Anti-idiotypic antibodies for neutralizing anti-D antibodies and uses thereof

Abstract

The present invention discloses an anti-idiotypic antibody for detecting and neutralizing anti-D antibodies, comprising 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 HCDR1 has the amino acid sequence shown in SEQ ID No. 1; the amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 2; the amino acid sequence of HCDR3 has the amino acid sequence shown in SEQ ID No. 3. The anti-idiotypic antibody and anti-D-mediated hemagglutination of red blood cells can be used to develop various diagnostic reagents, which can be used to detect mixed samples of unexpected antibodies of various blood types containing anti-D.

Description

Technical Field

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

[0002] In clinical transfusion therapy, the importance of the Rh blood group system is second only to the ABO blood group system. The main antigens of the Rh blood group are D, C, c, E and e antigens. The immunogenicity of the Rh blood group system antigens is very strong, among which the D antigen is the strongest. Red blood cell blood group antibodies follow the general principle of immunology: if an individual has / expresses a certain antigen, the corresponding antibody will not be produced; if a certain antigen is missing, the corresponding antibody may be produced. Under the stimulation of external red blood cell blood group antigens, the plasma cells differentiated by the proliferation of B lymphocytes or memory B lymphocytes will produce antibodies that can specifically bind to the corresponding red blood cell antigens. Studies have found that Rh-negative individuals may produce anti-D when they first transfuse more than 2ml of Rh-positive red blood cells. In recent years, with the advancement of experimental technology and the sharing of data, it has been found that some D variant individuals with incomplete expression of D antigen due to mutations in the RHD gene will also produce anti-D under the stimulation of D antigen due to the lack of D antigen epitopes. Anti-D is mainly IgG type immune antibodies, 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 or newborn (HDFN). Therefore, in clinical work, accurate detection of anti-D is the basic guarantee for transfusion safety and the basis for guiding the prevention, diagnosis and treatment of Rh-HDFN.

[0003] At present, the method used for red blood cell blood type antibody detection is the immunoserological method, the principle of which is to detect unknown antibodies with known antigens. Since it is currently impossible to obtain a single, purified red blood cell blood type antigen, it is only possible to select red blood cells carrying multiple antigens for antibody detection, and determine the specificity of unexpected antibodies according to a certain reaction pattern. The current method for detecting IgG type anti-D is the indirect anti-human globulin method, which uses an anti-Fc-terminal secondary antibody as an intermediary to cause agglutination of red blood cells bound to IgG antibodies; the gold standard for detecting antibodies is the indirect anti-human globulin method in a test tube, but it has the disadvantages of being cumbersome to operate and being greatly affected by the technical level of the operator. The concentration of anti-D is usually expressed in titer or titer, and the detection method is to dilute the antibody and detect it with the indirect anti-human globulin method, which cannot accurately detect the amount of anti-D. For patients who produce unexpected blood type antibodies, in order to eliminate the impact of the unexpected antibodies, only blood that is negative for the corresponding antigen can be used in blood transfusion therapy. In emergency situations, due to this limitation, it may be impossible to find matching blood, delaying the patient's treatment. For Rh-negative pregnant women who commonly produce anti-D, if the anti-D titer is high, the risk and severity of HDFN will increase, and stillbirth and miscarriage often occur in early pregnancy.

[0004] At present, the method used for red blood cell blood type antibody detection is the immunoserological method, the principle of which is to detect unknown antibodies with known antigens. Since it is currently impossible to obtain a single, purified red blood cell blood type antigen, it is only possible to select red blood cells carrying multiple antigens for antibody detection, and determine the specificity of unexpected antibodies according to a certain reaction pattern. The current method for detecting IgG type anti-D is the indirect anti-human globulin method, which uses an anti-Fc-terminal secondary antibody as an intermediary to cause agglutination of red blood cells bound to IgG antibodies; the gold standard for detecting antibodies is the indirect anti-human globulin method in a test tube, but it has the disadvantages of being cumbersome to operate and being greatly affected by the technical level of the operator.

[0005] To solve the above problems, it is necessary to obtain an "antigen" that can specifically bind to the Fab end of anti-D to capture the antibody. Since the RhD protein is a transmembrane protein, in addition to folding through the membrane, it also needs to interact with Rh-related proteins to form a spatial conformation to be antigenic. If the protein is obtained by extracting it from the red blood cell membrane, the quaternary structure of the RhD protein will be lost, and the RhD antigenicity will also disappear; similarly, by synthesizing proteins through genetic engineering, only amino acid chains without RhD antigenicity can be obtained. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-idiotypic antibody for neutralizing anti-D antibodies and its use, and to utilize the anti-idiotypic antibody and anti-D mediated erythrocyte agglutination to develop various diagnostic reagents, which can be used to detect mixed samples of unexpected antibodies of various blood types containing anti-D.

[0007] The present invention adopts the following technical scheme: an anti-idiotypic antibody for detecting and neutralizing anti-D antibodies, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, wherein:

[0008] The amino acid sequence of the HCDR1 has the amino acid sequence shown in SEQ ID No. 1;

[0009] The amino acid sequence of the HCDR2 has the amino acid sequence shown in SEQ ID No. 2;

[0010] The amino acid sequence of the HCDR3 has the amino acid sequence shown in SEQ ID No.3.

[0011] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, wherein:

[0012] The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 4;

[0013] The amino acid sequence of the LCDR2 has the amino acid sequence shown in SEQ ID No.5;

[0014] The amino acid sequence of the LCDR3 has the amino acid sequence shown in SEQ ID No.6.

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

[0016] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.23; the amino acid sequence of the light chain variable region is shown in SEQ ID No.24.

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

[0018] Furthermore, 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.

[0019] Furthermore, the antigen binding portion of the anti-idiotypic antibody is selected from a F(ab')2 fragment.

[0020] The present invention also discloses the use of the above-mentioned anti-idiotypic antibody for detecting and neutralizing anti-D antibodies, the use of the anti-idiotypic antibody in preparing a kit for detecting the content of anti-D antibodies in serum; or the use of the anti-idiotypic antibody in preparing a kit for neutralizing anti-D antibodies.

[0021] The beneficial effects of the present invention are as follows: 1. The anti-idiotypic antibody can specifically bind to the Fab end of the anti-D antibody and can play a role in simulating antigens. 2. The anti-idiotypic antibody can be used to neutralize the anti-D-mediated red blood cell agglutination, and then can be used to develop various diagnostic reagents, can be used to detect mixed samples of multiple blood type unexpected antibodies containing anti-D, can also be used for risk assessment and clinical diagnosis and treatment of Rh-HDFN, and can also lay the foundation for subsequent antibody humanization, and become a drug for blocking anti-D in the treatment of Rh-HDFN and Rh-negative blood transfusion, which has important application value for improving the safety of clinical blood transfusion and disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are test diagrams for the preparation of human anti-D antibodies and the preparation of immunogens by pepsin cleavage; wherein: A is the SDS-PAGE electrophoresis diagram of the crude protein of anti-D antibody red blood cell lysate before and after purification; B. is the SDS-PAGE electrophoresis diagram of the products of anti-D antibody before and after pepsin cleavage.

[0023] Figure 2 This is a graph showing the determination of serum antibody titers in mice immunized with anti-DF(ab')2 protein.

[0024] Figure 3 Screening of anti-idiotypic antibodies by ELISA.

[0025] Figure 4 It is the SDS-PAGE electrophoresis diagram of anti-idiotypic antibody; where: M is the pre-stained protein Marke; 2 represents 426# anti-idiotypic antibody.

[0026] Figure 5 Fitting plot for the anti-idiotypic antibody Fortebio assay.

[0027] Figure 6 This is a diagram showing the neutralizing effect of anti-idiotypic antibodies on red blood cell agglutination mediated by different anti-D antibodies, among which the anti-D antibodies selected were Shanghai anti-D antibody and Millipore anti-D antibody.

[0028] Figure 7 This is the identification of recombinant anti-idiotypic antibodies; A is the SDS-PAGE electrophoresis diagram of anti-idiotypic antibodies; B is the red blood cell deagglutination experiment of recombinant anti-idiotypic antibodies. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

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

[0031] The term "antigen binding portion" of an antibody refers to a portion or segment of a complete antibody molecule that is responsible for binding to an antigen. The antigen binding portion may comprise a heavy chain variable region, a light chain variable region, or both. The antigen binding portion of an antibody may be prepared from a complete antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques. 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 consisting of amino acid residues that mimic the hypervariable region of an antibody, such as isolated CDRs. The term "antigen binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetrabodies, and minibodies. For example, the Fd fragment in the present invention refers to an antibody fragment consisting of a VH and CH1 domain; the Fv fragment consists of a VL and VH domain in a single arm of an antibody; and the dAb fragment consists of a VH domain.

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

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

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

[0035] In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies constituting the population are identical except for the presence of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein particularly include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and the remainder of the heavy chain 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 include fragments of such antibodies, as long as they can exhibit the desired biological activity.

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

[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 thereto, wherein the purpose is to reverse, alleviate, ameliorate, inhibit or relieve or prevent symptoms, complications, conditions or progression, development, severity or recurrence associated with a disease. The term "prevention" refers to administration to a subject who does not have a disease in order to prevent the disease from occurring or, if present, to minimize its effects.

[0038] The present invention discloses an anti-idiotypic antibody for detecting and neutralizing anti-D antibodies, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, wherein:

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

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

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

[0042] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, wherein:

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

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

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

[0046] The anti-idiotypic antibody is a mouse antibody, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 23:

[0047] EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAPGKGPEWV VFISNLAYSIYYADTVTGRFTISRENAKNTLYLEMNSLRSEDTAMYYCTRVDH YTGRPWFAYWGQGTLVTVSA. 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. 24: QIVLTQSPAIMSASPGEKVTMTCSASSGVSYMHWYQQKSGTSPKRWIYDTSNL ASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQWSSHPPTFGSGTKLEIK, i.e. FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (106aa).

[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 mouse antibody. 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 kappa or lambda type light chain constant region.

[0052] As a specific example, the present invention provides a 426# mouse-derived anti-idiotypic antibody for detecting and neutralizing anti-D antibodies. 426# is an artificial number.

[0053] In the present invention, hybridoma cells are screened and secreted antibodies that can bind to anti-D antibody proteins. In the present invention, the anti-idiotypic antibody type and subtype can be determined using antigens specific to specific antibody types and subtypes, and the simulated RhD antigen isotype can be determined using ELISA assays.

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

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

[0056] The present invention also discloses the use of an anti-idiotypic antibody for detecting and neutralizing anti-D antibodies, the use of the anti-idiotypic antibody in preparing a kit for detecting the content of anti-D antibodies in serum, or the use of the anti-idiotypic antibody in preparing a kit for neutralizing anti-D antibodies.

[0057] Example 1

[0058] Purification of anti-D antibody: The crude product of anti-D antibody red blood cell lysate was purified by Protein A affinity purification and eluted to obtain anti-D antibody. The harvested antibody molecules were identified by SDS-PAGE electrophoresis, and the molecular weight was in line with expectations, such as Figure 1 As shown in A. The anti-D antibody was ultrafiltered to replace the solvent into 50mM citric acid pH 3.0 buffer, pepsinized at 37℃ for 2h, purified by Mabsure LX to remove the Fc fragment and the intact protein that was not cleaved, and the collected flow was passed through the Capto L purification column to remove pepsin and other antibody fragments, and the F(ab')2 protein was eluted. The harvested protein was identified by SDS-PAGE electrophoresis. Figure 1 As shown in Figure B, the reduced molecular weight of the F(ab')2 segment of the target protein is 25 kD, and the non-reduced molecular weight is about 100 kD, which is in line with expectations and can be used for subsequent immune experiments.

[0059] Example 2

[0060] Hybridoma preparation of anti-idiotypic antibodies:

[0061] 1. Take 6-8 week old female Balb / c mice (Beijing Huafukang Biotechnology Co., Ltd.), and subcutaneously inject anti-DF(ab')2 protein fully emulsified with immune adjuvant, 50μg / dose / mouse, and repeat three times. After three subcutaneous immunizations of mice, 150μl of blood was collected from the eye sockets, and serum was obtained after centrifugation. The anti-DF(ab')2 protein was coated on the enzyme-linked plate at a concentration of 1μg / ml, and the mouse serum antibody titer was detected by ELISA. The mouse serum dilution ratio started from 1:4000, and was diluted to five concentrations of 1:3. PBS was used as the negative control, and the optical density was detected by an enzyme reader at a wavelength of 450nm. Figure 2 It can be seen that the immunization effects of the three mice are comparable, and the abundance of serum antibody titers from high to low are 1#, 2#, and 3#.

[0062] 2. Before fusion, mice were intraperitoneally injected with 50 μg of anti-DF(ab')2 protein for three consecutive days. One day before fusion, intraperitoneal macrophages of ordinary Kunming mice (Beijing Huafukang Biotechnology Co., Ltd.) were taken as trophoblasts and inoculated into 96-well plates. The spleen of the immunized mice was fused with the non-secretory myeloma SP2 / 0 cell line, and the hybridoma cells were added to the 96-well plate with the trophoblast, and the fused hybridoma cells were selected by HAT.

[0063] 3. After the hybridoma cells grow in HAT medium for 12 days, take 50 μl of the hybridoma supernatant and detect the antibody titer in the supernatant by ELISA. Set up positive, negative and blank controls. Collect all positive clones, replace the HAT medium for two days and screen again by ELISA. Take the clones that are positive twice.

[0064] Example 3

[0065] Screening of anti-idiotypic antibodies: Screening was performed by measuring OD620 using an ELISA reader. The specific method is as follows: Take a 96-well ELISA plate, add 75μl of hybridoma supernatant and 5μl of anti-D antibody, mix, incubate at room temperature for 10 minutes, then add 20μl of type O red blood cells, keep the reaction system at 100μl, set up an agglutination positive control without hybridoma supernatant and a secondary antibody negative control, mix well and incubate in a 37°C incubator for 0.5h. Wash the red blood cells with saline, centrifuge at 200g for 5min, and repeat the wash twice. Add 100μl of anti-human IgG secondary antibody, centrifuge at 300g for 5min, shake gently, visually observe the agglutination state of the red blood cells, and measure the optical density value using an ELISA reader OD620. The results are as follows Figure 3 As shown, the secondary antibody cannot cause O-type red blood cell agglutination, and the red blood cells are evenly distributed in the plate wells; in the positive agglutination wells, the red blood cells aggregate on the side walls of the wells, resulting in increased transmittance of the wells. The target antibody is a clone with low transmittance and high OD620 value, and the wells marked in red are candidate target clones. After multiple screenings, the hybridoma cells were obtained. Candidate clones.

[0066] The candidate hybridoma cells were subcloned by limiting dilution method, and feeder cells were pre-plated in 96-well cell culture plates to provide nutritional support for the growth of monoclonal hybridoma cells. After the candidate hybridoma cells in the exponential growth phase were centrifuged, the concentration of the cell culture medium was adjusted to make the number of cells per well 0.8 / well. After the hybridoma cells formed clones, the positive clones were screened by ELISA method to obtain monoclonal hybridoma cells.

[0067] Example 4

[0068] Anti-idiotypic antibody and antigen affinity determination:

[0069] Preparation of anti-idiotypic antibodies: After hybridoma cells were expanded and cultured, the cell concentration was adjusted to 2e6 / ml with PBS and injected into the peritoneal cavity of male 6-8 week old Balb / c mice, 1 ml per mouse. The ascites was extracted on the 10th day. After centrifugation of the ascites, the supernatant was filtered with a 0.45μM filter to remove impurities. The antibodies in the supernatant were purified by Protein A. SDS-PAGE identification of the purified antibodies showed that the molecular weight of the light and heavy chains of the antibodies was correct. Figure 4 It can be seen that the antibody purity is greater than 95%.

[0070] The Fortebio method is used to detect the quantitative detection of the affinity between anti-idiotypic antibodies and immunogens. The AMC probe is used to bind to the Fc segment of the idiotypic antibody to detect its affinity and dissociation with the immunogen anti-DF(ab')2. The ForteBio Octetred 96e Molecular Interaction Analysis System is an advanced technology for non-labeled, real-time monitoring, mainly used for comprehensive quantitative analysis of interactions between biomolecules. The AMC probe is used to bind to the Fc segment of the idiotypic antibody to detect its affinity and dissociation with the immunogen anti-DF(ab')2. Figure 5 It is known that the anti-idiotypic antibody can bind to the anti-DF(ab')2 molecule, has a strong binding affinity with the anti-D antibody, hardly dissociates, and the dissociation curve is straight. The affinity (KD) obtained by fitting calculation is less than 1.0E-12.

[0071] Example 5

[0072] Anti-idiotypic antibody neutralization test on red blood cell agglutination:

[0073] Two sources of anti-D antibodies were selected: IgG-D and IgM / GD. IgG-D was selected from Shanghai Blood Biopharmaceutical Co., Ltd., with a national medical device registration number of 20223401104, and IgM / GD was selected from Millipore, with clones of TH-28 / MS-26. Take a 96-well plate, add anti-idiotypic antibody and anti-D antibody, the amount used is 5μl or 10μl, the final concentration of anti-idiotypic antibody is 500μg / ml, incubate at room temperature for 10min, then add 20μl O-type red blood cells, keep the reaction system at 100μl, set up a red blood cell blank control and a secondary antibody negative control, mix well, incubate at 37℃ incubator for 0.5h, wash the red blood cells with 200μl saline, centrifuge at 200g for 5min, repeat the wash twice, add 100μl anti-IgG secondary antibody, centrifuge at 300g for 5min, and shake. In order to present the agglutination state of red blood cells more intuitively, the automatic scanning and photographing function of the Elisapot instrument is used to display the agglutination state of red blood cells. Figure 6 As can be seen from a, compared with the secondary antibodies 5C and 5D, 5μl or 10μl of anti-D antibody can cause significant agglutination effect of red blood cells 5A, 5B and 5G, 5H; where: "5" represents the fifth column in the figure; A, B, C, D, G and H represent the row numbers respectively. Taking 5A as an example, it represents the picture at the intersection of the "5th" column and the Ath row.

[0074] according to Figure 6 It can be seen from the results of 1B, 2B, 3B, and 4B that the anti-idiotypic antibody has a neutralizing effect on Shanghai IgG-D; according to the results of 6B and 7B, it has no antagonistic effect on Millipore's IgM / GD, indicating that the anti-idiotypic antibody prepared by the present invention has selective neutralizing activity.

[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 with 1×PBST 1:50, and add the sample to be tested to the sample well of the strip, 50 μL / well. Then add 1× sheep anti-mouse IgM+IgG-HRP to the sample well, 50 μL / well. Mix gently on a mixer and incubate at room temperature for 1 hour. Discard the liquid in the well, wash the plate three times with 1×PBST, and pat dry on absorbent paper. Add the freshly prepared color development solution to the well, 100 μL / well, and color at room temperature in the dark for 10-20 minutes. Add the stop solution to each well, 100 μL / well. The microplate reader reads OD450, and the well with the darkest color or the highest OD value corresponds to the corresponding subtype. The results are shown in Table 1.

[0077] Table 1. Identification of anti-idiotypic antibody subtypes

[0078]

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

[0080] Example 7

[0081] Recombinant expression and identification of anti-idiotypic antibodies against B antibodies:

[0082] 7.1 Construction of mouse recombinant antibody vector:

[0083] The obtained heavy chain and light chain nucleic acid variable region sequences were synthesized by BNA (Jiangsu Saisuofei Biotechnology Co., Ltd.), and the sequences were cloned into the mouse IgG1 backbone eukaryotic expression heavy chain vector (Beijing ImmunoArk Pharmaceutical Technology Co., Ltd., Cat No. pQKXM14) using homologous recombination technology to obtain the corresponding heavy chain expression vector and light chain expression vector (Beijing ImmunoArk Pharmaceutical Technology Co., Ltd., Cat No. pQKXM15), and two anti-idiotypic antibody light chain expression vectors were obtained. HEK293 cells (ATCC, Cat No. CRL-1573) were cultured in OPM-293CB05 serum-free medium (Aopu Mai, Cat No. 81075-001), 36.5°C, 7.5% CO 2 , 120rpm suspension culture. During transfection, the recombinant light-paired heavy chain plasmid was mixed in 10mL OPM-293CB05 culture medium at a weight ratio of 1:1 (total BNA was 100μg), followed by adding 100μL PEI (concentration was 3mg / mL), vortexing quickly, and incubating at room temperature for 15 minutes. The mixture was then added to the above cell culture. After 7 days of cell culture, the antibodies in the supernatant were harvested to obtain a cell culture.

[0084] 7.2 Purification of recombinant antibodies:

[0085] The harvested cell culture was centrifuged at 3000×g for 20 min, and the supernatant was collected and filtered with a 0.45 μm filter. The antibody was eluted with 5 mL of Protein A affinity chromatography column (GE) 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 SBS-PAGE and Coomassie Brilliant Blue staining, as shown in FIG. Figure 7 As shown in A, the reduced molecular weight of the target protein is 50KB and 25kB, indicating that the target protein is of IgG type.

[0086] 7.3 ELISA identification of recombinant anti-idiotypic antibodies:

[0087] ELISA was used to detect the binding of anti-idiotypic antibodies to immunogens. The ELISA plate was coated with anti-DF(ab')2 protein at a concentration of 1 μg / ml, and the mouse recombinant antibody was added to the ELISA plate at a concentration of 1 μg / ml, 100 μl per well, and PBS solution was used as a blank control. The secondary antibody selected was HRP-labeled sheep anti-mouse IgG (Suzhou Botelon Immunotechnology Co., Ltd., Cat No. BF03001), and the optical density was detected by an ELISA reader at a wavelength of 450 nm. The results are shown in Table 2. The prepared recombinant anti-idiotypic antibody binds to the antigen, indicating that the variable region sequence is correct.

[0088] Table 2

[0089]

[0090] 7.4 Identification of erythrocyte deagglutination by recombinant anti-idiotypic antibodies:

[0091] Take a 96-well plate, add 50μg of recombinant anti-idiotypic antibody and 5μl IgG-D antibody, mix, incubate at room temperature for 10min, then add 20μl O-type red blood cells, keep the reaction system at 100μl, set up red blood cell blank control and secondary antibody negative control, mix well and incubate at 37℃ incubator for 0.5h, wash red blood cells with 200μl normal saline, centrifuge at 200g for 5min, repeat washing twice, add 100μl anti-IgG secondary antibody, centrifuge at 300g for 5min, shake gently, observe and record the red blood cell agglutination state under a microscope. Figure 7 As shown in Figure B, neither the red blood cell blank control nor the secondary antibody negative control group produced red blood cell agglutination, but 5 μl of anti-D antibody could cause obvious red blood cell agglutination. After premixing 50 μg of anti-idiotypic antibody with anti-D antibody, compared 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: DTS.

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.23; the amino acid sequence of the light chain variable region is shown in SEQ ID No.

24.

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. An anti-idiotypic antibody for detecting and neutralizing anti-D antibodies according to claim 4, characterized in that: The antigen binding portion of the anti-idiotypic antibody is selected from a F(ab')2 fragment.

6. Use of an anti-idiotypic antibody for detecting and neutralizing anti-D antibodies according to any one of claims 1 to 5, 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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