CN113721009B - Optical system calibration method for immunity analyzer - Google Patents
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- 230000003287 optical effect Effects 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000036039 immunity Effects 0.000 title claims description 5
- 239000000872 buffer Substances 0.000 claims abstract description 68
- 238000001514 detection method Methods 0.000 claims abstract description 39
- 238000003018 immunoassay Methods 0.000 claims abstract description 26
- 239000007853 buffer solution Substances 0.000 claims description 34
- 239000012528 membrane Substances 0.000 claims description 24
- 238000012360 testing method Methods 0.000 claims description 18
- 239000000020 Nitrocellulose Substances 0.000 claims description 16
- 229920001220 nitrocellulos Polymers 0.000 claims description 16
- 239000011248 coating agent Substances 0.000 claims description 14
- 238000000576 coating method Methods 0.000 claims description 14
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 12
- 238000011068 loading method Methods 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 7
- 241000283707 Capra Species 0.000 claims description 6
- 239000002250 absorbent Substances 0.000 claims description 6
- 230000002745 absorbent Effects 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000011156 evaluation Methods 0.000 claims description 3
- 239000012114 Alexa Fluor 647 Substances 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 description 14
- 238000007865 diluting Methods 0.000 description 11
- 238000002156 mixing Methods 0.000 description 9
- 238000011088 calibration curve Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000012123 point-of-care testing Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 241001494479 Pecora Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000003754 machining Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a calibration method of an optical system of an immunoassay analyzer, which comprises the following steps: 1. preparing a buffer standard; 2. preparing a calibration detection card; 3. detecting a sample; 4. calibrating; 5. calibrating and evaluating; according to the invention, a fitting algorithm is utilized to calculate a fitting curve between the calibrator and the standard machine, and the inter-table errors caused by nonlinear relations among different optical modules are controlled within a controllable range.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a method for calibrating an optical system of an immunoassay analyzer.
Background
The POCT in vitro diagnosis is a detection technology with great potential, has the advantages of rapidness, simplicity, convenience, high efficiency, low cost, short detection period, small sample dosage and the like, and has been widely applied to clinic. POCT has been rapidly developed in recent years as a new development direction.
However, since the domestic market starts later, there is a place where improvement is required in the process of generation and development of the optical module, the unified instrument optical system can cause the inter-stage error of the nonlinear optical module due to the difference between components themselves, the difference caused by machining tolerance or the difference caused by the adjustment process.
In view of the foregoing, there is a need to develop an optical system calibration method for an immunoassay analyzer to solve the above-mentioned problems.
Disclosure of Invention
Aiming at the defects existing in the prior art, the main purpose of the invention is to calculate the fitting curve between the calibrator and the standard machine by using a fitting algorithm, and control the inter-table errors caused by nonlinear relations among different optical modules within a controllable range.
To achieve the above objects and other advantages and in accordance with the purpose of the invention, there is provided an immunoassay analyzer optical system calibration method including:
step 1, preparing a buffer standard:
preparing 10 buffer standard substances with different calibration point concentrations into a buffer plate;
step 2, preparing a calibration detection card:
preparing a prepared rabbit IgG coating buffer solution, and coating the prepared rabbit IgG coating buffer solution on the nitrocellulose membrane; loading the sample loading pad, the absorbent paper and the nitrocellulose membrane on a PVC bottom plate, and cutting the sample loading pad, the absorbent paper and the nitrocellulose membrane into membrane strips; placing the membrane strip in a detection card shell;
step 3, sample detection:
recording calibration point information of a buffer standard into an immunoassay analyzer;
detecting buffer solution standard substances with 10 different calibration point concentrations by using an immunoassay analyzer to obtain corresponding calibration point fluorescence values;
step 4, calibrating:
obtaining standard calibration point fluorescence values of 10 buffer standard products according to the calibration point information of the buffer standard products, and calculating an F (x) equation under the standard condition through a regression function;
obtaining test calibration point fluorescence values corresponding to 10 buffer liquid standard substances according to a sample detection result, and calculating a G (x) equation under the detection condition through a regression function family;
obtaining a calibration equation Y (x) =f (x) -G (x);
calibrating an optical system of the immunity analyzer according to a calibration equation;
step 5, calibration evaluation:
and (3) testing by adopting a buffer solution with non-calibrated point concentration, comparing with a standard machine testing result, and correcting to be qualified when the accuracy deviation is within the allowable deviation degree.
Further, the buffer standard used in the step 1 is a goat anti-rabbit antibody solution labeled with Alexa Fluor 647.
Further, the sheep anti-rabbit antibody solution label coupling ratio is between 1.4 and 4.
Further, the nitrocellulose membrane in the step 2 has a width of 3.9-4.1mm.
Further, the nitrocellulose membrane in step 2 was coated with 0.5ul of rabbit IgG coating buffer per revolution.
Further, the concentration of the rabbit IgG coating buffer in the step 2 is 1.0mg/ml.
Further, the step of detecting the buffer standard by the immunoassay analyzer in the step 3 includes:
1) The mechanical arm takes TIP consumable (pipette TIP) and installs the TIP consumable to a pipette;
2) The pipettor moves to a buffer night plate;
3) The pipettor absorbs the buffer standard;
4) Discarding the used buffer plate;
5) Moving the detection card to a reaction position;
6) The liquid shifter shifts the buffer standard to the detection card;
7) Moving the detection card to a detection position for detection;
8) Displaying and storing the detection result;
9) Discarding the detection card;
10 TIP consumables (pipette TIPs) are discarded.
Further, the allowable deviation degree in the step 5 is 5%.
One of the above technical solutions has the following advantages or beneficial effects:
the method for calibrating the optical system of the immunity analyzer is simple to operate, high in detection speed, wide in raw material sources, easy to obtain and low in cost;
the invention calculates the fitting curve between the calibration machine and the standard machine by using a fitting algorithm, and controls the inter-table errors caused by nonlinear relations among different optical modules within a controllable range.
Drawings
FIG. 1 is a top view of a buffer plate 100 in a method for calibrating an optical system of an immunoassay analyzer according to an embodiment of the present invention;
FIG. 2 is a schematic view of a membrane strip in an optical system calibration method of an immunoassay analyzer according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a test card 200 in an optical system calibration method of an immunoassay device according to an embodiment of the present invention;
FIG. 4 is a diagram showing a sample detection step in the method for calibrating an optical system of an immunoassay device according to an embodiment of the present invention;
FIG. 5 is a graph of calibration curves obtained by calibration in an immunoassay analyzer optical system calibration method according to an embodiment of the present invention;
in the figure: 1-a first buffer; 2-a second buffer; 3-a third buffer; 4-fourth buffer; 5-fifth buffer; 6-sixth buffer; 7-seventh buffer; 8-eighth buffer; 9-a ninth buffer; 10-tenth buffer; 100-buffer plate 100; 200-detecting card; 211-loading pad; 212-absorbent paper; 213-nitrocellulose membrane; 214-PVC bottom plate; 220-detecting a cartridge;
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other examples, which a person of ordinary skill in the art would obtain without undue burden based on the embodiments of the invention, are within the scope of the invention.
In the drawings, the shape and size may be exaggerated for clarity, and the same reference numerals will be used throughout the drawings to designate the same or similar components.
In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, etc. are defined with respect to the configuration shown in the drawings, and in particular, "height" corresponds to the top-to-bottom dimension, "width" corresponds to the left-to-right dimension, and "depth" corresponds to the front-to-back dimension, are relative concepts, and thus may vary accordingly depending on the location and use of the terms, and therefore these or other orientations should not be interpreted as limiting terms.
Terms (e.g., "connected" and "attached") referring to an attachment, coupling, etc., refer to a relationship wherein these structures are directly or indirectly secured or attached to one another through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.
As can be seen in conjunction with the illustrations of fig. 1 and 2, according to an embodiment of the present invention, the immunoassay analyzer optical system calibration method includes:
examples:
1. preparing a buffer solution:
1.1Alexa Fluor 647 the goat anti-rabbit antibody is marked, the coupling ratio of the marking is detected to be between 1.4 and 4 by using a spectrophotometer, and the theoretical concentration of the marked goat anti-rabbit is 2.0mg/ml;
1.2 preparation of 10 buffer standards at different calibration point concentrations:
first buffer 1: diluting marked 2.0mg/ml goat anti-rabbit solution 80 times, diluting the concentration to 0.025mg/ml, obtaining a first buffer solution 1, and taking 6ml for later use;
second buffer 2: mixing 3ml of the first buffer solution 1 with 3ml of diluent, diluting the concentration of the diluent to 0.0125mg/ml to obtain a second buffer solution 2, and taking 6ml for later use;
third buffer 3: mixing 3ml of the second buffer solution 2 with 3ml of diluent, and diluting the concentration of the diluent to 6.25ug/ml to obtain a third buffer solution 3, wherein 6ml of the third buffer solution is taken for standby;
fourth buffer 4: mixing 3ml of the third buffer solution 3 with 3ml of diluent, and diluting the concentration of the third buffer solution to 3.125ug/ml to obtain a fourth buffer solution 4, and taking 6ml for later use;
fifth buffer 5: mixing 3ml of the fourth buffer solution 4 with 3ml of diluent, diluting the concentration of the fourth buffer solution to 1.5625ug/ml to obtain a fifth buffer solution 5, and taking 6ml for later use;
sixth buffer 6: mixing 3ml of the fifth buffer solution 5 with 3ml of diluent, diluting the concentration of the diluent to 0.78125ug/ml to obtain a sixth buffer solution 6, and taking 6ml for later use;
seventh buffer 7: mixing 3ml of the sixth buffer solution 6 with 3ml of diluent, diluting the concentration of the diluent to 0.390625ug/ml to obtain a seventh buffer solution 7, and taking 6ml for later use;
eighth buffer 8: mixing 3ml of the seventh buffer solution 7 with 3ml of diluent, diluting the concentration of the solution to 0.1953125ug/ml to obtain an eighth buffer solution 8, and taking 6ml for later use;
ninth buffer 9: mixing 3ml of the eighth buffer solution 8 with 3ml of diluent, diluting the concentration of the diluent to 0.0976563ug/ml to obtain a ninth buffer solution 9, and taking 6ml for later use;
tenth buffer 10: mixing 3ml of the solution of the ninth buffer solution 9 with 3ml of diluent, diluting the concentration of the solution to 0.0488282ug/ml to obtain a tenth buffer solution 10, and taking 6ml for later use;
1.3 10 buffer standards of different concentrations were filled into buffer plates 100, 60ul of buffer standard per well (as shown in fig. 1);
1.4 sealing by using a film sealing machine for standby.
2. Preparation of calibration test card 200
2.1 preparing 2ml of rabbit IgG coating buffer, wherein the concentration of the rabbit IgG is 1.0mg/ml;
2.2 coating rabbit IgG coating buffer solution to a nitrocellulose membrane 213 by using a full-automatic membrane-drawing production platform, wherein the width of the nitrocellulose membrane is 3.9-4.1mm, and each part of the nitrocellulose membrane 213 is coated with 0.5ul of rabbit IgG coating buffer solution, and drying is carried out at 60 ℃ for 2min;
2.3 assembling the test card 200, the assembly process is as follows:
2.3.1 preparation of materials: a loading pad 211, a water absorbing paper 212, a coated nitrocellulose membrane 213, a PVC base plate 214, and a detection cartridge 220;
2.3.2 assembling the three materials of the sample pad 211, the absorbent paper 212 and the coated nitrocellulose membrane 123 on the PVC bottom plate 214 to form a membrane strip, wherein the width of the membrane strip is 3.9-4.1mm (shown in figure 2);
2.3.3 the membrane strip 210 is placed in the test cassette 220 (as shown in fig. 3).
3. Sample detection
3.1, starting the instrument, and performing hardware self-checking;
3.2, logging in user software;
3.3 loading TIP consumables;
3.4 loading of calibration buffer: inputting calibration buffer information;
3.5, inputting calibration detection information and detection quantity in user software;
3.6 sample detection, namely repeatedly completing 10 different gradient calibration tests to obtain corresponding calibration point fluorescence values;
the immunoassay method comprises the following steps of:
3.6.1. the mechanical arm takes TIP consumable (pipette TIP) and installs the TIP consumable to a pipette;
3.6.2. the pipettor moves to a buffer night plate;
3.6.3. the pipettor absorbs the buffer standard;
3.6.4. discarding the used buffer plate 100;
3.6.5. moving the test card 200 to the reaction position;
3.6.6. the pipettor pipettes the buffer standard to the detection card 200;
3.6.7. moving the detection card 200 to detection position detection;
3.6.8. displaying and storing the detection result;
3.6.9. discarding the test card 200;
3.6.10. the TIP consumable (pipette TIP) was discarded.
3.7, repeating the calibration test of the buffer standard substance for detecting 10 different calibration point concentrations to obtain corresponding fluorescence values.
4. And (3) calibrating:
4.1. from the calibration buffer information, the F (X) equation is calculated by a regression function: f (X) = 203.49X 2 -1424.5x+1996.4;
4.2. After all calibration tests are completed, calibration point concentration values of 10 buffers are obtained, and a G (X) equation is calculated through a regression function: g (X) = 206.31X 2 -1411.1x+1953.6;
4.3, to obtain a new equation Delta Y (X) =f (X) -G (X), as shown in fig. 5:
Y(X)=2.8175x 2 -13.392x+42.885;
4.4Y (X) is the calibration curve of the optical system of the immunoassay analyzer, and the optical system of the immunoassay analyzer is calibrated according to the calibration curve.
The invention calculates the fitting curve between the calibration machine and the standard machine by using a fitting algorithm, and controls the inter-table errors caused by nonlinear relations among different optical modules within a controllable range.
5. Evaluation of calibration results
Performing test comparison and rechecking by using a standard machine and using a buffer test solution with non-calibrated point concentration, wherein the accuracy deviation is considered to be qualified within the allowable deviation degree, and the allowable deviation degree is 5%;
standard machine test results | G(x) | Y(x) | Post calibration results | Deviation of |
-41.99 | 52.50 | -94.30 | -41.80 | 0.00 |
10.36 | 108.35 | -97.80 | 10.55 | 0.02 |
152.13 | 266.36 | -107.03 | 159.33 | 0.05 |
1062.87 | 1204.44 | -151.34 | 1053.10 | -0.01 |
3200.77 | 3535.21 | -234.14 | 3301.07 | 0.03 |
6346.84 | 6785.58 | -328.36 | 6457.22 | 0.02 |
The test shows that the fluorescence reading is carried out on the buffer solution with the concentration of 6 non-calibrator materials, the deviation between the fluorescence reading and the standard machine is within the allowable deviation range, and the calibration is qualified.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (8)
1. An immunoassay analyzer optical system calibration method, comprising:
step 1, preparing a buffer standard:
preparing 10 buffer standard substances with different calibration point concentrations into a buffer plate;
step 2, preparing a calibration detection card:
preparing a prepared rabbit IgG coating buffer solution, and coating the prepared rabbit IgG coating buffer solution on the nitrocellulose membrane; loading the sample loading pad, the absorbent paper and the nitrocellulose membrane on a PVC bottom plate, and cutting the sample loading pad, the absorbent paper and the nitrocellulose membrane into membrane strips; placing the membrane strip in a detection card shell;
step 3, sample detection:
recording calibration point information of a buffer standard into an immunoassay analyzer;
detecting buffer solution standard substances with 10 different calibration point concentrations by using an immunoassay analyzer to obtain corresponding calibration point fluorescence values;
step 4, calibrating:
obtaining standard calibration point fluorescence values of 10 buffer standard products according to the calibration point information of the buffer standard products, and calculating an F (x) equation under the standard condition through a regression function;
obtaining test calibration point fluorescence values corresponding to 10 buffer liquid standard substances according to a sample detection result, and calculating a G (x) equation under the detection condition through a regression function family;
obtaining a calibration equation Y (x) =f (x) -G (x);
calibrating an optical system of the immunity analyzer according to a calibration equation;
step 5, calibration evaluation:
and (3) testing by adopting a buffer solution with non-calibrated point concentration, comparing with a standard machine testing result, and correcting to be qualified when the accuracy deviation is within the allowable deviation degree.
2. The method for calibrating an optical system of an immunoassay according to claim 1, wherein the buffer standard used in the step 1 is a goat anti-rabbit antibody solution labeled with Alexa Fluor 647.
3. The immunoassay analyzer optical system calibration method of claim 2, wherein the goat anti-rabbit antibody solution label coupling ratio is between 1.4 and 4.
4. The method for calibrating an optical system of an immunoassay analyzer according to claim 3, wherein the nitrocellulose membrane in the step 2 has a width of 3.9 to 4.1mm.
5. The method according to claim 4, wherein the nitrocellulose membrane in step 2 is coated with 0.5ul of rabbit IgG coating buffer per revolution.
6. The method for calibrating an optical system of an immunoassay according to claim 5, wherein the concentration of the rabbit IgG coating buffer in the step 2 is 1.0mg/ml.
7. The method for calibrating an optical system of an immunoassay analyzer according to claim 3, wherein the step of detecting a buffer standard in the immunoassay analyzer in step 3 comprises:
1) The mechanical arm takes TIP consumable material and installs the TIP consumable material to the pipettor;
2) The pipettor moves to a buffer night plate;
3) The pipettor absorbs the buffer standard;
4) Discarding the used buffer plate;
5) Moving the detection card to a reaction position;
6) The liquid shifter shifts the buffer standard to the detection card;
7) Moving the detection card to a detection position for detection;
8) Displaying and storing the detection result;
9) Discarding the detection card;
10 A TIP consumable is discarded.
8. The method of calibrating an optical system of an immunoassay according to claim 7, wherein the allowable deviation in the step 5 is 5%.
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