Disclosure of Invention
The invention aims to provide a CEA antibody which has high affinity and can be used for detecting carcinoembryonic antigen aiming at the defects in the prior art.
The invention provides the following technical scheme:
an anti-CEA monoclonal antibody, consisting of a heavy chain and a light chain, which comprise a variable region and a constant region, respectively, wherein the heavy chain variable region is selected from SEQ ID NO. 7 and the light chain variable region is selected from SEQ ID NO. 8.
The anti-CEA monoclonal antibody heavy and light chain variable region of the invention has 6 complementarity determining regions. The 3 CDR sequences of the heavy chain variable region of the anti-CEA monoclonal antibody are respectively SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; the variable region of light chain has 3 CDR sequences of SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6.
The heavy and light chains of the anti-CEA monoclonal antibody of the invention further comprise constant regions, and the antibody light chain constant region further comprises murine kappa and lambda chain sequences. The antibody heavy chain constant region further comprises a murine IgG1, IgG2a, IgG2b, or IgG3, or IgA or IgM sequence.
The invention also aims to provide a CEA quantitative detection kit, a preparation method and application thereof. The kit comprises the CEA-recognizing antibody, and is used for prognosis judgment, curative effect observation and diagnosis of monitoring metastasis or relapse of tumor lesions of colon cancer and rectal cancer patients.
The CEA quantitative detection kit comprises an enzyme label plate coated by an anti-CEA monoclonal antibody and an anti-CEA enzyme-labeled antibody.
The anti-CEA enzyme-labeled antibody is a CEA polyclonal antibody marked by horseradish peroxidase. The preparation method of the polyclonal antibody and the method of the enzyme-labeled antibody are all routine operations in the field.
Further, the quantitative detection kit also comprises a CEA standard substance, a concentrated washing solution, a sample diluent, a substrate solution and a stop solution.
The anti-CEA monoclonal antibody, a CEA standard substance, a horseradish peroxidase labeled anti-CEA polyclonal antibody, a concentrated washing solution, a sample diluent, a substrate solution and a stop solution are respectively put into each reagent bottle, and each reagent bottle is fixed by a sponge bracket and is arranged in a kit body together with an ELISA plate and a sealing plate membrane which are coated by the CEA antibody.
Advantageous effects
The invention has the following beneficial effects: provides a new CEA antibody with high affinity, which can be used for carcinoembryonic antigen detection; the CEA detection kit established by the antibody of the invention has the advantages of simple and easy operation, quick and sensitive detection, simple and popular enzyme labeling instrument and low price, and provides an effective tool for prognosis judgment, curative effect observation and diagnosis of monitoring metastasis or relapse of tumor lesions of patients with colon cancer and rectal cancer.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 anti-CEA antibody preparation and purification
Culturing CHO cells stably expressing CEA in DMEM medium, and culturing 107CHO-CEA+Cells were injected intraperitoneally with Freund's complete adjuvant in female Balb/c mice for primary immunization followed by 10 more times every 2 weeks7CHO-CEA+Cells were injected intraperitoneally into the female Balb/c mice and immunization was continued. Finally, the last immunization is carried out 3 days before the fusion with the myeloma cells, and the last immunization is 107CHO-CEA+Cells are immunized intravenously. Spleen cells of immunized Balb/c mice were fused with a myeloma Sp2/0 cell line, and the fused cells were diluted to an appropriate concentration in Iscove's medium (0.1mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.M thymidine) containing 10% serum, and then cultured in a 96-well plate. After 10 days, cell supernatants were taken and primary cultures showing positive reaction with recombinant CEA protein in the supernatants were examined by high throughput ELISA. Then hybridizing the wellsAnd (3) diluting the tumor cells for subcloning, and screening by an ELISA method to finally obtain a positive hybridoma cell strain. After expansion culture, the hybridoma cells were cryopreserved.
BALB/c mice were injected intraperitoneally with 0.5 ml/mouse, 1 week before hybridoma inoculation. After 1 week, each mouse was inoculated intraperitoneally at about 1X106(ii) individual hybridoma cells; and after 7-10 days, collecting ascites. Centrifuging ascites at 10000 Xg for 30min, removing precipitate, salting out with 50% ammonium sulfate, coarse extracting, dissolving with PBS, and dialyzing with flowing water for 5 hr; dialyzing and equilibrating with 0.1mol/L phosphate buffer (pH8.0) overnight; and (3) loading, eluting the hybrid protein by using 0.1mol/L phosphate buffer solution (pH8.0), eluting by using citrate eluents with different pH values, collecting elution peaks in sections, concentrating, analyzing and purifying the purity of the monoclonal antibody by 10 percent SDS-PAGE, and determining the titer by an indirect ELISA method. Through indirect ELISA detection, the titer of 8 mAbs can reach 1: 108。
Example 2 anti-CEA antibody subtype identification
The subtype of the anti-CEA monoclonal antibody Cab-207 obtained in example 1 was identified by ELISA (kit purchased from Proteintech). The microplate provided in the kit was already pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the anti-CEA antibody sample Cab-207 purified in example 1 was added to the sample well at 50. mu.l per well without incubation. Adding 1X goat anti-mouse IgA + IgM + IgG-HRP into sample wells, mixing the sample wells with 50 μ l each, and incubating for 1 h. And (4) deducting liquid in the holes, adding 1XPBST to wash the holes for 3 times, and absorbing the excessive moisture by absorbent paper. Adding color development solution, and developing 100 μ l per well in dark at room temperature for 15 min. The color reaction was stopped by adding 100. mu.l of stop solution. The OD value at 450nm was measured by a microplate reader, and the results are shown in FIG. 1, in which all the heavy chain subtypes and all the light chain subtypes of the obtained anti-CEA monoclonal antibody Cab-207 are IgG 1.
EXAMPLE 3 monoclonal antibody sequencing
After the hybridoma cell strain corresponding to Cab-207 is recovered, the hybridoma cell strain is cultured until the total number is 107The cells were centrifuged at 1000rpm for 5min to collect the cells, and RNA was extracted. TRNzol-A + was added to the cell pellet for lysis and allowed to stand at room temperature for 15 min. TRNzo per mlMu.l of chloroform was added to the solution of l-A +, and the mixture was vortexed for 15 seconds and allowed to stand for 3 minutes. After centrifugation at 13000rpm for 10 minutes at 4 ℃, Trizol-A + cell solution is divided into three layers: transferring the water phase dissolved with the RNA into a centrifuge tube, adding isopropanol with the same volume into the water phase, uniformly mixing, and standing at room temperature for 25 minutes. 13000rpm, 4 ℃ centrifugal 10 minutes, discarded waste liquid to get the bottom of the RNA precipitation. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in PEDC water and stored at-80 ℃. Synthesizing first chain cDNA by reverse transcription kit SMARTERRACE, and amplifying antibody variable region DNA sequence corresponding to hybridoma cell by using the first chain cDNA as subsequent template. Designing specific nested PCR primer, the primer sequence used in the amplification reaction is complementary with the first frame region and the constant region of the antibody variable region, and amplifying the target gene by adopting a conventional PCR method. Sequencing the amplified product to obtain the heavy chain variable region sequence SEQ ID NO:7 and light chain variable region sequences SEQ ID NO: 8; the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; the variable region of light chain has 3 CDR sequences of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.
Example 4 identification of antibody specificity
The monoclonal antibody purified in example 1 was cross-reacted with the CEA, MA, NCA coated ELISA plate by indirect ELISA, and the specificity was analyzed. The specific experimental process is to coat CEA, MA and NCA (all from Fitzgerald) respectively at a concentration of 0.1mg/ml, add the antibody obtained in example 1 to three different coating antigens respectively, and detect the degree of cross reaction by indirect ELISA. As shown in FIG. 2, the anti-CEA monoclonal antibody Cab-207 reacted only with CEA and did not cross-react with MA and NCA.
Example 5 affinity assay for anti-CEA antibodies
Coating an antibody capture Antibody (AHC) on the surface of a CM5 chip by an amino coupling mode, preparing chip activation buffer solution N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), AHC and ethanolamine for blocking according to the specifications of an amino coupling kit and an anti-capture kit, and selecting the coating in a Biacore3000 systemProcedure AHC amino groups were coupled to the CM5 chip surface. The anti-CEA antibody Cab-207 obtained in example 1 was captured on the chip surface. The antibody was diluted to 1. mu.g/mL with HBS-EP + buffer, set to a flow rate of 5. mu.L/min, and coated to a response value of 300 RU. CEA antigen was set up in 7 different concentration gradients and serially diluted in HBS-EP buffer in 2-fold gradients from 200nM to 3.125 nM. The flow rate of CEA antigen was set at 30. mu.L/min and the binding time was set at 3 min. The flow rate of HBS-EP + buffer is set to be 30 mu L/min, and the dissociation time is set to be 10 min. Use of 3M MgCl2As a regeneration buffer, the chip was regenerated according to the regeneration procedure. In addition, affinity assay was performed according to the same procedure with reference to antibody sanofi (antibody light and heavy chain variable region sequences are shown in SEQ ID NO:9, 10, respectively). Calculation of binding Rate (K) by Simultaneous fitting of binding and dissociation sensorgramsa) And dissociation Rate (K)d). Equilibrium dissociation constant (K)d) Using dissociation rate (K)d) Rate of binding (K)a) And (4) calculating. The results are shown in table one: compared with the control antibody, the antibody of the invention has higher affinity, and the affinity KDThe value reaches 1.55x10-9M。
Watch 1
Antibodies
|
Ka(1/Ms)
|
Kd(1/s)
|
KD(M)
|
Cab-207
|
6.91x104 |
1.07x10-4 |
1.55x10-9 |
sanofi
|
4.11x104 |
1.91x10-3 |
4.65x10-8 |
Example 6 preparation of CEA quantitative immunoassay kit (first) preparation of ELISA plate
The anti-CEA monoclonal antibody Cab-207 obtained in example 1 was diluted to 0.5. mu.g/ml with 0.01M phosphate buffer solution and the pH was 7.0 to 7.4 to obtain a coating solution. Adding 3% of skimmed milk powder into 0.01M phosphate buffer solution with the pH value of 7.0-7.4 to prepare confining liquid. Adding the prepared coating solution into the holes of an enzyme-labeled plate, and adding 100 mu L of the coating solution into each hole; the enzyme label plate is placed in an environment of 4 ℃ for coating overnight; adding the prepared confining liquid into the pores of an enzyme-labeled plate, adding 100 mu L of confining liquid into each pore, and placing the pores in a 37 ℃ incubator for 30 minutes; the enzyme label plate is taken out from the incubator, the confining liquid is discarded, and the temperature is kept constant at 37 ℃ for 30 minutes.
(II) preparing enzyme-labeled antibody solution
Immunizing a rabbit with the CEA antigen to obtain an anti-CEA polyclonal antibody, and labeling the anti-CEA polyclonal antibody with horseradish peroxidase to obtain an enzyme-labeled antibody. Firstly, weighing a proper amount of HRP enzyme, dissolving the HRP enzyme in triple distilled water, adding a newly-prepared sodium periodate solution, uniformly mixing, and then placing at 4 ℃ for 30 min; adding ethylene glycol solution, and standing at room temperature for 30 min; adding appropriate amount of purified antibody, mixing, adjusting pH to 9.0, standing at 4 deg.C overnight; adding sodium borohydride, mixing, and standing at 4 deg.C for 2 hr; adding the enzyme-labeled antibody mixed solution into an isovolumetric saturated ammonium sulfate solution, and standing at 4 ℃ for 30 min; after centrifugation, the mixture was dialyzed overnight against a phosphate buffer solution of pH 7.4.
(III) preparing CEA standard substance
CEA antigen: commercially available from Fitzgerald corporation.
(IV) preparation of concentrated Wash solution (20X 0.01M PBS)
Mixing 96 parts of sodium chloride, 2.4 parts of potassium chloride, 42.96 parts of disodium hydrogen phosphate dodecahydrate, 2.88 parts of potassium dihydrogen phosphate, 200.05 parts of tween-and 1000 parts of ultrapure water uniformly to obtain the finished product.
(V) substrate solution (TMB)
TMB Tetramethylbenzidine (3,3 ', 5, 5' -Tetramethylbenzidine), available from Tiangen, Beijing.
(VI) preparing stop solution (2mol/L sulfuric acid solution)
Diluting concentrated sulfuric acid and ultrapure water by 1: 8 to prepare stop solution.
Example 7 determination of the detection Range of the CEA quantitative immunoassay kit
CEA antigen was diluted with artificial serum in a gradient of 1000, 400, 200, 100, 50, 20, 5, 1, 0.2 ng/ml. Detection was performed using the kit of example 6. And (3) drawing a standard curve by taking the CEA antigen concentration as an abscissa and the corresponding A450 as an ordinate to obtain a fitting regression equation, and selecting the CEA antigen concentration range with the best linear relation as the optimal detection range of the detection reagent. The final data are shown in fig. 3, and the detection results show that when the CEA concentration is 0.2-400ng/ml, the linear relationship with the corresponding a450 value is good, R2 is 0.997, and the regression equation is: y is 0.0041x +0.216, and the lowest detection line is 0.09 ng/ml.
Example 8 kit stability assay
In order to test the stability and accuracy of the carcinoembryonic antigen CEA quantitative detection kit in example 6, the kit in example 6 was subjected to performance every 2 months, an ELISA plate was coated with an anti-CEA antibody Cab-207, stored at 4 ℃ for 3 months, subjected to standard protein detection again, and a standard curve was drawn. CEA antigen was diluted with artificial serum in a gradient manner to concentrations of 1000, 400, 200, 100, 50, 20, 5, 1, 0.2 ng/ml. Detection was performed using the kit of example 6. And (3) drawing a standard curve by taking the CEA antigen concentration as an abscissa and the corresponding A450 as an ordinate to obtain a fitting regression equation. The test results are shown in table 2, and the results show that the linear correlation between the carcinoembryonic antigen CEA detection kit of the embodiment 6 and the luminescence value is kept above 0.99 for 10 consecutive months, and the lowest detection limit is less than 0.15, which shows that the kit of the invention has higher stability and repeatability.
TABLE 2
Sequence listing
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