An antibody for inhibiting interference of cd38 mab on blood transfusion compatibility test

Abstract

The application discloses an antibody for inhibiting interference of CD38 monoclonal antibody on transfusion compatibility detection, a heavy chain amino acid sequence of the antibody is shown in sequence 1, and a light chain amino acid sequence of the antibody is shown in sequence 2. The anti-human CD38 rabbit monoclonal antibody can effectively combine with CD38 antigens on the surface of human red blood cells, so that the combination of human CD38 monoclonal antibody and the CD38 antigens is inhibited.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemistry, and particularly relates to an antibody for inhibiting interference of CD38 monoclonal antibody on transfusion compatibility detection. BACKGROUND

[0002] CD38 molecule is a type II transmembrane glycoprotein with receptor and exoglycosidase activity, and is widely involved in cell adhesion and transmembrane signal transduction. In various hematological malignancies including relapsed refractory multiple myeloma (MM), CD38 molecule is highly expressed, making it an ideal immunotherapy target. Daratumumab is the first full human CD38 monoclonal antibody of immunoglobulin G1κ (IgG1κ) type, which can specifically bind to CD38 antigen on the surface of tumor cells. However, the target of CD38 monoclonal antibody is not only highly expressed on tumor cells, but also has low level expression on red blood cells, resulting in positive indirect antiglobulin test (IAT) results in transfusion compatibility detection by binding to CD38 antigen on the surface of red blood cells. This binding can trigger the phenomenon of panagglutination, interfering with key transfusion detection links such as antibody screening, antibody identification, and cross matching. If the transfusion laboratory staff does not understand the interference mechanism of CD38 monoclonal antibody on transfusion detection, or fails to obtain the CD38 monoclonal antibody treatment history of the patient, it is easy to misjudge this interference as the presence of red blood cell "unexpected antibodies", and this misjudgment may lead to delayed transfusion, and even cover up the presence of allogeneic antibodies in the patient, resulting in missed detection, increasing the risk and safety hazards of transfusion. This interference effect can still last for up to 6 months after the patient stops using CD38 monoclonal antibody, further increasing the complexity and uncertainty of transfusion detection.

[0003] To address the interference of CD38 monoclonal antibodies in transfusion compatibility testing, researchers have proposed various strategies. However, there is currently no unified standard method domestically or internationally, and the methods employed still have many limitations in terms of operational difficulty, cost, and clinical application. Dithiothreitol (DTT) treatment of erythrocytes is currently the most commonly used method internationally. Its mechanism of action is to cleave the disulfide bonds in the extracellular region of the CD38 molecule, hydrolyzing the CD38 antigen structure on the surface of erythrocytes, thereby preventing CD38 monoclonal antibodies from binding to the CD38 antigen. However, the application of this method has significant limitations: DTT can destroy other clinically important antigens on the surface of erythrocytes, such as Kell antigens, therefore, in Caucasian patients, Kell homologous transfusion is required after DTT treatment. In addition, the DTT treatment procedure is cumbersome, requiring specific experimental conditions, increasing the difficulty of laboratory operation. Furthermore, because DTT causes denaturation of multiple erythrocyte antigens, its applicability in patients with different blood types is limited. The agglutination amine method is another approach used to replace the antiglobulin test for antibody screening, antibody identification, and crossmatching. It avoids interference from CD38 monoclonal antibodies. However, the agglutination amine method may miss low-level or poorly affinity antibodies, potentially leading to false negatives and threatening transfusion safety. The most direct way to eliminate CD38 monoclonal antibody interference is to neutralize the CD38 antigen on the surface of the donor's red blood cells before the indirect antiglobulin test (IAT). Summary of the Invention

[0004] The purpose of this invention is to provide an antibody for inhibiting the interference of CD38 monoclonal antibody on transfusion compatibility testing. The antibody is an anti-human CD38 rabbit monoclonal antibody, which can effectively bind to the CD38 antigen on the surface of human red blood cells, thereby inhibiting the binding of human CD38 monoclonal antibody to CD38 antigen.

[0005] The present invention adopts the following technical solution: an antibody for inhibiting the interference of CD38 monoclonal antibody on transfusion compatibility detection, wherein the amino acid sequence of the heavy chain variable region is shown in Sequence 1 and the amino acid sequence of the light chain variable region is shown in Sequence 2.

[0006] The present invention also discloses the use of the antibody described above for inhibiting the interference of CD38 monoclonal antibody on transfusion compatibility testing, and the use of the reagent for preparing the reagent for inhibiting the interference of CD38 monoclonal antibody on transfusion compatibility testing.

[0007] Furthermore, it is used to bind to the CD38 antigen on the surface of red blood cells.

[0008] The beneficial effects of this invention are: 1. By immunizing rabbits with CD38 antigen, an anti-human CD38 rabbit monoclonal antibody is obtained. This anti-human CD38 rabbit monoclonal antibody can effectively bind to the CD38 antigen on the surface of human erythrocytes, inhibiting the binding of human CD38 monoclonal antibody to the CD38 antigen on the surface of erythrocytes. Since it does not bind to anti-human globulin, it eliminates the interference of human CD38 monoclonal antibody in transfusion compatibility testing. 2. The anti-human CD38 rabbit monoclonal antibody inhibits the interference of human CD38 monoclonal antibody in pre-transfusion testing based on the species specificity of the antibody Fc fragment. Each 50 μl of cells requires 12 μg of anti-human CD38 rabbit monoclonal antibody, which is a small amount and avoids damage to other important erythrocyte antigens (such as Kell antigen), especially in Caucasian populations, avoiding problems related to Kell antigen. Furthermore, this method does not require complex experimental conditions, reducing operational difficulty and time costs; and it can avoid missing the detection of unexpected antibodies with low concentrations or poor affinity, reducing the occurrence of false negative results, thereby ensuring the accuracy of transfusion compatibility testing and transfusion safety. Attached Figure Description

[0009] Figure 1 The graph shows the expression and identification of hCD38-Fc protein, where:

[0010] A. DNA electrophoresis diagram of PCR products of hCD38 and Fc genes; M. DNA marker, Lane 1. hCD38 gene, Lane 2. hFc gene;

[0011] B. SDS-PAGE electrophoresis image of hCD38-Fc recombinant protein; M. Protein marker, N. Non-reduced protein, R. Reduced protein;

[0012] C. Fortebio detection curves of binding and dissociation between CD38 monoclonal antibody daratumumab and hCD38-Fc protein;

[0013] Figure 2 The image shows the ELISA results for the antibody titer in hCD38-Fc-immunized rabbit serum, where:

[0014] ELISA results of antibody titer in rabbit serum immunized with A.hCD38-Fc;

[0015] B. SDS-PAGE gel electrophoresis results of anti-human CD38 rabbit polyclonal antibody; M. protein marker, N. non-reducing, R. reducing;

[0016] C. The affinity of anti-human CD38 polyclonal antibody for hCD38-Fc protein was detected by ELISA, with an EC50 of 83.88 ng / ml;

[0017] Figure 3 This is a verification diagram showing how the anti-human CD38 rabbit polyclonal antibody overcomes interference from CD38 monoclonal antibodies; where:

[0018] A. Validation results of overcoming daratum interference;

[0019] B. Validation results of overcoming esartanoxicillin interference.

[0020] Figure 4 The detection map of anti-human CD38 rabbit polyclonal antibody overcoming daratum interference does not affect the detection of other unexpected antibodies, among which:

[0021] A. Results of the indirect anti-human globulin test on anti-D plasma samples;

[0022] Results of the indirect anti-human globulin test on BB anti-E plasma samples;

[0023] C. Results of the indirect anti-human globulin test on anti-Fya plasma samples;

[0024] D. Results of the indirect anti-human globulin test on anti-K plasma specimens;

[0025] Figure 5 Preparation, expression and identification of anti-human CD38 rabbit monoclonal antibody.

[0026] A. Rabbit single B cell flow cytometry sorting diagram, FITC and APC double-positive cells in the AE gate are target cells;

[0027] B. SDS-PAGE electrophoresis image of rabbit anti-hCD38 monoclonal antibody. M. Protein marker;

[0028] C. ELISA was used to detect the affinity of anti-hCD38 rabbit monoclonal antibody for hCD38-Fc protein;

[0029] D. Fortebio assay for the binding affinity of anti-hCD38 rabbit monoclonal antibody to hCD38-His protein;

[0030] E. Fortebio analysis of epitope competition between anti-hCD38 rabbit monoclonal antibody and clinical treatment antibody;

[0031] F. Flow cytometry detection of the binding of anti-hCD38-D2 rabbit monoclonal antibody to CD38 molecules on the surface of RBC membrane (Figure a);

[0032] G. Flow cytometry detection of the binding of anti-hCD38-D2 rabbit monoclonal antibody to CD38 molecules on the surface of RBC membrane (Figure b);

[0033] Figure 6 To overcome CD38 monoclonal antibody interference with anti-human CD38 rabbit monoclonal antibody, the following is included:

[0034] A. Validation results of overcoming daratum interference;

[0035] B. Validation results of overcoming esartanux interference;

[0036] Figure 7 The lowest concentration of rabbit monoclonal antibody against human CD38 that inhibits daratum interference;

[0037] Figure 8 The shortest incubation time for inhibiting daratum interference with anti-human CD38 rabbit monoclonal antibody;

[0038] Figure 9 A comparison of the effects of anti-human CD38 rabbit monoclonal antibody and DTT on erythrocyte blood group antigens;

[0039] Figure 10 The rabbit monoclonal antibody against human CD38 overcomes daratum interference without affecting the detection of other unexpected antibodies, including:

[0040] A. Results of the indirect anti-human globulin test on anti-D plasma samples;

[0041] B. Results of the indirect anti-human globulin test on anti-E plasma samples;

[0042] C. Results of the indirect anti-human globulin test on anti-Fya plasma samples;

[0043] D. Results of the indirect anti-human globulin test on anti-K plasma specimens. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0045] This invention discloses an antibody for inhibiting the interference of CD38 monoclonal antibodies on transfusion compatibility testing. The amino acid sequence of its heavy chain variable region is shown in Sequence 1, and the amino acid sequence of its light chain variable region is shown in Sequence 2.

[0046] Sequence 1 is as follows:

[0047] QSLEESGGRLVTPGTPLTLTCTASGFSLNNYAMNWVRQAPGKGLEWIGIISDVGMTVYANWASSRFTISKTSSTTVDLKIASPTTEDTATYFCARGLFGGSTFYLGWDGFDPWGPGTLVTISS.

[0048] Sequence 2 is as follows:

[0049] AYDMTQTPASVEVPVGGTVTINCQASQSIANQLSWYQQKPGQPPKLLMYWASTLASGVSSRFKGSRSGTELTLTISGVECADAATYYCQQAYTTDNVDNVFGGGTEVVVK.

[0050] The present invention also discloses the use of the antibody used to inhibit the interference of CD38 monoclonal antibody on transfusion compatibility testing, and the use of the reagent used to prepare the reagent to inhibit the interference of CD38 monoclonal antibody on transfusion compatibility testing.

[0051] Specifically, it is used to bind to the CD38 antigen on the surface of red blood cells to inhibit the interference of CD38 monoclonal antibody with transfusion compatibility testing.

[0052] In this invention, rabbits were immunized with CD38 antigen, and a rabbit spleen B-cell monoclonal antibody was obtained through flow cytometry sorting, B-cell sequencing, synthesis, expression, and purification techniques. The anti-human CD38 rabbit monoclonal antibody and the human CD38 monoclonal antibody (daratumum) bind to the same epitope on CD38; therefore, the anti-human CD38 rabbit monoclonal antibody blocks the binding of free daratumum antibody in serum to erythrocytes. Its working principle is based on the species specificity of the antibody's Fc fragment: rabbits are rodents, and rabbit-derived antibodies differ significantly from human-derived antibodies in their Fc fragments. Therefore, the Fc fragment of the rabbit-derived antibody does not bind to anti-human globulin. The anti-human globulin of this secondary antibody mainly binds to the Fc fragment of the human antibody. This characteristic ensures that the anti-human CD38 rabbit monoclonal antibody does not interfere with the anti-human globulin reaction during transfusion compatibility testing, thus effectively inhibiting the interference of the human CD38 monoclonal antibody on pre-transfusion testing, ensuring the accuracy of experimental results and transfusion safety. The method allows for standard pre-transfusion testing without interference from the use of daratumumab.

[0053] Example 1. Preparation of CD38 antigen

[0054] 1.1 Construction of eukaryotic expression vector for hCD38 extracellular region

[0055] Human CD38 gene vector (Accession # NP_001766) was purchased from Sinocare Biotechnology Co., Ltd. (Cat: HG10818-M). Primers were designed to amplify the extracellular region fragment of the human CD38 gene (AA Val 43-Ile300) using PCR. The PCR fragment was approximately 800 bp and recovered via gel extraction. Human antibody IgG1 constant region Fc gene DNA (Accession # ACK87036.1) was synthesized by Jiangsu Saisofe Biotechnology Co., Ltd. and inserted into the cloning vector pUC. Primers were designed to amplify the Fc segment using PCR. The PCR fragment was approximately 700 bp, recovered via gel extraction, and used for later use.Figure 1 As shown in Figure A, the extracellular region of the CD38 gene and Fc were cloned into the eukaryotic expression vector PQKX1 (Beijing ImmunoArk Pharmaceutical Technology Co., Ltd.) using homologous recombinase. The CD38 and Fc genes were expressed co-frames, resulting in the PQKX1-hCD38-Fc vector. Sequencing of the plasmid vector DNA confirmed its accuracy, and it was used for subsequent transient cell transfection.

[0056] 1.2 Transient transduction of hCD38 extracellular region eukaryotic expression vector

[0057] HEK293 cells (ATCC, Cat No. CRL-1573) were cultured under the following conditions: OPM-293 CD05 serum-free medium (OPM, Cat No. 81075-001), 36.5℃, 7.5% CO2, suspension culture at 120 rpm. For transfection, 100 mg of PQKX1-hCD38-Fc plasmid was mixed in 10 mL of OPM-293 CD05 serum-free medium, followed by the addition of 100 mL of PEI (3 mg / mL). The mixture was rapidly vortexed and incubated at room temperature for 15 minutes to obtain the final mixture. This mixture was then added to the cell culture. Antibodies were harvested from the supernatant after 7 days of cell culture.

[0058] 1.3 Purification of recombinant hCD38-Fc protein

[0059] The antibody in the harvested supernatant was centrifuged at 3000×g for 20 min. The supernatant was collected and filtered through a 0.45 μm filter to remove debris. Elution was performed using a 5 mL Protein A affinity chromatography column (GE) with 50 mM citrate buffer (pH 3.0) at a flow rate of 5 mL / min. The complete elution peak was collected, and the pH of the collected eluent was adjusted to approximately 7.0 using 1 M Tris HCl buffer (pH 9.0). The obtained hCD38-Fc protein was detected by SDS-PAGE and Coomassie Brilliant Blue staining. Figure 1 From B, we can see that the molecular weight of the non-reduced dimer CD38-Fc is about 140 kDa, and the molecular weight of the reduced CD38-Fc fragment is about 70 kDa, which is in line with expectations.

[0060] 1.4 Identification of recombinant hCD38-Fc protein

[0061] The ForteBio Octet red 96e molecular interaction analysis system is primarily used for the quantitative analysis of biomolecular interactions. This example uses the ForteBio method to quantitatively detect the affinity between an anti-human CD38 monoclonal antibody (daratumor, Gastrodin) and recombinant hCD38-Fc protein. A CH1 probe is used to bind to the CH1 region of the heavy chain of the anti-human CD38 monoclonal antibody (daratumor, Gastrodin), and its affinity and dissociation from the immunogen-derived recombinant hCD38-Fc protein are detected. Figure 1 According to the results from C, the hCD38-Fc protein can bind to the anti-human CD38 monoclonal antibody. The dissociation curve is sloping, and the affinity KD is calculated to be 8.449E-8, which proves that the obtained hCD38-Fc protein sequence is correct. The CD38 antigen can be prepared and used for subsequent immunization experiments.

[0062] Example 2. Preparation and identification of anti-human CD38 rabbit polyclonal antibody

[0063] 2.1 Immunization of New Zealand White Rabbits

[0064] Six-week-old female New Zealand white rabbits were selected and subcutaneously injected with hCD38-Fc protein, fully emulsified with an adjuvant, at a dose of 0.5 mg / rabbit, repeated four times. After three subcutaneous immunizations, 150 μl of blood was collected from the ear vein, centrifuged, and serum was obtained. An ELISA plate was coated with hCD38-Fc protein at a concentration of 1 μg / ml, and the antibody titer in rabbit serum was detected by ELISA. The rabbit serum was serially diluted five times, starting at 1:9000 and using a 1:2 gradient. PBS was used as a negative control, and the optical density was measured at 450 nm using a microplate reader. The immunization effect was determined by OD... 450nm Value is reflected in it. (By) Figure 2 As shown in A, after the three-stage exemption: OD after dilution of 243000 450nm The antibody concentration was higher than the background level, indicating excellent immunization efficacy. The background level was 0.375. Blood was collected from the heart after four subcutaneous immunizations to prepare serum for subsequent purification of the rabbit polyclonal antibody.

[0065] 2.2 Purification of anti-human CD38 rabbit polyclonal antibody

[0066] Peripheral blood collected from rabbit hearts was centrifuged at 3000×g, 4℃ for 15 min, and serum was collected. The serum was filtered through a 0.45 μm filter and eluted with a 5 mL Protein A affinity chromatography column (GE antibody), using 50 mM citrate buffer (pH 3.0), at a flow rate of 5 mL / min. The complete elution peak was collected, and the pH of the elution buffer was adjusted to 7.0 with 1 M Tris HCl buffer (pH 9.0). The obtained rabbit polyclonal antibody was detected by SDS-PAGE and Coomassie brilliant blue staining, and antibody quantification was performed using Nanodrop. Figure 2According to B, the molecular weight of the rabbit polyclonal antibody is 140kDa, with light and heavy chains of 50kDa and 25kDa respectively, which is consistent with the theoretical value and the purity is greater than 90%.

[0067] 2.3 Affinity Identification of Anti-Human CD38 Rabbit Polyclonal Antibody

[0068] Antibody affinity was detected using an ELISA method. The CD38-Fc protein coating concentration was 1 μg / ml. Rabbit polyclonal antibody was serially diluted from 10 mg / ml to a total of 15 concentrations. A PBS blank control was included. The secondary antibody was HRP-labeled goat anti-rabbit IgG at a dilution ratio of 1:5000. The absorbance of each well at 450 nm was measured using a microplate reader. Data were processed and analyzed using GraphpadPrism software. Figure 2 From C, we can see that at a concentration of 40 ng / ml, its OD 450nm All values ​​were above 0.5, with an EC50 of 83.88 ng / ml. 50 The value represents the half-maximal effective concentration; a low value indicates a high antibody titer. Therefore, it can be concluded that the rabbit polyclonal antibody has a high titer.

[0069] 2.4 Validation of the inhibition of CD38 monoclonal antibody by anti-human CD38 rabbit polyclonal antibody

[0070] Since the plasma concentrations of CD38 monoclonal antibodies, daratumum and esartanux, are typically no more than 0.5 mg / mL, daratumum and esartanux were added to antibody-screened negative plasma samples to achieve a final concentration of 0.5 mg / mL for validation. First, the concentration of anti-human CD38 rabbit polyclonal antibody was diluted to 1 mg / mL. 50 μL of 0.8% antibody screening reagent red blood cells were centrifuged to remove the supernatant, and then 20 μL of anti-human CD38 rabbit polyclonal antibody was added. The cells were incubated at room temperature for 30 minutes. After incubation, the cells were washed three times with PBS at pH 7.4, and then resuspended in PBS at pH 7.4, adjusting the concentration to 0.8%. Next, 50 μL of red blood cells treated with anti-human CD38 rabbit polyclonal antibody and 40 μL of serum containing daratumum or esartanux were added to an anti-human globulin detection card. The cells were incubated at 37°C for 15 minutes using a K37-24 blood typing card incubator. Then, using an automated two-phase centrifuge, centrifuge at 55g for 2 minutes and then at 200g for 3 minutes in an ID centrifuge. Finally, the agglutination reaction was visually evaluated. Figure 3 As shown in Figures A and B, erythrocytes treated with anti-human CD38 rabbit polyclonal antibody can effectively inhibit the panagglutination phenomenon caused by interference from daratumol and esartanoxicillin.

[0071] 2.5 The inhibition of CD38 monoclonal antibody-mediated CD38 monoclonal antibody daratumuria by anti-human CD38 did not affect the validation of the detection of other unexpected antibodies.

[0072] Plasma or serum samples containing known clinically relevant accidental antibodies (anti-D, anti-E), as well as commercial serum samples containing anti-K and anti-Fya (CE-IMMUNDIAGNOSTIKAH), were prepared. These samples were adjusted to the minimum detectable concentration for the indirect antiglobulin assay using an anti-human globulin detection card to simulate a sample with 1+ accidental antibodies. Then, daratumol (CD38 monoclonal antibody) was added to a concentration of 0.5 mg / ml. Following the above experimental procedure, 50 μL of 0.8% antibody screening reagent red blood cells were centrifuged to remove the supernatant, and 20 μL of anti-human CD38 rabbit polyclonal antibody was added. The cells were incubated at room temperature for 30 minutes. After incubation, the cells were washed three times with PBS (pH 7.4) and resuspended in PBS at pH 7.4, adjusting the concentration to 0.8%. Then, 50 μL of red blood cells treated with anti-human CD38 rabbit polyclonal antibody and 40 μL of daratumol serum containing accidental antibodies were added to an anti-human globulin detection card for the indirect antiglobulin assay. Figure 4 From A, B, C, and D, we can see that erythrocytes treated with anti-human CD38 rabbit polyclonal antibody can effectively inhibit panagglutination interference caused by daratumol, but will not affect previously added accidental antibodies, such as anti-D, anti-E, anti-K, and anti-Fy. a The detection was performed; meanwhile, compared with the internationally accepted DTT treatment method for red blood cells, the results showed that DTT treatment of red blood cells destroyed the K antigen, leading to missed detection of anti-K. In the figure, "+" indicates the addition of the corresponding component; "-" indicates the absence of the corresponding component.

[0073] Example 3. Preparation of anti-human CD38 rabbit monoclonal antibody

[0074] 3.1 Flow Cytometry Preparation and Sorting of Rabbit Spleen

[0075] Spleens were isolated from the peritoneal cavity of New Zealand white rabbits under aseptic conditions and placed in 6-well plates. The spleens were rinsed once with PRIM1640 culture medium. The spleens were then transferred to a 70 mm filter screen and gently crushed on the screen with a 10 ml syringe needle to prepare a single-cell suspension. The spleen cells were collected, resuspended in 20 ml of PRIM1640 culture medium, counted, and stored on ice for later use.

[0076] 3.2 Single B-cell flow cytometry sorting

[0077] Add 5 ml of Alexa Fluor 647 donkey anti-rabbit IgG (Biolegend Cat.406414) antibody to 2e6 / 500 ml cells, achieving a final CD38-FITC protein concentration of 10 mg / ml. Mix well and incubate on ice for 15 min. Wash once with 10 ml of PBS containing 2% FBS, centrifuge at 300g for 5 min, discard the supernatant, resuspend in 500 ml of 2% FBS / PBS, add 5 μL of propidium iodide dye (PI, 100x), mix well, and incubate on ice for 5 min. Then perform single-cell sorting. The flow cytometer used is a Sony SH800. The positive cell population is characterized by PI-negative APC-IgG and FITC-CD38 double-positive cell populations. The flow cytometry sorting procedure is as follows: Figure 5 A. Single B cells were collected in 0.2 ml PCR tubes in an eight-cell array. The collection solution was prepared according to the instructions of the Novizan Single Cell Specific Sequence Amplification Kit as shown in Table 1. The rabbit antibody light and heavy chain specific primers were provided by Beijing ImmunoArk Pharmaceutical Technology Co., Ltd.

[0078] Table 1. Rabbit single B cell RT-PCR reaction system

[0079]

[0080] 3.3 RT-PCR amplification of the variable regions of light and heavy chains of single B cell antibodies

[0081] After single-cell sorting, the eight-tube PCR arrays were placed at -80℃ for 2 min, centrifuged at 3000 rpm for 2 min, and immediately placed in a PCR instrument to begin the RT-primary PCR reaction. After the primary PCR, 20 ml of nuclease-free purified water was added to each well, mixed well, and 4 ml was used as template for the secondary PCR. The DNA polymerase used for the secondary PCR was GoldMix (Qingke Biotechnology Co., Ltd., Cat.TSE102), with a reaction volume of 25 ml. The PCR reaction program is shown in Table 2. After separation of the secondary PCR products by 1% agarose gel electrophoresis, the DNA fragments of the light and heavy chains were approximately 350 bp in size. The cloned DNA fragments that were successfully amplified in both light and heavy chains were recovered from the gel, cloned into a T vector, and sequenced. The sequencing results were analyzed using the Igblast program to obtain the variable region sequences of the light and heavy chains of the antibody.

[0082] Table 2. Rabbit single B cell RT-PCR reaction procedure

[0083]

[0084] 3.4 Synthesis, Expression, and Purification of Anti-human CD38 Rabbit Monoclonal Antibody

[0085] The antibody light and heavy chain DNA sequences were synthesized by General Biotechnology (Anhui) Co., Ltd., and cloned into pQKR22 and pQKR23, respectively. pQKR22 is a rabbit IgG heavy chain expression vector, and pQKR23 is a rabbit κ light chain expression vector. The light and heavy chain vectors were transiently transfected into HEK293 cells at a 1:1 mass ratio. After 7 days, the supernatant was harvested and purified with Protein A to obtain the recombinant rabbit anti-hCD38-D2 monoclonal antibody (IgG isotype). The rabbit monoclonal antibody was separated by SDS-PAGE gel electrophoresis. The light and heavy chain sizes were 25 kDa and 50 kDa, respectively, which was consistent with expectations. The antibody purity was greater than 95%, as shown in the results. Figure 5 As shown in B.

[0086] Example 4. Verification of anti-human CD38 rabbit monoclonal performance

[0087] 4.1 Affinity Identification of Anti-Human CD38 Rabbit Monoclonal Antibody

[0088] Antibody affinity was detected using ELISA. The CD38-Fc protein coating concentration was 1 μg / ml. Rabbit monoclonal antibody was serially diluted from 20 mg / ml to a total of 15 concentrations. A PBS blank control was included. The secondary antibody was HRP-labeled goat anti-rabbit IgG (Suzhou Botron Immunotechnology Co., Ltd., BF03008-1ml), diluted 1:5000. The absorbance of each well at 450 nm was measured using a microplate reader. Data were processed and analyzed using Graphpad Prism software. Figure 5 As shown in Figure C, the binding of the D2 rabbit monoclonal antibody to the immunogen CD38 occurs in the concentration range of 20 ng / ml to 20000 ng / ml, and the affinity fitting curve is close to a linear relationship.

[0089] 4.2 Fortebio Affinity Analysis of Anti-Human CD38 Rabbit Monoclonal Antibody

[0090] Human CD38-his protein was purchased from SinoBiological (Cat: 10818-H08H) in Beijing. Antibody binding activity was detected using a ForteBio molecular interaction analyzer. The antigen was labeled His, and a Ni-NTA sensor was used to immobilize the antigen at a working concentration of 10 mg / mL. After antigen binding equilibration, rabbit anti-human CD38-D2 antibody was bound, and the binding / dissociation curves were observed. Protein binding activity was analyzed using the instrument's built-in program. The results are shown in Table 3. Figure 5 As shown in Figure D, the results indicate that D2 binds to human CD38 protein with a KD value of 3.822E-10, demonstrating strong affinity.

[0091] Table 3. Detection of binding activity of anti-human CD38 rabbit monoclonal antibody to CD38 antigen

[0092]

[0093] 4.3 Epitope Competition Analysis of Anti-human CD38 Rabbit Monoclonal Antibody

[0094] The Fortebio method was used to detect the competitive binding of anti-human CD38 rabbit monoclonal antibody and the clinically used CD38 monoclonal antibody daratumum to the CD38 molecule. The CH1 probe bound to daratumum antibody, and after equilibration, it sequentially bound to the CD38 molecule and then to the rabbit monoclonal antibody. If the prepared anti-human CD38 rabbit monoclonal antibody and daratumum antibody bound to the same epitope of CD38, the binding curve of the anti-human CD38 rabbit monoclonal antibody disappeared; conversely, if the anti-human CD38 rabbit monoclonal antibody and daratumum antibody bound to different epitopes, a binding curve of the anti-human CD38 rabbit monoclonal antibody would appear. Figure 5 As can be seen from E, after daratumumab binds to CD38 molecules, it blocks the binding of hCD38-D2, indicating that D2 and daratumumab have the same binding site.

[0095] 4.4 FACS Detection of Binding of Anti-human CD38 Rabbit Monoclonal Antibody to CD8 Molecules on the Surface of Human Erythrocytes

[0096] The concentrated red blood cells (Shanghai Blood Biopharmaceutical Co., Ltd.) were washed once with PBS and then diluted to 5×10⁻⁶. 7 Cells / mL; dilute the anti-human CD38 rabbit monoclonal antibody to a final concentration of 1000 µg / mL. Using this as the stock solution, sequentially dilute the antibody 1:9 in three gradients. Add 10 μL of the antibody to 90 μL of the adjusted cell sample, mix well, and the final concentrations are 100 μg / mL, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL, respectively. Incubate at 4°C for 1 h, wash once with PBS, add FITC goat anti-rabbit IgG secondary antibody, incubate at room temperature for 0.5 h, wash once with PBS, add 200 μL of PBS, and analyze by flow cytometry. The results show that the anti-human CD38 rabbit monoclonal antibody D2 specifically binds to the CD38 molecule on the surface of human erythrocytes. Figure 5 As shown in F and G.

[0097] 4.5 Anti-human CD38 rabbit monoclonal antibody inhibits CD38 monoclonal antibody

[0098] To verify whether the recombinant rabbit anti-hCD38-D2 monoclonal antibody could inhibit the interference of daratumum and esartanux, 50 μL of 0.8% antibody screening reagent red blood cells were first centrifuged to remove the supernatant, and then 20 μg of rabbit anti-hCD38-D2 monoclonal antibody was added and incubated at room temperature for 30 minutes. After incubation, the cells were washed three times with PBS (pH 7.4) and resuspended in PBS at pH 7.4, adjusting the concentration to 0.8%. Next, 50 μL of red blood cells treated with rabbit anti-hCD38-D2 monoclonal antibody and 40 μL of serum supplemented with daratumum were taken, with a final concentration of 0.5 mg / mL. Finally, an antibody screening experiment was performed using an anti-human globulin detection card. Figure 6 As shown in Figures A and B, erythrocytes treated with rabbit monoclonal antibody against human CD38-D2 can inhibit the panagglutination phenomenon caused by interference from daratumol and esartanoxicillin.

[0099] Example 5. Determining the minimum concentration and shortest incubation time of an anti-human CD38 rabbit monoclonal antibody sufficient to inhibit daratum interference.

[0100] 5.1 Determine the minimum concentration of anti-human CD38 rabbit monoclonal antibody sufficient to inhibit daratum interference.

[0101] In the experiment, for ease of labeling, the rabbit anti-human CD38 monoclonal antibody was represented as rabbit anti-hCD38-D2 monoclonal antibody. First, the concentration of the recombinantly expressed rabbit anti-hCD38-D2 monoclonal antibody was adjusted to 1 mg / mL. To determine the minimum concentration of rabbit anti-hCD38-D2 monoclonal antibody required to inhibit daratumum interference, 50 μL of 0.8% antibody screening reagent red blood cells were taken, centrifuged to remove the supernatant, and then different amounts of monoclonal antibody were added, from 2 μL to 20 μL, and incubated at room temperature for 30 minutes. After incubation, the cells were washed three times with PBS at pH 7.4, and then resuspended in PBS at pH 7.4, adjusting the concentration to 0.8%. Except for the control group, all samples contained a fixed concentration of 0.5 mg / mL daratumum. Finally, the indirect antiglobulin experiment was performed according to the above method. Figure 7 Therefore, for 50 μL of 0.8% cells, at least 12 μL of rabbit anti-hCD38-D2 monoclonal antibody needs to be added to effectively inhibit daratum's interference.

[0102] 5.2 Determine the minimum incubation time for anti-human CD38 rabbit monoclonal antibodies sufficient to inhibit daratum interference.

[0103] An optimization experiment was conducted by gradually reducing the incubation time, from 30 minutes to 15 minutes, 10 minutes, 5 minutes, and 0 minutes. First, 50 μL of 0.8% erythrocytes and 12 μL of rabbit anti-hCD38-D2 monoclonal antibody were incubated at room temperature for 15 minutes, 10 minutes, 5 minutes, and 0 minutes, respectively. After incubation, the supernatant was removed by centrifugation, and the samples were resuspended in PBS at pH 7.4, adjusting the concentration to 0.8%. Except for the control group, all samples were supplemented with a fixed concentration of 0.5 mg / mL daratum. Finally, an indirect antiglobulin assay was performed according to the above method. Figure 8 It can be seen that for 50 μL of 0.8% cells, rabbit anti-hCD38-D2 monoclonal antibody needs to be incubated with red blood cells at room temperature for 10 minutes to effectively inhibit the interference of daratum.

[0104] Example 6. Verification of the inhibition of transfusion compatibility test interference by anti-human CD38 rabbit monoclonal antibody.

[0105] 6.1 Effects of anti-human CD38 rabbit monoclonal antibody on erythrocyte blood group antigens

[0106] Anti-K, Anti-k, Anti-D, Anti-M, Anti-N, Anti-S, Anti-s, Anti-Jk a Anti-Jk b Anti-Fy a Anti-Fy b Anti-Di a and anti-Wr a The IgG antibody reagent was purchased from CE immundiagnostika GmbH, Germany. Anti-E, anti-e, anti-C, and anti-c IgG antibodies were prepared in-house. The effects of anti-human CD38 rabbit monoclonal antibody and DTT treatment on erythrocyte antigens were compared. Figure 9 From A, 9B, 9C, and 9D, we can conclude that the anti-human CD38 rabbit monoclonal antibody has no effect on red blood cell blood group antigens, while DTT treatment will destroy the K and K antigens on red blood cells.

[0107] 6.2 The inhibition of daratum interference by anti-human CD38 rabbit monoclonal antibody did not affect the detection of other unexpected antibodies.

[0108] Following the procedure in 5.2 of Example 5, plasma or serum samples containing known clinically relevant incidental antibodies (anti-D, anti-E), as well as anti-K and anti-Fy, are... aCommercial serum samples (CE-IMMUNDIAGNOSTIKAH) were adjusted to the lowest detectable concentration for the indirect antiglobulin assay. Daratumol was then added to a concentration of 0.5 mg / ml. Next, following the above experimental procedure, 50 μL of 0.8% antibody screening reagent red blood cells (Oxend, USA) were centrifuged to remove the supernatant, and 12 μL of rabbit anti-hCD38-D2 monoclonal antibody was added. The cells were incubated at room temperature for 10 minutes. After incubation, the supernatant was removed again by centrifugation, and the cells were resuspended in PBS at pH 7.4, adjusting the concentration to 0.8%. Then, 50 μL of red blood cells treated with rabbit anti-hCD38-D2 monoclonal antibody and 40 μL of daratumol serum containing unexpected antibodies were added to an antiglobulin detection card for the indirect antiglobulin assay. Figure 10 From A, B, C, and D, we can see that rabbit anti-hCD38-D2 monoclonal antibody does not affect the detection of accidental antibodies, which include anti-D, anti-E, anti-K, and anti-Fy. a .

[0109] 6.3 Anti-human CD38 rabbit monoclonal antibody inhibits interference from daratum in clinical samples

[0110] This example involves 36 patients treated with daratum. Of these, 34 (94.5%) were diagnosed with multiple myeloma, 1 with non-Hodgkin's lymphoma, and 1 with myelodysplastic syndrome. Patient samples were obtained from multiple medical institutions. All patient samples, including EDTA plasma, serum, or apheresis, were stored at -80°C or below after collection until use. First, antibody titers were tested on plasma samples from 30 patients, ranging from 4 to 4096. Following the experimental procedures described above, 50 μL of 0.8% antibody screening reagent red blood cells were centrifuged to remove the supernatant, and then 12 μL of rabbit anti-hCD38-D2 monoclonal antibody was added and incubated at room temperature for 10 minutes. After incubation, the cells were centrifuged again to remove the supernatant, and then resuspended in PBS at pH 7.4, adjusting the concentration to 0.8%. Finally, an anti-human globulin detection card was used for antibody screening. The results showed that the rabbit anti-hCD38-D2 monoclonal antibody could inhibit the interference of daratum in clinical samples, as shown in Table 4.

[0111] Table 4. Rabbit anti-hCD38-D2 monoclonal antibody inhibits daratumumab interference in clinical samples

[0112]

[0113]

[0114] 9 a Patient 9 was identified as having IgG-Wra .

[0115] twenty four b Patient 27 was found to have IgG-cE in his body.

Claims

1. A rabbit monoclonal antibody against human CD38 for inhibiting interference from human CD38 monoclonal antibodies in transfusion compatibility testing, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human CD38 rabbit monoclonal antibody is shown in Sequence 1, and the amino acid sequence of the light chain variable region is shown in Sequence 2.

2. The use of the monoclonal antibody as described in claim 1 in the preparation of a reagent for inhibiting the interference of human CD38 monoclonal antibody on transfusion compatibility testing, wherein the human CD38 monoclonal antibody is selected from daratumumab and esartuximab.

3. The use as described in claim 2, characterized in that, The anti-human CD38 rabbit monoclonal antibody is used to bind to the CD38 antigen on the surface of red blood cells.

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