CN108588030A - 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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- CN108588030A CN108588030A CN201810294254.7A CN201810294254A CN108588030A CN 108588030 A CN108588030 A CN 108588030A CN 201810294254 A CN201810294254 A CN 201810294254A CN 108588030 A CN108588030 A CN 108588030A
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- 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
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- 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
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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- 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
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- 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
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 herpes simplex infections.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, more particularly to a kind of monoclonal antibody of anti-human IgM.
Background technology
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 types 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 diagnosis is particularly important, since body occurs at first after infection antibody is IgM type antibody, therefore detects morbidity just
Specific IgM antibodies have great importance in clinic early diagnoses in phase blood samples of patients.
In recent years, the research that there are some about anti-human IgM monoclonal antibody, such as 2010026758 A1 of patent document WO
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
Note in the anti-human IgM monoclonal antibody be used for external diagnosis reagent when 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 life biology is the anti-human IgM monoclonal antibody of common commercialization,
But its stability is not good enough, to Shortcomings when 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, is obtained with 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 the poor antibody of stability requires higher to preservation condition, operating condition, to reduce its practicality as diagnostic reagent
The reliability of property and result, and inevitably cause the rising of cost；Clinical test is then used for illustrating that antibody is diagnosing certain
Effect when specified disease.
Therefore, in order to diagnose the application of aspect in vitro, urgent need systemic evaluation result in this field is preferable, and is suitable for especially
The monoclonal antibody of the early diagnosis of pathogen infection.
Invention content
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 the kit and their purposes in detecting people IgM of monoclonal antibody or hybridoma cell strain.It is evaluated by systematicness,
The antibody has preferable performance in all respects, and external diagnosis reagent case is used to prepare to be suitable as immune diagnostic reagent,
It is especially suitable for preparing herpes simplex virus early diagnosis kit.
For this purpose, present inventor has performed numerous studies, mouse is immunized by people IgM, through limiting dilution after cell fusion
Method clone at least 4 times, until reaching monoclonal, screened from obtained numerous clones 1 plant can stably excreting antibody it is novel
Hybridoma cell strain (Hybridoma) IgM-6, and be preserved in China typical culture collection on March 8th, 2018
The heart, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University, deposit number are CCTCC NO:C201848, so as to complete this hair
It is bright.
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:C201848.
In the second aspect, the present invention provides a kind of monoclonal antibody, the monoclonal antibody can be special with people IgM
Property combine.
In one embodiment, the monoclonal antibody is not combined with human IgG, mouse IgG, rabbit igg or ox IgG.
In another embodiment, the middle titer of the monoclonal antibody is 194800 and in multigelation, long-term
It preserves, there is more preferably stability and reliability under thermal acceleration mal-condition.
In one preferred embodiment, the monoclonal antibody is by the anti-of the hybridoma cell strain secretion of the present invention
Body.
In terms of third, the present invention provides a kind of kit, the kit includes the hybridoma of the present 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.
At the 4th aspect, the use of the hybridoma cell strain or monoclonal antibody of the present 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 herpes simplex virus.
In addition, the monoclonal antibody for additionally providing hybridoma cell strain of the invention or the present invention is being prepared for simple blister
Purposes in the kit of exanthema virus diagnosis, is preferred for the kit of herpes simplex virus early diagnosis.
The advantageous effect of the monoclonal antibody of the present invention is that first, 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 reactions 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, to which cost be greatly saved；Finally, clinical trial shows that the monoclonal antibody of the present invention can be used for I type and II type
The enzyme linked immunological kit of herpes simplex virus I gM antibody tests, for detection sensitivity up to 100%, detection specificity is reachable
100%.
That is, the monoclonal antibody of the present invention is more in antibody titer, cross reactivity, stability and detection result 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.
Description of the drawings
Fig. 1 shows the SDS-PAGE electrophoresis of the antibody IgM-Ab6 of the present invention；
Fig. 2 Fig. 1 shows that the antibody IgM-Ab6 of the present invention and the antibody titer of two kinds of commercialization anti-human IgM antibodies are measured
As a result, wherein abscissa is Log (extension rate), ordinate OD450。
Specific implementation mode
Below in conjunction with specific implementation mode 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 implementation modes 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 and is led with belonging to the present invention
The identical meaning of general understanding of field technique personnel.If there are contradiction, this specification is preferential.
Antibody
As it is used herein, term " antibody " refers to immunoglobulin molecules, including but not limited to chimeric antibody, Ren Yuan
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 the synthetically antibody of generation/synthesis.
In a preferred embodiment, antibody refers to being produced from deposit number as CCTCC NO:The hybridoma of C201848 is thin
The antibody of born of the same parents' 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 being produced from deposit number as CCTCC NO:The antibody of the hybridoma cell strain of C201848.The reality of antibody fragment
Example includes but not limited to Fab, Fab', F (ab') 2 and Fv segments；Homodimer；Linear antibodies；Single-chain antibody molecules, for example,
sc-Fv；And the multi-specificity antibody formed by antibody fragment.In general, when based on mole come expression activity, segment retain 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 (be known 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 to constitute 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 is not polluted by other immunoglobulins substantially.
Kit
Diversified forms can be used in the detection kit of the present 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.
The present invention kit 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.
The kit of the present invention is illustrated for enzyme-linked immunosorbent assay in embodiment, but should not be construed as the present invention's
Kit is only limitted to enzyme-linked immunosorbent assay.
The kit of the present invention includes being produced from deposit number as CCTCC NO:The hybridoma cell strain of C201848 resists
Body can be existed in such a way that this field is conventional, for example, being present in container with dissolving 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 dissolving 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 are one of an important factor for restricting kit results.Shown in following article embodiment 9
, more conventional IgM antibody possesses more under extreme conditions as the raw material of enzyme linked immunological kit by antibody IgM-Ab6 of the invention
Good stability, to make the result reliability of kit enhance and in a disguised form reduce cost.
The antibody of the present invention can be used with the concentration of 0.1~10 μ g/ml, preferably 1~5 μ g/ml, more preferably 2.5 μ
g/ml。
It is used to carry out diagnosing/detecting required other materials in the kit of the present invention to include but not limited to remove the present invention
Antibody outside other anti-human IgM antibodies, the antigen that is combined 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 dissolving 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 dissolving or dried forms, but the present invention is not limited to
This.
For carrying out diagnosing/detecting required other structures including but not limited to for sampling knot in the kit of the present 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 the kit of the present invention to include but 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 in required other materials it is one or more can also
Detectably mark.
In one preferred embodiment, kit of the invention is the kit early diagnosed for cause of disease.
Purposes
The anti-human IgM antibodies or hybridoma cell strain of the present invention can be used for relevant any with the specific reaction of people IgM
Purpose.Preferably, antibody of the invention or hybridoma cell strain can be used for detecting people IgM.
The antibody or hybridoma cell strain of the present invention can be detected 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 for the early stage for coming from people.
The method of immunoassays can be used quantitative or the presence of qualitative detection people IgM, the immunoassays generally include
Biological sample is incubated together with the other materials needed for the antibody of the present invention and/or detection or is contacted successively, and by a variety of
The antibody that technology detection well known in the art combines.
Detection method includes but 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).
The people IgM that anti-human IgM monoclonal antibody can be generated with various pathogen infections is combined, but is preparing in-vitro diagnosis examination
When agent, since detection architecture is different or the nature difference of antibody itself, diagnosis effect inevitably can difference.It is real in following article
It applies shown in example 8, the antibody of the invention effect in IgM caused by detecting herpes simplex virus (HSV) is common better than at present
Commercially available detection kit.
Therefore, in one preferred embodiment, antibody of the invention or hybridoma are particularly suitable for detecting simple blister
The people IgM that exanthema virus initial infection generates, to diagnose whether individual infects herpes simplex virus.
The antibody or hybridoma of the present invention can be used for detecting whether subject infects herpes simplex virus, or for detecting
Whether subject is with herpe simplex caused by herpes simplex 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 companies, article No. SLBF-9338V), is injected with 1ml in equal volume
Device emulsification is oil emulsion, until the oil emulsion instilled in water does not disperse to stop emulsifying, only with 90 μ l/ by the lotion
It is immune for the first time that dosage four limbs armpit is subcutaneously applied to BALB/c mouse (Chengdu reaches large Experimental Animal Center, 6 week old female, 2)
Enhance immune, breast after taking people IgM to be mixed in equal volume with incomplete Freund's adjuvant (Sigma companies, article No. SLBM9367V) after 21 days
Change, immunizing dose is 45 μ l/, and enhancing is immune primary week about later, and tail blood is adopted before being immunized every time, detaches serum, uses
Indirect elisa method measures potency.After 5 times immune, 2 mice serum potency are more than 1:106, you can for merging.Fusion preceding 3
It, takes people IgM with normal saline dilution to 3.0mg/ml, then mix tail vein supplementary immunization, dosage in equal volume with physiological saline
Only for 45 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 macrophages.1 day before fusion, BALB/c takes blood
Neck is drawn to put to death, 0.1% bromogeramine impregnates 1 minute, is transferred to 75% alcohol and impregnates 1 minute, with cutting under sterile working in super-clean bench
Knife abdominal cut skin, exposure peritonaeum, with syringe Intraperitoneal injection RPMI1640 basic culture solution 7ml, after taken out with dropper, then
3ml RPMI1640 basic culture solutions are added to rinse repeatedly, recycle flushing liquor, 1000rpm, centrifugation stays precipitation in 5 minutes, with having added
The RPMI1640 culture solutions for entering 20% newborn bovine serum are resuspended, and adjustment cell concentration is 2.3 × 10596 orifice plates are added in a/ml,
100 holes μ l/, 37 DEG C, 5%CO2Culture.
The preparation of 2-2 immune spleen cells
Mouse supplementary immunization in embodiment 1 aseptically takes out spleen, is placed in plate respectively after three days,
RPMI1640 basic culture solutions rinse primary, shred, grind, filter after the splenocyte that is disperseed, 1000rpm, centrifugation 5 minutes
Precipitation is stayed to centrifuge, RPMI1640 basic culture solutions are 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 exponential phase, take 5 big bottle (75cm2) cell suspension is made, 1000rpm is centrifuged 5 minutes and is stayed precipitation, is used
RPMI1640 basic culture solutions are resuspended, count, by 0.6 × 105The cell concentration of a/ml carries out sub-bottle culture (ordinary circumstance every 1
Replace complete 1640 culture mediums of a 15~30ml within~2 days).
2-4 cell fusions and HAT selection culture hybridomas
Myeloma cell and immune spleen cell are pressed 1:1 ratio mixing, RPMI1640 is used in 50ml conical centrifuge tubes
Basic culture solution is washed 1 time, and 1000rpm is centrifuged 5 minutes and stayed precipitation.By cell mixing, it is slowly added to the PEG4000 of 1.5ml 50%
Fusion, 50ml is added in fusion RPMI1640 basic culture solutions after 1 minute terminate cell fusion.700rpm centrifugations weigh after five minutes
It is suspended from 1640 culture mediums containing 1%HAT and 20% newborn bovine serum, averagely instills 25 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 changes culture medium again after 7 days.
The screening of 2-5 positive cell strains
With 0.06M pH9.6 carbonate buffer solutions dilution people IgM, (Sichuan mikey biology new material technology Co., Ltd criticizes
Number 150127) to 5 μ g/ml, 100 μ l are coated with per hole in 96 hole elisa Plates, for detecting cells and supernatant.It is positioned in refrigerator
2~8 DEG C overnight, are abandoned liquid in hole for second day, ELISA washing lotions board-washing three times, pats dry, with the 0.01M 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 for use.The 9th day after splenocyte fusion, cell is taken
In the detection plate of above-mentioned 96 hole, 37 DEG C are incubated 40 minutes 100 μ l of supernatant, and 8000 times of dilutions are added in ELISA board-washings 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, 100 μ L is added per hole and contain 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 lemons
Acid phosphoric acid buffer solution, 37 DEG C are incubated 10 minutes, and 50 μ L 2M sulfuric acid solutions are added per hole and terminate reaction, survey 450nm absorption values.With
It is positive control that mice serum, which is diluted to 100 times, when fusion, and 1640 complete culture solutions of RPMI are as negative control, negative control
OD values ＜ 0.2, positive control OD values ＞ 1.8 are that detecting system is effective, when value >=2 sample OD × negative control OD value, for the positive,
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, screening
Method continuously clone four times, reach 100% monoclonal, are transferred to 24 holes and continue to cultivate, wait for that cell covers with 80% on positive Kong Yi
When be transferred to after cell bottle expands culture, sub-bottle passes on when cell covers with cell bottle 80%, the cell growth of passage to logarithmic phase
When, with appropriate serum-free RPMI-1640 culture mediums cell dispersion bottle inner cell, cell suspension is collected in conical centrifuge tube, note
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 centrifugations 5min, the cell number of precipitation 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, 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 25 plants of energy
Surely the hybridoma cell strain for secreting IgM monoclonal antibody antibody, see the table below 1.Wherein, hybridoma cell strain 9C11-C1-C9-A8-
Antibody secreted by the D8 effect in the core antibody IgM antibody for detecting hepatitis b virus infected early stage is optimal, this is miscellaneous
It hands over tumor cell strain to be denoted as IgM-6, is deposited in China typical culture collection center on March 8th, 2018, deposit number is
CCTCC NO:C201848。
Table 1 screens the hybridoma cell strain of the obtained anti-human IgM monoclonal antibody of stably excreting
| Hybridoma | Hybridoma | Hybridoma |
| 1G1-F2-C12-D1-G1 | 2G7-B8-C11-D2-F1 | 3C4-D6-F8-G9-H2 |
| 1G11-B12-C9-E2-G9 | 5B8-H8-H2-G7-G3 | 3E3-D2-F1-F4-G2 |
| 4B3-G1-D7-G1-D12 | 5E4-G2-B6-D6-E9 | 3A12-D2-G8-G4-C1 |
| 7C4-D8-H7-D2-C11-D8 | 5A10-A1-D4-A7-C12 | 6H7-G2-G6-A9-G10 |
| 7F5-E3-B4-D4-B11-B12 | 9C11-C1-C9-A8-D8 | 6E1-D5-A9-A10-H2-F3 |
| 18H7-F10-F9-G5-D2 | 19E6-H1-G1-C12-C12 | 18D3-C4-F6-G7-G4 |
| 18A1-F10-C12-G4-C11 | 19G3-B11-E12-F1-F11 | 19D1-H9-G5-G7-F5-F12 |
| 18A8-A5-A1-D2-E7 | 19E7-D2-H5-F2-G7 | 18E7-E9-C4-E8-D12 |
| 25G2-E2-D2-C2-B2 |
The preparation of 3 monoclonal antibody of embodiment
Select the BALB/c mouse of 6~8 weeks health, every mouse peritoneal injection 0.5ml atoleines (Tianjin Ke Miou), 7
Every mouse peritoneal injection 1.7 × 10 after it6A hybridoma.Inoculating cell can generate ascites, close observation after 7~10 days
The health status of animal and sign of ascites as, before waiting for that ascites is as more as possible, and mouse is dying, execution mouse, 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 precipitation, then is purified with Protein A affinity chromatography, and it is big that SDS-PAGE detects antibody purity
In 90% (being denoted as IgM-Ab6), electrophoresis result is as shown in Figure 1.
4 Hybridoma Cell Culture supernatant bioactivity of embodiment
With 0.06M pH9.6 carbonate buffer solutions dilution people IgM, (Sichuan mikey biology new material technology Co., Ltd criticizes
Number 150127) to 5 μ g/ml, 100 μ l are coated with per hole in 96 hole elisa Plates.It is positioned in refrigerator and stays overnight for 2~8 DEG C, abandon within second day
Liquid in hole, ELISA board-washings are machine-washed three times, are patted dry, with the PBS of the 0.01M pH7.2 containing 10% calf serum, 150 holes μ l/,
37 DEG C of closings abandon liquid in 2 hours, pat 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 the 5th hole to the tenth hole is diluted step by step with 2 times, and mice serum is diluted to 100 when 11-holes are to merge
Make positive control again, negative control is made in the 12nd hole with 1640 complete culture solutions of RPMI, is 100 μ l per hole sample volume.37℃
It is incubated 40 minutes, the sheep anti-mouse igg (four of 12000 times of diluted horseradish peroxidase labels is added in ELISA board-washings after machine-washing five times
River mikey biology new material technology Co., Ltd produces) 100 μ L, after 37 DEG C of incubations are ibid washed for 30 minutes, 100 μ L are added per hole and contain
0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C are incubated 10 minutes, per hole
50 μ L 2M sulfuric acid solutions are added and terminate reaction, survey 450nm absorption values.Negative control OD value ＜ 0.2, positive control OD values ＞ 1.8
It is effective for detecting system, it is on the contrary for feminine gender for the positive when value >=2 OD × negative control OD value.The minimum positive hole institute of detected value
Corresponding dilution ratio is Hybridoma Cell Culture supernatant potency, this Hybridoma Cell Culture supernatant antibody titer is more than 1:
1.6×103, it is shown in Table 2.
2 culture supernatant potency result of table
| It numbers in enzyme mark hole | 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-6 supernatants | 2.11 | 2.00 | 1.78 | 1.06 | 0.68 | 0.34 | 0.21 | 0.13 | 0.06 | 0.04 | 2.56 | 0.06 |
5 titer of ascites of embodiment detects
ELISA detection method is the same as embodiment 4.Dilution process is different, specially：First hole is former times ascites, with 0.01M
The PBS buffer solution of pH7.2 dilutes step by step again from the second hole to seven apertures in the human head 10, is diluted step by step with 2 times from octal to the tenth hole.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-6 institutes
The titer of ascites of preparation is more than 1:2×106。
3 titer of ascites of table
| It numbers in enzyme mark hole | 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-6 ascites | 2.35 | 2.10 | 2.08 | 2.06 | 1.68 | 0.77 | 0.27 | 0.13 | 0.06 | 0.04 | 2.64 | 0.05 |
6 antibody titer of embodiment detects
It is first that the antibody IgM-Ab6 after purification prepared in embodiment 3 is unified dilute with the PBS buffer solution of 0.01M pH7.2
Be interpreted as 1mg/ml, after dilute initial first hole of 100 times of conducts again, since the second hole to the tenth hole make 5 times dilute step by step, the tenth
Mice serum is diluted to 100 times and makees positive control when one hole is to merge, and negative control is made in the 12nd hole with PBS, per hole sample body
Product is 100 μ l.37 DEG C are incubated 50 minutes, and 8000 times of diluted horseradish peroxidase labels are added in ELISA board-washings after machine-washing five times
Sheep anti-mouse igg (production of Sichuan mikey biology new material technology Co., Ltd) 100 μ L, 37 DEG C are incubated after 1h ibid wash, per hole
100 μ L are added and contain 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C of incubations
15 minutes, 50 μ L 2M sulfuric acid solutions are added per hole and terminate reaction, detection 450nm absorption values (multi-functional readout instrument, manufacturer
Thermo, model Varioskan Flas), it is repeated 3 times and takes OD average values.Negative control OD value ＜ 0.2, positive control OD values ＞
1.8 is effective for detecting system.Antibody titer criterion：It is vertical sit with antibody mean OD value with LOG (dilution) for abscissa
It is denoted as curve, curvilinear equation is y=min+ (max-min)/(1+10^ ((logEC50-x) × Hillslope)), is passed through
Sigmaplot data processing software matched curves, take middle titer=10logEC50.As a result show that this antibody IgM-Ab6 intermediate values are imitated
Valence is 194800.Commercialized anti-human IgM antibodies B (being purchased from Xiamen Bo Sheng Bioisystech Co., Ltd) and C (safe sections of Luoyang City one hundred
Bioisystech Co., Ltd) purity is all higher than 90%, concentration is uniformly adjusted to 1mg/ml, in kind control test B, C
Potency is by being fitted, and titer is that titer is 8793 in 13600, C antibody in B antibody, and the hybridoma for being below the present invention is thin
The antibody of intracrine.Table 4 with Fig. 2 shows titration results.
4 antibody titer of table detects
7 cross reaction of embodiment measures
With 0.06M pH9.6 carbonate buffer solutions dilution human IgG, (Sichuan mikey biology new material technology Co., Ltd criticizes
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, 100 μ l are coated in 96 hole elisa Plates per hole.It is positioned in refrigerator and stays overnight for 2~8 DEG C, abandon within second day
Liquid in hole, ELISA board-washings 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 are abandoned liquid in 2 hours, are patted dry spare.By the anti-human IgM monoclonal antibody IgM-Ab6 HRP of the present invention
It is diluted to same concentrations (0.5mg/ml) after label, then dilutes 500 times, 1000 times, 2000 times, 4000 times, 8000 times, in addition
State in the ELISA Plate hole being coated with, reacted with four kinds of IgG respectively per 50 μ l of hole, 37 DEG C be incubated 30 minutes after ELISA board-washings
Machine washing five times, pats dry, and 100 μ L are added per hole and contain 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acids
Phosphate buffer, 37 DEG C are incubated 10 minutes, and 50 μ L 2M sulfuric acid solutions are added per hole and terminate reaction, survey 450nm absorption value (more work(
Energy readout instrument, manufacturer Thermo, model Varioskan Flas).Following table is the cross reaction measurement result of IgM-Ab6, with four
Kind IgG does not have cross reaction.
5 cross reaction of table measures
| The IgM-Ab6 dilutions of enzyme mark | 1:500 | 1：1000 | 1：2000 | 1：4000 | 1:8000 |
| People IgM | 1.709 | 1.321 | 0.911 | 0.8 | 0.542 |
| Human IgG | 0.064 | 0.053 | 0.095 | 0.054 | 0.08 |
| Mouse IgG | 0.099 | 0.085 | 0.067 | 0.264 | 0.163 |
| Ox IgG | 0.051 | 0.075 | 0.053 | 0.157 | 0.071 |
The clinical diagnosis of 8 cause of disease of embodiment
(1) prepared by kit
Kit uses enzyme linked immunosorbent assay, utilizes prize law principle.
With the μ of antibody IgM-Ab6 to 2.5 g/ml of the 0.06M pH9.6 carbonate buffer solutions dilution present invention, 96 hole elisa Plates
In per hole be coated with 100 μ l.It being positioned in refrigerator and stays overnight for 2~8 DEG C, abandon within second day liquid in hole, ELISA board-washings are machine-washed three times,
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 abandon liquid in 2 hours, pat dry standby
With.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 Healthy People
1000 times are diluted with the PBS buffer solution of the 0.01M pH7.2 containing 10% calf serum make negative control after serum filtration sterilization, with
Make positive control again with the PBS buffer solution dilution herpesviral IgM antibody 1000 of the 0.01M pH7.2 containing 10% calf serum,
Yin and yang attribute is set and compares each 2 hole and 1 hole of blank, injection volume is 100 μ l.37 DEG C are incubated 60 minutes, ELISA board-washings machine washing five
Secondary, the recombinant herpes simplex virus antigen that horseradish peroxidase (HRP) marks is added, and (Sichuan mikey biology new material technology has
Limit company produces) 100 μ L, after 37 DEG C of incubations are same as above board-washing in 30 minutes, 100 μ L are added per hole and contain 0.1% (M/V) o-phenylenediamine,
0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C are incubated 10 minutes, and 50 μ L 2M sulfuric acid solutions are added per hole
Reaction is terminated, 450nm absorption values (multi-functional readout instrument, manufacturer Thermo, model Varioskan Flas) are surveyed.Calculate cut
Off=1.1+ negative control average values are the positive when sample OD values are more than cut off, otherwise are feminine gender.
(2) sensitivity technique
The detection kit prepared in aforementioned manners examines 200 positives and 200 negative samples respectively.Table 6 is listed
Kit and commercial kit (beautiful limited public affairs of pearl reagent share in Zhuhai prepared by the monoclonal antibody IgM-Ab6 of the present invention
Department) comparative experiments, inspection result show its sensitivity of detection kit prepared by the monoclonal antibody IgM-Ab6 of the present invention and
Specificity is above commercial kit, and testing result is listed in table 6.
Table 6 is positive to be examined with negative sample
9 stability of embodiment is verified
The stability of monoclonal antibody of the present invention in the presence of a harsh environment is by the monoclonal antibody IgM-Ab6 of the present invention following
Under the conditions of handle：A-20 DEG C of multigelation 2 times；B-20 DEG C of multigelation 3 times；C-20 DEG C of multigelation 4 times；D-20 DEG C freezes repeatedly
Melt 5 times；E-20 DEG C preserves 7 months；37 DEG C of f thermal accelerations 7 days；37 DEG C of g thermal accelerations 14 days, other conditions are constant, by above-mentioned side
It is 100% that method, which is prepared into detection reagent and examines 15 parts of positive reference product and 15 parts of negative reference product, coincidence rate respectively,.Show this
Antibody stability is good, and good using the detection reagent accuracy of this Antibody preparation.It is anti-human to choose the higher commercialization of potency
IgM monoclonal antibody B (life of Xiamen wave) is prepared into detection reagent and detects 15 parts of positive ginsengs respectively after being handled with above-mentioned similarity condition
Examine product and 15 parts of negative reference product, as a result show its multigelation 5 times or -20 DEG C preserve 7 months 37 DEG C of thermal accelerations after 14 days
Beginning is degraded, 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 the monoclonal antibody stability of the present invention is high, I type and herpes simplex virus type 2 being made from it
IgM antibody detection reagent can also keep very high Stability and dependability, this point facing under the conditions of more rugged environment
It is very valuable progress in bed and laboratory applications.
Claims (10)
1. a kind of hybridoma cell strain IgM-6 is preserved in China typical culture collection center, deposit number CCTCC
NO:C201848。
2. a kind of monoclonal antibody, the monoclonal antibody is secreted by hybridoma cell strain described in claim 1.
3. a kind of kit, the kit includes the list described in hybridoma cell strain described in claim 1 or 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；It is preferred that
For enzyme linked immunological kit.
5. the monoclonal antibody described in hybridoma cell strain described in claim 1 or claim 2 is in reagent preparation box
Purposes.
6. purposes according to claim 5, the kit is the kit based on immune detection, it is preferred that the examination
Agent box is 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；More preferably enzyme linked immunological kit.
7. purposes according to claim 5, the kit is for detecting people IgM.
8. purposes according to claim 7, the people IgM is the people IgM generated at pathogen infection initial stage.
9. purposes according to claim 8, the cause of disease is herpes simplex virus.
10. the monoclonal antibody described in hybridoma cell strain described in claim 1 or claim 2 is being prepared for simple blister
Purposes in the kit of exanthema virus diagnosis.
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| 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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Address after: 611731 1st floor, block B, building 1, No.8, Anhe 2nd Road, high tech Zone, Chengdu, Sichuan Patentee after: Sichuan ankerei New Material Technology Co.,Ltd. Address before: 611730 floor 1, block B, building 1, No. 8, Anhe Second Road, high tech Zone, Chengdu, Sichuan Patentee before: SICHUAN MACCURA BIOLOGICAL NEW MATERIAL TECHNOLOGY Co.,Ltd. |