CN108949698B - Hybridoma cell strain C11-6F7 and its HCMV monoclonal antibody and application of generation - Google Patents
Hybridoma cell strain C11-6F7 and its HCMV monoclonal antibody and application of generation Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/088—Varicella-zoster virus
-
- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/04—Varicella-zoster virus
- G01N2333/045—Cytomegalovirus
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Abstract
The present invention provides hybridoma cell strain C11-6F7, and the HCMV monoclonal antibody and application that are generated by the cell strain.Hybridoma cell strain C11-6F7 is preserved in China typical culture collection center, and deposit number is CCTCC NO:C2017196.HCMV monoclonal antibody can be applicable in method of immunity, and the present invention also provides a kind of kit, the kit includes above-mentioned HCMV monoclonal antibody.The present invention utilizes 642 gene expression PP65-C214 albumen of truncated PP65 gene/C terminal, reduces cost while also reducing the expression difficulty of recombinant antigen, and the recombinant antigen purity obtained is higher；The monoclonal antibody sensitivity is higher, high specificity, applies in terms of immunofluorescence and Flow Cytometry, can detect to infection cell, and can reflect the active state of HCMV infection.
Description
Technical field
The present invention relates to a kind of monoclonal antibody art more particularly to hybridoma cell strain C11-6F7 and its generations
HCMV monoclonal antibody and application.
Background technique
Human cytomegalovirus (human cytomegalovirus, HCMV) is that β belongs to herpesviral subfamilies, double-stranded linear DNA
Virus.Route of transmission multiplicity can be infected through placenta or genital tract uplink, can also be propagated through milk, blood, saliva etc., after infection
Lifelong to carry, virus has extensive target cell, each organ of whole body can be invaded, so clinical symptoms are complicated and changeable.Giant cell disease
Poison is 90% or more in the infection rate of developing country, fairly common, although most of adult the infecteds have no apparent clinic
Symptom (latent infection), but to fetus, newborn or there is a cognition of immune deficiency to bring serious disease.HCMV is congenital
One of most common pathogen is infected, invading fetus by placenta can cause stillborn foetus, miscarriage, fetal anomaly etc. to be damaged, without obvious
There are about 10%-15% can generate apparent sequelae such as dysnoesia etc. in the congenital infection newborn of clinical symptoms；Simultaneously
HCMV is also that the crowd such as HIV for causing body's immunity low or Organ Transplantation Patients are complicated by infection dead encountered pathogenic
Body.Therefore, the early stage of HCMV is quickly detected to give patient's antiviral treatment in time and to seem most important.
Fluorescein activated cell sorter has become a hot spot of clinical laboratory medicine development in recent years, its working principle is that
Multi-parameter, high-throughput quantitative point are carried out to individual cells or other biological particle by monoclonal antibody on cellular and molecular level
Analysis from a cell can measure multiple parameters simultaneously, can be quickly obtained accurate experimental result, continuous with detection technique
Perfect, flow cytometry object develops from original cell membrane component to cellular content.The diagnosis master of viral disease
Will dependent on laboratory check, but cell be separately cultured virus it is very time-consuming, while virus specific antibody recall rate often
It is lower.It not only can detecte the viral antigen of cell surface absorption using Flow Cytometry, but also can detecte infection cell
Interior viral antigen can go out feel by the monoclonal antibody of the specific recognition viral antigen of fluorescent marker with rapid quantitative detection
The cell of dye.
The method of domestic detection HCMV mainly has the methods of quantitative fluorescent PCR or ELISA, kit for detecting nucleic acid at present
Although having the characteristics that high sensitivity high specificity, its signal detected is nucleic acid (including latent infection), cannot be distinguished and lives
Dynamic sexy dye and latent infection, can not solve the problems, such as Active infection.Antibody used in the immunizations such as ELISA is Anti-TNF-α
Body has the problems such as purity and stability can not ensure, so being applied in Active infection detection for it, there are sensitivity
The disadvantages of low, specific not high.Therefore, it is necessary to obtain high quality HCMV monoclonal antibody, to establish immunological method HCMV detection reagent
Box.
Summary of the invention
In order to solve the above technical problems, the present invention provides hybridoma cell strain C11-6F7, and generated by the cell strain
HCMV monoclonal antibody and application.The human cytomegalovirus monoclonal antibody sensitivity is higher, can specifically bind PP65-
C214 albumen；Application of the kit prepared with the monoclonal antibody on fluidic cell platform is able to satisfy clinical detection requirement, and detection is quasi-
Exactness is consistent with presently commercially available kit for detecting nucleic acid, and performance is better than import reagent box.
Hybridoma cell strain C11-6F7 provided by the invention, is preserved in China typical culture collection center, deposit number
For CCTCC NO:C2017196, the deposit date is on September 21st, 2017, preservation place was Luojiashan, Wuchang, Wuhan City, Hubei Province
Wuhan University.
The present invention also provides a kind of anti-HCMV monoclonal antibody of mouse, the antibody is CCTCC by deposit number
The hybridoma cell strain C11-6F7 of NO:C2017196, which secretes, to be generated, and identification antigen is the pp65 PROTEIN C end 214 of UL83 coding
A amino acid.
The present invention also provides application of the HCMV monoclonal antibody in method of immunity, are included in fluidic cell skill
Art, ELISA immunoassay, immunochormatography, immunocytochemical stain measuring method and immunohistochemical staining measuring method
Etc. application.
The present invention also provides a kind of kit, the kit includes above-mentioned HCMV monoclonal antibody.
The present invention provides high specifics to identify HCMV monoclonal antibody, applies in immunofluorescence and Flow Cytometry side
Face can detect infection cell, and can reflect the active state of HCMV infection.
The present invention utilizes 642 gene expression PP65-C214 albumen of truncated PP65 gene/C terminal, reduces cost simultaneously
The expression difficulty of recombinant antigen is also reduced, and the recombinant antigen purity obtained is higher；It is being flowed with kit prepared by the monoclonal antibody
Application on formula cell platform is able to satisfy clinical detection requirement, accuracy in detection and presently commercially available kit for detecting nucleic acid one
It causes, performance is better than import reagent box.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis result figure of recombinant antigen PP65-C214 protein purification；
Fig. 2 is Westernblot detection PP65-C214 monoclonal antibody specific outcome figure；
Fig. 3 is the mean fluorecence measured after antibody 6F7 and import reagent box antibody detect sample with indirect streaming method respectively
Intensity contrast figure；Wherein figure A is the average fluorescent strength figure that measures after antibody 6F7 detects sample with indirect streaming method, figure B be into
The average fluorescent strength figure that opening reagent box antibody measures after detecting sample with indirect streaming method.
Fig. 4 is that monoclonal antibody of the present invention and import reagent box are compared inspection to clinical sample respectively with indirect immunofluorescence
The result figure of survey；Wherein, the result figure that figure A detects clinical sample for the kit of monoclonal antibody of the present invention preparation；Scheme B be into
The result figure that opening reagent box detects clinical sample.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that the described embodiment is only a part of the embodiment of the present invention, rather than whole embodiments.Based on this hair
Embodiment in bright, all other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.The terms such as involved virus, cell strain and reagent illustrate such as in this specification embodiment
Under:
HCMV: human cytomegalovirus；
The antigen protein of PP65:UL83 coding；
6F7: anti-HCMV-pp65 monoclonal antibody；
VH-F/R mixture: single-chain antibody heavy chain amplimer；
VL-F/R mixture: single-chain antibody light chain amplimer；
Trizon: it is bought from Tiangeng company；
M-MLV: reverse transcriptase；
Taq: it is bought from Promega company.
The acquisition of embodiment 1:HCMV-pp65-C214 monoclonal antibody
Anti- HCMV-pp65-C214 monoclonal antibody is prepared to carry out as follows:
1. the preparation of immunogene: finding out the protein sequence of UL83 coding from NCBI, 214 amino acid of C-terminal are by original
Synthetic gene sequence after nuclear expression codon optimization is connected in prokaryotic expression carrier pet30a, through IPTG inducing expression.Forgive
Body is precipitated with the dissolution of 8M urea after ultrasonic disruption after ni-sepharose purification, 20mM imidazole wash foreign protein, collect respectively 50mM,
The albumen that 100mM, 200mM imidazoles elute, eluted product run SDS-PAGE identification, and electrophoresis result is as shown in Figure 1, figure acceptance of the bid
Note PP65-C214 is the destination protein that 200mM imidazoles elutes, the SDS-PAGE result electrophoretogram of purifying.It can by Fig. 1
See, obtained immunogen protein size and expected size are coincide, and target protein is initially identified as.The recombination egg of identified mistake
White -80 DEG C save for use.
2. renaturation: new bag filter distillation boiling 5-10min, taking-up are dried, dress water leak detection, for use.It is taken out from -80 DEG C
Recombinant protein about 6ml, it is known that OD about 0.678 is added in bag filter, saturating in 4 DEG C to 1L 0.02M PBS (pH7.2)+6M urea
Analysis overnight, is during which stirred continuously with magnetic stirring apparatus；It takes out, is further continued for each to its 4 DEG C dialysis with 4/3/2/1/0M urea+PBS
1-2 hours, recombinant protein centrifugation is taken out, 12000RPM is centrifuged 1min, takes supernatant into another clean EP pipe, surveys OD about
0.530, -80 DEG C save for use, which is referred to as recombinant antigen PP65-C214.
3. mouse is immunized with the recombinant antigen PP65-C214 of above-mentioned renaturation, monoclonal antibody is prepared, the specific steps are as follows:
Initial immunity and twice booster immunization are carried out to mouse with the immunogene after renaturation, prepare spleen cell, and by its in life
The murine myeloma cell SP2/0 of long logarithmic phase is merged, and hybridoma is obtained.Above-mentioned gained hybridoma is by having
It limits dilution method and carries out monoclonal, then positive monoclonal cell strain is filtered out by indirect ELISA.Cell after monoclonal is through injecting
Enter the mouse peritoneal that incomplete Freund's adjuvant is exempted from advance, induction generates ascites.
Embodiment 2: indirect ELISA surveys gained titer of ascites and sensitivity
2.1 titer of ascites: antigen PP65-C214 obtained in embodiment 1 uses carbonate buffer solution as coating buffer, by it
96 orifice plates, the hole 100ng/ are coated with after dilution, 37 DEG C of heat incubate 2h, PBS-T board-washing 1 time after incubation.It is added in embodiment 1 and obtains
Different extension rates 37 DEG C of incubation 30min of PP65-C214 ascites, setting negative control hole, Positive control wells (3E4) and sky
White control wells (antibody diluent), PBS-T board-washing 5 times after incubation.Enzyme mark dilution is by HRP labelled antibody (sheep anti mouse
IgG-HRP 1000 times) are diluted, every 100 μ l of hole.37 DEG C of incubation 30min.PBS-T board-washing 5 times after incubation.Colour developing, A liquid: B
Liquid=1: 1, it is ready-to-use.Every hole 100 μ l, 37 DEG C of incubation 15min；Terminate liquid, 50 holes μ l/, color development stopping reaction, detection is added
Absorbance is shown in Table 1, and table 1 is titer of ascites measurement result table.Seen from table 1, the 12 plants of titer of ascites measurement results screened
It is as follows, gained titer of ascites about 46, ascites substantially eliminated nonspecific reaction after 4,000,000 times of dilutions, and as the result is shown 1
: the antibody 6F7 is reacted with antigen PP65-C214 after 4,000,000 times of dilutions is still presented higher absorbance value, shows the antibody
Sensitivity and specificity with higher.
Table 1
| Ascites extension rate | 1: 1 ten thousand | 1: 5 ten thousand | 1: 10 ten thousand | 1: 50 ten thousand | 1: 100 ten thousand | 1: 200 ten thousand | 1: 400 ten thousand | Blank |
| C11-2F8 | 0.905 | 0.389 | 0.306 | 0.242 | 0.22 | 0.209 | 0.232 | 0.047 |
| C11-3A9 | 2.162 | 1.793 | 1.351 | 0.641 | 0.466 | 0.345 | 0.272 | 0.046 |
| C11-14B7 | 0.602 | 0.285 | 0.323 | 0.217 | 0.21 | 0.206 | 0.377 | 0.057 |
| C11-3E4 | 2.326 | 1.923 | 1.731 | 1.012 | 0.673 | 0.485 | 0.322 | 0.032 |
| C11-6F7 | 2.68 | 2.114 | 1.801 | 1.105 | 0.723 | 0.549 | 0.422 | 0.058 |
| C11-2E2 | 1.306 | 0.636 | 0.401 | 0.21 | 0.119 | 0.093 | 0.079 | 0.063 |
| C11-6A7 | 1.369 | 1.256 | 1.188 | 1.034 | 0.867 | 0.678 | 0.448 | 0.039 |
| C11-2E3 | 1.205 | 1.039 | 0.836 | 0.539 | 0.354 | 0.254 | 0.181 | 0.009 |
| C11-7E3 | 1.009 | 1.009 | 0.724 | 0.541 | 0.335 | 0.187 | 0.101 | 0.073 |
| C11-7E5 | 1.109 | 0.847 | 0.568 | 0.438 | 0.317 | 0.177 | 0.146 | 0.006 |
2.2 sensitivity determinations: 12 plants of ascites concentration of screening are adjusted to 1mg/ml, (are 1mg/ with commercial antibody
Ml) A (luxuriant and rich with fragrance roc biology)/B (Hytest Ltd)/C (the raw work in Shanghai) does indirect ELISA detection to different diluted concentration antigens simultaneously
Sensitivity, antigen PCT are coated with 96 orifice plates after being diluted with carbonate coating buffer, and 5 gradient dilutions are done in the hole 100ng/, and 37 DEG C of heat are incubated
2h, PBS-T board-washing 1 time after incubation.Monoclonal antibody, 37 DEG C of incubation 30min, setting blank control (antibody dilution is added
Liquid).After incubation, PBS-T board-washing 5 times.HRP labelled antibody (sheep anti-mouse igg-HRP) is diluted 1000 times by enzyme mark dilution,
Every 100 μ l of hole.37 DEG C of incubation 30min.After incubation, PBS-T board-washing 5 times.Develop the color A liquid: colour developing liquid=1 B: 1, now with existing
With.Every hole 100 μ l, 37 DEG C of incubation 15min；Terminate liquid, 50 holes μ l/ is added, color development stopping reaction detects absorbance, as a result sees
The following table 2, table 2 are ascites sensitivity determination result table, and as can be seen from Table 2,4 plants of ascites sensitivity such as 6F7 are above purchase
Several plants of commercialization monoclonal antibodies bought.
Table 2
| Antigen doubling dilution | The hole 100ng/ | The hole 50ng/ | The hole 25ng/ | The hole 12.5ng/ | 6.25ng/ hole | 3.125ng/ hole | Blank |
| A(1mg/ml) | 1.342 | 1.155 | 1.92 | 0.671 | 0.465 | 0.29 | 0.004 |
| B(1mg/ml) | 1.434 | 1.311 | 1.198 | 1.131 | 0.869 | 0.69 | 0.012 |
| C(1mg/ml) | 1.298 | 1.205 | 0.82 | 0.618 | 0.43 | 0.257 | 0.006 |
| C11-2F8 | 1.055 | 0.879 | 0.118 | 0.109 | 0.102 | 0.1 | 0.005 |
| C11-3A9 | 2.259 | 2.041 | 2.033 | 1.85 | 1.699 | 1.48 | 0.04 |
| C11-14B7 | 0.878 | 0.494 | 0.248 | 0.129 | 0.063 | 0.046 | 0.001 |
| C11-3E4 | 2.309 | 2.234 | 2.17 | 1.986 | 1.238 | 1.07 | 0.026 |
| C11-6F7 | 2.361 | 2.122 | 2.086 | 1.922 | 1.735 | 1.605 | 0.006 |
| C11-2E2 | 1.344 | 1.193 | 0.606 | 0.311 | 0.165 | 0.081 | 0.014 |
| C11-6A7 | 1.549 | 1.396 | 1.297 | 1.099 | 0.972 | 0.752 | 0.011 |
| C11-2E3 | 1.205 | 1.039 | 0.836 | 0.539 | 0.354 | 0.254 | 0.009 |
| C11-7E3 | 1.009 | 1.009 | 0.724 | 0.541 | 0.335 | 0.187 | 0.073 |
| C11-7E5 | 1.358 | 1.209 | 1.094 | 0.833 | 0.706 | 0.515 | 0.027 |
3 ascites specific detection of embodiment and Western Blot are further verified
3.1 specific detections: it is coated with the PP65-N347 antigen and PP65- of pp65 gene N-terminal 1041bp coding respectively
C214 antigen, the cross reaction using Inhibition ELISA detection 6F7 antibody to PP65-N347, detects antibody specificity with this,
Antigen is coated with 96 orifice plates, the every hole 100/50ng after being diluted with carbonate coating buffer, and 37 DEG C of heat incubate 2h, and PBS-T is washed after incubation
Plate 1 time.Monoclonal antibody, 37 DEG C of incubation 30min, blank control (antibody diluent) is added.After incubation, PBS-T board-washing 5
It is secondary.HRP labelled antibody (sheep anti-mouse igg-HRP) is diluted 1000 times by enzyme mark dilution, every 100 μ l of hole.37 DEG C of incubation 30min.
After incubation, PBS-T board-washing 5 times.Develop the color A liquid: colour developing liquid=1 B: 1, it is ready-to-use.Every 100 μ l of hole, 37 DEG C of incubations
15min；Terminate liquid, 50 holes μ l/ is added, color development stopping reaction detects absorbance, the results are shown in Table 3, table 3 is the inspection of ascites specificity
Table is surveyed, the result shows that, with PP65-N347 cross reaction, molecule of the antigen binding do not occur for antibody obtained by 6F7 etc. by table 3
PP65-C214。
Table 3
3.2 Western Blot further verify anti-HCMV monoclonal antibody specificity:
Sample-loading buffer is added in antigen PP65-N347 and PP65-C214, boils each 4 hole of point sample after 5min, carries out 15%
SDS-PAGE electrophoresis.The antigen protein PP65-C214 on SDS-PAGE glue is transferred on NC film through half-dried robin, be put into containing
The TBS-T of 5% skimmed milk power closes 30min.It takes out NC film to cut off by 8 ducts respectively, by 4 kinds of monoclonal antibody 3A9/6F7/
6A7/3E4 is incubated for above-mentioned NC film item after distinguishing 1:1000 dilution, while being placed in 37 DEG C of incubations 2h, TBS-T washing 3 times, 10min/
Secondary, each to be added secondary antibody sheep anti-mouse igg-HRP, 37 DEG C of incubations 1h, TBS-T are washed 3 times, and addition developer solution is placed in gel imager
In take pictures, as a result as shown in Fig. 2, Fig. 2 be Western blot detection PP65-C214 monoclonal antibody specific outcome figure；There is band to say
It is bright to there is antigen (PP65-C214) and antibody (ascites) to specifically bind, it is not combined without band explanation, from Figure 2 it can be seen that described
Other four kinds of monoclonal antibodies such as 6F7 can be specifically bound with antigen PP65-C214, non-specific without occurring with PP65-N347 antigen
Association reaction.
Embodiment 4: the sensitivity of monoclonal antibody and import reagent box more of the present invention
After MRC-5 cell (ATCC) is trained single layer, cell is collected in digestion, and 0.1M phosphate is used after being fixed with 4% formaldehyde
Buffer (PBS) washing, then cell suspension is made with PBS resuspension；CMV is inoculated into the long MRC-5 cell to single layer, and lesion generates
After collect cell, washed after being fixed with 4% formaldehyde with PBS, then with PBS resuspension cell suspension is made.Above-mentioned MRC-5 cell and
Cmv infection cell suspension is separately added into 6F7, import reagent box primary antibody (CMV Brite Turbo, IQ Products), after mixing
37 DEG C incubate 20 minutes.After being washed with PBS, then it is separately added into sheep anti-mouse igg-FITC and (it is sharp limited up to biotechnology is purchased from Guangzhou
Company), it is incubated 20 minutes for 37 DEG C after mixing.After PBS washing, detected with flow cytometer (ACEA NoveCyte).Testing result
See that Fig. 3, Fig. 3 are antibody 6F7 and import reagent box antibody is strong with the mean fluorecence measured after indirect streaming method detection sample respectively
Spend comparison diagram；Wherein figure A is the average fluorescent strength figure measured after antibody 6F7 detects sample with indirect streaming method, and figure B is import
The average fluorescent strength figure that kit antibody measures after detecting sample with indirect streaming method；The testing result analyzed by Fig. 3,
4 are shown in Table, table 4 is antibody 6F7 and import reagent box antibody comparison result table, it can be seen that, after indirect immunofluorescence dyeing,
The P/N that 6F7 is generated is greater than the P/N that import reagent box generates, more suitable for being used in indirect flow cytometry and indirect immunofluorescence
In detection, that is to say, that the kit that the present invention obtains monoclonal antibody preparation has lower background signal to pattern detection, higher
Accuracy, namely there is higher sensitivity.
Table 4
Embodiment 5: monoclonal antibody 6F7 is compared with import reagent box, in detection CMV recurrence Organ Transplantation Patients blood sample
Pp65 antigen
Blood sample is cracked with the erythrocyte cracked liquid in import reagent box (CMV Brite Turbo, IQ Products)
After red blood cell, leucocyte is collected, is counted, taking 200,000 cells to be coated on glass slide, (every piece of glass slide applies 200,000 cells, applies 2 altogether
Block glass slide), consolidated after dry with import reagent box (CMV Brite Turbo, IQ Products) fixer and penetrating liquid
It is fixed and penetrating.6F7 antibody and import reagent box primary antibody is added dropwise respectively again, 37 DEG C incubate 20 minutes.After being washed with PBS, then distinguish
Sheep anti-mouse igg-FITC (being purchased from Guangzhou Ruida Bioscience Co., Ltd.) and import reagent box secondary antibody (CMV Brite is added
Turbo, IQ Products), it is incubated 20 minutes for 37 DEG C after mixing.After PBS washing, with fluorescence microscope and take pictures.As a result
See that Fig. 4, Fig. 4 are that monoclonal antibody of the present invention and import reagent box are compared detection to clinical sample respectively with indirect immunofluorescence
Result figure；Wherein, the result figure that figure A detects clinical sample for the kit of monoclonal antibody of the present invention preparation；Figure B is import
The result figure that kit detects clinical sample.
Embodiment 6: with fluidic cell by monoclonal antibody 6F7 and human cytomegalovirus kit for detecting nucleic acid (being purchased from Kai Jie company)
The comparative test that CMV in Organ Transplantation Patients blood sample is detected
81 blood samples of patients samples are acquired, detect blood plasma by human cytomegalovirus kit for detecting nucleic acid (Kai Jie) specification
In CMV DNA；Blood sample extracts mono-nuclear leukocytes using Ficoll separating liquid, then presses 1 the method for embodiment, uses
6F7 and sheep anti-mouse igg-FITC (being purchased from Guangzhou Ruida Bioscience Co., Ltd.) carry out flow cytometer detection.By two kinds of detection methods
Result statistics be shown in Table 5.Table 5 is (to be purchased monoclonal antibody of the present invention and human cytomegalovirus kit for detecting nucleic acid with flow cytometry
From Kai Jie company) to the result data table of the comparative test detected of the CMV in Organ Transplantation Patients blood sample.According to
SPSS13.0 statistical result can obtain, and Kappa>0.75 illustrates that two methods diagnostic result consistency is preferable in p<0.05 simultaneously, instead
Same index is reflected, and the difference of dissimilar parts is not significant, the monoclonal antibody can be used for fluidic cell platform to clinic as the result is shown
The detection of sample, the accuracy of testing result are consistent with presently commercially available molecule diagnosis kit.
Table 5
Embodiment 7: single-chain antibody gene extracts
1, hybridoma RNA extract and cDNA synthesis: collect fresh 15ml grow to logarithmic phase secretion 6F7 it is miscellaneous
Oncocyte is handed over, after serum free medium washs, extracts cell total rna with Trizol method；Again using total serum IgE as template, with oligo
DT and random primer are primer, form cDNA through M-MLV reverse transcriptase.
2, it the amplification of single-chain antibody heavy chain and light chain gene: using above-mentioned cDNA as template, is saved respectively with this laboratory
VH-F/R, VL-F/R are that primer amplification obtains the heavy chain and light chain gene PCR product of variable region of mab.Reaction condition is
94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s 35 are recycled, last 72 DEG C of 10min totally.It will
Above-mentioned PCR product recycling is connected in carrier T, and sequencing obtains heavy chain and light chain gene sequence, is about 300bp or so.CMV-
VH-P and this room other source of mouse monoclonal antibody heavy chain variable amino acid alignment and blastp result are as follows:
Be sequenced resulting gene order by EXPASY translation on line at amino acid sequence, Blastp comparison result and its with
Other source of mouse monoclonal antibody heavy chains of this room and light-chain amino acid sequence carry out sequence analysis, as the result is shown 6F7-VH and 6F7-VL difference
For the heavy chain and light-chain variable sequence of source of mouse monoclonal antibody, wherein heavy chain variable amino acid sequence and other monoclonal antibodies of this room
The homology of heavy chain variable amino acid sequence is below 68%, and centre may be antibody there are the extremely low segment of several homologys
Hypervariable region sequence, CMV-VL-P and this room other source of mouse monoclonal antibody chain variable region amino acid alignment and blastp result are as follows
It is shown:
The result shows that described two monoclonal antibody variable region gene sequences that amplification obtains are not contaminated, it is newfound source of mouse
Single-chain antibody variable region fragment.The cell of secrete monoclonal antibody only generates a type of antibody molecule in principle, strictly
It says and only generates containing a kind of heavy chain variable region and a kind of antibody molecule of light chain variable region, therefore obtained gene is described
The corresponding source of mouse antibody of HCMV antigen.
The present invention is immunized mouse using the antigen PP65 of UL83 coding expression and obtains the Dan Ke with specific bond HCMV
Grand antibody is conducive to carry out the detection of pp65 antigenemia, it is often more important that, it is demonstrated by immunofluorescence and Flow Cytometry
Monoclonal antibody of the invention detects the water for reaching HCMV PP65 import detection kit in terms of specificity and sensitivity to HCMV
It is flat, it can not only combine fluorescence quantitative PCR method and detect to obtain more objective and accurate as a result, being also beneficial to improve simultaneously existing
HCMV antigenemia detection technique.The acquisition of monoclonal antibody and its single-chain antibody gene has the researchs such as HCMV clinical diagnosis
Potential significance.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principle of the present invention, several improvement and deformations can also be made, these improvement and deformations are also considered as
Protection scope of the present invention.
Claims (3)
1. hybridoma cell strain C11-6F7, which is characterized in that the hybridoma cell strain is preserved in China typical culture collection
Center, deposit number are CCTCC NO:C2017196.
2. a kind of anti-HCMV monoclonal antibody of mouse, which is characterized in that the antibody is CCTCCNO by deposit number:
The hybridoma cell strain C11-6F7 of C2017196, which secretes, to be generated.
3. a kind of kit, which is characterized in that the kit includes HCMV monoclonal antibody as claimed in claim 2.
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