CN108152491A - A kind of carcinomebryonic antigen (CEA) immunologic function test reagent and its preparation and detection method - Google Patents
A kind of carcinomebryonic antigen (CEA) immunologic function test reagent and its preparation and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of carcinomebryonic antigen (CEA) detection reagent and its preparation and detection methods, belong to field of immunodetection.More particularly to a kind of carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent and its preparation and detection method, the reagent includes：Carcinomebryonic antigen specific antibody, the indicator for detecting carcinomebryonic antigen specific antibody carcinomebryonic antigen compound；Above-mentioned indicator is selected from enzymatic reagent, and the enzymatic reagent is made of the substrate of carcinomebryonic antigen enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose dehydrogenase carcinomebryonic antigen enzyme mark conjugate, and the substrate of enzyme is glucose.Carcinomebryonic antigen immunologic function test reagent high sensitivity of the present invention, reproducible, stability is strong, and can measure multiple samples simultaneously on automatic clinical chemistry analyzer, realizes high-throughput, the rapid measure of carcinomebryonic antigen, is conducive to clinical large-scale promotion and uses.
Description
Technical field
The present invention relates to a kind of carcinomebryonic antigen detection reagent and its preparation and detection methods, and in particular to a kind of carcinomebryonic antigen
Homogeneous enzyme immunoassay detection reagent and its preparation and detection method.
Technical background
Carcinomebryonic antigen (Carcinoembryonic antigen, CEA) is to be equal to nineteen sixty-five head by Gold and Freeman
Inferior to being found in colon cancer and fetal gut tissue.In embryo development procedure, CEA is mainly formed in as cell surface protein
Gastrointestinal tract and pancreas, and secrete in body fluid.The synthesis of adult CEA does not stop completely, exists in many and stomach and intestine or lung
In the cancer cell of the related ectodermal histological in source.CEA is mainly inactivated in liver, half-life period 14d, and bioactivity is at present still
It is unclear, but may be related with the adherency of immunosuppressant cell.
The knurl tire glycoprotein that CEA is made of 45%-55% sugar and 50% protein, relative molecular mass 180-200kD,
There are 9 antigenic determinants, gene family includes 17 activating genes of 2 subgroups, and it is related to belong to non-organ specificity tumour
Antigen.Change of serum C EA raisings are mainly seen in：The adenocarcinoma of colon patient CEA of 70%-90% is positive, the positive in other malignant tumours
Rate sequence is gastric cancer, cancer of pancreas, intestinal adenocarcinoma, lung cancer, liver cancer, breast cancer, urinary system cancerous swelling.Benign tumour, inflammation and regression
Property disease（Such as cholestasis, polyp of colon, alcoholic cirrhosis patient accounts, chronic hepatitis, pancreatitis, ulcerative colitis, lung
Wind-puff）The meeting of CEA contents is slight or moderate rises, but typically not greater than 10ng/ml.There are about 30%CEA in smoker to be more than 5ng/
ml.CEA can be additionally operable to instruct oncotherapy and follow-up, energy as benign and malignant tumour antidiastole foundation, measure
Judge for the state of an illness, prognosis and observation of curative effect provide important evidence.
The common method of detection CEA has radioimmunology (RIA), chemoluminescence method, Electrochemiluminescince, golden labelled immune
Diafiltration chromatography, Timed resolved fluoroimmunoassay and enzyme linked immunosorbent assay (ELISA) etc..Radioimmunology is used for earliest
The detection of CEA, but radioactive pollution is easily generated in detection process, and accuracy is low, the radioimmunoassay instrument of profession is needed,
It is difficult to carry out in common lab, be rarely employed at this stage.In remaining method either complex steps or take it is longer or
Person needs the instrument and equipment of complex and expensive or needs professional operator etc., is all unfavorable for being widely used in clinical detection.
Homogeneous enzyme immunoassay detection method high sensitivity, reproducible, stability is strong, easy to operate and can automatically give birth to
Change and measure multiple samples simultaneously on analyzer, realize high-throughput, the rapid measure of carcinomebryonic antigen, be conducive to clinic and push away on a large scale
It is wide to use.
Invention content
The present invention using carcinomebryonic antigen specific antibody and indicator in order to overcome the shortcomings of the prior art, prepared
Carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent, the reagent can be combined with various types automatic biochemistry analyzer, help to realize cancer
Embryonal antigen high throughput, rapid detection, and it is of less demanding to testing staff, be conducive to clinical expansion.
The purpose of the present invention is to provide a kind of carcinomebryonic antigen homogeneous enzyme immunoassay detection reagents, it is characterised in that：Contain cancer
Embryonal antigen specific antibody and indicator.
The indicator is enzymatic reagent, is made of the substrate of carcinomebryonic antigen enzyme mark conjugate and enzyme, enzyme mark conjugate is
Glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate, the substrate of enzyme is glucose.
The enzyme mark conjugate is coupled to be formed for glucose dehydrogenase and carcinomebryonic antigen.
A kind of preparation method of carcinomebryonic antigen immunologic function test reagent, which is characterized in that include the following steps：
(1) preparation of homogeneous enzyme substrate solution：By NAD+With glucose TRIS buffer solutions, preparation obtains homogeneous enzyme bottom
Object；
(2) preparation of glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate：Glucose dehydrogenase solution (GDH) is prepared, by GDH
It is coupled with carcinomebryonic antigen, purifies coupled product；
(3) preparation of carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent：
Reagent 1 (R1)：It is mixed by TRIS buffer solutions, carcinomebryonic antigen specific antibody, homogeneous zymolyte；
Reagent 2 (R2)：It is mixed by TRIS buffer solutions, glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate.
A kind of preparation method of aforementioned carcinomebryonic antigen immunologic function test reagent, the step (1) detailed process are：
By the nicotinamide adenine dinucleotide NAD of oxidation state+, glucose 55mM, pH=8.0 TRIS buffer solution systems
Into homogeneous zymolyte.The NAD+Ultimate density with glucose is 22.50 mM.
A kind of preparation method of aforementioned carcinomebryonic antigen immunologic function test reagent, the step (2) detailed process are：
1) preparation of glucose dehydrogenase solution：
A. 8mg MgCl are weighed2, 100mg NaCl and 15.6mg glucose dehydrogenase（100KU）, it is molten in order at room temperature
Solution is in the TRIS buffer solutions of 18ml 50mM pH=9.0；
B. be sequentially added into 337.5mg reduction-states nicotinamide adenine dinucleotide NADH, 140.2mg glucose and
1.125mL carbitol；
C. the dimethyl sulfoxide of 3mL is added dropwise.
2) preparation of glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate：
Carcinomebryonic antigen is added dropwise in glucose dehydrogenase solution obtained above, 2-8 DEG C is stirred overnight.The carcinomebryonic antigen
Mass ratio with glucose dehydrogenase is 1:1000-1000:1.Finally, by G-25 gel chromatography column purification enzyme mark conjugates,
It is stored at 2-8 DEG C.
A kind of preparation method of aforementioned carcinomebryonic antigen immunologic function test reagent, the step (3) detailed process are：
Reagent 1：Carcinomebryonic antigen antibody is added in above-mentioned homogeneous zymolyte.Antibody is 1 with homogeneous zymolyte volume ratio:100-
1:10000, preferably 1:1000.
Reagent 2：Glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate is added to the TRIS buffer solutions of 120mM pH=8.2
In, the volume ratio of above-mentioned conjugate and TRIS buffer solutions is 1:100-1:10000, preferably 1:3000.
Utilize the detection method of carcinomebryonic antigen immunologic function test reagent, which is characterized in that include the following steps：
1) sample to be tested is contacted with carcinomebryonic antigen specific antibody；
2) according to the combination situation of carcinomebryonic antigen in sample to be tested and carcinomebryonic antigen specific antibody, judge sample using indicator
The content of carcinomebryonic antigen in this.
The sample to be tested is various physiology samples, such as serum, blood plasma, saliva or urine etc..Preferably, sample to be tested
For EDTA anti-freezing blood plasma.
The invention has the beneficial effects that：The carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent high sensitivity of the present invention repeats
Property it is good, stability is strong, easy to operate and can measure multiple samples simultaneously on automatic clinical chemistry analyzer, realize that cancer embryo resists
Former high-throughput, rapid measure is conducive to clinical large-scale promotion and uses.
Description of the drawings
Fig. 1 is carcinomebryonic antigen homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent range of linearity figure.
Fig. 3 is carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent correlation analysis figure.
Specific embodiment
The technology used in the present invention principle is：Antigen and enzyme-labelled antigen competitive binding antibody in sample, treat test sample
Amount of carcinomebryonic antigen is more in this, and the amount of the enzyme-labelled antigen to dissociate in homogeneous enzyme solutions is more, and enzymatic reaction is faster, causes to inhale
Shading value (OD) rises.
Signified " antibody " refers not only to complete antibody molecule in the present invention, also includes retaining complete antibody specificity knot
The antibody fragment or derivative of conjunction ability.It can also be monoclonal antibody that the antibody of the present invention, which can be polyclonal antibody, excellent
Select polyclonal antibody.
A kind of carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent, including：Carcinomebryonic antigen specific antibody resists for detecting cancer embryo
The indicator of former specific antibody-carcinomebryonic antigen compound.Indicator be selected from enzymatic reagent, radioactive isotope, fluorescent reagent or
One kind in luminescence reagent, preferably enzymatic reagent；The enzymatic reagent is made of the substrate of carcinomebryonic antigen enzyme mark conjugate and enzyme,
Enzyme mark conjugate is glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate, can be obtained by chemical synthesis process, the substrate of enzyme
For glucose.
The detection method of the above-mentioned homogeneous enzyme detection reagent of carcinomebryonic antigen, includes the following steps：
1) sample to be tested is contacted with carcinomebryonic antigen specific antibody；
2) according to the combination situation of carcinomebryonic antigen in sample to be tested and carcinomebryonic antigen specific antibody, judge sample using indicator
The content of carcinomebryonic antigen in this.
The sample to be tested is various physiology samples, such as serum, blood plasma, saliva or urine etc..Preferably, sample to be tested
For EDTA anti-freezing blood plasma.
With reference to specific embodiment, further illustrate the present invention.
Embodiment one：The preparation of homogeneous zymolyte：
By the nicotinamide adenine dinucleotide NAD of oxidation state+, glucose 55mM, pH=8.0 TRIS buffer solution systems
Into homogeneous zymolyte.The NAD+Ultimate density with glucose is 22.50mM.
Embodiment two：The preparation of glucose dehydrogenase solution：
A. 8mg MgCl are weighed2, 100mg NaCl and 15.6mg glucose dehydrogenase（100KU）, it is molten in order at room temperature
Solution is in the TRIS buffer solutions of 18ml 50mM pH=9.0；
B. be sequentially added into 337.5mg reduction-states nicotinamide adenine dinucleotide NADH, 140.2mg glucose and
1.125mL carbitol；
C. the dimethyl sulfoxide of 3mL is added dropwise.
Embodiment three：The preparation of glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate：
Carcinomebryonic antigen is added dropwise in glucose dehydrogenase solution obtained above, 2-8 DEG C is stirred overnight.The carcinomebryonic antigen
Mass ratio with glucose dehydrogenase is 1:1000-1000:1.Finally, by G-25 gel chromatography column purification enzyme mark conjugates,
It is stored at 2-8 DEG C.
Example IV：The preparation of carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent：
Carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent, including：Carcinomebryonic antigen specific antibody, for detect carcinomebryonic antigen specificity
The indicator of antibody-carcinomebryonic antigen compound.Indicator is selected from enzymatic reagent, radioactive isotope, fluorescent reagent or luminescence reagent
In one kind, preferably enzymatic reagent；The enzymatic reagent is made of the substrate of carcinomebryonic antigen enzyme mark conjugate and enzyme, the coupling of enzyme mark
Object is glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate, can be obtained by chemical synthesis process, and the substrate of enzyme is grape
Sugar.
Carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the enzyme mark conjugate in indicator and
The substrate of enzyme reacts, and the substrate of enzyme mark conjugate and enzyme is separated, is not mixed, thus by the substrate of enzyme with it is above-mentioned
Carcinomebryonic antigen antibody mixes, that is to say, that carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent was provided separately including two kinds
Reagent, it is specific as follows：
1. the preparation of reagent 1：Carcinomebryonic antigen specific antibody is added in above-mentioned homogeneous zymolyte, antibody and homogeneous zymolyte body
Product is than being 1:100-1:10000, specific ratio is 1 in the present embodiment:1000.
2. the preparation of reagent 2：Glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate is added to 120mM pH=8.2
In TRIS buffer solutions, the volume ratio of above-mentioned conjugate and TRIS buffer solutions is 1:100-1:10000, in the present embodiment specifically
Ratio is 1:3000.
The application method of above-mentioned carcinomebryonic antigen immunologic function test reagent, includes the following steps：
1) sample to be tested is contacted with carcinomebryonic antigen specific antibody；
2) according to the combination situation of carcinomebryonic antigen in sample to be tested and carcinomebryonic antigen specific antibody, judge sample using indicator
The content of carcinomebryonic antigen in this.
Specifically, during detection, sample to be tested is added in reagent 1, the carcinomebryonic antigen in sample to be tested and the cancer in reagent 1
Embryonal antigen antibody is specifically bound, generation carcinomebryonic antigen antibody-carcinomebryonic antigen compound；Reagent 2 is added, at this time reagent
Glucose dehydrogenase-carcinomebryonic antigen conjugate in 2 is mixed with the zymolyte in reagent 1, and enzymatic reaction occurs, and forms inspection
The indicator of carcinomebryonic antigen specific antibody-carcinomebryonic antigen compound is surveyed, indicator is according to carcinomebryonic antigen in sample to be tested and cancer
The combination situation of embryonal antigen specific antibody judges the content of carcinomebryonic antigen in sample to be tested.
Since glucose dehydrogenase-carcinomebryonic antigen conjugate and the carcinomebryonic antigen competitive binding cancer embryo in sample to be tested resist
Former specific antibody, so, the amount of carcinomebryonic antigen is more in sample to be tested, and the glucose dehydrogenase dissociated in homogeneous enzyme solutions-
The amount of carcinomebryonic antigen conjugate is more, and enzymatic reaction is faster, and absorbance value (OD) is caused to rise.
The sample to be tested is various physiology samples, such as serum, blood plasma, saliva or urine etc..
As a preferred solution, above-mentioned sample to be tested is EDTA anti-freezing blood plasma.
Embodiment five：Carcinomebryonic antigen homogeneous enzyme immunoassay detects
1. obtain calibration curve：Olympus AU400 automatic clinical chemistry analyzer response parameters are set, and operating process is：First plus
Reagent 1, then add calibration object, it is eventually adding reagent 2.After adding in reagent 2, extinction of the test wavelength for different time points at 340nm
Angle value calculates reaction rate during different calibration object concentration, needs constantly to adjust the body of reagent 1 and reagent 2 in actual mechanical process
Product ratio, while survey luminous point is adjusted, finally obtain more satisfactory reaction calibration graph, as shown in Figure 1.
2. Biochemical Analyzer detects：By taking the operation of Olympus AU400 automatic clinical chemistry analyzers as an example：Add in 15 μ L samples
This, then adds in 150 μ L reagents 1, and blending incubation 5min adds in 150 μ L reagents 2, then blending incubation 30s starts to read and inhale
Luminosity A1, then after being incubated 5min, absorbance A 2 is read, calculate Δ A=A2-A1.Reaction condition：Master/slave wavelength：340/410nm；
Reaction temperature：37℃；Methodology：End-point method；The Direction of Reaction：Rise；Calibration mode：Multiple spot is non-linear.Calibration object concentration：
0.00,10.00,20.00,40.00,80.00,160.00 ng/mL.
3. utilizing the basic, normal, high concentration Quality Control sample of the calibrating curve determining 10 times, above-mentioned Quality Control sample is：Cancer embryo is resisted
Former calibration object is dissolved in human serum, and concentration is respectively 2.50ng/ml, 50.00ng/ml, 100.00ng/ml.Detection data and
Data analysis is shown in Table 1.
1 sample measures of table and precision assessment
| Sample | It is low | In | It is high |
| Concentration of specimens (ng/ml) | 2.50 | 50.00 | 100.00 |
| 1 | 2.48 | 52.47 | 100.22 |
| 2 | 2.56 | 53.28 | 101.12 |
| 3 | 2.51 | 49.71 | 98.35 |
| 4 | 2.45 | 52.68 | 102.14 |
| 5 | 2.52 | 50.75 | 104.28 |
| 6 | 2.55 | 51.32 | 103.02 |
| 7 | 2.43 | 51.01 | 95.33 |
| 8 | 2.49 | 48.53 | 98.49 |
| 9 | 2.59 | 50.21 | 100.59 |
| 10 | 2.57 | 49.86 | 101.26 |
| Average value (ng/ml) | 2.52 | 50.98 | 100.48 |
| Standard deviation (SD) | 0.0530 | 1.4919 | 2.5769 |
| Precision (CV%) | 2.10 | 2.93 | 2.56 |
Testing result shows：The accuracy that the homogeneous enzyme immunoassay detection reagent of the present invention measures is high, and precision is good, and CV is below
3%。
Embodiment six：Carcinomebryonic antigen homogeneous enzyme immunoassay detects linear analysis
The evaluation result of the carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent range of linearity is shown in Table 2.
2 carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent range of linearity evaluation result of table
| Low value | High level | 1 | 2 | 3 | Mean value | Theoretical value | Absolute deviation | Relative deviation (%) |
| 1 | 0 | 0.21 | 0.18 | 0.23 | 0.21 | 0.16 | 0.05 | 31.25 |
| 0.9 | 0.1 | 20.87 | 20.32 | 20.63 | 20.61 | 21.30 | -0.69 | -3.24 |
| 0.8 | 0.2 | 42.87 | 43.28 | 42.61 | 42.92 | 42.44 | 0.48 | 1.13 |
| 0.6 | 0.4 | 85.25 | 87.73 | 85.19 | 86.06 | 84.72 | 1.34 | 1.58 |
| 0.4 | 0.6 | 120.11 | 121.18 | 123.36 | 121.55 | 127.00 | -5.45 | -4.29 |
| 0.2 | 0.8 | 176.93 | 175.09 | 179.28 | 177.10 | 169.28 | 7.82 | 4.62 |
| 0 | 1 | 210.28 | 208.57 | 205.15 | 208.00 | 211.55 | -3.55 | -1.68 |
The high concentration sample close to the range of linearity upper limit is diluted with the low concentration sample (0.21) close to range of linearity lower limit
(211.55), 6 diluted concentrations are mixed into, each concentration replication 3 times calculates average value.Recurrence side is calculated using software
Journey y=211.3922x+0.1620, R2=0.9972.The experimental results showed that carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent of the present invention
With the good range of linearity.Linear analysis curve is obtained by the homogeneous enzyme immunoassay detection reagent of the present invention, sees Fig. 2.
Embodiment seven：Carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent correlation analysis
60 clinical samples including 50 normal samples and 10 exceptional samples are used with the magnetic of Beijing Li Deman respectively
The homogeneous enzyme immunoassay detection reagent of particulate chemistry luminescence reagent box and the present invention carry out correlation analysis, and the data of measure are shown in Table 3.
3 authentic specimen measured value of table
| Catalogue number(Cat.No.) | Homogeneous enzyme immunoassay method measured value (ng/ml) | Magnetism particulate immuno chemistry luminescence method measured value (ng/ml) |
| 1 | 4.89 | 4.92 |
| 2 | 3.73 | 3.67 |
| 3 | 4.39 | 4.38 |
| 4 | 3.35 | 3.32 |
| 5 | 3.02 | 2.95 |
| 6 | 4.08 | 4.13 |
| 7 | 3.23 | 3.17 |
| 8 | 2.97 | 2.92 |
| 9 | 2.58 | 2.61 |
| 10 | 4.50 | 4.53 |
| 11 | 2.14 | 2.16 |
| 12 | 2.49 | 2.44 |
| 13 | 2.29 | 2.37 |
| 14 | 2.85 | 2.74 |
| 15 | 2.93 | 2.88 |
| 16 | 2.25 | 2.14 |
| 17 | 3.43 | 3.51 |
| 18 | 2.96 | 2.87 |
| 19 | 3.91 | 4.05 |
| 20 | 5.48 | 5.46 |
| 21 | 9.25 | 9.38 |
| 22 | 3.84 | 3.97 |
| 23 | 3.73 | 3.78 |
| 24 | 3.61 | 3.57 |
| 25 | 3.12 | 3.03 |
| 26 | 2.17 | 2.14 |
| 27 | 4.48 | 4.25 |
| 28 | 4.35 | 4.38 |
| 29 | 3.46 | 3.51 |
| 30 | 4.04 | 4.11 |
| 31 | 8.89 | 8.85 |
| 32 | 2.99 | 3.02 |
| 33 | 7.06 | 6.98 |
| 34 | 1.97 | 1.83 |
| 35 | 2.49 | 2.45 |
| 36 | 2.87 | 2.81 |
| 37 | 4.98 | 5.02 |
| 38 | 4.68 | 4.75 |
| 39 | 14.48 | 14.22 |
| 40 | 2.74 | 2.68 |
| 41 | 7.31 | 7.35 |
| 42 | 4.99 | 4.87 |
| 43 | 9.79 | 9.80 |
| 44 | 4.74 | 4.72 |
| 45 | 8.36 | 8.23 |
| 46 | 3.69 | 3.71 |
| 47 | 2.82 | 2.88 |
| 48 | 3.59 | 3.65 |
| 49 | 5.99 | 6.07 |
| 50 | 3.05 | 3.04 |
| 51 | 3.12 | 3.19 |
| 52 | 3.53 | 3.58 |
| 53 | 2.44 | 2.72 |
| 54 | 2.01 | 1.96 |
| 55 | 3.25 | 3.21 |
| 56 | 2.92 | 3.09 |
| 57 | 6.77 | 6.68 |
| 58 | 3.74 | 3.72 |
| 59 | 2.69 | 2.76 |
| 60 | 4.54 | 4.55 |
It maps to above-mentioned data, sees Fig. 3, obtained linear equation is：Y=0.9908x+0.0340, coefficient R2=0.9984,
Show that the carcinomebryonic antigen clinical samples accuracy that the detection reagent of the present invention measures is high.
Since the detection process of the present invention is completed by instrument full-automation, so to the of less demanding of testing staff, easily
In realizing and promote the use of.
It should be noted that the foregoing is merely the embodiment of the present invention, it is not intended to limit the scope of the invention,
Every equivalent structure or equivalent flow shift done using description of the invention and accompanying drawing content, is directly or indirectly used in
Other correlative technology fields, are included within the scope of the present invention.
Claims (8)
1. a kind of carcinomebryonic antigen (CEA) immunologic function test reagent, which is characterized in that including：Carcinomebryonic antigen specific antibody, for examining
Survey the indicator of carcinomebryonic antigen specific antibody-carcinomebryonic antigen compound.
2. carcinomebryonic antigen immunologic function test reagent according to claim 1, which is characterized in that described to be used to detect carcinomebryonic antigen
The indicator of specific antibody-carcinomebryonic antigen compound is in enzymatic reagent, radioactive isotope, fluorescent reagent or luminescence reagent
One kind, preferably enzymatic reagent；The enzymatic reagent is made of the substrate of carcinomebryonic antigen enzyme mark conjugate and enzyme, enzyme mark conjugate
For glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate, the substrate of enzyme is glucose.
3. carcinomebryonic antigen immunologic function test reagent according to claim 2, which is characterized in that the enzyme mark conjugate is grape
Glucocorticoid dehydrogenase and carcinomebryonic antigen are coupled to be formed.
4. a kind of preparation method of carcinomebryonic antigen immunologic function test reagent, which is characterized in that include the following steps：
(1) preparation of homogeneous enzyme substrate solution：By NAD+With glucose TRIS buffer solutions, preparation obtains homogeneous enzyme bottom
Object；
(2) preparation of glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate：Glucose dehydrogenase solution (GDH) is prepared, by GDH
It is coupled with carcinomebryonic antigen, purifies coupled product；
(3) preparation of carcinomebryonic antigen homogeneous enzyme immunoassay detection reagent：
Reagent 1 (R1)：It is mixed by TRIS buffer solutions, carcinomebryonic antigen specific antibody, homogeneous zymolyte；
Reagent 2 (R2)：It is mixed by TRIS buffer solutions, glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate.
5. the preparation method of a kind of carcinomebryonic antigen immunologic function test reagent according to claim 4, which is characterized in that described
Step (1) detailed process is：
By the nicotinamide adenine dinucleotide NAD of oxidation state+, glucose is made of the TRIS buffer solutions of 55mM, pH=8.0
Homogeneous zymolyte；The NAD+Ultimate density with glucose is 22.50 mM.
6. the preparation method of a kind of carcinomebryonic antigen immunologic function test reagent according to claim 4, which is characterized in that described
Step (2) detailed process is：
1) preparation of glucose dehydrogenase solution：
A. 8mg MgCl are weighed2, 100mg NaCl and 15.6mg glucose dehydrogenase (100KU), dissolve in order at room temperature
TRIS buffer solutions in 18ml 50mM pH=9.0；
B. be sequentially added into 337.5mg reduction-states nicotinamide adenine dinucleotide NADH, 140.2mg glucose and
1.125mL carbitol；
C. the dimethyl sulfoxide of 3mL is added dropwise；
2) preparation of glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate：
Carcinomebryonic antigen is added dropwise in glucose dehydrogenase solution obtained above, 2-8 DEG C is stirred overnight；The carcinomebryonic antigen
Mass ratio with glucose dehydrogenase is 1:1000-1000:1；Finally, by G-25 gel chromatography column purification enzyme mark conjugates,
It is stored at 2-8 DEG C.
7. the preparation method of a kind of carcinomebryonic antigen immunologic function test reagent according to claim 4, which is characterized in that described
Step (3) detailed process is：
Reagent 1：Carcinomebryonic antigen antibody is added in the homogeneous zymolyte described in claim 5, antibody and homogeneous zymolyte body
Product is than being 1:100-1:10000, preferably 1:1000；
Reagent 2：Glucose dehydrogenase-carcinomebryonic antigen enzyme mark conjugate is added in the TRIS buffer solutions of 120mM pH=8.2, on
The volume ratio for stating conjugate and TRIS buffer solutions is 1:100-1:10000, preferably 1:3000.
8. using the detection method of the carcinomebryonic antigen immunologic function test reagent described in 3 any one of claims 1 to 3, feature exists
In including the following steps：
1) sample to be tested is contacted with carcinomebryonic antigen specific antibody；
2) according to the combination situation of carcinomebryonic antigen in sample to be tested and carcinomebryonic antigen specific antibody, judge sample using indicator
The content of carcinomebryonic antigen in this；The sample to be tested is various physiology samples, such as serum, blood plasma, saliva or urine etc.；It is preferred that
, sample to be tested is EDTA anti-freezing blood plasma.
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| CN101358976A (en) * | 2008-04-28 | 2009-02-04 | 北京华大吉比爱生物技术有限公司 | Micro array-ELISA detecting kit for detecting six tumor markers |
| CN102768284A (en) * | 2012-08-01 | 2012-11-07 | 苏州博源医疗科技有限公司 | Immunodetection reagent of carbamazepine homogeneous enzyme and detection method thereof |
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| CN101358976A (en) * | 2008-04-28 | 2009-02-04 | 北京华大吉比爱生物技术有限公司 | Micro array-ELISA detecting kit for detecting six tumor markers |
| CN102768284A (en) * | 2012-08-01 | 2012-11-07 | 苏州博源医疗科技有限公司 | Immunodetection reagent of carbamazepine homogeneous enzyme and detection method thereof |
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