CN103376327A - Method for detecting concentration of antibody or fusion protein - Google Patents
Method for detecting concentration of antibody or fusion protein Download PDFInfo
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- CN103376327A CN103376327A CN2012101304467A CN201210130446A CN103376327A CN 103376327 A CN103376327 A CN 103376327A CN 2012101304467 A CN2012101304467 A CN 2012101304467A CN 201210130446 A CN201210130446 A CN 201210130446A CN 103376327 A CN103376327 A CN 103376327A
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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
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/525—Tumor necrosis factor [TNF]
Abstract
The invention relates to a method for detecting concentration of an antibody or fusion protein. The method comprises the following steps of providing a test strip comprising a sample part, a joint part, a detection part and a control part, wherein the joint part comprises a control protein capable of being combined with a crystallizable fragment of the antibody or the fusion protein and an identification matter combined with the control protein, the detection part comprises an antigen of the antibody or the fusion protein, and the control part comprises a control antibody corresponding to the control protein; contacting a sample solution comprising the antibody or the fusion protein with the sample part; transferring the sample solution from the sample part and passing through the control part; detecting the signal strength of the detection part; and determining the concentration of the antibody or the fusion protein in the sample solution according to a formula of the concentration and the signal strength of the antibody or the fusion protein.
Description
Technical field
The present invention relates to detect the method for the concentration of antibody or fusion.
Background technology
Antibody and fusion are used to treat disease.For example, trastuzumab (Trastuzumab) is the anti-HER2/neu acceptor monoclonal antibody of the very fast expansion breast cancer of a kind of ErbB2 of inhibition, and it is used to treat this type of cancer.Infliximab (Infliximab) is a kind of monoclonal antibody of resisting tumor necrosis factor-alpha, is used to treat autoimmune disease.
It is a kind of method of making antibody or fusion that cell is cultivated, but wants the long period.Whether in any time in this process, antibody or fusion all may produce the variation that changes its immunity, occur to observe to change so need to constantly monitor the process of cell cultivation.And one of method that the monitoring cell is cultivated is exactly to detect the concentration of antibody in the cell culture or fusion.
Most of prior art can only detect the existence of antibody or fusion, but can't detect their quantity (concentration).For example, United States Patent (USP) discloses a kind of signal that produces by the formation of detectable antigens-antibody complex for No. 6841159, confirms to exist at least in patient's humoral sample the antibody of an antagonism antigen of mycobacterium.
United States Patent (USP) discloses mobile two light sources that are radiated on the test-strips for No. 6394952 can obtain two light sources in the reflectance ratio of each point, it is plotted figure, can be respectively on the peak that test section and the control part of test-strips obtains reflectance ratio, can draw the concentration of the fetal fibronectin in the sufferer sample based on the area ratio on two peaks.Reflection coefficient reads by the reflection coefficient reader, and the signal of test section and control part all needs to read the concentration that could determine the fetal fibronectin in the sufferer sample.In addition, the reagent of joint portion is the specific antibodies for analyte (fetal fibronectin) on the test-strips that United States Patent (USP) is mentioned for No. 6394952, so, when analyte changes, the joint portion also needs to adjust accordingly, and this is very inconvenient in some specific applied environments.
Eva Engvall and Peter Perlmann in July, 1972 at THE JOURNAL OF IMMUNOLOGY magazine (Vol.109, No.1) deliver one piece of article that is called " ENZYME-LINKED IMMUNOSORBENT ASSAY; ELISA III.Quantitation of Specific Antibodies by Enzyme-Labeled Anti-Immunoglobulin in Antigen-Coated Tubes ", wherein mention a kind of method that detects antibody quantity.The method needs at least 22 hours culture period, so very consuming time.
Therefore, be necessary to develop method new or improved detection antibody or fusion concentration.
Summary of the invention
The purpose of this invention is to provide a kind of method new or improved detection antibody or fusion concentration.
On the one hand, the method that the present invention relates to comprises: the test-strips that comprises sample section, joint portion, test section and control part is provided, wherein said joint portion comprises the control albumen that can combine with the FC of described antibody or fusion and the sign material that combines with described control albumen, described test section comprises the antigen of described antibody or fusion, and described control part comprises the control antibody corresponding with described control albumen; The sample solution that will comprise described antibody or fusion contacts with described sample section; Allow described sample solution from described sample section's migration and by described control part; Detect the signal intensity of described test section; And the concentration of determining antibody described in the described sample solution or fusion according to concentration and the formula between the signal intensity of antibody or fusion.
Detection antibody involved in the present invention or the method for fusion concentration have solved the technical matters of prior art.
Description of drawings
Be described for embodiments of the invention in conjunction with the drawings, the present invention may be better understood, in the accompanying drawings:
Fig. 1 is the blast perspective diagram of the test-strips that provides according to one embodiment of present invention.
Fig. 2 is the schematic top plan view of test-strips among Fig. 1.
Fig. 3 is the chart that obtains in the example 5.
Fig. 4 is the chart that obtains in example 6 and the Comparative Examples 1.
Embodiment
Approximate term in the instructions is used for modifying quantity, and expression the present invention is not limited to this concrete quantity, also comprises the part of the correction of the change that can not cause relevant basic function with approach acceptable of this quantity.Accordingly, modify a numerical value with " approximately ", " pact " etc., mean and the invention is not restricted to this accurate numerical value.In some example, approximate term may be corresponding to the precision of the instrument of measuring numerical value.
In the following description book and claim, unless clearly point out in addition, single plural number is not limited.
Mentioned " can ", " can ", " possibility " and " can be " of the application is illustrated in the possibility that occurs under the certain environment; Character with appointment, the possibility of feature or function; And/or by showing that one or more ability, performance are suitable for another kind of action, the perhaps possibility relevant with this action that is fit to.Therefore, the term that is used for " can ", " can ", " possibility " and " can be " expression modification obviously suitable, can or be suitable for represented ability, function, perhaps purposes, consider simultaneously in some cases, the term of modifying may be not suitable for sometimes, can not or improper.For example, in some cases, event or ability may be desired, and in other cases, this event or ability can not occur.This difference is contained by term " can ", " can ", " possibility " and " can be ".
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings, will can not describe well-known function and structure in detail, to avoid becoming pondered-over because unnecessary details makes the present invention.
Please refer to the drawing 1 and Fig. 2, the test-strips 1 that provides according to one embodiment of the present of invention comprises: sample section 2, joint portion 3, test section 4, and control part 5.Joint portion 3 comprises the control albumen that can combine with the FC of antibody or fusion (not shown) and the sign material (not shown) that combines with control albumen.Test section 4 comprises the antigen (not shown) of antibody or fusion.Control part 5 comprises the control antibody (not shown) of controlling albumen.
In certain embodiments, test-strips 1 comprises absorbent portion 6, and the distance between itself and the test section 4 is greater than the distance between itself and the control part 5.
Test-strips 1 can be contain any absorbent material rectangular, and sample solution can adsorb thereon and along its migration.Sample section 2, joint portion 3, test section 4, control part 5 and absorbent portion 6 can be made by same or different materials.The sample section 2 of test-strips 1, joint portion 3, test section 4, control part 5 and absorbent portion 6 can be integrally formed, also can combine mutually respectively.
In certain embodiments, test-strips 1 can comprise by the picture polyester
The perhaps back pad made of the plastic material such as PET is to be used for supporting sample section 2, joint portion 3, test section 4, control part 5 and absorbent portion 6.
In certain embodiments, sample section 2 can be by a glass fibre bar being immersed the solution contain bovine serum albumin(BSA) (BSA), Tween 20 and phosphate buffered saline (PBS), then be dried and sample pad.
In certain embodiments, joint portion 3 can be dry absorbed behind the binding soln the glass fibre bar and pad.Binding soln comprises the control albumen that can combine with the FC of antibody or fusion and the sign material that combines with control albumen.
The sign material can be arbitrary physics or chemistry sign material, and it can be detected at solid carrier with optical instrument, and the detected antibody on test-strips 1 test section 4 or fusion can be distinguished with other compounds and material and be come.The sign material can be but be not limited to, by reacting colorific enzyme-substrate combination, coloured particulate (for example rubber particle), colloidal metal, metal melten gel, carbon melten gel, fluorescent material and lipid somatocyst or polymerization somatocyst.In certain embodiments, aurosol is used as the sign material in the binding soln.
Control albumen can be at least a in albumin A or the Protein G.Control albumen combines with the sign material, and when sample solution moved through joint portion 3, control albumen was combined with the FC of antibody or fusion.When detecting the concentration of antibody or fusion, change analyte, namely antibody or fusion must not cause controlling the replacing of albumen and sign material, so the joint portion can be general in the process that detects antibody or fusion concentration, therefore reduce the cost that designs and produces test-strips.
T line solution comprises the antigen of antibody or fusion.For example, in certain embodiments, antibody or fusion are tumor necrosis factor-alpha antibody, and antigen is tumor necrosis factor-alpha albumen.
C line solution comprises the control antibody corresponding with control albumen, and in certain embodiments, control albumen is albumin A, and control antibody is albumin A antibody (anti-albumin A).
In certain embodiments, sample section 2, joint portion 3, barrier film 8 and absorbent portion 6 are assembled together.For guaranteeing sample solution from 2 migrations of sample section and by control part 5,3 whiles and sample section 2 and film 8 crossovers of joint portion, film 8 is also overlapping with absorbent portion 6.
In certain embodiments, test-strips 1 can be contained in the plastic casing (not shown).In certain embodiments, sample section 2 may extend to outside the plastic casing.
Sample solution can be the solution that comprises antibody or fusion.Sample solution can be the solution that is made by antibody or fusion.In certain embodiments, sample solution can be got by the dilution of the cell culture in the cultured cell process.Except antibody or fusion, sample solution can comprise BSA and PBST (PB/NaCl that contains Tween20) damping fluid etc.
Sample solution and sample section 2 are by contacting such as sample solution being added to sample section 2 or sample section 2 being immersed the mode such as sample solutions.
Sample solution moves forward from sample section 2 under capillary action.When sample solution during through joint portion 3, antibody or fusion be in its FC and control protein combination that some have been combined with the sign material, thereby drive those control albumen and sign continues to move forward together.
When sample solution moved to test section 4, antibody or fusion combined with antigen, thereby stayed in test section 4.The unnecessary sign material that does not combine with antibody or fusion and control albumen is then along with sample solution continues to move forward, thereby combines and rest on control part 5 with control antibody.
The signal that produces at the sign material of test section 4 and control part 5 can be with the naked eye or optical instrument observation.The generation of signal shows that sample solution passes through and arrival control part 5 from test section 4 in the control part 5.
Available any optical instrument is measured the signal intensity of the sign material of test section 4.The signal intensity of test section 4 can be the output valve of optical instrument.In certain embodiments, signal intensity is the peak height of reading with the strip image analyzer.
Antibody or the fusion solution of concentration known are moved along test-strips, and detect the signal intensity of corresponding test section, thereby set up the concentration of antibody or fusion and the formula between the test section signal intensity.Signal intensity by formula and test section 4 calculates the concentration of antibody in the sample solution to be measured or fusion.
Make sample solution contact and move along it with test-strips 1, then use the concentration of antibody or fusion and the concentration that the formula between the test section signal intensity is determined antibody or fusion, whole process required time is shorter.Therefore, very simple and quick.Can find out that from the aftermentioned example result that the result that the method that the present invention relates to draws and enzyme linked immunosorbent assay (ELISA) method obtains is complementary, therefore very reliable.The control albumen of joint portion 3, for example at least a in albumin A and the Protein G can combine with the FC of antibody or fusion, so, the joint portion does not need to change because of the change of antibody or fusion, thereby the cost that manufactures and designs test-strips can be saved.
Experimental example
Following experimental example implements the invention provides further guidance for those skilled in the art.Example does not limit the scope of the present invention that defines in claims.
Example 1
Nano Au particle solution (Au-NP with 1 milliliter, 40nm) (from the fast clever bio tech ltd of Chinese Shanghai) joins in little centrifugal separating tube of 1.5 milliliters, then add the 0.2M solution of potassium carbonate (from Chinese Shanghai traditional Chinese medicines chemical reagent company limited) of 5ul, and rotate little centrifugal separating tube so that solution wherein fully mixes.After adding in addition 4ul albumin A (5mg/ml is from Hangzhou China Long Ji Bioisystech Co., Ltd), rotate immediately the abundant mixing of little centrifugal separating tube, then leave standstill and wait for 10 minutes.10% bovine serum albumin(BSA) (BSA is from the Belgian Acros Organics company) aqueous solution that then adds 100ul is rotated to leave standstill behind the abundant mixing of little centrifugal separating tube and is waited for 10 minutes.Then, with the speed centrifuging of 11000rpm 10 minutes.Remove the upper strata after the stillness of night, containing of 100uL is dissolved with 1/1000 bovine serum albumin(BSA) (BSA, from Belgian Acros Organics company) phosphate buffered saline (PBS) (PBS, pH value is 7.4, with the acquisition that is dissolved in the water of dibastic sodium phosphate, sodium dihydrogen phosphate and sodium chloride) again aaerosol solution add in a subtle way in the centrifugal separating tube, again to dissolve the aurosol particle (Protein A-AuNP) of being combined with albumin A, to obtain albumin A-AuNP solution.
With glass fibre bar (33 glass, wide 0.8cm, long 4cm, from the General Electric of N.J. medical treatment life science company) put into and 20uL albumin A-AuNP solution and 180uL are housed in conjunction with little centrifugal separating tube of dilution buffer liquid (0.01M PBS is dissolved with 3% trehalose, 0.5%BSA and 1%Tween 20), placed 1 to 2 minute until all solution are adsorbed on the glass fibre bar uniformly.Then the glass fibre bar is put into stove, with dry 3 hours of 37 ℃ of temperature, obtain pad.
Example 2
Glass fibre bar (33 glass, wide 1.5cm, long 4cm) is put into the sample pad solution (0.01M PBS is dissolved with 1%BSA and 0.5%Tween 20) of 350uL, until soak into, then the glass fibre bar is put into stove, with dry 3 hours of 37 ℃ of temperature, to obtain sample pad.
Example 3
A FF85 nitrocellulose membrane (from the General Electric medical treatment life science company of N.J.) is affixed on the wherein one side of DB-6 plastics adhesion back pad (from the outstanding biological Science and Technology Ltd. of Chinese Shanghai).
The Egg-white A antibody (3mg/ml, anti-albumin A is from Hangzhou Long Ji Bioisystech Co., Ltd) of 20ul is mixed with the 0.01M PBS (the pH value is 7.2) of 40ul, to obtain C line solution (the anti-albumin A solution of 1mg/ml).
With recombinant human tumor necrosis factor-alpha albumen (1mg/ml) solution (from the Bo Aosen of BeiJing, China scientific ﹠ technical corporation) as T line solution.
With the ratio of BioDot XYZ-3050 divider (from the BioDot Inc. in California, USA Irving city) with 0.5ul/cm T line solution and C line solution are added to respectively on the test section and control part of FF85 nitrocellulose membrane.
Example 4
470 cotton papers from the General Electric of N.J. medical treatment life science company are adhered to an end of DB-6 plastics adhesion back pad, as absorption pad, and make it cover 1 to 2 millimeter of FF85 nitrocellulose membrane one end.Distance between absorption pad and the test section is greater than the distance between itself and the control part.
The pad of preparation in the example 1 is positioned over the other end of DB-6 plastics adhesion back pad and 1 to 2 millimeter of the covering FF85 nitrocellulose membrane other end.
The sample pad of preparation in the example 2 is adhered to DB-6 plastics adhesion back pad and makes its 1-2 millimeter that covers pad one end, to obtain the combination bar.To make up bar with cutting machine (ZQ2000, Chinese Shanghai gold mark bio tech ltd) and be divided into the wide test-strips of 4mm.
Example 5
(be dissolved with 0.1%Tween 20 in the 0.1M PB/0.01M sodium chloride with the PBST damping fluid that is dissolved with 0.5%BSA, pH value is 7.4) dilute anti-tumor necrosis factor in the ELISA kit that Chinese Shanghai Mei En Bioisystech Co., Ltd provides-α sample, obtain that 8 parts of concentration are respectively 0, the sample solution of 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL and 80ng/mL.
This 8 duplicate samples solution (every part of 150 μ L) is added respectively in eight holes of 96 orifice plates.The sample pad of one test-strips is inserted also vertical test-strips in each hole so that sample solution is up mobile along it.After 10 minutes, test-strips is put into strip image analyzer (DT2030 of Chinese Shanghai gold mark bio tech ltd) to read the signal intensity of T line, namely the signal peak height value of test section.The concentration of anti-tumor necrosis factor-α is as transverse axis in the sample solution, take the signal intensity (peak height) of T line as the longitudinal axis, draw two-dimensional diagram as shown in Figure 3, obtain formula: y=16.825ln (x)-10.285 between the signal intensity of the concentration of anti-tumor necrosis factor-α and T line, wherein y is signal intensity, x is the concentration of anti-tumor necrosis factor-Alpha antibodies in the sample solution, the degree of correlation (R
2) be 0.9606.
Example 6
Collect tumor necrosis factor-alpha antibody cell culture samples at the second day (incubation time: 1 day) that cell is cultivated every day to the 7th day (incubation time: 6 days), (dilution ratio of second day and the 3rd day sample was as 1: 100 take different dilution ratios; The 4th day be 1: 200; The 5th day be 1: 400; The 6th day be 1: 600; The 7th day be 1: 800) dilute to prepare 6 duplicate samples solution with the PBST damping fluid that is dissolved with 0.5%BSA.
The sample solution of every part of 150uL is poured in the hole of 96 orifice plates.The sample pad of one test-strips is inserted also vertical test-strips in each hole so that sample solution is up mobile along it.After 10 minutes, test-strips is put into strip image analyzer (DT2030 of Chinese Shanghai gold mark bio tech ltd) to read the signal intensity of T line, namely the signal peak height value of test section.
The formula that obtains in the signal intensity of the T line that reads with the strip image analyzer and the example 5 calculates the concentration of tumor necrosis factor-alpha antibody in the sample solution in 96 orifice plates.The concentration of tumor necrosis factor-alpha antibody in the sample solution is obtained the concentration of tumor necrosis factor-alpha antibody in the corresponding cell culture sample divided by the dilution ratio when preparing corresponding sample solution.Corresponding different cell incubation times (my god) the cell culture sample in the concentration of tumor necrosis factor-alpha antibody be shown in lines 1 among Fig. 4.
Comparative Examples 1
With with example 6 in same mode make 6 duplicate samples solution.According to the Standard Operating Procedure of the ELISA kit of Chinese Shanghai Mei En Bioisystech Co., Ltd, come the concentration of tumor necrosis factor-alpha antibody in the test sample solution with this ELISA kit.The concentration of tumor necrosis factor-alpha antibody in the sample solution is obtained the concentration of tumor necrosis factor-alpha antibody in the corresponding cell culture sample divided by the dilution ratio when preparing corresponding sample solution.Corresponding different cell incubation times (my god) the cell culture sample in the concentration of tumor necrosis factor-alpha antibody be shown in lines 2 among Fig. 4.
As can be seen from Figure 4, the concentration data that obtains in the concentration data that obtains in the example 6 and the Comparative Examples 1 is complementary.
Although in embodiment, Partial Feature of the present invention is had been described in detail and describes, under the prerequisite that does not break away from spirit of the present invention, can carry out various changes and replacement to the present invention.Same, those skilled in the art also can obtain other change disclosed by the invention and equivalent according to normal experiment.All these change, and replacement and equivalent are all within the design and scope of the defined claim of the present invention.
Claims (10)
1. a method that detects the concentration of antibody or fusion is characterized in that, comprising:
The test-strips that comprises sample section, joint portion, test section and control part is provided, wherein said joint portion comprises the control albumen that can combine with the FC of described antibody or fusion and the sign material that combines with described control albumen, described test section comprises the antigen of described antibody or fusion, and described control part comprises the control antibody corresponding with described control albumen;
The sample solution that will comprise described antibody or fusion contacts with described sample section;
Allow described sample solution from described sample section's migration and by described control part;
Detect the signal intensity of described test section; And
The concentration of determining antibody described in the described sample solution or fusion according to concentration and the formula between the signal intensity of antibody or fusion.
2. the method for the concentration of detection antibody as claimed in claim 1 or fusion, it is characterized in that the concentration of described antibody or fusion and the formula between the signal intensity are by allowing the antibody of concentration known or fusion solution set up by test-strips and the signal intensity that detects corresponding test section.
3. the method for the concentration of detection antibody as claimed in claim 2 or fusion is characterized in that, described signal intensity is the signal peak height of strip image analyzer output.
4. such as the method for the concentration of the described detection antibody of arbitrary claim in the claims 1 to 3 or fusion, it is characterized in that described antibody or fusion are tumor necrosis factor-alpha antibody.
5. the method for the concentration of detection antibody as claimed in claim 4 or fusion is characterized in that, described antigen is tumor necrosis factor-alpha albumen.
6. such as the method for the concentration of the described detection antibody of arbitrary claim in the claims 1 to 3 or fusion, it is characterized in that it further comprises: the cell culture of collecting when using cell to cultivate prepares described sample solution.
7. such as the method for the concentration of the described detection antibody of arbitrary claim in the claims 1 to 3 or fusion, it is characterized in that described test-strips comprises absorbent portion, the distance between itself and the test section is greater than the distance between itself and the control part.
8. the method for the concentration of detection antibody as claimed in claim 1 or fusion is wherein controlled albumen and is at least a in albumin A or the Protein G.
9. the method for the concentration of detection antibody as claimed in claim 8 or fusion, wherein controlling albumen is albumin A.
10. the method for the concentration of detection antibody as claimed in claim 9 or fusion wherein identifies material and derives from aurosol.
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| CN2012101304467A CN103376327A (en) | 2012-04-28 | 2012-04-28 | Method for detecting concentration of antibody or fusion protein |
| PCT/SE2013/050449 WO2013162456A1 (en) | 2012-04-28 | 2013-04-24 | Method for detecting concentration of antibody or fusion protein |
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| CN104964973A (en) * | 2015-07-08 | 2015-10-07 | 邓双胜 | Test paper reading and analyzing method and system based on mobile terminal camera |
| CN109068971A (en) * | 2016-01-14 | 2018-12-21 | 黛尔格诺斯蒂尔有限公司 | The method for measuring the tear component in ocular fluid samples |
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| WO2016178083A2 (en) | 2015-05-01 | 2016-11-10 | Diagnostear, Ltd. | Method for measuring tear constitutents in a tear sample |
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| WO2013162456A1 (en) | 2013-10-31 |
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