WO2015007153A2

WO2015007153A2 - An immunoassay reading device and its calibration method

Info

Publication number: WO2015007153A2
Application number: PCT/CN2014/081062
Authority: WO
Inventors: Liu Wei; Zhu XINYU
Original assignee: Abon Biopharm (Hangzhou) Co., Ltd.
Priority date: 2013-07-15
Filing date: 2014-06-28
Publication date: 2015-01-22
Also published as: CN105518459A; WO2015007153A3; CN104297468A; CN105784985A; CN104297468B; CN104297469B; CN105759024B; CN105759024A; CN104297469A

Abstract

The present invention relates a calibration method for devices, especially a calibration method for devices reading immune test strips, comprising two calibrations for offset. The use of such calibration method can improve the consistency among devices. Particularly, when using such devices to test results in the test area of reagent strips, the results obtained are more accurate and the test accuracy is higher.

Description

An Immunoassay Reading Device and Its Calibration Method

Field of technology

The present invention relates to a calibration method for testing devices, particularly to a calibration method for devices having optical detection on immunoassay detection reagent strips. Background technology

Statement of the background of the present invention is only for the purpose of helping readers to understand this invention, instead of constituting any description or elaboration of the prior art of the present invention.

In the field of rapid diagnosis, there are many devices which detect the analyte in samples (such as saliva, urine or blood, etc) by making use of test strips or test boards. These test strips or test boards are used cooperatively with reading devices and they could be integrated as a non-detachable whole, or they could be split type. Such reading device reflects the result of the analyte contained in the detected sample on the test strip on an electronic device connected with it so that the result is obtained in a digitized way, which is more objective than the method of visual observation with naked eyes and the test result can be kept and transmitted electronically. For instance, a reading device incorporates an optical element, such as a CCD camera, which obtains the pattern on the testing device through optical elements, and is equipped with or linked with a calculating circuit through which the detection result data on the test strip (testing device) is further transformed and calculated to get much more easily identifiable results as described in the patent application US 10/741 , 416. In some other embodiments, a testing device is connected with a general computer which further transforms and reads the detection result data by setting relevant programs in the computer, thus being convenient for users with large amount of use, such as medical institutions. Such reading devices are described specifically in Chinese patent application 201210132692.6 and 201310025671.9, as well as US patent application US20050168747 and US20070134812. Generally, these electronic devices conducting digital processing on detection results on test strips comprise light emitting devices giving out light on test strips, photoelectric detectors detecting light reflected or scattered from reagent strips and some central processing units. Some reading devices also comprise CCD or COMS image collector which collects images and processes images to obtain test results on test strips. During the mass production of reading devices for reagent strips, it is an important factor affecting the stability of the devices on how to realize the consistency among reading devices and reduce their deviation. Due to mechanical errors of the parts of each device, such as electronic components, and the installation of components, there will always be some deviations after initial assembly of devices. If such deviations go beyond the acceptable range, they will give rise to such shortcomings as being not ready or low precision of test results when reading immunoassay test strips. Generally, it is required to calibrate these devices so that each device could conform to the uniform standard, and devices from the same batch and different batches could be kept within an error range as much as possible to conform to the same standard.

Summary of the invention

The present invention relates to a calibration method for reading devices that could calibrate devices reading the test results of immunoassay test strips, provide them with better consistency and improve their stability and test accuracy.

On the one hand, the present invention relates to a calibration method for reading devices, comprising a calibration method for reading devices reading immunoassay testing devices, wherein such method comprises the following steps:

(1) Use a standard device to read at least the first, the second and the third levels on a standard color card to obtain AOD original values;

(2) Use a device to be calibrated to read at least the said first, second and third levels on the standard color card to obtain AOD original values;

(3) Use AOD values of the first and the second levels of the standard device, as well as AOD values of the first and the second levels of the device to be calibrated to make the first calibration curve; and use the first calibration curve to conduct the first calibration on the device to be calibrated;

(4) Fit the second calibration curve with the AOD value of the second levels after the first calibration and AOD value of the original third levels of the device to be calibrated, as well as AOD original values of the second and the third levels of the standard device, and use the second calibration curve to conduct the second calibration on the device to be calibrated.

In some preferred embodiments, the second levels on the said color card are situated between the first and the third levels. In some other preferred embodiments, the said marking device and the device to be calibrated comprise optical reading components.

In some other preferred embodiments, the calibration method according to claim 3, wherein the said optical reading components comprise COMS or CCD components. In some other preferred embodiments, the said AOD value is the average value of multiple values. In some other preferred embodiments, wherein the said marking device is selected in the following way: use the first, the second and the third levels on the said standard color card to respectively test more than 3 sets of reading devices to obtain AOD values lasting for at least 1 day, analyze the data, and take the device with the minimum CV value and the minimum deviation with the AOD average value of other devices as the standard device.

In some other preferred embodiments, the said immunoassay testing device comprises a test area and a mark area. In some other preferred embodiments, the said test area comprises fixed antibodies or antigens, while the mark area comprises colored particles. In some other preferred embodiments, the said colored particles are gold colloid particles or emulsion colloid particles. In some other preferred embodiments, the said immunoassay testing device also comprises a sample area upstream of the mark area and a test result control area downstream of the test area. In some other preferred embodiments, the said test area is located on nitrate cellulose membrane.

In some other preferred embodiments, the color values of the first, the second and the third levels of the said standard color card are G3, G4, and G6 respectively.

On the other hand, a calibration method for reading devices reading immunoassay testing devices, wherein such method comprises the following steps:

(3) Use AOD values of the first and the second levels of the standard device, as well as AOD values of the first and the second levels of the device to be calibrated to make the first calibration curve; and use the first calibration curve to conduct the first calibration on the device to be calibrated to obtain the value of the second levels of the device to be calibrated;

It is preferred that if the value of the second levels obtained via step (3) is greater than the value of the second levels of the standard device, it is required to conduct the second calibration on the device to be calibrated. It is preferred that the second calibration method is as follows: fit the second calibration curve with AOD values of the second and the third levels after the first calibration of the device to be calibrated, as well as AOD original values of the second and the third levels of the standard device, and use the second calibration curve to conduct the second calibration on the device to be calibrated.

It is preferred that the second levels on the said color card are situated between the first and the third levels.

It is preferred that the said marking device and the device to be calibrated comprise optical reading components. The said optical reading components comprise COMS or CCD components.

It is preferred that the said AOD value is the average value of multiple values. It is preferred that the said marking device is selected in the following way: use the first, the second and the third levels on the said standard color card to respectively test more than 3 sets of reading devices to obtain AOD values lasting for at least 1 day, analyze the data, and take the device with the minimum CV value and the minimum deviation with the AOD average value of other devices as the standard device.

It is preferred that the color values of the first, the second and the third levels of the said standard color card are G3, G4, and G6 respectively.

On the other hand, the present invention relates to a calibration method for reading devices reading immunoassay testing devices, wherein such method comprises the following steps:

Use a device to be calibrated to obtain AOD values of the first, the second and the third original levels on a standard color card;

Substitute into the first calibration curve to obtain the first calibration value of the device to be calibrated; and if the value of the second levels obtained is greater than the value of the second levels of the standard device, it is required to substitute the obtained original values into the second calibration curve for calibration again.

In some preferred embodiments, the way to get the first calibration curve is:

Use a standard device to read at least the first, the second and the third levels on a standard color card to obtain AOD original values;

Use a device to be calibrated to read at least the said first, the second and the third levels on the standard color card to obtain AOD original values;

Use AOD values of the first and the second levels of the standard device, as well as AOD values of the first and the second levels of the device to be calibrated to make the first calibration curve.

In some preferred embodiments, the way to get the second calibration curve is: fit the second calibration curve with AOD values of the second and the third levels after the first calibration of the device to be calibrated, as well as AOD original values of the second and the third levels of the standard device.

In all the aforesaid embodiments, AOD original values obtained from reading at least the first, the second and the third levels on the standard color card with the standard device are stored in a storage carrier which can be any medium readable to the device, such as magnetic disk, 2D card, USB flash disk, 3D card, ID card or other media.

Beneficial effects

The use of the present invention can effectively improve the consistency among devices. Particularly, when using such device to test results in the test area of reagent strips, the results obtained are more accurate and the test accuracy is higher.

Brief description of the drawings

Figure 1 shows the structural view of an immunochromatography reagent strip in one specific embodiment of the present invention;

Figure 2 shows the schematic view of a standard color card, wherein Figure 2A shows the schematic view of a standard color card comprising 10 levels-free used in the present invention, and Figure 2B shows the schematic view of a standard color card used in specific embodiments of the present invention;

Figure 3 shows the fitted first calibration curve with three points G3, G4 and G6 of a standard device 0023 and a device to be calibrated 0018 in one embodiment, obtaining a calibration curve equation y(0023 ) =0.1811+1.020X(0018);

Figure 4 shows the fitted first calibration curve with three points G3, G4 and G6 of a standard device 0023 and a device to be calibrated 0021 in one embodiment, obtaining a calibration curve equation y(0023 ) =0.5113+0.9745X(0020);

Figure 5 shows the fitted first calibration curve with two points G3 and G6 of a standard device 0023 and a device to be calibrated 0018 in one embodiment, obtaining a calibration curve equation y(0023 ) =0.0.03819+1.026X(0018);

Figure 6 shows the fitted first calibration curve with two points G3 and G6 of a standard device 0023 and a device to be calibrated 0018 in one embodiment, obtaining a calibration curve equation y(0023 ) =-0.2304+1.017X(0020);

Figure 7 shows the linear relation diagram of levels of color card used versus AOD values in the present invention;

Figure 8 shows the fitted first calibration curve with two points G3 and G4 of a standard device 0023 and a device to be calibrated 0018 in one embodiment, obtaining a calibration curve equation y(0023 ) =0.4748+0.9365X(0018);

Figure 9 shows the fitted first calibration curve with two points G3 and G4 of a standard device 0023 and a device to be calibrated 0021 in one embodiment, obtaining a calibration curve equation y(0023 ) =-0.9206+1.398X(0020);

Figure 10 shows the fitted second calibration curve with the AOD average value of the G4 value after the first calibration and the original G6 value of a device 0018, as well as G4 and G6 of a standard device in one embodiment, obtaining a calibration curve equation y(0023 ) =-0.2194+1.045X(0018).

Figure 11 shows the fitted second calibration curve with the AOD average value of the G4 value after the first calibration and the original G6 value of a device 0021, as well as the AOD average value of G4 and G6 of a standard device in one embodiment.

Figure 12 shows the fitted first calibration curve with two points G3 and G4 of a standard device 0023 and a device to be calibrated 0018 in one embodiment;

Figure 13 shows the fitted first calibration curve with two points G3 and G4 of a standard device 0023 and a device to be calibrated 0021 in one embodiment;

Figure 14 shows the fitted second calibration curve with the AOD average value of the G4 value after the first calibration and the original G6 value of a device 0018, as well as the AOD average value of G4 and G6 of a standard device in one embodiment.

Figure 15 shows the fitted second calibration curve with the AOD average value of G4 and G6 after the first calibration of a device 0021, as well as G4 and G6 of a standard device in one embodiment. Figure 16 shows the flow chart to obtain the first and the second calibration curve in device calibration.

Figure 17 shows the flow chart of device calibration with software design.

Brief description of marks on the drawings

Test area 30, test result control area 40, sample absorption area 50, carrier 20, mark area 60, sample application area 10 and levels 100

Detailed description

Testing device

"Testing device" in the present invention refers to those devices that could detect or assay the analyte in samples via chemical or physical reaction. Such devices could be test strips (Figure 1), including testing devices with test strips or test reagents. On testing devices, there usually are some test reagents which will have direct or indirect reaction with the analyte and take on changes in color or other changes on testing devices, so that results or changes at the test area on testing devices could be judged or analyzed with naked eyes or machine to see if there is the analyte in samples, or the quantity of the analyte existing.

Many immunoassay testing devices are well known to technicians in this field to be used for detecting the analyte in samples. In respect of the detection of polypeptide or protein in patient samples, immunoassay testing devices and methods are frequently used. Please see US patent 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; and 5,480,792, all of which are completely listed in the references, including all the forms, diagrams and claims. These devices and methods can make use of various marker macromolecules for detection with sandwich technique, to generate signals relevant to existence or quantity of the target analyte in a competitive or non-competitive testing form. In addition, a kind of method and device, such as biosensor and optical immunoassay detection method, may not need marker macromolecules and can test the existence or existing quantity of the target analyte. Please refer to US patent 5,631,171 and 5,955,377, both of which are completely listed in the references, including all the forms, diagrams and claims. Skilled technicians in this field believe that testing devices include but not limited to Beckman, Abbott AxSym, Roche ElecSys and Dade Behring chromatographic system, the immunoassay detecting system of which can be used for the immunoassay detection hereof. Immunoassay detection method is preferred for the analysis of markers. Although other methods (such as measuring the RNA level of markers) are also well known to technicians in this field, the most preferred is sandwich immunoassay detection method. By binding specific antibodies of corresponding markers with their detection specificities, the existence or existing quantity of the markers can usually be detected. The immunoassay binding of markers with their specific antibodies can be directly or indirectly detected. Taking immunoassay detection method as an example, the most common method required for biological detection and analysis is the quantitative method, in which fluorophore or other macromolecular substances could form antibody-marker by binding a kind of enzyme. The detectable markers comprise macromolecular substances (such as fluorophore, electrochemical label and metal chelate, etc) that can be detected, as well as indirectly detectable molecules (such as enzyme like horseradish peroxidase and alkaline phosphatase, etc) with detectable reaction products, or a detectable specific binding of binding molecules (such as biotin, digoxin, maltose, oligohistidine, 2,4-dinitrobenzene, benzenearsonic acid, ssDNA and dsDNA, etc). The particularly preferred detectable marker is fluorescent emulsion particle as stated in US patent 5,763,189, 6,238,931 and 6,251,687 and international publication WO95/08772, all of which are completely listed in the references. Demonstration of conjugation in particles will be mentioned hereunder. Direct labels including fluorescent or luminescent label, metal, dye, radionuclide and the like are bound with antibodies, while indirect labels comprise various enzymes known in this field, such as alkaline phosphatase, horseradish peroxidase and the like.

The use of fixed antibodies to specifically detect the analyte is also a part of this invention. The term "solid phase" used here is a general material, including solid, semisolid, gel, film, membrane, network, felt, compound, particle, test paper and the like, and technicians in this field generally can use them to adsorb macromolecular substances. Solid matters can be nonporous or porous. Appropriate solid phase includes those mature and/or matters as solid in solid binding detection. For instance, the entire Immunoassay is taken as reference or a part of the present invention (please see the example: chapter 9 of Immunoassay, E. P. Dianiandis and T. K. Christopoulos eds., Academic Press: New York). Appropriate solid examples include membrane, filter, cellulosic paper, glass bead (including polymerized, emulsion and paramagnetic particles), glass, silicon wafer, microparticle and nano particle, such as Tenta gel, Agro gel, PEGA gel, SPOCC gel and porous disk (please see the example: Leon et al., Bioorg. Med. Chem. Lett. 8: 2997, 1998; Kessler et al., Agnew. Chem. Int. Ed. 40: 165, 2001; Smith et al, J. Comb. Med. 1: 326, 1999; Orain et al, Tetrahedron Lett. 42: 515, 2001; Papanikos et al, J. Am. Chem. Soc. 123: 2176, 2001; Gottschling et al, Bioorg. Med. Chem. Lett. 11: 2997, 2001). Antibodies can be fixed on various solid carriers, such as magnetic or chromatographic grade matrix granule, test board surface (such as microporous plate), solid substrate material or membrane (such as plastic, nylon and paper), etc. Test strips are formed by applying a kind of antibody or various antibodies in matrix arrangement. After being immersed in testing samples, these test strips will generate detectable signals, such as color mottles, after fast rinsing and detection procedure. When adopting multiple detection methods, a great many of locations that could be separately set with an address will be generated on a single solid carrier, with each location corresponding to different markers and comprising antibodies binding with such markers. The term "discrete" here refers to a discrete surface area. That is to say, if borders not belonging to any of the area completely enclose each of the two areas, then two surface areas are independent and discrete. The term "independent address" refers to discrete surface areas where specific signals can be obtained.

A chromatography immunoassay testing device generally comprises a test area 30 and a mark area 90. In some embodiments, a testing device also comprises a sample application area 10 and a water absorption area 50. There are marking matters at the mark area, such as colloidal gold, emulsion or color articles. In some other embodiments, the test area 30 is included on a solid carrier 20, such as membrane, filter, cellulosic paper, glass bead (including polymerized, emulsion and paramagnetic particles), glass, silicon wafer, microparticle and nano particle. In some other embodiments, the solid carrier is membrane, such as nitrocellulose membrane and nylon membrane, etc. In some embodiments, antibodies involved in the immunoassay reaction are fixed on the test area. In some embodiments, a control area 40 can be set near the test area to verify if test results at the test area are valid or not.

Test area

"Test area" said here refers to the area where the existence or existing quantity of the analyte in samples can be read. There can be multiple test areas on one testing device, aiming at the detection of different analytes. In some embodiments, the detection of different types of analyte can also be conducted at one test area. In some embodiments, the test area can be located on the solid carrier in the testing device, in such forms as line, dot, spot, block, geometrical shape or geometrical symbol, like a 0.5- 1.5cm long and 0.2-5mm wide line. On the test area, test results can be obtained with naked eyes or instrument, and can directly and/or indirectly indicate the existence or existing quantity of the analyte in the sample, and/or the variety of the analyte. Corresponding to the test area, there is a test control area where effectiveness of the test results and the testing device can be controlled.

In some embodiments, the existence or existing quantity of a kind or multiple kinds of analyte in the sample is indicated in color after chemical or physical changes. Common technicians in this field are acquainted with the display of colors in such test area. Generally, color particles appear or are fixed on the test area, such as colloidal gold, nano particles or emulsion particles, and will take on colors due to the accumulation or fixation of color particles. In general, the quantity of color particles is relevant to the quantity of the analyte existing in the sample. Except for color particles, chemical reaction may also occur at the test area and generate colors. For example, redox reaction will happen in case of oxidized substrate to change color of the substrate, in which way, the color will also appear at the test area. Similarly, the shade of the color after chemical reaction is also relevant to the concentration of the analyte existing in the sample.

The relevance here can be positive or negative. For instance, the deeper or the denser the color is, or the more the color particles are, or the stronger the emitting light is, the more the analyte in corresponding sample will be. Or on the contrary, the deeper or the denser the color is, or the more the color particles are, or the stronger the emitting light is, the less the analyte in corresponding sample will be or even does not exist.

Description of the preferred embodiment

The present invention will be further described next with specific embodiments. However, such descriptions will not constitute any restriction upon the present invention.

Materials:

1. Standard color card (Figure 2A and Figure 2B): the usage of such standard color card: in actual test, shade of the test area 30 on the test strip is generally used for qualitative comparison with the standard color card (with naked eyes) to see which color is close to it, and then the test result can be determined. In the double antibody or antigen sandwich method, if color on the test strip belongs to G1-G2, it is generally recognized as negative; if it belongs to G3, it is recognized as positive sometimes, or negative sometimes, or requiring another test sometimes; and if it belongs to G4-G11, it is generally recognized as positive. Of course, if the detection is conducted with competitive method, the color values are contrary to the test results. Generally speaking, the strength of the color is relevant to concentration of the sample and the stronger the color is, the higher concentration is in the sample; while in terms of competitive immunoassay detection method, the stronger the color is, the lower concentration is in the sample. In all the following embodiments of the present invention, the stated standard color card is the color card No. 0123.

When using a reading device to read the color value on the test area 30 of a test strip, if one also expects that the AOD value (AOD stands for the conversion value of image signal strength of color lines, and AOD value of the same line or levels read by the same device is substantially the same) of the test area read by the reading device is in linear relationship with the concentration value in the sample, one can have quantitative detection on the test value. If one also expects the linear correspondence between the color reading and the concentration in sample, one can also have qualitative detection on values in the sample. When detecting reading devices, a standard color card is usually employed for detection to see if the reading can discriminate color lines (levels) on the standard color card very well, such as being able to discriminate Gl and G2 very well, having at least 99.9% discrimination upon each color gradient on the color card, and being able to realize no normal intersection of positive and negative 3SD. If a good discrimination is realized upon each line (Gl-Gll) on the color card, then a good discrimination is realized upon the color on the test area of the test strip.

2. Reading device:

All reading devices in the present invention adopt COMS camera, together with software programming, program downloading and other hardware settings so that COMS collects AOD values of color lines on test strips, has conversion with the concentration, and finally obtains test values. All parts of the aforesaid devices are from the same batch.

Although the software and hardware employed are kept consistent as much as possible, it is still required to have uniform calibration on reading devices so that the variance among devices after calibration is less than 5% or within other acceptable range or value. Table 1: Definitions

Reference Description

G3,G4,G6,G8,G10 Levels of a standard color card (Figure 2)

C.V Coeffic ient of dispersion

SD Standai d deviation

Embodiment 1: selection of a standard device

Randomly select 5 sets of reading devices (No.: 0023, 0025, 0021, 0018 and 0020), use G3, G4 and G6 on a standard color card to respectively test 5 sets of multi-function immunoassay detectors and collect the test data AOD values for consecutive 5 days. Seeing from analysis on data in the 5 days, No. 0023 device has the minimum CV value (0.83%), and its SD deviation with AOD average value of the other 4 sets of devices is the minimum. At last, No. 0023 device is selected as the standard device (specific test data omitted).

Embodiment 2: collection of original data of a device to be calibrated and a standard device

Use a standard color card (Figure 2) to respectively obtain the original AOD values of 3 sets of reading devices (the standard device 0023 and the devices to be calibrated 0018 and 0020), with original values as shown in the following table:

Table 2: Original AOD Values of Different Devices

G4-6 4.879 4.637 4.229

G4-7 4.982 4.818 4.207

G4-8 4.919 4.798 4.215

G4-9 5.044 4.883 4.197

G4-10 4.927 4.777 4.254

Average 4.939 4.764 4.190

value

SD 0.053 0.082 0.057

CV 1.08% 1.73% 1.37%

G6-1 17.139 16.458 17.105

G6-2 16.928 16.562 17.102

G6-3 17.111 16.534 17.096

G6-4 17.048 16.627 17.014

G6-5 17.251 16.613 17.024

G6-6 17.071 16.557 17.048

G6-7 17.215 16.657 17.126

G6-8 17.069 16.643 16.981

G6-9 17.103 16.627 16.993

G6-10 17.195 16.578 17.233

Average 17.113 16.586 17.072

value

SD 0.094 0.061 0.076

CV 0.55 % 0.36 % 0.45 %

After the following calibration of the device to be calibrated with the standard device, compare gradient of the standard device and the device after calibration on the color card to see if they conform to the set requirements, such as the SD value, CV value or differ% value.

Embodiment 3: calibration method with three points for the device to be calibrated by using the standard device

1) Conduct linear fitting with AOD average value of three points G3, G4 and G6 of the standard device 0023, as well as AOD average value of three points G3, G4 and G6 of the devices 0018 and 0020 to be calibrated, obtaining the linear fitting calibration equation (as shown in Figure 3 and Figure 4).

Data analysis of the reading device after calibration of G3, G4 and G6:

After calibration of three points G3, G4 and G6, the differences (differ%) between AOD values of the device 0020 at G3 and G4 and AOD values of the standard device 0023 at G3 and G4 are still greater than 5% (respectively being 11.4% and 6.97%). The calibration results turn out to be not that ideal, although the difference between the test value of the device 018 after calibration and the standard equipment is up to the standard. AOD values are obtained through the device after calibration reading AOD values of the levels G3, G4 and G6 on the uniform standard color card, with the results in the following table.

Table 3: Comparative Analysis of AOD Values between Each Device after Calibration and the Standard Device

G6-10 17.195 16.578 17.091 0.61% 17.233 17.305 -0.64%

Average 17.113 16.586 17.098 0.08 % 17.072 17.148 -0.21% value

SD 0.094 0.061 0.062 0.076 0.074

CV 0.55% 0.36% 0.36 % 0.45 % 0.43%

Embodiment 4: comparison of calibration methods with two points I. Calibration method with two points G3 and G6

1) Conduct linear fitting with AOD average value of two points G3 and G6 of the standard device 0023 (AB 130023-SET1), as well as AOD average value of two points G3 and G6 of the devices 0018 (AB 130018-SET1) and 0020 (AB 130020-SET1) to be calibrated, obtaining the linear fitting calibration equation (as shown in Figure 5 and Figure 6).

2) Data analysis after calibration of G3 and G6:

After calibration of two points G3 and G6, the difference (differ%) between the AOD value of the device 0020 at G4 and the AOD value of the standard device 0023 at G4 is still greater than 5% (being 12.62%). The calibration results turn out to be not that ideal, although the value deviation of the device 0018 after calibration is within the acceptable range (less than 5%). AOD values are obtained through the device after calibration reading AOD values of the levels G3, G4 and G6 on the uniform standard color card, with the results in the following table.

Table 4: Comparison of AOD Values between Each Device after Calibration and the Standard Device

G4-2 4.9 4.692 5.040 -2.85% 4.207 4.332 11.59%

G4-3 4.958 4.851 5.201 -4.91% 4.223 4.348 12.30%

G4-4 4.974 4.647 4.994 -0.41% 4.112 4.238 14.80%

G4-5 4.866 4.767 5.116 -5.14% 4.065 4.191 13.87%

G4-6 4.879 4.637 4.984 -2.15% 4.229 4.354 10.76%

G4-7 4.982 4.818 5.168 -3.73% 4.207 4.332 13.04%

G4-8 4.919 4.798 5.147 -4.64% 4.215 4.340 11.77%

G4-9 5.044 4.883 5.234 -3.76% 4.197 4.322 14.31%

G4-10 4.927 4.777 5.126 -4.04% 4.254 4.379 11.12%

Average 4.939 4.764 5.113 -3.53% 4.190 4.315 12.62% value

SD 0.053 0.082 0.084 0.057 0.057

CV 1.08% 1.73% 1.63% 1.37% 1.32%

G6-1 17.139 16.458 16.982 0.91% 17.105 17.145 -0.04%

G6-2 16.928 16.562 17.088 -0.94% 17.102 17.142 -1.26%

G6-3 17.111 16.534 17.060 0.30% 17.096 17.136 -0.15%

G6-4 17.048 16.627 17.154 -0.62% 17.014 17.055 -0.04%

G6-5 17.251 16.613 17.140 0.65% 17.024 17.065 1.08%

G6-6 17.071 16.557 17.083 -0.07% 17.048 17.088 -0.10%

G6-7 17.215 16.657 17.184 0.18% 17.126 17.166 0.28%

G6-8 17.069 16.643 17.170 -0.59% 16.981 17.022 0.28%

G6-9 17.103 16.627 17.154 -0.30% 16.993 17.034 0.40%

G6-10 17.195 16.578 17.104 0.53% 17.233 17.272 -0.45%

Average 17.113 16.586 17.112 0.00% 17.072 17.113 0.00% value

SD 0.094 0.061 0.061 0.076 0.076

CV 0.55% 0.36% 0.36% 0.45% 0.44%

II. Calibration method with two points G3 and G4, and G4 and G6

Calibrate reading devices (0020 and 0018) to be calibrated with two points G4 and G6, and G3 and G4 by referring to the same method for G3 and G6 and take 0023 as the standard device.

After the calibration of two points G4 and G6, differences (differ%) between the AOD value of the device 0020 and 0018 at G3 and the AOD value of the standard device 0023 at G3 are still greater than 5%. The calibration results turn out to be not that ideal, wherein the difference (differ%) of 0018 at G3 is 7.35% and the difference (differ%) of 0020 at G3 is -24.98% (specific data omitted). However, their differences at other levels G4 and G6 are less than 5%.

After the calibration with two points G3 and G4, differences (differ%) between the AOD value of the device 0018 and 0020 at G6 and the AOD value of the standard device 0023 at G6 are still greater than 5%. The calibration results turn out to be not that ideal (specific data omitted), wherein the difference (differ%) of 0018 at G6 is 6.46% and the difference (differ%) of 0020 at G3 is -34.09%. Conclusions: After comparison of the aforesaid 4 calibration methods, we can draw the following conclusions:

(1) G3G4G6 calibration curve— G3 value and G4 value are higher after calibration, and their differences (differ%) with the average value of the standard device are greater than 5%.

(2) After calibration of G3 G6— the difference (differ%) between G4 value of the device after calibration and the average value of the standard device is greater than 5%.

(3) Calibration of G4 G6— the difference (differ%) between G3 value of the device after calibration and the average value of the standard device is greater than 5%.

(4) Calibration of G3 G4— the difference (differ%) between G6 value of the device after calibration and the average value of the standard device is greater than 5%.

Embodiment 5: repeated calibration method with two points

I. Analysis of AOD values on color card

To solve the problems above, we have determination on AOD of the standard color card used, and surprisingly find out that these AOD values of color card gradients are not in linear relationship. For example, in Figure 7, the color gradient distribution of G2-G3-G4-G6-G8 is not linear. The gradients of G3 and G4 are smaller and can be fitted to be linear, while the gradients of G4, G6 and G8 are larger and can be independently linear. Therefore, a second calibration can be conducted to solve the problems in the first calibration.

II. Second calibration for offset

Method 1:

1) Fit a calibration curve upon the device (0018 and 0020) to be calibrated with AOD values of the standard device 0023 at G3 and G4. Use the data of G3G4 to fit the calibration curve (as shown in Figure 8 and Figure 9).

(2) Then, fit a calibration curve with the AOD value of G4 after calibration and the original data of G6 of the device to be calibrated, as well as G4 and G6 of the standard device 0023 (as shown in Figure 10 and Figure 11).

After calibration of G3 and G4 of the device to be calibrated, fit a calibration curve with the data of G4 after calibration, the original data of G6 and the data of G4G6 of the standard device 0023, and it turns out that the differ% between the AOD average value of G3, G4 and G6 after calibration and the AOD value of G3, G4 and G6 of the standard device 0023 is less than 2%, being ideal and within the acceptable standard.

Table 5: Comparative Analysis of AOD of Levels on Standard Color Card between Device after Calibration and the Standard Device

value

SD 0.094 0.061 0.063 0.076 0.076

CV 0.55% 0.36% 0.37% 0.45% 0.45%

Method 2:

Fit a calibration curve with the data of G4 and G6 after the fitting calibration of G3 and G4 and G4G6 of the standard device— after calibration, G6 can be pulled back to the G6 value of the standard device to be less than 5%. This method is convenient and the average value of G3, G4 and G6 of the standard device is required to be recorded. Fit the first curve with G3 and G4 value of the device to be calibrated and G3 and G4 of the standard device, and then fit another curve with the data of G4 and G6, to realize full chroma approximate calibration.

1) Fit a calibration curve for data of the device to be calibrated with G3 and G4 of the standard device. Please see Figure 12 and Figure 13 for details.

2) Second calibration by having linear fitting with the data of G4 and G6 after the first calibration, as well as G4 and G6 of the standard device to obtain the standard curve as shown in Figure 15 and Figure 14.

After the calibration of G3 and G4, fit a calibration curve with the data of G4 and G6 after calibration and G4G6 data of the standard device 0023 and it turns out that differ% between the AOD average value of G3, G4 and G6 after calibration and the standard device 0023 is less than 2% with ideal results. Please see the following table for details:

Table 6: Comparative Analysis of AOD of Levels on Standard Color Card between Device after Calibration and the Standard Device

value

SD 0.051 0.042 0.039 0.041 0.057

CV 1.81% 1.68% 1.39% 1.52% 2.02%

G4-1 4.936 4.77 4.942 -0.12% 4.186 4.931 0.09%

G4-2 4.9 4.692 4.869 0.64% 4.207 4.961 -1.24%

G4-3 4.958 4.851 5.018 -1.21% 4.223 4.983 -0.51%

G4-4 4.974 4.647 4.827 2.96% 4.112 4.828 2.94%

G4-5 4.866 4.767 4.939 -1.50% 4.065 4.762 2.13%

G4-6 4.879 4.637 4.817 1.26% 4.229 4.992 -2.31%

G4-7 4.982 4.818 4.987 -0.10% 4.207 4.961 0.43%

G4-8 4.919 4.798 4.968 -1.00% 4.215 4.972 -1.08%

G4-9 5.044 4.883 5.048 -0.07% 4.197 4.947 1.93%

G4-10 4.927 4.777 4.948 -0.44% 4.254 5.026 -2.02%

Average 4.939 4.764 4.936 0.04% 4.190 4.936 0.04% value

SD 0.053 0.082 0.077 0.057 0.080

CV 1.08% 1.73% 1.56% 1.37% 1.63%

G6-1 17.139 16.458 16.988 0.88% 17.105 17.142 -0.02%

G6-2 16.928 16.562 17.095 -0.99% 17.102 17.140 0.00%

G6-3 17.111 16.534 17.066 0.26% 17.096 17.134 0.03%

G6-4 17.048 16.627 17.162 -0.67% 17.014 17.056 0.48%

G6-5 17.251 16.613 17.147 0.60% 17.024 17.066 0.43%

G6-6 17.071 16.557 17.090 -0.11% 17.048 17.089 0.29%

G6-7 17.215 16.657 17.193 0.13% 17.126 17.162 -0.14%

G6-8 17.069 16.643 17.178 -0.64% 16.981 17.025 0.66%

G6-9 17.103 16.627 17.162 -0.34% 16.993 17.037 0.60%

G6-10 17.195 16.578 17.111 0.49% 17.233 17.263 -0.73%

Average 17.113 16.586 17.113 0.00% 17.072 17.111 0.16% value

SD 0.094 0.061 0.062 0.076 0.072

CV 0.55% 0.36% 0.36% 0.45% 0.42%

Embodiment 6: sensitivity analysis

Use the device (0018) after calibration to test different levels of the same set of standard color cards, and it turns out to have 99.9% discrimination upon 6 gradients of G1-G8 and realize no normal intersection of +/-3SD. Please see the following table for results.

By measuring original data of the initial 3 sets of detectors and collecting data for consecutive

5 days, devices with comprehensive discreteness differ% within +/-15% can be calibrated. By establishing and verifying the calibration method model for multi-function immunoassay detectors, during the production of such devices, the consistency index differ% among devices between the AOD average value of G3, G4, G6 and the AOD average value of G3, G4, G6 of the standard device 0023 can be less than 2% through establishing the second calibration formula for the calibration test card. Therefore, during the production stage and function test stage, the consistency after calibration can be set as less than +1-5%.

To realize the second calibration upon testing devices, it is required to first establish a standard device to output the AOD average value of G3, G4 and G6 of the calibration test card tested on the standard device. During the production of devices, such calibration test card is tested 32 times on devices to be calibrated to obtain the AOD average value of G3, G4 and G6 of devices to be calibrated, and then the software is tested with the calibration method model to realize the second calibration of the devices to be calibrated, with calibration parameters kept.

For the sensitivity of multi-function immunoassay detectors, the test of levels of a standard test color card can realize the discrimination of 99.9% for G1-G8.

All the patents and publications referred to in the present invention indicate that they are public techniques in this field and can be used by this invention. All the patents and publications quoted here are listed in the references as well, like the independent reference of each publication. The present invention can be realized in case of the absence of any element or multiple elements, any restriction or multiple restrictions which are not specially explains hereof. For instance, any one of the terms "comprise" , "be substantially comprised of" and "be comprised of" can be replaced by the other two terms. Terms and means of expression used here are descriptive approaches rather than restrictions. Besides, there is no intent to indicate that these terms and explanations hereof exclude any equivalent characteristics. However, we can know that any appropriate changes or modifications within the scope of this invention and the claims hereof are allowed. It is understandable that embodiments in the present invention are preferred embodiments and characteristics, and based on the essence of this invention, any common technicians in this field can make some modifications and changes which are also recognized as within the scope of the present invention and the scope of the independent claims and dependent claims.

Claims

1. A calibration method for reading devices reading immunoassay testing devices, wherein such method comprises the following steps:

(1) using a standard device to read at least the first, the second and the third levels on a standard color card to obtain AOD original values;

(2) using a device to be calibrated to read at least the said first, second and third levels on the standard color card to obtain AOD original values;

(3) using AOD values of the first and the second levels of the standard device, as well as AOD values of the first and the second levels of the device to be calibrated to form the first calibration curve, obtain the first calibration equation, and use the first calibration curve to conduct the first calibration on the device to be calibrated;

(4) fitting the second calibration curve with the AOD value of the second levels after the first calibration and AOD value of the original third levels of the device to be calibrated, as well as AOD original values of the second and the third levels of the standard device, obtain the second calibration equation, and use the second calibration curve to conduct the second calibration on the device to be calibrated.

2. The calibration method according to claim 1, wherein the second levels on the said color card are situated between the first and the third levels.

3. The calibration method according to one of claims 1-2, wherein the said marking device and the device to be calibrated comprise optical reading components.

4. The calibration method according to claim 3, wherein the said optical reading components comprise COMS or CCD components.

5. The calibration method according to one of claims 1-4, wherein the said AOD value is the average value of multiple values.

6. The calibration method according to one of claims 1-5, wherein the said standard device is selected in the following way: use the first, the second and the third levels on the said standard color card to respectively test more than 3 sets of reading devices to obtain AOD values lasting for at least 1 day, analyze the data, and take the device with the minimum CV value and the minimum deviation with the AOD average value of other devices as the standard device.

7. The calibration method according to one of claims 1-6, wherein the said immunoassay testing device comprises a test area and a mark area.

8. The calibration method according to claim 7, wherein the said test area comprises fixed antibodies or antigens, while the mark area comprises colored particles.

9. The calibration method according to claim 8, wherein the said colored particles are gold colloid particles or emulsion colloid particles.

10. The calibration method according to one of claims 7-9, wherein the said immunoassay testing device also comprises a sample area upstream of the mark area and a test result control area downstream of the test area.

11. The calibration method according to one of claims 7-9, wherein the first, the second and the third levels on the said standard color card are respectively G3, G4 and G6.

12. The calibration method according to claim 1, wherein the AOD original values obtained from reading at least the first, the second and the third levels on the standard color card by the said standard device are stored in a storage medium.

13. An immunoassay reading device, wherein such device is calibrated as per the method according to one of claims 1-12.

14. A calibration method for reading devices reading immunoassay testing devices, wherein such method comprises the following steps:

(3) using AOD values of the first and the second levels of the standard device, as well as AOD values of the first and the second levels of the device to be calibrated to form the first calibration curve and obtain the first calibration equation; and use the first calibration curve to conduct the first calibration on the device to be calibrated and obtain the value of the second levels of the device to be calibrated; if the value of the second levels obtained via step (3) is greater than the value of the second levels of the standard device, it is required to conduct the second calibration on such device to be calibrated.

15. The method according to claim 14, wherein the second calibration method is as follows: fit the second calibration curve with AOD values of the second and the third levels after the first calibration of the device to be calibrated, as well as AOD original values of the second and the third levels of the standard device, obtain the second calibration equation, and use the second calibration curve to conduct the second calibration on the device to be calibrated.

16. The calibration method according to claim 15, wherein the second levels on the said color card are situated between the first and the third levels.

17. The calibration method according to one of claims 14-16, wherein the said marking device and the device to be calibrated comprise optical reading components.

18. The calibration method according to claim 17, wherein the said optical reading components comprise COMS or CCD components.

19. The calibration method according to one of claims 14-18, wherein the said AOD original value is the average value of multiple values.

20. The calibration method according to one of claims 14-18, wherein the said standard device is selected in the following way: use the first, the second and the third levels on the said standard color card to respectively test more than 3 sets of reading devices to obtain AOD values lasting for at least 1 day, analyze the data, and take the device with the minimum CV value and the minimum deviation with the AOD average value of other devices as the standard device.

21. The calibration method according to one of claims 14-20, wherein the said immunoassay testing device comprises a test area and a mark area.

22. The calibration method according to claim 22, wherein the said test area comprises fixed antibodies or antigens, while the mark area comprises colored particles.

23. The calibration method according to claim 23, wherein the said colored particles are gold colloid particles or emulsion colloid particles.

24. The calibration method according to one of claims 14-23, wherein the first, the second and the third levels on the said standard color card are respectively G3, G4 and G6.

25. The calibration method according to claim 14, wherein the AOD original values obtained from reading at least the first, the second and the third levels on the standard color card by the said standard device are stored in a storage medium.