CN108517315B - Anti-human IgM monoclonal antibody, its hybridoma cell strain and application - Google Patents
Anti-human IgM monoclonal antibody, its hybridoma cell strain and application Download PDFInfo
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- CN108517315B CN108517315B CN201810296837.3A CN201810296837A CN108517315B CN 108517315 B CN108517315 B CN 108517315B CN 201810296837 A CN201810296837 A CN 201810296837A CN 108517315 B CN108517315 B CN 108517315B
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to hybridoma cell strain and its secreted monoclonal antibody, the antibody can be specifically bound with people IgM, and can be used for the vitro detection of people IgM, be especially suitable for the early diagnosis of rubella virus infection.The invention further relates to the kits for including the hybridoma cell strain or monoclonal antibody.
Description
Technical field
The present invention relates to a kind of monoclonal antibody, in particular to a kind of monoclonal antibody of anti-human IgM.
Background technique
Immune diagnostic reagent is one of main Types of external diagnosis reagent.Its spy being combined with each other using antigen and antibody
The opposite sex reacts to carry out qualitative or quantitative diagnosis, either technology or market, and such reagent is in current all diagnostic reagents
It is with fastest developing speed in product.
After cause pathogeny imcrobe infection human body, IgM type antibody generates at first during stimulating induction body fluid immune response,
The early diagnosis of infectious diseases is the premise for carrying out early stage, effectively treating, especially for onset urgency, the dangerous infection of the state of an illness
Property disease, early diagnose particularly important, since the antibody that occurs at first of body is IgM type antibody after infection, therefore detect morbidity just
Specific IgM antibodies have great importance in clinic early diagnosis in phase blood samples of patients.
In recent years, there are some researchs about anti-human IgM monoclonal antibody, such as patent document WO 2010026758A1
In disclose a kind of anti-human IgM monoclonal antibody, which aims to solve the problem that the low problem of polyclonal antibody specificity, and resit an exam
It has examined the property being aggregated between monoclonal antibody induction people IgM and has demonstrated it and inhibited the effect of non-specific responding, but do not closed
Infuse when the anti-human IgM monoclonal antibody is for external diagnosis reagent systematicness evaluation (as, antibody titer, cross reactivity,
Stability, detection sensitivity and specificity etc.), thus, it is indefinite that whether which, which is suitable for preparing external diagnosis reagent,.
In another example the anti-human IgM monoclonal antibody of Xiamen wave biology is the anti-human IgM monoclonal antibody of common commercialization,
But its stability is not good enough, thus the Shortcomings when being used for immunodiagnosis antibody.
In fact, it is a complicated process that screening, which is used to prepare the monoclonal antibody of external diagnosis reagent, first have to obtain
Good antigen is obtained, to prepare enough antibody, the evaluation of system is then carried out to antibody, obtaining has clinical correlation
Candidate antibodies are redeveloped into detection reagent.Wherein, antibody titer, cross reactivity, stability and clinical effectiveness etc. are important
Factor of evaluation, as antibody titer height reflect under a certain concentration with antigen reactive minimum titre, titre more low liter more
It is high；Antibody cross reaction can influence the specificity of the antibody；The stability of antibody will have a direct impact on the reliability of final result,
And stability poor antibody preservation condition, operating condition are required it is higher, to reduce it as the practical of diagnostic reagent
The reliability of property and result, and inevitably cause the rising of cost；Clinical test is then used to illustrate that antibody is diagnosing certain
Effect when specified disease.
Therefore, in order to diagnose the application of aspect in vitro, it is preferable that systemic evaluation result is badly in need of in this field, and is suitable for especially
The monoclonal antibody of the early diagnosis of pathogen infection.
Summary of the invention
The object of the present invention is to provide a kind of anti-human IgM monoclonal antibody, the hybridoma cell strain for secreting the antibody, containing upper
State monoclonal antibody or hybridoma cell strain kit and they detection people IgM in purposes.It is evaluated by systematicness,
The antibody has preferable performance in all respects, so that being suitable as immune diagnostic reagent is used to prepare external diagnosis reagent case,
It is especially suitable for preparation rubella virus early diagnosis kit.
For this purpose, present inventor has performed numerous studies, it is immunized mouse by people IgM, through limiting dilution after cell fusion
Method is cloned at least 4 times, and until reaching monoclonal, the novel of 1 plant of energy stably excreting antibody is screened from obtained numerous clones
Hybridoma cell strain (Hybridoma) IgM-5, and be preserved in China typical culture collection on March 8th, 2018
The heart, the Chinese Wuhan Wuhan University, deposit number are CCTCC NO:C201847, so as to complete the present invention.
In the first aspect, the present invention provides a kind of hybridoma cell strain, it is preserved in China typical culture collection
Center, deposit number are CCTCC NO:C201847.
In the second aspect, the present invention provides a kind of monoclonal antibody, the monoclonal antibody can be special with people IgM
The opposite sex combines.
In one embodiment, the monoclonal antibody is not in conjunction with human IgG, mouse IgG, rabbit igg or ox IgG.
In another embodiment, the middle titer of the monoclonal antibody is 138900 and in multigelation, long-term
It saves, there is more preferably stability and reliability under thermal acceleration mal-condition.
In one preferred embodiment, the monoclonal antibody is by the anti-of hybridoma cell strain secretion of the invention
Body.
In the third aspect, the present invention provides a kind of kit, the kit includes hybridoma of the invention
Strain or monoclonal antibody.
In a specific embodiment, the kit is colloidal gold immunoassay kit, chemical luminescence reagent kit, radiation
Immune reagent kit, enzyme linked immunological kit or fluorescence immunoassay kit.
In one preferred embodiment, the kit is enzyme linked immunological kit.
In another embodiment, the kit is micro-fluid chip.
In the fourth aspect, the use of hybridoma cell strain or monoclonal antibody of the invention in reagent preparation box is provided
On the way.
In one embodiment, the kit is the kit based on immune detection, it is preferred that the kit is
Colloidal gold immunoassay kit, chemical luminescence reagent kit, radioimmunoassay kitss, enzyme linked immunological kit or fluorescence immunoassay reagent
Box.
In one preferred embodiment, the kit is enzyme linked immunological kit.
In another embodiment, the kit is micro-fluid chip, it is preferred that the micro-fluid chip is based on
Immune detection.
In one embodiment, the kit is for detecting people IgM.
In one preferred embodiment, the people IgM is the people IgM generated at pathogen infection initial stage.
In a specific embodiment, the cause of disease is rubella virus.
In addition, additionally providing hybridoma cell strain of the invention or monoclonal antibody of the invention in preparation for rubella
Purposes in the kit of poison diagnosis, is preferred for the kit of rubella virus early diagnosis.
The beneficial effect of monoclonal antibody of the invention is, firstly, its antibody titer is more usually used than in in-vitro diagnosis
At least high an order of magnitude of commercially available human IgM antibody, have more preferably immune effect；Secondly, antibody of the invention and human IgG,
Mouse IgG, rabbit igg, ox IgG no cross reaction have good specific binding capacity；Again, have than commercially available people IgM
Antibody is more preferably for a long time and thermal stability, that is to say, that extends service life and can receive condition of storage and the behaviour of relative loose
Make condition, so that cost be greatly saved；Finally, clinical trial shows that monoclonal antibody of the invention can be used for preparing rubella
The enzyme linked immunological kit of malicious IgM antibody detection, detection sensitivity detect specificity up to 100% up to 100%.
That is, monoclonal antibody of the invention is more in antibody titer, cross reactivity, stability and detection effect etc.
A aspect has shown good performance, to can be used for in-vitro diagnosis and comprehensive through systematicness evaluation the present invention provides a kind of
Conjunction ability anti-human IgM monoclonal antibody outstanding.
Detailed description of the invention
Fig. 1 shows the SDS-PAGE electrophoresis of antibody IgM-Ab5 of the invention；
It measures and ties Fig. 2 shows the antibody titer of antibody IgM-Ab5 of the invention and two kinds of commercialization anti-human IgM antibodies
Fruit, wherein abscissa is Log (extension rate), ordinate OD450。
Specific embodiment
Below in conjunction with specific embodiment and embodiment, it is specifically described the present invention, advantages of the present invention and various effects
It thus will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating
The present invention is not intended to limit the present invention.
Throughout the specification, unless otherwise specified, terms used herein are interpreted as usual in this field
Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has leads with belonging to the present invention
The identical meaning of the general understanding of field technique personnel.Contradiction if it exists, this specification are preferential.
Antibody
As it is used herein, term " antibody " refers to immunoglobulin molecules, including but not limited to chimeric antibody, source of people
Change antibody, human antibody, CDR grafted antibody and antibody construct, such as scFv (scFv) or antibody fusion protein；In addition,
Further relate to recombination ground or synthetically generation/synthesis antibody.
In a preferred embodiment, antibody refers to the hybridoma for being produced from that deposit number is CCTCC NO:C201847
The antibody of cell strain.
" antibody fragment " generally includes the antigen binding domain of parental generation antibody, light chain and/or heavy chain variable region, one or more
At least part of (such as six) CDR retains at least certain binding specificity of parental generation antibody.Particularly, herein
Parental generation antibody refers to the antibody for being produced from the hybridoma cell strain that deposit number is CCTCC NO:C201847.Antibody fragment
Example includes but is not limited to Fab, Fab', F (ab') 2 and Fv segment；Homodimer；Linear antibodies；Single-chain antibody molecules, example
Such as, sc-Fv；And the multi-specificity antibody formed by antibody fragment.In general, segment is protected when based on mole come expression activity
It is left to the few 50% combination activity to people IgM.Preferably compared with parental generation antibody, segment retain at least 60%, 70%,
80%, 90%, the 95% or 100% combination activity to people IgM.
Preferably, antibody fragment refers to antigen binding domain, light chain and the heavy chain variable region or six CDR of antibody.
" antibody derivatives " refer to may include antibody conserved amino acid substitutes (referred to as " conservative variant "), its life
Object activity does not change substantially compared with parental generation antibody.
The present invention provides the monoclonal in form of antibody.
As used herein, term " monoclonal antibody " refers to the antibody for being obtained from the substantially group of allo-antibody,
That is, except it is possible can be in addition to a small amount of existing abiogenous mutation, it is identical for constituting the individual antibody of group.For list
Antigen site, monoclonal antibody are high specials.Monoclonal antibody is advantageous, because they can pass through hybridoma
Strain culture obtains, and substantially not by the pollution of other immunoglobulins.
Kit
Diversified forms can be used in detection kit of the invention, for example, test paper, the test containing reagent needed for various tests
Box, micro-fluid chip etc. can manufacture kit according to standard step well known by persons skilled in the art.
Kit of the invention may include as needed container, chip, operation instructions, buffer, immune auxiliaries and/or
For carrying out diagnosing/detecting required other materials, structure and/or reagent.
Using being illustrated for enzyme-linked immunosorbent assay to kit of the invention in embodiment, but it should not be construed as this hair
Bright kit is only limitted to enzyme-linked immunosorbent assay.
Kit of the invention includes the anti-of the hybridoma cell strain for being produced from deposit number as CCTCC NO:C201847
Body can exist in such a way that this field is conventional, for example, being present in container with dissolution or dried forms, be coated on solid phase
On carrier (for example, film, plate, pearl, particle (such as magnetic particle)), it is present in the chamber of chip with dissolution or dried forms, but
The invention is not limited thereto.
Since objective factors, the kits such as transport and place to use generally require to be suitble to be showed in all kinds of complex environments
Field detecting, therefore, the stability of raw material be restrict kit results an important factor for one of.Shown in embodiment 9 as follows
, more conventional IgM antibody possesses more under extreme conditions as the raw material of enzyme linked immunological kit by antibody IgM-Ab5 of the invention
Good stability, so that making the result reliability of kit enhances and in a disguised form reduce costs.
Antibody of the invention can come to use preferably 1~5 μ g/ml, more preferably 1.5 μ with the concentration of 0.1~10 μ g/ml
g/ml。
It is used to carry out diagnosing/detecting required other materials in kit of the invention to include but is not limited to remove the present invention
Antibody outside other anti-human IgM antibodies, the antigen in conjunction with human IgM antibody and/or people IgM.Above-mentioned other materials can be with
The mode of this field routine exists, for example, be present in container with dissolution or dried forms, be coated on solid phase carrier (for example,
Film, plate, pearl, particle (such as magnetic particle)), it is present in the chamber of chip with dissolution or dried forms, but the present invention is not limited to
This.
For carrying out diagnosing/detecting required other structures including but is not limited to be used to sample knot in kit of the invention
Structure, the structure for carrying out contrast structure and/or for observing detection process or result.
It is used to diagnose/detect other required reagents in kit of the invention to include but is not limited to, detergent, show
Toner and/or terminator.
In one embodiment, the antibody in kit of the present invention detectably marks.Ability can be used
Any marker and labeling method known to field technique personnel.For example, the marker that can be used in the present invention includes enzyme, puts
Injectivity isotope, colloidal metal, fluorescent chemicals, chemiluminescence compound and bioluminescent compound, but the present invention is not limited to
This.
Common marker may include enzyme (such as horseradish peroxidase, beta galactosidase, alkaline phosphatase), radiation
Property isotope is (such as32P or125I) etc., biotin, digoxin, colloidal metal (such as colloidal gold), fluorescent dye (such as fluorescein, sieve
Red bright, texas Red etc.), chemiluminescence compound or bioluminescent compound (such as dioxetane, luminol or acridine
Deng).Any markers step well-known in the art can be used, such as enzyme or the covalent coupling of biotin group, iodate, phosphorus
Acidification, biotinylation etc..
In some embodiments, for diagnose/detect one of required other materials or it is a variety of can also be with
Detectably mark.
In one preferred embodiment, kit of the invention is the kit for cause of disease early diagnosis.
Purposes
Anti-human IgM antibodies or hybridoma cell strain of the invention can be used for relevant to the specific reaction of people IgM any
Purpose.Preferably, antibody of the invention or hybridoma cell strain can be used for detecting people IgM.
Antibody or hybridoma cell strain of the invention can detect the biological sample from the mankind.
As used herein, " biological sample " refers to sperm, lymph, serum, blood plasma, urine, synovia or spinal fluid.?
In preferred embodiment, biological sample refers to blood, serum or blood plasma.
Preferably, using the biological sample of the early stage from people.
The method of immunoassays can be used quantitatively or the presence of qualitative detection people IgM, the immunoassays generally include
Biological sample is incubated for together with other materials needed for antibody of the invention and/or detection or is successively contacted, and by a variety of
The antibody that technology detection well known in the art combines.
Detection method includes but is not limited to autoradiograph, fluorescence microscopy, enzymatic reaction directly or indirectly, puts
Injectivity isotope method or non radioactive isotope method etc..These methods especially include western blot, overlapping measures, RIA (is put
Penetrate immunoassays) and IRMA (immune radiating immunoassays), GIA (colloid gold immune measurement), EIA (enzyme immunoassay (EIA)), ELISA
(enzyme linked immunosorbent assay (ELISA)), FIA (fluorescence immunoassay) and CLIA (chemiluminescence immunoassay).
Anti-human IgM monoclonal antibody can be tried in conjunction with the people IgM that various pathogen infections generate, but in preparation in-vitro diagnosis
When agent, since detection architecture is different or the nature difference of antibody itself, diagnosis effect inevitably can difference.Middle reality as follows
It applies shown in example 8, for antibody of the invention when detecting IgM caused by rubella virus (RV), effect is commercially available better than common at present
Detection kit.
Therefore, in one preferred embodiment, antibody of the invention or hybridoma cell strain are particularly suitable for detecting
The people IgM that rubella virus infection initial stage generates, to diagnose whether individual infects rubella virus.
Embodiments of the present invention are described in detail below in conjunction with embodiment, actual conditions are not specified in embodiment
, it carries out according to conventional conditions or manufacturer's recommended conditions.Production business men is not specified in agents useful for same or instrument, for that can lead to
Cross commercially available conventional products.
1 mouse of embodiment is immunized
The people IgM (Sichuan mikey biology new material technology Co., Ltd, lot number 150127) that blood source is extracted uses physiology salt
Water is diluted to 3.0mg/ml, mixes with Freund's complete adjuvant (Sigma company, article No. SLBF-9338V), is infused in equal volume with 1ml
Emitter emulsification is oil emulsion, until the oil emulsion instilled in water does not disperse to stop emulsifying, by the lotion with 160 μ l/
Dosage four limbs armpit only is subcutaneously applied to BALB/c mouse (Chengdu reaches large Experimental Animal Center, 6 week old female, 2) for the first time
Enhance after 21 days immune and be immunized, takes people IgM and incomplete Freund's adjuvant (Sigma company, article No. SLBM9367V) mixed in equal volume
It is emulsified after conjunction, immunizing dose is 80 μ l/, and enhancing is immune primary every other week later, and tail blood is adopted before being immunized every time, separates blood
Clearly, potency is measured with indirect elisa method.After 5 times immune, all mice serum potency are greater than 1:106, can be used to merge.Melt
It closes first 3 days, takes people IgM with normal saline dilution to 3.0mg/ml, then mix tail vein supplementary immunization in equal volume with physiological saline,
Dosage is 80 μ l/.
The preparation of 2 hybridoma cell line of embodiment
The preparation of 2-1 feeder cells
Make feeder cells with normal 10 week old BALB/c mouse peritoneal macrophage.1 day before fusion, BALB/c takes eye
Blood draws neck to put to death, and 0.1% bromogeramine impregnates 1 minute, is transferred to 75% alcohol and impregnates 1 minute, uses under sterile working in super-clean bench
Scissors abdominal cut skin, exposure peritonaeum, with syringe Intraperitoneal injection RPMI1640 basic culture solution 6ml, after taken with dropper
Out, 4ml RPMI1640 basic culture solution repeated flushing is added, flushing liquor, 1000rpm are recycled, centrifugation stays precipitating in 5 minutes, uses
The RPMI1640 culture solution that 20% newborn bovine serum has been added is resuspended, and adjustment cell concentration is 3.2 × 10596 holes are added in a/ml
Plate, 100 holes μ l/, 37 DEG C, 5%CO2Culture.
The preparation of 2-2 immune spleen cell
Behind mouse supplementary immunization three days in embodiment 1, spleen is aseptically taken out respectively, is placed in plate,
RPMI1640 basic culture solution rinses primary, the splenocyte that is dispersed after shredding, grind, filter, 1000rpm, centrifugation 5 minutes
Precipitating is stayed to be centrifuged, RPMI1640 basic culture solution is resuspended, and the dilution of 3% acetic acid counts.
The preparation of 2-3 myeloma cell
Murine myeloma cell Sp2/0 (preservation of Sichuan mikey biology new material technology Co., Ltd) is sieved through 8-anaguanine
After choosing, cultivates to logarithmic growth phase, take 5 big bottle (75cm2) cell suspension is made, 1000rpm is centrifuged 5 minutes and stays precipitating, uses
RPMI1640 basic culture solution is resuspended, counts, by 2.0 × 105The cell concentration of a/ml carries out sub-bottle culture (ordinary circumstance every 1
~2 days complete 1640 culture mediums of 15~30ml of replacement).
2-4 cell fusion and HAT selection culture hybridoma
Myeloma cell and immune spleen cell are mixed in the ratio of 1:6, use RPMI1640 in 50ml conical centrifuge tube
Basic culture solution is washed 1 time, and 1000rpm is centrifuged 5 minutes and stays precipitating.Cell is mixed, the PEG4000 of 0.6ml 50% is slowly added to
Fusion, the RPMI1640 basic culture solution that 30ml is added after fusion 1 minute terminate cell fusion.700rpm centrifugation weighs after five minutes
It is suspended from 1640 culture mediums containing 1%HAT and 20% newborn bovine serum, averagely instills 12 set of 96 porocyte culture plates.37 DEG C,
5%CO2Culture, it is full to hole that next day adds 1640 culture mediums containing 1%HAT and 20% newborn bovine serum.5 days later half to change culture
Base partly changed culture medium after 7 days again.
The screening of 2-5 positive cell strain
With 0.06M pH9.6 carbonate buffer solution dilution people IgM, (Sichuan mikey biology new material technology Co., Ltd is criticized
Number 150127) to 5 μ g/ml, every hole is coated with 100 μ l in 96 hole elisa Plates, for detecting cells and supernatant.It is placed in refrigerator
2~8 DEG C overnight, are abandoned liquid in hole for second day, and ELISA washing lotion board-washing three times, pats dry, with the 0.01M for containing 10% calf serum
The PBS of pH7.2,150 holes μ l/, 37 DEG C are closed 2 hours, are patted dry, Vacuum Package is stand-by.The 9th day after splenocyte fusion, cell is taken
In the detection plate of above-mentioned 96 hole, 37 DEG C are incubated for 40 minutes 100 μ l of supernatant, and 8000 times of dilutions are added in ELISA board-washing after machine-washing five times
Horseradish peroxidase label sheep anti-mouse igg (production of Sichuan mikey biology new material technology Co., Ltd) 100 μ L, 37 DEG C incubate
It educates 30 minutes after ibid washing, every hole is added 100 μ L and contains 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 lemon
Acid phosphoric acid buffer, 37 DEG C are incubated for 10 minutes, and every hole is added 50 μ L 2M sulfuric acid solutions and terminates reaction, survey 450nm absorption value.With
Mice serum is diluted to 100 times as positive control when fusion, and 1640 complete culture solution of RPMI is as negative control, negative control
OD value < 0.2, positive control OD value > 1.8 are that detection system is effective, are positive when value >=2 sample OD × negative control OD value
Property, otherwise it is feminine gender.Secretory antibody positive cell hole is cloned on 96 well culture plates with limiting dilution assay with 1 cells/well, sieve
Method is continuously cloned four times on choosing positive Kong Yi, is reached 100% monoclonal, is transferred to 24 holes and continues to cultivate, cover with to cell
It is transferred to when 80% after cell bottle expands culture, sub-bottle passes on when cell covers with cell bottle 80%, and the cell of passage is grown to pair
When the number phase, with appropriate serum-free RPMI-1640 culture medium cell dispersion bottle inner cell, cell suspension is collected in conical centrifuge tube
In, cell suspension volume V is recorded, takes appropriate cell suspension to carry out cell count, obtains cell suspension density (a/ml), remaining
Supernatant is abandoned after 1000rpm centrifugation 5min, the cell number of precipitating is calculated according to cell density and centrifugation precursor, to cell precipitation
It is middle that appropriate frozen stock solution adjustment cell density is added to 4~8 × 106A/ml (takes and carries out counting confirmation in right amount, if cell number is not
In range, be then centrifuged again according to cell count total amount, rejoin appropriate frozen stock solution, finally make cell concentration 4~8 ×
106A/ml), it is then sub-packed in sterile cryopreservation tube, every cryopreservation tube refinement cytosol 0.5ml.It is steady that cell fusion obtains 12 plants of energy
Surely the hybridoma cell strain for secreting the anti-human IgM monoclonal antibody antibody of mouse, see the table below 1.Wherein, hybridoma cell strain 9E1-C1-
The effect in the IgM antibody for detecting rubella virus infection early stage of antibody secreted by C8-B3-G8 is optimal, this hybridoma is thin
Born of the same parents' strain is denoted as IgM-5, and China typical culture collection center is deposited on March 8th, 2018, and deposit number is CCTCC NO:
C201847。
Table 1 screens the hybridoma cell strain of the obtained anti-human IgM monoclonal antibody of stably excreting
| Hybridoma | Hybridoma | Hybridoma |
| 1A10-C1-D1-H12-C7 | 3H3-H10-D5-D12-D1 | 9E1-C1-C8-B3-G8 |
| 1D6-E1-C3-B7-F2 | 12F2-A12-C9-D12-D9 | 4F10-H7-C1-C12-D5 |
| 2A3-A5-E2-C12-G10 | 11C10-H7-H6-F8-E5 | 5H7-C1-C9-A8-G9 |
| 2E11-H2-A4-E11-G9 | 10H12-E1-H11-D11-G5 | 8H2-H1-E5-E5-E9 |
The preparation of 3 monoclonal antibody of embodiment
The BALB/c mouse of 6~8 weeks health is selected, every mouse peritoneal injects 0.5ml atoleine (Tianjin Ke Miou), and 7
Every mouse peritoneal injection 1.6 × 10 after it6A hybridoma.Inoculating cell can produce ascites, close observation after 7~10 days
The health status of animal and sign of ascites as, it is as more as possible to ascites, and before mouse is dying, mouse is put to death, with dropper by ascites
It sucks in test tube, a mouse can obtain 1~5ml ascites.The ascites centrifuging and taking supernatant of collection takes sample to be put in -20 DEG C of refrigerators and protects
It deposits.Ascites is respectively with after sulfate of ammoniac saturation precipitating, then is purified with Protein A affinity chromatography, and SDS-PAGE detects this antibody and (is denoted as
IgM-Ab5) purity is greater than 90%, and electrophoresis result is as shown in Figure 1.
4 Hybridoma Cell Culture supernatant bioactivity of embodiment
With 0.06M pH9.6 carbonate buffer solution dilution people IgM, (Sichuan mikey biology new material technology Co., Ltd is criticized
Number 150127) to 5 μ g/ml, every hole is coated with 100 μ l in 96 hole elisa Plates.It is placed in refrigerator and stays overnight for 2~8 DEG C, abandon within second day
Liquid in hole, ELISA board-washing machine-wash three times, pat dry, with containing 10% calf serum 0.01M pH7.2 PBS, 150 holes μ l/,
37 DEG C of closings, 2 hours abandoning liquid, pats dry, for detecting Hybridoma Cell Culture supernatant, ascites and antibody titer.Hybridoma
The first hole of culture supernatant bioactivity is former times supernatant culture solution, is buffered from the second hole to the 4th hole with the PBS of 0.01M pH7.2
Liquid 10 dilutes step by step again, and ten hole the 5th Kong Zhi is diluted step by step with 2 times.Mice serum is diluted to 100 when 11-holes are to merge
Make positive control again, negative control is made with 1640 complete culture solution of RPMI in the 12nd hole, and every hole sample volume is 100 μ l.37℃
It is incubated for 40 minutes, the sheep anti-mouse igg of 12000 times of diluted horseradish peroxidase labels is added in ELISA board-washing after machine-washing five times
(production of Sichuan mikey biology new material technology Co., Ltd) 100 μ L, after 37 DEG C are ibid washed for incubation 30 minutes, 100 μ are added in every hole
L contains 0.1% (M/V) o-phenylenediamine, and 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffer, 37 DEG C are incubated for 10 minutes,
Every hole is added 50 μ L 2M sulfuric acid solutions and terminates reaction, surveys 450nm absorption value.Negative control OD value < 0.2, positive control OD value
> 1.8 is that detection system is effective, on the contrary for feminine gender for the positive when value >=2 OD × negative control OD value.The minimum sun of detected value
Dilution ratio corresponding to property hole is Hybridoma Cell Culture supernatant potency, this Hybridoma Cell Culture supernatant antibody titer is big
In 1:8 × 103, it is shown in Table 2.
2 culture supernatant potency result of table
| Enzyme mark hole number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Extension rate | 1 | 10 | 100 | 1000 | 2000 | 4000 | 8000 | 16000 | 32000 | 64000 | Positive control | Negative control |
| IgM-5 supernatant | 1.91 | 1.92 | 1.58 | 1.28 | 0.79 | 0.59 | 0.25 | 0.04 | 0.04 | 0.04 | 2.35 | 0.04 |
The detection of 5 titer of ascites of embodiment
ELISA detection method is the same as embodiment 4.Dilution process is different, specifically: the first hole is former times ascites, with 0.01M
The PBS buffer solution of pH7.2 dilutes step by step again from the second Kong Zhi seven apertures in the human head 10, is diluted step by step from octal to the tenth hole with 2 times.Inspection
Dilution ratio corresponding to the minimum positive hole of measured value is titer of ascites, and table 3 is titer of ascites, this hybridoma IgM-5 institute
The titer of ascites of preparation is greater than 1:2 × 106。
3 titer of ascites of table
| Enzyme mark hole number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Extension rate | 1 | 10 | 100 | 1000 | 10000 | 100000 | 1000000 | 2000000 | 4000000 | 8000000 | Positive control | Negative control |
| IgM-5 ascites | 2.91 | 2.72 | 2.58 | 2.28 | 1.11 | 0.90 | 0.50 | 0.34 | 0.04 | 0.04 | 2.65 | 0.04 |
The detection of 6 antibody titer of embodiment
First antibody IgM-the Ab5 after purification prepared in embodiment 3 is diluted to the PBS buffer solution of 0.01M pH7.2
1mg/ml, after dilute again 100 times be used as initial first holes, since the second hole to the tenth hole make 5 times dilute step by step, 11-holes
Mice serum is diluted to 100 times and makees positive control when merging, and negative control is made with PBS in the 12nd hole, and every hole sample volume is
100μl.37 DEG C are incubated for 50 minutes, and the sheep of 8000 times of diluted horseradish peroxidase labels is added in ELISA board-washing after machine-washing five times
Anti- 100 μ L of mouse IgG (production of Sichuan mikey biology new material technology Co., Ltd), after 37 DEG C of incubation 1h are ibid washed, every hole is added
100 μ L contain 0.1% (M/V) o-phenylenediamine, and 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffer, 37 DEG C are incubated for 15
Minute, every hole is added 50 μ L 2M sulfuric acid solutions and terminates reaction, detects 450nm absorption value.Negative control OD value < 0.2 is positive right
It is that detection system is effective according to OD value > 1.8.Antibody titer criterion: with LOG (dilution) for abscissa, it is with antibody OD value
Ordinate makees curve, and curvilinear equation is y=min+ (max-min)/(1+10^ ((logEC50-x) × Hillslope)), passes through
Sigmaplot data processing software matched curve, takes middle titer=10logEC50.This antibody IgM-Ab5 intermediate value is imitated as the result is shown
Valence is 138900.In kind two kinds of commercialization anti-human IgM monoclonal antibody B and C of mouse of control test, by fitting, B is anti-
Titer is that titer is 8793 in 13600, C antibody in body, the antibody secreted lower than hybridoma of the invention.Table 4 with
Fig. 2 shows titration results.
The detection of 4 antibody titer of table
7 cross reaction of embodiment measurement
With 0.06M pH9.6 carbonate buffer solution dilution human IgG, (Sichuan mikey biology new material technology Co., Ltd is criticized
Number 140120), mouse IgG (Sichuan mikey biology new material technology Co., Ltd, lot number 140221), rabbit igg (Sichuan mikey biology
New material technology Co., Ltd, lot number 140225) and ox IgG (Sichuan mikey biology new material technology Co., Ltd, lot number
140311) to 2.5 μ g/ml, every hole is coated with 100 μ l in 96 hole elisa Plates.It is placed in refrigerator and stays overnight for 2~8 DEG C, abandon within second day
Liquid in hole, ELISA board-washing machine-wash three times, pat dry, make confining liquid with 0.5% casein of Tris-HCl containing 10mM (7.4),
150 holes μ l/, 37 DEG C of closings, 2 hours abandoning liquid, pat dry spare.Anti-human IgM monoclonal antibody IgM-Ab5 of the invention is used
It is diluted to same concentrations (0.5mg/ml) after HRP label, then dilutes 500 times, 1000 times, 2000 times, 4000 times, 8000 times, is added
Enter in the good ELISA Plate hole of above-mentioned coating, every 50 μ l of hole is reacted with four kinds of IgG respectively, 37 DEG C be incubated for 30 minutes after ELISA
Board-washing is machine-washed five times, is patted dry, and every hole is added 100 μ L and contains 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 lemon
Lemon acid phosphoric acid buffer, 37 DEG C are incubated for 10 minutes, and every hole is added 50 μ L 2M sulfuric acid solutions and terminates reaction, survey 450nm absorption value
(multi-functional readout instrument, manufacturer Thermo, model Varioskan Flas).Following table is the cross reaction measurement result of IgM-Ab5,
There is not cross reaction with four kinds of IgG.
5 cross reaction of table measurement
| Enzyme mark IgM-Ab5 dilution | 1:500 | 1:1000 | 1:2000 | 1:4000 | 1:8000 |
| People IgM | 1.911 | 1.775 | 1.798 | 1.705 | 1.105 |
| Human IgG | 0.251 | 0.126 | 0.104 | 0.102 | 0.07 |
| Mouse IgG | 0.060 | 0.118 | 0.111 | 0.137 | 0.1 |
| Ox IgG | 0.058 | 0.155 | 0.175 | 0.158 | 0.079 |
The clinical diagnosis of 8 cause of disease of embodiment
(1) prepared by kit
Kit uses enzyme linked immunosorbent assay, utilizes prize law principle.
The μ of antibody IgM-Ab5 to 1.5 g/ml of the invention, 96 hole enzyme marks are diluted with 0.06M pH9.6 carbonate buffer solution
Every hole is coated with 100 μ l in plate.It is placed in refrigerator and stays overnight for 2~8 DEG C, abandon within second day liquid in hole, ELISA board-washing machine washing three
It is secondary, it pats dry, with the PBS of the 0.01M pH7.2 containing 10% calf serum, 150 holes μ l/, 37 DEG C of closings, 2 hours abandoning liquid is patted dry
It is spare.Sample is diluted 500 times with the PBS buffer solution of 0.01M pH7.2, is added in the good enzyme mark hole of above-mentioned coating, with health
It is negative right with PBS buffer solution 1000 times of works of dilution of the 0.01M pH7.2 containing 10% calf serum after human serum filtration sterilization
According to the PBS buffer solution after rubella virus IgM antibody positive human serum inactivation with the 0.01M pH7.2 containing 10% calf serum
Positive control is made in 1000 times of dilution, and setting yin and yang attribute compares each 2 hole and 1 hole of blank, and injection volume is 100 μ l.37 DEG C are incubated for 60
Minute, ELISA board-washing is machine-washed five times, and the recombinant rubella virus antigen that horseradish peroxidase (HRP) is marked is added, and (Sichuan steps
The production of gram biological material Technology Co., Ltd.) 100 μ L, 37 DEG C are incubated for 30 minutes ibid after board-washing, and every hole is added 100 μ L and contains
0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffer, 37 DEG C be incubated for 10 minutes, often
Hole is added 50 μ L 2M sulfuric acid solutions and terminates reaction, surveys 450nm absorption value (multi-functional readout instrument, manufacturer Thermo, model
Varioskan Flas).Sample OD value/feminine gender value >=2.0 average control OD are the positive, otherwise are feminine gender.OD value is in 2.1-
It is low concentration region between 2.5, extension rate repetition measurement should be reduced.
(2) sensitivity technique
The detection kit prepared in aforementioned manners examines 200 positive samples and 200 negative samples respectively.Table 6 is listed
The kit and commercial kit (Zhuhai Special Economic Zone Haitai biology system of monoclonal antibody IgM-Ab5 preparation of the invention
Medicine Co., Ltd) comparative experiments, inspection result show monoclonal antibody IgM-Ab5 preparation of the invention detection kit its
Sensitivity and specificity are above commercial kit, list testing result in table 6.
Table 6 is positive to be examined with negative sample
The verifying of 9 stability of embodiment
The stability of monoclonal antibody of the present invention in the presence of a harsh environment by monoclonal antibody IgM-Ab5 of the invention with
Handled under the conditions of lower: a-20 DEG C multigelation 2 times；B-20 DEG C multigelation 3 times；C-20 DEG C multigelation 4 times；D-20 DEG C repeatedly
Freeze thawing 5 times；E-20 DEG C saves 7 months；37 DEG C of f thermal acceleration 7 days；37 DEG C of g thermal acceleration 14 days, other conditions are constant, by above-mentioned
Method is prepared into detection reagent and examines 15 parts of positive reference product and 15 parts of negative reference product respectively, and coincidence rate is 100%.Show
This Antibody stability is good, and good using the detection reagent accuracy of this Antibody preparation.It is anti-to choose the higher commercialization of potency
People's IgM monoclonal antibody B (Xiamen wave is raw), is prepared into detection reagent after being handled with above-mentioned similarity condition and detects 15 parts of positives respectively
Reference material and 15 parts of negative reference product, beginning after it is saved 7 months for multigelation 5 times or -20 DEG C repeatedly as the result is shown
Degradation, inspection result and reference material not in full conformity with.Testing result is listed in table 7.
7 stability result of table
It can be seen that monoclonal antibody stability of the invention is high, the rubella virus IgM antibody detection reagent being made from it
Very high Stability and dependability is also able to maintain under the conditions of more rugged environment, this point is in clinical and laboratory applications
In be very valuable progress.
Claims (13)
1. a kind of hybridoma cell strain IgM-5 is preserved in China typical culture collection center, deposit number CCTCC
NO:C201847。
2. a kind of monoclonal antibody, the monoclonal antibody is as secreted by hybridoma cell strain described in claim 1.
3. a kind of kit, the kit includes hybridoma cell strain described in claim 1 or list as claimed in claim 2
Clonal antibody.
4. kit according to claim 3, the kit be colloidal gold immunoassay kit, chemical luminescence reagent kit,
Radioimmunoassay kitss, enzyme linked immunological kit or fluorescence immunoassay kit or the kit are micro-fluid chips.
5. kit according to claim 4, wherein the kit is enzyme linked immunological kit.
6. hybridoma cell strain described in claim 1 or monoclonal antibody as claimed in claim 2 are in reagent preparation box
Purposes.
7. purposes according to claim 6, the kit is the kit based on immune detection.
8. purposes according to claim 7, wherein the kit is colloidal gold immunoassay kit, chemical illuminating reagent
Box, radioimmunoassay kitss, enzyme linked immunological kit or fluorescence immunoassay kit or the kit are micro-fluid chips.
9. purposes according to claim 8, wherein the kit is enzyme linked immunological kit.
10. purposes according to claim 6, the kit is for detecting people IgM.
11. purposes according to claim 10, the people IgM is the people IgM generated at pathogen infection initial stage.
12. purposes according to claim 11, the cause of disease is rubella virus.
13. hybridoma cell strain described in claim 1 or monoclonal antibody as claimed in claim 2 are used for rubella in preparation
Purposes in the kit of poison diagnosis.
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| CN112094352B (en) * | 2020-09-27 | 2021-03-16 | 南京妙迪生物科技有限公司 | anti-IgM monoclonal antibody |
| WO2023088443A1 (en) * | 2021-11-20 | 2023-05-25 | 东莞市朋志生物科技有限公司 | Anti-human igm antibody and preparation method therefor and use thereof |
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