CN106950382A - MMP3 determines reagent and preparation method thereof - Google Patents
MMP3 determines reagent and preparation method thereof Download PDFInfo
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- CN106950382A CN106950382A CN201710171773.XA CN201710171773A CN106950382A CN 106950382 A CN106950382 A CN 106950382A CN 201710171773 A CN201710171773 A CN 201710171773A CN 106950382 A CN106950382 A CN 106950382A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- 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
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96486—Metalloendopeptidases (3.4.24)
- G01N2333/96491—Metalloendopeptidases (3.4.24) with definite EC number
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Abstract
Reagent, including pretreating reagent, antibody latex reagent, calibrating reagent are determined present invention is disclosed a kind of MMP3；Pretreating reagent includes buffer, preservative, sensitizer, inorganic salts, surfactant, and the pH value of pretreating reagent is 7.2 7.6；Antibody latex reagent includes buffer, the latex microsphere for being combined with the antibody of antihuman substance metal protease 3, preservative, protective agent, a diameter of 100 400nm of latex microsphere；Calibrating reagent includes quantitative MMP3 antigen.Present invention further teaches the preparation method that a kind of MMP3 determines reagent.The present invention is combined by the antibody of antihuman substance metal protease 3 with latex microsphere, the content of MMP 3 in human serum can be determined using latex enhancing immune turbidimetry, the reagent operation is easy, the degree of accuracy is high, it is reproducible, suitable for high flux detection, it can be used on automatic clinical chemistry analyzer, the result for exempting from quantitative approach measure with enzyme has good accordance.
Description
Technical field
The present invention relates to biological technical field, and in particular to one kind can use latex enhancing immune turbidimetry for Determination human serum
The Transin-1 of the content of matrix metalloproteinase -3 determines reagent and preparation method thereof.
Background technology
MMP-3 is one of member of matrix metalloproteinase superfamily.Matrix metalloproteinase is an extended familys, because of it
Need Ca2+、Zn2+Gained the name Deng metal ion as confactor.Its family member has similar structure, typically by 5 work(
Domain that can be different is constituted:(1) hydrophobic signal peptide sequence；(2) propetide area, main function is to maintain the stabilization of proenzyme, when this
After region is cut off by exogenous enzymes, MMPs proenzymes are activated；(3), there is zinc ion binding site in catalytic activity area, and enzymatic is made
Performance is most important；(4) hinge area of Pro-rich；(5) carboxyl terminal area, it is relevant with the substrate specificity of enzyme.Its
Middle catalytic activity area and propetide area have well-conserved.
MMP-3 is the protease that a class is capable of degradation of cell epimatrix, is present in normal human, participates in the drop of cartilage matrix
Solution, bone information, metalloproteinases 3 not only can directly be degraded II, IX in extracellular base wherein, and XI Collagen Type VIs can also be adjusted
Other MMPs activity.
MMP-3 can act on PG, LN, FN, III type familial combined hyperlipidemia collagen and gelatin, and its activity is adjusted by three levels,
That is gene transcription level, inactive enzyme precursor is activated through proteolysis and the work of specificity suppressioning factor (TIMP)
With.
MMP-3 can shear substrate molecule decorin, and then conversion of the release with biological activity grows
The factor-β (TGF-β), the increment of TGF-β not only regulating cell, and also have effect to angiogenesis.Therefore MMP-3 and mammary gland
The generation of cancer, development relationship closely, and infiltration in breast cancer, play an important role in transfer process.MMP-3 can degrade poly-
The remarkable activity of the collagen of the articulationes cartilagineae azelon desmin of collection, core protein and IV, VII, Ⅸ type, can cause cartilage
Degraded.Patients with ankylosing spondylitis serum MMP-3 levels are significantly higher than normal population, can effectively reflect the disease activity of patient
And Bones and joints destructiveness, can be that clinical judgment as disease progressions and curative effect provide scientific basis.
MMP-3 is a kind of metastable protein, and sample can be preserved for a long time at -20 DEG C.There is chromatography qualitative at present
Exempt from quantitative detection method with enzyme can use, chromatograph qualitative be only capable of in concentration of specimens more than simply determination whether there is after test limit, it is impossible to
Accurate quantitative analysis, it is impossible to reflect the light and heavy degree of the state of an illness and discovery and treatment early is carried out to the state of an illness, and there is larger vacation
Cloudy or false sun, enzyme exempts from method and is mainly applied to scientific research by scientific researcher, due to each carrying out the preparation of detection reagent, does not have
Testing conditions are not furtherd investigate, cause different batches reagent testing result to differ greatly by unified formula and technique.
The content of the invention
Reagent and preparation method thereof is determined it is an object of the invention to provide a kind of Transin-1.
One of for achieving the above object, the present invention is adopted the following technical scheme that：
A kind of Transin-1 determines reagent, including pretreating reagent, antibody latex reagent, calibrating reagent；
The pretreating reagent includes buffer, preservative, sensitizer, inorganic salts, surfactant, the pretreatment examination
The pH value of agent is 7.2-7.6；
The antibody latex reagent include buffer, be combined with the antibody of antihuman substance metal protease -3 latex microsphere,
Preservative, protective agent, a diameter of 100-400nm of latex microsphere；
The calibrating reagent includes quantitative Transin-1 antigen.
As further improved technical scheme of the present invention, the inorganic salts in the pretreating reagent include sodium chloride, chlorine
Change potassium, buffer is one kind in phosphate, trishydroxymethylaminomethane, glycine, 4- hydroxyethyl piperazineethanesulfonic acids, sensitizer
For one kind in Macrogol 4000, Macrogol 6000, PEG 8000, sodium fluoride.
As further improved technical scheme of the present invention, what is combined in the antibody latex reagent with latex microsphere is anti-human
Transin-1 antibody is the monoclonal antibody of mouse antihuman substance metal protease -3, goat-anti people's Transin-1
At least one of monoclonal antibody, rabbit-anti people's Transin-1 monoclonal antibody, or the anti-human matrix metal egg of mouse
The white polyclonal antibody of enzyme -3, goat-anti people's Transin-1 polyclonal antibody, many grams of rabbit-anti people's Transin-1
At least one of grand antibody.
As further improved technical scheme of the present invention, the protective agent in the antibody latex reagent is bovine serum albumin
In vain, the one or more in casein, quaternary ammonium salt, reduced glutathione.
To realize above-mentioned another goal of the invention, the present invention is adopted the following technical scheme that：
A kind of Transin-1 determines the preparation method of reagent, and pretreating reagent, antibody latex examination are prepared respectively
Agent, calibrating reagent；
The preparation method of the pretreating reagent comprises the following steps：By buffer, preservative, sensitizer, inorganic salts, table
Face activating agent is that the solution that pH value is 7.2-7.6 is produced with water mixing preparation；
The preparation method of the antibody latex reagent comprises the following steps successively：
S1, by a diameter of 100-400nm latex microsphere containing carbodiimides and N- hydroxy thiosuccinimides
Activated in cushioning liquid, pH of cushioning fluid is 6.1；
S2, centrifugation or tangential flow filtration remove the component in addition to latex microsphere is activated being made in step S1 in reaction solution,
Plus the pH7.0-7.4 buffer solution resuspended scattered latex microsphere of ultrasound, the antibody of antihuman substance metal protease -3 is added, is taken the photograph 25
Family name's degree continues, slightly mixed 1 hour；
S3, centrifugation or tangential flow filtration remove removing in obtained reaction solution in step S2 and are combined with anti-human matrix metalloprotease
Component outside the latex microsphere of the antibody of enzyme -3, plus the pH7.5-8.0 buffer solution containing bovine serum albumin(BSA) or casein are ultrasonic
Resuspended scattered latex microsphere, continues at 25 degrees Celsius, slightly mixes 1 hour；
S4, centrifugation or tangential flow filtration remove the component in addition to latex microsphere being made in step S3 in reaction solution, plus
The pH7.5-8.0 buffer solution containing protective agent, preservative is produced with the resuspended scattered latex microsphere of ultrasound；
The preparation method of the calibrating reagent comprises the following steps：It is using dilution that Transin-1 antigen is dilute
Required concentration is released to produce.
As further improved technical scheme of the present invention, the latex in the preparation process S1 of the antibody latex reagent is micro-
Ball is carboxyl polystyrene latex microspheres.
As further improved technical scheme of the present invention, the buffer solution in the preparation process S1 of the antibody latex reagent
For 2- (N- morpholines) ethanesulfonic acid buffer, and concentration is between 20-50mmol/L.
As further improved technical scheme of the present invention, the latex in the preparation process S1 of the antibody latex reagent is micro-
Ball is single particle diameter, or is mixed by the latex microsphere of different-grain diameter size.
As further improved technical scheme of the present invention, the carbonization two in the preparation process S1 of the antibody latex reagent
Imines is 1- (3- dimethylaminopropyls) -3- ethyl-carbodiimide hydrochlorides.
As further improved technical scheme of the present invention, the protective agent in the preparation process S4 of the antibody latex reagent
For the one or more in bovine serum albumin(BSA), casein, quaternary ammonium salt, reduced glutathione.
Relative to prior art, the technical effects of the invention are that：
The present invention is combined by the antibody of antihuman substance metal protease -3 with latex microsphere, can use latex enhancing immune ratio
Turbid method determines the content of MMP-3 in human serum, and the reagent operation is easy, and the degree of accuracy is high, reproducible, suitable for high flux inspection
Survey, can be used on automatic clinical chemistry analyzer, the result for exempting from quantitative approach measure with enzyme has good accordance.
Embodiment
Below with reference to embodiment, the present invention will be described in detail.But these embodiments are not intended to limit this hair
Bright, structure that one of ordinary skill in the art is made according to these embodiments, method or conversion functionally are included
Within the scope of the present invention.
The invention provides a kind of Transin-1 determine reagent, including pretreating reagent, antibody latex reagent,
Calibrating reagent；
The pretreating reagent includes buffer, preservative, sensitizer, inorganic salts, surfactant, the pretreatment examination
The pH value of agent is 7.2-7.6；
The antibody latex reagent include buffer, be combined with the antibody of antihuman substance metal protease -3 latex microsphere,
Preservative, protective agent, a diameter of 100-400nm of latex microsphere；
The calibrating reagent includes quantitative Transin-1 antigen.
Further, the inorganic salts in the pretreating reagent include sodium chloride, potassium chloride, and buffer is phosphate, three
One kind in hydroxymethyl aminomethane, glycine, 4- hydroxyethyl piperazineethanesulfonic acids, sensitizer is Macrogol 4000, poly- second two
One kind in alcohol 6000, PEG 8000, sodium fluoride.
Further, the antibody of antihuman substance metal protease -3 combined in the antibody latex reagent with latex microsphere is
The monoclonal antibody of mouse antihuman substance metal protease -3, goat-anti people's Transin-1 monoclonal antibody, rabbit-anti people's matrix
At least one of monoclonal antibody of metalloproteinases -3, or the polyclonal antibody of mouse antihuman substance metal protease -3, goat-anti
At least one of people's Transin-1 polyclonal antibody, rabbit-anti people's Transin-1 polyclonal antibody.
Further, the protective agent in the antibody latex reagent is bovine serum albumin(BSA), casein, quaternary ammonium salt, reduction
One or more in type glutathione.
Further, the Transin-1 antigen that the calibrating reagent includes is Transin-1 weight
The native matrix metalloprotease -3 that histone is either extracted from human serum.
Present invention also offers the preparation method that a kind of Transin-1 determines reagent, pretreatment examination is prepared respectively
Agent, antibody latex reagent, calibrating reagent；
The preparation method of the pretreating reagent comprises the following steps：By buffer, preservative, sensitizer, inorganic salts, table
Face activating agent is that the solution that pH value is 7.2-7.6 is produced with water mixing preparation；
The preparation method of the antibody latex reagent comprises the following steps successively：
S1, by a diameter of 100-400nm latex microsphere containing carbodiimides and N- hydroxy thiosuccinimides
Activated in cushioning liquid, pH of cushioning fluid is 6.1；
S2, centrifugation or tangential flow filtration remove the component in addition to latex microsphere is activated being made in step S1 in reaction solution,
Plus the pH7.0-7.4 buffer solution resuspended scattered latex microsphere of ultrasound, the antibody of antihuman substance metal protease -3 is added, is taken the photograph 25
Family name's degree continues, slightly mixed 1 hour；
S3, centrifugation or tangential flow filtration remove removing in obtained reaction solution in step S2 and are combined with anti-human matrix metalloprotease
Component outside the latex microsphere of the antibody of enzyme -3, plus the pH7.5-8.0 buffer solution containing bovine serum albumin(BSA) or casein are ultrasonic
Resuspended scattered latex microsphere, continues at 25 degrees Celsius, slightly mixes 1 hour；
S4, centrifugation or tangential flow filtration remove the component in addition to latex microsphere being made in step S3 in reaction solution, plus
The pH7.5-8.0 buffer solution containing protective agent, preservative is produced with the resuspended scattered latex microsphere of ultrasound；
The preparation method of the calibrating reagent comprises the following steps：It is using dilution that Transin-1 antigen is dilute
Required concentration is released to produce.
Further, the latex microsphere in the preparation process S1 of the antibody latex reagent is that carboxyl polystyrene latex is micro-
Ball.
Further, the buffer solution in the preparation process S1 of the antibody latex reagent buffers for 2- (N- morpholines) ethyl sulfonic acid
Liquid, and concentration is between 20-50mmol/L.
Further, the latex microsphere in the preparation process S1 of the antibody latex reagent is single particle diameter, or by not
Latex microsphere with particle size is mixed.The detection performance of reagent can be made by the latex microsphere mixing of different-grain diameter size
Reach higher sensitivity and the linear upper limit.
Further, the carbodiimides in the preparation process S1 of the antibody latex reagent is 1- (3- dimethylaminos
Propyl group) -3- ethyl-carbodiimide hydrochlorides.
Further, the protective agent in the preparation process S4 of the antibody latex reagent be bovine serum albumin(BSA), casein,
One or more in quaternary ammonium salt, reduced glutathione.
The principle that the present invention carries out the assay of sample matrix metalloproteinase -3 is：It is first using antigen-antibody reaction
Pretreating reagent is added, makes the antigen site exposure of the Transin-1 in sample, when addition is combined with anti-human matrix
The latex microsphere reagent of the antibody of metalloproteinases -3 is antibody latex reagent, forms the antigen-antibody cross-linking reaction in reaction solution
Antigen antibody complex, makes latex microsphere aggegation, so that certain turbidity is produced, the content of sample matrix metalloproteinase -3
The turbidity with generation is calculated into positive correlation, then by calibration curve within the specific limits, so as to obtain matrix metalloprotease
The content of enzyme -3.
The present invention compared with prior art, with following features：
1) Transin-1 of the invention, which determines reagent, has high detection sensitivity, and lowest detection can reach
10.0ng/ml, it is easy to operate, quickly, at most only needed to 10 minutes from result is detected out, in addition it is shorter.
2) preparation of the latex microsphere of the binding antibody in antibody latex reagent is due to using two step chemical coupling methods, confrontation
Body effect fanout free region, joint efficiency is high, and good stability, differences between batches are small, can at least be preserved 1 year at 2-8 degrees Celsius.And adopt
With the reagent of physisorphtion, differences between batches are than larger, and stability is bad.
3) Transin-1 of the invention determines reagent to the content detection of sample matrix metalloproteinase -3
Data statistical analysis, exempting from method with the enzyme of scientific research, there was no significant difference, and testing result is reliable, and clinical evaluation is good, available for facing
Bed application.
Below in conjunction with an embodiment, the present invention will be described
Embodiment one
First, antibody latex reagent is prepared
By 1g particle sizes for 200nm Carboxylated latex microballoon be added to 100ml contain 0.2g 1- (3- dimethyl
Aminopropyl) -3- ethyl-carbodiimide hydrochlorides and 2g N- hydroxy thiosuccinimides PH6.1,0.05mol/L, 2-
In (N- morpholines) ethanesulfonic acid buffer, react 1 hour.
Reaction solution is divided into 10ml/ pipes, after centrifugation during rotating speed is 10000 revs/min of high speed freezing centrifuge 30 minutes,
Remove supernatant, often pipe plus concentration are 0.05mol/L, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid buffer solutions 10ml carries out ultrasound
It is resuspended.Continue after centrifugation during rotating speed is 10000 revs/min of high speed freezing centrifuge 30 minutes, remove supernatant, often pipe enriching
Spend for 0.05mol/L, it is resuspended that pH7.4 4- hydroxyethyl piperazineethanesulfonic acid buffer solutions 10ml carries out ultrasound.Often pipe addition mouse is anti-human
Transin-1 antibody 10mg, continues at 25 degrees Celsius, slightly mixes 1 hour.
After centrifugation during rotating speed is 10000 revs/min of high speed freezing centrifuge 30 minutes, remove supernatant, often pipe enriching
Spend for 0.01mol/L, it is resuspended that pH7.5 phosphate buffer 1 0ml carries out ultrasound.Continue in the height that rotating speed is 10000 revs/min
After being centrifuged 30 minutes in fast refrigerated centrifuge, remove supernatant, often pipe adds concentration for 0.01mol/L, pH7.5, pure containing ox blood
Protein 10 g/L, glycine 10g/L phosphate buffer 1 0ml progress ultrasounds are resuspended, continue at 25 degrees Celsius, slightly mix 1
Hour.
After centrifugation during rotating speed is 10000 revs/min of high speed freezing centrifuge 30 minutes, remove supernatant, often pipe enriching
Spend for 0.01mol/L, pH7.5,10g/L containing bovine serum albumin(BSA), quaternary ammonium salt 10g/L, proclin3000.1% phosphate delay
Fliud flushing 10ml progress ultrasounds are resuspended, produce antibody latex reagent.
2nd, pretreating reagent is prepared
Prepare 0.05mol/L, pH7.4,40g/L containing Macrogol 6000, sodium chloride 8g/L, potassium chloride 2g/L,
Proclin300 0.1%, the TRIS buffer of polysorbas20 0.1% is produced.
3rd, calibrating reagent is prepared
Prepare 0.05mol/L, pH7.4,10g/L containing bovine serum albumin(BSA), sodium chloride 8g/L, potassium chloride 2g/L,
Proclin300 0.1%, the phosphate buffer of polysorbas20 0.1% produces dilution.
The Transin-1 antigen diluent purified concentration needed for is produced using dilution.
The assay method that Transin-1 determines reagent is illustrated below
Calibrating reagent concentration：1200ng/ml、600ng/ml、300ng/ml、150ng/ml、75ng/ml、0ng/ml；
Determine wavelength：570nm；
Pretreating reagent：Antibody latex reagent：Sample=μ the l of l: 50 μ of 150 μ l: 3；
Calibrating mode：spline.
Detection process：Pretreating reagent is added with sample, is reacted 5 minutes, is then added after antibody latex reagent 20 seconds, reads
Absorbance A 1 is extracted reaction solution, is added after antibody latex reagent 5 minutes, then reads reaction solution absorbance A 2, the change of absorbance is calculated
Change Δ A=A2-A1.
Transverse axis is made of calibrating reagent concentration, Δ A does the longitudinal axis, sets up calibration curve.The concentration of unknown sample is reacted by it
Δ A from calibration curve calculate obtain.
The performance indications that Transin-1 of the present invention determines reagent are illustrated below
1. blank test limit is determined
Reagent is determined to dummy (calibrating reagent dilution) replication 20 times, blank with Transin-1
Test limit concentration is that average concentration adds two standard deviations, obtains blank test limit concentration requirement≤2ng/ml.
2. range of linearity 10-1200ng/ml.
3. the correlation with the kit measurement result of enzyme-linked immunoassay method.
By the examination for determining 100 sample (concentration of specimens is uniformly distributed in the range of 10-1200ng/ml) this reagent preparations
The coefficient R of the kit of agent box and enzyme-linked immunoassay method2=0.980, dependent equation is：Y=1.07X-0.962.
Wherein, X is the detected value of this reagent, and Y exempts from method detected value for enzyme.
4. precision
Same reagent detects same sample 10 times, is repeated variation lines with the standard deviation divided by average value of 10 measured values
Number ,≤5%.
Finally it should be noted that：Embodiment of above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to aforementioned embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing each embodiment can so be modified, or which part technical characteristic is equal
Replace；And these modifications or replacement, the essence of appropriate technical solution is departed from each embodiment technical scheme of the invention
Spirit and scope.
Claims (10)
1. a kind of Transin-1 determines reagent, it is characterised in that including pretreating reagent, antibody latex reagent, school
Quasi- reagent；
The pretreating reagent includes buffer, preservative, sensitizer, inorganic salts, surfactant, the pretreating reagent
PH value is 7.2-7.6；
The antibody latex reagent includes buffer, the latex microsphere for being combined with the antibody of antihuman substance metal protease -3, anti-corrosion
Agent, protective agent, a diameter of 100-400nm of latex microsphere；
The calibrating reagent includes quantitative Transin-1 antigen.
2. Transin-1 according to claim 1 determines reagent, it is characterised in that in the pretreating reagent
Inorganic salts include sodium chloride, potassium chloride, buffer be phosphate, trishydroxymethylaminomethane, glycine, 4- hydroxyethyl piperazines
One kind in ethyl sulfonic acid, sensitizer is one kind in Macrogol 4000, Macrogol 6000, PEG 8000, sodium fluoride.
3. Transin-1 according to claim 1 determines reagent, it is characterised in that the antibody latex reagent
In the antibody of antihuman substance metal protease -3 that is combined with latex microsphere be the monoclonal antibody of mouse antihuman substance metal protease -3,
At least one of goat-anti people's Transin-1 monoclonal antibody, rabbit-anti people's Transin-1 monoclonal antibody,
Or the polyclonal antibody of mouse antihuman substance metal protease -3, goat-anti people's Transin-1 polyclonal antibody, rabbit-anti
At least one of people's Transin-1 polyclonal antibody.
4. Transin-1 according to claim 1 determines reagent, it is characterised in that the antibody latex reagent
In protective agent be bovine serum albumin(BSA), casein, quaternary ammonium salt, reduced glutathione in one or more.
5. a kind of Transin-1 determines the preparation method of reagent, it is characterised in for preparing pretreating reagent respectively, resist
Body emulsion reagent, calibrating reagent；
The preparation method of the pretreating reagent comprises the following steps：Buffer, preservative, sensitizer, inorganic salts, surface are lived
Property agent is that the solution that pH value is 7.2-7.6 is produced with water mixing preparation；
The preparation method of the antibody latex reagent comprises the following steps successively：
S1, by a diameter of 100-400nm latex microsphere in the buffering containing carbodiimides and N- hydroxy thiosuccinimides
Activated in solution, pH of cushioning fluid is 6.1；
S2, centrifugation or tangential flow filtration remove the component in addition to latex microsphere is activated being made in step S1 in reaction solution, plus
The pH7.0-7.4 buffer solution resuspended scattered latex microsphere of ultrasound, adds the antibody of antihuman substance metal protease -3, Celsius 25
Degree continues, slightly mixed 1 hour；
S3, centrifugation or tangential flow filtration remove removing in obtained reaction solution in step S2 and are combined with antihuman substance metal protease -3
Component outside the latex microsphere of antibody, plus the pH7.5-8.0 buffer solution containing bovine serum albumin(BSA) or casein are resuspended with ultrasound
Scattered latex microsphere, continues at 25 degrees Celsius, slightly mixes 1 hour；
S4, centrifugation or tangential flow filtration remove the component in addition to latex microsphere being made in step S3 in reaction solution, plus pH7.5-
8.0 buffer solution containing protective agent, preservative is produced with the resuspended scattered latex microsphere of ultrasound；
The preparation method of the calibrating reagent comprises the following steps：Transin-1 antigen diluent is arrived using dilution
Required concentration is produced.
6. Transin-1 according to claim 5 determines the preparation method of reagent, it is characterised in that described anti-
Latex microsphere in the preparation process S1 of body emulsion reagent is carboxyl polystyrene latex microspheres.
7. Transin-1 according to claim 5 determines the preparation method of reagent, it is characterised in that described anti-
Buffer solution in the preparation process S1 of body emulsion reagent is 2- (N- morpholines) ethanesulfonic acid buffer, and concentration is in 20-50mmol/L
Between.
8. Transin-1 according to claim 5 determines the preparation method of reagent, it is characterised in that described anti-
Latex microsphere in the preparation process S1 of body emulsion reagent is single particle diameter, or is mixed by the latex microsphere of different-grain diameter size
Form.
9. Transin-1 according to claim 5 determines the preparation method of reagent, it is characterised in that described anti-
Carbodiimides in the preparation process S1 of body emulsion reagent is 1- (3- dimethylaminopropyls) -3- ethyl carbodiimide hydrochlorides
Salt.
10. Transin-1 according to claim 5 determines the preparation method of reagent, it is characterised in that described
Protective agent in the preparation process S4 of antibody latex reagent is bovine serum albumin(BSA), casein, quaternary ammonium salt, reduced glutathione
In one or more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710171773.XA CN106950382A (en) | 2017-03-22 | 2017-03-22 | MMP3 determines reagent and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710171773.XA CN106950382A (en) | 2017-03-22 | 2017-03-22 | MMP3 determines reagent and preparation method thereof |
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| Publication Number | Publication Date |
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| CN106950382A true CN106950382A (en) | 2017-07-14 |
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| CN201710171773.XA Pending CN106950382A (en) | 2017-03-22 | 2017-03-22 | MMP3 determines reagent and preparation method thereof |
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Cited By (13)
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| CN108152501A (en) * | 2018-01-08 | 2018-06-12 | 山东畜牧兽医职业学院 | A groups of bovine rota detection kits and the antiviral antibody colloid gold reagent preparation method |
| CN108663516A (en) * | 2018-03-22 | 2018-10-16 | 吉林基蛋生物科技有限公司 | A kind of preparation method and kit of sensitizing latex |
| CN110058015A (en) * | 2019-04-08 | 2019-07-26 | 深圳优普生物技术有限公司 | The measuring method and application of reagent, kit, platelet response index |
| CN110068680A (en) * | 2019-05-27 | 2019-07-30 | 杭州创新生物检控技术有限公司 | A kind of detection reagent plate and the kit and its detection method for detecting MMP-3 content |
| CN111320696A (en) * | 2020-02-27 | 2020-06-23 | 上海捷门生物技术有限公司 | MMP-3 antibody compound based on streptavidin latex and kit thereof |
| CN111830254A (en) * | 2020-07-29 | 2020-10-27 | 武汉生之源生物科技股份有限公司 | Matrix metalloproteinase-3 determination kit and preparation method thereof |
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| CN108152501A (en) * | 2018-01-08 | 2018-06-12 | 山东畜牧兽医职业学院 | A groups of bovine rota detection kits and the antiviral antibody colloid gold reagent preparation method |
| CN108663516A (en) * | 2018-03-22 | 2018-10-16 | 吉林基蛋生物科技有限公司 | A kind of preparation method and kit of sensitizing latex |
| CN110058015A (en) * | 2019-04-08 | 2019-07-26 | 深圳优普生物技术有限公司 | The measuring method and application of reagent, kit, platelet response index |
| CN110068680A (en) * | 2019-05-27 | 2019-07-30 | 杭州创新生物检控技术有限公司 | A kind of detection reagent plate and the kit and its detection method for detecting MMP-3 content |
| CN112904027A (en) * | 2019-12-04 | 2021-06-04 | 苏州普瑞斯生物科技有限公司 | Detection kit for oxidized low-density lipoprotein and preparation method thereof |
| CN111320696A (en) * | 2020-02-27 | 2020-06-23 | 上海捷门生物技术有限公司 | MMP-3 antibody compound based on streptavidin latex and kit thereof |
| CN111856009A (en) * | 2020-02-28 | 2020-10-30 | 安徽大千生物工程有限公司 | Kit for determining MMP-3 based on latex enhanced immunoturbidimetry, and preparation and use methods thereof |
| CN111830254B (en) * | 2020-07-29 | 2022-07-26 | 武汉生之源生物科技股份有限公司 | Matrix metalloproteinase-3 determination kit and preparation method thereof |
| CN111830254A (en) * | 2020-07-29 | 2020-10-27 | 武汉生之源生物科技股份有限公司 | Matrix metalloproteinase-3 determination kit and preparation method thereof |
| CN113281513A (en) * | 2021-05-17 | 2021-08-20 | 上海执诚生物科技有限公司 | MMP-3 kit, and preparation method and application thereof |
| CN113281513B (en) * | 2021-05-17 | 2024-05-28 | 上海执诚生物科技有限公司 | MMP-3 kit and preparation method and application thereof |
| CN114137204A (en) * | 2021-11-24 | 2022-03-04 | 安徽大千生物工程有限公司 | KL-6 determination kit and preparation and detection methods thereof |
| CN114137204B (en) * | 2021-11-24 | 2023-08-15 | 安徽大千生物工程有限公司 | KL-6 determination kit and preparation and detection method thereof |
| CN114200129A (en) * | 2021-12-09 | 2022-03-18 | 北京利德曼生化股份有限公司 | Hematuria simultaneous detection kit of tissue metalloproteinase inhibitor-2 latex immunoturbidimetry |
| CN114200129B (en) * | 2021-12-09 | 2023-04-14 | 北京利德曼生化股份有限公司 | Hematuria simultaneous detection kit of tissue metalloproteinase inhibitor-2 latex immunoturbidimetry |
| CN115032402A (en) * | 2022-05-30 | 2022-09-09 | 上海科华生物工程股份有限公司 | Matrix metalloproteinase-3 latex detection kit and preparation method thereof |
| CN115327122A (en) * | 2022-08-09 | 2022-11-11 | 江西乐成生物医疗有限公司 | Matrix metalloproteinase-3 detection kit and preparation method thereof |
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