CN104374911B - A kind of detection by quantitative rheumatoid factor, the detection kit of antistreptolysin O (ASO) - Google Patents
A kind of detection by quantitative rheumatoid factor, the detection kit of antistreptolysin O (ASO) Download PDFInfo
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- CN104374911B CN104374911B CN201410738142.8A CN201410738142A CN104374911B CN 104374911 B CN104374911 B CN 104374911B CN 201410738142 A CN201410738142 A CN 201410738142A CN 104374911 B CN104374911 B CN 104374911B
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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Abstract
The present invention relates to field of biological detection, particularly relate to detection kit of a kind of detection by quantitative rheumatoid factor, antistreptolysin O (ASO) and its production and use.The present invention provides a kind of detection kit, including the most independent rheumatoid factor Test paper card and antistreptolysin O (ASO) Test paper card, described rheumatoid factor Test paper card and antistreptolysin O (ASO) Test paper card the most each include base plate and be positioned at backplate surface start, from sample-adding end, the sample pad, fluorescent marker pad, nitrocellulose filter and the adsorptive pads that are arranged in order.Rheumatoid factor in rheumatism three, antistreptolysin O (ASO) are detected by detection kit provided by the present invention first by fluorescent micro-ball immune chromatography technology simultaneously, the content of rheumatoid factor in sample, antistreptolysin O (ASO) just can be detected by being once loaded operation, simplify operating process, have susceptiveness and specificity, quick and precisely assessment rheumatoid arthritis clinical symptoms concurrently.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of detection by quantitative rheumatoid factor, antistreptolysin O (ASO)
Detection kit and its production and use.
Background technology
Rheumatoid factor (rheumatoid factor, RF) can be divided into IgM, IgA, IgG, IgD, IgE five type (note: facing
It is four types described in bed internal medicine, there is no IgD type；But it is 5 types described in the laboratory diagnosis), it is rheumatoid arthritis blood
For a class autoantibody of epitope in IgG FC fragment in Qing, rheumatoid factor positive patient is more with joint appearance
Existing, such as subcutaneous nodule and vasculitis etc..IgM type RF positive rate is 60%-78%.
After body infects A group B streptococcus because of pharyngitis, tonsillitis, scarlet fever, erysipelas, pyoderma, rheumatic fever etc., can produce
Streptolysin O antibody, i.e. " Anti-Streptolysin O (ASO) ".
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of detection by quantitative rheumatoid factor, anti-chain
Detection kit of pneumoniae pneumolysin O and its production and use, is used for solving the problems of the prior art.
For achieving the above object and other relevant purposes, the present invention provides a kind of detection by quantitative rheumatoid factor (RF), anti-hammer
The detection kit of bacterium hemolysin O (ASO), including the most independent rheumatoid factor Test paper card and streptococcus haemolysis
Element O Test paper card, described rheumatoid factor Test paper card and antistreptolysin O (ASO) Test paper card the most each include the end
Plate and be positioned at the sample pad starting to be arranged in order from sample-adding end of backplate surface, fluorescent marker pad, nitrocellulose filter
And adsorptive pads, the fluorescent marker pad of described rheumatoid factor Test paper card comprises people degeneration IgG, described anti-hammer
Hammer bacteriolysin O (SLO), described each celluloid is comprised on the fluorescent marker pad of bacterium hemolysin O Test paper card
Being coated with detection line and nature controlling line on film, hammer bacteriolysin O and people degeneration IgG on described fluorescent marker pad use glimmering
Light microsphere labelling.
Preferably, so that test kit has more preferably sensitivity and color developing effect, heretofore described fluorescent marker knot
Close pad also pre-treatment buffer used in pretreatment, pretreatment and include following component: stachyose, Alumen, fructose two
Sodium phosphate, sodium hexameta phosphate and glycine, and stachyose, Alumen, Fructose Diphosphate sodium, sodium hexameta phosphate, glycine is total
Concentration is 3.5-7.5g/L, and the pH value of buffer is 7.2-7.6.
Preferably, each component concentration in buffer is:
The solvent of described pre-treatment buffer is water.
Concretely comprising the following steps of described pretreatment: fluorescent marker pad soaks in pretreatment fluid 1.5-2.5h, and taking-up is put in
36-38 DEG C of drying.
Described pre-treatment buffer can use the various conventional pH adjusting agent in this area to carry out the regulation of pH value.
Preferably, described base plate is PVC base plate.
Preferably, on described nitrocellulose filter, detection line is positioned at side close to sample-adding end, and nature controlling line is positioned at from sample-adding end relatively
Remote side.
Preferably, the detection line of described rheumatoid factor Test paper card is coated with people degeneration IgG..
Preferably, the detection line of described antistreptolysin O (ASO) Test paper card is coated with streptolysin O.
Preferably, nature controlling line is coated sheep anti-mouse antibody.
Preferably, also include getting stuck, described in get stuck and include that back card and upper cover, described back card are provided with two parallel test card draw-in grooves,
Described test card is embedded in described test card draw-in groove respectively, described on be covered with two testing windows and two wells, said two
Match with detection line and the position of nature controlling line of two test cards respectively in the position of testing window, the position of said two well with
Match in the position of the sample pad of two test cards.
Get stuck described in it is furthermore preferred that and get stuck for plastics.
It is furthermore preferred that be additionally provided with the translot connecting two wells between said two well.
Preferably, described detection kit detects rheumatoid factor, the content of antistreptolysin O (ASO) for simultaneous quantitative.
Detection by quantitative rheumatoid factor provided by the present invention, the detection kit of antistreptolysin O (ASO) use double when detection
Antigen sandwich immunochromatographic method, supporting immune quantitative analytical tool uses.Immunoassay instrument passes through acquisition testing line (T) and matter
Control line (C) band fluorescence signal, calculates T/C signal value.First various criterion product are added drop-wise on test card before using, analyze
Calibration curve (T/C signal value and the relation of standard substance actual value) is set up in process, then by the T/C value obtained when detecting sample and mark
Directrix curve compares, and can obtain the rheumatoid factor in detection sample, the content of antistreptolysin O (ASO).
Second aspect present invention provides the preparation of the detection kit of described detection by quantitative rheumatoid factor, antistreptolysin O (ASO)
Method, comprises the steps:
1) SLO and people's degeneration IgG solution with fluorescent microsphere labelling spray antistreptolysin O (ASO), rheumatoid factor respectively
Fluorescent marker pad；
2) on the detection line and nature controlling line of antistreptolysin O (ASO) nitrocellulose filter, SLO and sheep anti-mouse antibody are sprayed respectively,
The detection line and nature controlling line of rheumatoid factor nitrocellulose filter spray people degeneration IgG and sheep anti-mouse antibody respectively；
3) nitrocellulose filter prepared by the fluorescent marker pad two set sample pad, steps 1 prepared, step 2, water suction
Pad is pasted onto on respective base plate successively, and cutting prepares Test paper card；Finally being snapped fits into by Test paper gets stuck prepares detection examination
Agent box.
Preferably, so that test kit has more preferably sensitivity and color developing effect, heretofore described fluorescent marker knot
Close pad also pre-treatment buffer used in pretreatment, pretreatment and include following component: stachyose, Alumen, fructose two
Sodium phosphate, sodium hexameta phosphate and glycine, and stachyose, Alumen, Fructose Diphosphate sodium, sodium hexameta phosphate, glycine is total
Concentration is 3.5-7.5g/L, and the pH value of buffer is 7.2-7.6.
Preferably, each component concentration in buffer is:
The solvent of described pre-treatment buffer is water.
Concretely comprising the following steps of described pretreatment: fluorescent marker pad soaks in pretreatment fluid 2h, takes out and is put in 37 DEG C
Dry.
Third aspect present invention provide described detection by quantitative rheumatoid factor, antistreptolysin O (ASO) detection kit at class wind
The wet factor, the purposes of antistreptolysin O (ASO) detection field.
Detection by quantitative rheumatoid factor provided by the present invention, antistreptolysin O (ASO) detection kit first by rheumatism three
In rheumatoid factor, antistreptolysin O (ASO) detected, by once adding by fluorescent micro-ball immune chromatography technology simultaneously
Sample operation just can detect the content of rheumatoid factor in sample, antistreptolysin O (ASO), simplifies operating process, has spirit concurrently
Quick property and specificity, quick and precisely assessment rheumatoid arthritis clinical symptoms.Additionally, described detection kit has operation quickly
The advantages such as easy, result is accurate, economic and practical, little by the serious blood fat of serum (or blood plasma), haemolysis interference, when serum (or
Blood plasma) hemoglobin≤600mg/L, triglyceride≤100mg/dL time, bilirubin≤20mg/dL, the impact on accuracy
Variation ＜ 10%.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by disclosed by this specification
Content understand other advantages and effect of the present invention easily.The present invention can also be added by the most different detailed description of the invention
To implement or application, the every details in this specification can also be based on different viewpoints and application, in the essence without departing from the present invention
Various modification or change is carried out under god.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific
Specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments,
Rather than in order to limit the scope of the invention；In description of the invention and claims, unless literary composition additionally clearly refers to
Going out, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, two end points of each numerical range with
And any one numerical value all can be selected between two end points.Unless otherwise defined, all technology used in the present invention and section are academic
The same meaning that language and those skilled in the art of the present technique are generally understood that.In addition to the concrete grammar used in embodiment, equipment, material,
According to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use and the embodiment of the present invention
Described in method, any method, equipment and the material of the similar or equivalent prior art of equipment, material realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use the art normal
Rule molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell cultivate, recombinant DNA technology and
The routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the series
METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS
IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
The preparation of test card of the present invention:
1) using pre-treatment buffer that fluorescent marker pad is carried out pretreatment, pre-treatment buffer is: stachyose 2g/L,
Alumen 0.5g/L, Fructose Diphosphate sodium 1.5g/L, sodium hexameta phosphate 0.3g/L, the aqueous solution of glycine 1.88g/L, pH=7.4,
Concretely comprising the following steps of pretreatment: fluorescent marker pad soaks in pretreatment fluid 2h, takes out and is put in 37 DEG C of drying；With
SLO and people's degeneration IgG buffer solution of appropriate fluorescent microsphere labelling spray pretreated fluorescent marker pad respectively,
Preparing two kinds of fluorescent marker pads, in solution, fluorescent microsphere is 5:1 with the mass ratio of label, and the concentration of solution is
10mg/ml, quantity for spray is 4ul/cm；
2) on the detection line and nature controlling line of nitrocellulose filter, appropriate SLO and sheep anti-mouse antibody solution are sprayed respectively, and
Be coated with appropriate people degeneration IgG and sheep anti-mouse antibody solution, prepare two kinds be coated after nitrocellulose filter, the concentration of spray solution
For 1mg/ml, quantity for spray is 1ul/cm；
3) nitrocellulose filter, adsorptive pads prepared by the fluorescent marker pad sample pad, step 1 prepared, step 2 depend on
Secondary being pasted onto on respective PVC base plate, cutting prepares rheumatoid factor Test paper card and the streptococcus haemolysis of wide 3-5mm
Element O Test paper card；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
Normal line curve:
Respectively by concentration be 0,20,50,100,150,200,250,300,400, the rheumatoid factor of 500ng/mL delays
Dissolved drop is added in sample pad, and each concentration sets 5 repetitions (testing result takes 5 meansigma methodss repeated), film chromatography 10
After minute, immunoassay instrument is used to pass through acquisition testing line (T) and nature controlling line (C) band fluorescence signal, analyser
Detection range to fluorescence signal is AD value 0-10000, calculates T/C signal value, sets up rheumatoid factor calibration curve, wherein
Y-axis is T/C signal value, and X-axis is standard substance actual value.
Respectively by concentration be 0,20,50,100,150,200,250,300,400, the streptococcus haemolysis of 500pg/mL
Element O buffer solution drips in sample pad, and each concentration sets 5 repetitions (testing result takes 5 meansigma methodss repeated), film
After chromatographing 10 minutes, immunoassay instrument is used to pass through acquisition testing line (T) and nature controlling line (C) band fluorescence signal,
Calculating T/C signal value, set up antistreptolysin O (ASO) calibration curve, wherein Y-axis is T/C signal value, and X-axis is standard substance
Actual value.
Rheumatoid factor, the detection of antistreptolysin O (ASO) content anti-interference:
Being added in sample pad by detection sample drop, each sample sets 5 repetitions (testing result takes 5 meansigma methodss repeated), film
After chromatographing 10 minutes, the T/C value obtained during by detection sample compares with standard curve, it is thus achieved that detect rheumatoid in sample because of
Son, the detection data of antistreptolysin O (ASO) content, then rheumatoid factor, the antistreptolysin O (ASO) of detection acquisition are contained
Amount data contrast with true rheumatoid factor, antistreptolysin O (ASO) content data, it is thus achieved that accuracy affects deviation value.
Sample 1:50ng/mL rheumatoid factor, 300pg/mL antistreptolysin O (ASO) content, 600mg/L hemoglobin,
100mg/dL triglyceride, 10mg/dL bilirubin；
Sample 2:100ng/mL rheumatoid factor, 200pg/mL antistreptolysin O (ASO) content, 500mg/L hemoglobin,
50mg/dL triglyceride, 15mg/dL bilirubin；
Sample 3:150ng/mL rheumatoid factor, 150pg/mL antistreptolysin O (ASO) content, 80mg/L hemoglobin,
20mg/dL triglyceride, 20mg/dL bilirubin；
Sample 4:200ng/mL rheumatoid factor, 100pg/mL antistreptolysin O (ASO) content, 150mg/L hemoglobin,
30mg/dL triglyceride, 4mg/dL bilirubin；
Sample 5:300ng/mL rheumatoid factor, 50pg/mL antistreptolysin O (ASO) content, 300mg/L hemoglobin,
80mg/dL triglyceride, 9mg/dL bilirubin；
Blank control sample: 300mg/L hemoglobin, 80mg/dL triglyceride, 9mg/dL bilirubin serum sample.
The rheumatoid factor content data of the detection that sample 1-5 is obtained be respectively 48ng/mL, 102ng/mL, 149ng/mL,
198ng/mL, 315ng/mL, antistreptolysin O (ASO) content be respectively 290pg/mL, 202pg/mL, 145pg/mL,
95pg/mL, 52pg/mL, accuracy affect variation ＜ 10%, blank does not finds that obvious fluorescence signal changes.
Embodiment 2
The preparation of comparative example test card: only changing pre-treatment buffer formula, the pre-treatment buffer of comparative example test card is 25mM
Glycine buffer, pH=7.4, other steps are all identical with preparation process in embodiment 1.
Rheumatoid factor, the susceptiveness of antistreptolysin O (ASO) and detection limit contrast experiment:
Judging with fluorescent instrument, the detection range to fluorescence signal of analyser is AD value 0-10000, according to the property of instrument
Can, CUTOFF value is 50, under certain concentration, and more than 90% detection example AD value >=50, i.e. think that test kit can be used in
Detection under this concentration.
Use 5%BSA normal saline solution should not contain measured object as dummy, dummy.Take 0.3-5.3ng/mL ladder
The known blood sample of rheumatoid factor of degree concentration carries out susceptiveness detection, arranges a gradient at interval of 0.2ng/mL, and each gradient sets
Put 20 samples, record testing result.Result display test card lowest detection prepared by embodiment 1 is limited to 0.5ng/mL,
The lowest detectable limit of comparative example test card is higher than 5.3ng/mL.
Use 5%BSA normal saline solution should not contain measured object as dummy, dummy.Take the anti-of 1-50pg/mL
The known blood sample of streptolysin O carries out susceptiveness detection, arranges a gradient at interval of 1pg/mL, and each gradient arranges 20
Individual sample, records testing result.Result display test card lowest detection prepared by embodiment 1 is limited to 3pg/mL, and comparative example is tried
The lowest detectable limit of paper card is higher than 50pg/mL.
In sum, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any it is familiar with this skill
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage of art.Therefore, such as
All that in art, tool usually intellectual is completed under without departing from disclosed spirit and technological thought etc.
Effect is modified or changes, and must be contained by the claim of the present invention.
Claims (6)
1. a detection kit for detection by quantitative rheumatoid factor, antistreptolysin O (ASO), including the most independent rheumatoid because of
Sub-Test paper card and antistreptolysin O (ASO) Test paper card, described rheumatoid factor Test paper card and streptococcus
Hemolysin O Test paper card the most each include base plate and be positioned at backplate surface start, from sample-adding end, the sample that is arranged in order
Pad, fluorescent marker pad, nitrocellulose filter and adsorptive pads, the fluorescent labeling of described rheumatoid factor Test paper card
Comprise people degeneration IgG on thing pad, the fluorescent marker pad of described antistreptolysin O (ASO) Test paper card wraps
Containing hammer bacteriolysin O, described each nitrocellulose filter is coated with detection line and nature controlling line, described fluorescent marker pad
On hammer bacteriolysin O and people degeneration IgG use fluorescent microsphere labelling, the detection of described rheumatoid factor Test paper card
It is coated with people degeneration IgG on line, the detection line of described antistreptolysin O (ASO) Test paper card is coated with streptococcus haemolysis
Element O, described each nature controlling line is coated sheep anti-mouse antibody；
Described each fluorescent marker pad also pre-treatment buffer used in pretreatment, pretreatment includes following group
Point: stachyose, Alumen, Fructose Diphosphate sodium, sodium hexameta phosphate and glycine, and stachyose, Alumen, Fructose Diphosphate sodium,
Sodium hexameta phosphate, the total concentration of glycine are 3.5-7.5g/L, and the pH value of buffer is 7.2-7.6, and each component is in buffer
Concentration be:
The solvent of described pre-treatment buffer is water.
2. detection by quantitative rheumatoid factor, the detection kit of antistreptolysin O (ASO) as claimed in claim 1, its feature exists
In, on described each nitrocellulose filter, detection line is positioned at side close to sample-adding end, and nature controlling line is positioned at from sample-adding end farther out
Side.
3. detection by quantitative rheumatoid factor, the detection kit of antistreptolysin O (ASO) as claimed in claim 1, its feature exists
In, also include getting stuck, described in get stuck and include that back card and upper cover, described back card are provided with two parallel test card draw-in grooves, described
Test card is embedded in described test card draw-in groove respectively, described on be covered with two testing windows and two wells, said two is surveyed
Match with detection line and the position of nature controlling line of two test cards respectively in the position of examination window, the position of said two well with
Match in the position of the sample pad of two test cards.
4. detection by quantitative rheumatoid factor, the detection kit of antistreptolysin O (ASO) as claimed in claim 3, its feature exists
In, it is additionally provided with the translot connecting two wells between said two well.
5. detection by quantitative rheumatoid factor, the detection kit of antistreptolysin O (ASO) as claimed in claim 1, its feature exists
In, described detection kit detects rheumatoid factor, the content of antistreptolysin O (ASO) for simultaneous quantitative.
6. the preparation method of the detection kit as described in claim 1-5 any claim, comprises the steps:
1) with the streptolysin O of fluorescent microsphere labelling and people's degeneration IgG solution spray respectively antistreptolysin O (ASO),
Rheumatoid factor fluorescent marker pad；
2) spray respectively on the detection line and nature controlling line of antistreptolysin O (ASO) nitrocellulose filter streptolysin O and
Sheep anti-mouse antibody, sprays people degeneration IgG and sheep anti mouse on the detection line and nature controlling line of rheumatoid factor nitrocellulose filter respectively
Antibody；
3) nitrocellulose filter prepared by the fluorescent marker pad two set sample pad, steps 1 prepared, step 2, water suction
Pad is pasted onto on respective base plate successively, and cutting prepares Test paper card；Finally being snapped fits into by Test paper gets stuck prepares detection examination
Agent box.
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| CN104931706B (en) * | 2015-05-02 | 2017-06-13 | 王贤俊 | A kind of antistreptolysin O detection kit and its preparation |
| CN112285358A (en) * | 2020-09-17 | 2021-01-29 | 上海基灵生物科技有限公司 | Reagent card for dog C-reactive protein and pancreas specific lipase duplex detection |
| CN114236135A (en) * | 2021-12-15 | 2022-03-25 | 上海捷门生物技术有限公司 | Test strip for detecting anti-streptolysin O antibody |
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| CN102707050A (en) * | 2012-05-24 | 2012-10-03 | 蓝十字生物药业（北京）有限公司 | Colloidal gold test strip for fast detecting rheumatoid factors |
| CN203732542U (en) * | 2013-10-17 | 2014-07-23 | 上海科新生物技术股份有限公司 | Test paper card and diagnostic kit for detecting rheumatoid arthritis |
| CN203838159U (en) * | 2014-02-26 | 2014-09-17 | 郑州浪峰生物技术有限公司 | Colloidal gold test paper strip for detecting anti-cyclic citrullinated peptide antibodies and rheumatoid factors |
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Effective date of registration: 20180111 Address after: 400000 Taikang Road Dadukou District of Chongqing Bridge Industrial Park C District No. 6 Building 30 layer 1-4 Patentee after: Chongqing Zhongyuan Ji Ji Biological Technology Co., Ltd. Address before: Jiulongpo Branch Park four street 400039 Chongqing city 70-1, 70-2 block J No. six Patentee before: CHONGQING QIANDE BIOTECHNOLOGY CO., LTD. |
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Address after: Floor 1-4, building 30, no.6, Taikang Road, Zone C, Jianqiao Industrial Park, Dadukou District, Chongqing 400000 Patentee after: Zhongyuan Huiji Biotechnology Co.,Ltd. Address before: Floor 1-4, building 30, no.6, Taikang Road, Zone C, Jianqiao Industrial Park, Dadukou District, Chongqing 400000 Patentee before: CHONGQING ZHONGYUAN HUIJI BIOTECHNOLOGY Co.,Ltd. |