CN106771203A - For carcinoma of the rectum external diagnosis reagent case and its detection method - Google Patents
For carcinoma of the rectum external diagnosis reagent case and its detection method Download PDFInfo
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- 108010035532 Collagen Proteins 0.000 claims abstract description 35
- 102000008186 Collagen Human genes 0.000 claims abstract description 34
- 229920001436 collagen Polymers 0.000 claims abstract description 34
- 239000000427 antigen Substances 0.000 claims description 49
- 102000036639 antigens Human genes 0.000 claims description 49
- 108091007433 antigens Proteins 0.000 claims description 49
- 238000002360 preparation method Methods 0.000 claims description 32
- 241000124008 Mammalia Species 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 18
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- 238000000576 coating method Methods 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 15
- 238000004140 cleaning Methods 0.000 claims description 14
- 238000000746 purification Methods 0.000 claims description 13
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 12
- 238000012215 gene cloning Methods 0.000 claims description 11
- 210000004962 mammalian cell Anatomy 0.000 claims description 11
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 6
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- 239000007853 buffer solution Substances 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 210000002966 serum Anatomy 0.000 description 14
- 206010028980 Neoplasm Diseases 0.000 description 13
- 239000000523 sample Substances 0.000 description 11
- 208000015634 Rectal Neoplasms Diseases 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 206010038038 rectal cancer Diseases 0.000 description 8
- 229910019142 PO4 Inorganic materials 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
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- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100036217 Collagen alpha-1(X) chain Human genes 0.000 description 1
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- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
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- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
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- 210000000664 rectum Anatomy 0.000 description 1
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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Abstract
It is used for carcinoma of the rectum external diagnosis reagent case, including capture antibody the invention discloses one kind, the capture antibody is monoclonal antibody obtained in X-type collagen α 1；The kit also includes detection antibody, and the detection antibody is polyclonal antibody obtained in X-type collagen α 1.Beneficial effects of the present invention are as follows：With sensitivity high accuracy it is strong the characteristics of, its sensitivity reaches more than 70%.
Description
Technical field
It is specially a kind of to be used for carcinoma of the rectum external diagnosis reagent the present invention relates to the kit field of cancer diagnosis detection
Box and its detection method.
Background technology
The carcinoma of the rectum is one of most common malignant tumour, the annual people of new cases 33.1 ten thousand of colorectal cancer of China, morbidity
Rate is number four position in whole malignant tumours；The death rate then occupies cancer mortality reason the 5th.The crowd of health, there is a large amount of
Polyp of colon patient carries out the antidiastole of colorectal cancer, and its average attack rate reaches 10%.Clinical treatment finds that the first phase is straight
Survival rate after intestinal cancer treatment is closer absolutely up to the survival rate after 80 percent, zero phase rectum cancer treatment therefore early
Phase finds and treats extremely important.
Tumor markers (tumor markers, TM) refers in tumour generation and breeding, by tumour cell in itself
Synthesis, release, or tumour cell is reacted and the signal tumor presence of generation and a class material of growth by body.These materials
The level for not existing in adult normal or occurring in cancer patient is significantly higher than normal person.Current tumor-marker quality testing
Survey technology is considered as the unique channel of the asymptomatic micro- stove tumour of early detection, this detection technique can prior to X-ray, ultrasound,
The PEs such as CT, MRI or PET-CT find tumour.Can be used for the examination of people at highest risk's malignant tumour, diagnosing tumor and discriminating
Diagnosis, assesses the effect for the treatment of, prediction or monitoring tumor recurrence or transfer.At present, the carcinoma of the rectum diagnostic kit that hospital occurs
All it is to detect some common tumor markers, sensitivity and accuracy are all relatively low.Because of mainly selected tumor marker list
Item detection often has significant limitation, it is difficult to meet the requirement of Rapid＆Early diagnosis.
At present, rapidly and efficiently diagnostic kit of the in the market also not for the carcinoma of the rectum comes out, and badly influences the carcinoma of the rectum
Early detection and treatment.
The content of the invention
The purpose of the present invention is directed to the deficiency existed with above-mentioned existing carcinoma of the rectum diagnosis, there is provided a kind of sensitivity is high, standard
True property is strong and easy to use efficiently for carcinoma of the rectum external diagnosis reagent case.
Another object of the present invention is to provide a kind of detection method for carcinoma of the rectum external diagnosis reagent case.
In order to realize the object of the invention, the present invention is adopted the technical scheme that：For carcinoma of the rectum external diagnosis reagent case, bag
Capture antibody is included, the capture antibody is monoclonal antibody obtained in X-type collagen α 1.
The kit also includes detection antibody, and the detection antibody is polyclonal antibody obtained in X-type collagen α 1.
The capture antibody is that X-type collagen α 1 is cloned into carrier for expression of eukaryon, and real in mammalian cell
The expression of existing albumen, obtains corresponding antigen, the corresponding monoclonal antibody that the antigen immune mammal is obtained after purification.
The preparation method of the capture antibody comprises the following steps：
（1）The preparation of antigen：The gene cloning of X-type collagen α 1 to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, antigen is obtained after purification, this antigen can also be used as standard items；
（2）Capture the preparation of antibody：By above-mentioned antigen immune mammal, it is capture antibody to obtain corresponding monoclonal antibody.
The detection antibody is to carrier for expression of eukaryon and thin in mammal by the gene cloning of X-type collagen α 1
The expression of albumen is realized in born of the same parents, corresponding antigen is obtained after purification, corresponding many grams that the antigen immune mammal is obtained
Grand antibody.
The preparation method of the detection antibody comprises the following steps：
（1）The preparation of antigen：The gene cloning of X-type collagen α 1 to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, antigen is obtained after purification, this antigen can also be used as standard items；
（2）The preparation of detection antibody：By above-mentioned antigen immune mammal, corresponding polyclonal antibody is obtained for detection antibody.
The capture antibody is coated in the hole of microtiter plate in advance, can simplify step, improves detection efficiency.
For the preparation method of carcinoma of the rectum external diagnosis reagent case, it is comprised the following steps：
（1）The preparation of antigen：By the gene cloning of X-type collagen α 1 to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, required antigen is obtained after purification, wherein, the antigen can also be used as standard items；
（2）Capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal resists
Body as this kit capture antibody；
（3）The preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
（4）Capture antibody coating：
1. the capture antibody that concentration is 1 μ g/ml is coated with micro drop by carbonate/bicarbonates buffer solution (pH9.6)
The hole of fixed board；2. covers microtiter plate and the overnight incubation at 4 DEG C；3. discards coating buffer（Carbonate/bicarbonate
The capture antibody of buffer solution dilution）, and wash microtiter plate twice with cleaning solution, 200 μ l are added in micropore every time
PBST（Phosphate Tween buffer）, the gently whipping microtiter plate above tank removes cleaning solution, patted on paper handkerchief micro-
Amount titer plate, removes remaining drop, dry be put in it is standby in 4 DEG C of environment；
（5）Closing and sample-adding：200 μ l Block buffers are added per hole（1.2%BSA/PBS）, it is remaining in closing coating hole
Protein binding site.
For the detection method of carcinoma of the rectum external diagnosis reagent case, it is comprised the following steps：
（1）The preparation of antigen：By the gene cloning of X-type collagen α 1 to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, required antigen is obtained after purification；
（2）Capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal resists
Body as this kit capture antibody；
（3）The preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
（4）Capture antibody coating：
1. the capture antibody that concentration is 1 μ g/ml is coated with microtitration by carbonate/bicarbonates buffer solution (pH9.6)
The hole of plate；
2. covers microtiter plate and the overnight incubation at 4 DEG C；
3. discards coating buffer（The capture antibody of carbonate/bicarbonate buffer solution dilution）, and wash microtitration with cleaning solution
Plate adds 200 μ l PBST twice, every time in micropore（Phosphate Tween buffer）, gently whipping is micro above tank
Titer plate, removes cleaning solution, pats microtiter plate on paper handkerchief, removes remaining drop, dry be put in it is standby in 4 DEG C of environment
With；
（5）Closing
1. adds 200 μ l Block buffers in each hole of microtiter plate（1.2%BSA/PBS）, it is surplus in closing coating hole
Remaining protein binding site；
2. covers microtiter plate and is incubated 1 hour at 37 DEG C；
（6）Sample-adding
1. the sample of 100 μ l is added to each hole by, is incubated 60 minutes at 37 DEG C;Accurate quantitative result is obtained, is led to
Normal way is to compare the signal of unknown sample and standard curve.Each ELISA Plate must bioassay standard product（Double measure or three
It is fixed to resurvey）And blank sample, to ensure accuracy；
2. discards sample, and washs microtiter plate three times, adds 200 μ l PBST in micropore every time（Phosphate tween delays
Fliud flushing）；
3. the detection antibody that 100 μ l concentration are 0.5 μ g/ml is added to each hole by；
4. covers microtiter plate and is incubated 1 hour at 37 DEG C；
5. washs microtiter plate with PBST four times；
6. adds 100 μ l mark secondary antibodies；
7. covers microtiter plate and is incubated 1 hour at 37 DEG C；
8. washs microtiter plate with PBST four times；
(7) detect
1. is by TMB（3,3', 5,5'- tetramethyl benzidine）Solution is added to each hole, is incubated 15-30 minutes, addition etc.
The terminate liquid of volume, then reads optical density at 450 nm.
2. draws standard curve by the data that serial dilutions are obtained, and concentration is marked on X axles（Logarithmic scale）On, and inhale
Photometric scale is in Y-axis（Lineal scale）On.Sample concentration is drawn on this standard curve by interpolation method.
The present invention filters out new tumor marker COL10A1 (X-type collagen α 1) from numerous tumor markers
Composition carcinoma of the rectum quick diagnosis reagent kit.X-type collagen α 1 is a kind of novel collagen of discovered in recent years, is into lamellar structure
Collagen, they are assembled into lamellar structure in extracellular matrix (ECM).Generally triple helix knot is constituted by three polypeptide chains
Structure, during embryonic development, tissue reconstruction, injury repair etc., the table of growth factor and differentiation factor to collagen gene
Up to important regulating and controlling effect.Many diseases have close relationship with the exception of X-type collagen.Nearest research also confirms that it
It is the ideal mark thing of the carcinoma of the rectum.
Compared with prior art, beneficial effects of the present invention are as follows：With sensitivity high accuracy it is strong the characteristics of, its is sensitive
Degree reaches more than 70%.
Brief description of the drawings
Fig. 1 is schematic diagram of the present invention for carcinoma of the rectum external diagnosis reagent case；
Fig. 2 is comparative selection figure of the present invention for the detection antibody best effort concentration of carcinoma of the rectum external diagnosis reagent case；
Fig. 3 is that the present invention says for diagnosis and the antidiastole contrast to clinical serum sample of carcinoma of the rectum external diagnosis reagent case
Bright figure；
Fig. 4 is detection comparison diagram of the present invention for carcinoma of the rectum external diagnosis reagent case sensitiveness；
Fig. 5 is the present invention for the specific detection comparison diagram of carcinoma of the rectum external diagnosis reagent case；
Fig. 6 is that the present invention detects X-type collagen α for carcinoma of the rectum external diagnosis reagent case in blood before and after rectum cancer treatment
1 change comparison diagram.
Specific embodiment
Detailed retouching is carried out for carcinoma of the rectum external diagnosis reagent case to the present invention below in conjunction with the drawings and specific embodiments
State bright.
For carcinoma of the rectum external diagnosis reagent case, including capture antibody, capture antibody Block buffer, standard items, mark
Antibody, cleaning solution and nitrite ion, the labelled antibody mark secondary antibody from HRP；The capture antibody is that X-type collagen α 1 makes
The monoclonal antibody for obtaining；Its sensitivity and accuracy reach more than 70%.
The capture antibody is that X-type collagen α 1 is cloned into carrier for expression of eukaryon, and real in mammalian cell
The expression of existing albumen, obtains corresponding antigen, the corresponding monoclonal antibody that the antigen immune mammal is obtained after purification.
The preparation method of the capture antibody comprises the following steps：
（1）The preparation of antigen：By the method for molecular cloning the gene clonings of X-type collagen α 1 to carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, antigen is obtained after purification, this antigen can also be used as standard items；The antigen
Preparation can also be prepared using existing conventional method, herein no longer burden.
（2）Capture the preparation of antibody：By above-mentioned antigen immune mammal, corresponding monoclonal antibody is obtained for capture is anti-
Body；The mammal is mouse and rabbit, preferably mouse；The preparation of the capture antibody can also use existing conventional method
Prepare, it is no longer burdensome herein.
The detection antibody includes polyclonal antibody obtained in X-type collagen α 1.
It is described catch detection antibody be the gene cloning by X-type collagen α 1 to carrier for expression of eukaryon, and in mammal
The expression of albumen is realized in cell, corresponding antigen is obtained after purification, by the antigen immune mammal obtain it is corresponding many
Clonal antibody.The preparation method of the detection antibody comprises the following steps：
（1）The preparation of antigen：By the method for molecular cloning the gene clonings of X-type collagen α 1 to carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, antigen is obtained after purification, this antigen can also be used as standard items；
（2）The preparation of detection antibody：By above-mentioned antigen immune mammal, corresponding polyclonal antibody is obtained for detection antibody,
The mammal is mouse and rabbit, preferably rabbit.
The capture antibody can be coated in the hole of the microtiter plate of PVC materials in advance, can simplify step, carry
High detection efficiency.
For the preparation method of carcinoma of the rectum external diagnosis reagent case, it is comprised the following steps：
（1）The preparation of antigen：By the method for molecular cloning by the gene clonings of X-type collagen α 1 to carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, required antigen is obtained after purification, wherein, the antigen also can be used as standard
Product are used；
（2）Capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal resists
Body as this kit capture antibody；
（3）The preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
（4）Capture antibody coating：
1. the capture antibody that concentration is 1 μ g/ml is coated in micro drop by carbonate/bicarbonates buffer solution (pH9.6)
The hole of fixed board；
2. covers microtiter plate and the overnight incubation at 4 DEG C with bond plastic product；
3. discards coating buffer（The capture antibody of carbonate/bicarbonate buffer solution dilution）, and wash microtitration with cleaning solution
Plate adds 200 μ l PBST twice, every time in micropore（Phosphate Tween buffer）, gently whipping is micro above tank
Titer plate, removes cleaning solution, pats microtiter plate on paper handkerchief, removes remaining drop, dry be put in it is standby in 4 DEG C of environment
With；The cleaning solution is PBS（Phosphate buffer）It is middle to add a certain amount of Tween 20, the quality percentage of the Tween 20
Specific concentration is 0.05%；
（5）Closing
1. adds 200 μ l Block buffers in every hole of microtiter plate（It is containing 1.2%BSA（Bovine serum albumin(BSA)）'s
PBS（Phosphate buffer））, for remaining protein binding site in closing coating hole；
2. bond plastic products cover microtiter plate and are incubated 1 hour at 37 DEG C；
For the detection method of carcinoma of the rectum external diagnosis reagent case, it is comprised the following steps：
（1）The preparation of antigen：By the method for molecular cloning by the gene clonings of X-type collagen α 1 to carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, required antigen is obtained after purification, wherein, the antigen also can be used as standard
Product are used；
（2）Capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal resists
Body as this kit capture antibody；
（3）The preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
（4）Capture antibody coating：
1. the capture antibody that concentration is 1 μ g/ml is coated with micro drop by carbonate/bicarbonates buffer solution (pH9.6)
The hole of fixed board；
2. covers microtiter plate and the overnight incubation at 4 DEG C with bond plastic product；
3. discards coating buffer（The capture antibody of carbonate/bicarbonate buffer solution dilution）, and wash microtitration with cleaning solution
Plate adds 200 μ l PBST twice, every time in micropore（Phosphate Tween buffer, can be containing 0.05% Tween-20
The phosphate buffer of pH7.4）, the gently whipping microtiter plate above tank removes cleaning solution, patted on paper handkerchief micro
Titer plate, removes remaining drop, dry be put in it is standby in 4 DEG C of environment；The cleaning solution is PBS（Phosphate buffer）In plus
Enter a certain amount of Tween 20, the mass percent concentration of the Tween 20 is 0.05%；
（5）Closing
1. adds 200 μ l Block buffers in each hole of microtiter plate（1.2%BSA/PBS）, it is surplus in closing coating hole
Remaining protein binding site；
2. bond plastic products cover microtiter plate and are incubated 1 hour at 37 DEG C；
（6）Sample-adding
1. suitably dilutes 100 μ l（20 times of dilution）Sample be added to each hole, at 37 DEG C be incubated 60 minutes;
Obtain accurate quantitative result, it is common practice that compare the signal of unknown sample and standard curve.Each ELISA Plate is necessary
Bioassay standard product（Double measure or triplicate）And blank sample, to ensure accuracy；
2. discards sample, and washs microtiter plate three times, adds 200 μ l PBST in micropore every time（Phosphate tween delays
Fliud flushing）；
3. the detection antibody that 100 μ l concentration are 0.5 μ g/ml is added to each hole by；
4. bond plastic products cover microtiter plate and are incubated 1 hour at 37 DEG C；
5. washs microtiter plate with PBST four times；
6. adds 100 μ l mark secondary antibodies, and it is just diluting 10000 times in PBS using preceding（1：10000）, the mark
Note secondary antibody can mark goat-anti rabbit for HRP；
7. bond plastic products cover microtiter plate and are incubated 1 hour at 37 DEG C；
8. washs microtiter plate with PBST four times；
(7) detect
1. is by TMB（3,3', 5,5'- tetramethyl benzidine）Solution is added to each hole, is incubated 15-30 minutes, addition etc.
Terminate liquid (the 2 M H of volume2SO4), optical density is then read at 450 nm with enzyme-linked immunosorbent assay instrument；
2. draws standard curve by the data that serial dilutions are obtained, and concentration is marked on X axles（Logarithmic scale）On, and absorbance
It is marked on Y-axis（Lineal scale）On, sample concentration is drawn on this standard curve by interpolation method.
The present invention raises X-type collagen α 1 as the diagnosis early metaphase carcinoma of the rectum for carcinoma of the rectum external diagnosis reagent case
Standard, Cleaning Principle is as shown in Figure 1；X-type collagen α 1 declines the standard as rectum cancer treatment recruitment evaluation.Can use
The height of tumor marker levels carries out dynamic evaluation to the therapeutic effect of the carcinoma of the rectum in clinically by detection blood.In addition also
Can be used for the application of relapse and metastasis and Index for diagnosis clinically to the carcinoma of the rectum；Can also be used for carcinoma of the rectum diagnosis.
Test effect explanation
The selection of double-antibody sandwich elisa optimum experimental condition
It is coated with the selection of the anti-monoclonal antibody best effort concentration of X-type collagen α 1 of mouse：Determine that coating concentration is 1ug/ according to square formation method
During mL, the OD values of monoclonal antibody are 1.05, so its optimal coating concentration is 1ug/mL.As shown in Fig. 2 rabbit-anti X-type collagen
The selection of the anti-best effort concentration of protein alpha more than 1：With the increase of monoclonal antibody extension rate, carcinoma of the rectum case serum to be measured and normal
Human serum OD values have the trend successively decreased, when AC is 1：When 200, (positive control OD values subtract blank to positive control
Control OD values) and the ratio between normal control (normal control OD values subtract blank OD values) A450nm (abbreviation P/N values
) higher, therefore selection rabbit-anti human antibody best effort concentration is 1：200.The best effort concentration that serum is groped is 1：25.Closing
Liquid gropes best effort solubility for 1.2%BSA.
Clinical serum Samples detection
50 parts of serum specimens are have detected altogether, through definitive pathological diagnosis are rectal cancer patient serum as positive controls with hospital（34（Its
Middle Early rectal tumor 15, advanced rectal cancer 19））, non-rectal cancer patient is feminine gender including rectal polyp, normal population serum
Control group（85（Normal 50, rectal polyp 35））, PBST is blank, is entered by above-mentioned double crush syndrome method
The qualitative and quantitative determination clinical serum sample of row.As shown in Fig. 2 with P/N values>2 is that double crush syndrome is positive judges mark
Standard, detects by standard of pathological diagnosis to clinical serum sample, X-type collagen in Fig. 3 explanation rectal cancer patient serum
The level of α 1 is significantly higher than normal person（P<0.01）.Early rectal tumor result detection sensitivity（SN）It is 58%（9/15）, late period
Carcinoma of the rectum detection sensitivity（SN）It is 73%（14/19）；Normal person's detection specificity（SP）It is 72%（36/50）, rectum breath
Meat patient detection specificity（SP）It is 61%（21/35）.See Fig. 4-5.
The clinical therapeutic effect to the carcinoma of the rectum carries out dynamic evaluation
To determine that can this kit be used to carry out dynamic evaluation to the therapeutic effect of the carcinoma of the rectum, we have collected 27 parts of carcinoma of the rectum
Serum before and after patient's treatment.As a result 27 testing results of patient show the serum before and after rectal cancer patient treatment referring to Fig. 6
There were significant differences for the level of middle X-type collagen α 1（P<0.05）.Effectively after treatment in serum X-type collagen α 1 level meeting
It is greatly reduced, points out this kit to be used to carry out dynamic evaluation to the therapeutic effect of the carcinoma of the rectum.
Claims (1)
- It is 1. a kind of to be used for carcinoma of the rectum external diagnosis reagent case, including capture antibody, it is characterised in that：The capture antibody is X-type Monoclonal antibody obtained in collagen α 1, the kit also includes detection antibody, and the detection antibody is X-type collagen Polyclonal antibody obtained in α 1；Its preparation method comprises the following steps：（1）The preparation of antigen：By the gene cloning of X-type collagen α 1 to carrier for expression of eukaryon, and in mammalian cell The expression of albumen is realized, required antigen is obtained after purification, wherein, the antigen is used as standard items；（2）Capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal resists Body as this kit capture antibody；（3）The preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α Body as this kit detection antibody；（4）Capture antibody coating：1. the capture antibody that concentration is 1 μ g/ml is coated with carbonate/bicarbonates buffer solution the hole of microtiter plate； 2. covers microtiter plate and the overnight incubation at 4 DEG C；3. discards coating buffer, and washs microtiter plate with cleaning solution Twice, 200 μ l PBST are added in micropore every time, the gently whipping microtiter plate above tank removes cleaning solution, Pat microtiter plate on paper handkerchief, remove remaining drop, dry be put in it is standby in 4 DEG C of environment；（5）Closing and sample-adding：200 μ l Block buffers, remaining protein binding site in closing coating hole are added per hole.
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| CN111742221A (en) * | 2017-07-27 | 2020-10-02 | 北欧生物科技公司 | Type X Collagen α1 Assay |
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| WO2013052480A1 (en) * | 2011-10-03 | 2013-04-11 | The Board Of Regents Of The University Of Texas System | Marker-based prognostic risk score in colon cancer |
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