CN105132377B - The hybridoma that anti-CA724 monoclonal antibodies generate - Google Patents
The hybridoma that anti-CA724 monoclonal antibodies generate Download PDFInfo
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- CN105132377B CN105132377B CN201510454053.5A CN201510454053A CN105132377B CN 105132377 B CN105132377 B CN 105132377B CN 201510454053 A CN201510454053 A CN 201510454053A CN 105132377 B CN105132377 B CN 105132377B
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Abstract
The preparation for the hybridoma that anti-sugar antigens CA724 monoclonal antibodies generate, belongs to field of immunoassay medicine.The present invention provides a kind of monoclonal antibody of specific recognition sugar antigens CA724, the hybridoma for generating the monoclonal antibody and the highly sensitive method using monoclonal antibody detection sugar antigens CA724.
Description
【Technical field】
The present invention relates to the hybridomas that sugar antigens CA724 monoclonal antibodies generate.
【Background knowledge】
Sugar antigen CA72-4 is the glycoprotein of mucin sample high molecular weight that a kind of molecular weight is about 220-400kDa, it is
The marker of a kind of relatively special gastrointestinal tract and ovarian neoplasm can detect that its raising in 85%~95% patients with gastric cancer,
It therefore can be as the reliability index of diagnosis of gastric cancer.It is clinical most of it is found by researches that the height of CA72-4 values and the knurl of gastric cancer are big
Small, infiltration degree, histological type, positive correlation is presented in lymphatic metastasis and DISTANT METASTASES IN, therefore the index can be used as stomach cancer development
One reliable Testing index of degree.CA72-4 has colorectal carcinoma certain diagnosis and discriminating to examine as tumor markers
Disconnected value.CA72-4 can preferably differentiate benign colorectal disease and malignant tumour.In addition, the clinic of CA72-4 and colorectal cancer
By stages, tumor cell differentiation is related；Clinical stages, is higher, and differentiation degree is lower, and the positive markers rate is higher, so CA72-4
There is certain meaning for monitoring post operative colo-rectal cancer curative effect and whether recurring.CA72-4 or epithelial ovarian cancer are especially glutinous
The preferable blood serum tumor markers of fluidity oophoroma are higher even if its positive rate of borderline mucinous neoplasms.It is to ovary
Cancer observation of curative effect is of great significance, can be as the dynamic detection index after ovarian cancer post operation and chemotherapy.CA72-4 is in a variety of evils
Property tumour in have expression, it is a kind of tumor markers of wide spectrum, measure change of serum C A72-4 to oophoroma, gastric cancer, the carcinoma of the rectum,
Liver cancer and lung cancer have certain clinical value.
【Invention content】
The purpose of the present invention is to provide a kind of anti-CA724 monoclonal antibodies and establish a kind of simple and tool high sensitivity
Detection CA724 contents method, this method can be applied to the detection of cancer.To solve existing monoclonal antibody detection
The sensitivity of CA724 is inadequate or expensive, is not suitable for clinical large-scale the shortcomings that applying.
Present invention obtains the hybridoma cell strain 1- for the monoclonal antibody (mAb) that can generate specific recognition CA724
5-2 and hybridoma cell strain 1-19-1, this 2 plants of cell strains are preserved in Chinese Typical Representative culture guarantor on July 17th, 2015 respectively
Tibetan center (CCTCC), preservation address are Chinese Wuhan Wuhan Universitys, and deposit number is CCTCC NO：C201588 and CCTCC
NO：C201589.In addition, by identification, two plants of monoclonal antibodies identify two different epitopes of CA724, pass through 1-5-2 respectively
And the combination of 1-19-1 establishes double antibody sandwich enzyme immune response method, is a kind of highly sensitive and high-throughput detection
System.
Therefore, the present invention provides described below 1 to 3 content：
1. the hybridoma cell strain of anti-CA724, preserving number is respectively CCTCC NO：C201588 and CCTCC NO：
C201589。
2. the monoclonal antibody of anti-CA724 is respectively CCTCC NO by preserving number：C201588 and CCTCC NO：
Secreted by the hybridoma cell line of C201589, the monoclonal antibody is named as 1-5-2 and 1-19-1.
3. the application of anti-CA724 monoclonal antibodies as claimed in claim 2 utilizes the double of the monoclonal antibody
Antibody sandwich ELISA detects CA724, and the antibody sources that sandwich method matches two-by-two are CCTCC NO in preserving number：C201588 is miscellaneous
It is CCTCC NO to hand over the antibody 1-5-2 of knurl secretion and preserving number：The antibody 1-19-1 of C201589 hybridomas secretion, step packet
It includes：
(1) it is coated with the monoclonal antibody 1-5-2 matched two-by-two；
(2) sample to be tested is added in be incubated；
(3) using another plant of monoclonal antibody 1-19-1 of the HRP matched two-by-two the labels as secondary antibody, reaction is added in
System；
(4) enzyme reaction substrate is added in after washing, OD values are read with 450nm；
(5) the result shows that the sensitivity of detection is very high.
【Description of the drawings】
Attached drawing 1 be shown the present invention in hybridoma caused by anti-CA724 monoclonal antibodies dripped in ELISA method
Spend measurement result.
The anti-CA724 monoclonal antibodies titre measurement result of label HRP is shown in attached drawing 2.
The detection sensitivity of sandwich method ELISA (S-ELISA) system is shown in attached drawing 3.
【Specific embodiment】
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Furthermore, it is to be understood that after present disclosure is elaborated, those skilled in the art
It can make various changes and modification to the present invention, such equivalent forms are equally models as defined in the claims in the application
It encloses.
Embodiment 1：Animal immune
The male Balb/C healthy mices of selection 8 week old homologous with myeloma cell used or so, antigen is albumen
It measures and is mixed for the sugar antigens CA724 of 1KU with Freund's complete adjuvant, PBS, completely after emulsification, 1KU every every time, takes back more
Point and oxter, groin are immunized.Immune programme：Second of same dose was carried out after 15 days to mix with incomplete Freund's adjuvant
It is immune；Third time same dose and Freund's complete adjuvant mixed immunity are carried out after 15 days again；Tail portion takes blood with indirectly after 10 days
ELISA method surveys serum titer, and adjuvant booster immunization is not added with the pure antigen of same dose；Extracting spleen cell is merged after 3 days.
Embodiment 2：The structure of hybridoma
1st, the culture and preparation of myeloma cell strain
(1) for the present invention using SP2/0 myeloma cell strains, the cell strain growth and fusion efficiencies are good, during multiplication
Between be 10-12 hours.Selection is in exponential phase, cellular morphology and active good cell during fusion.Myeloma cell is being melted
It should first make to adapt to culture on culture medium before conjunction, make cell growth to best state (i.e. exponential phase)；
(2) SP2/0 of culture is drawn in the pipe of 50mL, centrifuges, abandon supernatant, hang, add the culture medium of 10mL, drawn few
10 times of dilutions of amount count.
2nd, the preparation of splenocyte
(1) mouse is placed in hermetic bag, fills CO2Treat its death by suffocation；
(2) mouse disinfection is fixed on dissection plate, takes spleen in superclean bench, be placed on the culture dish of 12mL culture mediums
In, it peels adhesion organization off, grinds spleen, until surplus white tissues, suction pipe all picks up, then slowly getting glues tissue block
On tube wall even, supernatant is abandoned in centrifugation, the erythrocyte cracked liquid of 10mL is added to crack 10min, then the culture medium of 20-25mL is added to terminate
It is reacted, and after centrifugation, abandons supernatant, adds the culture medium of 10mL, is drawn a small amount of 10 times of dilutions and is counted.
3rd, cell fusion
Do cell fusion within 3 days after booster immunization.
Cell fusion is the key link of hybridoma technology, and basic step is to take the Sp2/0 cells in exponential phase
It is mixed with splenocyte 1: 10, by polyethylene glycol (PEG) method to obtain hybridoma, is named as 1-5-2 and 1-19-1.It is obtained
The hybridoma obtained is suspended in the HAT culture mediums containing feeder cells, is then added in 96 orifice plates, at 37 DEG C, 5%
CO2Incubator in closing culture 12 days.
Embodiment 3：The preparation and screening of monoclonal antibody
1st, the preparation of monoclonal antibody
The supernatant of culture medium is recycled in the cell of hybridoma obtained in from embodiment 2, is chosen at ELISA side
In method with the antigen reactive monoclonal antibodies of CA724.
2nd, the screening of monoclonal antibody
(1) by each cell of the CA724 antigens of a concentration of 100IU/mL of 100uL to 96 orifice plates, make after being stayed overnight in 4 DEG C
It is fixed on solid phase；
(2) closing 2 hours is carried out with the bovine serum albumin(BSA) of 150uL a concentration of 1%；
(3) medium supernatant of 100uL hybridomas is added in each cell, in 37 DEG C react 2 hours, so
The sheep anti-mouse antibody for adding in the horseradish peroxidase for diluting 10000 times afterwards reacts 1 hour in 37 DEG C；
(4) colour developing 20min is carried out using tetramethyl benzidine microwell peroxidase substrate (TMB) as substrate；
(5) after the sulfuric acid of a concentration of 0.1mol/L of addition 50uL terminates reaction, the absorbance of 450nm is measured；
(6) it selects absorbance and is about 3 1-5-2 and 1-19-1, and pass through limiting dilution assay and be subcloned.
3rd, a large amount of of monoclonal antibody prepare and and purify
Cell after subclone is enlarged culture with cell-culturing rotating bottle, after about 20 days, supernatant is collected, with grape ball
Bacterium A albumen (Protein A) carries out affinitive layer purification.Obtained monoclonal antibody is respectively designated as 1-5-2 and 1-19-1.
4th, the measure of antibody titer
The potency of 2 kinds of mAb filtered out is measured by ELISA method.It is separately added into 1-5-2,1-19-1 (10ug/
ML), after the reaction, developed the color using the anti-mouse antibody of horseradish peroxidase with TMB, two kinds of mAb potency reach
10-9Above (shown in attached drawing 1).
Embodiment 4：The label of monoclonal antibody and the measure of titre
To be purified into each antibody carries out HRP labels according to a conventional method, the titre of the monoclonal antibody marked is under
The method in face measures, and the CA724 antigens of a concentration of 100IU/mL are fixed on 96 hole microplates (100uL/ holes).Use 1%
Bovine serum albumin(BSA) carries out closing 2 hours, and the monoclonal antibody (the first hole dilutes 100 times) of marking does 4 since the second hole
It dilutes, reacts 2 hours at room temperature again.After adding TMB, reaction carries out 20 minutes at room temperature, in the sulfuric acid of 0.1mol/L
Only react.The absorbance in 450nm is measured, the titre for the antigen being fixed in solid phase is obtained in the way of in embodiment 3.
The result shows that with effective titre (shown in attached drawing 2).
Embodiment 5：Double-antibody sandwich elisa detects the foundation of CA724 methods
(1) with one of the monoclonal antibody matched two-by-two coating, the monoclonal antibody of 0.5ug/mL is with 100uL/
The amount in hole is added to microwell plate soil, is incubated 24 hours in 4 DEG C and is fixed on solid phase；
(2) cell is using the 20mM PBS (PBST) that the pH value containing 0.1%Tween 20 is 7.4, with 200uL/ holes
Amount washing 2 times.1% bovine serum albumin(BSA) is added in the amount in 150uL/ holes and carries out closing 2 hours；
(3) cell is washed 4 times with PBST with the amount in 200uL/ holes, is added in by the continuous 4 times of dilutions of initial concentration 10IU/mL
CA724 antigens in incubation at room temperature 2 hours, then add in another strain antibody (1: 5000 of HRP labels；100uL/ holes) and in room
Temperature incubates 2 hours；
(4) after adding TMB, reaction carries out 20 minutes at room temperature, adds in the sulfuric acid of 0.1mol/L to terminate reaction and survey
Measure the absorbance of 450nm；
(5) the result shows that the sensitivity of detection is very high (shown in attached drawing 3).
Claims (2)
1. the hybridoma cell strain of anti-CA724 matched, preserving number is respectively CCTCC NO：C201588 and CCTCC NO：
C201589。
2. the monoclonal antibody of anti-CA724 matched, one plant of preserving number is CCTCC NO：The hybridoma cell line of C201588
Secreted monoclonal antibody is named as 1-5-2；Another plant of preserving number is CCTCC NO：The hybridoma cell line institute of C201589
The monoclonal antibody of secretion is named as 1-19-1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104931703A (en) * | 2015-06-29 | 2015-09-23 | 上海交通大学 | Immunity magnetic bead test strip for testing tumor marker CA72-4 and preparing method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104931703A (en) * | 2015-06-29 | 2015-09-23 | 上海交通大学 | Immunity magnetic bead test strip for testing tumor marker CA72-4 and preparing method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Is CA72-4 a Useful Biomarker in Differential Diagnosis between Ovarian Endometrioma and Epithelial Ovarian Cancer?";Anastasi E等;《Disease Markers》;20131231;第35卷(第5期);第331-335页，参见第332页右栏末段 * |
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