang Bioscience Co., Ltd.) at 6 - 8 weeks old were subcutaneously injected with anti-D-F(ab')2 protein fully emulsified with an adjuvant, 50 μg / dose / animal, and repeated three times. After the mice were subcutaneously immunized three times, 150 μl of blood was collected from the orbital cavity, and the serum was obtained after centrifugation. The ELISA plates were coated with anti-D-F(ab')2 protein at a concentration of 1 μg / ml, and the antibody titers in the mouse sera were detected by ELISA. The mouse sera were diluted starting from 1:4000, with five dilutions of 1:3, using PBS as the negative control, and the optical density was detected at a wavelength of 450 nm with an ELISA reader. As Figure 2 can be seen, the immune effects of the three mice were comparable, and the abundances of the serum antibody titers from high to low were No. 1, No. 2, and No. 3.

[0061] II. Before fusion, the mice were intraperitoneally injected with a boost of 50 μg of anti-D-F(ab')2 protein for three consecutive days. One day before fusion, peritoneal macrophages from ordinary Kunming mice (Beijing Huafukang Bioscience Co., Ltd.) were taken as the feeder layer and inoculated into 96-well plates. The spleens of the immunized mice were fused with the non-secreting myeloma cell line SP2 / 0, and the hybridoma cells were added to the 96-well plates with the feeder layer, and HAT selection was performed on the fused hybridoma cells.

[0062] III. After the hybridoma cells had grown in HAT medium for 12 days, 50 μl of the hybridoma supernatant was taken, and the antibody titer in the supernatant was detected by ELISA, with positive, negative, and blank controls set. All positive clones were pooled, and after changing the HAT culture medium for two days, ELISA screening was performed again, and the clones that were positive in both tests were taken.

[0063] Example 3

[0064] Screening of anti-idiotypic antibodies: Screening was performed by measuring OD620 with an ELISA reader. The specific method was as follows: One 96-well ELISA plate was taken, 75 μl of the hybridoma supernatant and 5 μl of anti-D antibody were added and incubated at room temperature for 10 min, then 20 μl of type O red blood cells were added, and the reaction system was kept at 100 μl. A positive control for agglutination without the hybridoma supernatant and a negative control for the secondary antibody were set. After mixing, the mixture was incubated in an incubator at 37°C for 0.5 h. The red blood cells were washed with physiological saline, centrifuged at 200 g for 5 min, and the washing was repeated twice. 100 μl of anti-human IgG secondary antibody was added, and after centrifugation at 300 g for 5 min, it was gently shaken, and the agglutination state of the red blood cells was visually observed, and the optical density value was measured at OD620 with an ELISA reader. The results were as Figure 3 shown. The secondary antibody could not cause agglutination of type O red blood cells, and the red blood cells were evenly distributed in the wells of the plate; in the positive agglutination wells, the red blood cells aggregated on the side walls of the wells, resulting in increased light transmittance in this well. The target antibody was the clone with low light transmittance and high OD620 value, and the wells marked in red were the candidate target clones. After multiple screenings of the hybridoma cells, candidate clones were obtained.

[0065] The candidate hybridoma cells were subcloned by the limiting dilution method. The feeder cells were pre-plated in a 96-well cell culture plate to provide nutritional support for the growth of monoclonal hybridoma cells. After centrifugation of the candidate hybridoma cells in the exponential growth phase, the cell culture medium was adjusted in concentration so that the number of cells per well was 0.8 cells / well. When the hybridoma cells formed clones, positive clones were screened by the ELISA method, and monoclonal hybridoma cells were obtained.

[0066] Example 4

[0067] Determination of the affinity between the anti-idiotype antibody and the antigen:

[0068] Preparation of the anti-idiotype antibody: After the hybridoma cells were amplified and cultured, the cell concentration was adjusted to 2e6 / ml with PBS and injected into the abdominal cavity of male 6-8-week-old Balb / c mice, 1 ml was injected into the abdominal cavity of each mouse, and ascites was collected on the 10th day. After centrifugation of the ascites, the supernatant was filtered through a 0.45 μM filter to remove impurities, and the antibody in the supernatant was purified by Protein A. SDS-PAGE identification of the purified antibody showed that the molecular weights of the heavy and light chains of the antibody were correct. It was Figure 4 known that the antibody purity was greater than 95%.

[0069] The Fortebio method was used to quantitatively detect the affinity between the anti-idiotype antibody and the immunogen. An AMC probe was used to bind to the Fc segment of the anti-idiotype antibody to detect its affinity and dissociation with the immunogen anti-D-F(ab')2. The ForteBio Octetred 96e molecular interaction analysis system is an advanced non-labeled and real-time monitoring technology mainly used for the comprehensive quantitative analysis of the interactions between biomolecules. An AMC probe was used to bind to the Fc segment of the anti-idiotype antibody to detect its affinity and dissociation with the immunogen anti-D-F(ab')2. It was Figure 5 known that the anti-idiotype antibody could bind to the anti-D-F(ab')2 molecule, had a strong binding force with the anti-D antibody, hardly dissociated, the dissociation curve was flat, and the affinity (KD) calculated by fitting was less than 1.0E-12.

[0070] Example 5

[0071] Experiment on the neutralizing effect of the anti-idiotype antibody on erythrocyte aggregation:

[0072] The anti-D antibodies were selected from two sources: IgG-D and IgM / G-D. IgG-D was selected from Shanghai Blood Biopharmaceutical Co., Ltd., with the national medical device registration approval number: 20223401104, and IgM / G-D was selected from Millipore Corporation, Clones: TH-28 / MS-26. Take a 96-well plate, add the anti-idiotypic antibody and the anti-D antibody, with the usage amount being 5 μl or 10 μl, and the final concentration of the anti-idiotypic antibody being 500 μg / ml. Incubate at room temperature for 10 min, then add 20 μl of type O red blood cells, and keep the reaction system at 100 μl. Set up a red blood cell blank control and a secondary antibody negative control. After mixing, incubate in an incubator at 37 °C for 0.5 h. Wash the red blood cells with 200 μl of normal saline and centrifuge at 200 g for 5 min, and repeat the washing 2 times. Add 100 μl of anti-IgG secondary antibody, centrifuge at 300 g for 5 min, and then shake. In order to more intuitively present the state of red blood cell agglutination, use the automatic scanning and photographing function of the Elisapot instrument to display the state of red blood cell agglutination. As can be seen from Figure 6 a in the figure, compared with the secondary antibodies 5C and 5D, both 5 μl or 10 μl of the anti-D antibody can cause significant red blood cell agglutination effects 5A, 5B and 5G, 5H; among them: "5" represents the fifth column in the figure; A, B, C, D, G and H represent the row numbers in sequence. Taking 5A as an example, it represents the picture at the intersection of the "5" column and the A row.

[0073] According to Figure 6 the results observed in 1D, 2D, 3D, and 4D, the anti-idiotypic antibody has a neutralizing effect on Shanghai IgG-D; according to the results of 6D and 7D, it has no antagonistic effect on IgM / G-D of Millipore Corporation, indicating that the anti-idiotypic antibody prepared by the present invention has selective neutralizing activity.

[0074] In order to further verify whether the obtained anti-idiotypic antibody has a de-agglutination effect on clinical serum samples, the Dorner1 serum sample with a relatively high anti-D antibody titer was selected for detection, and the usage concentration of the 153# anti-idiotypic antibody was 500 μg / ml. As can be seen from Figure 6Shown in Figure b, where columns 1 and 2 are the anti-D serum group, and columns 3 and 4 are the neutralizing anti-idiotypic antibody intervention group. The anti-D serum was serially diluted from row A to row H, and the dilution ratios were 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280 in sequence. Compared with the secondary antibodies 5A and 5B, after serial dilution of columns 1 and 2 of the anti-D serum, the serum antibodies caused varying degrees of agglutination of RBCs. As the serum concentration decreased, the RBC agglutination gradually weakened. Among them: "5" represents the fifth column in the figure; A, B, C, D, G, and H represent the row numbers in sequence. Taking 5A as an example, it represents the picture at the intersection of the "5" column and row A. In the anti-idiotypic antibody intervention group of columns 3 and 4, according to the results of 3B and 4B, the 153# anti-idiotypic antibody showed an antagonistic effect on RBC agglutination; the deagglutination of RBC cells shown in 3C and 4C was obvious, indicating that the 153# idiotype antibody can be used for neutralizing anti-D antibodies in clinical serum samples.

[0075] Example 6

[0076] The hybridoma antibody subtype was determined using a mouse monoclonal antibody subtype identification kit (Proteintech, Cat. PK20002). Take 50 μl of the hybridoma supernatant and dilute it 1:50 with 1×PBST. Add the test sample to the well of the strip, 50 μL / well. Then add 1×goat anti-mouse IgM+IgG-HRP to the well, 50 μL / well. Gently mix on a mixer and incubate at room temperature for 1 h. Discard the liquid in the well, wash the plate three times with 1×PBST, and pat dry on absorbent paper. Add the freshly prepared chromogenic solution to the well, 100 μL / well, and develop color at room temperature in the dark for 10 - 20 min. Add the stop solution to each well, 100 μL / well. Read the OD450 with an enzyme-linked immunosorbent assay reader. The well with the darkest color or the highest OD value corresponds to the corresponding subtype, and the results are shown in Table 1.

[0077] Table 1. Identification of the subtype of murine anti-D idiotype antibody

[0078]

[0079] The experimental steps for sequencing the variable region gene of the anti-idiotypic antibody are briefly described as follows: Total RNA was extracted from hybridoma cells, and the first strand of cDNA was synthesized using reverse transcriptase (TaKaRa, Cat#2690A). According to the antibody subtype of the hybridoma cells, the corresponding primers were selected for PCR amplification of the light / heavy chain. The specific bands amplified by PCR were excised and recovered, cloned into the TA / Blunt-Zero (Nanjing Novoprotein Science & Technology Co., Ltd., Cat No. C601-01) vector, and at least five colonies of the antibody were sequenced. The sequencing results were analyzed by IgBLAST to determine the CDR region sequence of the antibody.

[0080] Example 7

[0081] Recombinant Expression and Identification of Anti-idiotypic Antibody of Anti-D Antibody

[0082] 7.1 Construction of Murine Recombinant Antibody Vector

[0083] The obtained heavy-chain and light-chain nucleic acid variable region sequences were synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. Using homologous recombination technology, the sequences were cloned into the eukaryotic expression heavy-chain vector of murine IgG1 backbone (Beijing Immunark Pharmaceutical Technology Co., Ltd., Cat No. pQKXM14) respectively to obtain the corresponding heavy-chain expression vector, and the light-chain expression vector (Beijing Immunark Pharmaceutical Technology Co., Ltd., Cat No. pQKXM15) to obtain two anti-idiotypic antibody light-chain expression vectors. The culture conditions of HEK293 cells (ATCC, Cat No. CRL-1573) were suspension culture in OPM-293CD05 serum-free medium (OPM, Cat No. 81075-001) at 36.5 °C, 7.5% CO2, and 120 rpm. At the time of transfection, the recombinant light-paired heavy-chain plasmid was mixed in 10 mL of OPM-293CD05 medium at a weight ratio of 1:1 (total DNA amount was 100 μg), and then 100 μL of PEI (concentration was 3 mg / mL) was added, and quickly vortexed and mixed evenly, and incubated at room temperature for 15 minutes. Then this mixture was added to the above cell culture. The antibody in the supernatant was harvested 7 days after cell culture to obtain the cell culture.

[0084] 7.2 Purification of Recombinant Antibody

[0085] The harvested cell culture was centrifuged at 3000×g for 20 min, the supernatant was collected and filtered through a 0.45 μm filter. The antibody was purified with a 5 mL Protein A affinity chromatography column (GE), eluted with 50 mM citric acid (pH 3.0) buffer at a flow rate of 5 mL / min, and the complete elution peak was collected. At the same time, the pH of the collected eluate was adjusted to about 7.0 with 1 M Tris HCl (pH 9.0) buffer. The obtained protein was detected by SDS-PAGE and Coomassie brilliant blue staining. As shown in A below, the reduced molecular weights of the target proteins were 50 KD and 25 kD, indicating that the target proteins were of IgG type. Figure 7 As shown in A below, the reduced molecular weights of the target proteins were 50 KD and 25 kD, indicating that the target proteins were of IgG type.

[0086] 7.3 ELISA Identification of Recombinant Anti-idiotypic Antibody

[0087] The binding of the anti-idiotypic antibody to the immunogen was detected by ELISA. The ELISA plate was coated with anti-D-F(ab')2 protein at a concentration of 1 μg / ml, and the murine recombinant antibody was at a concentration of 1 μg / ml. 100 μl of each was added to the ELISA plate, and PBS solution was used as the blank control. The secondary antibody was HRP-labeled goat anti-mouse IgG (Botelong Immunotech Co., Ltd., Suzhou, Cat No. BF03001). The optical density was detected at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader. The results are shown in Table 2. The prepared recombinant anti-idiotypic antibody bound to the antigen, indicating that the variable region sequence was correct.

[0088] Table 2

[0089]

[0090] 7.4 Identification of the recombinant anti-idiotypic antibody for red blood cell deagglutination:

[0091] Take a 96-well plate, add 50 μg of the recombinant anti-idiotypic antibody and 5 μl of IgG-D antibody and mix them. Incubate at room temperature for 10 min, then add 20 μl of type O red blood cells. The reaction system was maintained at 100 μl. Set up a red blood cell blank control and a secondary antibody negative control. After mixing, incubate in an incubator at 37 °C for 0.5 h. Wash the red blood cells with 200 μl of normal saline and centrifuge at 200 g for 5 min. Repeat the washing twice. Add 100 μl of anti-IgG secondary antibody, centrifuge at 300 g for 5 min, then gently shake. Observe and record the red blood cell agglutination state under a microscope. As Figure 7 shown in B, no red blood cell agglutination occurred in the red blood cell blank control and the secondary antibody negative control groups; 5 μl of anti-D antibody could cause obvious red blood cell agglutination. After premixing 50 μg of the anti-idiotypic antibody against anti-D antibody and comparing it with the corresponding anti-D antibody group, it can be seen that the recombinant anti-idiotypic antibody has the effect of neutralizing the red blood cell deagglutination mediated by anti-D antibody.

Claims

1. An anti-idiotypic antibody for detecting and neutralizing anti-D antibodies, characterized in that It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, wherein: The amino acid sequence of the HCDR1 is the amino acid sequence shown in SEQ ID No.1; The amino acid sequence of the HCDR2 is the amino acid sequence shown in SEQ ID No. 2; The amino acid sequence of the HCDR3 is the amino acid sequence shown in SEQ ID No.3; The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, wherein: The amino acid sequence of LCDR1 is the amino acid sequence shown in SEQ ID No.4; The amino acid sequence of LCDR2 is the amino acid sequence shown in SEQ ID No.5; The amino acid sequence of the LCDR3 is the amino acid sequence shown in SEQ ID No.6; The SEQ ID No. 5 is as follows: LVS.

2. An anti-idiotypic antibody for detecting and neutralizing anti-D antibodies according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.7; the amino acid sequence of the light chain variable region is shown in SEQ ID No.

8.

3. An anti-idiotypic antibody for detecting and neutralizing anti-D antibodies according to claim 2, characterized in that: The anti-idiotypic antibody is a mouse antibody.

4. An anti-idiotypic antibody for detecting and neutralizing anti-D antibodies according to claim 3, characterized in that: The constant region of the heavy chain of the anti-idiotypic antibody is the heavy chain constant region of mouse IgM, IgG1, IgG2a, IgG2b or IgG3, and the light chain constant region of κ or λ type.

5. Use of an anti-idiotypic antibody for detecting and neutralizing anti-D antibodies according to any one of claims 1 to 4, characterized in that: The anti-idiotypic antibody is used in preparing a kit for detecting the content of anti-D antibodies in serum; or is used in preparing a kit for neutralizing anti-D antibodies.

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