Calibrator for fluorescence spot imaging quantity of fluorescence spot enzyme-linked immunosorbent assay analyzer

Abstract

The fluorescent spot imaging quantity calibrator for the fluorescent spot enzyme-linked immunosorbent assay analyzer comprises an elisa plate and a calibration assembly, the calibration assembly comprises a sieve plate with micropores, a fluorescent polymer film, a reflective film and a sealing film, when the calibration assembly is used for top detection, the lower end of the sieve plate with the micropores is covered and bonded with the fluorescent polymer film, and the lower end of the sieve plate with the micropores is bonded with the reflective film. The lower end of the fluorescent polymer film is covered and bonded with a reflecting film, the upper end of the sieve plate with the micropores is covered and bonded with a sealing film, the diameter of the calibration components is smaller than that of plate holes of the elisa plate, and the calibration components are respectively placed in 4-6 plate holes near the center of the elisa plate in parallel according to the bonding positions. The sieve plates with the micropores are arranged in the plate holes and are respectively fixed in the plate holes through a binder, the number of the micropores in the sieve plates with the micropores is known, the elisa plate is black, the bottom of the elisa plate is a transparent or non-transparent elisa plate for visible light, and the sealing film is a transparent sealing film in a visible light area.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and in vitro diagnosis, and in particular to a calibrator for the fluorescent spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay (ELISA) analyzer. Background Art

[0002] The Fluorescence ELISPOT Analyzer is a medical research instrument that combines cell culture technology with enzyme-linked immunosorbent assay (ELISA), enabling detection of cytokine secretion at the single-cell level. The device features dual ELISPOT (enzyme-linked immunosorbent spot) and FluoroSpot (fluorescent immunospot) analysis capabilities, supporting two-color and multi-color spot detection, with advantages such as high sensitivity and the ability to detect live cells. Its core technology uses antibodies to capture secreted cytokines and presents the results using colorimetric or fluorescent labeling. It is widely used in basic scientific research, clinical immune monitoring, and other fields.

[0003] The instrument cultures cells in microplate wells coated with specific antibodies, utilizes the secreted cytokines to bind to labeled antibodies, and then uses enzyme-catalyzed substrate color development or fluorescent labeling to form visual spots. Combining cell culture technology with enzyme-linked immunosorbent assay (ELISA), the device can detect cytokine secretion at the single-cell level. This involves using antibodies to capture cytokines secreted by cultured cells and displaying them as enzyme-linked spot color development or fluorescence. It has the advantages of high sensitivity, single-cell level / live cell detection, and simple and economical operation. Fluorescence imaging technology can accurately identify multi-color factor co-secretion spots.

[0004] The spot enzyme-linked immunosorbent assay (ELISA) has important application value in cytokine detection and cell activity and function research. This technology can accurately quantify a variety of cytokines (such as IL-2, IFN-γ, TNF-α, etc.) in cell culture supernatants or biological samples through high-sensitivity fluorescence signal detection, providing key data for immune response mechanism research, inflammatory response assessment, and analysis of disease occurrence and development mechanisms. In terms of cell function analysis, the instrument can be used to detect the activation state and effector function of immune cells (such as T cells and NK cells), for example, by detecting the cytokine secretion spectrum (Th1 / Th2 / Th17 type cytokines) to evaluate the polarization direction of immune cells, or by detecting effector molecules such as granzyme B and perforin to detect cytotoxic activity. In addition, combined with multi-color fluorescent labeling technology, the platform supports the simultaneous detection of multiple cytokines in a single well, significantly improving experimental efficiency, and is suitable for drug screening (such as the effect of immunomodulators on cytokine networks) or functional quality control of cell therapy products (such as CAR-T cells). Its high specificity and low sample requirements make it an efficient tool in cellular immunology research and translational medicine applications.

[0005] The accuracy of the number of spots in a spot-based enzyme-linked immunosorbent assay directly determines the reliability and repeatability of the test results. In quantitative analysis, each spot represents the binding signal of a specific target molecule (such as an antibody, antigen, or cytokine). Inaccurate spot counts may lead to false positives or false negatives, affecting the accuracy of clinical diagnosis or scientific research conclusions. For example, in cytokine detection, if the spot count deviation is too high, the intensity of the immune response may be misjudged, misleading the judgment of disease status or analysis of drug efficacy. In addition, during multiple detection, the spots of different targets need to be accurately distinguished, and the error in the number of spots will cause cross-interference, affecting the validity of multi-parameter data. For low-abundance targets, spot loss will reduce detection sensitivity; while excessive background spots may mask the true signal. In standardized applications (such as vaccine efficacy evaluation or biological product quality control), the repeatability of the spot count is a key quality indicator. Therefore, optimizing image analysis algorithms, calibrating optical systems, and standardizing operating procedures are core measures to ensure accurate spot counting, which directly affect the accuracy, sensitivity, and comparability of results between laboratories.

[0006] The accuracy of the number of spots in a fluorescent spot enzyme-linked immunosorbent assay (ELISA) needs to be ensured through a systematic calibration and evaluation process. Currently, standard fluorescent microspheres or biomarker standards of known concentrations are mainly used. Through automatic imaging and software analysis of the instrument, a linear relationship between the number of spots and the analyte is established, and the performance of the optical module (such as the excitation light source and CCD camera) is regularly verified. The usual calibration and evaluation methods include: (1) repeatability testing, which calculates the coefficient of variation (CV) of the number of spots by multiple tests on the same sample, with a requirement of CV < 10%; (2) linearity verification, which uses a gradient dilution standard to confirm the linear range of the number of spots and the concentration (usually R 2 ≥0.98); (3) Specificity evaluation, using negative control samples to eliminate interference from nonspecific binding spots; (4) Multicenter comparison, using unified standards to evaluate the consistency between different instruments. In addition, it is necessary to combine image analysis algorithm optimization (such as threshold segmentation and spot deconvolution) to distinguish overlapping or weak signal spots. Automated calibration software can monitor the instrument status in real time, and manual review of suspicious results (such as missing spots caused by edge effects) can further reduce errors. These measures together ensure the accuracy of spot counting and provide a reliable basis for accurate detection such as cytokine spectrum analysis.

[0007] Although the current spot enzyme-linked immunosorbent assay is calibrated during use, the current calibration system still has several key technical bottlenecks, which restrict the accuracy and reliability of the calibration results. When using fluorescent microspheres for calibration, there are challenges in tracing the standard value of the microspheres. Due to the particle size heterogeneity, differences in the distribution of fluorescent dyes, and agglomeration during storage during the microsphere preparation process, the actual number of microspheres may deviate significantly from the nominal value, sometimes even up to 15%-20%. At the same time, the optical properties of the microspheres (such as excitation / emission wavelengths) are essentially different from those of real biological samples, making it difficult for microsphere-based calibration to truly reflect the detection conditions of complex biological samples. In terms of standard calibration, its limitations are even more prominent: first, there are differences in post-translational modifications (such as glycosylation) between recombinant protein standards and natural proteins, resulting in different binding affinities; second, the cell culture status (such as the number of passages, culture medium components) will significantly affect the physical and chemical properties of secreted cytokines, causing systematic errors in the standard curve and actual sample detection. Experimental data show that even after strict calibration, the detection error of certain low-abundance cytokines may still exceed 30%, which poses a serious problem for application scenarios that require precise quantification, such as Th1 / Th2 cell balance research. In addition, the existing calibration methods do not adequately compensate for the dynamic range of multiple detections. When high / low concentration indicators are detected simultaneously, high-concentration signals often mask adjacent low-concentration spots, and traditional linear calibration cannot effectively correct such nonlinear interference. The existence of these problems urgently requires the development of a new generation of calibration technology. Only by improving the reliability of the calibration results can we truly achieve high-precision (CV<5%) cellular immune function assessment and meet the stringent requirements of precision medicine and translational research for data reliability. Summary of the Invention

[0008] The purpose of the present invention is to fill the gap in the calibration standard of fluorescent spot enzyme-linked immunosorbent assay (ELISA) and to provide a calibrator for the fluorescent spot imaging quantity of fluorescent spot enzyme-linked immunosorbent assay (ELISA) that is highly stable, maintenance-free, easy to operate, inexpensive, and applicable to multiple wavelengths.

[0009] In order to achieve the invention purpose of this application, the present invention adopts the following technical solutions:

[0010] The present invention discloses a calibrator for the fluorescent spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay instrument, comprising an enzyme labeling plate and a calibration component, wherein the calibration component comprises a sieve plate with micropores, a fluorescent polymer film, a reflective film and a sealing film, wherein: when used for top detection, the lower end of the sieve plate with micropores is covered and bonded with the fluorescent polymer film, the lower end of the fluorescent polymer film is covered and bonded with a reflective film, and the upper end of the sieve plate with micropores is covered and bonded with a sealing film, the diameter of the calibration component is smaller than the plate well diameter of the enzyme labeling plate, the calibration components are respectively placed in parallel in 4-6 plate wells located near the center of the enzyme labeling plate according to the above-mentioned pasting positions, and are respectively fixed in the plate wells by an adhesive, the number of micropores on the sieve plate with micropores is known, the enzyme labeling plate is black and its bottom is transparent or opaque to visible light, and the sealing film is a sealing film transparent in the visible light region.

[0011] The present invention discloses a calibrator for the fluorescent spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay (ELISA) instrument, comprising an ELISA plate and a calibration component, wherein the calibration component comprises a sieve plate with micropores, a fluorescent polymer film, a reflective film, and a sealing film. When used for bottom detection, the upper end of the sieve plate with micropores is sequentially covered and bonded with the fluorescent polymer film, the reflective film, and the sealing film. The diameter of the calibration component is smaller than the diameter of the plate wells of the ELISA plate. The calibration components are respectively placed in parallel in 4-6 plate wells located near the center of the ELISA plate according to the above-mentioned pasting positions and fixed in the plate wells by an adhesive. The number of micropores on the sieve plate with micropores is known. The ELISA plate is black and its bottom is transparent to visible light. The sealing film is black.

[0012] The calibrator for the fluorescent spot imaging quantity of the fluorescent spot enzyme-linked immunosorbent assay of the present invention is characterized in that: the diameter of the calibration component is 99%-85% of the plate hole diameter of the enzyme labeling plate.

[0013] The present invention provides a calibrator for the fluorescent spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay instrument, wherein: the number of micropores on the sieve plate with micropores in the standard components installed on the same ELISA plate is different, and the number of micropores on the sieve plate with micropores is 50-1000.

[0014] The present invention provides a calibrator for fluorescent spot imaging of a fluorescent spot enzyme-linked immunosorbent assay instrument, wherein the diameter of the micropores on the sieve plate with micropores is 30-100 μm, and the micropores are evenly distributed on the sieve plate with micropores.

[0015] The calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay instrument of the present invention comprises the following steps: the sieve plate with micropores is made of metal, hard plastic or paper.

[0016] The calibrator of the fluorescent spot imaging quantity for the fluorescent spot enzyme-linked immunosorbent assay analyzer of the present invention comprises the following: the reflective film is made of aluminum foil, glass plated with aluminum or glass with a silver reflective film.

[0017] The present invention is a calibrator for the fluorescence spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay, wherein: the fluorescent polymer film comprises: a fluorescent dye solution, a polymerization solvent, a polymerization cross-linking agent and a polymerization initiator, wherein: the fluorescent dye is quantum dots, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 800, Alexa Fluor 801, Alexa Fluor 802, Alexa Fluor 803, Alexa Fluor 804, Alexa Fluor 805, Alexa Fluor 806, Alexa Fluor 807, Alexa Fluor 808, Alexa Fluor 809, Alexa Fluor 810, Alexa Fluor 811, Alexa Fluor 812, Alexa Fluor 813, Alexa Fluor 814, Alexa Fluor 815, Alexa Fluor 816, Alexa Fluor 817, Alexa Fluor 818, Alexa Fluor 819, Alexa Fluor 820, Alexa Fluor 821, Alexa Fluor 822, Alexa Fluor 823, Alexa Fluor 824, Alexa Fluor 825, Alexa Fluor 826 Fluor790, quinine sulfate, fluorescein, Cy2, Cy3, Cy3B, Cy5, Cy7 and Eu ions, the molar ratio of each component is 0.1 to 10, the fluorescent dye is dissolved in water, methanol, acetonitrile, isopropanol, cyclohexane or chloroform to prepare a fluorescent dye solution of 1 nmol / L to 100 μmol / L; the polymerization solvent is methanol, acetonitrile, isopropanol, cyclohexane or chloroform, and the volume ratio of the fluorescent dye solution to the polymerization solvent is 1:5 to 1:50; the polymerization cross-linking agent is EGDMA (ethylene glycol dimethacrylate), TEGDMA (diethylene glycol dimethacrylate), DEGDMA (diethylene glycol dimethacrylate) The polymerization initiator is AIBN (azobisisobutyronitrile), ACVA (azobisisoheptanenitrile, 2,2'-Azobis(2,4-dimethylvaleronitrile)), AMBN (azobisisobutyric acid dimethyl ester, 2,2'-Azobis(2-methylbutyronitrile)), BPO (benzoyl peroxide, Benzoyl peroxide) and the like. Peroxide), LPO (Lauroyl Peroxide), TBPB (tert-Butyl Peroxybenzoate), APS (Ammonium Persulfate), Irgacure 184 or Irgacure 2959, with a dosage of (1-50) mg / L and a polymerization time within the range of 1-72 hours; the thickness of the fluorescent polymer film after polymerization is 10-1000 μm.

[0018] The present invention provides a calibrator for fluorescent spot imaging quantity of a fluorescent spot enzyme-linked immunosorbent assay instrument, wherein the sealing film is a polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), polyethylene (PE) or polypropylene (PP) film.

[0019] The calibrator for the fluorescent spot imaging quantity of the fluorescent spot enzyme-linked immunosorbent assay (ELISA) of the present invention is suitable for calibrating fluorescent spot enzyme-linked immunosorbent assay (ELISA) analyzers with excitation wavelengths from 300 nanometers to 700 nanometers without the need to replace the standard device. At the same time, one standard device has the function of calibrating fluorescence and time-resolved fluorescence simultaneously, and can also meet the calibration requirements of top detection and bottom detection instruments. It has the characteristics of wide application range, low cost, simple structure and accurate measurement value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the structure of a calibrator for the fluorescent spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay (ELISA) of the present invention when used for top detection. For the sake of clarity, the calibration component is enlarged in the figure;

[0021] Figure 2 The figure is a schematic structural diagram of a calibrator for the fluorescent spot imaging quantity of a fluorescent spot enzyme-linked immunosorbent assay instrument according to the present invention when used for bottom detection. For the sake of clarity, the calibration component is enlarged in the figure.

[0022] exist Figures 1 to 2 In the figure, number 1 is an enzyme labeling plate; number 2 is a plate well; number 3 is a sieve plate with micropores; number 4 is a fluorescent polymer film; number 5 is a reflective film; and number 6 is a sealing film. DETAILED DESCRIPTION

[0023] Example 1

[0024] like Figure 1 As shown, the calibrator of the fluorescent spot imaging amount for the fluorescent spot enzyme-linked immunosorbent assay of the present invention includes: an enzyme-labeled plate 1 and a calibration component, the calibration component includes: a sieve plate 3 with micropores, a fluorescent polymer film 4, a reflective film 5 and a sealing film 6. When used for top detection, the lower end of the sieve plate 3 with micropores is covered and bonded with the fluorescent polymer film 4, the lower end of the fluorescent polymer film 4 is covered and bonded with the reflective film 5, and the upper end of the sieve plate 3 with micropores is covered and bonded with the sealing film 6. The diameter of the above-mentioned calibration component is 93% of the diameter of the plate well 2 of the enzyme-labeled plate 1. The above-mentioned calibration components are placed in parallel in the four plate wells 2 located near the center of the enzyme-labeled plate 1 according to the above-mentioned pasting positions, and are respectively fixed in the plate wells 2 by adhesives. The enzyme-labeled plate 1 is black and its bottom is an enzyme-labeled plate that is transparent or opaque to visible light. The sealing film 6 is a sealing film that is transparent in the visible light region.

[0025] The number of micropores on the microporous frit 3 is known. The number of micropores on the microporous frit 3 varies across the standard assembly installed in the same ELISA plate 1. The number of micropores on the four different assemblies is 50, 100, 250, and 512, respectively. The diameter of the micropores on the microporous frit 3 is (50±1) μm, and the micropores are evenly distributed across the frit. The microporous frit 3 is made of stainless steel.

[0026] The reflective film 5 is aluminum foil. The fluorescent polymer film comprises a fluorescent dye solution, a polymerization solvent, a polymerization crosslinker, and a polymerization initiator. The fluorescent dyes are quinine sulfate, fluorescein, Alexa Fluor 532, Alexa Fluor 594, Alexa Fluor 647, Cy7, and Eu ions, with the molar ratio of each component being 1:1. The fluorescent dye is dissolved in methanol to prepare a 10 nmol / L fluorescent dye solution. The polymerization solvent is methanol, with a volume ratio of the fluorescent dye solution to the polymerization solvent of 1:10. The polymerization crosslinker is EGDMA (ethylene glycol dimethacrylate), with a volume ratio of the polymerization solvent to the crosslinker of 10:1. The polymerization initiator is AIBN (azobisisobutyronitrile), with a dosage of 5 mg / L. The polymerization time is 24 hours. After polymerization, the thickness of the fluorescent polymer film is 50 μm. The sealing film 6 is a polycarbonate (PC) film transparent in the visible light region.

[0027] Example 2

[0028] like Figure 2 As shown, Example 2 is basically the same as Example 1, and the same parts are not repeated here. The difference is that the calibrator of the fluorescent spot imaging amount for the fluorescent spot enzyme-linked immunosorbent assay in this embodiment is used for bottom detection, and the upper end of the sieve plate 3 with micropores is sequentially covered and bonded with a fluorescent polymer film 4, a reflective film 5 and a sealing film 6. The diameter of the above-mentioned calibration component is smaller than the diameter of the plate hole 2 of the ELISA plate 1. The above-mentioned calibration components are placed in parallel in the 4-6 plate holes 2 located near the center of the ELISA plate 1 according to the above-mentioned pasting positions, and are fixed in the plate holes 2 by adhesives. The ELISA plate 1 is black and its bottom is an ELISA plate transparent to visible light. The sealing film 6 is a black polycarbonate (PC) film.

[0029] The calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay of the present invention can be used to calibrate or evaluate the indication error and repeatability measurement characteristic index of the fluorescent spot counting of the instrument. Before the evaluation, the 488nm channel is selected and the fluorescence gain is set to 128, and the four plate wells 2 with the calibration component installed in the enzyme labeling plate 1 of the calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay of the present invention are analyzed respectively to obtain clear and bright micropore imaging results. The number of fluorescent spots obtained by the analysis is counted using the software provided by the fluorescent spot enzyme-linked immunosorbent assay, and each plate well 2 is measured continuously for 11 times. The average value of the fluorescent spot counting result is T, which is compared with the known number of micropores T on the sieve plate 3 with micropores placed in the corresponding well. s The comparison was made and the indication error of the spot number analysis was calculated.

[0030] ΔT=TT S (1)

[0031] When evaluating the repeatability of the instrument, the operation is the same as that of the indication error calibration evaluation. The number of micro-well imaging spots in the four plate wells 2 equipped with the calibration component is measured using a fluorescent spot enzyme-linked immunosorbent assay (ELISA). Each plate well 2 is measured 11 times continuously. The results are T i , the relative standard deviation was calculated according to Bessel formula (2) as the representation of instrument repeatability.

[0032]

[0033] T in formula (2) is the average value of the results of 11 consecutive measurements of plate well 2.

[0034] The indication error and repeatability calculation results of the measurement results are shown in Table 1.

[0035] Table 1 Fluorescent spot enzyme-linked immunosorbent assay spot counting error calibration results

[0036]

[0037]

[0038] The above is a schematic description of the present invention and its embodiments, which is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by this and, without departing from the spirit of the present invention, designs methods and embodiments similar to this technical solution without creatively designing them, they shall fall within the scope of protection of this patent.

Claims

1. A calibrator for the fluorescence spot imaging amount of a fluorescence spot enzyme-linked immunosorbent assay (ELISA), comprising: An enzyme labeling plate (1) and a calibration component, the calibration component comprising: a sieve plate with micropores (3), a fluorescent polymer film (4), a reflective film (5) and a sealing film (6), characterized in that: when used for top detection, the lower end of the sieve plate with micropores (3) is covered and bonded with the fluorescent polymer film (4), the lower end of the fluorescent polymer film (4) is covered and bonded with the reflective film (5), and the upper end of the sieve plate with micropores (3) is covered and bonded with the sealing film (6), the diameter of the above-mentioned calibration component is smaller than the diameter of the plate holes (2) of the enzyme labeling plate (1), the above-mentioned calibration components are respectively placed in parallel in 4-6 plate holes (2) located near the center of the enzyme labeling plate (1) according to the above-mentioned pasting positions, and are respectively fixed in the plate holes (2) by adhesive, the number of micropores on the sieve plate with micropores (3) is known, the enzyme labeling plate (1) is black and its bottom is an enzyme labeling plate that is transparent or opaque to visible light, and the sealing film (6) is a sealing film that is transparent in the visible light region.

2. A calibrator for the fluorescent spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay, comprising: An enzyme labeling plate (1) and a calibration component, wherein the calibration component comprises: a sieve plate (3) with micropores, a fluorescent polymer film (4), a reflective film (5) and a sealing film (6), characterized in that: when used for bottom detection, the upper end of the sieve plate (3) with micropores is sequentially covered and bonded with the fluorescent polymer film (4), the reflective film (5) and the sealing film (6), the diameter of the calibration component is smaller than the diameter of the plate holes (2) of the enzyme labeling plate (1), the calibration components are respectively placed in parallel in 4-6 plate holes (2) located near the center of the enzyme labeling plate (1) according to the above-mentioned pasting positions, and are respectively fixed in the plate holes (2) by adhesive, the number of micropores on the sieve plate (3) with micropores is known, the enzyme labeling plate (1) is black and its bottom is an enzyme labeling plate transparent to visible light, and the sealing film (6) is black.

3. The calibrator for the fluorescent spot imaging amount of a fluorescent spot enzyme-linked immunosorbent assay as claimed in claim 1 or 2, characterized in that: The diameter of the calibration component is 99%-85% of the diameter of the plate hole (2) of the ELISA plate (1).

4. The calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay as claimed in claim 3, characterized in that: The number of micropores on the sieve plate (3) with micropores in the standard components installed on the same ELISA plate (1) is different, and the number of micropores on the sieve plate (3) with micropores is 50 to 1000.

5. The calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay as claimed in claim 4, characterized in that: The diameter of the micropores on the microporous sieve plate (3) is 30 to 100 μm, and the micropores are evenly distributed on the microporous sieve plate (3).

6. The fluorescent spot imaging calibrator for a fluorescent spot enzyme-linked immunosorbent assay (ELISA) according to claim 5, characterized in that: The sieve plate (3) with micropores is made of metal, hard plastic or paper.

7. The calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay as claimed in claim 6, characterized in that: The reflective film (5) is made of aluminum foil, glass plated with aluminum, or glass with a silver reflective film.

8. The calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay as claimed in claim 7, characterized in that: The fluorescent polymer film comprises: a fluorescent dye solution, a polymerization solvent, a polymerization cross-linking agent and a polymerization initiator, wherein the fluorescent dye is quantum dots, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 80 ... Fluor790, quinine sulfate, fluorescein, Cy2, Cy3, Cy3B, Cy5, Cy7 and Eu ions, the molar ratio of each component is 0.1 to 10, the fluorescent dye is dissolved in water, methanol, acetonitrile, isopropanol, cyclohexane or chloroform to prepare a fluorescent dye solution of 1 nmol / L to 100 μmol / L; the polymerization solvent is methanol, acetonitrile, isopropanol, cyclohexane or chloroform, and the volume ratio of the fluorescent dye solution to the polymerization solvent is 1:5 to 1:50; the polymerization cross-linking agent is EGDMA (ethylene glycol dimethacrylate), TEGDMA (diethylene glycol dimethacrylate), DEGDMA (diethylene glycol dimethacrylate) The polymerization initiator is AIBN (azobisisobutyronitrile), ACVA (azobisisoheptanenitrile, 2,2'-Azobis(2,4-dimethylvaleronitrile)), AMBN (azobisisobutyric acid dimethyl ester, 2,2'-Azobis(2-methylbutyronitrile)), BPO (benzoyl peroxide, Benzoyl peroxide) and the like. Peroxide), LPO (Lauroyl Peroxide), TBPB (tert-Butyl Peroxybenzoate), APS (Ammonium Persulfate), Irgacure 184 or Irgacure 2959, with a dosage of (1-50) mg / L and a polymerization time within the range of 1-72 hours; the thickness of the fluorescent polymer film after polymerization is 10-1000 μm.

9. The calibrator for the fluorescent spot imaging amount of the fluorescent spot enzyme-linked immunosorbent assay as claimed in claim 8, characterized in that: The sealing film (6) is a polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), polyethylene (PE) or polypropylene (PP) film.

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