阅读说明：本技术 一种免疫分析仪光学系统校准方法 (Calibration method for optical system of immunoassay analyzer ) 是由 席秋子 孙虎 张瑞娟 朱烨欣 张运平 于 2021-03-05 设计创作，主要内容包括：本发明公开了一种免疫分析仪光学系统校准方法,包括：1.制备缓冲液标准品；2.制备校准检测卡；3.样本检测；4.校准；5.校准评估；根据本发明,其利用拟合算法,计算出校准机与标准机之间的拟合曲线,把不同光学模块之间用于非线性关系引起的台间误差控制住可控范围之内。(The invention discloses a method for calibrating an optical system of an immunoassay analyzer, which comprises the following steps: 1. preparing a buffer solution standard substance; 2. preparing a calibration detection card; 3. detecting a sample; 4. calibrating; 5. evaluating the calibration; according to the invention, a fitting algorithm is utilized to calculate a fitting curve between the calibration machine and the standard machine, and the inter-station error caused by the nonlinear relation between different optical modules is controlled within a controllable range.)

1. A method for calibrating an optical system of an immunoassay analyzer, comprising:

step 1, preparing a buffer solution standard:

preparing 10 buffer solution standard products with different calibration point concentrations into a buffer solution 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 a nitrocellulose membrane; loading the sample loading pad, the absorbent paper and the nitrocellulose membrane to a PVC (polyvinyl chloride) bottom plate, and cutting into membrane strips; placing the membrane strip in a detection card shell;

step 3, sample detection:

inputting calibration point information of a buffer solution standard substance into an immunoassay analyzer;

detecting buffer solution standard substances with 10 different calibration point concentrations by using an immunoassay analyzer to obtain corresponding fluorescence values of the calibration points;

step 4, calibration:

obtaining standard calibration point fluorescence values of 10 buffer solution standard products according to calibration point information of the buffer solution standard products, and calculating an equation F (x) under a standard condition through a regression function;

obtaining the fluorescence values of the test calibration points corresponding to the 10 buffer solution standard products according to the detection result of the sample, and calculating a G (x) equation under the detection condition through a regression function;

obtaining a calibration equation y (x) ═ f (x) -g (x);

calibrating an optical system of the immunoassay analyzer according to a calibration equation;

step 5, calibration evaluation:

and (4) testing by adopting a buffer solution with a non-calibration point concentration, comparing the test result with a test result of a standard machine, and correcting to be qualified when the accuracy deviation is within an allowable deviation degree.

2. The method for calibrating an optical system of an immunoassay analyzer according to claim 1, wherein the buffer standard used in step 1 is a solution of goat anti-rabbit antibody labeled with Alexa Fluor 647.

3. The method for calibrating an optical system of an immunoassay analyzer according to 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 width of the nitrocellulose membrane in step 2 is 3.9 to 4.1 mm.

5. The method for calibrating an optical system of an immunoassay analyzer according to claim 4, wherein the nitrocellulose membrane of step 2 is coated with 0.5ul of rabbit IgG coating buffer per cm.

6. The method for calibrating an optical system of an immunoassay analyzer according to claim 5, wherein the concentration of the rabbit IgG coating buffer in the step 2 is 1.0 mg/ml.

7. The method for calibrating an optical system of an immunoassay analyzer of claim 3, wherein the step of detecting a buffer standard by the immunoassay analyzer in step 3 comprises:

1) the mechanical arm takes TIP consumables (pipette TIPs) and installs the TIP consumables into a pipette;

2) moving the pipettor to a buffer night plate;

3) sucking a buffer solution standard substance by a liquid shifter;

4) discarding the used buffer liquid plate;

5) moving the detection card to a reaction position;

6) the pipettor pipettes the buffer solution standard substance 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 test card;

10) TIP consumables (pipette TIPs) were discarded.

8. The method for calibrating an optical system of an immunoassay analyzer of claim 7, wherein the allowable deviation degree in the step 5 is 5%.

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to a calibration method for an optical system of an immunoassay analyzer.

Background

The in vitro diagnosis POCT is a detection technology with great potential, has the advantages of rapidness, simplicity, convenience, high efficiency, low cost, short detection period, small sample consumption and the like, and is widely applied to clinic. POCT has been rapidly developed in recent years as a new development direction.

However, since the domestic market starts late, where improvement is needed in the production and development processes of the optical module, the unified instrument optical system may cause the inter-stage error of the non-linear optical module due to the difference between the components, the difference caused by machining tolerance or the difference caused by the installation and adjustment process.

In view of the above, it is necessary to develop a calibration method for an optical system of an immunoassay analyzer to solve the above problems.

Disclosure of Invention

Aiming at the defects in the prior art, the invention mainly aims to calculate the fitting curve between the calibration machine and the standard machine by using a fitting algorithm and control the inter-station errors caused by the nonlinear relation between different optical modules within a controllable range.

To achieve the above objects and other advantages in accordance with the present invention, there is provided an immunoassay analyzer optical system calibration method including:

step 1, preparing a buffer solution standard:

preparing 10 buffer solution standard products with different calibration point concentrations into a buffer solution 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 a nitrocellulose membrane; loading the sample loading pad, the absorbent paper and the nitrocellulose membrane to a PVC (polyvinyl chloride) bottom plate, and cutting into membrane strips; placing the membrane strip in a detection card shell;

step 3, sample detection:

inputting calibration point information of a buffer solution standard substance into an immunoassay analyzer;

detecting buffer solution standard substances with 10 different calibration point concentrations by using an immunoassay analyzer to obtain corresponding fluorescence values of the calibration points;

step 4, calibration:

obtaining standard calibration point fluorescence values of 10 buffer solution standard products according to calibration point information of the buffer solution standard products, and calculating an equation F (x) under a standard condition through a regression function;

obtaining the fluorescence values of the test calibration points corresponding to the 10 buffer solution standard products according to the detection result of the sample, and calculating a G (x) equation under the detection condition through a regression function;

obtaining a calibration equation y (x) ═ f (x) -g (x);

calibrating an optical system of the immunoassay analyzer according to a calibration equation;

step 5, calibration evaluation:

and (4) testing by adopting a buffer solution with a non-calibration point concentration, comparing the test result with a test result of a standard machine, and correcting to be qualified when the accuracy deviation is within an allowable deviation degree.

Further, the buffer standard used in step 1 is a solution of goat anti-rabbit antibody labeled with Alexa Fluor 647.

Further, the sheep anti-rabbit antibody solution labeling coupling ratio is between 1.4 and 4.

Further, the width of the nitrocellulose membrane in the step 2 is 3.9-4.1 mm.

Further, in the step 2, the nitrocellulose membrane is coated with 0.5ul of rabbit IgG coating buffer per centimeter.

Further, the concentration of the rabbit IgG coating buffer in the step 2 is 1.0 mg/ml.

Further, the step of detecting the buffer solution standard by the immunoassay analyzer in the step 3 comprises:

1) the mechanical arm takes TIP consumables (pipette TIPs) and installs the TIP consumables into a pipette;

2) moving the pipettor to a buffer night plate;

3) sucking a buffer solution standard substance by a liquid shifter;

4) discarding the used buffer liquid plate;

5) moving the detection card to a reaction position;

6) the pipettor pipettes the buffer solution standard substance 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 test card;

10) TIP consumables (pipette TIPs) were 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 immunoassay analyzer has the advantages of simple operation, high detection speed, wide and easily obtained raw material sources and low cost;

the invention uses the fitting algorithm to calculate the fitting curve between the calibrator and the standard machine, and controls the inter-station error caused by the nonlinear relation between different optical modules within the controllable range.

Drawings

Fig. 1 is a top view of a buffer plate 100 in a calibration method for an optical system of an immunoassay analyzer according to an embodiment of the present invention;

FIG. 2 is a view showing the structure of a membrane strip in a calibration method for an optical system of an immunoassay analyzer according to an embodiment of the present invention;

fig. 3 is a view showing the structure of a detection card 200 in the calibration method of an optical system of an immunoassay analyzer according to an embodiment of the present invention;

FIG. 4 is a diagram illustrating the steps of sample detection in the calibration method of the optical system of the immunoassay analyzer according to one embodiment of the present invention;

FIG. 5 is a calibration graph obtained by calibration in a calibration method for an optical system of an immunoassay analyzer according to an embodiment of the present invention;

in the figure: 1-a first buffer; 2-a second buffer; 3-a third buffer; 4-a fourth buffer; 5-fifth buffer; 6-sixth buffer; 7-a seventh buffer; 8-eighth buffer; 9-ninth buffer; 10-tenth buffer; 100-buffer plate 100; 200-detection card; 211-loading pad; 212-absorbent paper; 213-nitrocellulose membrane; 214-PVC base plate; 220-detection of the card shell;

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present 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 configurations shown in the respective drawings, and in particular, "height" corresponds to a dimension from top to bottom, "width" corresponds to a dimension from left to right, "depth" corresponds to a dimension from front to rear, which are relative concepts, and thus may be varied accordingly depending on the position in which it is used, and thus these or other orientations should not be construed as limiting terms.

Terms concerning attachments, coupling and the like (e.g., "connected" and "attached") refer to a relationship wherein structures are secured or attached, either directly or indirectly, to one another through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.

According to an embodiment of the present invention, with reference to the illustrations of fig. 1 and 2, it can be seen that the method for calibrating the optical system of the immunoassay analyzer comprises:

example (b):

1. preparing a buffer solution:

1.1Alexa Fluor 647 is used for marking the goat anti-rabbit antibody, a spectrophotometer is used for detecting that the coupling ratio of the mark is between 1.4 and 4, and the theoretical concentration of the marked goat anti-rabbit is 2.0 mg/ml;

1.2 prepare buffer standards at 10 different calibration point concentrations:

first buffer 1: diluting the labeled 2.0mg/ml goat anti-rabbit solution by 80 times, diluting the labeled goat anti-rabbit solution to 0.025mg/ml to obtain a first buffer solution 1, and taking 6ml for later use;

second buffer 2: mixing 3ml of the first buffer solution 1 solution with 3ml of diluent, diluting the concentration of the first buffer solution 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 solution with 3ml of diluent, diluting the concentration of the diluent to 6.25ug/ml to obtain a third buffer solution 3, and taking 6ml for later use;

fourth buffer 4: 3ml of the third buffer solution 3 solution is mixed with 3ml of diluent, the concentration of the diluent is diluted to 3.125ug/ml, a fourth buffer solution 4 is obtained, and 6ml of the fourth buffer solution is taken for standby;

fifth buffer 5: mixing 3ml of the fourth buffer solution 4 solution with 3ml of diluent, diluting the concentration of the fourth buffer solution 4 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 solution 5 of the fifth buffer solution 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 solution 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 solution with 3ml of diluent, diluting the concentration of the diluent 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 solution 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 ninth buffer solution 9 solution with 3ml of diluent, diluting the concentration of the diluent to 0.0488282ug/ml to obtain a tenth buffer solution 10, and taking 6ml for later use;

1.3 filling 10 buffer standard products with different concentrations into a buffer plate 100, and filling 60ul of buffer standard products in each hole (as shown in figure 1);

and 1.4, sealing by using a film sealing machine for later use.

2. Preparation of calibration test card 200

2.1 preparing 2ml of rabbit IgG coating buffer solution, wherein the concentration of the rabbit IgG is 1.0 mg/ml;

2.2 coating the rabbit IgG coating buffer solution to a nitrocellulose membrane 213 by using a full-automatic scribing production platform, wherein the width of the nitrocellulose membrane is 3.9-4.1mm, each centimeter of the nitrocellulose membrane 213 is coated with 0.5ul of the rabbit IgG coating buffer solution, and drying is carried out for 2min at 60 ℃;

2.3 assembling the test card 200, the assembly process is as follows:

2.3.1 Material preparation: a sample loading pad 211, absorbent paper 212, a coated nitrocellulose membrane 213, a PVC base plate 214, and a detection card shell 220;

2.3.2 assembling three materials of a loading pad 211, absorbent paper 212 and a coated nitrocellulose membrane 123 on a PVC base 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 Place the membrane strip 210 in the test card housing 220 (as shown in FIG. 3).

3. Sample detection

3.1 starting the instrument and performing hardware self-test;

3.2 logging in user software;

3.3 loading TIP consumables;

3.4 Loading calibration buffer: inputting information of a calibration buffer solution;

3.5 inputting calibration detection information and detection quantity in user software;

3.6 detecting the sample, and repeatedly finishing detection of 10 different gradient calibration tests to obtain corresponding fluorescence values of the calibration points;

the detection process of the immunoassay analyzer is as shown in fig. 4:

3.6.1. the mechanical arm takes TIP consumables (pipette TIPs) and installs the TIP consumables into a pipette;

3.6.2. moving the pipettor to a buffer night plate;

3.6.3. sucking a buffer solution standard substance by a liquid shifter;

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 solution standard substance to the detection card 200;

3.6.7. moving the detection card 200 to a detection position for detection;

3.6.8. displaying and storing the detection result;

3.6.9. a discard detection card 200;

3.6.10. TIP consumables (pipette TIPs) were discarded.

3.7 repeating the calibration test of the buffer solution standard substance for detecting 10 different calibration point concentrations to obtain the corresponding fluorescence value.

4. Calibration:

4.1. from the calibration buffer information, the f (x) equation was calculated by the regression function: f (x) 203.49x²-1424.5x+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) ═ 206.31x²-1411.1x+1953.6；

4.3 subtracting the two equations to obtain a new equation Delta Y (X) ═ f (X) -g (X), as shown in fig. 5:

Y(X)＝2.8175x²-13.392x+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 uses the fitting algorithm to calculate the fitting curve between the calibrator and the standard machine, and controls the inter-station error caused by the nonlinear relation between different optical modules within the controllable range.

5. Evaluation of calibration results

Performing test comparison and retest by using a standard machine and using buffer test solution with non-calibration point concentration, wherein the accuracy deviation is qualified within the allowable deviation degree which is 5%;

test results of the Standard machine	G(x)	Y(x)	Results after calibration	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 selected buffer solution with 6 non-calibrator concentrations is used for fluorescence reading, and the standard deviation is found to be within the allowable deviation degree range, so that the calibration is qualified.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.