Composition, kit and detection method for enhancing the photobleaching stability of phycobiliprotein

Abstract

This application relates to the technical field of flow cytometry detection. Specifically, it relates to a composition, a kit, and a detection method for enhancing the photobleaching stability of phycobiliprotein. The composition for enhancing the photobleaching stability of phycobiliprotein includes a pigment and a fluorescent probe containing phycobiliprotein, and the absorption wavelength range of the pigment is 400 - 700 nm. This application uses a pigment with an absorption wavelength range of 400 - 700 nm to be compounded with a fluorescent probe containing phycobiliprotein. The pigment with an absorption wavelength range of 400 - 700 nm can effectively improve the photobleaching stability of phycobiliprotein in the fluorescent probe, so that phycobiliprotein is not easily subjected to the photobleaching effect even under the condition of not requiring light avoidance. Furthermore, the fluorescent probe formed by phycobiliprotein still has a stable and strong fluorescent signal under the condition of not requiring light avoidance, which is beneficial to greatly improving the convenience of fluorescence immunoassay.

Description

Technical Field

[0001] This application relates to the technical field of flow cytometry detection. Specifically, it relates to a composition, a kit, and a detection method for enhancing the photobleaching stability of phycobiliprotein. Background Art

[0002] The Stokes shift is an important parameter in fluorescence immunoassay, and its magnitude determines the reliability and sensitivity of the fluorescent probe. If a fluorescent label has a large Stokes shift, the interference from Rayleigh scattering and Raman scattering during fluorescence detection is smaller. The larger the Stokes shift, the closer the emitted fluorescence is to the red end of the visible spectrum, which is more conducive to the analysis of biochemical samples. Because the fluorescence interference caused by biochemical matrices and serum components in the red region is significantly reduced, the detection sensitivity can be significantly improved.

[0003] Phycobiliprotein is a large protein molecule derived from algae such as cyanobacteria and dinoflagellates. Phycobiliprotein is a new type of fluorescent label that can label antibodies, biotin esters, or streptavidin to form fluorescent probes, and has broad application prospects in fluorescence immunoassay. Phycobiliprotein has a large molecule (for example, the molecular weight of phycoerythrin PE is 240 kD), a large Stokes shift (75 - 200 nm, for example, the Stokes shift of phycoerythrin PE is as high as 80 nm), and a stable emission spectrum. Therefore, the fluorescent probe formed by phycobiliprotein labeling has high reliability and sensitivity, and is very suitable for flow cytometry in quantitative analysis.

[0004] However, phycobiliprotein is sensitive to ambient light and is easily affected by the photobleaching effect, resulting in a reduction in the fluorescence signal of fluorescence immunoassay such as flow cytometry. Therefore, the operation steps need to be carried out in a dark environment, which causes poor convenience in detection and analysis operations. Summary of the Invention

[0005] The purpose of this application is to provide a composition for enhancing the photobleaching stability of phycobiliprotein, a kit for flow cytometry, and a detection method based on flow cytometry, aiming to improve the technical problem of poor photobleaching stability of existing phycobiliprotein.

[0006] In the first aspect, this application provides a composition for enhancing the photobleaching stability of phycobiliprotein. The composition includes a pigment and a fluorescent probe containing phycobiliprotein; the absorption wavelength range of the pigment is 400 - 700 mm.

[0007] In this application, a pigment with an absorption wavelength range of 400 - 700 nm is combined with a fluorescent probe containing phycobiliprotein. The pigment with an absorption wavelength range of 400 - 700 nm can be effectively wrapped on the surface of phycobiliprotein, effectively improving the photobleaching stability of phycobiliprotein in the fluorescent probe, so that phycobiliprotein is not easily subject to the photobleaching effect even without light avoidance conditions. Furthermore, the fluorescent probe formed by phycobiliprotein still has a stable and strong fluorescent signal without light avoidance conditions, which is beneficial to greatly improving the operational convenience of fluorescence immunoassay (including flow cytometry, etc.).

[0008] In some embodiments of the first aspect of this application, the phycobiliprotein includes at least one of phycoerythrin, phycocyanin, phycoerythrocyanin, allophycocyanin, and chlorophyll protein.

[0009] The fluorescent probe formed by the above substances has high reliability and sensitivity, and is very suitable for flow cytometry in quantitative analysis, which is beneficial to improving the reliability and sensitivity of flow cytometry analysis; and the above substances can effectively combine with the pigment to improve the photobleaching stability of phycobiliprotein substances in the fluorescent probe.

[0010] In some embodiments of the first aspect of this application, the pigment includes at least one of fruit green, carmine, lemon yellow, sunset yellow, and allura red.

[0011] Selecting the above substances as the pigment can effectively improve the photobleaching stability of phycobiliprotein in the fluorescent probe.

[0012] In some embodiments of the first aspect of this application, the mass ratio of phycobiliprotein to the pigment is (1 - 5):(50 - 500).

[0013] Under the above conditions, it is beneficial to further improve the photobleaching stability of phycobiliprotein in the fluorescent probe. If the proportion of phycobiliprotein is relatively large, some phycobiliprotein cannot be effectively wrapped by the pigment, resulting in some phycobiliprotein being affected by a certain photobleaching effect; if the proportion of phycobiliprotein in the composition is relatively small, it will lead to a low content of the fluorescent probe and affect the fluorescence intensity.

[0014] In some embodiments of the first aspect of this application, the pigment is selected from fruit green, and the phycobiliprotein is selected from phycoerythrin.

[0015] The fluorescent probe formed by phycoerythrin labeling has higher reliability and sensitivity, and after combining with fruit green pigment, it can further effectively improve the photobleaching stability of phycoerythrin.

[0016] Optionally, the mass ratio of phycoerythrin to fruit green is (2 - 3):(100 - 200).

[0017] In some embodiments of the first aspect of the present application, the fluorescent probe containing phycobiliprotein includes at least one of an antibody labeled with phycobiliprotein, a biotin ester labeled with phycobiliprotein, and streptavidin labeled with phycobiliprotein.

[0018] In some embodiments of the first aspect of the present application, the composition further includes ultrapure water.

[0019] Optionally, the composition further includes bovine serum albumin.

[0020] In a second aspect, the present application provides a kit for flow cytometry, including the composition provided in the first aspect as described above.

[0021] The kit for flow cytometry provided by the present application can achieve stable and strong fluorescence signals of the fluorescent probe even under the condition of not requiring light avoidance, which is beneficial to greatly improving the operation convenience of flow cytometry.

[0022] Optionally, the kit further includes a fixing solution, a buffer solution, a permeabilization solution, a blocking solution, a washing solution, and a biotinylated antibody solution.

[0023] In a third aspect, the present application provides a detection method based on flow cytometry, including: detecting by using the kit provided in the second aspect as described above.

[0024] In some embodiments of the third aspect of the present application, the detection is carried out under the condition of not requiring light avoidance.

[0025] The detection method based on flow cytometry provided by the present application can perform detection with stable and strong fluorescence signals under the condition of not requiring light avoidance, greatly improving the operation convenience of the detection. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 The scatter plot of the detection results of the flow cytometry provided in Comparative Example 1 of the present application is shown.

[0028] Figure 2 The scatter plot of the detection results of the flow cytometry provided in Comparative Example 2 of the present application is shown.

[0029] Figure 3 The scatter plot of the detection results of the flow cytometry provided in the embodiment of the present application is shown.

[0030] Figure 4 Shows the histogram of the detection results of flow cytometry provided by Comparative Example 1 of the present application.

[0031] Figure 5 Shows the histogram of the detection results of flow cytometry provided by Comparative Example 2 of the present application.

[0032] Figure 6 Shows the histogram of the detection results of flow cytometry provided by the examples of the present application. Detailed implementation manners

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments whose manufacturers are not indicated are all conventional products that can be obtained by purchasing in the market.

[0034] The present application provides a composition for enhancing the photobleaching stability of phycobiliprotein. The composition includes a pigment and a fluorescent probe containing phycobiliprotein; wherein, the absorption wavelength range of the pigment is 400 - 700 mm.

[0035] In the present application, by compounding a pigment with an absorption wavelength range of 400 - 700 mm with a fluorescent probe containing phycobiliprotein, the pigment with an absorption wavelength range of 400 - 700 mm can be effectively wrapped on the surface of phycobiliprotein to effectively improve the photobleaching stability of phycobiliprotein in the fluorescent probe, so that phycobiliprotein is not easily subject to photobleaching effect under the condition of not requiring light avoidance. Furthermore, the fluorescent probe formed by phycobiliprotein still has a stable and strong fluorescent signal under the condition of not requiring light avoidance, which is beneficial to greatly improving the operation convenience of fluorescence immunoassay (including flow cytometry, etc.).

[0036] In the present application, phycobiliprotein includes at least one of phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin. The fluorescent probes formed by the above substances have high reliability and sensitivity and are very suitable for flow cytometry for quantitative analysis, which is beneficial to improving the reliability and sensitivity of flow cytometry analysis; and the above substances can effectively compound with the pigment to improve the photobleaching stability of phycobiliprotein substances in the fluorescent probe.

[0037] In the present application, the pigment includes at least one of fruit green (CAS No.: 012111-09-7, absorption wavelength range: 400 - 700 nm), carmine (CAS No.: 915-67-3, wavelength corresponding to the maximum absorption peak: 506 - 510 nm), lemon yellow (CAS No.: 1934-21-0, wavelength corresponding to the maximum absorption peak: 426 - 430 nm), sunset yellow (CAS No.: 2783-94-0, wavelength corresponding to the maximum absorption peak: 480 - 484 nm), and allura red (CAS No.: 25956-17-6, wavelength corresponding to the maximum absorption peak: 497 - 501 nm). By selecting the above substances as the pigment, the photobleaching stability of phycobiliprotein in the fluorescent probe can be effectively improved.

[0038] It should be noted that the pigment is not limited to the above substances and can be either natural pigment or synthetic pigment, as long as the absorption wavelength range of the pigment is within 400 - 700 nm.

[0039] In the present application, the mass ratio of phycobiliprotein to pigment in the composition for enhancing the photobleaching stability of phycobiliprotein is (1 - 5):(50 - 500). Under the above conditions, it is beneficial to further improve the photobleaching stability of phycobiliprotein in the fluorescent probe. If the proportion of phycobiliprotein is relatively large, some phycobiliprotein cannot be effectively encapsulated by the pigment, resulting in some phycobiliprotein being affected by the photobleaching effect to a certain extent; if the proportion of phycobiliprotein in the composition is relatively small, it will lead to a low content of the fluorescent probe and affect the fluorescence intensity.

[0040] Exemplarily, the mass ratio of phycobiliprotein to pigment in the composition for enhancing the photobleaching stability of phycobiliprotein can be 1:50, 2:300, 3:100, 1:100, 2.5:200, 3:200, 3:500, etc.

[0041] Further, in the embodiments of the present application, the pigment is selected from fruit green and the phycobiliprotein is selected from phycoerythrin. The reliability and sensitivity of the fluorescent probe formed by phycoerythrin labeling are higher, and after compounding with fruit green pigment, the photobleaching stability of phycoerythrin can be further effectively improved.

[0042] Still further, the pigment is selected from fruit green, the phycobiliprotein is selected from phycoerythrin, and the mass ratio of phycoerythrin to fruit green in the composition for enhancing the photobleaching stability of phycobiliprotein is (2 - 3):(100 - 200). Under the above conditions, the photobleaching stability of phycoerythrin can be effectively improved.

[0043] Exemplarily, the mass ratio of phycoerythrin to phycocyanin in the composition for enhancing the photobleaching stability of phycobiliprotein can be 2:100, 2.5:100, 3:100, 1:100, 2.5:200, 3:200, 2:150, and so on.

[0044] Exemplarily, the fluorescent probe containing phycobiliprotein includes at least one of a phycobiliprotein-labeled antibody, a phycobiliprotein-labeled biotin ester, and a phycobiliprotein-labeled streptavidin. Further, the fluorescent probe containing phycobiliprotein is a phycobiliprotein-labeled streptavidin. Still further, in the embodiments of the present application, the fluorescent probe containing phycobiliprotein is a phycoerythrin-labeled streptavidin (PE-SA).

[0045] In the present application, the composition for enhancing the photobleaching stability of phycobiliprotein further includes ultrapure water, so that the composition is in a solution state, facilitating direct use as a fluorescent probe solution in fluorescence immunoassays such as flow cytometry.

[0046] Exemplarily, in the embodiments of the present application, in the composition for enhancing the photobleaching stability of phycobiliprotein, the pigment is phycocyanin and the mass concentration of phycocyanin in the composition is 0.2 - 0.4 g / 1000 mL, the fluorescent probe containing phycobiliprotein is PE-SA, and the mass concentration of PE-SA in the composition is 4.0 - 6.0 μg / mL.

[0047] Further, the composition for enhancing the photobleaching stability of phycobiliprotein further includes bovine serum albumin (BSA). Exemplarily, the mass concentration of BSA in the composition is 10 g / 1000 mL.

[0048] The present application also provides a kit for flow cytometry, which includes the composition for enhancing the photobleaching stability of phycobiliprotein provided above.

[0049] The kit for flow cytometry provided by the present application can achieve a stable and strong fluorescent signal of the fluorescent probe even under non-light-shielded conditions due to containing the composition for enhancing the photobleaching stability of phycobiliprotein provided above, which is beneficial to greatly improving the operational convenience of flow cytometry.

[0050] In the present application, the kit for flow cytometry further includes a fixing solution, a buffer solution, a permeabilization solution, a blocking solution, a washing solution, and a biotinylated antibody solution.

[0051] The fixing solution consists of NaH2PO4·2H2O, Na2HPO4, paraformaldehyde (PFA), and ultrapure water; among them, the final mass concentrations of NaH2PO4·2H2O, Na2HPO4, and PFA in the fixing solution are 2.9 g / 1000 mL, 19.24 g / 1000 mL, and 40 g / 1000 mL, respectively.

[0052] The buffer solution is a phosphate buffer solution, which consists of Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, and ultrapure water; among them, the final mass concentrations of Na2HPO4·12H2O, NaH2PO4·2H2O, and NaCl in the phosphate buffer solution are 174.09 g / 3000 mL, 17.79 g / 3000 mL, and 27 g / 3000 mL, respectively.

[0053] The permeabilization solution consists of Triton X-100 (polyethylene glycol octyl phenyl ether), the above-mentioned phosphate buffer solution, and ultrapure water; each 1000 mL of the permeabilization solution contains 1 mL of Triton X-100, 100 mL of the above-mentioned phosphate buffer solution, and the balance of ultrapure water.

[0054] The blocking solution consists of BSA (bovine serum albumin), the above-mentioned phosphate buffer solution, and ultrapure water; each 1000 mL of the blocking solution contains 10 g of BSA, 100 mL of the above-mentioned phosphate buffer solution, and the balance of ultrapure water.

[0055] The washing solution consists of Tween 20 (polysorbate-20), the above-mentioned phosphate buffer solution, and ultrapure water; each 1000 mL of the washing solution contains 1 mL of Tween 20, 100 mL of the above-mentioned phosphate buffer solution, and the balance of ultrapure water.

[0056] The biotinylated antibody solution consists of BSA (bovine serum albumin), biotinylated antibody, and ultrapure water; each 1000 mL of the biotinylated antibody solution contains 10 g of BSA, 10 mL of biotinylated antibody, and the balance of ultrapure water. Exemplarily, the biotinylated antibody is selected as sialidase (NEU1 / 3) on the sperm membrane for measuring semen samples.

[0057] The present application also provides a detection method based on flow cytometry, including: using the kit for flow cytometry provided above for detection.

[0058] In the present application, the detection based on flow cytometry can be carried out under non-light-shielding conditions. The detection method based on flow cytometry provided by the present application can perform detection with stable and strong fluorescence signals without the need for light shielding, greatly improving the convenience of detection operations.

[0059] Exemplarily, the flow cytometry-based detection includes the following steps:

[0060] (1) Washing: Take 0.5 mL of the sample to be tested (such as a semen sample), add 1 mL of phosphate buffer for washing, centrifuge at 3000 rpm for 5 min, discard the supernatant, and repeat the washing 3 times; add 1 mL of phosphate buffer to make a suspension of the sample to be tested.

[0061] (2) Fixing: Take 200 μl of the above-mentioned suspension of the sample to be tested, centrifuge at 3000 rpm for 5 min, discard the supernatant; add 200 μl of fixing solution, fix at room temperature for 10 min, centrifuge at 3000 rpm for 5 min, discard the supernatant; then add 200 μl of phosphate buffer for washing once, centrifuge at 3000 rpm for 5 min, discard the supernatant, to obtain a precipitate of the sample to be tested.

[0062] (3) Permeabilization: Add 200 μl of permeabilization solution to the precipitate of the sample to be tested obtained in step (2), permeabilize at room temperature for 10 min, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a precipitate of the sample to be tested.

[0063] (4) Washing: Add 200 μl of phosphate buffer to the precipitate of the sample to be tested obtained in step (3) for washing, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a precipitate of the sample to be tested.

[0064] (5) Blocking: Add 200 μl of blocking solution to the precipitate of the sample to be tested obtained in step (4), block at room temperature for 30 min, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a precipitate of the sample to be tested.

[0065] (6) Incubation: Add 100 μl of biotinylated antibody solution to the precipitate of the sample to be tested obtained in step (5), incubate at room temperature for 2 h, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a precipitate of the sample to be tested.

[0066] (7) Washing: Add 200 μl of washing solution to the precipitate of the sample to be tested obtained in step (6) for washing, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a precipitate of the sample to be tested.

[0067] (8) Incubation: Add 100 μl of the composition provided above for enhancing the photobleaching stability of phycobiliprotein to the precipitate of the sample to be tested obtained in step (7), incubate at room temperature under non-light-blocking (i.e., natural light) conditions for 1 h, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a precipitate of the sample to be tested.

[0068] (9) Washing: Under non-light-blocking (i.e., natural light) conditions, add 200 μl of washing solution to the precipitate of the sample to be tested obtained in step (8) for washing, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a precipitate of the sample to be tested.

[0069] (10) Detection: Add 300 μL of phosphate buffer to the precipitate of the sample to be tested obtained in step (9) under non-light-shielded (i.e., natural light) conditions to prepare a suspension of the sample to be tested, and then perform detection on a flow cytometer.

[0070] Example

[0071] This example provides a method for flow cytometry detection of semen samples, including the following steps:

[0072] (1) Washing: Take 0.5 mL of semen sample, add 1 mL of phosphate buffer for washing, centrifuge at 3000 rpm for 5 min, discard the supernatant, and repeat the washing 3 times; add 1 mL of phosphate buffer to make a sperm suspension.

[0073] (2) Fixation: Take 200 μl of the above sperm suspension, centrifuge at 3000 rpm for 5 min, discard the supernatant; add 200 μl of fixative, fix at 25 °C for 10 min, centrifuge at 3000 rpm for 5 min, discard the supernatant; then add 200 μl of phosphate buffer for washing once, centrifuge at 3000 rpm for 5 min, discard the supernatant, to obtain a sperm precipitate mass.

[0074] (3) Permeabilization: Add 200 μl of permeabilization solution to the sperm precipitate mass obtained in step (2), permeabilize at 25 °C for 10 min, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a sperm precipitate mass.

[0075] (4) Washing: Add 200 μl of phosphate buffer to the sperm precipitate mass obtained in step (3) for washing, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a sperm precipitate mass.

[0076] (5) Blocking: Add 200 μl of blocking solution to the sperm precipitate mass obtained in step (4), block at 25 °C for 30 min, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a sperm precipitate mass.

[0077] (6) Incubation: Add 100 μl of biotinylated antibody solution to the sperm precipitate mass obtained in step (5), incubate at 25 °C for 2 h, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a sperm precipitate mass.

[0078] (7) Washing: Add 200 μl of washing solution to the sperm precipitate mass obtained in step (6) for washing, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a sperm precipitate mass.

[0079] (8) Incubation: Add 100 μl of the composition to the sperm precipitate mass obtained in step (7), incubate at 25 °C under non-light-shielded (i.e., natural light) conditions for 1 h, centrifuge at 3000 rpm for 5 min to discard the supernatant, to obtain a sperm precipitate mass.

[0080] (9) Washing: Under the condition of not avoiding light (i.e., natural light), add 200 μl of washing solution to the sperm pellet obtained in step (8) for washing, centrifuge at 3000 rpm for 5 min to remove the supernatant, and obtain the sperm pellet.

[0081] (10) Detection: Under the condition of not avoiding light (i.e., natural light), add 300 μL of phosphate buffer solution to the sperm pellet obtained in step (9) to prepare a sperm suspension, and perform detection on a machine (flow cytometer).

[0082] Among them, the fixing solution is composed of NaH2PO4·2H2O, Na2HPO4, paraformaldehyde, and ultrapure water, and each 1000 mL of the fixing solution contains 2.9 g of NaH2PO4·2H2O, 19.24 g of Na2HPO4, 40 g of PFA, and the balance of ultrapure water. The phosphate buffer solution is composed of Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, and ultrapure water, and each 3000 mL of the phosphate buffer solution contains 174.09 g of Na2HPO4·12H2O, 17.79 g of NaH2PO4·2H2O, 27 g of NaCl, and the balance of ultrapure water. The permeabilization solution is composed of Triton X-100, the above-mentioned phosphate buffer solution, and ultrapure water, and each 1000 mL of the permeabilization solution contains 1 mL of Triton X-100, 100 mL of the above-mentioned phosphate buffer solution, and the balance of ultrapure water. The blocking solution is composed of BSA, the above-mentioned phosphate buffer solution, and ultrapure water, and each 1000 mL of the blocking solution contains 10 g of BSA, 100 mL of the above-mentioned phosphate buffer solution, and the balance of ultrapure water. The washing solution is composed of Tween 20, the above-mentioned phosphate buffer solution, and ultrapure water, and each 1000 mL of the washing solution contains 1 mL of Tween 20, 100 mL of the above-mentioned phosphate buffer solution, and the balance of ultrapure water. The biotinylated antibody solution is composed of BSA, NEU1 / 3, and ultrapure water, and each 1000 mL of the biotinylated antibody solution contains 10 g of BSA, 10 mL of NEU1 / 3, and the balance of ultrapure water. The composition is composed of BSA, fruit green, PE-SA, and ultrapure water, and each 1000 mL of the composition contains 10 g of BSA, 0.2 g of fruit green, 4 mg of PE-SA, and the balance of ultrapure water.

[0083] Comparative Example 1

[0084] This comparative example provides a method for performing flow cytometry detection on semen samples. The difference between this comparative example and the example is that step (6) is not performed, and steps (8)-(10) are performed under light avoidance conditions.

[0085] Comparative Example 2

[0086] This comparative example provides a method for flow cytometry detection of semen samples. The difference between this comparative example and the example is that the composition does not contain phthalein green.

[0087] Experimental Example

[0088] The same semen samples were detected respectively according to the detection methods provided in the example and Comparative Examples 1-2. After the compositions in the example and Comparative Example 2 were prepared, they were placed under natural light conditions for 12 h before the experiment; the scatter plots of the detection results of Comparative Examples 1-2 and the example are respectively as Figures 1 to 3 shown, and the histograms of the detection results are respectively as Figures 4 to 6 shown.

[0089] Since the incubation step of adding biotinylated antibody solution was not performed in Comparative Example 1, Comparative Example 1 was used as the negative control (NTC) for the example and Comparative Example 2. From Figures 1 to 6 the results, it can be seen that the positive proportion in the example (49.76%) is higher than that in Comparative Example 2 (20.72%), indicating that adding phthalein green in the example can effectively improve the photobleaching stability of phycoerythrin.

[0090] In summary, the composition for improving the photobleaching stability of phycobiliprotein provided in this application can effectively improve the photobleaching stability of phycobiliprotein in the fluorescent probe, so that the phycobiliprotein is not easily subjected to the photobleaching effect under the condition of not requiring light avoidance. Furthermore, the fluorescent probe formed by the phycobiliprotein still has a stable and strong fluorescent signal under the condition of not requiring light avoidance, which is beneficial to greatly improving the convenience of fluorescence immunoassay.

[0091] The above are only the preferred embodiments of this application and are not intended to limit this application. For those skilled in the art, various changes and modifications can be made to this application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims

1. A composition for enhancing the photo-bleaching stability of phycobiliprotein, characterized in that, The composition comprises a pigment and a fluorescent probe containing phycobiliprotein; the absorption wavelength range of the pigment is 400 - 700 nm; The pigment is selected from fruit green, and the phycobiliprotein is selected from phycoerythrin; The mass ratio of the phycoerythrin to the fruit green is (2 - 3):(100 - 200).

2. The composition according to claim 1, characterized in that, The fluorescent probe containing phycobiliprotein comprises at least one of an antibody labeled with phycobiliprotein, a biotin ester labeled with phycobiliprotein, and a streptavidin labeled with phycobiliprotein.

3. The composition according to claim 1, wherein The composition further comprises ultrapure water.

4. The composition according to claim 3, wherein The composition further comprises bovine serum albumin.

5. A kit for flow cytometry, characterized in that, Comprising the composition according to any one of claims 1 - 4.

6. The kit according to claim 5, wherein The kit further comprises a fixing solution, a buffer solution, a permeabilizing solution, a blocking solution, a washing solution, and a biotinylated antibody solution.

7. A detection method based on flow cytometry, characterized in that Comprising: Detecting by using the kit according to claim 5.

8. The detection method according to claim 7, wherein The detection is carried out under non-light-shielding conditions.

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