A fully automatic analysis method for enzyme-linked immunosorbent assay

Abstract

The present invention provides a fully automatic analysis method for an enzyme-linked immunosorbent assay. The fully automatic analysis method for an enzyme-linked immunosorbent assay comprises: an analysis system and an automatic sample loading module, wherein the analysis system comprises a porous plate, the analysis system comprises a liquid storage module, a reaction module, a cleaning module, and a detection module, and the analysis system further comprises a temperature control module, a multichannel pipette system, and a detection system; the liquid storage module utilizes the porous plate strips to store the corresponding reaction reagents; the automatic sample loading module mainly relies on a multichannel pipette to perform liquid addition and liquid removal operations, and automatically completes the addition and removal of the relevant liquids. The fully automatic analysis method for an enzyme-linked immunosorbent assay provided by the present invention is easy to use, can reduce the production and use costs of the instrument, reduce the amount of solution used and the generation of waste liquid, thereby reducing the workload of staff and improving detection efficiency and accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a fully automatic analysis method for enzyme-linked immunosorbent assay (ELISA). Background Art

[0002] As a method for measuring trace amounts of antigens and antibodies, enzyme-linked immunosorbent assay (ELISA) or microarray biochips are widely used in food safety testing, clinical testing, and scientific research experiments due to their high sensitivity, high specificity, and low cost. ELISA and microplate array chips are usually tested on multi-well plates. The basic process includes sample addition, sample incubation, plate washing, substrate color development, data reading, etc. In the related art, an ELISA simulation method is disclosed, including the existing standard ELISA method. The method is to replace the coated ELISA plate with a 48-well blank ELISA plate, use distilled water or physiological saline instead of serum, use a blue indicator instead of the enzyme reagent and substrate reagent, and use hydrochloric acid solution instead of the stop solution. Finally, the existing standard ELISA method is used to perform a simulation test, which can reflect the objective phenomena visible in the standard ELISA test. The present invention uses cheap distilled water and a 48-well blank ELISA plate to simulate the operation method and visible objective phenomena of the enzyme-linked immunosorbent assay (ELISA). Students can each have a 48-well blank ELISA plate and perform plate washing training twice before and after the experiment; 50 μl of distilled water is added to each well of the 48-well blank ELISA plate instead of serum, which not only saves costs but also reduces the chance of laboratory contamination or infection caused by serum brought from the clinic.

[0003] However, there are still some shortcomings in the above structure. Compared with immunochromatographic test strips, microplate array chips are a relatively labor-intensive, time-consuming and energy-intensive experiment. Moreover, due to the large processing volume and multiple procedures, it is very easy to omit, over-add or wrongly add reagents, which may affect the experimental results. In addition, since there is always a sequence when adding reagents, there are differences in incubation or washing time, especially the final color development time, which will directly affect the experimental results and make the results unreliable. During the operation, it is also necessary to strictly prevent external contamination and cross-contamination between different wells.

[0004] Therefore, it is necessary to provide a new ELISA fully automatic analysis method to solve the above technical problems Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a fully automatic analysis method for enzyme-linked immunosorbent assay, which is easy to use, can reduce the production and use costs of the instrument, reduce the amount of solution used and the generation of waste liquid, thereby reducing the workload of staff and improving the detection efficiency and accuracy.

[0006] To solve the above technical problems, the present invention provides a fully automatic analysis method for enzyme-linked immunosorbent assay, comprising: an analysis system and an automatic sample loading module, wherein the analysis system comprises a porous plate, the analysis system comprises a liquid storage module, a reaction module, a cleaning module and a detection module, and the analysis system further comprises a temperature control module, a multi-channel pipetting system and a detection system; the liquid storage module utilizes the porous plate strips to store the corresponding reaction reagents; the automatic sample loading module mainly relies on a multi-channel pipette to perform liquid adding and liquid removal operations, automatically completing the addition and removal of relevant liquids; the reaction module and the temperature control module enable the relevant reactions to be carried out under temperature-controlled conditions; the detection module detects the well strips after the reaction and automatically analyzes and provides a detection report.

[0007] As a further embodiment of the present invention, the porous plate is divided into zone 1, zone 2, zone 3, zone 4, zone 5 and zone 6.

[0008] As a further solution of the present invention, each solution in the liquid storage module is stored in a liquid storage area in a multi-well plate, and the solutions include antibody reagent, cleaning solution, color developing solution A and color developing solution B respectively.

[0009] As a further solution of the present invention, the automatic sample loading module performs sample loading operations through a multi-channel electric pipette, and array points of different items to be detected are fixed in the 5-zone well plate.

[0010] As a further solution of the present invention, the temperature control module is located at the bottom of the fixed porous plate and is used for heating during the reaction with the reagent to provide the temperature required for the reaction.

[0011] As a further solution of the present invention, the cleaning template is cleaned by removing the washing solution with a multi-channel electric pipette, and the detection module mainly uses CCD to take pictures of the microarray chip in the 5 areas to obtain images, and then the software extracts the signals and provides a detection report.

[0012] As a further embodiment of the present invention, the analysis system comprises the following steps:

[0013] (1) Add the standard or sample to each reaction well in the 5 zones of the multi-well plate;

[0014] (2) Pipette the antibody working solution from zone 1 to zone 5 and heat for 15 minutes;

[0015] (3) Aspirate the reacted solution from zone 5 to zone 6;

[0016] (4) Pour the cleaning solution from zone 4 to the reacted zone 5 and clean for 10 seconds;

[0017] (5) Aspirate the cleaning fluid from zone 5 to zone 6;

[0018] (6) Repeat (4) and (5) once;

[0019] (7) Pipette color development solution A from area 2 into color development solution B in area 3 and mix for 5 seconds;

[0020] (8) Pipette the mixed color developing solution from zone 3 to zone 5 and heat for color development reaction for 10 minutes;

[0021] (9) Pipette the color-developing solution after reaction from zone 5 to zone 6;

[0022] (10). Repeat (4) and (5) once.

[0023] Compared with related technologies, the fully automatic analysis method for enzyme-linked immunosorbent assay provided by the present invention has the following beneficial effects:

[0024] The present invention provides a fully automatic analysis method for enzyme-linked immunosorbent assay:

[0025] 1. By setting up a liquid storage module, an automatic sample addition module, a reaction module, a temperature control module, a cleaning template and a detection module, the system can be operated automatically and used more conveniently, thereby reducing the workload of the staff and improving the detection efficiency and accuracy;

[0026] 2. The storage, reaction, and cleaning of cleaning liquids and waste liquids are all completed in the multi-well plate, saving space. The pipetting is performed by an 8-channel electric pipette and an automatic translation device, eliminating the need for structures such as peristaltic pumps or syringes. While ensuring functionality, it saves instrument space and reduces the space occupied by the analyzer, thereby reducing the production and use costs of the instrument, the amount of solution used, and the generation of waste liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0028] Figure 1 Schematic diagram of the porous plate partitioning of the porous plate in the present invention. DETAILED DESCRIPTION

[0029] Please refer to Figure 1 ,in, Figure 1 Schematic diagram of the porous plate partitioning of the porous plate in the present invention.

[0030] Example 1:

[0031] A fully automatic analysis method for enzyme-linked immunosorbent assay (ELISA) comprises an analysis system and an automatic sample loading module, wherein the analysis system comprises a multi-well plate, a liquid storage module, a reaction module, a cleaning module, and a detection module, and further comprises a temperature control module, a multi-channel pipetting system, and a detection system.

[0032] The liquid storage module will use porous strips to store corresponding reaction reagents;

[0033] The automatic sample adding module mainly relies on a multi-channel pipette to perform liquid adding and liquid taking operations, automatically completing the addition and removal of relevant liquids;

[0034] The reaction module and the temperature control module enable the relevant reactions to be carried out under temperature control conditions;

[0035] The detection module will detect the well strip after the reaction and automatically analyze and provide a detection report.

[0036] The multi-well plate is divided into zone 1, zone 2, zone 3, zone 4, zone 5 and zone 6.

[0037] Each solution in the liquid storage module is stored in a liquid storage area in a multi-well plate, and the solutions include an antibody reagent, a cleaning solution, a color developing solution A, and a color developing solution B.

[0038] The automatic sample loading module performs sample loading operations through a multi-channel electric pipette, and array points of different items to be detected are fixed in the 5-area medium-well plate.

[0039] The temperature control module is located at the bottom of the fixed porous plate and is used for heating during the reaction with the reagent to provide the temperature required for the reaction.

[0040] The cleaning template is cleaned by taking washing solution with a multi-channel electric pipette. The detection module mainly takes pictures of the microarray chip in the 5 zones through CCD to obtain images, and then the software extracts the signals and provides a detection report.

[0041] The analysis system comprises the following steps:

[0042] (1) Add the standard or sample to each reaction well in the 5 zones of the multi-well plate;

[0043] (2) Pipette the antibody working solution from zone 1 to zone 5 and heat for 15 minutes;

[0044] (3) Aspirate the reacted solution from zone 5 to zone 6;

[0045] (4) Pour the cleaning solution from zone 4 to the reacted zone 5 and clean for 10 seconds;

[0046] (5) Aspirate the cleaning fluid from zone 5 to zone 6;

[0047] (6) Repeat (4) and (5) once;

[0048] (7) Pipette color development solution A from area 2 into color development solution B in area 3 and mix for 5 seconds;

[0049] (8) Pipette the mixed color developing solution from zone 3 to zone 5 and heat for color development reaction for 10 minutes;

[0050] (9) Pipette the color-developing solution after reaction from zone 5 to zone 6;

[0051] (10). Repeat (4) and (5) once.

[0052] The working principle of the fully automatic analysis method for enzyme-linked immunosorbent assay provided by the present invention is as follows:

[0053] Each solution in the liquid storage module is stored in the liquid storage area of ​​the multi-well plate. The solutions include antibody reagent, color developing solution A, color developing solution B, and cleaning solution. The volume of each solution is 80-300μl. The automatic loading module performs loading operations through a multi-channel electric pipette. The well plate in the reaction module is fixed with array points of different items to be detected. The heating module is located at the bottom of the fixed multi-well plate and is used to heat during the reaction with the reagent to provide the temperature required for the reaction. The cleaning template is cleaned by transferring the washing solution through a multi-channel electric pipette. The detection module mainly uses CCD to take pictures to obtain images, and then the software extracts the signal and provides a detection report. All operating procedures are set through the operation panel.

[0054] Compared with related technologies, the fully automatic analysis method for enzyme-linked immunosorbent assay provided by the present invention has the following beneficial effects:

[0055] The present invention provides a fully automatic analysis method for enzyme-linked immunosorbent assay, which is equipped with a liquid storage module, an automatic sample addition module, a reaction module, a temperature control module, a cleaning template and a detection module, so that the method can be operated automatically and used more conveniently, thereby reducing the workload of the staff and improving the detection efficiency and accuracy.

[0056] The storage, reaction, and cleaning of cleaning liquids and waste liquids are all completed in the multi-well plate, saving space. The pipetting is performed using an 8-channel electric pipette and an automatic translation device, eliminating the need for structures such as peristaltic pumps or syringes. While ensuring functionality, it saves instrument space and reduces the space occupied by the analyzer, thereby reducing the production and use costs of the instrument, the amount of solution used, and the generation of waste liquid.

[0057] Example 2: The present invention also provides a fully automatic analysis method for enzyme-linked immunosorbent assay.

[0058] (1) Weigh 1 g of crushed vegetables and add 10 mL of PBST buffer for subsequent analysis;

[0059] (2) Add nanosilver-labeled carbendazim, thiophanate-methyl, and carbofuran mixed antibody working solution, 80 μL / well of color development solution A and 80 μL / well of color development solution B to microwell storage area 1, 2, and 3. Add 350 μL of cleaning solution / well to microwell area 4.

[0060] (3) In the wells of zone 5 of the microwell, goat anti-mouse IgG was used as a positive control, bovine serum albumin was used as a blank control, and the target substances, carbendazim, thiophanate-methyl, and carbofuran antigens, were spotted in the microwells through the spotting preparation system and fixed at 37°C for 2 hours.

[0061] (4) Microwell zone 6 is a blank well strip used to store waste liquid.

[0062] (5) Microplate according to Figure 1 After the pattern is placed, follow the steps below to react:

[0063] 1) Pipette 50 μL of standard or sample into each reaction well in zone 5 of the microplate;

[0064] 2) Pipette 50 μL of the mixed antibody working solution of carbendazim, thiophanate-methyl and carbofuran from microwell zone 1 into the well plate with the antigen array spots of carbendazim, thiophanate-methyl and carbofuran in zone 5, and heat at 37°C for 15 minutes;

[0065] 3) The reacted solution is sucked back from zone 5 to the waste liquid area in zone 6;

[0066] 4) Pipette 50 μL of cleaning solution from area 4 into the reacted area 5 and wash for 10 seconds;

[0067] 5). Aspirate the cleaning fluid from zone 5 to zone 6;

[0068] 6). Repeat (4) and (5) once;

[0069] 7) Pipette 50 μL of color development solution A from area 2 into color development solution B in area 3 and mix for 5 seconds;

[0070] 8) Pipette 50 μL of the mixed color development solution from zone 3 into zone 5 and heat at 37°C for 10 minutes;

[0071] 9) Pipette the color-developing solution from zone 5 to zone 6;

[0072] 10). Repeat (4) and (5) once.

[0073] (6) After cleaning, the color development results of the microarray chip in zone 5 are photographed and images are obtained through CCD, and then the signal is extracted by software. The signal value is proportional to the depth of the color development point. Quantification is achieved through the external standard curve and a test report is provided.

[0074] The detection limits for the pesticide residues carbendazim, chlorothalonil, and carbofuran in vegetable samples were 0.3 mg / kg, 0.3 mg / kg, and 0.02 mg / kg, respectively, all below the national maximum allowable residue limits. The relative standard deviation was less than 15%. A single fully automated analysis can test up to 18 samples, essentially meeting the daily testing needs of grassroots organizations. The fully automated analysis equipment also reduces reliance on grassroots testing personnel.

Claims

1. A fully automatic analysis method for enzyme-linked immunosorbent assay, characterized in that: include: An analysis system and an automatic sample loading module, wherein the analysis system includes a porous plate, a liquid storage module, a reaction module, a cleaning module, and a detection module, and further includes a temperature control module, a multi-channel pipetting system, and a detection system; The liquid storage module will use porous strips to store corresponding reaction reagents; The automatic sample adding module mainly relies on a multi-channel pipette to perform liquid adding and liquid taking operations, automatically completing the addition and removal of relevant liquids; The reaction module and the temperature control module enable the relevant reactions to be carried out under temperature control conditions; The detection module will detect the well strip after the reaction and automatically analyze and provide a detection report; The multi-well plate is divided into zone 1, zone 2, zone 3, zone 4, zone 5 and zone 6; Each solution in the liquid storage module is stored in a liquid storage area in a multi-well plate, and the solutions include antibody reagent, cleaning solution, color development solution A and color development solution B respectively; The automatic loading module performs loading operation by a multi-channel electric pipette, and array points of different items to be tested are fixed in the 5-area medium-well plate; The temperature control module is located at the bottom of the fixed porous plate and is used for heating during the reaction with the reagent to provide the temperature required for the reaction; The cleaning template is cleaned by pipetting washing solution with a multi-channel electric pipette, and the detection module mainly uses CCD to take pictures of the microarray chip in the 5 areas to obtain images, and then the software extracts the signals and provides a detection report; The analysis system comprises the following steps: (1) Add the standard or sample to each reaction well in the 5 zones of the multi-well plate; (2) Pipette the antibody working solution from zone 1 to zone 5 and heat for 15 minutes; (3) Aspirate the reacted solution from zone 5 to zone 6; (4) Pour the cleaning solution from zone 4 to the reacted zone 5 and clean for 10 seconds; (5) Aspirate the cleaning fluid from zone 5 to zone 6; (6) Repeat (4) and (5) once; (7) Pipette color development solution A from area 2 into color development solution B in area 3 and mix for 5 seconds; (8) Pipette the mixed color developing solution from zone 3 to zone 5 and heat for color development reaction for 10 minutes; (9) Pipette the color-developing solution after reaction from zone 5 to zone 6; (10). Repeat (4) and (5) once.

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