CN107085110A - One kind is used for thyroid cancer external diagnosis reagent case and its detection method - Google Patents
One kind is used for thyroid cancer external diagnosis reagent case and its detection method Download PDFInfo
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- CN107085110A CN107085110A CN201710399343.3A CN201710399343A CN107085110A CN 107085110 A CN107085110 A CN 107085110A CN 201710399343 A CN201710399343 A CN 201710399343A CN 107085110 A CN107085110 A CN 107085110A
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- microtiter plate
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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
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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
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/046—Thyroid disorders
Abstract
It is used for thyroid cancer external diagnosis reagent case and its detection method, including capture antibody the invention discloses one kind, the capture antibody is L selectins（L‑SELECTIN）Obtained monoclonal antibody；The kit also includes detection antibody, and the detection antibody is polyclonal antibody made from L selectins.The present invention using L selectins tumor cell surface height expression, be a kind of molecular marked compound of new malignant tumour high specific, with sensitivity high accuracy it is strong the characteristics of, its sensitivity and accuracy reach more than 65%.
Description
Technical field
It is specially that one kind is examined in vitro for thyroid cancer the present invention relates to the kit technical field of cancer diagnosis detection
Disconnected kit and its detection method.
Background technology
Thyroid cancer Professional Committee of Chinese Anti-Cancer Association General Board is brilliant, and professor claims, and in recent years, China's thyroid cancer is occurred frequently,
Growth rate most fast malignant tumour is turned into, the incidence of disease increases nearly 5 times for 10 years.Thyroid cancer is especially apt to occur in young and middle-aged female
Property, women and masculinity proportion are 3：1, it is most fast swollen into the female malignant rate of climb in Jin20Nian Lai China cancer spectrum
Knurl.Nowadays in the female group of one, tier 2 cities, the sick incidence of disease ranks front three substantially, and what is had has even leapt to the umber one.Face
Thyroid benign and malignant tumour are often showed only as thyroid nodule on bed, and expert reminds thyroid cancer patients key to be morning
It was found that, early treatment, thyroid nodule malignant and benign lesion diagnosis is even more important.The report of Endocrine Society of Chinese Medical Association shows
Show, the illness rate of thyroid nodule is 18.6%, it means that just with the presence of nearly 1 people thyroid nodule in every 5 people of China.Also
It is to say, China has nearly 2.8 hundred million people to carry out the malignant and benign lesion diagnosis of thyroid nodule.
Clinical treatment finds that the early diagnosis and treatment of thyroid cancer are extremely important.
Tumor markers (tumor markers, TM) refer to tumour occur and breeding in, by tumour cell in itself
Synthesis, release, or the signal tumor presence reacted tumour cell by body and produced and a class material of growth.These materials
The level either occurred in cancer patient is not present in normal adult and 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.Available for the examination of people at highest risk's malignant tumour, diagnosing tumor is with differentiating
Diagnosis, assesses the effect for the treatment of, prediction or monitoring tumor recurrence or transfer.At present, in the market is not directed to thyroid cancer also
Rapidly and efficiently diagnostic kit comes out, and badly influences thyroid cancer early detection and treatment.
The content of the invention
It is an object of the invention to provide a kind of sensitivity height, accuracy are strong and easy to use efficiently for thyroid cancer
External diagnosis reagent case and its detection method, with asking for the diagnosis of thyroid cancer Shortcomings that solve to propose in above-mentioned background technology
Topic.
To achieve the above object, the present invention provides following technical scheme：One kind is used for thyroid cancer external diagnosis reagent case,
Including capture antibody, the capture antibody is monoclonal antibody made from L selectins.
It is preferred that, the step of preparation method of the capture antibody includes is as follows：
S1：The preparation of antigen；The gene clonings of L selectins to carrier for expression of eukaryon, and realize in mammalian cell egg
White expression, obtains antigen after purification；
S2：Capture the preparation of antibody；By above-mentioned antigen immune mammal, it is capture antibody to obtain corresponding monoclonal antibody.
It is preferred that, the kit also includes detection antibody, and the detection antibody is Anti-TNF-α made from L selectins
Body.
It is preferred that, the step of preparation method of the detection antibody includes is as follows：
S1：The preparation of antigen；The gene clonings of L selectins to carrier for expression of eukaryon, and realize in mammalian cell egg
White expression, obtains antigen after purification；
S2：Detect the preparation of antibody；By above-mentioned antigen immune mammal, it is detection antibody to obtain corresponding polyclonal antibody.
It is preferred that, the capture antibody is coated in the hole of microtiter plate in advance；
The present invention also provides a kind of technical scheme：A kind of preparation method for thyroid cancer external diagnosis reagent case, including with
Lower step：
S1：The preparation of antigen：By L selectin gene clonings to carrier for expression of eukaryon, and realize in mammalian cell albumen
Expression, required antigen is obtained after purification；
S2：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；
S3：Detect the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
S4：Capture antibody coating：
S4.1：Concentration is coated with to the hole of microtiter plate with carbonate/bicarbonate buffer solution for 1 μ g/ml capture antibody；
S4.2：Cover microtiter plate and be incubated overnight at 4 DEG C；
S4.3：Coating buffer is discarded, and microtiter plate is washed twice with cleaning solution, 200 μ l PBST are added in micropore every time,
Cleaning solution and remaining drop are removed, dries and is put in 4 DEG C of environment；
S5：Closing：Remaining protein binding site in 200 μ l Block buffers, closing coating hole is added per hole.
The present invention provides another technical scheme：A kind of detection method for thyroid cancer external diagnosis reagent case, bag
Include following steps：
S1：The preparation of antigen：By L selectin gene clonings to carrier for expression of eukaryon, and realize in mammalian cell albumen
Expression；
S2：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；
S3：Detect the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
S4：Capture antibody coating：
S4.1：Concentration is coated with to the hole of microtiter plate with carbonate/bicarbonate buffer solution for 1 μ g/ml capture antibody；
S4.2：Cover microtiter plate and be incubated overnight at 4 DEG C；
S4.3：Coating buffer is discarded, and microtiter plate is washed twice with cleaning solution, 200 μ l PBST are added in micropore every time,
Cleaning solution and remaining drop are removed, dries and is put in 4 DEG C of environment；
S5：Closing：
S5.1：Remaining protein in 200 μ l Block buffers, closing coating hole is added in each hole of microtiter plate to combine
Site；
S5.2：Cover microtiter plate and be incubated 1 hour at 37 DEG C；
S6：Sample-adding：
S6.1：100 μ l samples are added to each hole, are incubated 60 minutes at 37 DEG C；
S6.2：Sample is discarded, and washs microtiter plate three times, 200 μ l PBST are added in micropore every time；
S6.3：100 μ l concentration are added to each hole for 0.5 μ g/ml detection antibody；
S6.4：Cover microtiter plate and be incubated 1 hour at 37 DEG C；
S6.5：Microtiter plate is washed with PBST four times；
S6.6：Add 100 μ l mark secondary antibodies；
S6.7：Cover microtiter plate and be incubated 1 hour at 37 DEG C；
S6.8：Microtiter plate is washed with PBST four times；
S7：Detection：
S7.1：TMB solution is added to each hole, is incubated 15-30 minutes, isometric terminate liquid is added, then at 450nm
Read optical density；
S7.2：The data obtained by serial dilutions draw standard curve, and concentration is marked on X-axis（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.
Compared with prior art, the beneficial effects of the invention are as follows：
This is used for thyroid cancer external diagnosis reagent case and its detection method, from numerous tumor markers, filters out L selections
Element（L-SELECTIN）Constitute thyroid cancer quick diagnosis reagent kit；L selectins are main to be selected by one kind of Expressions In Lymphocytes
Plain adhesion molecule, can be combined with being expressed in the CD34 and GlyCAM-1 of HEV endothelial cells, unactivated lymphocyte is entered
Enter peripheral lymphoid tissue；L-selectin most early in being found on lymphocyte as homing receptor, is found various white thin later
All expressed on born of the same parents；The part of selectin identification is all some oligosaccharides groups, is distributed in vivo relatively broad；Be distributed in leucocyte,
Endothelial cell, hematoblastic selectin, by the identification and combination of mediated leucocytes and endothelial cell, participate in leucocyte and cross blood
Pipe enters the process that scorching district's groups are knitted and lymphocyte is gone back to the nest and followed again, turns in glomerulonephritis, multiple sclerosis, blood coagulation, tumour
Played an important role in a variety of physiology such as shifting and insulin-dependent diabetes mellitus or pathologic event；L selectins are in tumor cell surface
High expression, be a kind of molecular marked compound of new malignant tumour high specific, with sensitivity high accuracy it is strong the characteristics of, its
Sensitivity and accuracy reach more than 65%.
Brief description of the drawings
Fig. 1 is that the present invention is used for the schematic diagram of diagnosis of thyroid cancer kit；
Fig. 2 is comparative selection figure of the present invention for the detection antibody best effort concentration of diagnosis of thyroid cancer kit；
Fig. 3 is diagnosis and antidiastole comparative illustration of the present invention for diagnosis of thyroid cancer kit to clinical serum sample
Figure；
Fig. 4 is the detection of diagnosis of thyroid cancer kit sensitiveness of the present invention；
Fig. 5 is the specific detection of diagnosis of thyroid cancer kit of the present invention；
Fig. 6 is the change that the present invention detects L selectins for diagnosis of thyroid cancer kit in blood before and after treatment of thyroid carcinoma
Change comparison diagram.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
One kind is used for diagnosis of thyroid cancer kit, including capture antibody, captures antibody Block buffer, standard items, mark
Remember antibody, cleaning solution and nitrite ion etc.；Labelled antibody marks secondary antibody from HRP；It is monoclonal made from L selectins to capture antibody
Antibody；Its sensitivity and accuracy reach more than 65%；Capture antibody is that L selectins are cloned into carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, corresponding antigen is obtained after purification, the phase that antigen immune mammal is obtained
Answer monoclonal antibody；The step of preparation method of capture antibody includes is as follows：
The first step：The preparation of antigen；By the method for molecular cloning L selectin gene clonings 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 use existing conventional method to prepare, no longer burdensome herein.
Second step：Capture the preparation of antibody；By above-mentioned antigen immune mammal, obtain corresponding monoclonal antibody to catch
Obtain antibody；Mammal is mouse and rabbit, preferably mouse, and the preparation of capture antibody can also use existing conventional method system
It is standby, it is no longer burdensome herein.
Detect that antibody includes polyclonal antibody made from L selectins；It is to very by the gene cloning of L selectins to detect antibody
Nuclear expression carrier, and the expression of albumen is realized in mammalian cell, corresponding antigen is obtained after purification, and antigen immune is fed
The corresponding polyclonal antibody that newborn animal obtains；The step of preparation method of detection antibody includes is as follows：
The first step：The preparation of antigen；By the method for molecular cloning L selectin gene clonings 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；
Second step：Detect the preparation of antibody；By above-mentioned antigen immune mammal, corresponding polyclonal antibody is obtained anti-for detection
Body, mammal is mouse and rabbit, preferably rabbit.
Wherein, capture antibody can be coated in the hole of the microtiter plate of PVC materials in advance, can be simplified step, be carried
High detection efficiency.
A kind of preparation method for diagnosis of thyroid cancer kit, it comprises the following steps：
The first step：The preparation of antigen；By the method for molecular cloning by L selectin gene clonings to carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, required antigen is obtained after purification, wherein, antigen can also be used as standard items；
Second step：Capture the preparation of antibody；Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, monoclonal resists
Body as this kit capture antibody；
3rd step：Detect the preparation of antibody；Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, Anti-TNF-α
Body as this kit detection antibody；
4th step：Antibody coating is captured, is comprised the following steps that：
（1）Concentration is coated in microtiter plate for 1 μ g/ml capture antibody with carbonate/bicarbonate buffer solution (pH9.6)
Hole；
（2）Microtiter plate is covered with bond plastic product and is incubated overnight at 4 DEG C；
（3）Discard coating buffer（The capture antibody of carbonate/bicarbonate buffer solution dilution）, and wash microtitration with cleaning solution
Plate adds 200 μ l PBST in micropore twice, every time（Phosphate Tween buffer）, the gently micro drop of whipping above tank
Fixed board, 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；
Its cleaning solution is PBS（Phosphate buffer）It is middle to add a certain amount of Tween20, the mass percent concentration of the Tween20
For 0.05%；
5th step：Closing；First 200 μ l Block buffers are added in every hole of microtiter plate（For containing 1.2%BSA（Cow's serum
Albumin）PBS（Phosphate buffer））, for closing remaining protein binding site in coating hole；Then, with bonding
Plastic products cover microtiter plate and are incubated 1 hour at 37 DEG C.
A kind of detection method for diagnosis of thyroid cancer kit, it comprises the following steps：
The first step：The preparation of antigen；By the method for molecular cloning by L selectin gene clonings to carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, required antigen is obtained after purification, wherein, antigen can also be used as standard items；
Second step：Capture the preparation of antibody；Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, monoclonal resists
Body as this kit capture antibody；
3rd step：Detect the preparation of antibody；Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, wherein, it is many
Clonal antibody as this kit detection antibody；
4th step：Antibody coating is captured, specific method is as follows：
（1）Concentration is coated with microtiter plate for 1 μ g/ml capture antibody with carbonate/bicarbonate buffer solution (pH9.6)
Hole；
（2）Microtiter plate is covered with bond plastic product and is incubated overnight at 4 DEG C；
（3）Discard coating buffer（The capture antibody of carbonate/bicarbonate buffer solution dilution）, and wash microtitration with cleaning solution
Plate adds 200 μ l PBST in micropore twice, every time（Phosphate Tween buffer, can contain 0.05% Tween-20
PH7.4 phosphate buffer）, 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, Tween20 mass percent concentration is 0.05%；
5th step：Closing；First, 200 μ l Block buffers are added in each hole of microtiter plate（1.2%BSA/PBS）, envelope
Closure is by remaining protein binding site in hole；Then, cover microtiter plate with bond plastic product and be incubated 1 at 37 DEG C
Hour；
6th step：Sample-adding, specific method is as follows：
（1）100 μ l are suitably diluted（20 times of dilution）Sample be added to each hole, at 37 DEG C be incubated 60 minutes, to obtain
Accurate quantitative result, it is common practice that compare the signal of unknown sample and standard curve, each ELISA Plate must determine mark
Quasi- product（Double measure or triplicate）And blank sample, to ensure accuracy；
（2）Sample is discarded, and washs microtiter plate three times, 200 μ l PBST are added in micropore every time（Phosphate tween delays
Fliud flushing）；
（3）100 μ l concentration are added to each hole for 0.5 μ g/ml detection antibody；
（4）Microtiter plate is covered with bond plastic product and is incubated 1 hour at 37 DEG C；
（5）Microtiter plate is washed with PBST four times；
（6）100 μ l mark secondary antibodies are added, it is just diluting 10000 times in PBS before（1：10000）, it marks secondary antibody
Goat-anti rabbit can be marked for HRP；
（7）Microtiter plate is covered with bond plastic product and is incubated 1 hour at 37 DEG C；
（8）Microtiter plate is washed with PBST four times；
7th step：Detection；First, by TMB（3,3', 5,5'- tetramethyl benzidine）Solution is added to each hole, is incubated 15-30
Minute, isometric terminate liquid (2MH2SO4) is added, optical density is then read at 450nm with enzyme-linked immunosorbent assay instrument；So
Afterwards, the data obtained by serial dilutions draw standard curve, and concentration is marked on X-axis（Logarithmic scale）On, and absorbance is marked on Y-axis
（Lineal scale）On, sample concentration is drawn on this standard curve by interpolation method.
The present invention raises L selectins for diagnosis of thyroid cancer kit the mark as diagnosis early metaphase thyroid cancer
Standard, Cleaning Principle is as shown in Figure 1；L selectins decline the standard as treatment of thyroid carcinoma recruitment evaluation；It can be used for clinically
By detecting that the height of tumor marker levels in blood carries out dynamic evaluation to the therapeutic effect of thyroid cancer；It can in addition contain with
In relapse and metastasis clinically to thyroid cancer and the application of Index for diagnosis；It 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-L selectins monoclonal antibody best effort concentration of mouse：It is single when determining coating concentration according to square formation method for 1ug/mL
The OD values of clonal antibody are 1.05, so its optimal coating concentration is 1ug/mL；As shown in Fig. 2 resisting rabbit-anti L selectins optimal more
The selection of working concentration：With the increase of antibody extension rate, cases of thyroid cancer serum and normal human serum OD values to be measured have
The trend successively decreased, when antibody concentration is 1：When 200, positive control (positive control OD values subtract blank control OD values) with it is normal
The ratio between control (normal control OD values subtract blank control OD values) A450nm (abbreviation P/N values) is higher, therefore selection rabbit-anti human antibody
Best effort concentration is 1：200；The best effort concentration that serum is groped is 1：25；Confining liquid gropes best effort solubility
1.2%BSA.
Clinical serum Samples detection：
91 parts of serum specimens are have detected altogether, through definitive pathological diagnosis are thyroid cancer patients serum as positive controls using hospital（36
（Wherein early stage thyroid cancer 16, patients with late-stage thyroid carcinoma 20））, Non-thyrogenous cancer patient include thyroid adenoma, normal population
Serum is negative control group（55（Normal 30, thyroid adenoma 25））, PBST is blank control, passes through above-mentioned double antibodies sandwich
ELISA method carries out qualitative and quantitatively detects clinical serum sample；As shown in Fig. 2 with P/N values>2 be that double crush syndrome is positive
Criterion, is detected by standard of pathological diagnosis to clinical serum sample；Fig. 3 illustrates that L is selected in thyroid cancer patients serum
The level for selecting element is significantly higher than normal person（P<0.05）；Early stage thyroid cancer result detection sensitivity（SN）For 44%（7/16）,
Patients with late-stage thyroid carcinoma detection sensitivity（SN）For 65%（13/20）；Normal person's detection specificity（SP）For 80%（24/30）, first
Shape adenoma patients detection specificity（SP）For 60%（15/25）；See Fig. 4-5.
The clinical therapeutic effect to thyroid cancer carries out dynamic evaluation：
To determine that can this kit be used to carry out dynamic evaluation to the therapeutic effect of thyroid cancer, 12 parts of thyroid cancers are have collected
As a result serum before and after patient's treatment, the testing result of 12 patients shows the blood before and after thyroid cancer patients treatment referring to Fig. 6
There were significant differences for the level of L selectins in clear（P<0.05）；Effectively the level of L selectins can be greatly reduced in serum after treatment,
This kit is pointed out to be used to carry out dynamic evaluation to the therapeutic effect of thyroid cancer.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Claims (8)
1. one kind is used for thyroid cancer external diagnosis reagent case, it is characterised in that including capture antibody, the capture antibody is L
Monoclonal antibody made from selectin.
2. it is according to claim 1 a kind of for thyroid cancer external diagnosis reagent case, it is characterised in that the capture resists
The step of preparation method of body includes is as follows：
S1：The preparation of antigen；The gene clonings of L selectins to carrier for expression of eukaryon, and realize in mammalian cell egg
White expression, obtains antigen after purification；
S2：Capture the preparation of antibody；By above-mentioned antigen immune mammal, it is capture antibody to obtain corresponding monoclonal antibody.
3. it is according to claim 1 a kind of for thyroid cancer external diagnosis reagent case, it is characterised in that the kit
Also include detection antibody, the detection antibody is polyclonal antibody made from L selectins.
4. it is according to claim 3 a kind of for thyroid cancer external diagnosis reagent case, it is characterised in that the detection resists
The step of preparation method of body includes is as follows：
S1：The preparation of antigen；The gene clonings of L selectins to carrier for expression of eukaryon, and realize in mammalian cell egg
White expression, obtains antigen after purification；
S2：Detect the preparation of antibody；By above-mentioned antigen immune mammal, it is detection antibody to obtain corresponding polyclonal antibody.
5. it is according to claim 1 or 2 a kind of for thyroid cancer external diagnosis reagent case, it is characterised in that described to catch
Antibody is obtained to be coated in the hole of microtiter plate in advance.
6. a kind of preparation method according to claim 5 for thyroid cancer external diagnosis reagent case, it is characterised in that
Comprise the following steps：
S1：The preparation of antigen：By L selectin gene clonings to carrier for expression of eukaryon, and realize in mammalian cell albumen
Expression, required antigen is obtained after purification；
S2：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；
S3：Detect the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
S4：Capture antibody coating：
S4.1：Concentration is coated with to the hole of microtiter plate with carbonate/bicarbonate buffer solution for 1 μ g/ml capture antibody；
S4.2：Cover microtiter plate and be incubated overnight at 4 DEG C；
S4.3：Coating buffer is discarded, and microtiter plate is washed twice with cleaning solution, 200 μ lPBST is added in micropore every time, removes
Cleaning solution and remaining drop are removed, dries and is put in 4 DEG C of environment；
S5：Closing：Remaining protein binding site in 200 μ l Block buffers, closing coating hole is added per hole.
7. a kind of detection method according to claim 5 for thyroid cancer external diagnosis reagent case, it is characterised in that
Comprise the following steps：
S1：The preparation of antigen：By L selectin gene clonings to carrier for expression of eukaryon, and realize in mammalian cell albumen
Expression；
S2：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；
S3：Detect the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the Anti-TNF-α
Body as this kit detection antibody；
S4：Capture antibody coating：
S4.1：Concentration is coated with to the hole of microtiter plate with carbonate/bicarbonate buffer solution for 1 μ g/ml capture antibody；
S4.2：Cover microtiter plate and be incubated overnight at 4 DEG C；
S4.3：Coating buffer is discarded, and microtiter plate is washed twice with cleaning solution, 200 μ l PBST are added in micropore every time,
Cleaning solution and remaining drop are removed, dries and is put in 4 DEG C of environment；
S5：Closing：
S5.1：Remaining protein in 200 μ l Block buffers, closing coating hole is added in each hole of microtiter plate to combine
Site；
S5.2：Cover microtiter plate and be incubated 1 hour at 37 DEG C；
S6：Sample-adding：
S6.1：100 μ l samples are added to each hole, are incubated 60 minutes at 37 DEG C；
S6.2：Sample is discarded, and washs microtiter plate three times, 200 μ l PBST are added in micropore every time；
S6.3：100 μ l concentration are added to each hole for 0.5 μ g/ml detection antibody；
S6.4：Cover microtiter plate and be incubated 1 hour at 37 DEG C；
S6.5：Microtiter plate is washed with PBST four times；
S6.6：Add 100 μ l mark secondary antibodies；
S6.7：Cover microtiter plate and be incubated 1 hour at 37 DEG C；
S6.8：Microtiter plate is washed with PBST four times；
S7：Detection：
S7.1：TMB solution is added to each hole, is incubated 15-30 minutes, isometric terminate liquid is added, then at 450nm
Read optical density；
S7.2：The data obtained by serial dilutions draw standard curve, and concentration is marked on X-axis（Logarithmic scale）On, and absorbance
It is marked on Y-axis（Lineal scale）On.
8. sample concentration is drawn on this standard curve by interpolation method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261494A (en) * | 2018-11-01 | 2019-09-20 | 青岛大学附属医院 | A kind of method and its detection kit for identifying thyroid malignancy biomarker |
| CN113075403A (en) * | 2021-03-18 | 2021-07-06 | 长治医学院 | Molecular marker and kit for gastric cancer diagnosis |
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| CN110261494A (en) * | 2018-11-01 | 2019-09-20 | 青岛大学附属医院 | A kind of method and its detection kit for identifying thyroid malignancy biomarker |
| CN113075403A (en) * | 2021-03-18 | 2021-07-06 | 长治医学院 | Molecular marker and kit for gastric cancer diagnosis |
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