CN106501523A - Stromatin enzyme 3 determines test kit - Google Patents
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Abstract
The present invention provides a kind of stromatin enzyme 3 and determines test kit, it is related to technical field of medical detection, using double antibody sandwich method, the micropore endoperidium HBP monoclonal antibodies of enzyme mark coating plate, during primary incubation, combined with coated HBP monoclonal antibodies in solid phase as antigenic substance by the use of the HBP in Sample dilution diluting plasma, formed insolubilized antibody antigenic compound；Fully wash to remove unconjugated composition using lavation buffer solution, add enzyme conjugates incubation, enzyme conjugates to be combined with insolubilized antibody antigenic compound；Further washing, removing unconjugated composition, add substrate incubation, substrate is become color products by enzyme catalysiss, terminate liquid added with terminating reaction, detect each hole optical density value by microplate reader；The size of optical density value is with HBP concentration in blood plasma into positive correlation.The present invention can Quantitative in vitro determine human plasma in MMP 3 content, adjuvant clinical diagnosis, treatment, be a kind of new immunoassay kit.
Description
Technical field
A kind of the present invention relates to technical field of medical detection, more particularly to stromatin enzyme 3 measure test kit.
Background technology
The degraded of extracellular matrix and the generation of numerous disease development have important relationship, are affecting many of substrate degradation
In factor, matrix metalloproteinase（MMPs）Play an important role, be research emphasis in recent years.MMPs is one group of Zn2+Rely on
Protease family, it has now been found that just have kind more than 20, four big class can be divided into according to substrate difference：Collagenase, gelatinase,
Stromlysin and model matrix metalloproteinase.The major function of MMPs is degraded and moulds extracellular matrix again, maintains carefully
The dynamic equilibrium of extracellular matrix, participates in the multiple pathology of human body and physiological process.Many research shows, MMPs participates in tissue and occur,
The process such as injury repairing, tumor-infiltrated and transfer, inflammatory reaction, signal transduction.Wherein, MMP3（MMP-3）Can
To activate other MMPs, play a crucial role in MMPs activation processs, be one of important object of study, serum MMP-3 levels
The order of severity of multiple diseases can be reflected, have important clinical meaning.
MMPs is the protease family that one group of zinc ion is relied on, including collagenase, gelatinase, stromelysins and membranous type base
4 type of matter metalloproteases.More than 20 kinds of MMPs is found at present at least, they have similar 26S Proteasome Structure and Function, common participation
A series of physiology, the pathological processes such as tissue generation, injury repairing, inflammatory reaction, signal transduction.MMP-3 is also known as stromelysins
1st, front gelatinase, is the important member of MMP families.In chromosome 11q22.3 areas, length is about for the MMP-3 gene mapping of the mankind
1.8kb.MMP-3 is synthesized with precursor forms proMMP-3 in the cell, contains 477 aminoacid, molecular weight about 57KDa altogether.
The structure of MMP-3 albumen can be divided into amino terminal propetide area, 3 part of amino-terminal catalytic area and carboxy-terminal domains.Ammonia
The front portion of Ji Moduanqiantaiqu has a signal sequence being made up of 17 amino acid residues, can guide new and into
MMP-3 is moved to cell membrane.Amino-terminal catalytic area is connected with aminoterminal propetide area by a hinge region, and its structure is included
There is a zinc ion motif, be the active site of whole protease.Carboxy-terminal domains aminoacid sequence is combined with haemachrome
Albumen is much like, is the position that MMP-3 is combined with substrate.
Research shows that various kinds of cell can express MMP-3 after by appropriate stimulation, and such as fibroblast, cartilage is thin
Born of the same parents, endotheliocyte, macrophage, vascular smooth muscle cell, osteoblast, keratinocyte etc..Body is in 3 aspects pair
MMP-3 activity is strictly regulated and controled.MMP-3 expression is in very low level, environmental stimuli, the change of interior environment etc. under normal circumstances
The change that MMP-3 can be caused to express, these regulations occur in transcriptional level, are first aspects that body adjusts MMP-3.Induction
MMP-3 secretion cytokine and somatomedin spectrum widely as INF- β, INF- γ, IL-1 α, IL-1 β, IL-6, IL-8,
IL-10, IL-17, IL-18, transforminggrowthfactor-α, TNF-α etc., wherein TNF-α are that a very important MMP-3 induction is scorching
Sex factor, not only directly induced fibroblast secretes MMP-3 for it, but also produces more IL-1, IL- by stimulating IL-6
1 stimulates the more MMP-3 of generation again.The factor of MMP-3 expression is suppressed to include transforming growth factor, glucocorticoid etc..Second
Individual aspect is post translational modification, new and into MMP-3 not active, in its catalytic domain, Zn2+ is not matched somebody with somebody for one with amino terminal
To cysteine with coordinate bond combine, it is impossible to expose its catalysis activity, only this coordinate bond by plasminogen or other change
When learning material damage, MMP-3 could really be converted into activity form.The tissue inhibitor of metalloproteinase existed in tissue
（TIMPs）It is last aspect that body adjusts MMP-3 activity, it has now been found that TIMPs in 4, wherein TIMP-1 can be with
The catalysis region of MMP-3 combines to form complex, so as to suppress the activity of MMP-3.MMP3 at least 3 kinds forms in vivo, bag
Include MMP-3 precursors, activity MMP-3 and MMP-3/TIMPs complex.
The topmost functions of MMP-3 are to adjust the reconstruction of extracellular matrix, the III that can degrade, IV, V, IX, X-type collagen, fibre
The most extracellular matrixs such as dimension connection albumen, laminin，LN, elastin laminin, Dan Baiduotang proteoglycan PG, and various by hydrolyzing
Extracellular matrix components activate propagation and the migration that the various ways such as other MMPs adjust cell.That most study is MMP- at present
3 play a significant role in tumor, atherosclerosiss and arthritis.Tumor, especially malignant tumor always endanger the mankind
Health major disease, malignant tumor reduces intercellular coherent condition by MMP-3 degradation of cell epimatrixs so that
Tumor cell is easy to invasion and attack migration, causes the transfer of tumor.Research confirms, after suppressing tumor cell secretion MMP-3, cell invasion
Substantially weaken with transfer ability, illustrate that MMP-3 is relevant with tumor invasion and metastasis；Zucker and Vacirca are in colon cancer tissue
It was found that the up-regulated of MMP-3 promotes the transfer of tumor cell and points out prognosis malas；Zhang Junyong etc. has found MMP-3 in gastric cancer group
Expression in knitting is significantly higher than normal gastric mucosa；The research such as Huang Gangding shows that metastases in local lymph node person's MMP-3 expressions are bright
Aobvious is higher than the non-transferrer of lymph node；Detection MMP-3 serological levels contribute to judging the grade malignancy of tumor and prognosis, and point out
MMP-3 expression is suppressed to carry out antineoplaston.MMP-3 destroys chondrocyte by its proteolytic activity, participates in rheumatic and closes
Section inflammation and the generation of osteoarthritis, the index that clinically commonly uses at present have erythrocyte sedimentation rate（ESR）, rheumatoid factor（RF）, C reaction
Albumen（CRP）, anti-cyclic citrullinated peptide（CCP）, and the destructiveness of MMP-3 reflection articular cartilage, serum MMP-3 levels and ESR,
RF, CRP are proportionate, and, can carry if there is high-caliber MMP-3 in serum as the efficiency index of reflection state of an illness activity
Show there has been the destruction of articular cartilage, should treat in time.Atherosclerosiss（AS）It is the pathologic basis of cardiovascular and cerebrovascular disease, greatly
Quantity research finds that MMPs participates in a series of pathophysiological processes such as blood vessel wall reconstruct, plaque rupture, thrombosiss.AS extensively involves
Big-and-middle tremulous pulse, vessel walls and different degrees of narrow of lumen of vessels is caused, corresponding organ may occur in which ischemic lesions.The disease of AS
Reason process is broadly divided into 4 stages：Fat stricture of vagina phase, fibrous plaque phase, atheromatous plaque phase and compound pathological changes phase.The generation of AS develops
A kind of complex process for being related to multifactor, too many levels, is by various kinds of cell such as blood tubule, leukocyte, smooth muscle cell and multiple
The inflammatory reaction process that the factor is participated in jointly.MMP-3 is the important member that MMPs families are expressed in AS specklees, is widely present in
AS speckle lesions, can the most of compositions of selective degradation extracellular matrix, such as collagen protein, Dan Baiduotang proteoglycan PG, elastin laminin, fibre
Even the Multiple components such as albumen, gelatin, contain substantial amounts of macrophage and MMP-3, and macrophage can be secreted in AS specklees
MMP-3, research confirm that the generation of AS Plaque instabilities is closely related with the secretion of MMP-3.In AS initial periods, MMP-3 joins
With the formation of fibrous cap, the progress of AS is promoted.And the overexpression of MMP-3 can promote the rupture of AS specklees, cause then
Thrombosiss and block blood vessel, cause serious consequence, such as acute coronary syndrome（ACS）, research display, serum MMP-3 levels
Compared with hs-CRP（hs-CRP）, MMP-9 more effectively can predict Stable coronary heart disease adverse cardiac events at a specified future date send out
Raw rate is the influence prognosis factor of Stable coronary heart disease independence, and serum MMP-3 levels can effectively point out ACS patient's AS plaque ruptures
Generation, be the important blood serum designated object of ACS, the timely diagnosis and treatment of ACS played an important role.
In addition, current research show MMP-3 can also degrade characteristic speckle in Alzheimer brain main into
Divide amyloid beta（AD）, Parkinsonian（PD）The α core synapsins that exception is gathered in vivo.Participating in nervus centraliss
When the generation of systemic disease develops, MMP-3 or a kind of important signal transduction molecule, neuron synthesized in apoptosis early stage
MMP-3 is discharged into extracellular MMP-3 and can activate microglia, promotes microglia to make the phagocytosis of Apoptotic neuron
With this effect is conducive to removing AD specklees, but exacerbates the death of PD patient's dopamine neuron.Research has confirmed MMP-
3 can occur in nucleus, and pass through its catalysis activity inducing cell apoptosis.Also research shows, MMP-3 participates in intrauterine
The occurrence and development of endometriosis, endometriosis patients MMP-3 levels are significantly higher than healthy control group, MMP-3 and
The detection by quantitative of TIMP-1 is significant to the early diagnosiss of endometriosis.Research both at home and abroad also show MMP-3 with
Diabetes are closely related.
In sum, the function of MMP-3 is extremely diversified, participates in numerous disease and develops, detection by quantitative MMP-3 water
Flat clinically significant, have wide practical use.It is contemplated that, MMP3 determines test kit（Enzyme-linked
Immunization）It will be following study hotspot.
Content of the invention
It is an object of the invention to provide a kind of stromatin enzyme 3 determines test kit, to solve above-mentioned technical problem.Be with
For the purpose of Quantitative in vitro determines the stromatin enzyme 3 in human plasma, develop stromatin enzyme 3 and determine test kit（Enzyme linked immunological
Method）Formula.
The technical problem to be solved employs the following technical solutions to realize：
A kind of stromatin enzyme 3 determines test kit, it is characterised in that include：Enzyme mark coating plate, Sample dilution, washing buffer
Liquid, terminate liquid, enzyme conjugates, substrate, calibration object, quality-control product；
The test kit adopts double antibody sandwich method, the micropore endoperidium of enzyme mark coating plate MMP-3 monoclonal antibodies, incubates for the first time
During educating, by the use of the MMP-3 in Sample dilution diluting plasma as antigenic substance and coated MMP-3 monoclonal antis in solid phase
Body is combined, and forms insolubilized antibody antigenic compound；
Fully wash to remove unconjugated composition using lavation buffer solution, add enzyme conjugates incubation, enzyme conjugates and solid phase
Antigen-antibody complex is combined；
Further wash, remove unconjugated composition, add substrate incubation, substrate is become color products by enzyme catalysiss, then plus
Enter terminate liquid with terminating reaction, each hole optical density value is detected by microplate reader；The size of optical density value and MMP-3 concentration in blood plasma
Into positive correlation.
Preferably, MMP-3 monoclonal antibody of the enzyme conjugates for enzyme labelling.
Preferably, potassium dihydrogen phosphate of the Sample dilution by 0.01mol/L, the phosphoric acid hydrogen of 0.01mol/L
The Klorvess Liquid composition of two sodium solutions, the sodium chloride solution of 0.01mol/L and 0.01mol/L, pH7.2.
Preferably, Tris-HCl buffer of the lavation buffer solution for 0.01mol/L.
Preferably, the terminate liquid：The H of 2mol/L2SO4Solution.
Preferably, the substrate is 3,3', 5,5'- tetramethyl benzidines.
The method that stromatin enzyme 3 is determined using mentioned reagent box, it is characterised in that comprise the following steps：
（1）Preparation：Other components in enzyme mark coating plate and test kit are balanced about 30min at 18-25 DEG C, each reagent is used
Front fully shake up.Lavation buffer solution 25mL in test kit is diluted using 225mL10 times of purification stand-by.Dilution quality-control product and sample
This：1 is pressed with 390 μ L of Sample dilution:40 dilution low value quality-control products, intermediate value quality-control product, 10 μ L of high level quality-control product and sample, fill
Divide and mix；
（2）Sample-adding：Plus 100 μ L calibration object 1-6, pre-dilution low value quality-control product, intermediate value quality-control product, high level quality-control product and sample are to phase
In the hole that answers, two repetitions are respectively done；
（3）Incubation：Under the conditions of 18-25 DEG C, 60 min are incubated；
（4）Washing：Quick flippase mark coating plate, falls off the solution in reacting hole, by enzyme mark coating plate in absorbent paper
Back-off is patted dry, and is at least added per hole 300 μ L lavation buffer solutions to rinse reacting hole, is fallen off the solution in reacting hole, be inverted in
Pat dry in absorbent paper, make liquid and the bubble being visible by naked eyes in reacting hole.Press aforesaid operations, repeated washing 3 times；
（5）Enzyme-added conjugate：Add the enzyme conjugates of 100 μ L per hole, under the conditions of 18-25 DEG C, be incubated 60 min；
（6）Washing：Same step（5）；
（7）Colour developing：Add 100 μ L substrates per hole, under the conditions of 18-25 DEG C, be incubated 10 min；
（8）Terminating reaction：Add per hole 100 μ L terminate liquids, jog ELISA Plate to mix, it is ensured that the bubble being visible by naked eyes, terminate anti-
Should；
（9）Read absorbance：Optical density value is read under microplate reader under 450nm；
（10）Result of calculation：Average optical density value with calibration object 1-6 as vertical coordinate, the concentration value with calibration object as abscissa,
Drawn using microplate reader systems soft ware, calibration curve is drawn using linear regression fit and pass through sample optical density value, you can in school
The concentration of specimens value corresponding to which is found on directrix curve.
Preferably, potassium dihydrogen phosphate of the Sample dilution by 0.01mol/L, the phosphoric acid hydrogen of 0.01mol/L
The Klorvess Liquid composition of two sodium solutions, the sodium chloride solution of 0.01mol/L and 0.01mol/L, pH7.2.
Preferably, Tris-HCl buffer of the lavation buffer solution for 0.01mol/L.
Preferably, H of the terminate liquid for 2mol/L2SO4Solution；The substrate be 3,3', 5,5'- tetramethyl biphenyls
Amine.
Ultimate principle：
Antigen or antibodies are made to certain surface of solid phase carriers, and keeps its immunocompetence.
Antigen or antibody is made to connect into enzyme-labelled antigen or antibody with certain enzyme, this enzyme-labelled antigen or antibody had both retained which and exempted from
Epidemic disease activity, retains the activity of enzyme again.When determining, by inspection specimen（Determine antibody therein or antigen）With enzyme-labelled antigen or anti-
Antigen or antibody of the body by different steps with surface of solid phase carriers react.Formation on solid phase carrier is made with the method for washing
Antigen antibody complex is separated with other materials, finally combine the amount of enzyme amount on solid phase carrier and tested substance in specimen into
Certain ratio.After adding the substrate of enzyme reaction, substrate is changed into color products, the amount of product and examined object in specimen by enzyme catalysiss
The amount of matter is directly related, therefore can carry out qualitative or quantitative analysis according to the depth of color reaction.Due to enzyme catalysis frequency very
Height, thus can greatly iodine effect so that assay method reaches very high sensitivity.
ELISA can be used to determine antigen, it can also be used to determine antibody.There are 3 kinds of necessary reagents in this assay method：
The antigen or antibody of solid phase
The antigen or antibody of enzyme labelling
The substrate of enzyme effect.
The invention has the beneficial effects as follows：
The present invention can Quantitative in vitro determine human plasma in MMP-3 content, adjuvant clinical diagnosis, treatment, be a kind of new
Immunoassay kit.It is that medical test and human health contribute a strength.
Specific embodiment
In order that technological means, creation characteristic, reached purpose and effect that the present invention is realized are easy to understand, tie below
Specific embodiment is closed, the present invention, but following embodiments only the preferred embodiments of the present invention are expanded on further, and not all.
Embodiment in based on embodiment, those skilled in the art's other realities obtained on the premise of creative work is not made
Example is applied, protection scope of the present invention is belonged to.
Embodiment 1
MMP3 determination box（Euzymelinked immunosorbent assay (ELISA)）, including enzyme mark coating plate, Sample dilution, lavation buffer solution, end
Only liquid, enzyme conjugates, substrate, calibration object, quality-control product.The test kit adopts double antibody sandwich method, the micropore endoperidium of ELISA Plate
MMP-3 monoclonal antibodies, during primary incubation, the MMP-3 in diluting plasma is coated with solid phase as antigenic substance
MMP-3 monoclonal antibodies are combined, and form insolubilized antibody antigenic compound.Fully wash to remove unconjugated other compositions, plus
Enter enzyme conjugates incubation, the MMP-3 monoclonal antibodies of enzyme labelling are combined with the antigen-antibody complex with solid phase.Further wash
Washing, removing unconjugated composition, add substrate incubation, substrate is become color products by enzyme catalysiss, terminate liquid added with end
Only react, each hole optical density value is detected by microplate reader.The size of optical density value is with MMP-3 concentration in blood plasma into positive correlation.
Wherein：
Sample dilution：By the potassium dihydrogen phosphate of 0.01mol/L, the disodium phosphate soln of 0.01mol/L, 0.01mol/
The Klorvess Liquid composition of the sodium chloride solution and 0.01mol/L of L, pH7.2；
Lavation buffer solution：0.01mol/L Tris-HCl buffer；
Terminate liquid：The H of 2mol/L2SO4；
Enzyme conjugates：The anti-human MMP-3 antibody of enzyme mark；
Substrate：3,3', 5,5'- tetramethyl benzidine.
Prepare material
50 spectrophotometers of Infinite, KDC-2046 low speed refrigerated centrifuges, 0.5-50 μ l, 50-200 μ l, 200-1000 μ l
Pipettor, sample-adding pipette tips, sodium citrate anticoagulant vacuum tube, EP pipes, disposable plastic gloves, test tube rack.
Sample collection
After collecting and having recorded clinical data, peripheric venous blood 4mL is gathered, is placed in sodium citrate anticoagulant vacuum tube, 3000r/
Blood plasma is isolated after min centrifugation 5min standby.
Technology path
（1）Preparation：Other components in enzyme mark coating plate and test kit are balanced about 30min at 18-25 DEG C, each reagent is used
Front fully shake up.By the lavation buffer solution in test kit（25mL）Using purification（225mL）10 times of dilutions are stand-by.Dilution quality-control product
And sample：Use Sample dilution（390μL）Press 1:40 dilution low value quality-control products, intermediate value quality-control product, high level quality-control product and sample
（10 μL）, fully mix.
（2）Sample-adding：Plus 100 μ L calibration object 1-6, pre-dilution low value quality-control product, intermediate value quality-control product, high level quality-control product and sample
This respectively does two repetitions into corresponding hole.
（3）Incubation：Under the conditions of 18-25 DEG C, 60 min are incubated.
（4）Washing：Quick flippase mark coating plate, falls off the solution in reacting hole, by enzyme mark coating plate in water suction
On paper, back-off is patted dry, and is at least added per hole 300 μ L lavation buffer solutions to rinse reacting hole, is fallen off the solution in reacting hole,
It is placed in absorbent paper and pats dry, makes liquid and the bubble being visible by naked eyes in reacting hole.Press aforesaid operations, repeated washing 3 times.
（5）Enzyme-added conjugate：Add the enzyme conjugates of 100 μ L per hole, under the conditions of 18-25 DEG C, be incubated 60 min.
（6）Washing：Same step（5）.
（7）Colour developing：Add 100 μ L substrates per hole, under the conditions of 18-25 DEG C, be incubated 10 min.
（8）Terminating reaction：100 μ L terminate liquids, jog ELISA Plate are added to mix per hole, it is ensured that the bubble being visible by naked eyes, eventually
Only react.
（9）Read absorbance：Optical density value is read under microplate reader under 450nm.
（10）Result of calculation：, as vertical coordinate, the concentration value with calibration object is as horizontal stroke for average optical density value with calibration object 1-6
Coordinate, is drawn using microplate reader systems soft ware, is drawn calibration curve using linear regression fit and is passed through sample optical density value, you can
The concentration of specimens value corresponding to which is found on calibration curve.
The Multiple components such as MMP-3 degradable collagen protein, Dan Baiduotang proteoglycan PG, sugar albumen, fibronectin, gelatin, can be with
Other MMPs are activated, is played an important role in the multiple pathology of human body and physiological process.Test kit inspection is determined using the MMP-3 of research and development
Survey human plasma in MMP-3 content, for clinical diagnosises significant.
(1) MMP-3 and tumor
Tumor refers to body under the effect of the various tumorigenesis factors, and the neoplasm formed by local organization hyperplasia, because this
Being in occupancy block-like protrusions neoplasm, also referred to as vegetation is current clinically commonly encountered diseases, frequently-occurring disease more.According to neoplastic
Cell characteristics and the extent of injury to body, and tumor is divided into benign tumor and malignant tumor, and cancer is malignant tumor
General name, be at present endanger human health most serious a class disease, talk cancer complexion changed be also just derived from its high fatality rate.But cancer
Disease is simultaneously not equal to death, and World Health Organization (WHO) is set to chronic disease tumor, at present due to the development of medical science, the cure rate of tumor
More and more higher, and more early discovery, more early treatment, cure rate are higher.
Malignant tumor is malignant tumor energy local infiltration and metastasis with the most basic difference of benign tumor, and this is also to dislike
The main cause of property tumor causing death.The invasion and attack of tumor and the multistep that transfer is tumor and the interphase interaction of host cell
Suddenly, too many levels process, comes off from primary tumor including tumor cell, invades surrounding tissue, enters blood circulation, breaks through in the portion of being far apart
Blood capillary forms metastasis, and wherein the formation of new vesselses, the destruction of basement membrane and the degraded of extracellular matrix are tumor invasions
The important step of transfer.MMPs is one group of zinc ion dependence protein hydrolytic enzyme, can pass through degradation of cell epimatrix, destruction substrate
The integrity of film, reduction is intercellular to stick state, so as to promote the infiltration and transfer of tumor cell.MMP-3 becomes substrate again
Lysin -1, the fibronectin for constituting extracellular matrix and basement membrane of can degrading, mucoprotein, laminin，LN etc..Mesh
After front existing a lot of correlational studyes, such as Mercapide etc. have found to suppress tumor cell secretion MMP-3, cell invasion and transfer energy
Power substantially weakens, and illustrates that MMP-3 is relevant with tumor invasion and metastasis；Zucker and Vacirca have found MMP-3 in colon cancer tissue
Up-regulated promote the transfer of tumor cell and point out prognosis malas；Zhang Junyong etc. has found tables of the MMP-3 in stomach organization
Reach and be significantly higher than normal gastric mucosa；The research such as Huang Gangding shows metastases in local lymph node person's MMP-3 expressions apparently higher than pouring
Fawn on non-transferrer；Sun Dong etc. has carried out Serologic detection to MMP-3, it is found that the postoperative serum MMP-3 levels of patients with gastric cancer are notable
Less than preoperative, and with the horizontal no significant difference of normal group, point out detection serum MMP-3 levels contribute to whether judging postoperative tumor
Recurrence, while finding that serum MMP levels are in concordance with tissue MMP-3 expression, has noinvasive due to determining serum MMP-3 levels
Wound, easy to operate the advantages of, it is possible to use determine serum MMP-3 levels and replace cancerous tissue MMP-3 immunohistochemistries to judge
Malignancy and prognosis.
（2）MMP-3 and arthritis
Arthritis refer to generation in human synovial and its inflammatory diseasess of surrounding tissue, can be divided into tens of kinds, the arthritis of China
Patient has more than 100,000,000, and number is being continuously increased.Clinical manifestation for joint red, swollen, hot, bitterly, dysfunction and joint abnormal
Shape, severe patient cause joint deformity, affect patients ' life quality.Research shows that MMP-3 participates in the inflammatory reaction process of body,
There is substantial connection with arthritis.
Rheumatoid arthritis（RA）It is a kind of chronic auto-immune disease, serious harm human health, faces for a long time
In bed work, point out its early stage destruction of joint and judge that the index of state of an illness activity is extremely limited, it is impossible to which directly reflection rheumatoid is closed
The scorching cartilage of section and the destructiveness of bone.MMPs as a kind of important paathogenic factor, by directly degraded cartilage and sclerotin to closing
Section produces destruction.Wherein, MMP-3 is the most important protease for causing cartilage degradation.RA early stages, the synovial tissue of hypertrophy is to pass
Section cartilage surface grows and corrodes articular cartilage, forms pannuss.Pannuss contain a large amount of fibroblasts, releasable collagen protein
Enzyme, MMP-3 etc. and cause the destruction of osseous tissue, MMP-3 can be also secreted by synovial cell and chondrocyte.Research finds RA pathology
Group serum MMP-3 levels are significantly higher than matched group, in inflammation phase, the synovial membrane of early stage RA is mainly shown as that inflammatory cell soaks
Profit, though patient has morning stiffness, arthralgia, swelling, hydrops or limitation of activity etc. to show, closes without involvement on joint deformity, X-ray film
Saving any structure can be without exception, but a large amount of secretions of this phase existing MMP-3.In addition, research confirms MMP-3 levels with x-ray point
Phase is relevant, and with increasing for MMP-3 levels, x-ray destruction is also more serious, and up to bony fusion, MMP-3 levels can reflect cartilage
Destructiveness, can be used as the index of reflection RA state of an illness activities.
Osteoarthritis（OA）It is a kind of common chronic, gradual, degenerated joint pathological changes, can behave as in various degree
Arthralgia, the symptom such as limitation of activity, based on articular cartilage and the destruction of subchondral bone progressive, joint sliding formwork inflammatory reaction
Want pathological characters, early diagnosiss are difficult, the diagnostic means that clinically commonly uses often are directed to middle and advanced stage patient, early stage patient is used as one
Individual special population, its impact of early diagnosiss level to its Health outcome are more direct and obvious.MMP-3 can by chondrocyte,
Synovial cell secretes, and especially based on synovial tissue, research finds that MMP-3 is significantly raised in OA blood samples of patients and joint fluid, and
Lesion degree with OA is in steady-state growth trend into positive correlation, i.e. MMP-3 in OA course advancements, and this explanation MMP-3 is at OA
There is during hair growth promoting exhibition important function.There are some researches show, MMP-3 participates in O cartilage heavy damage generating processes, synovial tissue is sent out
During raw inflammatory reaction, MMP-3 expressions are significantly increased and are proportionate with lesion degree, and the MMP-3 of overexpression can activate MMP-
1st, 9,13, produce waterfall enlarge-effect, the type i collagen of common degraded cartilage matrix, II Collagen Type VIs, gelatin and fibronectin
The composition such as white, and then promote the degraded of articular cartilage matrix, cause the generation of OA.Study and confirm in terms of diagnosis early stage OA,
MMP-3 is sensitive biomarker, can be used as the important indicator of OA early diagnosiss.
（3）MMP-3 and atherosclerosiss
Atherosclerosiss（AS）It is the pathologic basis of cardiovascular and cerebrovascular disease, the stability of AS specklees is mainly by fibrous cap and fat core
Composition, wherein the size of fat core, the thickness of fibrous cap determine the stability of AS specklees, and fat core is bigger, the thinner speckle of fibrous cap
More unstable, fat core is less, the thicker speckle of fibrous cap is more stable.Numerous studies find that MMPs take part in blood vessel during AS
A series of pathophysiological processes such as wall reconstruct, plaque rupture, thrombosiss, are carried out to the stability of AS specklees according to MMPs levels
Comprehensive assessment has affirmative meaning.MMP-3 is the important member that MMPs families are expressed in AS specklees, is widely present AS pathological changes
Place, most of compositions of energy selective degradation extracellular matrix, while activate the common degradation of cell epimatrixs of other MMPs.AS speckles
The instable generation of block is closely related with the secretion of MMP-3, and in the key position of speckle, the overexpression of MMP-3 can promote
The rupture of AS specklees, causes then thrombosiss and blocks blood vessel.Research also finds that the rising of serum MMP-3 levels indicates AS
The rupture of speckle, is the blood serum designated object of important cardiovascular and cerebrovascular disease, clinically the timely diagnosis to cardiovascular and cerebrovascular disease
Significant with treating.
Project risk analysis
1st, security feature issue list
Issue list of the inventory according to 0316 normative annexes C of YY/T, supplements relevant MMP3 and determines examination
Agent box（Euzymelinked immunosorbent assay (ELISA)）Distinctive safety issue.
2nd, hazard analysis, including foreseeable sequence of events, harm situation and generable infringement
3rd, risk assessment, risk control measure log
Ultimate principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technical staff of the industry
It should be appreciated that the present invention is not restricted to the described embodiments, described in above-described embodiment and description is only the excellent of the present invention
Example is selected, the present invention is not intended to limit, without departing from the spirit and scope of the present invention, the present invention also has various change
And improvement, these changes and improvements are both fallen within scope of the claimed invention.The claimed scope of the invention is by appended
Claims and its equivalent thereof.
Claims (10)
1. a kind of stromatin enzyme 3 determines test kit, it is characterised in that include：Enzyme mark coating plate, Sample dilution, washing are slow
Rush liquid, terminate liquid, enzyme conjugates, substrate, calibration object, quality-control product；
The test kit adopts double antibody sandwich method, the micropore endoperidium of enzyme mark coating plate MMP-3 monoclonal antibodies, incubates for the first time
During educating, by the use of the MMP-3 in Sample dilution diluting plasma as antigenic substance and coated MMP-3 monoclonal antis in solid phase
Body is combined, and forms insolubilized antibody antigenic compound；
Fully wash to remove unconjugated composition using lavation buffer solution, add enzyme conjugates incubation, enzyme conjugates and solid phase
Antigen-antibody complex is combined；
Further wash, remove unconjugated composition, add substrate incubation, substrate is become color products by enzyme catalysiss, then plus
Enter terminate liquid with terminating reaction, each hole optical density value is detected by microplate reader；The size of optical density value and MMP-3 concentration in blood plasma
Into positive correlation.
2. stromatin enzyme 3 according to claim 1 determines test kit, it is characterised in that：The enzyme conjugates are enzyme mark
The MMP-3 monoclonal antibodies of note.
3. stromatin enzyme 3 according to claim 1 determines test kit, it is characterised in that：The Sample dilution by
The potassium dihydrogen phosphate of 0.01mol/L, the disodium phosphate soln of 0.01mol/L, the sodium chloride solution of 0.01mol/L and
The Klorvess Liquid composition of 0.01mol/L, pH7.2.
4. stromatin enzyme 3 according to claim 1 determines test kit, it is characterised in that：The lavation buffer solution is
The Tris-HCl buffer of 0.01mol/L.
5. stromatin enzyme 3 according to claim 1 determines test kit, it is characterised in that：The terminate liquid：2mol/L's
H2SO4Solution.
6. stromatin enzyme 3 according to claim 1 determines test kit, it is characterised in that：The substrate be 3,3', 5,
5'- tetramethyl benzidines.
7. using the method for kit measurement stromatin enzyme 3 described in claim 1, it is characterised in that comprise the following steps：
（1）Preparation：Other components in enzyme mark coating plate and test kit are balanced about 30min at 18-25 DEG C, each reagent is used
Front fully shake up.Lavation buffer solution 25mL in test kit is diluted using 225mL10 times of purification stand-by.Dilution quality-control product and sample
This：1 is pressed with 390 μ L of Sample dilution:40 dilution low value quality-control products, intermediate value quality-control product, 10 μ L of high level quality-control product and sample, fill
Divide and mix；
（2）Sample-adding：Plus 100 μ L calibration object 1-6, pre-dilution low value quality-control product, intermediate value quality-control product, high level quality-control product and sample are to phase
In the hole that answers, two repetitions are respectively done；
（3）Incubation：Under the conditions of 18-25 DEG C, 60 min are incubated；
（4）Washing：Quick flippase mark coating plate, falls off the solution in reacting hole, by enzyme mark coating plate in absorbent paper
Back-off is patted dry, and is at least added per hole 300 μ L lavation buffer solutions to rinse reacting hole, is fallen off the solution in reacting hole, be inverted in
Pat dry in absorbent paper, make liquid and the bubble being visible by naked eyes in reacting hole.Press aforesaid operations, repeated washing 3 times；
（5）Enzyme-added conjugate：Add the enzyme conjugates of 100 μ L per hole, under the conditions of 18-25 DEG C, be incubated 60 min；
（6）Washing：Same step（5）；
（7）Colour developing：Add 100 μ L substrates per hole, under the conditions of 18-25 DEG C, be incubated 10 min；
（8）Terminating reaction：Add per hole 100 μ L terminate liquids, jog ELISA Plate to mix, it is ensured that the bubble being visible by naked eyes, terminate anti-
Should；
（9）Read absorbance：Optical density value is read under microplate reader under 450nm；
（10）Result of calculation：Average optical density value with calibration object 1-6 as vertical coordinate, the concentration value with calibration object as abscissa,
Drawn using microplate reader systems soft ware, calibration curve is drawn using linear regression fit and pass through sample optical density value, you can in school
The concentration of specimens value corresponding to which is found on directrix curve.
8. stromatin enzyme 3 according to claim 7 determines test kit, it is characterised in that：The Sample dilution by
The potassium dihydrogen phosphate of 0.01mol/L, the disodium phosphate soln of 0.01mol/L, the sodium chloride solution of 0.01mol/L and
The Klorvess Liquid composition of 0.01mol/L, pH7.2.
9. stromatin enzyme 3 according to claim 7 determines test kit, it is characterised in that：The lavation buffer solution is
The Tris-HCl buffer of 0.01mol/L.
10. stromatin enzyme 3 according to claim 1 determines test kit, it is characterised in that：The terminate liquid is 2mol/L
H2SO4Solution；The substrate be 3,3', 5,5'- tetramethyl benzidines.
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