CN116284368A - Anti-human MxA antibodies or antigen-binding portions thereof - Google Patents

Anti-human MxA antibodies or antigen-binding portions thereof Download PDF

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CN116284368A
CN116284368A CN202210674638.8A CN202210674638A CN116284368A CN 116284368 A CN116284368 A CN 116284368A CN 202210674638 A CN202210674638 A CN 202210674638A CN 116284368 A CN116284368 A CN 116284368A
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seq
antibody
mxa
variable region
chain variable
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易维京
汤定斌
蒋会会
徐小霞
张亘
粟瑞
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Chongqing Aishengsi Bioengineering Co ltd
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Chongqing Aishengsi Bioengineering Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention relates to the field of biotechnology, in particular to an anti-human MxA antibody or an antigen binding part thereof and application thereof, and discloses a group of anti-human MxA antibodies or antigen binding parts thereof which can be matched for use, wherein the anti-human MxA antibody has stable antibody performance, can avoid interference of other impurities, is combined with MxA with high specificity, realizes high affinity, high sensitivity and high precision measurement of MxA, and has great significance in research and development of auxiliary medical diagnostic reagents.

Description

Anti-human MxA antibodies or antigen-binding portions thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-human MxA antibody or an antigen binding portion thereof.
Background
The myxovirus resistance protein A (Myxovirus resistance protein, mxA) is a protein produced by human cells and has the function of resisting virus infection. Studies have shown that the MxA protein has activity against a variety of viruses. Infection of human body by many viruses, such as Influenza virus (Influenza virus), parainfluenza virus (PIV), adenovirus (ADV), herpes virus (herps virus), respiratory Syncytial Virus (RSV), epstein barr virus, rhinovirus (RhV) etc. induces MxA protein in human cells, whereas bacterial infection does not induce high expression of MxA protein. In clinic, bacteria or virus infection needs to be rapidly distinguished, so as to guide the clinical treatment direction, and detection of the expression condition of human MxA protein can help to rapidly distinguish virus infection from bacterial infection.
In emergency, rapid discrimination of viral and bacterial infections is of great value for timely and correct administration and life saving, while the sensitivity and linear width of detection in immunodiagnosis are often determined by the antibody or antibody pair used, so that the specificity, sensitivity and linear width of detection of the antibody are particularly important. At present, the MxA antibodies reported at home and abroad are few, but the effect of the antibodies for clinical diagnosis is not fully confirmed. Therefore, the development of the antibody with high recognition sensitivity to MxA protein induced by virus infection of human body has great clinical medical application value.
Disclosure of Invention
The invention provides two antibodies with very high recognition reactivity to MxA protein, which are specifically as follows: an anti-human MxA antibody or an antigen-binding portion thereof comprising:
(a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 1; a heavy chain variable region CDR2 as set forth in SEQ ID No. 2; a heavy chain variable region CDR3 as set forth in SEQ ID No. 3; light chain variable region CDR1 as set forth in SEQ ID No. 4; light chain variable region CDR2 as set forth in SEQ ID No. 5; light chain variable region CDR3 as set forth in SEQ ID No. 6;
or (b)
(b) A heavy chain variable region CDR1 as set forth in SEQ ID No. 7; a heavy chain variable region CDR2 as set forth in SEQ ID No. 8; a heavy chain variable region CDR3 as set forth in SEQ ID No. 9; light chain variable region CDR1 as set forth in SEQ ID No. 10; light chain variable region CDR2 as set forth in SEQ ID No. 11; light chain variable region CDR3 as set forth in SEQ ID No. 12.
Preferably, the anti-MxA antibody or antigen-binding portion thereof is a monoclonal antibody.
Preferably, the said
(a) Comprises the monoclonal antibodies of SEQ ID NOs: 13, a heavy chain variable region shown in figure 13; and SEQ ID NO:14, a light chain variable region shown in seq id no;
(b) Comprises the monoclonal antibodies of SEQ ID NOs: 15, and a heavy chain variable region shown in seq id no; and SEQ ID NO:16, and a light chain variable region shown in seq id no;
in another aspect, the invention features polynucleotides encoding the antibodies or antigen binding portions thereof.
The nucleotide heavy chain variable region of the monoclonal antibody of the (a) is coded by a sequence shown in SEQ ID NO:17, the sequence of the light chain variable region is shown in SEQ ID NO: shown at 18;
the nucleotide heavy chain variable region of the monoclonal antibody of (b) has a sequence shown in SEQ ID NO:19, the sequence of the light chain variable region is shown in SEQ ID NO: shown at 20;
in another aspect, the invention features an antigen-antibody mixture that includes any of the anti-human MxA antibodies or antigen-binding portions thereof described above.
In another aspect, the invention discloses a kit for detecting MxA, comprising the anti-human MxA antibody or an antigen-binding portion thereof or the antigen-antibody mixture of claim.
In another aspect, the invention discloses a conjugate comprising the anti-human MxA antibody or antigen-binding portion thereof of claim covalently linked to a chemical label or biomarker.
In another aspect, the invention features a conjugate formed from the anti-human MxA antibody or antigen-binding portion thereof, and/or the conjugate coupled to a solid medium or semi-solid medium.
In another aspect, the invention discloses the use of said anti-human MxA antibodies or antigen binding portions thereof and/or said conjugates for the preparation of a product for detecting MxA expression.
Advantageous effects
The antibody sequence disclosed by the invention has stable antibody performance, and can realize specific and efficient combination of MxA in a complex environment with a plurality of interference factors in a whole blood cell lysate. The human MxA immunochromatographic diagnostic reagent developed based on the antibody can realize rapid identification of virus infection or bacterial infection, and has important clinical practical value.
Drawings
FIG. 1 shows the interaction of the antibodies disclosed in the present invention with MxA proteins;
FIG. 2 is an MxA assay of clinical whole blood samples for parainfluenza virus (PIV) infection using antibodies of the present invention;
FIG. 3 immunochromatographic reagent detects the amount of MxA protein in a virus-infected sample.
Detailed Description
Example 1MxA antibody preparation
1. Immunogen preparation
Human MxA full-length protein (10-662 Aa, P20591) is constructed to a eukaryotic expression vector to obtain an MxA expression plasmid. Serum-free acclimatized 293 cells were cultured in suspension to a cell density of 2.0X10 6 The expression plasmid was transfected into 293 cells in suspension culture using PEI (polyethylenimine) -mediated plasmid transfection method at a rate of per ml, and culture was continued for 1 week after transfection, with medium and feed addition every other time. After fermentation was completed, the cell pellet was collected by centrifugation at 4000g for 30min at 10 ℃. Cell pellet was resuspended in PBS and then broken using an ultrasonicator. After the wall breaking is completed, supernatant is collected by centrifugation and used for purifying target protein.
2. Mouse immunity and antibody detection
5 SPF-class female BALB/c mice of 6-8 weeks old were selected, and Freund's complete adjuvant was mixed with MxA protein at a concentration of 1mg/ml in equal volumes and emulsified. The emulsified antigen was used to immunize 6-8 week old SPF grade female BALB/c mice, and each mouse was injected with 40. Mu.g antigen protein by plantar injection. Two weeks after the primary immunization was completed, the antigen protein was mixed with Freund's incomplete adjuvant to emulsify, and 40. Mu.g of antigen protein was injected into each mouse again by plantar injection or dorsal subcutaneous injection. After two weeks, blood was collected through the tail vein, the supernatant was collected by centrifugation and serum titers were detected by ELISA. Immunization was performed every two weeks and serum titers were measured. After 2 immunizations, the serum titer after million-fold dilution was up to 2.0. Screening bloodClean potency 10 6 In the above mice, lymphocytes were isolated from the lymph for cell fusion.
3. Hybridoma antibody screening
Antibody screening protocols are key to the successful development of antibodies of the invention. In the antibody screening stage, firstly, biotin is used for marking MxA protein to obtain MxA-biotin, after a potential target antibody is combined with the MxA-biotin, an avidin-HRP is used for matching with ELISA chromogenic liquid for color development, and hybridoma clones with high reactivity are screened out. Secondly, collecting peripheral blood whole blood of a patient with confirmed virus infection such as PIV, RSV, ADV, EBV and the like, and using the peripheral blood whole blood as a positive sample; peripheral blood whole blood of healthy persons free from the above virus infection was collected and used as a negative sample. The collected clinical samples were lysed using 1% np40 lysate (CA 630) for 10min to obtain whole blood lysates. The positive whole blood lysate is used to screen hybridoma clones with high reactivity to the sample, while the negative whole blood lysate is used to screen hybridoma clones with low reactivity to the sample.
The target clone obtained by screening needs to meet the following conditions: 1. has better reactivity to recombinant MxA protein; 2. has better reactivity to positive whole blood lysate; 3. has no obvious reactivity to negative whole blood lysate. The invention screens MxA-A cell strain and MxA-B cell strain for preparing the antibody A and the antibody B.
4. Production and purification of monoclonal antibodies
Two groups of 6-8 week BALB/c mice were selected and injected intraperitoneally with 500. Mu.L paraffin oil to suppress the immune response in the mice. One week after injection, 0.5ml of MxA-A hybridoma cells were intraperitoneally injected into a group of mice, the number of cells being about 1×106; another group of mice was intraperitoneally injected with 0.5ml of MxA-B hybridoma cells. Ascites collection was started after two weeks. The collected ascites is precipitated by ammonium sulfate and affinity purified by protein G to obtain the target antibody.
5. Subtype identification and gene sequence cloning of monoclonal antibodies
The subtype of monoclonal antibody heavy and light chains was identified by the procedure of the specification using the SBA Clonotyping System-HRP kit from southern Biotech. The specific operation is as follows:
1. the capture antibody was diluted to 1. Mu.g/mL with coating solution (0.05M carbonate and bicarbonate buffer pH 9.5), and the ELISA plate was added at 100. Mu.L/well and coated overnight at 4 ℃. Plates were washed 3 times with PBS buffer (plate wash) containing 0.05% Tween-20.
2. Culture supernatants of hybridoma cells to be examined were diluted 1:1 with a diluent (1% BSA,0.1% PBST), and an ELISA plate was added at 100. Mu.L/well, and incubated at 37℃for 30 minutes. The corresponding enzyme-labeled antibodies (Ig-HRP, igG1-HRP, igG2a-HRP, igG2b-HRP, igG3-HRP, igM-HRP, kappa-HRP, lamda-HRP) were diluted 1:3000 with the diluent.
3. After 3 washes with plate wash, 100 μl of diluted enzyme-labeled antibody was added to each well and incubated at 37 ℃ for 30 minutes. After washing the plate again 3 times, the color-developing solution was added, and after about 5 minutes (depending on the reaction intensity), 2M sulfuric acid was added to terminate the reaction, and the OD450 absorbance was read. The heavy chain subtype of the MxA-A antibody and the MxA-B antibody are identified as IgG1, and the light chain is Kappa. According to the result of the antibody subtype, the antibody gene sequence is cloned by adopting a method based on the RACE technical route. Collecting hybridoma cells with good growth state, obtaining total RNA of the hybridoma cells by using a total RNA extraction kit, performing reverse transcription on mRNA into cDNA according to the operation method of SMART RACE instruction of Takara company, and amplifying the full-length sequence of the target antibody.
Example 2 antibody Performance validation
(1) Interaction of antibodies with MxA proteins
This example demonstrates the binding properties of the antibodies of the invention by their interaction with MxA proteins.
The specific operation steps are as follows: antibody a/antibody B was diluted to 1ug/mL using a coating solution (0.05 m carbonate and bicarbonate buffer pH 9.5) and coated onto a conventional 96 well ELISA plate. The MxA protein purified after recombinant expression was subjected to biotin modification using a biotinylation reagent (final commercial reagent). The biotin-modified MxA protein was diluted to each target concentration (20 ng/. Mu.l, 40 ng/. Mu.l, 60 ng/. Mu.l, …,180 ng/. Mu.l). Specific concentrations of MxA protein were added to ELISA wells coated with the antibody of interest, and 100 μl per well was added and incubated at 37 ℃ for 30min. After washing the plates with plate washing solution (150 mM NaCl solution containing 0.05% Tween-20), 1:5000 dilutions of streptavidin-horseradish peroxidase conjugate (finished commercial reagent) were added to each well with 100 μl and incubated for 15min at 37 ℃. Finally, ELISA color development is performed, and an enzyme-labeled instrument is used for detecting OD450 absorbance.
The results are shown in FIG. 1, and the results of experiments in which antibodies interact with recombinant expressed purified human MxA protein were examined using enzyme-linked immunosorbent assay (ELISA) experiments. The abscissa in the figure indicates the concentration of MxA protein used, and the ordinate indicates the detection result of the ELISA experiment, i.e. the absorbance value detected by the spectrophotometer at 450nm (OD 450), the higher the value of OD450, the stronger the detected interaction. The results show that the interaction between the antibody A and the antibody B is strong, and the antibody is suitable for detecting the MxA protein.
(2) Clinical verification
The specific operation is as follows: antibody a was diluted to 1ug/mL using a coating solution (0.05 m carbonate and bicarbonate buffer ph 9.5) and coated onto a conventional 96-well ELISA plate. After lysis of each clinical sample collected using 1% NP-40, it was diluted 10-fold with sample dilution (1%BSA,0.1%Tween 20,1X PBS) and added to ELISA wells and incubated at 37℃for 30min. Antibody B-HRP conjugated double-core is prepared by label coupling of antibody B using horseradish peroxidase labeling reagent (finished commercial reagent). Antibody B-HRP conjugate replicates were diluted to 1ug/mL using dilution. After ELISA wells incubated with samples were washed with plate wash (150 mM NaCl solution containing 0.05% Tween-20), diluted antibody B-HRP conjugate multiplex was added and incubated at 37℃for 30min. Finally, ELISA color development is performed, and an enzyme-labeled instrument is used for detecting OD450 absorbance.
The results are shown in FIG. 2, wherein the abscissa indicates different whole blood samples, and the ordinate indicates the detection results obtained using an enzyme-linked immunosorbent assay (ELISA) experiment, i.e. absorbance (OD 450) detected at 450nm by a spectrophotometer. Higher values of OD450 indicate stronger interactions detected, indicating higher MxA protein concentrations. For the same sample, there was the same MxA protein concentration. The same MxA detection experiment is carried out by using the antibody or other anti-MxA antibodies, and the OD450 detection results of the antibody are all larger than 0.6, which indicates that the antibody has excellent detection effect.
EXAMPLE 3 application of MxA antibodies to immunochromatographic detection
(1) Treatment of nitrocellulose membranes
a. Preparation of detection line
0.5g/mL of MxA antibody A was formulated with PBS, and a test line was drawn in the middle of the nitrocellulose membrane in an amount of 0.5 ul.
b. Drying
Baking at 37deg.C in a drying oven for 2 hr.
(2) Preparation of sample pad
The glass cellulose film was cut into 20 x 30cm strips.
(3) Preparation of absorbent pad
The absorbent paper was cut into strips of 30 x 2.7cm each.
(4) Assembly
The plastic base lining, the sample pad and the water absorption pad are common materials in the field, and the sample pad, the nitrocellulose membrane and the water absorption pad are tightly lapped on the plastic base lining at one time. The applied intermediate was cut into test strips 0.5cm wide by a chopper.
(5) Application method of test strip
a. The sample to be tested and the antibody B-colloidal gold complex are mixed according to the following formula 4:1, stirring and mixing uniformly;
b. dripping 60ul of mixed solution into the sample adding hole of the test strip prepared in the above way;
c. and (3) carrying out chromatography on the sample mixed solution until the test strip detection line is combined with the MxA antibody A.
(6) Kit performance verification
The experiment was performed as in (5) above, and the results are shown in FIG. 3, in which the arrow indicates the aggregation of dark red colloidal gold that occurs at this position on the chromatographic strip when the MxA protein is present in the test sample. The concentration of MxA protein in the sample correlated positively with the extent of aggregation of colloidal gold (the shade of color) at the arrow indication. The higher the MxA protein concentration in the sample, the darker the color development. The experimental result shows that the MxA lateral flow immunochromatography reagent prepared by using the antibody has good detection reaction effect on clinical samples.
Sequence listing
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<400> 20
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gaactagtca gaccattgta catagtaatg gaaacaccta tttagaatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300
cccacgttcg gtgctgggac caagctggag ctgaaa 336

Claims (9)

1. An anti-human MxA antibody or antigen-binding portion thereof comprising:
(a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 1; a heavy chain variable region CDR2 as set forth in SEQ ID No. 2; a heavy chain variable region CDR3 as set forth in SEQ ID No. 3; light chain variable region CDR1 as set forth in SEQ ID No. 4; light chain variable region CDR2 as set forth in SEQ ID No. 5; light chain variable region CDR3 as set forth in SEQ ID No. 6;
or (b)
(b) A heavy chain variable region CDR1 as set forth in SEQ ID No. 7; a heavy chain variable region CDR2 as set forth in SEQ ID No. 8; a heavy chain variable region CDR3 as set forth in SEQ ID No. 9; light chain variable region CDR1 as set forth in SEQ ID No. 10; light chain variable region CDR2 as set forth in SEQ ID No. 11; light chain variable region CDR3 as set forth in SEQ ID No. 12.
2. The anti-human MxA antibody or antigen-binding portion thereof according to claim 1, wherein the anti-MxA antibody or antigen-binding portion thereof is a monoclonal antibody.
3. The anti-human MxA antibody or antigen-binding portion thereof according to claim 2, wherein the
(a) Comprises the monoclonal antibodies of SEQ ID NOs: 13, a heavy chain variable region shown in figure 13; and SEQ ID NO:14, a light chain variable region shown in seq id no;
(b) Comprises the monoclonal antibodies of SEQ ID NOs: 15, and a heavy chain variable region shown in seq id no; and SEQ ID NO:16, and a light chain variable region shown in seq id no.
4. A polynucleotide encoding the antibody or antigen-binding portion thereof of any one of claims 1-3.
5. An antigen-antibody mixture comprising the anti-human MxA antibody or antigen-binding portion thereof according to any one of claims 1-3.
6. A kit for detecting MxA, comprising an anti-human MxA antibody or an antigen-binding portion thereof according to any one of claims 1-3 or an antigen-antibody mixture of claim 5.
7. A conjugate comprising an anti-human MxA antibody or an antigen-binding portion thereof according to any one of claims 1-3 covalently linked to a chemical label or biomarker.
8. A conjugate formed from the anti-human MxA antibody or antigen-binding portion thereof of any one of claims 1-3, and/or the conjugate of claim 7 coupled to a solid or semi-solid medium.
9. Use of an anti-human MxA antibody or antigen binding portion thereof according to any one of claims 1-3 and/or a conjugate according to claim 7 and/or a conjugate according to claim 8 for the preparation of a product for detecting MxA expression.
CN202210674638.8A 2022-06-15 2022-06-15 Anti-human MxA antibodies or antigen-binding portions thereof Pending CN116284368A (en)

Priority Applications (1)

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CN202210674638.8A CN116284368A (en) 2022-06-15 2022-06-15 Anti-human MxA antibodies or antigen-binding portions thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210674638.8A CN116284368A (en) 2022-06-15 2022-06-15 Anti-human MxA antibodies or antigen-binding portions thereof

Publications (1)

Publication Number Publication Date
CN116284368A true CN116284368A (en) 2023-06-23

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Country Status (1)

Country Link
CN (1) CN116284368A (en)

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