Anti-erythrocyte membrane antibodies and uses thereof

Abstract

The application discloses an anti-red blood cell membrane antibody and application thereof, and relates to a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO:1, or is a conservative mutant obtained by adding, deleting, replacing or modifying one or more amino acids of the sequence; the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO:2, or is a conservative mutant obtained by adding, deleting, replacing or modifying one or more amino acids of the sequence; the sequences of CDR1, CDR2 and CDR3 of the variable region of the heavy chain are shown as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; the sequences of CDR1, CDR2 and CDR3 of the variable region of the light chain are shown as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; and the anti-red blood cell membrane antibody is used in the construction of a bifunctional antibody reagent. The anti-red blood cell membrane antibody is used in chromatography detection, and rbc can be intercepted on a sample pad by processing on the sample pad, so that whole blood can be detected.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular to an anti-erythrocyte membrane antibody and its application. Background Technology

[0002] Anti-erythrocyte membrane antibodies (RBC antibodies) were first reported by Donatht and Landsteiner in 1904 and were the first autoantibodies elucidated in the human body, now known as Donath-Landsteiner (DL) antibodies. They are classified into three main categories: warm antibodies (WAS), cold agglutinin antibodies (CAs), and DL antibodies. These antibodies can cause autoimmune hemolytic anemia (AHA). Antiglobulin tests are used to detect anti-erythrocyte membrane antibodies. There are two types of antiglobulin tests: direct antiglobulin tests detect antibodies on the surface of erythrocytes; indirect antiglobulin tests detect free anti-erythrocyte antibodies. The anti-erythrocyte antibodies in SLE and idiopathic autoimmune hemolytic anemia are warm antibodies, the vast majority of which are IgG type. Cold antibodies are most commonly IgM type antibodies, and SLE with hemolytic anemia is associated with cold antibodies. Anti-erythrocyte membrane antibodies cause the destruction of erythrocytes, reducing the erythrocyte count.

[0003] Anti-erythrocyte membrane antibodies are closely associated with autoimmune hemolytic anemia (AIHA), which can be spontaneous, primary, or secondary to other diseases. Secondary AIHA mostly presents with polyclonal warm antibody autoimmune responses, related to HLA-A1, A8, and B8; however, the pathogenesis of AIHA induced by anti-Cas and anti-DL antibodies is not yet fully understood. AIHA is diagnosed by detecting anti-RBC antibodies in conjunction with other hemolytic anemia markers (such as elevated LDH, increased reticulocytes, and decreased haptoglobin). Erythrocyte destruction caused by CAs and anti-DL antibodies can lead to paroxysmal cold hemoglobinuria (PCH). Widespread infections are associated with Was induction; Was is often detectable in viral infections, especially in children. Cas can be found in the serum of 50%–80% of patients with mycoplasma pneumonia and 30%–50% of patients with EBV infection. Anti-BrRBC antibodies are associated with primary AIHA and hemolytic anemia caused by SLE, chronic lymphocytic leukemia, and hairy cell leukemia.

[0004] Anti-erythrocyte membrane antibodies, after modification and conjugation with antigens or other antibodies, can form bispecific conjugates, namely bifunctional antibodies that have developed rapidly in recent years. These are non-agglutinating antibodies that can bind to red blood cells (RBCs) of various human blood types without causing hemagglutination. However, under the bridging of antiglobulin assays, they can strongly agglutinate RBCs. If the blood sample contains the corresponding antibody or antigen, the anti-RBC antibody binds to the RBC, and the antigen or antibody in the conjugate binds to the corresponding antibody or antigen in the blood sample, causing the RBCs to bridge and agglutinate. The operation method is as convenient, rapid, and highly specific and sensitive as blood typing. Therefore, this method has broad application prospects and has been successfully used in the diagnosis of viral diseases such as HIV and HBV. Current technologies using anti-erythrocyte membrane antibodies have low detection sensitivity, and the conventional concentration used in chromatographic detection platforms is 0.5 mg / ml, requiring large quantities. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-erythrocyte membrane antibody, which has good stability, high sensitivity, and can reduce the concentration used to 0.25 mg / ml.

[0006] Another object of the present invention is to provide the application of the said red blood cell membrane antibody, such as in chromatography detection, where it is applied to a sample pad to retain RBCs in the sample pad, and whole blood can be detected.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] An anti-erythrocyte membrane antibody comprising a heavy chain and a light chain, wherein the variable region amino acid sequence of the heavy chain is as shown in SEQ ID NO: 1, or a conserved variant of the sequence obtained by conserved mutations involving the addition, deletion, substitution, or modification of one or more amino acids;

[0009] The variable region amino acid sequence of the light chain is shown in SEQ ID NO: 2, or it is a conserved variant obtained by conserved mutations of one or more amino acid additions, deletions, substitutions, or modifications.

[0010] The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0011] The sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0012] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 9; the amino acid sequence of the light chain is shown in SEQ ID NO: 10.

[0013] A polynucleotide fragment encoding an anti-erythrocyte membrane antibody belonging to any of the above.

[0014] A diagnostic reagent containing the aforementioned anti-erythrocyte membrane antibody.

[0015] A test kit containing the aforementioned anti-erythrocyte membrane antibody.

[0016] The use of the anti-erythrocyte membrane antibody in the construction of bifunctional antibody reagents.

[0017] Anti-erythrocyte membrane antibodies are used in chromatography detection. By processing the sample pad, RBCs can be retained on the sample pad, and whole blood can be detected.

[0018] Beneficial effects:

[0019] The anti-erythrocyte membrane antibody provided by this invention has good stability and high sensitivity. The concentration can be reduced to 0.25 mg / ml. Using 0.25 mg / ml is sufficient to keep erythrocytes on the sample pad, and the background is relatively clear and clean, resulting in smooth plate running. Detailed Implementation

[0020] Example 1

[0021] 1. Antigen Immunization Process

[0022] Antigen preparation procedure: Dilute red blood cells to 2*10^7 cells / ml. For the first immunization, use 50μl of cell solution and 100μl of Freund's complete adjuvant. For subsequent immunizations, use 50μl of antigen solution and 50μl of Freund's incomplete adjuvant. Use two 2ml syringes to draw up the antigen solution and adjuvant respectively, expelling as much air as possible from the syringes and connecting them with a T-connector. First, push the antigen into the adjuvant, and then repeatedly push the connected syringes for about 20-40 minutes until the emulsified antigen does not diffuse in water.

[0023] Immunization of mice: Select healthy laboratory mice with no obvious external injuries, good appetite, and active behavior; mice aged 6 to 8 weeks are preferred. Immunization is performed by subcutaneous injection of antigen at multiple sites. After the first immunization, a second immunization is given at an interval of 14 days. Subsequent immunizations are given at intervals of 7 days. After the third immunization, blood is collected from the tail on the 7th day after each immunization to test the titer. Immunization is stopped when the required titer is reached, and fusion is initiated.

[0024] 2. Preparations

[0025] Prepare HAT medium and dispense DMEM and PEG. Sterilize surgical instruments, 50ml plastic centrifuge tubes, and 50ml glass centrifuge tubes. Resuscitate SP2 / 0 cells one week in advance and culture them to a good growth state. One day in advance, collect feeder cells in HAT medium and plate them. Five feeder cell plates are needed for one confluent mouse.

[0026] 3. Integration

[0027] Processing SP2 / 0: Disperse the supernatant, transfer to a 50ml plastic centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add DMEM.

[0028] Disperse the contents, centrifuge at 1000 rpm for 5 minutes, add DMEM to about 10 ml, disperse again, count the contents, and set aside.

[0029] Extracting spleen cells

[0030] 3.1. Blood was collected from the eye sockets of mice, and the mice were euthanized by dislocation of the neck. The blood samples were kept as positive controls.

[0031] 3.2 The mouse spleen was isolated. The outside of the spleen was rinsed with DMEM, and the inside of the spleen was rinsed with DMEM. The rinsing fluid was collected and transferred to a 10 ml glass centrifuge tube and counted.

[0032] 3.3 Centrifuge at 1000 rpm for 5 min, discard the supernatant, add DMEM, blow away the supernatant and transfer to a 50 ml glass centrifuge tube. Simultaneously transfer the previously treated SP2 / 0 to a 50 ml glass centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and grind it evenly on the back of your hand.

[0033] 3.4 Place in a 37℃ warm water bath, add 1ml of PEG within 1 minute while shaking, and let stand for 90 seconds.

[0034] 3.5 Add 1 ml DMEM within 1 min, add 1 ml DMEM within 30 s, gradually accelerate, add DMEM to 40-50 ml, let stand at 37℃ for 10 min, centrifuge at 800 r for 6 min, discard the supernatant, add HAT and plate.

[0035] 3.6 Change the medium halfway after 3 days, and change it completely after one week, then use HAT medium for culturing.

[0036] 4. Subclone selection process

[0037] 4.1 Observe the cell status. When cell clusters are visible to the naked eye in the 96 wells, perform whole-plate testing. Select wells with higher positive rates for subcloning. One positive well is placed on one plate.

[0038] 4.2 Once cell clusters are visible to the naked eye in 6 wells, subcloning is performed. 12 wells with single cell clusters are selected from each plate for subcloning. Cells with high positivity and good cell condition are selected for the next subcloning step. Subcloning is performed at least three times. Finally, single-clone cell clusters with high positivity and good cell condition are selected for final establishment.

[0039] 4.3 Transfer the selected cell clusters to 24-well culture, and after the cells in the 24-well culture are well grown, transfer them to small square flasks for further culture.

[0040] 4.4 After the cells have grown well in the small flask, they are cryopreserved and ascites is prepared.

[0041] 5. Ascites preparation process

[0042] 5.1 One week in advance, inject the mice with paraffin, 0.5 ml per mouse.

[0043] 5.2 After the cells in the small flasks have grown well, disperse them by blowing and transfer them to 10ml glass centrifuge tubes. Centrifuge at 1000r for 5min, discard the supernatant, add 1ml DMEM, count the cells, and inject 0.8×10⁻⁶ cells per mouse. 6 One cell was diluted and injected into each mouse with 0.5 ml of the diluted cells, and then injected into the mouse's peritoneum.

[0044] 5.3 Observe the condition of the mice. Once the mice's abdomens are noticeably swollen, begin to drain and collect the ascites fluid.

[0045] 6. Antibody purification process

[0046] Affinity chromatography purification using Protein A

[0047] 6.1 Preparation:

[0048] 6.1.1 Reagent preparation: Equilibration buffer: 10mM Tris-NaCl (pH 8.3)

[0049] Dissociation buffer: 0.1M citric acid (pH 5.0)

[0050] Regeneration buffer: 0.1M glycine (pH 2.7)

[0051] Dialysis buffer: 20 mM Tris-NaCl (pH 7.5) or 10 mM PBS (pH 7.5)

[0052] pH neutralization solution: 2M Tris (pH 8.0)

[0053] 6.1.2 Equipment preparation: Protein A column, low temperature high speed centrifuge, balance, peristaltic pump.

[0054] 6.2 Operating Procedures

[0055] 6.2.1 Sample preparation: The frozen ascites fluid was thawed overnight at 10-15℃ the day before, and the oil was removed by filtering with degreased cotton soaked in purified water. The filtered ascites fluid was then filtered through a 0.55μm capsule filter membrane.

[0056] Column preparation: Pack an appropriate amount of Protein A packing material, wash with purified water for 5-10 column volumes, rinse to remove ethanol, and the packing material volume should not exceed 2 / 3 of the column volume.

[0057] 6.2.2 Equilibration: The Protein column was equilibrated with 5 column volumes of 10 mM Tris-NaCl (pH 8.3) equilibration buffer. After the column was equilibrated, the protein UV detector reading was adjusted to 0 as the baseline.

[0058] 6.2.3 Sample loading: The ascites fluid was diluted with two volumes of equilibration buffer 10mM Tris-NaCl (pH 8.3) as the sample for loading. The sample was loaded slowly using a peristaltic pump, and the number of times the column packing was used was recorded.

[0059] 6.2.4 Equilibration: After sample loading, equilibrate with 10 mM Tris-NaCl (pH 8.3) using equilibration buffer and wash with 10-12 column volumes until no protein flows out as detected by Coomassie Brilliant Blue.

[0060] 6.2.5 Dissociation: After equilibration, add 10% of the sample volume of pH neutralization solution to the receiving vessel and use 0.1M citric acid (pH 5.0) for dissociation. Start collecting protein when the protein UV detector value is greater than 0.1 and stop collecting protein when the protein UV detector value is less than 0.1.

[0061] 6.2.6 Regeneration: After dissociation, wash with 2 column volumes of 0.1M glycine (pH 2.7) buffer;

[0062] 6.2.7 Equilibration: After regeneration, equilibrate the column with 5-10 column volumes of 10mM Tris-NaCl (pH 8.3) equilibration buffer.

[0063] 6.2.8 Dialysis: Combine protein-containing components and perform dialysis using 20mM Tris-NaCl (pH 7.5) or 10mM PBS (pH 7.5) dialysis buffer, depending on product requirements. The dialysis ratio is 1:10, and dialysis is performed 5 times, with an interval of no less than 5 hours between each buffer change.

[0064] 6.2.9 Antibody treatment: After dialysis, the antibody was recovered and filtered through a 0.22 μm filter membrane. The concentration was then adjusted and 0.1% Proclin 300 was added to preserve the protein.

[0065] 6.3 Protein A column storage: If the Protein A column is not used for more than one week, flush the column with 5 column volumes of 20% ethanol and store at 5-8℃.

[0066] 6.4 Cleaning of peristaltic pump inlet tubes: When using the same peristaltic pump to process other samples, the silicone tubes should be rinsed 5-10 times with the same sample loading buffer before proceeding with other operations.

[0067] 7. Antibody sequencing process

[0068] Cell samples were reverse transcribed to obtain cDNA; the variable regions of the heavy and light chains were amplified and obtained; the cDNA was cloned into the pMD18-T vector and sequenced; the sequencing results were compared using IMGT / V-QUEST to obtain the complete sequence. Antibody sequencing is a technique known to those skilled in the art. Specific sequences are shown in the sequence listing. The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 2. The sequences of CDR1, CDR2, and CDR3 of the variable region of the heavy chain are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. The sequences of CDR1, CDR2, and CDR3 of the variable region of the light chain are shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0069] RBC monoclonal antibody testing standards

[0070] Physicochemical properties

[0071] Visual inspection: The outer packaging is intact, the labeling is clear and the information is accurate, and the contents are a clear liquid.

[0072] Antibody concentration detection: See "Protein Concentration Experimental Measurement Record".

[0073] Antibody purity detection: see the "Standard Operating Procedures for Electrophoresis Detection".

[0074] The test results are shown in Table 1.

[0075] Table 1

[0076]

[0077] RBC testing is performed on the HBsAg test.

[0078] 1. Gold Standard

[0079] HBsAg was used to label the HBsAg antibody, and the total OD value of the gold spray solution was 150.

[0080] 2. NC membrane

[0081] HBsAg antibody was coated with HBsAg at a concentration of 1.0 mg / mL.

[0082] HBsAg-coated antibody was used to scratch the NC membrane at a concentration of 1.0 mg / ml and a scratching volume of 1.0 μl / cm. Sample pads: The sample pads were treated with the RBCs to be detected. The components and contents of the sample pad treatment solution are shown in Table 2.

[0083]

[0084]

[0085] The above sample pad treatment solution was dispensed into 5 ml tubes, and 0.25 mg / ml of RBC was added to each tube.

[0086] The sample pads were treated with the above sample pad treatment solution and dried overnight in an oven at 37°C.

[0087] The above materials are assembled and then cut into reagent strips, 3mm / strip.

[0088] Test samples: Whole blood samples will be drawn on-site for later use.

[0089] Using 0.25 mg / ml is sufficient to keep red blood cells on the sample pad, and the background is relatively clear and clean, allowing for smooth plate running.

[0090] Functional testing

[0091] Test samples: 3 fresh clinical whole blood samples, each sample is tested for 1 person.

[0092] Sample addition method: Place the test strip flat on the table, add 65ul of sample to the bottom of the test strip using a pipette, and start the stopwatch at the same time.

[0093] Result interpretation: All tests should be interpreted within 15 minutes.

[0094] Stability: Repeat the freeze-thaw cycle 3 times to prepare reagent strips.

[0095] Select a benchmark batch and the inspection batch for testing together.

[0096]

[0097] The red blood cell membrane antibody detection of this invention has a clean background and high sensitivity. The concentration used in conventional commercial products is 0.5 mg / ml, while the antibody of this invention can achieve the same result with a concentration as low as 0.25 mg / ml.

Claims

1. An anti-erythrocyte membrane antibody, characterized in that: It comprises a heavy chain and a light chain, wherein the variable region amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the variable region amino acid sequence of the light chain is shown in SEQ ID NO:

2.

2. The anti-erythrocyte membrane antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 9; the amino acid sequence of the light chain is shown in SEQ ID NO:

10.

3. A polynucleotide fragment, characterized in that, It encodes the anti-erythrocyte membrane antibody as described in claim 1 or 2.

4. A test kit, characterized in that, It contains the anti-erythrocyte membrane antibody as described in claim 1 or 2.

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