Protein digital detection method based on fluorescence immunoadsorption
Through the digital detection method based on fluorescent immunosorbent as well as the digital imaging technology of fluorescence microscopy system, the problem of insufficient sensitivity and flux in the existing technology is solved, and high sensitivity and high throughput digital detection of proteins is achieved, which is suitable for large-scale applications in the market.
Patent Information
- Application Number
- CN202510091320.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-06-06
AI Technical Summary
The existing protein digital detection technology has shortcomings in terms of sensitivity and throughput, which is difficult to meet the market's demand for high sensitivity and high throughput detection.
The digital detection method based on fluorescent immunosorbent assay is adopted, and the digital imaging and analysis are combined with the fluorescent microscopy system to achieve high sensitivity and high throughput detection of various proteins.
It realizes economical, convenient operation, high sensitivity, medium and high throughput digital detection of proteins, suitable for large-scale applications in the market, and can effectively detect multiple proteins, with good linear relationships and high correlations.
Smart Images
Figure CN120102896A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a protein digital detection method based on fluorescent immunoadsorption. Background Art
[0002] Fluorescence immunoadsorption protein digital detection technology is based on a plate-based platform design and complies with the operating habits of conventional detection technology. Through an open detection platform, digital imaging, analysis and detection of target proteins are achieved. It has the advantages of economy, easy operation, high sensitivity, medium and high throughput, and shows great application potential in the field of life and health. Summary of the invention
[0003] To solve the above problems, the present invention provides a protein digital detection method based on fluorescent immunoadsorption. The method technology has the advantages of economy, convenient operation, high sensitivity, medium and high throughput, etc.
[0004] The present invention is achieved through the following technical solutions:
[0005] A protein digital detection method based on fluorescent immunoadsorption comprises the following steps:
[0006] Step 1: Add the capture antibody dilution corresponding to the target protein into the wells of the ELISA plate to prepare an ELISA plate coated with the capture antibody corresponding to the target protein for later use;
[0007] Step 2: preparing fluorescent particles coated with detection antibodies corresponding to the target protein;
[0008] Step 3: Add the standard sample, quality control sample or body fluid sample containing the target protein to the well of the ELISA plate coated with the capture antibody corresponding to the target protein, thereby preparing an ELISA plate containing the target protein;
[0009] Step 4: dilute the fluorescent particles coated with the detection antibody corresponding to the protein and add them to the wells of the ELISA plate containing the target protein, so as to use the fluorescent particles coated with the detection antibody corresponding to the target protein to perform fluorescence amplification and digital encoding on the target protein;
[0010] Step 5: Use a fluorescence microscopy system to digitally image the target protein, and combine image recognition and data processing software to generate analysis results.
[0011] Furthermore, in step 1, the capture antibody dilution solution is prepared by diluting the capture antibody concentration corresponding to the target protein to 1 to 5 ug / mL with an ELISA plate buffer, the ELISA plate to which the capture antibody dilution solution corresponding to the target protein is added is placed in a low temperature environment for reaction, and then the plate is washed several times with a washing buffer, and patted dry to obtain an ELISA plate coated with the capture antibody corresponding to the target protein.
[0012] Furthermore, the ELISA plate buffer was prepared by weighing 1.59 g of sodium carbonate and 2.94 g of sodium bicarbonate in a beaker, then adding ultrapure water to a volume of 1 L, and stirring thoroughly to dissolve;
[0013] The washing buffer solution was prepared by weighing 1.59 g of sodium carbonate, 2.94 g of sodium bicarbonate and 5 mL of Tween-20 in a beaker, then adding ultrapure water to make the volume 1 L, and stirring thoroughly to dissolve.
[0014] Furthermore, the specific method of step 2 is:
[0015] S1. Add the fluorescent particle dispersion into the fluorescent particle coupling buffer, mix by ultrasonication, remove the supernatant by centrifugation, and then repeat the ultrasonication and centrifugation washing for several times in the fluorescent particle coupling buffer;
[0016] S2, add EDC and NHS solution in sequence, mix by ultrasonication, and then place on a shaker in the dark for reaction, then centrifuge to remove the supernatant, then add to the fluorescent particle coupling buffer, mix by ultrasonication, and centrifuge to remove the supernatant;
[0017] S3, then add the fluorescent particle coupling buffer, after ultrasonic mixing, add the detection antibody, whose solvent is the fluorescent particle coupling buffer, vortex mix, and place on a shaker in the dark for reaction, then add the fluorescent particle blocking solution, vortex mix, and place on a shaker in the dark for reaction, then centrifuge and remove the supernatant;
[0018] S4. Add the fluorescent particle coupling buffer, mix by ultrasonication, centrifuge and remove the supernatant, then add the fluorescent particle preservation solution and store it in a low temperature environment for later use.
[0019] Furthermore, the preparation method of the fluorescent particle coupling buffer is as follows: 19.5 g of 2-(N-morpholino)ethanesulfonic acid and 52.5 mg of Proclin300 are dissolved in 80 mL of ultrapure water, and then the pH is adjusted to 6.3 with a 280 mg / mL KOH ultrapure aqueous solution, and finally dissolved in 100 mL with ultrapure water.
[0020] Furthermore, the EDC solution was prepared by dissolving 100 mg of EDC in 10 mL of fluorescent particle coupling buffer;
[0021] The preparation method of NHS solution is as follows: dissolve 100 mg of NHS in 10 mL of fluorescent particle coupling buffer.
[0022] Furthermore, the fluorescent particle blocking solution was prepared by dissolving 31.0 g of boric acid, 427 mg of sodium tetraborate decahydrate, 500 uL of Tween-20, 2.00 g of bovine serum albumin, and 480 mg of ethanolamine in 100 mL of ultrapure water;
[0023] The preparation method of the fluorescent particle preservation solution is as follows: dissolve 60.6 g of tris(hydroxymethyl)aminomethane, 87.7 g of sodium chloride, 250 uL of Tween-20, 1.00 g of bovine serum albumin, 5.00 g of trehalose and 100 mg of Proclin300 in 5 mL of ultrapure water.
[0024] Furthermore, step three is specifically as follows: adding a standard substance, a quality control substance or a body fluid sample containing the target protein into the well of the ELISA plate coated with the capture antibody corresponding to the target protein, placing it on a shaker, reacting at 37° C., and then washing with phosphate buffer several times;
[0025] The phosphate buffer washing solution was prepared by dissolving 3.40 g of sodium hydrogen phosphate, 6.32 g of sodium dihydrogen phosphate and 5 mL of Tween-20 in 1 L of ultrapure water.
[0026] Furthermore, the method for diluting the fluorescent particles in step 4 is as follows: the fluorescent particles coated with the detection antibody corresponding to the target protein are diluted 20 times with the fluorescent particle storage solution, added to the wells of the ELISA plate containing the target protein and placed on a shaker for reaction at 37°C; then washed several times with phosphate buffer, patted dry, and the target protein was fluorescently amplified and digitally encoded.
[0027] Furthermore, step five is specifically as follows: placing the ELISA plate on the sample stage of the fluorescence microscope system, using a 20x, 40x or 50x objective lens, performing digital imaging by transmission or reflection, using image recognition and data processing software to draw a standard curve, establishing a corresponding equation, substituting the signal values of the quality control product and the body fluid sample into the standard curve, and calculating the target protein concentration in the quality control product and the body fluid sample.
[0028] The beneficial effects of the present invention are:
[0029] (1) The fluorescent immunoadsorption protein digital detection technology used in the present invention is combined with a plate-type platform, retains the original operating habits, and uses fluorescent coding and digital amplification, a fluorescent microscope system for digital imaging, and digital analysis to achieve protein detection. This technology has the advantages of being economical, easy to operate, highly sensitive, and having medium to high throughput, and is very conducive to large-scale application in the market.
[0030] (2) The detection method provided by the present invention can realize the detection of multiple proteins (CRP, PCT, IL6, IL8, IL1β, D-Dimer, MYO, NT-proBNP).
[0031] (3) The protein (CRP, PCT, IL6, IL8, IL1β, D-Dimer, MYO, NT-proBNP) detection standard curves drawn by the method provided by the present invention have a good linear relationship, R 2All are greater than 0.99. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 The following is a schematic diagram of the basic principle of the technology;
[0033] Figure 2 It is the standard curve for human C-reactive protein (CRP) detection based on fluorescent immunosorbent protein digital detection technology;
[0034] Figure 3 The standard curve for human calcitonin (PCT) detection based on fluorescent immunosorbent protein digital detection technology;
[0035] Figure 4 It is the standard curve for human interleukin 6 (IL6) detection based on fluorescent immunosorbent protein digital detection technology;
[0036] Figure 5 Standard curve for human interleukin 8 (IL8) detection based on fluorescent immunosorbent protein digital detection technology;
[0037] Figure 6 Standard curve for human interleukin 1β (IL1β) detection based on fluorescent immunosorbent protein digital detection technology;
[0038] Figure 7 It is the standard curve of human D-dimer detection based on fluorescent immunosorbent protein digital detection technology;
[0039] Figure 8 It is the standard curve of human myoglobin (MYO) detection based on fluorescent immunosorbent protein digital detection technology;
[0040] Fig. 9 This is the standard curve for the detection of human amino-terminal pro-brain peptide (NT-proBNP) based on fluorescent immunosorbent protein digital detection technology. DETAILED DESCRIPTION
[0041] like Figure 1 As shown, a protein digital detection method based on fluorescent immunoadsorption comprises the following steps:
[0042] Step 1: Add the capture antibody dilution corresponding to the target protein into the wells of the ELISA plate to prepare an ELISA plate coated with the capture antibody corresponding to the target protein for later use; the ELISA plate is a 96-well or 384-well transparent substrate ELISA plate made of glass, polystyrene or polymethyl acrylate.
[0043] The capture antibody diluent was prepared by diluting the capture antibody corresponding to the target protein to a concentration of 1 to 5 ug / mL with ELISA plate buffer, and 100 uL of the capture antibody diluent was added to each well of the ELISA plate. The reaction was allowed to proceed in a 4°C refrigerator for 12 h, and the plate was then washed 5 times with washing buffer and patted dry for later use.
[0044] The preparation method of ELISA plate buffer is as follows: weigh 1.59g of sodium carbonate and 2.94g of sodium bicarbonate in a beaker, then add ultrapure water to make up to 1L, and stir thoroughly to dissolve;
[0045] The washing buffer solution was prepared by weighing 1.59 g of sodium carbonate, 2.94 g of sodium bicarbonate and 5 mL of Tween-20 in a beaker, then adding ultrapure water to make the volume 1 L, and stirring thoroughly to dissolve.
[0046] Step 2: Prepare fluorescent particles coated with detection antibodies corresponding to the target protein. The fluorescent particles can generate fluorescent signals under the excitation of light sources, and the particle size is between 50 and 300 nanometers. The specific preparation method is:
[0047] S1. Add 50uL of fluorescent particle dispersion (10mg / mL) to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, then add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min;
[0048] S2, add EDC and NHS solution in sequence, mix by ultrasonication, and incubate on a shaker in the dark at 37°C for 30 min, then centrifuge at 2000 g for 10 min, remove the supernatant, add 1 mL of fluorescent particle coupling buffer, mix by ultrasonication for 3 min, centrifuge at 2000 g for 10 min, and remove the supernatant;
[0049] S3, then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3 minutes, add 250uL of detection antibody with a concentration of 0.1-0.3mg / mL (the solvent is fluorescent particle coupling buffer), vortex for 1 minute to mix, and then protect from light and place on a shaker at 37℃ for 2 hours, then add 500uL of fluorescent particle blocking solution, vortex for 1 minute to mix, and then protect from light and place on a shaker at 37℃ for 1 hour, then centrifuge at 2000g for 10 minutes, and remove the supernatant;
[0050] S4. Add 1 mL of fluorescent particle coupling buffer, mix by ultrasonication for 3 min, centrifuge at 2000 g for 10 min, remove the supernatant, and then add 500 uL of fluorescent particle preservation solution, place in a 4 °C refrigerator, and store for later use.
[0051] Furthermore, the preparation method of the fluorescent particle coupling buffer is as follows: 19.5 g of 2-(N-morpholino)ethanesulfonic acid and 52.5 mg of Proclin300 are dissolved in 80 mL of ultrapure water, and then the pH is adjusted to 6.3 with a 280 mg / mL KOH ultrapure aqueous solution, and finally dissolved in 100 mL with ultrapure water.
[0052] Furthermore, the EDC solution was prepared by dissolving 100 mg of EDC in 10 mL of fluorescent particle coupling buffer;
[0053] The preparation method of NHS solution is as follows: dissolve 100 mg of NHS in 10 mL of fluorescent particle coupling buffer.
[0054] Furthermore, the fluorescent particle blocking solution was prepared by dissolving 31.0 g of boric acid, 427 mg of sodium tetraborate decahydrate, 500 uL of Tween-20, 2.00 g of bovine serum albumin, and 480 mg of ethanolamine in 100 mL of ultrapure water;
[0055] The preparation method of the fluorescent particle preservation solution is as follows: dissolve 60.6 g of tris(hydroxymethyl)aminomethane, 87.7 g of sodium chloride, 250 uL of Tween-20, 1.00 g of bovine serum albumin, 5.00 g of trehalose and 100 mg of Proclin300 in 5 mL of ultrapure water.
[0056] Step 3: Add 45uL of standard, quality control or body fluid sample containing the target protein to the well of the ELISA plate coated with the capture antibody corresponding to the target protein, place on a shaker, react at 37°C for 1h, and then wash several times with 300uL phosphate buffer;
[0057] The phosphate buffer washing solution was prepared by dissolving 3.40 g of sodium hydrogen phosphate, 6.32 g of sodium dihydrogen phosphate and 5 mL of Tween-20 in 1 L of ultrapure water.
[0058] Step 4: Dilute the fluorescent particles coated with the detection antibody corresponding to the target protein 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0059] Step 5. Place the ELISA plate on the sample stage of the fluorescence microscope system. Without the liquid path detection module, use a 20x, 40x or 50x objective lens to perform digital imaging by transmission or reflection. Use image recognition and data processing software to draw a standard curve, establish the corresponding equation, substitute the signal values of the quality control product and body fluid sample into the standard curve, and calculate the target protein concentration in the quality control product and body fluid sample.
[0060] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby.
[0061] Example 1
[0062] CRP detection method based on fluorescent immunoadsorption protein digital detection technology:
[0063] Dilute the capture antibody concentration corresponding to CRP to 2ug / mL with ELISA plate buffer, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0064] 50uL of fluorescent particle dispersion (10mg / mL) was added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, 10uL of EDC and NHS solution were added in sequence, ultrasonically mixed for 1min, placed on a shaker in the dark, reacted at 37℃ for 30min, then centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 1 0min, remove the supernatant, and then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3min, add 250uL of detection antibody corresponding to CRP at a concentration of 0.2mg / mL (the solvent is fluorescent particle coupling buffer), vortex for 1min, mix, and place on a shaker in the dark at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, and place on a shaker in the dark at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, ultrasonically mix for 3min, centrifuge at 2000g for 10min, remove the supernatant, and then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0065] 45uL of CRP standards with concentrations of 10, 3.33, 1.11, 0.37, 0.12, 0.04, and 0.013mg / mL were added to the wells of the ELISA plate coated with the capture antibody corresponding to CRP, placed on a shaker, and reacted at 37°C for 1h; then washed 5 times with 300uL phosphate buffer and patted dry.
[0066] After diluting the fluorescent particles coated with the CRP detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0067] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0068] The counting and data processing software results showed that the CRP standard concentration had a good correlation with the signal value through four-parameter fitting, R 2 is 0.997.
[0069] Example 2
[0070] PCT detection method based on fluorescent immunoadsorption protein digital detection technology:
[0071] Dilute the capture antibody concentration corresponding to PCT to 1.5ug / mL with ELISA plate buffer, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0072] 50uL of fluorescent particle dispersion (10mg / mL) was added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, 10uL of EDC and NHS solution were added in sequence, ultrasonically mixed for 1min, placed on a shaker in the dark, reacted at 37℃ for 30min, then centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 1 0min, remove the supernatant, and then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3min, add 250uL of detection antibody corresponding to PCT at a concentration of 0.2mg / mL (the solvent is fluorescent particle coupling buffer), vortex for 1min, mix, and place on a shaker in the dark at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, and place on a shaker in the dark at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, ultrasonically mix for 3min, centrifuge at 2000g for 10min, remove the supernatant, and then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0073] 45uL of PCT standards with concentrations of 33.3, 11.1, 3.7, 1.23, 0.41, and 0.14ng / mL were added to the wells of the ELISA plate coated with the capture antibody corresponding to PCT in sequence, placed on a shaker, and reacted at 37°C for 1h; then washed 5 times with 300uL phosphate buffer and patted dry.
[0074] After diluting the fluorescent particles coated with the PCT detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0075] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0076] The counting and data processing software results showed that the PCT standard concentration had a good correlation with the signal value through four-parameter fitting, R 2 is 0.997.
[0077] Example 3
[0078] IL6 detection method based on fluorescent immunoadsorption protein digital detection technology:
[0079] Dilute the capture antibody concentration corresponding to IL6 to 1.5ug / mL with ELISA plate buffer, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0080] 50uL of fluorescent particle dispersion (10mg / mL) was added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, 10uL of EDC and NHS solution were added in sequence, ultrasonically mixed for 1min, placed on a shaker in the dark, reacted at 37℃ for 30min, then centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 1 0min, remove the supernatant, and then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3min, add 250uL of 0.2mg / mL IL6 corresponding detection antibody (solvent is fluorescent particle coupling buffer), vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, ultrasonically mix for 3min, centrifuge at 2000g for 10min, remove the supernatant, and then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0081] Add 45uL of IL6 standards with concentrations of 5000, 1666, 555, 185, 61.7, 20.6, 6.86, and 2.29pg / mL into the wells of the ELISA plate coated with the capture antibody corresponding to IL6, place on a shaker, and react at 37°C for 1h; then wash 5 times with 300uL phosphate buffer and pat dry.
[0082] After diluting the fluorescent particles coated with the IL6 detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0083] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0084] The counting and data processing software results showed that the IL6 standard concentration had a good correlation with the signal value through four-parameter fitting, R 2 is 0.998.
[0085] Example 4
[0086] IL8 detection method based on fluorescent immunoadsorption protein digital detection technology:
[0087] Dilute the capture antibody concentration corresponding to IL8 to 1.5ug / mL with ELISA plate buffer, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0088] 50uL of fluorescent particle dispersion (10mg / mL) was added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, 10uL of EDC and NHS solution were added in sequence, ultrasonically mixed for 1min, placed on a shaker in the dark, reacted at 37℃ for 30min, then centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 1 0min, remove the supernatant, and then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3min, add 250uL of 0.2mg / mL IL8 corresponding detection antibody (solvent is fluorescent particle coupling buffer), vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, ultrasonically mix for 3min, centrifuge at 2000g for 10min, remove the supernatant, and then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0089] 45uL of IL8 standards with concentrations of 6000, 2733, 911, 303, 101, 33.7, 11.2, and 3.75pg / mL were added to the wells of the ELISA plate coated with the capture antibody corresponding to IL8 in sequence, placed on a shaker, and reacted at 37°C for 1h; then washed 5 times with 300uL phosphate buffer and patted dry.
[0090] After diluting the fluorescent particles coated with the IL8 detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0091] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0092] The counting and data processing software results showed that the IL8 standard concentration had a good correlation with the signal value through four-parameter fitting, R 2It is 0.993.
[0093] Example 5
[0094] IL1β detection method based on fluorescent immunoadsorption protein digital detection technology:
[0095] Dilute the capture antibody concentration corresponding to IL1β to 2.5ug / mL with ELISA plate buffer, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0096] 50uL of fluorescent particle dispersion (10mg / mL) was added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, 10uL of EDC and NHS solution were added in sequence, ultrasonically mixed for 1min, placed on a shaker in the dark, reacted at 37℃ for 30min, then centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 1 0min, remove the supernatant, and then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3min, add 250uL of 0.2mg / mL IL1β corresponding detection antibody (solvent is fluorescent particle coupling buffer), vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, ultrasonically mix for 3min, centrifuge at 2000g for 10min, remove the supernatant, and then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0097] 45uL of IL1β standards with concentrations of 333, 111, 37, 12.3, 4.1, and 1.4pg / mL were added to the wells of the ELISA plate coated with the capture antibody corresponding to IL1β, placed on a shaker, and reacted at 37°C for 1h; then washed 5 times with 300uL phosphate buffer and patted dry.
[0098] After diluting the fluorescent particles coated with the IL1β detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0099] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0100] The counting and data processing software results showed that the concentration of IL1β standard had a good correlation with the signal value through four-parameter fitting, R 2 is 0.999.
[0101] Example 6
[0102] D-Dimer detection method based on fluorescent immunoadsorption protein digital detection technology:
[0103] Dilute the capture antibody concentration corresponding to D-Dimer to 3ug / mL with ELISA plate buffer, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0104] Add 50uL of fluorescent particle dispersion (10mg / mL) to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, then add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, then add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, add 10uL of EDC and NHS solution in turn, mix by ultrasonication for 1min, place on a shaker away from light, react at 37℃ for 30min, then centrifuge at 2000g for 10min, remove the supernatant, add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10m in, remove the supernatant, then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3min, add 250uL of detection antibody corresponding to D-Dimer at a concentration of 0.2mg / mL (the solvent is fluorescent particle coupling buffer), vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, protect from light on a shaker, react at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, ultrasonically mix for 3min, centrifuge at 2000g for 10min, remove the supernatant, and then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0105] Add 45uL of D-Dimer standards with concentrations of 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.013, 0.0045, and 0.0015mg / L into the wells of the ELISA plate coated with the capture antibody corresponding to D-Dimer, place on a shaker, and react at 37°C for 1h; then wash 5 times with 300uL phosphate buffer and pat dry.
[0106] After diluting the fluorescent particles coated with the D-Dimer detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37℃ for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0107] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0108] The counting and data processing software results showed that the concentration of D-Dimer standard had a good correlation with the signal value through four-parameter fitting, R 2 It is 0.994.
[0109] Example 7
[0110] MYO detection method based on fluorescent immunoadsorption protein digital detection technology:
[0111] Dilute the capture antibody corresponding to MYO to 3ug / mL with ELISA plate buffer, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0112] 50uL of fluorescent particle dispersion (10mg / mL) was added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, 10uL of EDC and NHS solution were added in sequence, ultrasonically mixed for 1min, placed on a shaker in the dark, reacted at 37℃ for 30min, then centrifuged at 2000g for 10min, the supernatant was removed, and then added to 1mL of fluorescent particle coupling buffer, ultrasonically mixed for 3min, centrifuged at 2000g for 1 0min, remove the supernatant, and then add 750uL of fluorescent particle coupling buffer, ultrasonically mix for 3min, add 250uL of MYO corresponding detection antibody with a concentration of 0.2mg / mL (the solvent is fluorescent particle coupling buffer), vortex for 1min, mix, and place on a shaker in the dark at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, and place on a shaker in the dark at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, ultrasonically mix for 3min, centrifuge at 2000g for 10min, remove the supernatant, and then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0113] 45uL of MYO standards with concentrations of 3000, 1000, 333, 37, 4.1, and 1.4ng / mL were added to the wells of the ELISA plate coated with the capture antibody corresponding to MYO in sequence, placed on a shaker, and reacted at 37°C for 1h; then washed 5 times with 300uL phosphate buffer and patted dry.
[0114] After diluting the fluorescent particles coated with the MYO detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0115] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0116] The counting and data processing software results showed that the MYO standard concentration had a good correlation with the signal value through four-parameter fitting, R 2 is 0.998.
[0117] Example 8
[0118] NT-proBNP detection method based on fluorescent immunoadsorption protein digital detection technology:
[0119] Use ELISA plate buffer to dilute the capture antibody concentration corresponding to NT-proBNP to 3ug / mL, add 100uL of capture antibody dilution to each well of the ELISA plate, and react in a 4°C refrigerator for 12h. Then wash the plate 5 times with washing buffer, pat dry and set aside.
[0120] Add 50uL of fluorescent particle dispersion (10mg / mL) to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, then add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10min, remove the supernatant, then add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, add 10uL of EDC and NHS solution in turn, mix by ultrasonication for 1min, place on a shaker away from light, react at 37℃ for 30min, then centrifuge at 2000g for 10min, remove the supernatant, add to 1mL of fluorescent particle coupling buffer, mix by ultrasonication for 3min, centrifuge at 2000g for 10mi n, remove the supernatant, then add 750uL of fluorescent particle coupling buffer, mix ultrasonically for 3 minutes, add 250uL of detection antibody corresponding to NT-proBNP with a concentration of 0.2mg / mL (the solvent is fluorescent particle coupling buffer), vortex for 1min, mix, place on a shaker in the dark, react at 37℃ for 2h, then add 500uL of fluorescent particle blocking solution, vortex for 1min, mix, place on a shaker in the dark, react at 37℃ for 1h, then centrifuge at 2000g for 10min, remove the supernatant, add 1mL of fluorescent particle coupling buffer, mix ultrasonically for 3min, centrifuge at 2000g for 10min, remove the supernatant, then add 500uL of fluorescent particle preservation solution, place in a 4℃ refrigerator for storage.
[0121] Add 45uL of NT-proBNP standards with concentrations of 11666, 3889, 1296, 432, 144, and 48pg / mL into the wells of the ELISA plate coated with the capture antibody corresponding to NT-proBNP, place on a shaker, and react at 37°C for 1h; then wash 5 times with 300uL phosphate buffer and pat dry.
[0122] After diluting the fluorescent particles coated with the NT-proBNP detection antibody 20 times with the fluorescent particle storage solution, take 100uL and add it to the wells of the ELISA plate containing the target protein in turn, place it on a shaker, and react at 37°C for 1h; then wash it 5 times with 300uL phosphate buffer and pat dry.
[0123] Place the ELISA plate on the sample stage of the fluorescence microscope system, use a 40x objective lens, perform digital imaging through reflection, and use counting and data processing software to establish a standard curve correlation equation. If the signal values of the quality control product and body fluid sample are substituted into the standard curve equation, the target protein concentration in the quality control product and body fluid sample can be calculated.
[0124] The counting and data processing software results showed that the concentration of NT-proBNP standard had a good correlation with the signal value through four-parameter fitting, R 2 is 0.997.
[0125] The specific configurations of the corresponding buffers involved in the above Examples 1-8 are as follows:
[0126] Preparation of ELISA plate washing buffer: Weigh 1.59 g of sodium carbonate, 2.94 g of sodium bicarbonate and 5 mL of Tween-20 in a beaker, then add ultrapure water to make up to 1 L and stir thoroughly to dissolve.
[0127] Preparation of ELISA plate coating buffer: Weigh 1.59 g of sodium carbonate and 2.94 g of sodium bicarbonate into a beaker, then add ultrapure water to make up to 1 L and stir thoroughly to dissolve.
[0128] Fluorescent particle coupling buffer preparation: 19.5 g of 2-(N-morpholino)ethanesulfonic acid and 52.5 mg of Proclin300 were dissolved in 80 mL of ultrapure water, and then the pH was adjusted to 6.3 with a 280 mg / mL KOH ultrapure aqueous solution, and finally dissolved in 100 mL with ultrapure water.
[0129] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution preparation: Dissolve 100 mg of EDC in 10 mL of fluorescent particle coupling buffer.
[0130] N-hydroxysuccinimide (NHS) solution preparation: Dissolve 100 mg of NHS in 10 mL of fluorescent particle coupling buffer.
[0131] Preparation of fluorescent particle blocking solution: Dissolve 31.0 g of boric acid, 427 mg of sodium tetraborate decahydrate, 500 uL of Tween-20, 2.00 g of bovine serum albumin and 480 mg of ethanolamine in 100 mL of ultrapure water.
[0132] Preparation of fluorescent particle preservation solution: dissolve 60.6 g of tris(hydroxymethyl)aminomethane, 87.7 g of sodium chloride, 250 uL of Tween-20, 1.00 g of bovine serum albumin, 5.00 g of trehalose and 100 mg of Proclin300 in 5 mL of ultrapure water.
[0133] Preparation of phosphate buffered saline: Dissolve 3.40 g of sodium hydrogen phosphate, 6.32 g of sodium dihydrogen phosphate and 5 mL of Tween-20 in 1 L of ultrapure water.
[0134] It should be noted that although the present invention is described by the above embodiments, the present invention may also have other various embodiments. Without departing from the spirit and scope of the present invention, it is obvious that those skilled in the art may make various corresponding changes and deformations to the present invention, but these changes and deformations should all fall within the scope of protection of the appended claims of the present invention and their equivalents.
Claims
1. A protein digital detection method based on fluorescent immunoadsorption, characterized in that: The following steps are involved: Step 1: Add the capture antibody dilution corresponding to the target protein into the wells of the ELISA plate to prepare an ELISA plate coated with the capture antibody corresponding to the target protein for later use; Step 2: preparing fluorescent particles coated with detection antibodies corresponding to the target protein; Step 3: Add the standard sample, quality control sample or body fluid sample containing the target protein to the well of the ELISA plate coated with the capture antibody corresponding to the target protein, thereby preparing an ELISA plate containing the target protein; Step 4: dilute the fluorescent particles coated with the detection antibody corresponding to the protein and add them to the wells of the ELISA plate containing the target protein, so as to use the fluorescent particles coated with the detection antibody corresponding to the target protein to perform fluorescence amplification and digital encoding on the target protein; Step 5: Use a fluorescence microscopy system to digitally image the target protein, and combine image recognition and data processing software to generate analysis results.
2. A protein digital detection method based on fluorescent immunoadsorption according to claim 1, characterized in that: In step 1, the capture antibody dilution solution is prepared by diluting the capture antibody corresponding to the target protein to a concentration of 1 to 5 ug / mL with an ELISA plate buffer, and the ELISA plate to which the capture antibody dilution solution corresponding to the target protein is added is placed in a low temperature environment for reaction, and then the plate is washed several times with a washing buffer, and patted dry to obtain an ELISA plate coated with the capture antibody corresponding to the target protein.
3. A protein digital detection method based on fluorescent immunoadsorption according to claim 2, characterized in that: The preparation method of ELISA plate buffer is as follows: weigh 1.59g of sodium carbonate and 2.94g of sodium bicarbonate in a beaker, then add ultrapure water to make up to 1L, and stir thoroughly to dissolve; The washing buffer solution was prepared by weighing 1.59 g of sodium carbonate, 2.94 g of sodium bicarbonate and 5 mL of Tween-20 in a beaker, then adding ultrapure water to make the volume 1 L, and stirring thoroughly to dissolve.
4. A protein digital detection method based on fluorescent immunoadsorption according to claim 1, characterized in that: The specific method of step 2 is: S1. Add the fluorescent particle dispersion into the fluorescent particle coupling buffer, mix by ultrasonication, remove the supernatant by centrifugation, and then repeat the ultrasonication and centrifugation washing for several times in the fluorescent particle coupling buffer; S2, add EDC and NHS solution in sequence, mix by ultrasonication, and then place on a shaker in the dark for reaction, then centrifuge to remove the supernatant, then add to the fluorescent particle coupling buffer, mix by ultrasonication, and centrifuge to remove the supernatant; S3, then add the fluorescent particle coupling buffer, after ultrasonic mixing, add the detection antibody, whose solvent is the fluorescent particle coupling buffer, vortex mix, and place on a shaker in the dark for reaction, then add the fluorescent particle blocking solution, vortex mix, and place on a shaker in the dark for reaction, then centrifuge and remove the supernatant; S4. Add the fluorescent particle coupling buffer, mix by ultrasonication, centrifuge and remove the supernatant, then add the fluorescent particle preservation solution and store it in a low temperature environment for later use.
5. A protein digital detection method based on fluorescent immunoadsorption according to claim 4, characterized in that: The preparation method of fluorescent particle coupling buffer is as follows: dissolve 19.5 g of 2-(N-morpholino)ethanesulfonic acid and 52.5 mg of Proclin300 in 80 mL of ultrapure water, then adjust the pH to 6.3 with 280 mg / mL KOH ultrapure water solution, and finally dissolve in 100 mL with ultrapure water.
6. A protein digital detection method based on fluorescent immunoadsorption according to claim 1, characterized in that: The EDC solution was prepared by dissolving 100 mg of EDC in 10 mL of fluorescent particle coupling buffer; The preparation method of NHS solution is as follows: dissolve 100 mg of NHS in 10 mL of fluorescent particle coupling buffer.
7. A protein digital detection method based on fluorescent immunoadsorption according to claim 1, characterized in that: The fluorescent particle blocking solution was prepared by dissolving 31.0 g of boric acid, 427 mg of sodium tetraborate decahydrate, 500 uL of Tween-20, 2.00 g of bovine serum albumin, and 480 mg of ethanolamine in 100 mL of ultrapure water; The preparation method of the fluorescent particle preservation solution is as follows: dissolve 60.6 g of tris(hydroxymethyl)aminomethane, 87.7 g of sodium chloride, 250 uL of Tween-20, 1.00 g of bovine serum albumin, 5.00 g of trehalose and 100 mg of Proclin300 in 5 mL of ultrapure water.
8. The protein digital detection method based on fluorescent immunoadsorption according to claim 1, characterized in that: Step 3 is as follows: add the standard, quality control or body fluid sample containing the target protein into the well of the ELISA plate coated with the capture antibody corresponding to the target protein, place on a shaker, react at 37°C, and then wash several times with phosphate buffer; The phosphate buffer washing solution was prepared by dissolving 3.40 g of sodium hydrogen phosphate, 6.32 g of sodium dihydrogen phosphate and 5 mL of Tween-20 in 1 L of ultrapure water.
9. The method for digital protein detection based on fluorescent immunoadsorption according to claim 1, characterized in that: The method for diluting the fluorescent particles in step 4 is as follows: the fluorescent particles coated with the detection antibody corresponding to the target protein are diluted 20 times with the fluorescent particle storage solution, added to the wells of the ELISA plate containing the target protein and placed on a shaker at 37°C for reaction; then washed several times with phosphate buffer, patted dry, and the target protein was fluorescently amplified and digitally encoded.
10. The protein digital detection method based on fluorescent immunoadsorption according to claim 1, characterized in that: Step five is as follows: place the ELISA plate on the sample stage of the fluorescence microscope system, use a 20x, 40x or 50x objective lens to perform digital imaging through transmission or reflection, use image recognition and data processing software to draw a standard curve, establish a corresponding equation, substitute the signal values of the quality control product and body fluid sample into the standard curve, and calculate the target protein concentration in the quality control product and body fluid sample.