CN109959787A - A kind of rheumatoid arthritis auxiliary diagnostic box and detection method - Google Patents
A kind of rheumatoid arthritis auxiliary diagnostic box and detection method Download PDFInfo
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- CN109959787A CN109959787A CN201910311968.9A CN201910311968A CN109959787A CN 109959787 A CN109959787 A CN 109959787A CN 201910311968 A CN201910311968 A CN 201910311968A CN 109959787 A CN109959787 A CN 109959787A
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Abstract
The present invention provides a kind of rheumatoid arthritis auxiliary diagnostic box and detection methods, relationship of the technical solution probed between RA patient gene expression and pathological characters first, research is found, the mRNA relative expression levels of ATG5, ATG8, Beclin1 gene during different activities in RA peripheral blood mononuclear cells are in different degrees of differential expression, show the expression variation of the related autophagygene such as peripheral blood mononuclear cells Beclin1 and Atg8 to the prognosis and activity of RA with certain correlation and directive property.Based on the above positive discovery, the present invention develops a kind of RA auxiliary diagnostic box based on autophagy genetic test, the kit is using the serum of people, blood plasma or other body fluid as biological material, wherein ATG5, ATG8, Beclin1 content is quantitative determined using Competitive assays ELISA method, it can realize on this basis the auxiliary examination of RA, and evaluate the pathological characters and development process of RA.
Description
Technical field
The present invention relates to kit for diagnosing diseases technical fields, further to the disease auxiliary diagnosis based on genetic test
Technology, and in particular to a kind of rheumatoid arthritis auxiliary diagnostic box and detection method.
Background technique
Rheumatoid arthritis (RA) is a kind of unknown systemic disease chronic, based on inflammatory synovitis of cause of disease.RA
Arthritic pathology mainly has synovial membrane stave cell hyperplasia, the new life of interstitial massive inflammatory cells infiltrated and capilary, blood vessel
The formation of screen and the destruction of cartilage and bone tissue etc..Symptom shows as hand, the multi-joint of sufficient Minor articulus, symmetry, invasion more
Arthritis is often accompanied by organ outside joint and is involved the serum rheumatoid factor (SRF) positive, and joint deformity and function can be caused to lose
It loses.The morbidity of RA may be related with heredity, infection, sex hormone etc., and diagnostic criteria includes a series of feature: 1) morning stiffness >=30
Minute；2) it is greater than the arthritis of 3 hinge areas；3) cheirarthritis；4) rheumatoid factor (RF) is positive；5) antiCCP antibody is positive.
14 hinge areas include: bilateral elbow, wrist, metacarpophalangeal, proximal interphalangeal, knee, ankle and articulationes metatarsophalangeae；Features above meets 3 or more can
It is diagnosed as RA.
In recent years, with the development of molecular biology and Medical Imaging, other detections are according to the diagnosis for being introduced into RA
In, for example, the detection of RA patient's autoantibody, is that RA is different from other inflammatory arthritis, as psoriatic arthritis, reactivity are closed
Scorching one of the mark with osteoarthritis of section, current clinically used autoantibody includes rheumatoid factor (RF-IgM), antioxidant cyclic
Citrulling (CCP) antibody, rheumatoid factor IgG and IgA, APF ELISA, anti-keratin antibody and antinuclear antibodies, anti-ENA
Antibody etc., meanwhile, it further include anti-SARS-coro navirus antibody, anti-glucose-6-phosphate isomerase (GPI) antibody, anti-P68 antibody etc..In addition,
Pass through the articular surface cystis degeneration after the visible soft tissue swelling of X-ray, osteoporosis and disease progression, aggressive osteoclasia, joint
Face mould paste, joint space is narrow, joint fusion and dislocation.MRI checks that swivel of hand and carpal MRI inspection can prompt early stage
Synovitis lesion, helpful to the early stage destruction of joint of discovery rheumatoid arthritis patients, ultrasonic joint ultrasound is easy
Woundless testing has identification meaning for synovitis, hydrops articuli and destruction of joint.
RA lacks direct, effective prevention and treatment strategy at present, lacks hypersensitivity, specific diagnosis index.Face
Though a bed upper joint synovial tissue can leave and take by performing the operation or puncturing, invasive due to materials, make its application by a fixed limit
System.Some researches show that in the pathogenic process of RA, along with the change of gene expression, in this case, if can definitely obtain
The relationship between expression conditions and RA pathological characters is obtained, then is expected to become the new foundation of RA diagnosis, is based on expression conditions
RA diagnostic method, it is relatively reliable in terms of stability and accuracy, however, not slapped still at present due to the missing of correlative study
The gene classification that there is definite directive property to RA is held, does not also know the relationship between its expression and RA pathological characters.
Summary of the invention
The present invention is directed to be directed to the technological deficiency of the prior art, a kind of rheumatoid arthritis auxiliary diagnostic box is provided
And detection method, to solve to lack a kind of technology based on genetic test means, RA auxiliary diagnostic box in the prior art
Problem.
Another technical problem to be solved by the present invention is that between the pathogenesis and patient's autophagy expression conditions of RA
Relationship is still unobvious.
To realize the above technical purpose, the invention adopts the following technical scheme:
A kind of rheumatoid arthritis auxiliary diagnostic box, comprising: be coated with respectively ATG5 antigen, ATG8 antigen,
The orifice plate of Beclin1 antigen；Positive quality control: ATG5 positive serum, ATG8 positive serum, Beclin1 positive serum；Negative matter
Control: Healthy People negative serum；Working solution R: being marked with the anti-human ATG5 antibody of HRP, is marked with the anti-human ATG8 antibody of HRP, mark
Note has the anti-human Beclin1 antibody of HRP；Dilution: deionized water；Tmb substrate；Terminate liquid: the H of 1mol/L2SO4Solution；Concentration
Clean cleaning solution: PBS-Tween buffer.
Preferably, the orifice plate is 96 orifice plates.
Preferably, the ATG5 positive serum, ATG8 positive serum, Beclin1 positive serum are respectively 50 μ L.
Preferably, the Healthy People negative serum is 50 μ L.
Preferably, described mark the anti-human ATG5 antibody having, it is marked with the anti-human ATG8 antibody of HRP, is marked with
The anti-human Beclin1 antibody of HRP is respectively 150 μ L.
Preferably, the volume of the deionized water is 100 times of working solution R total volume.
Preferably, the tmb substrate has 15 parts, every part is 100 μ L.
Preferably, the terminate liquid has 15 parts, every part is 50 μ L.
Preferably, the concentration cleaning cleaning solution has 20 parts, every part is 250 μ L.
A method of rheumatoid arthritis is detected using mentioned reagent box, comprising the following steps:
1) it is loaded: the hole on orifice plate being divided into gauge orifice, sample to be tested hole, blank well, wherein the gauge orifice has 3
Hole is separately added into 3 kinds of positive quality controls, each 50 μ L of every kind of positive quality control into 3 gauge orifices, and blank well adds 50 μ L feminine gender Quality Controls,
50 μ L of sample to be tested is added in sample to be tested hole；Every hole machined makees liquid R150 μ L, and vibration mixes, and orifice plate adds overlay film, in 37 DEG C of temperature
It educates 1 hour；
2) liquid in hole is discarded, every hole is cleaned cleaning solution with the concentration of 250 μ L and washed, impregnates 1 minute, then in cleaning hole
All liq；It repeats board-washing 3 times, after last time is washed, pours out remaining concentration cleaning cleaning solution, ELISA Plate is tipped upside down on into suction
On water paper, the liquid remained in hole is all blotted；
3) every hole adds 100 μ L of tmb substrate, and ELISA Plate adds overlay film, is protected from light 10~30min of colour developing in 37 DEG C；
4) every hole adds 50 μ L of terminate liquid, terminates reaction.
This kit measures the content of ATG5, ATG8, Beclin1 in sample using Competitive assays enzyme-linked immunosorbent assay.
Monoclonal antibody is coated with microwell plate, solid phase carrier is made, the anti-of biotin labeling is added simultaneously into the micropore of coated antibody
Former and determined antigen (standard items or sample), determined antigen and biotin labeling antigen are at war with combination to specific antibody.
Remove unbonded object through washing after incubation, the Avidin of HRP label is then added, by incubating and substrate thoroughly being added after washing
TMB colour developing.TMB converts au bleu under the catalysis of peroxidase, and is converted to final yellow under the action of an acid.It is to be measured
Sample concentration is higher, and the combination of labelled antigen and antibody is more suppressed, and develops the color more shallow.The depth and enzyme amount of colour developing are in positive
It closes, and it is negatively correlated with test substance content in sample.It is measured absorbance (OD value), is calculated under 450nm wavelength with microplate reader
Sample concentration.
The present invention provides a kind of rheumatoid arthritis auxiliary diagnostic box and detection methods, and the technical solution is with head
The relationship between RA patient gene expression and pathological characters is first probed into, the study found that during different activities outside RA patient
The mRNA relative expression levels of ATG5, ATG8, Beclin1 gene in all blood mononuclear cells are poor in different degrees of expression
It is different, show prognosis and work of the expression variation of the related autophagygene such as peripheral blood mononuclear cells Beclin1 and Atg8 to RA
It is dynamic that there is certain correlation and directive property.Based on the above positive discovery, the present invention develops a kind of based on autophagy gene
The RA auxiliary diagnostic box of detection, the kit is using the serum of people, blood plasma or other body fluid as biological material, using competition
Inhibit ELISA method quantitative determination wherein ATG5, ATG8, Beclin1 content, can realize the auxiliary examination of RA on this basis,
And evaluate the pathological characters and development process of RA.The present invention is simple and easy to do, and detection process is smaller to human body wound, and Appreciation gist is more
It is reliable and stable.
Specific embodiment
Below by specific embodiments of the present invention will be described in detail.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.Approximation used in following embodiment
Language can be used for quantitative expression, show to allow quantity to have certain variation in the case where not changing basic function.It is fixed except having
Adopted outer, technical and scientific term used in following embodiment has the phase being commonly understood by with those skilled in the art of the invention
Same meaning.
Embodiment 1
1, materials and methods
1.1 research object
Case group: selection moves in attached hospital, the Zhangzhou City blood wind of Medical University Of Fujian from January, 2014 in June, 2015
The patient of wet internal medicine 60, female 40, male 20, patient's average age is 46.75 ± 13.52 years old, by rheumatoid arthritis
Diagnostic criteria is American Society of Rheumatism (ACR) and European antirheumatic alliance (RULAR) combines newest RA diagnosis in release 2009
Standard.By RA disease activity index (DAS28) be divided into low active stage (DAS28 < 3.2), the moderately active phase (DAS28 3.2~
5.1) with high activity phase (DAS28 > 5.1), wherein low active stage L group 22, moderately active phase M group 20, high activity
Phase H group 18.Control group: for same period outpatient service physical examination of healthy population, 25, female 18, male 7, average age is 45.35 ±
14.02 years old.
1.2 instrument reagent
Real-time PCR instrument, 7500 models, ABI company；Supercentrifuge, model 5424R, Thermo company；It is super
Pure water system, model Direct-Q 5, Millipore company.Main primer sequence: ATG5 (F:5 '-
AGAAGCTGTTTCGTCCTGTG-3’,R:5’-AGGTGTTTCCAACATTGGCT-3’ATG8:(F:5’-
ATAGAATAGAAAGTGCATCTCC-3',R:5'-TCTATTTATTGGTTCAGTCG-3')；Beclin1:(F:5'-
CAAGATCCTGGA-3',R:5'-CCGTGTCA-3')；GAPDH:(F:5'-AATGAAGGGGTCATTGATGG-3',R:5'-
AAGGTGAAGGTCGGAGTCAA-3 '), it is synthesized by Shanghai Sangon Biotech Company.
1.3 method
2ml venous blood is acquired, is placed in through being used for control group and experimental group in the processed vacuum blood collection tube of anti-coagulants
The separation of PBMCS.The extraction of total serum IgE is referring to Beijing Quanshijin Biotechnology Co., Ltd (TransGen) RNA extracts kit
The operation of TransZol Up Plus RNA Kit (Cat.No.:ER501-01) specification.Had using Beijing Quan Shijin biotechnology
Limit company (TransGen) PCR kit for fluorescence quantitative 2 × TransStarTM Top Green qPCR SuperMix
(Cat.No.:AQ131-02) mRNA of Real-time qPCR detection ATG5, ATG8, Beclin1 is carried out, after reaction, with
GAPDH is reference gene, and 2- △ △ Ct method analyzes gene expression dose, the differential expression result of more each gene.
1.4 statistical analysis
Data processing and clinical analysis are carried out using statistics SPSS software, measurement data is indicated with ± s, is used between group
Kruskal Wallis H is examined, and is compared two-by-two after having statistical difference with Nemenyi inspection, the correlation analysis between index
Using linear regression；Inspection level α=0.05 is that difference is statistically significant, and inquires into they and disease not with p ﹤ 0.05
With contacting between activity group.
2, result
The mRNA expression situation of the ATG5 of 2.1 each group RA patient PBMC: in low activity group (L), middle activity group (M)
The mRNA of ATG5 is above normal healthy controls, and P is respectively 0.035,0.013, respectively less than 0.05, and difference is statistically significant.Height is living
Dynamic group (H) and control group difference are little, p=0.078.
The mRNA expression situation of the ATG8 of 2.2 each group RA patient PBMC: low activity group (L), middle activity group (M), height
The mRNA of ATG8 is above normal healthy controls 0.49 ± 0.09 in activity group (H), and P is respectively 0.001,0.002,0.001 to be respectively less than
0.05, difference is statistically significant.Middle activity group (M) is lower than low activity group (L), high activity group (H), P is respectively 0.044,
0.043。
The mRNA expression situation of the Beclin1 of 2.3 each group RA patient PBMC
Low activity group (L), middle activity group (M), the mRNA of Beclin1 is above normal healthy controls 0.76 in high activity group (H)
± 0.10, P are respectively 0.012,0.013,0.015 respectively less than 0.05, and difference is statistically significant.Specific data are shown in Table 1.
MRNA relative expression's situation of ATG5, ATG8, Beclin1 in 1 RA patient PBMC of table
Note: * P < 0.05 compared with healthy control group
3, it discusses
Rheumatoid arthritis (RA) is a kind of with the chronic inflammation hyperplasia of synovial membrane and with the systemic of inflammatory cell infiltration
Autoimmune disease.Its reason may be the unbalance hair for leading to autoimmunity disease and auto-inflammatory of autophagy mechanism in body
It is raw, the understanding of autophagy molecular mechanism is started from found in yeast autophagy related gene (autophagy-related gene,
Atg).Currently, it has been found that more than 30 kinds of Atg in eucaryote.In human body, the multiple protein compound of autophagygene coding exists
It plays an important role in the formation of autophagosome.The autophagygenes such as ATG5, ATG8, Beclin1 take part in a series of autophagy and regulated and controled
Journey, Atg5 play important regulating and controlling effect in autophagy process, mainly by forming Atgl2- in conjunction with conservative protein Atgl2
Atg5 compound protein system promotes the extension of autophagosome film.Atg3 plays E2 enzyme catalysis, Atg8 and phosphorus in entire autophagy process
Acyl inositol (IP3) combines, and takes part in the growth course of autophagosome formation, autophagic vacuole.Autophagy has in the dead path of RASFs
There is double action.ATG8 gene is the homologue of LC3, and LC3 is the specific marker proteins of autophagosome, participates in the shape of autophagosome
At.The extension of autophagy occurs for these autophagygenes starting, autophagosome, maturation play a significant role, any one ring
The mistake of section can all lead to the exception of autophagy.Circumferential edge research is also shown, and ATG5, ATG8 exist in peripheral blood mononuclear cells
It is in different variations, the mRNA expression situation of the ATG5 of each group RA patient PBMC: L group, M in different RA patient activity's groups
The mRNA of ATG5 is respectively 3.38 ± 0.45,4.46 ± 1.28 in group, and being above normal healthy controls 2.06 ± 0.25, (P is respectively
0.035,0.013, respectively less than 0.05, have statistical difference).H group is 2.36 ± 0.96, p=s little with control group difference
0.078.There is research to illustrate that the missing of ATG5 will affect the removing of dead cell and the presentation of antigen, autoimmune response can be caused
And inflammation.And the mRNA expression situation of the ATG8 of each group RA patient PBMC: L group, M group, the mRNA of ATG8 is respectively in H group
1.98 ± 0.28,1.58 ± 0.81,2.07 ± 0.32, be above normal healthy controls 0.49 ± 0.09, P is respectively 0.001,0.002,
0.001 is respectively less than 0.05, there is statistical difference.It can be seen that ATG5, ATG8, Beclin1 gene have both participated in the autophagy row of RA patient
For the mRNA and albumen that experimental result shows the ATG8 in different activity group RA peripheral blood mononuclear cells are in different degrees of
Differential expression, show ATG5, ATG8 gene in peripheral blood mononuclear cells may take part in RA autoimmunity adjust, with
The progress of RA disease, ATG5, ATG8 prompt autophagy that may participate in the generation of RA in trend is gradually risen.
Beclinl gene is RA patient peripheral's blood lymphocyte autophagy Research of predicting markers, studies have found that in the osteoclastic of RA
In cell, autophagy is activated, and Beclin1 and Atg8 expression are increased.Beclin1 in conjunction with Bcl-2 by inhibiting PI3KC3 compound
Object activity, JNK1 and DAPK lead to Beclin1 phosphorylation, and Bcl-2 and Beclin1 is made to dissociate and induce autophagy.Mammal energy
Amount can also make ULK1 phosphorus by activated adenyl cyclase (AMPactivated protein kinase, AMPK) when lacking
It is acidified and activates autophagy.Some researches show that in the osteoclast of RA, autophagy activation, Beclin1 expression is increased, overexpression
Beclin1 can induce the generative capacity and reabsorption function of osteoclast.Can by targeted drug adjust gene level to
Inhibit autophagy, hinder the further differentiation of osteoclast, thus to TNF-α induction bone erosion, mucin loss and cartilage
The death of cell has protective effect.The expression quantity of Beclin1 and the osteoclastic situation of RA patient are closely related, it is possible thereby to as broken
The inspection target of bone cell differentiation.This experimental studies results shows the mRNA expression of the Beclin1 of each group RA patient PBMC
Situation: L group, M group, the mRNA of Beclin1 is respectively 2.08 ± 0.66,2.19 ± 0.30,1.89 ± 0.55 in H group, is above
Normal healthy controls 0.76 ± 0.10 (P is respectively 0.012,0.013,0.015, respectively less than 0.05, there is statistical difference).Autophagy is more
Number situations under to body have protective effect, especially starvation, anoxic etc. stress initial stage, since the shortage of amino acid intracellular can swash
MTOR starts autophagy in living cells, so that mismatching proteins matter or damaging cells device is degraded, Cycle of nutrients utilizes, to maintain cell
Balance.However, overstimulation and prolonged stress situation, can make cytoplasmic components excessive degradation lead to the death of cell, cell
Autophagy may have correlation with the occurrence and development of RA.
The exception that autophagy is adjusted will lead to the generations of many diseases, and studying more is autophagy in tumor development
Effect.More and more evidences show that autophagy is played an important role in congenital immunity and adaptive immunity now.Detection at present
The various autophagygene albumen of synovial cell, which mainly pass through operation or puncture acquisition joint fluid extraction synovial cell, to be detected, this
Belong to invasive, time-consuming while also bringing pain to patient, and be easily complicated by infection sometimes etc., this research is mentioned by acquiring peripheral blood
The total serum IgE in mononuclearcell is taken, the differential expression of autophagy related gene ATG5, ATG8, Beclin1 are detected, for peripheral blood
Middle correlation autophagygene is used for RA auxiliary examination, and this simple and easy to do, noninvasive inspection project has certain research significance.
Based on the above result of study, the present invention provides a kind of rheumatoid arthritis auxiliary diagnostic box, the reagents
Box using ATG5, ATG8, Beclin1 as test object, the kit include: be coated with respectively ATG5 antigen, ATG8 antigen,
The orifice plate of Beclin1 antigen；Positive quality control: ATG5 positive serum, ATG8 positive serum, Beclin1 positive serum；Negative matter
Control: Healthy People negative serum；Working solution R: being marked with the anti-human ATG5 antibody of HRP, is marked with the anti-human ATG8 antibody of HRP, mark
Note has the anti-human Beclin1 antibody of HRP；Dilution: deionized water；Tmb substrate；Terminate liquid: the H of 1mol/L2SO4Solution；Concentration
Clean cleaning solution: PBS-Tween buffer.
Embodiment 2
A kind of rheumatoid arthritis auxiliary diagnostic box, the kit is for detecting human serum, blood plasma or other bodies
The content of ATG5, ATG8, Beclin1 in liquid；The ingredient of the kit is as shown in the following Table 2:
2 Kit components list of table
The application method of the kit is as follows:
1) it is loaded: setting gauge orifice, sample to be tested hole, blank well respectively.
I sets 3 hole of gauge orifice, sequentially adds the positive quality control of 50 μ L various concentrations；
II blank well adds 50 μ L feminine gender Quality Controls；
III adds 50 μ L of sample to be tested to gaging hole, and every hole machined makees 150 μ L of liquid immediately after, gently vibrates, and mixes, pays attention to not
There is bubble, ELISA Plate adds overlay film, 37 DEG C, incubates 1 hour.
2) liquid in hole is discarded, every hole is washed with the cleaning solution of 250 μ L, impregnates 1 minute, ELISA Plate is patted on blotting paper
To remove all liq in hole.
It repeats board-washing 3 times.After last time is washed, remaining washing buffer is poured out, ELISA Plate is tipped upside down on into blotting paper
On, the liquid remained in hole is all blotted.
3) every hole adds 100 μ L of substrate solution, and ELISA Plate adds overlay film, and 37 DEG C are protected from light colour developing (reaction time, control was in 10-20
Minute, it does not exceed 30 minutes.It when gauge orifice has apparent gradient blue, can terminate).
4) every hole adds 50 μ L of stop bath, terminates reaction, and blue is vertical at this time turns yellow.The addition sequence of terminate liquid should be as far as possible
It is consistent with the addition sequence of substrate solution.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It is not intended to limit the invention.All any modifications, equivalent replacements, and improvements etc. done in application range of the invention, should all
It is included within protection scope of the present invention.
Claims (10)
1. a kind of rheumatoid arthritis auxiliary diagnostic box, characterized by comprising:
It is coated with the orifice plate of ATG5 antigen, ATG8 antigen, Beclin1 antigen respectively；
Positive quality control: ATG5 positive serum, ATG8 positive serum, Beclin1 positive serum；
Negative Quality Control: Healthy People negative serum；
Working solution R: being marked with the anti-human ATG5 antibody of HRP, is marked with the anti-human ATG8 antibody of HRP, is marked with the anti-human of HRP
Beclin1 antibody；
Dilution: deionized water；
Tmb substrate；
Terminate liquid: the H of 1mol/L2SO4Solution；
Concentration cleaning cleaning solution: PBS-Tween buffer.
2. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that the orifice plate
For 96 orifice plates.
3. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that the ATG5
Positive serum, ATG8 positive serum, Beclin1 positive serum are respectively 50 μ L.
4. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that the health
People's negative serum is 50 μ L.
5. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that the label
There is the anti-human ATG5 antibody of HRP, be marked with the anti-human ATG8 antibody of HRP, the anti-human Beclin1 antibody for being marked with HRP is respectively 150
μL。
6. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that it is described go from
The volume of sub- water is 100 times of working solution R total volume.
7. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that the bottom TMB
Object has 15 parts, and every part is 100 μ L.
8. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that the termination
Liquid has 15 parts, and every part is 50 μ L.
9. a kind of rheumatoid arthritis auxiliary diagnostic box according to claim 1, it is characterised in that the concentration
Cleaning cleaning solution has 20 parts, and every part is 250 μ L.
10. a kind of method using kit described in claim 1 detection rheumatoid arthritis, it is characterised in that including following
Step:
1) it is loaded: the hole on orifice plate being divided into gauge orifice, sample to be tested hole, blank well, wherein the gauge orifice has 3 holes, to 3
3 kinds of positive quality controls, each 50 μ L of every kind of positive quality control are separately added into a gauge orifice, blank well adds 50 μ L feminine gender Quality Controls, to test sample
50 μ L of sample to be tested is added in sample wells；Every hole machined makees liquid R150 μ L, and vibration mixes, and orifice plate adds overlay film, and it is small to incubate 1 in 37 DEG C
When；
2) liquid in hole is discarded, every hole is cleaned cleaning solution with the concentration of 250 μ L and washed, and impregnates 1 minute, then owns in cleaning hole
Liquid；It repeats board-washing 3 times, after last time is washed, pours out remaining concentration cleaning cleaning solution, ELISA Plate is tipped upside down on into blotting paper
On, the liquid remained in hole is all blotted；
3) every hole adds 100 μ L of tmb substrate, and ELISA Plate adds overlay film, is protected from light 10~30min of colour developing in 37 DEG C；
4) every hole adds 50 μ L of terminate liquid, terminates reaction.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111007262A (en) * | 2019-12-25 | 2020-04-14 | 漳州卫生职业学院 | Kit for auxiliary diagnosis of RA and detection method |
| WO2021120827A1 (en) * | 2019-12-20 | 2021-06-24 | 江苏品升医学科技有限公司 | Application of serum protein hfrep1 in diagnosis of rheumatoid arthritis disease state |
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