CN101870999B

CN101870999B - Activator assay kit of human matrix metalloprotease-13

Info

Publication number: CN101870999B
Application number: CN201010201875XA
Authority: CN
Inventors: 杨林; 郭艾
Original assignee: Beijing Friendship Hospital
Current assignee: Beijing Friendship Hospital
Priority date: 2010-06-09
Filing date: 2010-06-09
Publication date: 2013-09-18
Anticipated expiration: 2030-06-09
Also published as: CN101870999A

Abstract

The invention discloses an activator detection method of human matrix metalloprotease-13 and an assay kit. The detection method and the kit can rapidly and effectively detect the type of an MMP-13 activator to provide powerful assistance for clinical experiments and the like, thus having good application prospect and authorization prospect.

Description

The activator assay kit of human matrix metalloprotease enzyme-13

Technical field

The present invention relates to a kind of detection kit, specifically a kind of human matrix metalloprotease enzyme-13 activator assay kit.

Background technology

Osteoarthritis (OA) is to cause more than 50 years old crowd's functional disability, economic loss and affect one of principal disease of social development, finds at present to have many factors to participate in morbidity, but Etiological and concrete pathogenesis are not clear.Its pathology essence is that the more change of microcosmic has occured for articular chondrocytes, extracellular matrix aspect, and the damage on the macroscopic view is the external cause that microcosmic changes.Tool feature in the joint cartilage matrix, what content was maximum is the II Collagen Type VI.Matrix metalloproteinase (MMPs) is that a class extensively is present in each reticular tissue, the proteolytic enzyme superfamily that plays an important role in the physiological of extracellular matrix and pathologic degradation process.Wherein MMP-13 expresses in the OA joint cartilage, and specificity degraded II Collagen Type VI, and other many MMP hypotypes need to work by them to the degraded of II Collagen Type VI, becomes the targeted molecular of present primary study.MMP-13 is synthesized with the form of non-activity, cysteine residues unpaired in the conserved sequence has on-off action, it can form coordinate bond with the zine ion of reactive site, makes enzyme be in the non-activity state, is activated after extracellular N-terminal propetide is cut away by other molecules.MMP-13 can be by activation such as interleukin-, tumour necrosis factors.In addition, also have many molecules that the effect that activates MMP-13 may be arranged, need further research.This test kit is applicable to the activator of examination MMP-13 in the medical research, is the pathogenesis service of research OA.

At present, method for detection of the activator of MMP-13 comprises nucleic detection method and fluorescence detection, the two uses respectively nucleic and fluorescein-labelled collagen substrate, be activated rear decomposition substrate of MMP-13 discharges nucleic ray or fluorescence, comes the activity of indirect analysis MMP-13 by sense radiation intensity or fluorescence intensity.The major defect of nucleic detection method: the nucleic radioactivity has radiation injury to human body; The Decay Rate of nucleic self causes time limit of service limited, and is can not prolonged preservation for subsequent use; The unstable of nucleic self causes the experimental result systematic error larger; Need special ray detection instrument.The major defect of fluorescence detection: the cancellation of fluorescein self causes time limit of service limited, and is can not prolonged preservation for subsequent use; The unstable of fluorescein self causes the experimental result systematic error larger; Fluorescence intensity a little less than, need highly sensitive fluoroscopic examination instrument.

Therefore, those skilled in the art are badly in need of a kind of a kind of method that can accurately detect the MMP-13 activator that can effectively avoid above-mentioned technological deficiency and make.

Summary of the invention

The object of the present invention is to provide a kind of method and detection kit that detects the activator of human matrix metalloprotease enzyme-13.

To achieve these goals, invention thinking of the present invention is:

II Collagen Type VI substrate is made fiber membrane, covering also is fixed on the 96 brown orifice plates, MMP-13 with non-activity adds in the micropore again, at last the factor to be measured is added wherein, will the decomposes collagen fiber membrane if MMP-13 is activated, observe the damaged degree of fiber membrane and can analyze the activity of MMP-13, thereby judge the activation of this factor pair MMP-13.

To achieve these goals, the present invention adopts following concrete technical scheme:

A kind of activator assay kit of human matrix metalloprotease enzyme-13, described kit components is as follows:

MMP-13 10-15 microgram, the best are 10 micrograms;

Trypsinase 1-1.5 milliliter, the best are 1 milliliter;

Damping fluid 60-100 milliliter, the best are 60 milliliters;

Reaction terminating liquid 1-1.5 milliliter, the best are 1 milliliter;

2 of 96 orifice plates of rubber cover fibril ghost.

Described damping fluid is 50mM Tris-HCl (9.025 ℃ of pH), 1.5mM MgCl ₂, 15mM (NH ₄) ₂SO ₄, 0.1%Triton X-100.

Described reaction terminating liquid 10mM phenanthrolene.

The recommendation method of mentioned reagent box is:

(1) brown 96 orifice plates of rubber cover fibril ghost is divided into blank group, positive controls and experimental group.

(2) dilute MMP-13 by 1: 10 usefulness damping fluid, give institute porose MMP-13 and the 80 microlitre damping fluids that respectively add after 10 microlitres dilute.

(3) blank group adds 10 microlitre damping fluids, and positive controls adds 10 microlitre trypsinase, and experimental group adds the 10 microlitres factor to be measured, hatches 90 minutes under 37 ℃.

(4) give the porose 10 microlitre reaction terminating liquids that respectively add.

(5) flushing is taken pictures with digital camera behind the microwell plate, observes the digestible degree of collagen fiber membrane with image analysis software again and compares.

The factor to be measured comprises S-nitrosoglutathione-N-ethanoyl Trolovol, nf (NF)-κ B, IL-6, IL-8, NOC-9; 6-(2-hydroxyl-1-methyl-2-nitroso-group diazanyl)-N-methyl isophthalic acid-normal hexyl Amine (NOC-9,6-(2-Hydroxy-1-methyl-2-nitroso-hydrazino)-N-methyl-1-hexanamine), S-nitrosoglutathione (S-nitrosoglutathione), APMA (APMA) or Sodium Nitroprusside etc.

Except 96 orifice plates, can also use 48 orifice plates and 24 orifice plates.

Advantage of the present invention and benefit:

The inventive method is easy, and test kit comprises the recombinant human MMP-13 of non-activity and the microwell plate of rubber cover fibril ghost, and working method is simple, only needs the factor to be measured is added MMP-13, and the digestible degree of observing collagen fiber membrane on the microwell plate gets final product.Compared with prior art, the present invention has following advantage: direct-detection does not need to come indirect analysis by gamma intensity, fluorescence intensity; "dead", to human body without injury; But prolonged preservation is for subsequent use; The tunica fibrosa stable in properties, the systematic error of experimental result is little; Do not need the special detection instrument.

Description of drawings

Fig. 1 is the embodiment 296 orifice plate photos that digital camera is taken pictures;

Fig. 2 is for using the brown area pixel value of Photoshop CS computed in software of Adobe company.

Embodiment

Material and source:

The purchase producer of non-activity recombinant human MMP-13: U.S. Chondrex company;

Tryptic purchase producer: U.S. Sigma company;

The purchase producer of damping fluid and reaction terminating liquid: 96 orifice plates of the outstanding U.S. gene in Shanghai Pharmaceutical Technology Co., Ltd rubber cover fibril ghost: wherein brown 96 orifice plates are purchased from Shanghai just intelligent industry and trade company limited, II collagen type and polyvinyl alcohol, glycerine, water behind mixing under the solution state, are filmed and made; The ratio of I type i collagen albumen and polyvinyl alcohol, glycerine, water is 1: 3: 6: 6.

96 orifice plates of overlay film also can prepare with additive method, for example: with II collagen type, boric acid, glycerine, licorice polysaccharide leach liquor and water by 1: 1: 6: film and form after the preparation in 6: 10.

Embodiment 1 test kit of the present invention forms and using method

The composition of test kit of the present invention:

Non-activity recombinant human MMP-13 10 micrograms;

1 milliliter in trypsinase;

60 milliliters of damping fluids;

1 milliliter of reaction terminating liquid;

2 of 96 orifice plates of rubber cover fibril ghost.

Using method:

(1) 96 orifice plates of rubber cover fibril ghost is divided into blank group, positive controls and experimental group.

Wherein, damping fluid is 50mM Tris-HCl (9.025 ℃ of pH), 1.5mM MgCl ₂, 15mM (NH ₄) ₂SO ₄, 0.1%Triton X-100.Reaction terminating liquid 10mM phenanthrolene.

The concrete application examples of embodiment 2 test kits of the present invention

The factor to be measured is APMA (APMA), is purchased from Shanghai outstanding U.S. gene Pharmaceutical Technology Co., Ltd the present embodiment and uses the composition of test kit and method with embodiment 1:

As a result interpretation: as shown in Figure 1, front 10 holes that 1-3 is capable are respectively blank group, positive controls and experimental group, and collagen fiber membrane is damaged after by MMP-13 digestion, and brown microwell plate appears under the film, calculate brown area with image analysis software, namely represent the active size of MMP-13.

Use the brown area of Photoshop CS computed in software of Adobe company, pixel value as shown in Figure 2, the MMP-13 of positive controls is activated fully, reach maximum activity, the average brown area in each hole represents the maximum activity value of MMP-13, namely 100%, the average brown area in each hole of experimental group is compared the relative reactivity value that resulting per-cent is experimental group MMP-13 with positive controls.

The result shows, sees Table 1.

The brown area in each hole of table 1 (pixel value bytes)

The relative reactivity value of experimental group MMP-13 is 99.7%.

Adopt the check of two independent sample t, compare with the blank group, the damaged area of experimental group collagen fiber membrane is larger, statistical significance is arranged, p＜0.05; Compare with positive controls, the damaged area of collagen fiber membrane is similar, no difference of science of statistics, p＞0.05.

Utilize fluorescence detection method checking the present embodiment result:

Concrete grammar:

(1) 96 orifice plates is divided into blank group, positive controls and experimental group.

(3) blank group adds 10 microlitre damping fluids, and positive controls adds 10 microlitre trypsinase, and experimental group adds the 10 microlitres factor to be measured, hatches 60 minutes under 37 ℃.

(4) give the porose fluorescently-labeled II Collagen Type VI of the 100 microlitres substrate that respectively adds, hatched 30 minutes under 25 ℃.

(5) give the porose 10 microlitre reaction terminating liquids that respectively add.

(6) detect the fluorescence intensity in each hole with highly sensitive fluoroscopic examination microplate reader, testing conditions: λ em=450nm, λ ex=365nm.

As a result interpretation: see Table 2

The fluorescence intensity in each hole of table 2

The relative reactivity value of experimental group MMP-13 is 99.2%.

Principal advantage of the present invention is: by observing collagen fiber membrane by the activator of the postdigestive damaged degree direct-detection of MMP-13 MMP-13, need not to reduce the harm of systematic error and nucleic or fluorescein by the indirectly interpretation such as nucleic or fluorescein.

Claims

1. the activator assay kit of a human matrix metalloprotease enzyme-13 is characterized in that, described kit components is as follows:

Non-activity recombinant human MMP-13, the 10-15 microgram;

Trypsinase 1-1.5 milliliter;

Damping fluid 60-100 milliliter;

Reaction terminating liquid 1-1.5 milliliter;

96 orifice plates of rubber cover fibril ghost;

Described damping fluid is that 50mM is 9.0 Tris-HCl, 1.5mM MgCl at 25 ℃ of lower pH ₂, 15mM (NH ₄) ₂SO ₄, 0.1%Triton X-100;

Described reaction terminating liquid 10mM phenanthrolene;

96 orifice plates of described rubber cover fibril ghost, it is to make in accordance with the following methods:

Brown 96 orifice plates behind mixings under the solution state, are filmed II collagen type and polyvinyl alcohol, glycerine, water to make; The ratio of II collagen type and polyvinyl alcohol, glycerine, water is 1: 3: 6: 6;

Or make in accordance with the following methods:

Brown 96 orifice plates, with II collagen type, boric acid, glycerine, licorice polysaccharide leach liquor and water by 1: 1: 6: film and form after the preparation in 6: 10.

2. method of utilizing test kit claimed in claim 1 to detect the activator of human matrix metalloprotease enzyme-13, it is characterized in that, II Collagen Type VI substrate is made the collegen filament film, covering also is fixed on the carrier, non-activity recombinant human MMP-13 and the factor to be measured are added described carrier, in the situation that MMP-13 is activated, the collegen filament film is decomposed, observe the damaged degree of collegen filament film, analyze the activity of described MMP-13, judge the activation of the described MMP-13 of this factor pair to be measured.

3. the method for the activator of detection human matrix metalloprotease enzyme-13 according to claim 2 is characterized in that, described method comprises the steps:

(1) covering of II type collagen fiber film is fixed on the carrier;

(2) carrier of rubber cover fibril ghost is divided into blank group, positive controls and experimental group;

(3) with damping fluid dilution non-activity MMP-13;

(4) add the 10 microlitres factor to be measured, hatched 90 minutes under 37 ℃;

(5) add the reaction terminating liquid termination reaction;

(6) take pictures with digital camera, collagen fiber membrane is rear damaged by MMP-13 digestion, and carrier appears under the film, calculates displaying area with image analysis software, namely represents the active size of MMP-13.

4. the method for the activator of detection human matrix metalloprotease enzyme-13 according to claim 3 is characterized in that, described blank group adds MMP-13 and the damping fluid with the damping fluid dilution.

5. the method for the activator of detection human matrix metalloprotease enzyme-13 according to claim 3 is characterized in that, described positive controls adds trypsinase and dilutes MMP-13 with damping fluid.

6. the method for the activator of the described detection human matrix metalloprotease of any one enzyme-13 in 5 according to claim 2; it is characterized in that; the described factor to be measured comprises S-nitrosoglutathione-N-ethanoyl Trolovol, nf (NF)-κ B, IL-6, IL-8, NOC-9,6-(2-hydroxyl-1-methyl-2-nitroso-group diazanyl)-N-methyl isophthalic acid-normal hexyl Amine, S-nitrosoglutathione, APMA or Sodium Nitroprusside.