CN105132377A - Anti-CA724 (carbohydrate antigen 724) monoclonal antibody generation hybridoma - Google Patents

Abstract

The invention belongs to the medical field of immunoassay and relates to preparation of an anti-CA724 monoclonal antibody generation hybridoma. The invention further provides a monoclonal antibody for specific recognition of CA724, the hybridoma for generation of the monoclonal antibody and a method of using the monoclonal antibody for detection of high sensitivity of CA724.

Description

The hybridoma that anti-CA724 monoclonal antibody produces

[technical field]

The present invention relates to the hybridoma that carbohydrate antigen CA724 monoclonal antibody produces.

[background knowledge]

CA72-4 is the glycoprotein that a kind of molecular weight is about the mucoprotein sample high molecular of 220-400kDa, it is the mark of a kind of relatively special gi tract and ovarian tumor, can detect in the patients with gastric cancer of 85% ~ 95% that it raises, therefore can be used as the reliability index of diagnosis of gastric cancer.Clinical great majority find according to the study, the height of CA72-4 value and the tumorous size of cancer of the stomach, infiltration degree, histological type, and nodus lymphoideus transferring rate and distant metastasis present positive correlation, therefore this index can be used as a reliable Testing index of stomach cancer development degree.CA72-4, as tumor markers, has certain diagnosis and differential diagnosis to be worth to colorectal carcinoma.CA72-4 can differentiate optimum colorectal disease and malignant tumour preferably.In addition, the clinical stages of CA72-4 and colorectal cancer, tumor cell differentiation are relevant; Clinical stages, is higher, and differentiation degree is lower, and this positive markers rate is higher, so CA72-4 has certain meaning for monitoring post operative colo-rectal cancer curative effect and whether recurring.CA72-4 or epithelial ovarian cancer be the good blood serum tumor markers of mucus ovarian cancer especially, even if also higher at its positive rate of borderline mucinous neoplasms.It is significant to ovarian cancer observation of curative effect, can be used as the detection of dynamic index after ovarian cancer post operation and chemotherapy.CA72-4 all has expression in Several Kinds of Malignancy, and it is a kind of tumor markers of wide spectrum, and measuring change of serum C A72-4 has certain clinical value to ovarian cancer, cancer of the stomach, the rectum cancer, liver cancer and lung cancer.

[summary of the invention]

The object of the present invention is to provide a kind of anti-CA724 monoclonal antibody and set up a kind of simple and method of tool highly sensitive detection CA724 content, the method can be applicable to the detection of cancer.To solve the insufficient sensitivity or expensive that existing monoclonal antibody detects CA724, be not suitable for the shortcoming of clinical large-scale application.

Present invention obtains hybridoma cell strain 1-5-2 and the hybridoma cell strain 1-19-1 of the monoclonal antibody (mAb) that can produce specific recognition CA724, this 2 strain cell strain is preserved in China typical culture collection center (CCTCC) on July 17th, 2015 respectively, preservation address is, China. Wuhan. Wuhan University, deposit number is CCTCCNO:C201588 and CCTCCNO:C201589.In addition, through qualification, two strain monoclonal antibodies identify two different epi-positions of CA724 respectively, establish DASELISA immune response method by the combination of 1-5-2 and 1-19-1, and it is a kind of highly sensitive and high-throughout detection system.

Therefore, the invention provides the content of described below 1 to 3:

1. the hybridoma cell strain of anti-CA724, its preserving number is respectively CCTCCNO:C201588 and CCTCCNO:C201589.

2. the monoclonal antibody of anti-CA724, respectively by preserving number secreted by the hybridoma cell line of CCTCCNO:C201588 and CCTCCNO:C201589, described monoclonal antibody called after 1-5-2 and 1-19-1.

3. the application of anti-CA724 monoclonal antibody as claimed in claim 2, namely the DAS-ELISA of described monoclonal antibody is utilized to detect CA724, the antibody sources that sandwich assay matches between two in preserving number be CCTCCNO:C201588 hybridoma secretion antibody 1-5-2 and preserving number be CCTCCNO:C201589 hybridoma secretion antibody 1-19-1, its step comprises:

(1) with the described monoclonal antibody 1-5-2 bag quilt matched between two;

(2) add testing sample to hatch;

(3) another strain monoclonal antibody 1-19-1 marked using the described HRP matched between two resists as two, adds reaction system;

(4) add enzyme reaction substrate after washing, read OD value with 450nm;

(5) result shows that the sensitivity detected is very high.

[accompanying drawing explanation]

What accompanying drawing 1 showed is anti-CA724 monoclonal antibody titre measuring result in ELISA method that the hybridoma in the present invention produces.

What accompanying drawing 2 showed is the anti-CA724 monoclonal antibody titre measuring result marking HRP.

What accompanying drawing 3 showed is the detection sensitivity of sandwich method ELISA (S-ELISA) system.

[embodiment]

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition, should be understood that, after having set forth content of the present invention, those skilled in the art can do various change and amendment to the present invention, these equivalent form of values are the scope in the application defined in claims equally.

Embodiment 1: animal immune

Male Balb/C healthy mice about 8 week age of selection and myeloma cell's homology used, the carbohydrate antigen CA724 of antigen to be protein content be 1KU mixes with Freund's complete adjuvant, PBS, and after complete emulsification, 1KU is every only each, take back multiple spot, and oxter, inguinal region immunity.Immune programme for children: carried out second time same dose and Freund's incomplete adjuvant mixed immunity after 15 days; Third time same dose and Freund's complete adjuvant mixed immunity is carried out again after 15 days; After 10 days, afterbody is got blood indirect ELISA method and is surveyed serum titer, does not add adjuvant booster immunization with the pure antigen of same dose; After 3 days, extracting spleen cell merges.

Embodiment 2: the structure of hybridoma

1, the cultivation of myeloma cell strain and preparation

(1) what the present invention adopted is SP2/0 myeloma cell strain, and this cell strain growth and fusion efficiencies are all good, and the doubling time is 10-12 hour.Select during fusion to be in the good cell of logarithmic phase, cellular form and activity.Myeloma cell should first do to adapt to cultivate before fusion on substratum, makes Growth of Cells arrive best state (i.e. logarithmic phase);

(2) SP2/0 of cultivation is drawn in the pipe of 50mL, centrifugal, abandon supernatant, hang, add the substratum of 10mL, draw a small amount of 10 times of dilution countings.

2, the preparation of splenocyte

(1) mouse is placed in sealing bag, fills CO ₂treat its death by suffocation;

(2) mouse sterilization is fixed on dissection plate, in Bechtop, gets spleen, be placed in the culture dish of 12mL substratum, peel adhesion organization off, grinding spleen, till surplus white tissues, suction pipe all picks up, more slowly get make tissue block adhesion tube wall on, centrifugal, abandon supernatant, add the erythrocyte cracked liquid cracking 10min of 10mL, then the substratum adding 20-25mL stops its reaction, after centrifugal, abandon supernatant, add the substratum of 10mL, draw a small amount of 10 times of dilution countings.

3, cytogamy

Within after booster immunization 3 days, do cytogamy.

Cytogamy is the key link of hybridoma technology, and basic step gets the Sp2/0 cell that is in logarithmic phase and splenocyte 1: 10 mixes, by polyoxyethylene glycol (PEG) method to obtain hybridoma, and called after 1-5-2 and 1-19-1.The hybridoma obtained is suspended in the HAT substratum containing feeder cell, then joins in 96 orifice plates, at 37 DEG C, and 5%CO ₂incubator in close cultivation 12 days.

Embodiment 3: the preparation of monoclonal antibody and screening

1, the preparation of monoclonal antibody

The Kong Gezhong of the hybridoma obtained from embodiment 2 reclaims the supernatant liquor of substratum, to be chosen in ELISA method the antigen reactive monoclonal antibody with CA724.

2, the screening of monoclonal antibody

(1) by 100uL concentration be each Kong Gezhong of CA724 antigen to 96 orifice plates of 100IU/mL, after spending the night in 4 DEG C, make it be fixed on solid phase;

(2) closed 2 hours are carried out with the bovine serum albumin that 150uL concentration is 1%;

(3) medium supernatant of 100uL hybridoma is joined each Kong Gezhong, in 37 DEG C of reactions 2 hours, then add the sheep anti-mouse antibody of the horseradish peroxidase of dilution 10000 times in 37 DEG C of reactions 1 hour;

(4) tetramethyl benzidine microwell peroxidase substrate (TMB) is used to carry out colour developing 20min as substrate;

(5) adding 50uL concentration is after the sulfuric acid termination reaction of 0.1mol/L, measures the absorbancy of 450nm;

(6) select 1-5-2 and 1-19-1 that absorbancy is approximately 3, and carry out subclone by limiting dilution assay.

3, a large amount of preparation of monoclonal antibody and and purifying

Cell cell-culturing rotating bottle after subclone is carried out enlarged culturing, after about 20 days, collects supernatant, carry out affinitive layer purification with staphylococcal protein A,SPA (ProteinA).The monoclonal antibody obtained is called after 1-5-2 and 1-19-1 respectively.

4, the mensuration of antibody titer

Tiring of the 2 kinds of mAb filtered out is measured by ELISA method.Add 1-5-2,1-19-1 (10ug/mL) respectively, after the reaction, use the anti-mouse antibody of horseradish peroxidase and TMB to develop the color, two kinds of mAb tire and reach 10 ^-9above (shown in accompanying drawing 1).

Embodiment 4: the mark of monoclonal antibody and the mensuration of titre

To be purified into each antibody carries out HRP mark according to a conventional method, the titre of the monoclonal antibody marked is measured by method below, is that the CA724 antigen of 100IU/mL is fixed on (100uL/ hole) on 96 hole microplates by concentration.Use the bovine serum albumin of 1% to carry out closed 2 hours, tagged monoclonal antibody (the first hole dilutes 100 times), from the second hole, do 4 times of dilutions, react 2 hours under room temperature.After adding TMB, reaction at room temperature carries out 20 minutes, by the sulfuric acid stopped reaction of 0.1mol/L.Measure the absorbancy at 450nm, obtain the titre for the antigen be fixed in solid phase by the mode in embodiment 3.Result shows to have effective titre (shown in accompanying drawing 2).

Embodiment 5: double-antibody sandwich elisa detects the foundation of CA724 method

(1) with one of the described monoclonal antibody of matching between two bag quilt, the monoclonal antibody of 0.5ug/mL is added to microwell plate soil with the amount in 100uL/ hole, hatches be fixed on solid phase in 24 hours in 4 DEG C;

(2) pH value of lattice use in hole containing 0.1%Tween20 is the 20mMPBS (PBST) of 7.4, washs 2 times with the amount in 200uL/ hole.Add 1% bovine serum albumin with the amount in 150uL/ hole and carry out closed 2 hours;

(3) Kong Geyong PBST washs 4 times with the amount in 200uL/ hole, adds by the continuous 4 times of dilution CA724 antigens of initial concentration 10IU/mL, in incubation at room temperature 2 hours, then adds another strain antibody (1: 5000 of HRP mark; 100uL/ hole) and in incubation at room temperature 2 hours;

(4), after adding TMB, reaction at room temperature carries out 20 minutes, and the sulfuric acid adding 0.1mol/L carrys out termination reaction and measures the absorbancy of 450nm;

(5) result shows the sensitivity of detection very high (shown in accompanying drawing 3).

Claims (3)

1. the hybridoma cell strain of anti-CA724, its preserving number is respectively CCTCCNO:C201588 and CCTCCNO:C201589.

2. the monoclonal antibody of anti-CA724, respectively by preserving number secreted by the hybridoma cell line of CCTCCNO:C201588 and CCTCCNO:C201589, described monoclonal antibody called after 1-5-2 and 1-19-1.

3. the application of anti-CA724 monoclonal antibody as claimed in claim 2, namely utilize described monoclonal antibody DAS-ELISA detect CA724, the antibody sources that sandwich assay matches between two in preserving number be CCTCCNO:C201588 hybridoma secretion antibody 1-5-2 and preserving number be CCTCCNO:C201589 hybridoma secretion its step of antibody 1-19-1 comprise:

(1) with the described monoclonal antibody 1-5-2 bag quilt matched between two;

(2) add testing sample to hatch;

(3) another strain monoclonal antibody 1-19-1 marked using the described HRP matched between two resists as two, adds reaction system;

(4) add enzyme reaction substrate after washing, read OD value with 450nm;

(5) result shows that the sensitivity detected is very high.