CN115819591A - Anti-erythrocyte membrane antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-erythrocyte membrane antibody and application thereof, which comprises a heavy chain and a light chain, wherein the variable region amino acid sequence of the heavy chain is shown as SEQ ID NO:1 or conservative variant obtained by conservative mutation of the sequence through one or more amino acid addition, deletion, substitution and modification; the variable region amino acid sequence of the light chain is shown as SEQ ID NO:2 or conservative variant obtained by conservative mutation of the sequence through one or more amino acid addition, deletion, substitution and modification. The sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, respectively. The sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO: shown in fig. 8. The application of the anti-erythrocyte membrane antibody in constructing a bifunctional antibody reagent. When the anti-erythrocyte membrane antibody is used in chromatographic detection, rbc can be trapped on a sample pad after the antibody is treated on the sample pad, and whole blood can be detected.

Description

Anti-erythrocyte membrane antibody and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an anti-erythrocyte membrane antibody and application thereof.

Background

Anti-erythrocyte membrane antibodies (RBC antibodies) were first reported in 1904 by Donath and Landsteiner as the first autoantibodies elucidated in humans, now called Donath-Landsteiner (DL) antibodies. The antibody is divided into three categories, namely Warm Antibody (WAS), cold agglutinin antibody (CAs) and DL antibody. The antibody can cause Autoimmune Hemolytic Anemia (AHA). The antiglobulin test is used to detect anti-erythrocyte membrane antibodies. There are two direct and indirect antiglobulin tests: direct antiglobulin test for detecting antibodies on the surface of erythrocytes; the indirect antiglobulin test is used to detect free anti-erythrocyte antibodies. Anti-erythrocyte antibodies of SLE and idiopathic autoimmune hemolytic anemia are warm antibodies, most of which are of the IgG type. Cold antibodies are most commonly antibodies of the IgM type, and SLE with hemolytic anemia is associated with cold antibodies. Anti-erythrocyte membrane antibodies cause destruction of erythrocytes, resulting in a decrease in the number of erythrocytes.

Anti erythrocyte membrane antibodies are closely related to autoimmune hemolytic anemia, and can be spontaneous, primary or secondary to other diseases. The majority of secondary AIHA has polyclonal warm antibody autoimmune response, and is related to HLA-A1, A8 and B8; the pathogenesis of anti-Cas, anti-DL induced AIHA is less clear. Diagnosing AIHA by detecting anti-RBC antibodies in combination with other hemolytic anemia indicators (e.g., elevated LDH, reticulocyte proliferation, haptoglobin depletion, etc.); destruction of erythrocytes by CAs and anti-DL antibodies can cause Paroxysmal Cold Hemoglobinuria (PCH); widespread infection is associated with the induction of Was, which is often detectable in patients with viral infections, especially pediatric viral infections; cas can appear in serum from 50% to 80% Mycoplasma pneumonia, 30% to 50% EBV infected patients. anti-BrRBC antibodies are associated with both primary AIHA and hemolytic anemia arising from SLE, chronic lymphocytic leukemia, hairy cell leukemia.

Anti erythrocyte membrane antibodies are engineered to conjugate with antigens or other antibodies to form bispecific conjugates, i.e., bifunctional antibodies that have evolved rapidly in recent years. It is a non-agglutinated antibody which can be combined with human blood group Red Blood Cells (RBC) and does not cause hemagglutination, but can strongly agglutinate RBC under the condition of anti-globulin test bridge, if the blood sample contains corresponding antibody or antigen, the anti-RBC antibody is combined with RBC, the antigen or antibody in the combination is combined with corresponding antibody or antigen in the blood sample, so that RBC bridge together to cause agglutination. The operation method is as simple and convenient as the blood-checking type, and has the advantages of rapidness, strong specificity and high sensitivity. Therefore, the method has wide application prospect and is successfully used for diagnosing virus diseases such as HIV, HBV and the like. The anti-erythrocyte membrane antibody adopted in the prior art has low detection sensitivity, the conventional use concentration of the anti-erythrocyte membrane antibody on a chromatographic detection platform is 0.5mg/ml, and the use amount is large.

Disclosure of Invention

The invention aims to provide an anti-erythrocyte membrane antibody which has good stability and high sensitivity and can reduce the use concentration to 0.25mg/ml.

Another purpose of the invention is to provide the application of the erythrocyte membrane antibody, such as the application in chromatographic detection, rbc can be trapped on a sample pad after the antibody is processed on the sample pad, and whole blood can be detected.

In order to achieve the purpose, the invention adopts the technical scheme that:

an anti-erythrocyte membrane antibody, which comprises a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:1 or conservative variant obtained by conservative mutation of the sequence through one or more amino acid addition, deletion, substitution and modification;

the variable region amino acid sequence of the light chain is shown as SEQ ID NO:2 or conservative variant obtained by conservative mutation of the sequence through one or more amino acid addition, deletion, substitution and modification.

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

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

The amino acid sequence of the heavy chain is shown as SEQ ID NO:9 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 10.

A polynucleotide fragment encoding the anti-erythrocyte membrane antibody of any one of the above.

A detection reagent containing the anti-erythrocyte membrane antibody.

A detection kit contains the anti-erythrocyte membrane antibody.

The application of the anti-erythrocyte membrane antibody in constructing a bifunctional antibody reagent.

When the anti-erythrocyte membrane antibody is used in chromatographic detection, rbc can be trapped on a sample pad after the antibody is treated on the sample pad, and whole blood can be detected.

Has the advantages that:

the anti-erythrocyte membrane antibody provided by the invention has good stability and high sensitivity, can reduce the use concentration to 0.25mg/ml, can stop erythrocytes on a sample pad by using 0.25mg/ml, has clear and clean background and runs smoothly.

Detailed Description

Example 1

1. Antigen immunization process

Antigen preparation process: diluting erythrocytes to 2 x 10^ 7/ml, and immunizing for the first time by taking 50 mu l of cell solution and 100 mu l of Freund complete adjuvant; subsequent immunizations took 50. Mu.l of antigen solution and 50. Mu.l of Freund's incomplete adjuvant. Two 2ml syringes are used for respectively sucking the antigen solution and the adjuvant, air in the syringes is discharged as much as possible and linked by a tee joint, the antigen is pushed into the adjuvant, and the connected syringes are repeatedly pushed for about 20-40 min until the emulsified antigen is not diffused in water.

The mouse immunization process comprises the following steps: healthy experimental mice are selected, the mice with no obvious trauma on the body surface, active food and 6 to 8 weeks old are suitable. The immunization method is subcutaneous multi-point injection of antigen for immunization. After the first immunization, the second immunization was performed at an interval of 14 days. Immunizations were performed 7 days later. And after the third immunization, cutting off the tail and taking blood to detect the titer 7 days after each immunization, and stopping the immunization when the titer meets the requirement for fusion.

2. Preparation work

Preparing HAT culture medium, and subpackaging DMEM and PEG. Surgical instruments, 50ml plastic centrifuge tubes, 50ml glass centrifuge tubes were sterilized. SP2/0 cells were revived one week earlier, cultured and allowed to grow well. Feeder cells were harvested and plated one day in advance with HAT medium and 5 feeder cell plates were plated for one mouse.

3. Fusion

Treatment SP2/0: blowing, transferring to 50ml plastic centrifuge tube, centrifuging at 1000r for 5min, discarding supernatant, adding DMEM,

blowing, centrifuging at 1000r for 5min, adding DMEM to about 10ml, blowing, and counting for use.

Extraction of splenocytes

3.1. Mice were bled from their eye sockets, sacrificed by cervical dislocation, and blood samples were reserved as positive controls.

3.2 separating out the spleen of the mouse, washing the outside of the spleen by DMEM, then washing the inside of the spleen by DMEM, collecting washing liquid, transferring the washing liquid to a 10ml glass centrifuge tube, and counting.

3.3 Centrifuging at 1000r for 5min, discarding the supernatant, adding DMEM, blowing off and transferring to a 50ml glass centrifuge tube, simultaneously transferring the SP2/0 treated previously to a 50ml glass centrifuge tube, centrifuging at 1000r for 5min, discarding the supernatant, and grinding evenly on the back of the hand.

3.4 Warm water bath at 37 deg.C, adding 1ml PEG within 1min while shaking, and standing for 90s.

3.5 Adding 1ml DMEM in 1min, adding 1ml DMEM in 30s, gradually accelerating, adding DMEM to 40-50ml, standing at 37 ℃ for 10min, centrifuging at 800r for 6min, discarding supernatant, and adding HAT for plating.

3.6 Half the medium change after 3 days, and full medium change after one week, and HAT medium change.

4. Subclone selection process

4.1 observing the cell state, carrying out whole plate detection after a cell mass which can be observed by naked eyes exists in 96 holes, selecting the hole with higher positive for subcloning, and carrying out subcloning one positive hole by one plate.

4.2 when the 6 holes have cell clusters which can be observed by naked eyes, performing subcloning and picking, selecting 12 holes of single cell clusters for each plate, performing the next subcloning on the cells with high positive and good cell state, performing the subcloning at least three times, and finally selecting the single cell cluster fixed strains with high positive and good cell state.

4.3 transferring the picked cell mass to 24-hole culture, and transferring to a small square flask for culture after 24-hole cells grow well.

4.4 after the cells in the small square bottle grow well, freezing and storing and preparing ascites.

5. Process for the preparation of ascites

5.1 mice were injected one week earlier with paraffin, 0.5ml per mouse.

5.2 after the cells in the flask are well grown, blowing off, transferring to a 10ml glass centrifuge tube, centrifuging for 5min at 1000r, discarding the supernatant, adding 1ml DMEM, counting, injecting 0.8 × 10 per mouse ⁶ Each mouse was diluted by injecting 0.5ml of each cell, and then the mouse was intraperitoneally injected with the cells.

5.3 observing the state of the mouse, and beginning to extract and collect the ascites when the abdomen of the mouse is obviously enlarged.

6. Antibody purification process

Affinity chromatography purification Using Protein A

6.1 preparation:

6.1.1 reagent preparation: and (3) an equilibrium buffer: 10mM Tris-NaCl (pH8.3)

Dissociation buffer: 0.1M citric acid (pH5.0)

Regeneration of buffer solution: 0.1M Glycine (pH2.7)

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

pH neutralization solution: 2M Tris (pH8.0)

6.1.2 Equipment preparation: protein A column, low-temperature high-speed centrifuge, balance and peristaltic pump.

6.2 working procedure

6.2.1 sample preparation: thawing frozen ascites at 10-15 deg.C overnight on the previous day, filtering with absorbent cotton soaked with purified water to remove oil, and filtering the ascites with 0.55 μm filter membrane;

column preparation: filling a proper amount of Protein A filler, washing the mixture with purified water by 5-10 times of the column volume, and leaching to remove ethanol, wherein the filler volume should not exceed the column volume by 2/3.

6.2.2 balance: the Protein column is balanced by 5 times of column volume with balance buffer solution 10mM Tris-NaCl (pH 8.3), and the display value of the Protein ultraviolet detector is adjusted to 0 after the column is balanced, which is taken as a base line;

6.2.3 Loading: diluting ascites with two volumes of equilibration buffer solution 10mM Tris-NaCl (pH8.3) to be used as sample loading, slowly loading by using a peristaltic pump, and recording the using times of the column packing;

6.2.4 balance: after the sample loading is finished, 10mM Tris-NaCl (pH8.3) is used for balancing with the balance buffer solution to elute 10-12 times of column volume until no protein flows out through Coomassie brilliant blue detection;

6.2.5 dissociation: after the balance is finished, adding a pH neutralization solution with the sample collection volume of 10% into the sample collection dish, dissociating by using 0.1M citric acid (pH 5.0), starting to collect protein when the display value of the protein ultraviolet detector is more than 0.1, and stopping collecting protein when the display value of the protein ultraviolet detector is less than 0.1;

6.2.6 regeneration: after dissociation was complete, the column was washed 2 column volumes with 0.1M glycine (pH 2.7) buffer;

6.2.7 balance: after regeneration, the column is equilibrated by 5-10 column volumes with equilibration buffer 10mM Tris-NaCl (pH8.3);

6.2.8 dialysis: combining protein-containing components, and dialyzing with a dialysis buffer solution of 20mM Tris-NaCl (pH7.5) or 10mM PBS (pH7.5) according to the requirement of a product, wherein the dialysis proportion is 1;

6.2.9 antibody treatment: after completion of dialysis, the antibody was recovered and filtered through a 0.22 μm filter, and then the concentration was adjusted to 0.1% by adding Proclin300 preserved protein.

6.3Protein A column preservation: the Protein A column was not used for more than one week, and the column was washed with 5 column volumes of 20% ethanol and stored at 5-8 ℃.

6.4 cleaning of a peristaltic pump sampling tube: when the same peristaltic pump is used for processing other samples, the sample loading buffer solution is used for washing the silicone tube for 5-10 times, and then other operations are carried out.

7. Antibody sequencing process

Carrying out reverse transcription on a cell sample to obtain cDNA; amplifying and obtaining heavy chain and light chain variable regions; cloning to pMD18-T vector, sequencing; alignment of the sequencing results was performed using IMGT/V-QUEST, after which the entire sequence was obtained. Antibody sequencing procedures are well known to those skilled in the art. The specific sequence is shown in a sequence table. The amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO:1, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO:2 is as shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, respectively. The sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO: shown in fig. 8.

RBC monoclonal antibody test standard

Physical and chemical properties

And (3) appearance inspection: the external package is perfect, the identification is clear, the information is accurate, and the content is clear liquid.

Detection of antibody concentration: see "protein concentration assay records".

Detection of antibody purity: see "standard operating procedures for electrophoresis detection".

The results are shown in Table 1.

TABLE 1

Testing of RBC testing was performed on HBsAg project

1. Gold label

HBsAg was labeled with HBsAg antibody, and total OD was 150 using gold-spraying solution.

NC film

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

The NC membrane was streaked with HBsAg-coating antibody at a coating concentration of 1.0mg/ml and a streaking amount of 1.0. Mu.l/cm. Sample pad: the sample pad was treated with the RBC to be tested, and the composition and content of the sample pad treatment solution are shown in Table 2

Packaging the sample pad treatment solution into 5 ml/tube, and adding RBC0.25mg/ml

And respectively treating the sample pad with the sample pad treatment solution, and drying in an oven at 37 ℃ overnight.

The above materials were assembled and cut into reagent strips 3 mm/strip.

And (3) testing a sample: a whole blood sample is drawn on site for use.

The erythrocyte can stay on the sample pad by using 0.25mg/ml, the background is clear and clean, and the running board is smooth.

Functional assay

Test samples 3 fresh clinical whole blood samples were tested, 1 person per sample.

Sample adding mode: the reagent strip was placed flat on the table top, 65ul of sample was added to the lower end of the strip by a pipette, and a stopwatch was started to start timing.

Results interpretation all tests should be interpreted within 15 minutes.

And (3) stability, namely repeatedly freezing and thawing for 3 times, and repeating the steps to prepare the reagent strip.

And selecting a batch of reference batches and testing batches for testing.

The erythrocyte membrane antibody has clean detection background and high sensitivity, the use concentration of the conventional commercial product is 0.5mg/ml, and the use concentration of the antibody can be reduced to 0.25mg/ml.

Claims (8)

1. An anti-erythrocyte membrane antibody, characterized in that: comprises a heavy chain and a light chain, wherein the variable region amino acid sequence of the heavy chain is shown as SEQ ID NO: 1. the sequence is shown in the specification, or conservative variant obtained by conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions and modifications;

the variable region amino acid sequence of the light chain is shown as SEQ ID NO: 2. the sequence is shown, or conservative variant obtained by conservative mutation of one or more amino acid additions, deletions, substitutions and modifications.

2. An anti-erythrocyte membrane antibody according to claim 1, which is characterized in that: the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5. as shown.

3. The anti-erythrocyte membrane antibody of claim 1, wherein: the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8. as shown.

4. The anti-erythrocyte membrane antibody of claim 1, wherein: the amino acid sequence of the heavy chain is shown as SEQ ID NO:9 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: 10. as shown.

5. A polynucleotide fragment encoding an anti-erythrocyte membrane antibody according to any one of claims 1 to 4.

6. A detection reagent containing the anti-erythrocyte membrane antibody according to any one of claims 1 to 4.

7. A test kit comprising the anti-erythrocyte membrane antibody according to any one of claims 1 to 4.

8. Use of the anti-erythrocyte membrane antibody of any one of claims 1-4 for the construction of a bifunctional antibody reagent.