CN117467016B - Antibodies to human IgA, antibody combinations and uses thereof - Google Patents
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- G01N33/53—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
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- 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
-
- 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]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
Abstract
The invention relates to the technical field of cell immunology, in particular to an antibody of human IgA, an antibody combination and application. The antibodies (4C 3 and/or 1D 9) for specifically detecting human IgA have high sensitivity and high specificity to human IgA, each have a special spatial epitope, and show high affinity and high specificity to human IgA. The antibody 4C3 and the antibody 1D9 provided by the invention are commonly used for detecting the paired antibody of the human IgA, the content of the human IgA in a culture medium and serum can be specifically detected through a sandwich method, the affinity is high, the antibody does not react with other similar proteins, and the antibody is an ideal detection antibody combination of the content of the human IgA protein.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to an antibody of human IgA, an antibody combination and application thereof.
Background
Immunoglobulin a (IgA) is produced primarily by mucosa-associated lymphoid tissues, most of which are synthesized by gastrointestinal lymphoid tissues and a small fraction by respiratory, salivary and genital tract mucosal tissues. The content of the extract is closely related to a plurality of diseases: igA increases in cases of chronic liver disease, subacute or chronic infectious diseases (e.g., tuberculosis, fungal infection, etc.), autoimmune diseases (e.g., SLE, rheumatoid arthritis), cystic fibrosis, familial neutropenia, breast cancer, igA nephropathy, igA myeloma, etc.; igA is reduced in cases of hereditary or acquired antibody deficiency, immunodeficiency, selective IgA deficiency, agammaglobulinemia, protein loss bowel disease, burns, anti-IgA antibody syndrome, immunosuppressant therapy, late gestation, etc. In blood products, scientific experiments and clinical diagnosis, the rapid and accurate detection of the IgA content in human serum has great reference value. IgA has two subclasses, igA1 and IgA 2. IgA1 is mainly present in serum, and accounts for about 85% of IgA in serum, and the alpha 1 chain molecular weight is 56kD; igA2 is mainly present in exocrine fluids such as saliva, tears, colostrum, nasal and bronchial secretions, gastrointestinal fluids, urine, sweat, etc., and is the main antibody for local anti-infective immunity of the mucous membrane of the organism, and a small part of IgA exists as serotype, which accounts for about 15% of IgA in serum, and the alpha 2 chain lacks a hinge region and has a molecular weight of 52kD. IgA in serum is in the form of dimers or trimers covalently linked by J chains, in addition to monomeric forms. There are 2 antigen binding sites per IgA monomer. IgA cannot pass through the placenta and neonate serum is free of IgA antibodies, but secretory IgA is available from breast milk. IgG antibodies extracted from blood plasma of a donor are major factors mediating mucosal immunity of the organism, and can effectively prevent external antigens (including soluble antigens, pathogenic microorganisms and carcinogens existing in food) from adhering to or invading mucosal epithelium in mucosal secretion, thereby preventing damage of the organism caused by such foreign substances, keeping the mucosa of the organism intact and preventing the occurrence of gastrointestinal diseases, respiratory diseases and various diseases of genitourinary tract.
The currently commonly used IgA measurement method is an immunoturbidimetry method, wherein the immunoturbidimetry method utilizes the property that an antigen and an antibody can be specifically combined to generate a complex with a certain size in a liquid phase to form refraction or absorption of light, and the content of the light is calculated by measuring transmitted light or scattered light after refraction or absorption. The defects are as follows: the sample amount is more, and the key reagent usage amount is more (anti-human IgA serum); the large-batch detection is difficult, the operation is complex, and a specific analysis instrument is needed; the experimental repeatability is poor; the complex molecules formed in the liquid are not stable enough and are large enough to have accurate reading values, and the accuracy is poor when the content of the sample is low; sodium azide dangerous goods are also needed in the used buffer system. The above-mentioned disadvantages are also present in the UV-spectrophotometric measurement of IgA content.
The other detection methods also have immune diffusion experiments, and have the defects of more experimental operation influencing factors, long detection time and poor sensitivity, and are suitable for roughly estimating the content in a sample; HPLC and capillary electrophoresis have the disadvantage of requiring specialized equipment and experienced personnel to operate, which is expensive.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the technical problems, the invention provides an antibody of human IgA, an antibody combination and application. The antibody can accurately, rapidly and high-flux specifically detect the content of IgA in human serum or other serum analogues containing human IgA.
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides an antibody or antigen-binding fragment thereof to human IgA, the antibody or antigen-binding fragment thereof having the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
Preferably, the amino acid sequence of the heavy chain variable region of the antibody is as set forth in SEQ ID NO:15 or with SEQ ID NO:15 has at least 80% sequence similarity; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16 or with SEQ ID NO:16 has at least 80% sequence similarity.
Preferably, the antibody or antigen binding fragment thereof described above is Fab, fab ', F (ab') 2 Fd, fv, dAb, complementarity determining region fragments, single chain antibodies, monoclonal antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies or multispecific antibodies.
In some embodiments of the invention, an antibody composition is provided, consisting of 4C3 and 1D9. The two antibodies can be used as the paired antibodies of human IgA and respectively used as the coating antibody and the detection antibody in the double-antibody sandwich ELISA. Wherein the detection antibody may also carry a detectable label. The detectable label is not particularly limited in the present invention, and any label conventionally used in the art may be used.
In some embodiments of the invention, an antibody composition is provided, consisting of 4C3 and 1D9. Wherein, 4C3 is used as a coating antibody in the double-antibody sandwich ELISA, and 1D9 is used as a detection antibody after being labeled by HRP.
The antibody composition provided by the invention has good stability, can be used for rapidly detecting the IgA content of a person by adopting a double-antibody sandwich ELISA method, and has the advantages of simplicity in operation, high sensitivity, high specificity, high accuracy and high precision.
In a second aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the production of the antibodies or antigen binding fragments thereof are within the scope of the invention.
In a third aspect, the invention provides a biological material comprising a nucleic acid molecule as described above. Such biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.
Preferably, the biological material is an expression cassette, vector or host cell.
The above-mentioned expression cassette can be obtained by ligating a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and/or ligating a transcription or translation regulatory element such as a terminator downstream thereof.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
The host cells include microbial cells, insect cells, or other animal cells.
In a fourth aspect, the present invention provides an antibody conjugate obtained by coupling the antibody or antigen binding fragment thereof to a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.
In a fifth aspect, the present invention provides a composition of human IgA antibodies comprising an antibody or antigen-binding fragment thereof as shown in (1) below; also included are antibodies or antigen-binding fragments thereof as shown in (2) below;
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4.5 and 6;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
In a sixth aspect, the invention provides the use of any one of the following (1) - (5) of the antibody or antigen binding fragment thereof or the nucleic acid molecule or the biological material or the antibody conjugate or the antibody composition:
(1) Use in the preparation of a product for detecting the presence or level of human IgA in a sample;
(2) Use of detecting the presence or level of human IgA in a sample for non-diagnostic and therapeutic purposes;
(3) Use in the preparation of a product for neutralising human IgA activity in a sample;
(4) Use in the manufacture of a medicament for neutralising in vivo human IgA activity;
(5) Use in quality control or production of a human IgA-containing product.
Preferably, the application described in the above (1) or (2) includes: the antibodies or antigen-binding fragments thereof of the invention are prepared as a product for detecting the presence or level of human IgA in a sample, which is used to detect the presence or level of human IgA in a sample. For ease of detection, the antibodies or antigen-binding fragments thereof of the invention also preferably comprise a detectable label.
The invention also provides a method of detecting the presence or level of human IgA in a sample, the method comprising detecting the presence or level of human IgA in a sample using an antibody or antigen-binding fragment thereof of the invention. The above-described methods may be for diagnostic purposes (e.g., the sample is a sample from a patient, including blood samples, etc.), or for non-diagnostic and therapeutic purposes (e.g., the sample is a culture cell supernatant, etc., not a sample from a patient).
As a method for detecting human IgA by using the antibody or antigen-binding fragment thereof of the present invention, detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and the like can be used.
In some embodiments of the invention, human IgA is detected in a double antibody sandwich method using an antibody or antigen binding fragment thereof of the invention. The double antibody sandwich method is used for detecting the human IgA by using one antibody or antigen binding fragment thereof provided by the invention as a coated antibody, using the other antibody or antigen binding fragment thereof provided by the invention as a detection antibody, or using one antibody or antigen binding fragment thereof provided by the invention as a coated antibody, using other known anti-human IgA antibodies as detection antibodies, or using other known anti-human IgA antibodies as coated antibodies, and using one antibody or antigen binding fragment thereof provided by the invention as a detection antibody.
Preferably, in the above (3) and (4), the activity of neutralizing human IgA is specifically binding to human IgA in a sample by using the antibody or the antigen-binding fragment thereof provided by the present invention, and thereby neutralizing human IgA activity.
Preferably, in the above (5), the human IgA-containing products include foods, medicines, etc.
In a seventh aspect, the invention provides a kit comprising an antibody or antigen binding fragment thereof as described above, or comprising an antibody conjugate as described above, or comprising an antibody composition as described above.
Some embodiments of the invention provide an ELISA detection kit for human IgA comprising an antibody or antigen-binding fragment thereof as described above, or an antibody composition as described above. The kit may also contain other reagents for ELISA detection including, but not limited to, human IgA standards, PBST wash, blocking solution, chromogenic solution, stop solution, and the like.
In an eighth aspect, the invention provides a method of human IgA detection for non-diagnostic and therapeutic purposes, the method comprising: and detecting whether the human IgA exists in the sample to be detected and/or the existence level thereof by using a double-antibody sandwich ELISA method, wherein the coated antibody and the detection antibody in the double-antibody sandwich ELISA method are respectively 4C3 and 1D9.
The principle of detecting the human IgA content by adopting the enzyme-linked immunosorbent assay technology based on a double antibody sandwich method is as follows: coating an anti-human IgA monoclonal antibody on an ELISA plate; respectively adding a gradient diluted standard substance and a pre-diluted sample, wherein human IgA in the standard substance and the sample can be fully combined with a coating antibody on an ELISA plate; after washing the plate, adding HRP-labeled anti-human IgA antibody, wherein the antibody can specifically bind with the standard substance captured by the coated antibody on the plate and the human IgA in the sample; adding a chromogenic substrate TMB after washing the plate, if the sample in the reaction hole has different concentrations of human IgA, the HRP can change colorless TMB into blue substances with different depths (positive correlation), and after adding a stop solution, the reaction hole can change into yellow; finally, the absorbance (OD) of the reaction well sample was measured at λmax=450 nm (od=450 nm), the concentration of human IgA in the sample was proportional to OD, and the concentration of human IgA in the sample was calculated from the standard curve. The method uses an enzyme chromogenic amplifying system, has higher detection sensitivity and can detect samples with lower content.
The ELISA detection method provided by the invention has higher detection sensitivity (pg level) than other methods such as an immunoturbidimetry (mg level), an ultraviolet-spectrophotometry (mg level) and an HPLC (mg level), and the detection sensitivity is in the range of 12.5-800 ng/ml. The detection time was short (2.75 h) and high throughput (compared to 70 parts detection time) compared to other methods such as immunoturbidimetry (about 4 h), ultraviolet-spectrophotometry (about 4 h), HPLC (about 4 h).
Through the similar protein crossing experiment, the ELISA detection method provided by the invention has good performance, and does not recognize pig IgG, rabbit IgG, rat IgG, mouse IgG, duck IgG and human IgG.
Based on the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention provides an antibody for specifically detecting human IgA and application thereof. The antibodies (4C 3 and/or 1D 9) for specifically detecting human IgA have high sensitivity and high specificity to human IgA, each have a special spatial epitope, and show high affinity and high specificity to human IgA. The antibody 4C3 and the antibody 1D9 provided by the invention are commonly used as the paired antibodies of the human IgA, the content of the human IgA in a culture medium and serum can be specifically detected through a sandwich method, the affinity is high, the antibody does not react with other similar proteins, and the antibody is an ideal detection antibody combination of the content of the human IgA protein.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be described below.
FIG. 1 is a diagram showing the electrophoresis detection of monoclonal antibodies 4C3 and 1D9 in example 1 of the present invention, wherein M is Marker, lane 1 is 1D9, and lane 5 is 4C3.
FIG. 2 is a standard curve of detection of IgA content in human by the double antibody sandwich ELISA method in example 2 of the present invention.
FIG. 3 shows the results of the detection of IgA in human serum by antibody 1D9 of Experimental example 3 of the present invention as a primary antibody applied to WB.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the materials, reagents and the like used are all commercially available.
EXAMPLE 1 preparation of anti-human IgA monoclonal antibodies
1. Immunization of animals
Female Balb/c mice with the age of 6-8 weeks are selected, and are subjected to immunization after being emulsified by using natural extracted human IgA antigen and an equal volume of Freund's adjuvant, wherein the immunization period is two weeks, blood is taken after 3 times of immunization, the titer is measured, and the immunization is enhanced again three days before fusion.
2. Cell fusion
Mice were sacrificed by cervical scission, spleens were removed by aseptic manipulation, and spleen cell suspensions were prepared by squeeze milling in a plate. The prepared syngeneic myeloma cells and the spleen cells of the mice are mixed according to a certain proportion, and a fusogenic agent polyethylene glycol (PEG) is added. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells. The specific operation is as follows:
(1) Preparation of immune spleen cells:
taking one BALB/c mouse with enhanced immunity, killing the eye socket by bleeding (collecting serum, namely positive serum), soaking in 75% alcohol for 5-10 min for sterilization, then fixing the BALB/c mouse on an dissecting plate for dissection, taking out spleen, shearing the spleen, and placing the mouse in a sterilized homogenizer; grinding and cell suspension preparation method are as described in SP2/0, and counting for later use.
(2) Preparation of feeder cells:
one non-immunized BALB/c mouse was taken, the orbit was exsanguinated, and serum was collected as negative serum. 2-3 mL 1640 basic solution is injected into the abdominal cavity of the mouse, and is sucked out and placed in another centrifuge tube for standby after being blown, wherein the basic solution contains abdominal macrophages. Spleen cell suspensions were prepared and placed into the peritoneal macrophage tube in the same manner as above. 1000 Centrifuging at r/min for 10min to remove supernatant, suspending cells in HAT medium, and standing at 37deg.C and 5% C0 2 And (5) placing the mixture in an incubator for later use.
(3) Fusion:
will be 1-2×10 7 SP2/0 and 10 8 The individual immunocytes were mixed well in a 50 mL centrifuge tube, 1000 r/min and centrifuged for 8 min. After the supernatant was discarded, the centrifuge tube containing the cell mixture was placed in a 37℃water bath, followed by addition of 50% PEG 0.8 ml pre-warmed to 37℃and allowed to stand for 30 s after stirring. After standing, a base solution 1640 (10) and ml were added at 37℃to the mixture. Centrifuging at 1000 r/min for 5min, discarding supernatant, and standing at 37deg.C for 5-8 min. Subsequently mixed with feeder cell suspension, seeded in 96-well plates, 250 μl/well, and incubated in a 5% CO2 incubator at 37 ℃. HT medium was changed for continued culture on day 4 after fusion. And (4) when the colony of the fused cells grows to 1/4 of the culture hole and the culture medium turns yellow slightly, detecting the antibody.
3. Selection of hybridoma positive clones and cloning of cells
The purpose of the selective culture is to screen the fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells lack hypoxanthine-guanine-phosphoribosyl transferase and cannot synthesize DNA by salvage pathways to die. Unfused lymphocytes have hypoxanthine-guanine-phosphoribosyl transferase, but do not survive in vitro for long periods and die. Only fused hybridoma cells survive and proliferate in HAT medium due to the hypoxanthine guanine phosphoribosyl transferase obtained from spleen cells and the unlimited proliferation of myeloma cells. The specific operation is as follows:
(1) Positive hybridoma cells were screened by indirect ELISA as follows:
coating known antigens: diluting the purified coating antigen to 1-10 mu l/ml with coating buffer; 100 μl of each well was added to the microwells, gently shaken, and cooled overnight at 4deg.C in a refrigerator or at 37deg.C in a1 h oven; the liquid in the hole is thrown away (the liquid in the hollow is beaten as much as possible); washing for 2-3min each time 3 times.
Blocking the positions of the enzyme-labeled wells not coated with antigen: 200 μl of blocking solution (5% skimmed milk powder or 0.1% BSA) was added to each well, gently shaken well, and allowed to stand at 37deg.C 1 h; throwing away the liquid in the hole; washing buffer solution according to Kong Jiaman, standing for 2-3min, throwing away liquid in the hole, beating, and washing 3 times with the washing buffer solution. Sample adding: 50 μl of the supernatant from each well of the hybridoma to be tested was sequentially added to the microplate wells, gently shaken well, washed and patted dry at 37deg.C 1 h.
Adding enzyme-labeled anti-antibody: diluting the enzyme-labeled secondary antibody to a proper working concentration according to instructions by using a diluent, adding 100 mu l of the enzyme-labeled secondary antibody into each hole, gently shaking the mixture, and placing the mixture at 37 ℃ for 1 h; then washing and beating to dry. Adding a color development liquid: each well was added with 100. Mu.l of freshly prepared color development solution, gently shaken well, at 37℃for 10min. Terminating the reaction: mu.l of stop solution was added to each well.
Determination result: the enzyme label instrument OD450 reads 3 times larger than the negative hole, and the positive result is judged.
(2) Cloning of hybridoma cells was performed by limiting dilution, as follows:
preparing a mouse feeder cell layer before cloning; gently blowing the hybridoma cells to be cloned from the culture well, and counting the number of living cells by using a blood cell counting plate; diluting cells to 5, 10, 30 cells/ml with complete medium;
the three concentrations of cell suspension were added to the prepared 96-well culture plate of feeder cells at 100. Mu.l/well, so that each well contained 0.5, 1 and 3 cells, respectively. Culturing until 4 days of fluid infusion is one drop, carefully observing the growth condition of cells in each hole on 5-6 days, and recording;
detection of specific antibodies: cell clones can be detected when 1/3-1/2 field of vision is full of cell clones on days 7-9 after cloning; cells in the positive holes can be transferred to a 24-hole culture plate, and when the cells in the 24-hole plate grow well, the mice can be inoculated in the abdominal cavity to collect ascites.
4. Cell supernatant affinity data:
human IgA antigen was coated onto the ELISA plate and blocked. After PBST washing, the cell supernatant of the monoclonal cells is diluted to a saturation concentration and added to an ELISA plate, 100 mu L/well, and the plate is left standing at room temperature for incubation for 2 hours. After PBST plate washing, 60 μl/well of NaSCN solution of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mol/L was added sequentially, and the mixture was left standing at room temperature for 15min. After PBST plate washing, HRP-labeled goat anti-mouse IgG is added, and the plate is incubated at room temperature for 45min for chromogenic detection. The concentration of sodium thiocyanate corresponding to the decrease of OD value at 450nm after elution to 50% without elution is the relative affinity constant of the antibody, which is expressed by M (mol/L). The results are shown in Table 1. Table 1 shows: the affinity of anti-human IgA antibodies secreted by monoclonal cells clone numbers 8C3, 8B4, 7E8, 7C11, 6G3, 6B4, 6A9, 4C3, 4B7, 3C12, 1E4, 1D9, 1B12 is better. Can be used as the first monoclonal antibody for detecting human IgA by the antibody composition.
TABLE 1
5. Verification of pairing Effect of different monoclonal antibody combinations
(1) Primary pairing screening of monoclonal antibody pairs: monoclonal antibodies were diluted to 2. Mu.g/ml with 0.05M PH9.6 carbonate coating buffer, respectively. 0.1 μl was added to the 96-well polystyrene reagent plate reaction wells and incubated overnight at 4deg.C. The next day, the solution in the wells was discarded, and washed 3 times with wash buffer for 1 min each time. After the above procedure, each well of the reaction plate was blocked with a solution, and incubated at 0.3 μl per well for 2h. The solution in the wells was discarded, the plates were washed 3 times and dried. Mu.l of human IgA standard at a concentration of 100ng/ml was added and 0 wells were made. After sealing plates, incubate 2h at room temperature, wash plates 4 times and spin-dry. Adding 100 μl enzyme-labeled antibody working solution into the reaction well, sealing, incubating at room temperature for 45min, washing the plate 5 times, and spin-drying. Adding 100 μl of chromogenic substrate TMB into the reaction well, sealing the plate, developing at room temperature in dark for 5-20 min, adding 50 μl of stop solution, performing dual-wavelength detection by using an enzyme-labeled instrument, measuring the OD value at 450nm maximum absorption wavelength and 630 nm reference wavelength, and subtracting the OD value of 630 nm from the OD value of 450 nm. An antibody combination with a high point (100 ng/ml human IgA added) higher than 0 wells was screened for alternatives to the detection of human IgA antibody pairs.
(2) Primary pairing-specific screening of monoclonal antibody pairs: monoclonal antibodies were diluted to 2. Mu.g/ml with 0.05M PH9.6 carbonate coating buffer, respectively. 0.1 μl was added to the 96-well polystyrene reagent plate reaction wells and incubated overnight at 4deg.C. The next day, the solution in the wells was discarded, and washed 3 times with wash buffer for 1 minute each time. After the above procedure, each well of the reaction plate was blocked with a solution, and incubated at 0.3 μl per well for 2h. The solution in the wells was discarded, the plates were washed 3 times and dried. 100 μl of high concentration (10 μg/ml) human IgM standard and human IgG standard were added separately and 0 wells were made. After sealing plates, incubate 2h at room temperature, wash plates 4 times and spin-dry. Adding 100 μl enzyme-labeled antibody working solution into the reaction well, sealing, incubating at room temperature for 45min, washing the plate 5 times, and spin-drying. Adding 100 μl of chromogenic substrate TMB into the reaction well, sealing the plate, developing at room temperature in dark for 5-20 min, adding 50 μl of stop solution, performing dual-wavelength detection by using an enzyme-labeled instrument, measuring the OD value at 450nm maximum absorption wavelength and 630 nm reference wavelength, and subtracting the OD value of 630 nm from the OD value of 450 nm. Because of the similar structure of immunoglobulins, the immunoglobulins with the highest serum and plasma content are IgG, igM and IgA, which account for about 99.9 percent of the total immunoglobulin, and the combination of antibody pairs with low OD values for high-concentration (10 mug/ml) human IgG and human IgM reactions is selected for the alternative of specifically detecting the human IgA antibody pairs.
(3) Monoclonal antibody pairing optimization: from the above pairs, 12 pairs were selected which had higher OD values for low concentration IgA and lower OD values for high concentration IgM and IgG (10. Mu.g/ml). Further verifying the adjustment of the crossover and preliminary mark, the results showed that: the combination of 4C3 and 1D9, the placebo was lowest, reacted higher with IgA, and bound weaker to IgM and IgG (table 2).
TABLE 2
6. Monoclonal antibody 4C3 and 1D9 variable region sequencing
Collecting hybridoma cell number greater than 10 6 And sending to Beijing engine family organism for sequencing. Obtaining the result of gene sequencing. The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody 4C3 are shown as SEQ ID NO.1, 2 and 3 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.4, 5 and 6 respectively; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14; the coding gene sequence of the heavy chain variable region is shown as SEQ ID NO.17, and the coding gene sequence of the light chain variable region is shown as SEQ ID NO. 18. The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody 1D9 are shown as SEQ ID NO.7, 8 and 9 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.10, 11 and 12 respectively; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16; the coding gene sequence of the heavy chain variable region is shown as SEQ ID NO.19, and the coding gene sequence of the light chain variable region is shown as SEQ ID NO. 20.
7. Mass production of monoclonal antibodies 4C3 and 1D9
The hybridoma cells after the strain establishment are injected into the abdominal cavity of a mouse, ascites is collected about 7 days, and Protein G affinity chromatography is adopted to purify the antibody. The results are shown in FIG. 1.
Example 2 establishment of double antibody sandwich ELISA kit for human IgA content
1. HRP labelling of monoclonal antibody 1D9
Antibody 1D9 was added to the dialysis bag and dialyzed overnight at 4 ℃ in CB buffer 0.01M.14 mg of HRP was dissolved in 2.8. 2.8 ml water. Preparation of fresh NaIO 4 Solution 0.1, M, add 0.56 ml 0.1M NaIO 4 In 2.8 ml HRP solution, the mixture was stirred, left at room temperature and protected from light for 20min, immersed in NaAc buffer of 1 mM, and dialyzed overnight at 4 ℃. The overnight dialyzed antibody and HRP solution were removed with 0.168 ml,0.2M PH9.5 carbonate buffer, then immediately the removed antibody was added to the HRP solution and gently stirred at room temperature in the dark for 2 hours. Weigh 0.04 g NaBH 4 Dissolving in 10 ml water, adding 0.02: 0.02 ml into the reaction solution, mixing, and standing at 4deg.C for 2 hr. Taking out, placing in a dialysis bag, dialyzing in 0.01M PBS, changing the solution once after two hours, and dialyzing at 4deg.C overnight. Taking out the overnight dialyzed labeling solution, adding equal volume of glycerol, and storing at-20deg.C.
2. Preparation of monoclonal antibody 4C3 ELISA plate
Monoclonal antibody 4C3 was diluted to 2. Mu.g/ml with 0.05M PH9.6 carbonate coating buffer. 0.1 μl was added to the 96-well polystyrene reagent plate reaction wells and incubated overnight at 4deg.C. The next day, the solution in the wells was discarded, and washed 3 times with wash buffer for 1 minute each time. After the above procedure, each well of the reaction plate was blocked with a solution, and incubated at 0.3 μl per well for 2h. The solution in the hole is discarded, and the drying room is dried and vacuumized and stored at 4 ℃.
3. Establishment of double antibody sandwich ELISA method
30 min before the experiment, taking out the ELISA plate coated with the monoclonal antibody 4C3, recovering to room temperature, washing the plate for 3 times, and spin-drying. 100 μl of human IgA standard at various dilution concentrations of 800ng/ml, 400 ng/ml, 200 ng/ml, 100ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml was added and a blank was placed. After sealing plates, incubate 2h at room temperature, wash plates 4 times and spin-dry. 100 μl of enzyme-labeled antibody HRP-1D9 working solution was added to the wells, the plates were incubated at room temperature for 45min after sealing plates, washed 5 times and spin-dried. 100. Mu.l of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added, dual wavelength detection was performed using an ELISA reader, the OD at the 450nm maximum absorption wavelength and 630 nm reference wavelength was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. Taking human IgA standard substances with different concentrations asAnd drawing a standard curve by taking the corresponding OD value as the ordinate on the abscissa, and establishing a regression equation. The result shows that the detection range is 12.5-800 ng/ml, R 2 1.00000 (fig. 2).
4. Double-antibody sandwich ELISA sensitivity detection
The average of 20 zero standard concentrations OD was measured plus two standard deviations, and the corresponding detectable concentration was calculated with a sensitivity of 20 pg/ml (Table 3).
TABLE 3 Table 3
5. Double-antibody sandwich ELISA specific detection
30 min before the experiment, taking out the ELISA plate coated with the monoclonal antibody 4C3, recovering to room temperature, washing the plate for 3 times, and spin-drying. Human IgA standard or high concentration human IgA structural analog proteins including pig IgG, mouse IgG, rat IgG, duck IgG, rabbit IgG, human IgG were added at different dilution factors of 100. Mu.l. After sealing the plates, incubate 2h in a room temperature incubator, wash the plates 4 times and spin-dry. Adding 100 μl enzyme-labeled antibody HRP-1D9 working solution into the reaction well, sealing, incubating in a room temperature incubator for 45min, washing the plate 5 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added, dual wavelength detection was performed using an ELISA reader, the OD at the 450nm maximum absorption wavelength and 630 nm reference wavelength was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. The results showed that the antibody pair did not react with other similar proteins (table 4).
TABLE 4 Table 4
Example 3 human serum IgA content determination
30 min before the experiment, taking out the ELISA plate coated with the monoclonal antibody 4C3, recovering to room temperature, washing the plate for 3 times, and spin-drying. 16 parts of voluntary blood donation human serum are selected, 100 mul of diluted human serum sample or standard substances with different concentrations are added, the dilution multiple of the sample is 2 ten thousand times, the dilution concentration of the human IgA standard substances is 800ng/ml, 400 ng/ml, 200 ng/ml, 100ng/ml, 50 ng/ml, 25 ng/ml and 12.5 ng/ml, and blank control is arranged. After sealing the plates, incubate 2h in a room temperature incubator, wash the plates 4 times and spin-dry. Adding 100 μl enzyme-labeled antibody HRP-1D9 working solution into the reaction well, sealing, incubating in a room temperature incubator for 45min, washing the plate 5 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added, dual wavelength detection was performed using an ELISA reader, the OD at the 450nm maximum absorption wavelength and 630 nm reference wavelength was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. According to the report of the literature, the IgA content range in normal human serum is 0.71-3.85 mg/ml (according to different contents of age and individuals), and the detection result shows that the method can accurately measure the IgA content in human serum (Table 5).
TABLE 5
Experimental example 1 comparison with other brands of the same type of kit test results
The experimental example selects domestic brand X (Elabscience) to carry out analogy test, and experiments are carried out according to the optimal mode of each brand (the operation is carried out according to the specification of the experimental example). The performance of the 2 kits was compared by the following two aspects.
a. Standard curve comparison: the result shows that the fitting curve of the kit is higher than that of other brands of kits (R 2 =1.00000) and the gradient of the kit is obvious, the background performance is excellent (zero hole value is 0.008) (table 6), the color development of the brand X mark is low, and the gradient is poor.
TABLE 6
b. Sample measurement result comparison: 16 serum samples of voluntary blood donators were randomly selected and simultaneously measured (in mg/ml), and IgA content in normal human serum was reported to be in the range of 0.71-3.85 mg/ml (different contents according to ages and individuals) according to literature. The measurement result shows that the content of the sample measured by the kit is lower than that of the sample measured by the brand X, and the content of the sample measured by the kit is closer to that reported by a literature (table 7).
TABLE 7
Experimental example 2 double antibody sandwich ELISA stability detection
The test example carries out stability assessment on the kit through a 37 ℃ acceleration test:
the acceleration stability test was performed by placing the monoclonal antibody 4℃ 3 coated ELISA plate, HRP-labeled monoclonal antibody HRP-1D9, and human IgA standard at 37℃for 12 days (equivalent to about 18 months at 4 ℃). And then taking out for detection, wherein the detection method is that the ELISA plate is taken out for washing the plate for 3 times and spin-drying. 100 μl of standard with different dilution concentrations of 800ng/ml, 400 ng/ml, 200 ng/ml, 100ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml was added, and a blank was placed. After sealing the plates, incubate 2h in a room temperature incubator, wash the plates 4 times and spin-dry. Adding 100 μl enzyme-labeled antibody HRP-1D9 working solution into the reaction well, sealing, incubating in a room temperature incubator for 45min, washing the plate 5 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the wells, which were sealed off and developed at room temperature in the dark for 15min, and 50. Mu.l of stop solution was added. Dual wavelength detection was performed using an enzyme-labeled instrument, the OD values at 450nm maximum absorption wavelength and 630 nm reference wavelength were determined, and the OD measurement at 630 nm was subtracted from the OD measurement at 450 nm. And drawing a standard curve by taking standard substances with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The results show that at 152 th, the high point (at 800 ng/ml) OD values of the standard curve were still greater than 1.5 and the standard curve OD gradient was good, indicating good stability of the kit (table 8).
TABLE 8
Experimental example 3
The experimental example verifies the IgA effect of antibody 1D9 as primary antibody in WB detection in human serum: serum samples from volunteer donors were prepared, subjected to SDS-PAGE, and transferred; placing the NC membrane after membrane transfer in a diluent containing a 1D9 antibody for incubation; after washing the membrane, placing the membrane in a diluent containing HRP-labeled goat anti-mouse IgG-labeled secondary antibody for incubation; after washing the film, ECL luminous liquid is added, and development and exposure are carried out. The results are shown in FIG. 3.
Fig. 3 shows: a deeper band at about 65kDa indicates that monoclonal antibody 1D9 can bind to IgA heavy chains in serum.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (9)
1. A composition of human IgA antibodies, comprising an antibody or antigen-binding fragment thereof as shown in (1) below; also included are antibodies or antigen-binding fragments thereof as shown in (2) below;
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4.5 and 6;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
2. The composition of human IgA antibodies according to claim 1, wherein for the antibody shown in (1): the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15 or with SEQ ID NO:15 has at least 80% sequence similarity; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16 or with SEQ ID NO:16 has at least 80% sequence similarity.
3. The composition of human IgA antibodies according to claim 1 or 2, wherein for the antibody shown in (1): the antibody or antigen binding fragment thereof is selected from the group consisting of Fab, fab ', F (ab') 2 Fv, complementarity determining region fragments, single chain antibodies, monoclonal antibodies, animal-derived antibodies, or humanized antibodies.
4. A nucleic acid molecule composition encoding the antibody or antigen-binding fragment thereof of the composition of any one of claims 1 to 3.
5. A biological material comprising the nucleic acid molecule composition of claim 4; the biological material is an expression cassette, a vector or a host cell.
6. An antibody conjugate, characterized in that the antibody or antigen binding fragment thereof in the composition of the human IgA antibody of any one of claims 1-3 is conjugated with a label, wherein the label is one or more selected from the group consisting of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label and a radioactive label.
7. Use of the composition of any one of claims 1 to 3 of a human IgA antibody or the nucleic acid molecule composition of claim 4 or the biomaterial of claim 5 or the antibody conjugate of claim 6, in any one of the following (1) to (5):
(1) Use in the preparation of a product for detecting the presence or level of human IgA in a sample;
(2) Use of detecting the presence or level of human IgA in a sample for non-diagnostic and therapeutic purposes;
(3) Use in the preparation of a product for neutralising human IgA activity in a sample;
(4) Use in the manufacture of a medicament for neutralising in vivo human IgA activity;
(5) Use in quality control or production of a human IgA-containing product.
8. A kit comprising the composition of any one of claims 1 to 3 of a human IgA antibody, or comprising the antibody conjugate of claim 6, or comprising the composition of claim 7.
9. The kit according to claim 8, which is an enzyme-linked immunoassay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.
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