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CN105759024A - Calibration method of reading equipment for reading immunity test device - Google Patents

Calibration method of reading equipment for reading immunity test device Download PDF

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Publication number
CN105759024A
CN105759024A CN201610117982.1A CN201610117982A CN105759024A CN 105759024 A CN105759024 A CN 105759024A CN 201610117982 A CN201610117982 A CN 201610117982A CN 105759024 A CN105759024 A CN 105759024A
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equipment
color
value
standard
aod
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CN105759024B (en
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刘伟
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ABON Biopharm Hangzhou Co Ltd
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ABON Biopharm Hangzhou Co Ltd
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/29—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using visual detection
    • G01N21/293—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using visual detection with colour charts, graduated scales or turrets
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/4875—Details of handling test elements, e.g. dispensing or storage, not specific to a particular test method
    • G01N33/48771—Coding of information, e.g. calibration data, lot number

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
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  • Cell Biology (AREA)
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  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Spectrometry And Color Measurement (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

The invention provides a calibration method of reading equipment for reading an immunity test device. The calibration method is characterized by comprising the following steps: (1) reading at least first color gradation, second color gradation and third color gradation on a standard color card by using standard equipment to obtain AOD original values; (2) reading at least the first color gradation, the second color gradation and the third color gradation on the standard color card by using to-be-calibrated equipment to obtain the AOD original values; and (3) drawing a first calibration curve by using the AOD values of the first color gradation and the second color gradation of the standard equipment and the AOD values of the first color gradation and the second color gradation of the calibrated equipment; and carrying out first-time calibration on the calibrated equipment by use of the first calibration curve to obtain the value of the second color gradation of the calibrated equipment. When the equipment is used for testing the tested result of a test area on a test reagent strip, the obtained result is more accurate, and the test precision is higher.

Description

A kind of calibration steps reading equipment reading immunoassay device
This application is Chinese patent application, the divisional application of application number 201310332981.5.
Technical field
The present invention is about the calibration steps of a kind of detecting device, especially, the present invention relates to the calibration steps that immunologic function test reagent bar carries out optical detection apparatus.
Background technology
The statement of background of the present invention is only used to help the reader understanding present invention, and does not constitute the description to prior art of the present invention or elaboration.
In quick diagnosis field, existing much utilizes test strips or test board to detect sample (such as saliva, urine or blood etc.) in the device of analyte, these test strips or test board and reading equipment with the use of, may be integrally incorporated to is a non-removable entirety together, it is also possible to be split-type structural.The result of analyte contained in the sample detected in test strips is reflected on coupled electronic equipment by these reading equipment, the digitized mode of result is made to obtain, the method of relative gross visualization is more objective, and test result have can preserve, the advantage such as electric transmission.Such as, reading equipment includes optical element, such as CCD camera, obtain the figure on test device by optical element, install or be connected to counting circuit, passing through counting circuit, testing result in test strips (test device) is carried out further data conversion and calculating, obtain result more easy to identify, such as patent application US10/741, described in 416.In other embodiments, detecting device is connected with general computer, is undertaken converting further and digital independent by testing result by the relative program in setting computer, so conveniently make the user of large usage quantity, such as medical institutions etc..These read equipment in such as Chinese invention patent application 201210132692.6, and 201310025671.9, and U.S. Patent application US20050168747, US20070134812 have concrete description.These electronic equipments that testing result in test strip is digitized process generally comprise light-emitting device and send light and be irradiated in test strip and photoelectric detector detection light of reflection or scattering from reagent strip, and some CPU.The reading equipment also having includes CCD or COMS image acquisition device, gathers image by image acquisition device, then carries out image processing the test result obtained in test strip.
At the reading equipment carrying out batch production reagent strip, how realizing the concordance between reading equipment, the error as far as possible reduced between arranging is the important factor affecting stabilization of equipment performance.Every equipment is due to each parts; such as electronic component; the machine error etc. of the establishment that each element is installed; the equipment produced often is allowed to there is certain error at initial loading for after completing; if error exceedes acceptable scope between these equipment; the test result read on immunoassay bar can be caused and do not prepare, the shortcomings such as precision is not high.It is generally required to equipment is calibrated, allow every equipment all meet unified standard, so allow the equipment between same lot number and the equipment between different lot number be maintained in a range of error as far as possible, and meet a standard.
Summary of the invention
The present invention provides a kind of calibration steps reading equipment, it is possible to be calibrated between the reading device of the testing result of reading immunoassay bar, it is possible to allow and have good concordance between these equipment, improve the stability of detection equipment and the accuracy of test.
On the one hand, the present invention provides a kind of calibration steps reading equipment, and the method includes: a kind of calibration steps reading equipment reading immunoassay device, it is characterised in that the method comprises the following steps:
(1), read at least first, second, and third color range on standard color card with standard device and obtain AOD original value；
(2), read first, second, and third color range at least described on standard color card with the equipment intending being calibrated and obtain AOD original value；
(3), the first calibration curve is done by the AOD value of the AOD value of the first and second the two of standard device color ranges with the first and second color ranges being calibrated equipment；With the first calibration curve, the equipment being calibrated is done first time to calibrate；
(4), intend the equipment that is calibrated by the data of the AOD value of the second color range after calibrating for the first time and the AOD data on original tertiary color rank and Standard Machine second and the 3rd original AOD value matching the second calibration curve, of the second calibration curve the equipment being calibrated made and calibrate for the second time.
In some preferred schemes, on described colour atla, the second color range is between first and tertiary color rank.
In other preferred modes, described marking arrangement and the equipment intending being calibrated include optical pickup unit.
In other preferred modes, calibration steps according to claim 3, it is characterised in that described optical pickup unit includes COMS or CCD element.In other preferred modes, described AOD value is the meansigma methods of multiple value.It is characterized in that in other preferred modes, the selection of described marking arrangement carries out by the following method: respectively the reading equipment of more than 3 is tested by first, second, and third color range on described standard colour examining card, continues the AOD value of at least 1 day；By the analysis result of data, CV value is minimum and minimum as standard device with the deviation of other device A OD averages.
In other preferred modes, described immunoassay device includes test zone and marked region.In other preferred modes, described test zone includes antibody or the antigen fixed, marked region includes be with coloured granule.In other preferred modes, the coloured granule of described band is gold colloid particles or latex colloidal solid.In other preferred modes, described immunoassay device also includes being positioned at the sample areas of marked region upstream and being positioned at the testing result control area in test zone downstream.In other preferred modes, wherein said test zone is positioned on cellulose nitrate film.
In other preferred modes, the color value of the first color range of described standard color card is G3, and the color value of the second color range is G4, and the color value on tertiary color rank is G6.
On the other hand, the calibration steps reading equipment reading immunoassay device is planted, it is characterised in that the method comprises the following steps:
(1), read at least first, second, and third color range on standard color card with standard device and obtain AOD original value；
(2), read first, second, and third color range at least described on standard color card with the equipment intending being calibrated and obtain AOD original value；
(3), the first calibration curve is done by the AOD value of the AOD value of the first and second the two of standard device color ranges with the first and second color ranges being calibrated equipment；With the first calibration curve, the equipment being calibrated is done first time and calibrate the value of the second color range obtaining the equipment that is calibrated.
Preferably, if the second color range value obtained by step (3) is more than the second color range value of standard device；Then this equipment being calibrated is carried out secondary calibration.Preferably, the method for secondary calibration is as follows: with the AOD value and Standard Machine second and the 3rd original AOD value matching the second calibration curve that are calibrated the second color range after equipment is calibrated by first and tertiary color rank；With the second calibration curve, the equipment being calibrated is done second time to calibrate.
Preferably, on described colour atla, the second color range is between first and tertiary color rank.
Preferably, described marking arrangement and the equipment intending being calibrated include optical pickup unit.Described optical pickup unit includes COMS or CCD element.
Preferably, described AOD value is the meansigma methods of multiple value.Preferably, the selection of described marking arrangement carries out by the following method: respectively the reading equipment of more than 3 is tested by first, second, and third color range on described standard colour examining card, continues the AOD value of at least 1 day；By the analysis result of data, CV value is minimum and minimum as standard device with the deviation of other device A OD averages.
Preferably, the color value of the first color range of described standard color card is G3, and the color value of the second color range is G4, and the color value on tertiary color rank is G6.
On the other hand, the present invention provides a kind of calibration steps reading equipment reading immunoassay device, it is characterised in that the method comprises the following steps:
The AOD value of first, second, and third original color range of standard color card is obtained with the equipment intending being calibrated；
Bring first time calibration curve into and obtain the first calibration value of the machine of being calibrated；If the second value obtained is more than the second value of standard device, the original value obtained is brought into the second calibration curve and again calibrates.
In some preferred modes, it is thus achieved that the mode of the first calibration curve is:
Read at least first, second, and third color range on standard color card with standard device and obtain AOD original value；
Read first, second, and third color range at least described on standard color card with the equipment intending being calibrated and obtain AOD original value；The first calibration curve is done by the AOD value of the AOD value of the first and second the two of standard device color ranges with the first and second color ranges being calibrated equipment.
In some preferred modes, it is thus achieved that the mode of the second calibration curve is: with the AOD value and Standard Machine second and the 3rd original AOD value matching the second calibration curve that are calibrated the second color range after equipment is calibrated by first and tertiary color rank.
In aforementioned all of embodiment, read, with standard device, the AOD original value that on standard color card, at least first, second, and third color range obtains and be stored in a memory carrier.Memory carrier is any medium that can be read by equipment, for instance disk, (3 dimension cards, on the medium such as one-dimensional card for 2D two dimension card, USB disk.
Beneficial effect
The method of invention, it is possible to be effectively improved the concordance between instrument.Especially, when with such come instrument carry out the test result of test zone on test agent bar time, it is thus achieved that result more accurate, measuring accuracy is higher.
Accompanying drawing explanation
Fig. 1 is the structural representation of the immunochromatography reagent bar in one detailed description of the invention of the present invention
Fig. 2 is standard color card schematic diagram, and wherein Fig. 2 A used in the present invention includes 10 schematic diagrams without the standard color card of color range；Fig. 2 B is the standard color card schematic diagram used in the specific embodiment of the invention；
Fig. 3 is 3 primary calibration curves of G3, G4, G6 of an embodiment standard equipment 0023 and the 0018 equipment matching being calibrated, it is thus achieved that calibrating curve equation formula be y (0023)=0.1811+1.020X (0018)；
Fig. 4 is that an embodiment Plays sets 0023 standby 3 primary calibration curves of G3, G4, G6 with the 0021 equipment matching being calibrated；The calibrating curve equation formula obtained is y (0023)=0.5113+0.9745X (0020)
Fig. 5 is that an embodiment Plays sets 0023 standby 2 primary calibration curves of G3, G6 with the 0018 equipment matching being calibrated；The calibrating curve equation formula obtained is y (0023)=0.0.03819+1.026X (0018)
Fig. 6 is that an embodiment Plays sets 0023 standby 2 primary calibration curves of G3, G6 with the 0018 equipment matching being calibrated；The calibrating curve equation formula obtained is y (0023)=-0.2304+1.017X (0020)
Fig. 7 is the linear relationship chart between the color range of colour atla used in the present invention and AOD value；
Fig. 8 is that an embodiment Plays sets 0023 standby G3, G4 2 the first time calibration curve with the 0018 equipment matching being calibrated；The calibrating curve equation formula obtained is y (0023)=0.4748+0.9365X (0018)
Fig. 9 is that an embodiment Plays sets 0023 standby G3, G4 2 the first time calibration curve with the 0021 equipment matching being calibrated；The calibrating curve equation formula obtained is y (0023)=-0.9206+1.398X (0020)
Figure 10 is the G4 of 0018 equipment AOD average by the G4 value after calibrating for the first time and original G6 and reference instrument in an embodiment, the second time calibration curve of G6 matching, it is thus achieved that calibrating curve equation formula be y (0023)=-0.2194+1.045X (0018).
Figure 11 be in an embodiment 0021 equipment by the second time calibration curve of the AOD average of the G4 value after calibrating for the first time and original G6 with the AOD average matching of G4, the G6 of reference instrument.
Figure 12 is G3, G4 2 the first time calibration curve of an embodiment standard equipment 0023 and equipment 0018 matching being calibrated；
Figure 13 is G3, G4 2 the first time calibration curve of an embodiment standard equipment 0023 and equipment 0021 matching being calibrated；
Figure 14 be in an embodiment 0018 equipment by the second time calibration curve of the G4 value after calibrating for the first time, AOD average and the AOD average matching of G4, the G6 of standard device of G6.
Figure 15 be in an embodiment 0021 equipment by the second time calibration curve of the AOD average of G4, G6 after calibrating for the first time with G4, the G6 matching of reference instrument.
Figure 16 is the flow chart that equipment Alignment obtains first time and the second calibration curve.
Figure 17 is that software design is to carry out the flow chart of equipment Alignment.
Description of reference numerals
Test zone 30, testing result control area 40, sample absorbance region 50, carrier 20, marked region 60, sample applies region 10, color range 100.
Detailed description
Test device
" test device " described in the present invention refers to that those pass through reaction chemically or physically, it is possible to detection or the equipment of analyte in assay samples, such device can be test strip (Fig. 1), includes test device or the test agent of test strip.Test device generally comprises some test agent, these test agent and analyte carry out direct or indirect reaction, then color change or other changes occur on test device, the change thereby through naked eyes or machine, the result of the test zone on test device judged or test zone is occurred is analyzed, thus obtaining the quantity representing in sample, whether analyte exists or exist.
A lot of immunoassay device are well known to those skilled in the art for detecting analyte in sample.It is commonly used about the polypeptide in detection patient's sample or albumen, immunoassay device and method, sees United States Patent (USP) 6,143,576；6,113,855；6,019,944；5,985,579；5,947,124；5,939,272；5,922,615；5,885,527；5,851,776；5,824,799；5,679,526；5,525,524；With 5,480,792, each patent content is by complete listed in reference, including all of form, figure and claim.These equipment and method can utilize various labelling macromole in sandwich assay, produce the signal existed or quantity is relevant to target analyte with competition or non-competing test format.It addition, someway and equipment, for instance biosensor and optics immunodetection can not need the macromole of label just can detect the quantity of presence of analyte or existence.See United States Patent (USP) 5,631,171；With 5,955,377, each patent content is by complete listed in reference, including all of form, figure and claim.Those skilled in the art think that test device includes but not limited to Beckman, Abbott Laboratories AxSym, Roche ElecSys, and the immune detection system of DadeBehring tomographic system can carry out immune detection described here.
Preferably, immunodetection evaluation of markers thing, although additive method is also (such as measurement markers thing rna level) well known to those skilled in the art, but most preferably sandwich immunoassay.By the specific antibody of correspondence markings thing and the quantity detecting the specific binding existence that can usually detect label or existence thereof.The immunity of label and specific antibody combines and can be detected directly or indirect detection.Such as immunodetection, biological detection analysis needs the method for detection, and the most frequently used quantitative method is to combine a kind of enzyme, fluorophor or other macromolecular complex mass-energy to form antibody-label thing.Detectable label thing includes the macromolecular substances (such as fluorophor, electrochemical label, metallo-chelate etc.) inherently can being detected, also include produce detectable response product indirect detectable molecule (such as enzyme is as horseradish peroxidase, alkali phosphatase etc.) or by detectable binding molecule specific bond (such as biotin, digoxin, a maltose, oligohistidine, 2,4-dinitro benzenes, phenylarsonic acid, ssDNA, dsDNA etc.).Particularly preferred detectable is such as United States Patent (USP) 5,763,189,6,238,931, and 6,251,687 and international publication WO95/08772 described in fluorescent latex grain, above-mentioned patent and publication are all by complete listed in reference.Demonstration conjugation in granule can be mentioned hereinafter.Including fluorescence or luminescence label, metal, dyestuff, radionuclide and analog direct label by and antibodies, indirect labels includes the enzyme of various this areas numerical value, for instance alkali phosphatase, horseradish peroxidase and analog.
Utilize the antibody fixed to carry out specific detection analyte and fall within the part of the present invention.Terminology used here " solid phase " is a Generalized Material, and it includes solid, semi-solid, gel, film, thin film, net, felts, complex, microgranule, reagent paper and analog etc., and those skilled in the art are generally used for the material of absorption macromole.Solid matter can atresia or porose.Suitable solid phase include those maturations and/or in solid phase binding detects as the material of solid phase.Such as, the reference entirely as the present invention of " immunoassay " or a part (see example chapter9ofImmunoassay, E.P.DianiandisandT.K.Christopouloseds., AcademicPress:NewYork).Suitable solid phase example includes film, filter, cellulose paper, bead (includes polymerization, the granule with paramagnetic of latex), glass, silicon chip, microgranule, nanoparticle, such as Tenta gel, Agro gel, PEGA gel, SPOCC gel, and porous disc (see, example, Leonetal., Bioorg.Med.Chem.Lett.8:2997,1998；Kessleretal.,Agnew.Chem.Int.Ed.40:165,2001；Smithetal.,J.Comb.Med.1:326,1999；Orainetal.,TetrahedronLett.42:515,2001；Papanikosetal.,J.Am.Chem.Soc.123:2176,2001；Gottschlingetal.,Bioorg.Med.Chem.Lett.11:2997,2001).Antibody can be fixed on various solid carrier, for instance magnetic or chromatographic grade matrix granule, detection plate surface (such as microwell plate), solid substrate material or film (such as plastics, nylon, paper) etc..By coating a kind of antibody or the antibody of multiple matrix arrangement on solid phase carrier, form test strip.These test strip are subsequently dipped in detection sample, then pass through to get express developed and can measure signal with detecting step generation, for instance mottle.When adopting Through Several Survey Measure, can producing a lot of addressable position dividually on single solid phase carrier, the corresponding different label in each position, each position includes the antibody being combined with these labels.Term " discrete " described here refers to region, discontinuous surface.That is to say, if being not belonging to the border in any one region entirely around each region in two regions, namely region, two pieces of surfaces is independent from, discrete.Terminology used here " absolute address " refers to mutually discrete region, surface, can obtain nonspecific signal on these areas.
In chromatography immunoassay device, generally comprise test zone 30, marked region 90.In some modes, test device also includes sample and applies region 10 and suction zone 50.Marked region includes mark substance, for instance gold colloidal, latex or colored particle.In other modes, test zone 30 is included on solid phase carrier 20, for instance film, filter, cellulose paper, and bead (includes polymerization, the granule with paramagnetic of latex), and glass, silicon chip, microgranule, on nanoparticle.In other modes, solid phase carrier is film, for instance nitrocellulose filter, nylon membrane etc..In some modes, test zone is fixed with the immunoreactive antibody of participation.In some modes, closing on of test zone can also arrange control area 40, and this region is for being made whether effective checking to the test result on test zone.
Test zone
Here say " test zone " refers to by the reading to test zone, it is possible to obtain represent whether analyte exists or there is the region of quantity in sample.Test device can there is multiple test zone, for the detection of different analytes on each test zone.In some modes, can also for the detection of different types of analyte on a test zone.In some embodiments, test zone may be located on the solid carrier in test device.The form of test section can be lines, point, speckle, block, the pattern of geometry shape or geometry symbol, for instance long for 0.5-1.5 centimetre, width is the lines of 0.2-5 millimeter.On test zone, can obtaining test result by human eye or instrument, this test result can directly and/or indirectly represent the quantity that whether there is analyte or existence in sample and/or the kind of analyte.Corresponding with test zone is exactly testing and control region, this region can to the result of test whether effectively and test device whether effectively control.
In some modes, by change Show Color chemically or physically, test zone represents whether one or more analytes in sample exist or and the quantity that exists.On these test zones, how Show Color is persons skilled in the art is known.Conventional is occur or fixing colored particle on test zone, for instance gold colloidal, nano-particle or latex particle, due to the accumulation or fixing of colored particle, occurs as soon as color at test zone.Generally, there is dependency in the quantity that there is analyte in the number of colored particle and sample.Except colored particle occurs, chemical reaction can also be occurred to produce color at test zone, for instance redox reaction to occur when having oxidation substrates, allows substrate generation color change.So also occur in that color at test zone.Equally, after chemical reaction, the depth of color also has dependency with the concentration of analyte in sample.
Here dependency can be positive correlation, it is also possible to for negative correlation.Such as, color is more deep or more dense, or colored particle is more many, or the light sent is more strong, and in corresponding sample, the quantity of analyte is also more many.On the contrary, color is more deep or more dense, or colored particle is more many, and the light sent is more strong, and in corresponding sample, the quantity of analyte also exists more less or not even.
Detailed description of the invention
By specific embodiment, the present invention is described further below, but the present invention is not constituted any restriction by these explanations.
Material:
1. standard color card (Fig. 2 A and Fig. 2 B): the using method of above standard color card: in reality is tested, generally the shade on test zone 30 on test agent bar is compared (passing through naked eyes) qualitatively with standard color card, close with which color in standard color card, it is how many for being considered as test result.In double antibody or antigen sandwich FAXIA, if the color in fruit test strip belongs to G1-G2, it is generally recognized that negative, if belonging to G3, sometimes thinking the positive, sometimes thinking feminine gender, sometimes need again to test, if test result belongs to G4-G11, it is considered that be positive result.Certainly, if competing method detection, color value is contrary with test result.Generally, the power of color is relevant to concentration in sample, and color is more strong, and in sample, concentration is more big；If competition immunologic detection method, color is more strong, and in sample, concentration is more little.Below this fermentation in all of examples of implementation, the standard color card described be all be numbered 0123 colour atla.
Time if, with the color value read on equipment read test bar on test zone 30, (AOD represents the transforming numerical that the picture signal of colored line is strong and weak to the AOD value of the test zone also being intended to allow reading equipment read, the basic essence of AOD value reading same lines or color range with same equipment is the same) and sample in the value of concentration be linear relationship, can detection by quantitative test value, it also is intended to by color reads the concentration in linear corresponding sample, it is of course also possible to the value in qualitative detection sample.When reading equipment is detected, general standard color card detects, see whether reading has good discrimination to the colored line (color range) on standard color card, such as can well distinguish G1 and G2, such as each color gradient in colour atla is had the differentiation of at least 99.9%, it is possible to achieve positive and negative 3SD does not have normal state intersection etc..If achieved, each lines (G1-G11) on colour atla had good discrimination, then the color on the test zone in test strip can also be had good discrimination.
2. read equipment:
The reading equipment of the present invention all adopts COMS photographing unit and coordinates software programming and program write and the setting of other hardware, allows the AOD value of colored line on COMS collecting test bar, then passes through the conversion with concentration, final obtains the value tested.All parts that apparatus above adopts are all same batch.
Although the software and hardware that they adopt all is tried one's best, maintenance is consistent, but need nonetheless remain for reading equipment is carried out unified calibration, allows the variance between the equipment after being calibrated less than 5% or other acceptable scope or values.
Table 1: definition
Reference Describe
G3,G4,G6,G8,G10 The color range grade (Fig. 2) of standard color card
C.V Coefficient of dispersion
SD Standard deviation
Examples of implementation 1: the selection of standard device
Randomly select 5 reading equipment (numbering: 0023,0025,0021,0018,0020), respectively 5 multifunction immunity detectors are tested with G3, G4, the G6 on standard colour examining card, continue within 5 days, to collect test data AOD value.Being seen by the analysis result of 5 day data, the CV value of No. 0023 machine is minimum (0.83%), and minimum with the SD deviation of other 4 instrument AOD averages.Finally selected No. 0023 machine is as Standard Machine (concrete test data are slightly).
Examples of implementation 2: the collection of the initial data of the equipment being calibrated and calibrator (-ter) unit
3 are read equipment (standard device 0023 and the equipment 0018 and 0020 being calibrated) and use standard test panel to survey card (Fig. 2) respectively to obtain original AOD value, it is thus achieved that raw value such as following table:
Table 2: the original AOD value of distinct device
After the equipment being calibrated being calibrated following with standard device, standard of comparison equipment and the number of degrees to colour atla of the equipment after being calibrated, see whether they meet the requirement of setting, for instance SD, CV value or differ% value.
Examples of implementation 3: use the standard device three-point calibration method to being calibrated equipment
1), do linear fit with 3 AOD averages of G3, G4, G6 and 3 the AOD averages of G3, G4, G6 being calibrated machine 0018 and 0020 of Standard Machine 0023, obtain linear fit calibration equation (such as Fig. 3 and 4).
The data analysis reading equipment after being calibrated by G3, G4, G6:
After G3, G4, G6 3 calibrates, No. 0020 equipment is at G3, the AOD value of G4 and 0023 Standard Machine G3, the difference (differ%) of G4AOD is still above 5% (respectively 11.4% and 6.97%), calibration result is less desirable, although the difference of the test value after No. 018 equipment Alignment and standard device meets the requirements.The equipment after calibrating that is obtained by of the value of AOD reads the AOD value of G3, G4, G6 color range on unified standard colour atla, and result is shown in following table.
Table 3: the comparative analysis of the AOD value of each equipment and standard device after calibration
The comparison of examples of implementation 4: two point calibration method
One, G3, G6 two point calibration method
1), with 2 AOD averages of G3, G6 of Standard Machine 0023 (AB130023-SET1) be calibrated machine 0018 (AB130018-SET1) and the G3 of 0020 (AB130020-SET1), 2 AOD averages of G6 do linear fit, obtain linear fit calibration equation (such as Fig. 5 and 6).
2), the data analysis after G3, G6 calibration:
After the calibration of G3, G6, No. 0020 equipment is at the AOD value of G4 and the difference differ% of the G4AOD of 0023 Standard Machine still above 5% (12.62%), and calibration result is less desirable.Although the numerical bias after the calibration of No. 0018 equipment is within the acceptable range (less than 5%).The equipment after calibrating that is obtained by of the value of AOD reads the AOD value of G3, G4, G6 color range on unified standard colour atla, and result is shown in following table.
Table 4: the comparison of the AOD value of each equipment and standard device after calibration
Two, G3 and G4；G4 and G6 two point calibration method
With reference to the above method that G3 and G6 is identical, to G4, G6；With G3, G4 2, the reading equipment (0020 and 0018) being calibrated being calibrated, 023 as standard machine.
After G4, G6 2 calibrates, 0020 and 0018 at the AOD value of G3 and the difference differ% of 0023 Standard Machine G3AOD still above 5%, calibration result is less desirable.Wherein, the difference differ% of 0018 is 7.35% in the level of G3, and the difference differ% of 0020 is-24.98% (concrete data is slightly) in the level of G3, but the difference on other color range G4 and G6 level is less than 5%.
After the calibration of G3, G4,0018,0020 at the difference differ% of G6AOD value and 0023 Standard Machine G6AOD still above 5%, calibration result less desirable (concrete data are slightly).Wherein, the difference differ% of 0018 is 6.46% in the level of G6, and the difference differ% of 0020 is-34.09% in the level of G3.
Conclusion:
Through the contrast of above 4 kinds of calibration steps, we can obtain as drawn a conclusion:
(1), G3G4G6 calibration curve--after calibration, the value of G3 and G4 is driven high, with the differ% of the average of adjusting machine more than 5%.
(2), after G3G6 calibration--after calibration, the value of the G4 of equipment and the differ% of the average of adjusting machine are more than 5%.
(3), G4G6 calibration--after calibration, the value of the G3 of equipment and the differ% of the average of adjusting machine are more than 5%.
(4), G3G4 calibration--after calibration, the value of the G6 of equipment and the differ% of the average of adjusting machine are more than 5%.
Examples of implementation 5: two repeatedly calibration steps
One, colour atla AOD value is analyzed
In order to solve problem above, the standard color card used has been carried out the mensuration of AOD by us, and we have surprisingly found that, the AOD value of these colour atla gradients is not linear relationship.Such as, in the figure 7, the color ladder distribution of G2-G3-G4-G6-G8 itself is not linear, and G3 and G4 gradient is little can be fitted to linearly, and G4 and G6, the gradient of G8 can be individually for greatly linearly, it is possible to the problem that secondary calibration on probation makes up primary calibration.
Two, second compensation calibration
Method one,
1), first it is fitted calibration curve with the AOD value of G3, the G4 of standard device 0023 to being calibrated equipment (0018 and 0020).By data fitting calibration curve part (Fig. 8 and Fig. 9) of G3G4.
2), then with G4, G6 fitted calibration curve of data and the original G6 data Yu Standard Machine 0023 that are calibrated equipment of the AOD value being calibrated the G4 after the calibration of equipment.(such as Figure 10 and 11):
The equipment being calibrated is after G3 and G4 calibrates, it is the G3 after the calibration that fitted calibration curve obtains again by the G4G6 data of the data of the G4 after calibration with original G6 data with Standard Machine 0023, G4, the G3 of G6AOD average and Standard Machine 0023, G4, the AOD value differ% of G6 is less than 2%, and calibration result is desirable, for acceptable standard.
Table 5: the equipment after calibration and standard device are to the comparative analysis of the AOD of color range on each standard color card
Method two,
With the G6 data value realization < 5% that the data of G6 can be withdrawn into Standard Machine by G4, the G6 data after G3, G4 fitted calibration after being calibrated with Standard Machine G4G6 fitted calibration curve.This method is more convenient.Need the average of record standard machine G3, G4, G6.Article 1 curve is simulated with Standard Machine G3, G4, for data one curve of matching again of G4 and G6 by being calibrated the value of machine G3, G4.Realize panchromatic degree approximate calibration.
1) by the data fitting calibration curve of G3, G4 value of the standard device equipment to being calibrated, it is specifically shown in Figure 12 and 13.
2) secondary calibration, does linear fit with primary standard machine G4, G6 again by the data after G4, G6 primary calibration, it is thus achieved that standard curve such as Figure 15 and 14.
Instrument is after G3 and G4 calibrates, then is the differ% with Standard Machine 0023 of G3, G4, the G6AOD average after the calibration that fitted calibration curve obtains less than 2% by the G4G6 data of G4 and the G6 data after calibration with Standard Machine 0023, and calibration result is desirable.It is specifically shown in following table.
Table 6: the equipment after calibration and standard device are to the comparative analysis of the AOD of color range on each standard color card
Embodiment 6, sensitive analysis
Utilize the equipment (0018) after calibration that the different color ranges of same group of standard color card carry out test and the 6 of G1-G8 gradient can be had the discrimination of 99.9%.Realizing +/-3SD does not have normal state to intersect.Result is shown in following table.
G1 G2 G3 G4 G6 G8
AVG_AOD 0.903 1.554 2.304 4.930 17.431 45.892
STDEV 0.072 0.044 0.063 0.076 0.152 0.069
CV 7.958% 2.799% 2.734% 1.540% 0.872% 0.150%
+3SD 1.119 1.685 2.493 5.158 17.886 46.098
-3SD 0.688 1.424 2.115 4.702 16.975 45.685
Data collection by the measurement of the initial data to initial 3 detectors and lasting 5 days, it is possible to comprehensive discrete differ% instrument within +/-15% is calibrated.
By to the foundation of multifunction immunity detector calibration steps model and checking, in the instrument production phase by the foundation to calibration test card secondary calibration formula, it is achieved inter-instrument agreement index G3, G4, G3, G4, the G6AOD average differ% of G6AOD average and Standard Machine 0023 is less than 2%.Concordance after production phase functional detection-phase could be arranged to calibration is less than +/-5%
Realizing the secondary calibration to detector, it is necessary to first Criterion machine, output calibration test is stuck in the AOD average of G3, G4, the G6 of Standard Machine test.Be stuck in, with this calibration test, the machine that is calibrated when producing instruments to test 32 times and obtain, be calibrated machine G3, G4, G6 aod average after realized the secondary calibration being calibrated machine by calibration steps model measurement software, and preserve calibration parameter.
The sensitivity of multifunction immunity detector, for the test of standard testing colour atla color range, it is possible to achieve from the discrimination of the 99.9% of G1-G8.
The all patents mentioned in description of the present invention and publication all represent that these are the public technologies of this area, and the present invention can use.All patents referred to herein and publication are all listed in list of references equally, with concrete being individually referenced equally of each publication.The present invention described here can lack any element or multiple element, realizes when a kind of restriction or multiple restriction, and this restriction here is not particularly illustrated.Such as term " comprising " in each example here, " essence is by ... composition " and " by ... composition " can use all the other 2 terms replacements of one of both.Here the term adopted and expression way are done describing mode, and be not limited except as, here these terms indicating the description of this book also without any intention eliminate any equivalent feature with explaining, but it is known that, it is possible in the present invention and scope of the claims, make any suitable change or amendment.It is appreciated that, examples of implementation described in the invention are all some preferred embodiment and features, any one of ordinary skill in the art can according to doing some changes and change under the marrow that the present invention describes, in the scope that these changes and change are recognized as belonging to the scope of the present invention and independent claims and appended claims limit.

Claims (12)

1. the calibration steps reading equipment reading immunoassay device, it is characterised in that the method comprises the following steps:
(1), read at least first, second, and third color range on standard color card with standard device and obtain AOD original value；
(2), read first, second, and third color range at least described on standard color card with the equipment intending being calibrated and obtain AOD original value；
(3), the first calibration curve is done by the AOD value of the AOD value of the first and second the two of standard device color ranges with the first and second color ranges being calibrated equipment；With the first calibration curve, the equipment being calibrated is done first time and calibrate the value of the second color range obtaining the equipment that is calibrated.
2. method according to claim 1, if the second color range value of the second little standard device of color range value obtained by step (3)；Then this equipment being calibrated is carried out concentration fit equation.
3., according to the method one of claim 1-2 Suo Shu, on described colour atla, the second color range is between first and tertiary color rank.
4., according to the method one of claim 1-3 Suo Shu, described marking arrangement and the equipment intending being calibrated include optical pickup unit；Described optical pickup unit includes COMS or CCD element.
5. according to the calibration steps one of claim 1-4 Suo Shu, it is characterised in that described AOD value is the meansigma methods of multiple value.
6. according to the calibration steps one of claim 1-5 Suo Shu, it is characterized in that, the selection of described standard device carries out by the following method: respectively the reading equipment of more than 3 is tested by first, second, and third color range on described standard colour examining card, continues the AOD value of at least 1 day；By the analysis result of data, CV value is minimum and minimum as standard device with the deviation of other device A OD averages.
7. according to the calibration steps one of claim 1-6 Suo Shu, it is characterised in that described immunoassay device includes test zone and marked region.
8. according to the calibration steps one of claim 1-7 Suo Shu, it is characterised in that described test zone includes antibody or the antigen fixed, marked region includes be with coloured granule.
9. the calibration steps according to claim 7 or 8, it is characterised in that the coloured granule of described band is gold colloid particles or latex colloidal solid.
10. according to the calibration steps one of claim 7-9 Suo Shu, it is characterised in that described immunoassay device also includes being positioned at the sample areas of marked region upstream and being positioned at the testing result control area in test zone downstream.
11. according to the calibration steps one of claim 1-10 Suo Shu, it is characterised in that first, second, and third color range on described standard color card is corresponding G3, G4 and G6 respectively.
12. according to the calibration steps one of claim 1-11 Suo Shu, it is characterised in that described standard device reads the AOD original value that on standard color card, at least first, second, and third color range obtains and is stored in a storage medium.
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