CN103874925A - Method for detecting matrix metalloproteinase-3 by latex agglutination method - Google Patents
Method for detecting matrix metalloproteinase-3 by latex agglutination method Download PDFInfo
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- C12N9/6489—Metalloendopeptidases (3.4.24)
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Abstract
The purpose of the present invention is to provide a method for detecting matrix metalloproteinase-3 (MMP-3) by an LTIA method, which utilizes an anti-MMP-3 monoclonal antibody having high reactivity in a low value zone and having no cross-reactivity with other MMP family member such as MMP-10, can achieve a rapid measurement and has high sample throughput. It is found that the purpose can be achieved by providing: a detection method by a latex agglutination method, in which human matrix metalloproteinase-3 in a blood sample is detected employing, as a signal, the occurrence of agglutination as a result of an antigen-antibody reaction that is carried out by bringing latex particles each having a monoclonal antibody 14B1 supported thereon and latex particles each having a monoclonal antibody 3D3 supported thereon into contact with the blood sample; and a detection reagent and a detection kit both of which can be used for carrying out the detection method.
Description
Technical field
The present invention relates to the invention of the detection method of specific live body material, more specifically relates to detect according to latex agglutination the invention of the method for Transin-1.
Background technology
Matrix metalloproteinase (MMP)-the 3rd, the protein decomposition enzyme producing in cartilage cell or synovial cell increases in the time that synovium of joint is bred, and has the decomposition of the extracellular matrix of proteoglycans, fibronectin, collagen, laminin etc.Thereby, the destruction of joint of confirming in MMP-3 concentration in blood reflection rheumatoid arthritis, thus can be used as the index of arthritis activity and useful.Measure with in reagent at now commercially available MMP-3, have the reagent of use enzymoimmunoassay (ELISA method) and the reagent of use latex agglutination (LTIA method).
In addition, as the associated document of existing patent of the detection method about MMP-3, can enumerate International Publication separate edition (WO2010/090079) (patent documentation 1), Unexamined Patent 4-237499 communique (patent documentation 2), Unexamined Patent 11-318449 communique (patent documentation 3).In addition, as non-patent literature, can enumerate PRESKY, DH., et al., Biochemical and Biophysical Research Communications, 1993, vol.193, No.1, pp.364-370(non-patent literature 1).
Prior art document
Patent documentation
Patent documentation 1: International Publication WO2010/090079 separate edition
Patent documentation 2: Unexamined Patent 4-237499 communique
Patent documentation 3: Unexamined Patent 11-318449 communique
Patent documentation 4: JP 63-65369 communique
Patent documentation 5: JP 53-24015 communique
Non-patent literature
Non-patent literature 1:PRESKY, DH., et al., Biochemical and Biophysical Research Communications, 1993, vol.193, No.1, pp.364-370
Non-patent literature 2: " monoclonal antibody experimental implementation introduction " (the talk Scientific of society, peace Teng Minwei, Chiba zhang work), distribution on August 20th, 1993
Summary of the invention
The technical matters that invention will solve
Carry out like this detection of MMP-3 in blood, but also existed the problem in practicality in above-mentioned existing mensuration reagent.That is, use the reagent of ELISA method having problem aspect testing sample processing power (property rapidly), use latex agglutination (LTIA method) though reagent confirmed rapid property, the phenomenon that known existence is not inconsistent in the measured value of low value region and ELISA method.
The present invention, when measure in blood MMP-3 based on latex agglutination, has found not occur the not corresponding monoclonal antibody of measured value in low value region, to provide by using the detection means of MMP-3 in the blood of latex agglutination of this antibody as problem.
Can solve the monoclonal antibody of above-mentioned problem, it is generally acknowledged in low value region reactivity high, and, be from confirm less than with the obtaining for confirming the monoclonal antibody of MMP-3 of the cross reactivity of other MMP families such as MMP-10, be difficult but set up concrete prediction.
The technical scheme of dealing with problems
The inventor is for solving the above problems, by the latex particle of the latex particle of load monoclonal antibody 14B1 and load monoclonal antibody 3D3 is contacted with blood testing sample, using because of with blood testing sample in the aggegation that produces of the antigen-antibody reaction of Transin-1 as signal, found people's Transin-1 in the described blood testing sample of quantitative detection, can solve the above problems according to the detection method of latex agglutination (following, also referred to as detection method of the present invention).
2 kinds of above-mentioned monoclonal antibodies based on conventional method, for example, " the monoclonal antibody experimental implementation introduction " of non-patent literature 2 manufactured.
In addition, the latex agglutination (LTIA method) using in detection method of the present invention, typically can the disclosing of patent documentation 4,5 based on above-mentioned carry out.
; by the latex particle and the blood testing sample that are coated with respectively 2 kinds of above-mentioned monoclonal antibodies are coexisted; carry out the combination by the monoclonal antibody on the latex particle of people MMP-3 and these in described testing sample; detect the LTIA method of the aggegation of described latex particle, the people MMP-3 in can quantitative described testing sample.
The 2nd, the invention provides detection reagent and detection kit for carrying out above-mentioned detection method.In described detection reagent and detection kit, contain at least " latex particle of the latex particle of load monoclonal antibody 14B1 and load monoclonal antibody 3D3 ".In addition, also may contain as required dilution, cleaning fluid etc.
As hereinafter described, the hybridoma that produces these 2 kinds of monoclonal antibodies is deposited in the biological preservation of Independent Administrative Leged Industrial Technology Complex Inst's patent center separately, monoclonal antibody 3D3 preservation is " mouse-Mouse Hybridoma Cells 3D3(preserving number: FERMP-22223) ", and monoclonal antibody 14B1 preservation is " mouse-Mouse Hybridoma Cells 14B1(preserving number: FERMP-22225) ".
In the present invention, " blood testing sample " refers to the testing sample of serum, blood plasma, whole blood etc., is suitably especially serum testing sample or blood plasma testing sample, then is suitably serum testing sample.
Have again, in this manual, " matrix metalloproteinase " or " MMP ", only otherwise special instruction refers to " people's matrix metalloproteinase " or " people MMP ".
Invention effect
By the invention provides: used the LTIA method that testing sample processing power is high based on measuring rapidly of following antibody MMP-3 detection method and for carrying out detection reagent and the detection kit of described detection method.Described antibody be reactive in low value zone maintenance and confirm less than with other MMP families of MMP-10 etc. or with it similarly the cross reactivity of protein, for 2 kinds of monoclonal antibodies-" monoclonal antibody 14B1 " and " the monoclonal antibody 3D3 " of MMP-3.
Accompanying drawing explanation
Fig. 1: the figure of repeatability and diurnal inequality repeatability when showing detection method of the present invention.
Fig. 2: show the detection gauge in detection method of the present invention and dilute the figure of linear measurement range.
Fig. 3: the figure that shows the front band in detection method of the present invention.
Fig. 4: the figure that shows the impact of the coexisting substances in detection method of the present invention.
Fig. 5: the figure that shows and use the correlativity of the commercially available detection reagent of detection method of the present invention and ELISA method.
Fig. 6: show the blood testing sample that obtains negative value in the commercially available detection reagent to using LTIA method, the figure of the result of inquiring into by detection method of the present invention.
Fig. 7: show in the commercially available detection reagent to using LTIA method, obtain on the occasion of blood testing sample, use the figure of the result of inquiring into the correlativity of detection method of the present invention.
Embodiment
1. monoclonal antibody
Monoclonal antibody, by according to the method for recording in above-mentioned non-patent literature 2, obtains by using the immunity of recombinant protein to prepare hybridoma manufacture.
(1) selecting for the first time of the clone of generation monoclonal antibody
Selected for the first time through (i) following～process (iv) by hybridoma obtained above.As a result, 32 clones have been locked.
(i) by the screening of ELISA
The hole of 96 orifice plates of the hybridoma of originating from the mouse containing separately, selects the upper hole of reacting by force with rMMP3 solid phase ELISA.That is, will implement upper hole among the mouse of each immunization method, cultivating the hole that also remains positive after amplifying etc., adding up to 56 holes clones in addition.
(ii) by the cloning of limiting dilution assay
While carrying out the cloning by limiting dilution assay for the cell in 56 holes of above-mentioned middle selection, can set up 34 clones.
(iii) the subclass somatotype of antibody
The monoclonal antibody subclass obtaining uses mouse monoclonal antibody isotype to measure with kit (ISO-STRIP mouse IgG parting kit, 1-493-027, Boehringer mannheim), measures according to sequence of operation subsidiary in kit.
Subclass of antibody by 34 clones of above-mentioned limiting dilution assay cloning is that IgG1k is that 17 kinds of clones, IgG2ak are that 5 kinds of clones, IgG2bk are that 10 kinds of clones, IgMk are 2 kinds of clones.
(iv) select
Subclass of antibody IgM is because antibody purification process etc. is loaded down with trivial details, so get rid of above-mentioned 2 kinds of clones of IgM class in discussion backward.
(2) selecting for the second time of monoclonal antibody clone
32 clones that select are as described above expelled to separately to the abdominal cavity of the BALBc mouse (male 8 week age) of using pristane, before and after 1 week and take out ascites backward, use ammonium sulfate precipitation method and albumin A Sepharose to carry out purifying, obtain the monoclonal antibody corresponding to clone separately.For monoclonal antibody separately, use is inquired into the reactivity to people MMP-3 and MMP-10 in conjunction with the EIA method of the plate of each protein.
Next, 19 monoclonal antibody contacts of locking are here fixed on to the medicinal latex particle of commercially available in-vitro diagnosis, the performance while inquiring into as latex product, carries out the locking of further monoclonal antibody., carry out using confirm less than nonspecific latex agglutination test (with single monoclonal antibody not with antigen-reactive) as the result of the screening of condition, 12 monoclonal antibodies meet described condition.Next, inquire into the group of 66 kinds for 2 monoclonal antibodies of these 12 clone's combinations, using the group of 19 kinds that MMP-3 is obtained to specific latex agglutination test as candidate.For the group of these 19 kinds of monoclonal antibodies, screening is the IgG1 type of the subclass of the antibody when being suitable for as latex agglutination goods preparation, and do not show the nonspecific agglutination of learning while measuring as the physiological saline of blind check, while measuring MMP-3, show the combination of sufficient specific agglutination.Finally, scrutinizing and the result of the specific reaction of chronic rheumatoid arthritis patient's serum, finally select the combination of monoclonal antibody name " 14B1 ", " 3D3 " and " 8A6 ".
(3) sign of monoclonal antibody
Carry out as the sign of above-mentioned definite monoclonal antibody.
(i) subclass
14B1 antibody: IgG1/ κ
3D3 antibody: IgG1/ κ
8A6 antibody: IgG1/ κ
(ii) dissociation constant (M)
14B1 antibody: 5.34 × 10
-10
3D3 antibody: 1.91 × 10
-9
8A6 antibody: 2.20 × 10
-9
(iii) binding constant (1/Ms)
14B1 antibody: 2.45 × 10
5
3D3 antibody: 2.09 × 10
5
8A6 antibody: 3.20 × 10
5
(iv) atopy
By the results verification of the discussion of above-mentioned EIA method, 3 kinds of monoclonal antibodies are all to MMP-3 reaction, but substantially confirm less than the cross reactivity to MMP-10.
(4) preservation of hybridoma
The hybridoma that produces these 3 kinds of monoclonal antibodies is deposited in the biological preservation of Independent Administrative Leged Industrial Technology Complex Inst's patent center separately, monoclonal antibody 3D3 preservation is " mouse-Mouse Hybridoma Cells 3D3(preserving number: FERMP-22223) ", monoclonal antibody 14B1 preservation is " mouse-Mouse Hybridoma Cells 14B1(preserving number: FERMP-22225) ", and monoclonal antibody 8A6 preservation is " mouse-Mouse Hybridoma Cells 8A6(preserving number: FERMP-22224) ".
2. detection method of the present invention
Use the coated latex particle of above-mentioned monoclonal antibody " 14B1 " and monoclonal antibody " 3D3 " coated latex particle, the evaluation of MMP-3 detection system (also referred to as this product).Moreover combine the test that uses above-mentioned monoclonal antibody " 8A6 ", verify the independently superiority of the detection method of the present invention that relates to monoclonal antibody " 14B1 " and " 3D3 ".
As the further reagent of use relatively, use by the commercially available MMP-3 of enzymoimmunoassay (ELISA method) and detect reagent (パ Na Network リ ア MMP-3 " plate " first length of schooling that becomes more meticulous: also referred to as by approval products A) and detect reagent (パ Na Network リ ア MMP-3 " latex " first length of schooling that becomes more meticulous: also referred to as by approval product B) by the commercially available MMP-3 of LTIA method.Testing sample uses serum testing sample.
Use following machine.
7170S type automatic analysing apparatus (Hitachi, Ltd's system) (device using in the mensuration of LTIA reagent)
JCA-BM8060 automatic analysing apparatus (NEC company system) (device using in the mensuration of LTIA reagent)
AccuFLEXEL4000(ア ロ カ company system) (device using in the mensuration of ELISA reagent)
(1) repeatability test
Use detection system of the present invention, carry out the test to while repeatability and diurnal inequality repeatability.The results are shown in Fig. 1.The left representation result of repeatability simultaneously in Fig. 1, right coordinate graph diurnal inequality repeatability.Particularly, use the sample of 2 kinds of concentration, measure while each 10 times of repeatability, measured each 5 times of diurnal inequality repeatability through 5 days.
As shown in Figure 1, be no matter in repeatability test at the same time, or in the test of diurnal inequality repeatability, coefficient of alteration CV(%) all in 3%, the repeatability of detection system of the present invention is good.
(2) discussion of measurement range
Fig. 2 is the figure showing by the measurement range of the detection gauge in detection system of the present invention (left coordinate diagram) and dilution rectilinearity (right coordinate diagram).
Particularly, about detecting gauge, sample is carried out to the dilution of 10 stages with physiological saline, respectively measure 10 times.Then calculate mean value and standard deviation (SD), obtain detection gauge by ± 2SD method.About dilution rectilinearity, the high value sample of about 1300ng/ml is carried out to the dilution of 10 stages and formation determination sample with physiological saline.
As shown in Figure 2, the detection gauge to MMP-3 of learning detection system of the present invention is the left coordinate diagram of 8.9ng/ml(), the upper limit is 1200ng/ml.
(3) confirmation of front band
Fig. 3 is the figure that shows the result of the confirmation of carrying out prozone phenomenon in detection system of the present invention (suppressing phenomenon by the reaction due to antigen or antibody excessive).Particularly, superelevation value sample is carried out to the dilution of 5 stages and formation determination sample.
As shown in Figure 3, to MMP-3 concentration 5000ng/ml, measured value does not fall within the scope of said determination, can confirm that prozone phenomenon does not affect measurement result conventionally in people originates the mensuration of blood testing sample.
(4) discussion of the impact of coexisting substances
Fig. 4 is the figure that shows the result of the discussion of the coexisting substances in detection system of the present invention.
Particularly, add and interfere material to the sample of 2 kinds of concentration, calculate mean value ± 10% of the measured value of adding concentration 0 concentration (measuring for 10 times).In the time that the measured value of each interpolation concentration is being added in mean value ± 10% of concentration 0 concentration, be judged as measured value without impact.
As shown in Figure 4, do not find the impact coexisting to following concentration: sequestered cholerythrin is 20.0mg/dl, put together type cholerythrin is that 20.0mg/dl, haemolysis haemoglobin are that 500.0mg/dl, chyle (having the solution of the lipid of high concentration) are that 200 ℉ TU, rheumatoid factor are 500.0IU/ml.
(5) with the correlativity of being approved products A that uses ELISA method
Fig. 5 is the figure that inquires into detection system of the present invention and used the correlativity of being approved products A of ELISA method.
By testing sample normal saline dilution, while inquiring into its dilution rectilinearity, MMP-3 extremely about 1200ng/ml obtains good rectilinearity.This product can be confirmed as well (testing sample number 60) by regression equation y=1.002x-1.2, correlation coefficient r=0.995 with the correlativity of being approved products A.
(6) with the relation (1) of being approved product B that uses LTIA method
Fig. 6 is the blood testing sample that is obtained negative value in approval product showing by LTIA method, the figure of the result of inquiring into by detection method of the present invention.
With repeatedly being confirmed that by approval product B the measured value of MMP-3 becomes the example of negative value in low value scope.Thereby, other MMP-3 detection systems of 28 testing samples that show negative value in described goods are measured, carry out result comparison.
As the coordinate diagram in the left side from Fig. 6, by approve in product B be negative value testing sample whole this product and use ELISA method approved products A in become on the occasion of.In addition, right side coordinate diagram shows the result of the testing sample of described negative value being inquired into the correlativity of being approved products A and this product with normal saline dilution.Result is learnt, in this product of this scope and good by the correlationship of approval products A.By approval products A due to using ELISA method as measuring principle, thereby it is good to measure sensitivity, but confirms to have problem in the rapid property of measuring and testing sample processing power.Result learns, good in mensuration sensitivity and the ELISA method equal extent of detection system of the present invention, and confirms rapid property and testing sample processing power by the good mensuration of LTIA method.
(7) with the relation (2) of being approved product B that uses LTIA method
Fig. 7 is by detection system of the present invention (this product) with by the correlationship of approval product B, for confirm 60 testing samples less than negative value, the figure obtaining in the dilution with saline solution by physiology in using by the mensuration of approval product B.
As shown in the figure, learn in these 60 testing samples, in by approval product B and this product, confirm good correlationship.
Use the feature of the detection method of the present invention of LTIA method to be confirmed to be, measurement range is with the roughly wide scope of 10～1200ng/ml of MMP-3 densimeter.10～800ng/ml left and right by approval products A.Also learn in addition, also can carry out being diluted by the testing sample of physiological saline.Thereby measure (reexamining rate) that minimizing exceedes boundary by mensuration becomes possibility again, can expect in the rising that checks on-the-spot operating efficiency.In addition, detection system of the present invention with use that to measure the correlativity by approval products A of the good ELISA method of sensitivity good, and, as use LTIA method by approval product B, confirm to become less than MMP-3 the phenomenon of negative value in low value region.Thereby learn, detection method of the present invention has simplicity and correctness concurrently, there is the applicable reagent performance being suitable for very much in daily inspection.
(8) discussion of the further conspicuousness of detection system of the present invention
In the manufacturing process of the above-mentioned monoclonal antibody for MMP-3, for the present invention in the monoclonal antibody " 3D3 " that relates to and " 14B1 " together, among the final candidate of working load, the situation of the latex particle of residual " 8A6 " is inquired into.
; although be negative value what use LTIA method in approving product B; the use monoclonal antibody " 3D3 " relating in the present invention and the above-mentioned latex particle system of " 14B1 " and being approved in products A by ELISA method; for 3 testing samples that MMP-3 detected, in the pilot system that combines respectively the latex particle of " 3D3 " and the latex particle of " 14B1 " to the latex particle of load monoclonal antibody " 8A6 ", carry out the detection of MMP-3.And, carry out the combination by the latex particle of " 3D3 " of the present invention and the latex particle of " 14B1 ", utilize the detection of being approved product B of being approved products A, use LTIA method of ELISA method.
The results are shown in table 1.Have, while using 7170S type automatic analysing apparatus (Hitachi, Ltd's system) to measure, negative value represents with " 0 ", in by approval product B, has confirmed negative value again.
Table 1
The result of table 1 can be confirmed: compared with pilot system of the present invention, use the detected value of the pilot system of monoclonal antibody " 8A6 " to have the tendency uprising brokenly.As mentioned above, monoclonal antibody " 8A6 " be with the present invention in the final candidate that together screens of the monoclonal antibody " 3D3 " that relates to and " 14B1 ".That is, the result demonstration of this test, the detection method that uses latex particle of the present invention is not that the monoclonal antibody in the hybridoma source by being obtained by disclosed screening technique realizes uniformly.
Claims (3)
1. according to the detection method of latex agglutination, the people Transin-1 of the aggegation wherein following antigen-antibody reaction being caused in input blood testing sample, the aggegation of described antigen-antibody reaction is that the latex particle by load being had to monoclonal antibody 14B1 has the latex particle of monoclonal antibody 3D3 to contact generation with blood testing sample with load.
2. detection reagent, it is claimed in claim 1 according to the detection method of latex agglutination for carrying out.
3. detection kit, it is claimed in claim 1 according to the detection method of latex agglutination for carrying out.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011-255600 | 2011-11-22 | ||
| JP2011255600 | 2011-11-22 | ||
| PCT/JP2012/080097 WO2013077332A1 (en) | 2011-11-22 | 2012-11-20 | Method for detecting matrix metalloproteinase-3 by latex agglutination method |
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| Publication Number | Publication Date |
|---|---|
| CN103874925A true CN103874925A (en) | 2014-06-18 |
| CN103874925B CN103874925B (en) | 2016-08-31 |
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| CN201280050617.3A Expired - Fee Related CN103874925B (en) | 2011-11-22 | 2012-11-20 | Method according to latex agglutination detection Transin-1 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105974105A (en) * | 2016-05-16 | 2016-09-28 | 河北艾驰生物科技有限公司 | Matrix metalloproteinase-3(MMP3) detection kit |
| CN106872685A (en) * | 2017-03-31 | 2017-06-20 | 上海华臣生物试剂有限公司 | The preparation method of MMP3 kits |
| CN106950382A (en) * | 2017-03-22 | 2017-07-14 | 苏州普瑞斯生物科技有限公司 | MMP3 determines reagent and preparation method 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 |
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| US20060257420A1 (en) * | 2002-04-26 | 2006-11-16 | Cel-Sci Corporation | Methods of preparation and composition of peptide constructs useful for treatment of autoimmune and transplant related host versus graft conditions |
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| US7659125B2 (en) * | 2003-03-24 | 2010-02-09 | Mitsubishi Kagaku Latron, Inc. | Latex reagent for adiponectin analysis and method of adiponectin analysis |
| KR101433974B1 (en) * | 2006-07-24 | 2014-08-25 | 세키스이 메디칼 가부시키가이샤 | Method of measuring anticoagulation and reagent for measuring anticoagulation |
| JP4853666B2 (en) * | 2007-05-30 | 2012-01-11 | Ｊｓｒ株式会社 | Target substance detection method, mixed particles, and target substance detection reagent |
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| US20060257420A1 (en) * | 2002-04-26 | 2006-11-16 | Cel-Sci Corporation | Methods of preparation and composition of peptide constructs useful for treatment of autoimmune and transplant related host versus graft conditions |
| CN101283278A (en) * | 2005-10-06 | 2008-10-08 | 霍夫曼-拉罗奇有限公司 | Anti-CCP and antinuclear antibodies in diagnosis of rheumatoid arthritis |
| WO2007074860A1 (en) * | 2005-12-28 | 2007-07-05 | Sekisui Medical Co., Ltd. | Reagent for measuring aggregation and method of measuring aggregation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105974105A (en) * | 2016-05-16 | 2016-09-28 | 河北艾驰生物科技有限公司 | Matrix metalloproteinase-3(MMP3) detection kit |
| CN106950382A (en) * | 2017-03-22 | 2017-07-14 | 苏州普瑞斯生物科技有限公司 | MMP3 determines reagent and preparation method thereof |
| CN106872685A (en) * | 2017-03-31 | 2017-06-20 | 上海华臣生物试剂有限公司 | The preparation method of MMP3 kits |
| 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 |
Also Published As
| Publication number | Publication date |
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| WO2013077332A1 (en) | 2013-05-30 |
| JPWO2013077332A1 (en) | 2015-04-27 |
| CN103874925B (en) | 2016-08-31 |
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