SP10 / ACP / PSA triple test paper and its preparation process

Abstract

The application discloses a SP10 / ACP / PSA triple detection test strip and a preparation process thereof, wherein a nitrocellulose membrane in the test strip is provided with a first detection line T1, a second detection line T2, a third detection line T3 and a quality control line C; the first detection line T1 is marked with SP10 monoclonal antibody 1; the second detection line T2 is marked with ACP monoclonal antibody 1; the third detection line T3 is marked with PSA monoclonal antibody 1; the quality control line C is marked with avidin SA; and the combination pad is provided with quantum dot microspheres marked with SP10 monoclonal antibody 2, ACP monoclonal antibody 2 and PSA monoclonal antibody 2; the test strip can simultaneously detect three indexes, and has simple product structure and convenient operation; the marking process in the preparation process adopts a competitive inhibitor and a quencher, solves the hook effect, and simultaneously obviously improves the specificity of each index.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of rapid diagnosis, and particularly relates to a SP10 / ACP / PSA triple detection test strip and a preparation process thereof. BACKGROUND

[0002] Male infertility is often manifested as abnormal semen, so semen examination occupies an important position in clinic. It plays an important guiding role in the diagnosis and treatment of male infertility, and is an indispensable method for estimating fertility, guiding treatment and judging prognosis.

[0003] Sperm concentration is an important indicator for measuring male infertility. When the concentration of sperm is less than 15 x 10 6 Based on anatomy, genetics and biochemical characteristics, SP10 protein has been proved to be used as a biomarker for indicating the concentration of male sperm, which is only expressed in the testis and is a marker that differentiates in the last step of the spermatogenesis process. According to research, the concentration of SP10 protein shows a certain linear relationship, that is, the number of sperm can be quantitatively analyzed by detecting the concentration of SP10 protein.

[0004] In the discharge, survival, vitality, transport and fertilization of sperm, various components of seminal plasma play a key role. About 30% of seminal plasma is prostatic fluid, so the secretory function of the prostate affects the quality of sperm. Acid phosphatase (ACP) and prostate specific antigen (PSA) are characteristic secretions of the prostate and important components of seminal plasma. ACP and PSA can be used as experimental indicators reflecting the decrease of sperm density and sperm vitality and the viscosity of semen.

[0005] As an indicator of prostate secretory function, the level of ACP can reflect the secretory function of the prostate. ACP is involved in the metabolism and regulation of sperm. If sperm vitality, semen viscosity and density are abnormal, ACP will be significantly reduced.

[0006] Human semen will coagulate immediately after being discharged outside the body and liquefy within a short time. PSA plays a major role in the liquefaction process. Its physiological function is to degrade the main proteins in semen gel, so that the semen liquefies within 5-15 minutes and releases sperm. When the viscosity of sperm increases, the content of PSA will decrease significantly. PSA can also increase the content of bradykinin or bradykinin-like metabolites in semen through its bradykinin-releasing enzyme effect, so as to enhance sperm vitality. When sperm vitality decreases, PSA decreases significantly. In summary, the contents of ACP and PSA in seminal plasma are closely related to sperm quality and can be used as experimental diagnostic indicators for infertility. At present, the above three indicators can only be detected one by one in clinic, which is low in detection efficiency and also causes waste of consumables and increases detection cost. SUMMARY

[0007] Based on the above defects, the application discloses a SP10 / ACP / PSA triple detection test strip and a preparation process thereof, which can simultaneously detect three indexes, and has simple product structure, convenient operation, and saving of labor cost and time cost.

[0008] In a first aspect, the application provides a SP10 / ACP / PSA triple detection test strip, which adopts the following technical scheme: a SP10 / ACP / PSA triple detection test strip comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption membrane, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption membrane are sequentially and adhesively connected to the bottom plate, the nitrocellulose membrane is provided with a first detection line T1, a second detection line T2, a third detection line T3 and a quality control line C, the first detection line T1 is marked with SP10 monoclonal antibody 1, the second detection line T2 is marked with ACP monoclonal antibody 1, the third detection line T3 is marked with PSA monoclonal antibody 1, and the quality control line C is marked with avidin SA, and the combination pad is provided with quantum dot microspheres marked with SP10 monoclonal antibody 2, ACP monoclonal antibody 2 and PSA monoclonal antibody 2.

[0009] In a second aspect, the application provides a preparation process of a SP10 / ACP / PSA triple detection test strip, which adopts the following technical scheme:

[0010] The preparation process of the SP10 / ACP / PSA triple detection test strip comprises the following steps: in the coupling process of the quantum dot microsphere marking, a competitive inhibitor containing biotin BSA is added; and in the blocking process of the quantum dot microsphere marking, a quenching agent containing ethanolamine is added.

[0011] In some specific embodiments, the quantum dot microsphere marking comprises the following steps:

[0012] (1) quantum dot microspheres are added into an activation solution to form a solution 1, EDC and NHS are respectively added into the activation solution to form a solution 2 and a solution 3, the solution 1, the solution 2 and the solution 3 are uniformly mixed to obtain a mixed solution;

[0013] (2) the mixed solution is uniformly mixed with a coupling solution, then SP10 monoclonal antibody 2, ACP monoclonal antibody 2 and PSA monoclonal antibody 2 are added, and coupling is performed for 20-40 min, the competitive inhibitor is uniformly mixed, and coupling is continuously performed for 1.5-2 h, and the microsphere solution is obtained after coupling;

[0014] (3) the quenching agent is added into the microsphere solution, and reaction is performed for 30-60 min, the supernatant is discarded by centrifugation, the blocking solution is added, and reaction is continuously performed for 1-1.5 h, and the labeled quantum dot microspheres are obtained.

[0015] In some embodiments, the competitive inhibitor is biotin BSA and tergitol NP9 in a solution of hydroxyethylpiperazine ethanesulfonic acid.

[0016] The biotin BSA in the solution can participate in labeling. When the quantum dot microspheres are combined with the labeled antibody, some carboxyl groups on the microspheres can not be combined with the labeled antibody in time. At this time, the added biotin BSA can be combined with the carboxyl groups on the microspheres, thereby forming a combination of quantum dot microspheres-labeled antibody-biotin-BSA. The biotin BSA and avidin SA have high affinity and can form an independent C-line system without being interfered by samples.

[0017] The solution contains tergitol NP9 as a non-ionic surfactant. The tergitol NP9 does not interfere with the activity of the quantum dot microspheres and the covalent bond combination but can remove excess EDC and free proteins in the microsphere solution, thereby promoting the coupling efficiency of biotin BSA and the microspheres. The combination can improve the hook effect phenomenon of the labeling curve.

[0018] Generally, after a test substance is combined with the labeled antibody 1 on the microspheres, the test substance is chromatographed to the T-line and combined with the marked antibody 2, thereby obtaining the value of the T-line. The more the test substances, the higher the signal value of the T-line. However, when the concentration of the test substance is high, the number of the combined proteins on the T-line of the nitrocellulose membrane is limited due to the spatial structure of the nitrocellulose membrane. When the number of the combined proteins exceeds the upper limit, the excess proteins are not combined or are not firmly combined, and even the proteins are accumulated, thereby causing the accumulation of the quantum dot microspheres on the T-line and forming a spatial steric effect, which leads to the inaccuracy of the light excited from the T-line, that is, the signal value of the T-line detection is no longer increased or even decreased, thereby forming the hook effect.

[0019] After the chromatography on the T-line is completed, the excess quantum dot microspheres continue to be chromatographed to the C-line. The Biotin-BSA on the microspheres has high affinity with the biotin SA on the C-line, and the two are combined, thereby obtaining the value of the C-line. Generally, the microspheres are excessive, and the combination rate of the Biotin-BSA and the SA is high, thereby the signal of the C-line is high and the signal values are basically consistent. However, when the concentration of the test substance is high, a large number of microspheres are combined with the T-line, and the microspheres combined with the C-line are less, thereby the C-line is weak, and the difference between the values of the T-line and the C-line is large. The hook effect can be solved by processing the values of the T-line and the C-line.

[0020] In some embodiments, the competitive inhibitor is 100-200 μg of biotin BSA and 0.8-1.2% of tergitol NP9 in a solution of 40-60 mM hydroxyethylpiperazine ethanesulfonic acid.

[0021] In some embodiments, the preparation method of the competitive inhibitor comprises the following steps:

[0022] (1) preparing an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid and adjusting the pH to neutral to obtain a HEPES solution;

[0023] (2) mixing tergitol NP9 into the HEPES solution to obtain a mixed solution;

[0024] adding biotin-BSA into the mixed solution to obtain the competitive inhibitor.

[0025] In some embodiments, the quenching agent is ethanolamine dissolved in a PVA / PVP / PEG Tween-20 solution with an adjusted pH being alkaline.

[0026] The quenching agent can make the microsphere particles in the solution uniformly distributed in the suspension due to the macromolecular structure, and can also be used for improving the dispersion stability of the microsphere particles by coordination bonding of the shared electron pairs, thereby avoiding the crosslinking of the microspheres and the resulting sedimentation, and making the microspheres more stable.

[0027] The ethanolamine in the quenching agent has both N atoms and -OH groups, and has both amine and alcohol chemical properties, and can react with the excess carboxyl groups on the microspheres after the addition of proteins. In addition, the Tween-20 can be used in combination with these quenching agents to better elute the miscellaneous proteins that are not specifically bound to the surface of the microspheres, can reduce the non-specific adsorption of proteins, avoid the cross-reactions caused by non-specific adsorption of miscellaneous proteins in quantum dot testing, and significantly improve the specificity of the product.

[0028] In some embodiments, the quenching agent is 2-4M ethanolamine dissolved in a PVA / PVP / PEG Tween-20 solution with a mass concentration of 0.8-1.2% and an adjusted pH being alkaline.

[0029] In some embodiments, the quenching agent is prepared by the following method:

[0030] (1) placing the weighed PVA into water and continuously stirring during dissolution to make the PVA fully dispersed in the water; (2) heating through water bath, and after the water temperature is increased to 60-70℃, fully dissolving and then standing at room temperature; and (3) adding ethanolamine, fully dissolving, and storing at 2-8℃.

[0031] In summary, the present application has at least the following beneficial effects:

[0032] 1. The present application discloses a SP10 / ACP / PSA triple detection test strip, which solves the problem of only SP10 single detection test strips and PSA single detection test strips on the market at present.

[0033] 2、The application adds competitive inhibitors in the process of quantum dot labeling, in the prior art, when the test sample is high in concentration, a hook-shaped effect may occur, that is, the signal value may not increase or decrease, and the addition of competitive inhibitors can effectively solve the hook-shaped effect; 3、The application adds a quenching agent in the process of quantum dot labeling, when three performance indicators are tested at the same time, other performance indicators may also be tested when a certain performance indicator is tested, and the specificity of testing multiple performance indicators is obviously improved by changing the closure. DETAILED DESCRIPTION

[0034] In order to make the application purposes, technical effects and advantages of the present application more clear, the present application will be described in detail below. It should be noted that the aspects, features, embodiments and advantages described in the present application can be compatible and / or combined together.

[0035] Unless otherwise specified, the meanings of the scientific and technical terms in the specification are the same as those generally understood by those skilled in the art; definitions of abbreviations and key terms: SP10: human sperm SP10 protein, PSA: acid phosphatase, ACP: prostate specific antigen, BB: boric acid, Tween-20; Tween-20, HEPES: 4-hydroxyethylpiperazine ethanesulfonic acid, casein-Na: sodium caseinate, PVP-K30: polyvinylpyrrolidone-K30, S9: propylene oxide-ethylene oxide-vinyl dimethyl amine copolymer, PB: phosphate buffer, NaOH: sodium hydroxide, KH2PO4: potassium dihydrogen phosphate, Na2HPO4: disodium hydrogen phosphate, KCL: potassium chloride, NaCl: sodium chloride, Na2HPO4: disodium hydrogen phosphate, NaH2PO4: sodium dihydrogen phosphate, PVA: polyvinyl alcohol, PVP: polyvinylpyrrolidone, PEG: polyethylene glycol, EDC: 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, NHS: N-hydroxysuccinimide, biotin Biotin-BSA: bovine serum albumin conjugated biotin, tergitol NP9: nonylphenol polyoxyethylene ether NP-9.

[0036] The application relates to an SP10 / ACP / PSA triple detection test strip and a preparation process thereof.

[0037] The application will be specifically described below.

[0038] An SP10 / ACP / PSA triple detection test strip

[0039] The first aspect of the application provides a SP10 / ACP / PSA triple test paper strip, comprising a base plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption membrane, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption membrane are sequentially and adhesively connected on the base plate, the nitrocellulose membrane is provided with a first detection line T1, a second detection line T2, a third detection line T3 and a quality control line C; the first detection line T1 is marked with SP10 monoclonal antibody 1; the second detection line T2 is marked with ACP monoclonal antibody 1; the third detection line T3 is marked with PSA monoclonal antibody 1; the quality control line C is marked with avidin SA; the combination pad is provided with quantum dot microspheres marked with SP10 monoclonal antibody 2, ACP monoclonal antibody 2 and PSA monoclonal antibody 2.

[0040] A preparation process of a SP10 / ACP / PSA triple test paper strip

[0041] The second aspect of the application provides a preparation process of a SP10 / ACP / PSA triple test paper strip, which is prepared by the following steps: step one: quantum dot microsphere marking:

[0042] 1) 1-2 mL of μmol / L quantum dot microspheres are added into 10-20 mL of an activation solution to form a solution 1, 300-500 ug of EDC and 3000-50000 ug of NHS are respectively added into 1 mL of the activation solution to form a solution 2 and a solution 3, the solution 1, the solution 2 and the solution 3 are mixed and activated in a shaking bed for 30-60 min, and then centrifuged for 15-30 min to discard the supernatant;

[0043] 2) 10-20 mL of 50 mM HEPES+ (0.01-0.05%) Tween-20 coupling solution is added and ultrasonically resuspended, 100-200 ug of SP10 monoclonal antibody 2, 150-200 ug of ACP monoclonal antibody 2 and 100-200 ug of PSA monoclonal antibody 2 are added into the solution, and then the solution is coupled in a shaking bed for 30 min, 1-2 mL of a competitive inhibitor is added and mixed, and then the solution is coupled in the shaking bed for 1.5 h-2 h;

[0044] 3) Microsphere blocking: 100-200 ul of a quenching agent is added into the coupled microsphere solution, and then the solution is reacted in a shaking bed for 30-60 min, and then centrifuged for 15-30 min to discard the supernatant, and then 10-20 mL of a blocking solution is added and the solution is reacted in a shaking bed for 1 h-1.5 h;

[0045] 4) Microsphere preservation: the blocked microsphere solution is centrifuged for 15-30 min to discard the supernatant, and then 10-20 mL of a preservation solution is added.

[0046] Step two: combination pad preparation:

[0047] The 20mm*30mm glass cellulose membrane is soaked in 20mM PB treatment solution at room temperature for 1h, then taken out and dried in a 37℃ oven overnight.

[0048] The glass cellulose membrane is placed on the AUTOKUNHGS510 gold spraying platform, and the quantum dot immunofluorescence microspheres marked with SP10, ACP, and PSA antibodies are sprayed on the glass cellulose membrane at a spraying amount of 1-2uL / cm. After drying at 37℃ for 2 hours, the product is obtained.

[0049] Step three: sample pad preparation:

[0050] The polyester cellulose membrane is cut into 20mm*30mm, soaked in 20mM PB treatment solution at room temperature for 1h, then taken out and dried in a 37℃ oven overnight.

[0051] Step four: nitrocellulose membrane preparation:

[0052] SP10, ACP, and PSA antibodies and avidin SA are respectively diluted to a concentration of 0.5-2mg / ml with the line dilution solution, and are respectively formed into SP10, ACP, and PSA detection lines and avidin SA quality control lines on the nitrocellulose membrane on the AUTOKUNHGS510 gold spraying platform at a line amount of 0.5-2ul / cm, and are dried for standby use.

[0053] Step five: test strip assembly:

[0054] The sample pad, the binding pad, the nitrocellulose membrane, and the water absorption membrane are sequentially and partially overlappedly pasted on the PVC bottom plate, and are cut into strips with a length of 6-8cm and a width of 3-4mm, to obtain the quantum dot immunofluorescence method for detecting SP10 / ACP / PSA triple test strips.

[0055] In some embodiments, a biotin BSA-containing competitive inhibitor is added during the coupling process of quantum dot microsphere labeling; an ethanolamine-containing quencher is added during the blocking process of quantum dot microsphere labeling.

[0056] In some specific embodiments, the quantum dot microsphere labeling includes the following steps:

[0057] (1) The quantum dot microspheres are added to the activation solution to form solution 1, and then EDC and NHS are respectively added to the activation solution to form solution 2 and solution 3. The solution 1, solution 2, and solution 3 are mixed to obtain a mixed solution;

[0058] (2) adding the coupling solution into the mixed solution, mixing, then adding SP10 monoclonal antibody 2, ACP monoclonal antibody 2, PSA monoclonal antibody 2, coupling for 20-40 min, adding the competition inhibitor, mixing, and continuing to couple for 1.5-2 h, to obtain a microsphere solution;

[0059] (3) adding the quenching agent into the microsphere solution, reacting for 30-60 min, centrifuging to discard the supernatant, adding the blocking solution, and continuing to react for 1-1.5 h, to obtain the labeled quantum dot microspheres.

[0060] In some specific embodiments, the competition inhibitor is biotin Biotin-BSA and tergitol NP9 added in a solution of hydroxyethylpiperazine ethanesulfonic acid.

[0061] In some specific embodiments, the competition inhibitor is 100-200 μg of biotin Biotin-BSA and 0.8-1.2% tergitol NP9 by mass concentration added in a solution of 40-60 mM hydroxyethylpiperazine ethanesulfonic acid.

[0062] In some specific embodiments, the preparation method of the competition inhibitor comprises the following steps:

[0063] (1) preparing an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid, and adjusting the pH to neutral, to obtain a HEPES solution;

[0064] (2) adding tergitol NP9 into the HEPES solution, mixing, to obtain a mixed solution;

[0065] (3) adding biotin Biotin-BSA into the mixed solution, to obtain the competition inhibitor.

[0066] In some specific embodiments, the quenching agent is ethanolamine dissolved in a PVA / PVP / PEG Tween-20 solution, and the pH is adjusted to be alkaline.

[0067] In some specific embodiments, the quenching agent is 2-4 M ethanolamine dissolved in a PVA / PVP / PEG Tween-20 solution with a mass concentration of 0.8-1.2%, and the pH is adjusted to be alkaline.

[0068] In some specific embodiments, the quenching agent is prepared by the following method: (1) weighing PVA into water, continuously stirring during dissolving, and fully dispersing the PVA in the water; (2) heating by water bath, and fully dissolving after the water temperature is increased to 60-70 °C, and then standing at room temperature; (3) adding ethanolamine, fully dissolving, and storing at 2-8 °C.

[0069] Detection method:

[0070] 1) standard curve drawing: there are different concentrations of SP10 / ACP / PSA standard, respectively, 75 μL-100 ul is added to the sample diluent, drop two drops to the sample pad of the combined detection test strip, 10 min after the test strip is placed under the fluorescence immunoassay analyzer, the fluorescence intensity of T line (detection line) and C line (quality control line) is detected, the standard curve reflecting the content of SP10 / ACP / PSA is drawn according to the ratio of fluorescence intensity of T line and C line;

[0071] 2) sample detection: the collected sample, 75 μL-100 ul is added to the sample diluent, drop two drops to the sample pad of the combined detection test strip, 10 min after the test strip is placed under the same excitation light source as step 1), the fluorescence intensity of T line and C line is detected, respectively, according to the ratio of fluorescence intensity of T line and C line, the standard curve is substituted, the concentration of SP10 / ACP / PSA in the sample is calculated.

[0072] The performance evaluation of SP10 / ACP / PSA triple detection kit is as follows:

[0073] 1, the evaluation of linearity adopts sample diluent to prepare SP10 quality control product into standard solution with concentration of 5*10 6 / ml, 10*10 6 / ml, 15*10 6 / ml, 20*10 6 / ml, 40*10 6 / ml, 80*10 6 / ml, 160*10 6 / ml, 320*10 6 / mL, 600*10 6 / ml, 80 ul is added to the sample diluent, drop two drops to the sample pad of the combined detection test strip, 10 min after the test strip is placed under the fluorescence immunoassay analyzer, the fluorescence intensity of T line (detection line) and C line (quality control line) is detected, the standard curve reflecting the content of SP10 is drawn according to the ratio of fluorescence intensity of T line and C line, each concentration is detected for 3 times, the average value is the detection concentration, the correlation coefficient is calculated by linear equation of preparation concentration and detection concentration.

[0074] 2、Linearity evaluation ACP quality control was prepared into standard solution with concentrations of 10U / ml, 20U / ml, 40U / ml, 100U / ml, 200U / ml, 400U / ml, 800U / mL, 1000U / mL by sample diluent, 80ul was added into sample diluent and mixed, two drops were added to the sample pad of the combined test strip, after 10min, the test strip was placed under the fluorescence immunoassay analyzer, the fluorescence intensity of T line (detection line) and C line (quality control line) was detected, the standard curve reflecting the ACP content was drawn according to the ratio of the fluorescence intensity of T line and C line, each concentration was repeated 3 times, the average value was taken as the detection concentration, the correlation coefficient was calculated by linear equation of the prepared concentration and the detection concentration.

[0075] 3、Linearity evaluation PSA quality control was prepared into standard solution with concentrations of 100mg / L, 300mg / L, 500mg / L, 1000mg / L, 2000mg / L, 4000mg / L, 6000mg / L, 8000mg / L by sample diluent, 80ul was added into sample diluent and mixed, two drops were added to the sample pad of the combined test strip, after 10min, the test strip was placed under the fluorescence immunoassay analyzer, the fluorescence intensity of T line (detection line) and C line (quality control line) was detected, the standard curve reflecting the PSA content was drawn according to the ratio of the fluorescence intensity of T line and C line, each concentration was repeated 3 times, the average value was taken as the detection concentration, the correlation coefficient was calculated by linear equation of the prepared concentration and the detection concentration.

[0076] Embodiment

[0077] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application.

[0078] Embodiment 1

[0079] A SP10 / ACP / PSA triple test strip includes a bottom plate, a sample pad, a combined pad, a nitrocellulose membrane and a water absorption membrane, the sample pad, the combined pad, the nitrocellulose membrane and the water absorption membrane are sequentially and sequentially bonded on the bottom plate, the nitrocellulose membrane is provided with a first detection line T1, a second detection line T2, a third detection line T3 and a quality control line C; the first detection line T1 is marked with SP10 monoclonal antibody 1; the second detection line T2 is marked with ACP monoclonal antibody 1; the third detection line T3 is marked with PSA monoclonal antibody 1; the quality control line C is marked with avidin SA; the combined pad is provided with quantum dot microspheres of SP10 monoclonal antibody 2, ACP monoclonal antibody 2 and PSA monoclonal antibody 2.

[0080] The triple test reagent strip is prepared by the following steps:

[0081] I. Solution Preparation:

[0082] 1. Activation solution preparation:

[0083] Add 500 mL of ultrapure water in a beaker, weigh 0.143 g of borax, 0.2165 g of boric acid, 0.2 g of Tween-20 in water, add 0.1 M HCL to adjust the solution pH to 8.0, and store at 2-8 °C.

[0084] 2. Coupling solution preparation:

[0085] Add 500 mL of ultrapure water in a beaker, weigh 5.9575 g of 4-hydroxyethylpiperazine ethanesulfonic acid and 0.32 g of Tween-20 in water, add 0.1 M NaOH to adjust the solution pH to 7.0. Store at 2-8 °C.

[0086] 3. Blocking solution preparation:

[0087] Add 500 mL of ultrapure water in a beaker, weigh 5 g of BSA in water, mix well, and store at 2-8 °C.

[0088] 4. Microsphere storage solution preparation:

[0089] Add 500 mL of ultrapure water in a beaker, weigh 0.2165 g of boric acid, 0.143 g of borax, 5 g of casein-Na, 50 g of glucose in water, add 0.1 M HCL to adjust the pH to 8.0, and store at 2-8 °C.

[0090] 5. Streak dilution solution preparation:

[0091] Add 500 mL of ultrapure water in a beaker, weigh 0.12 g of KH2PO4, 0.72 g of Na2HPO4, 0.1 g of KCL, 4 g of NaCL, 10 g of trehalose, 25 g of NaCL, dissolve in 500 mL of water, mix well, pH value is 7.4, and store at 2-8 °C.

[0092] 6. Binding pad treatment solution preparation:

[0093] Add 500 mL of ultrapure water in a beaker, weigh 1.15 g of Na2HPO4, 0.228 g of NaH2PO4, 1 g of casein-Na, 7.5 g of PVP-K30, adjust the solution pH to 7.5, and store at 2-8 °C.

[0094] 7. Sample pad treatment solution preparation:

[0095] Add 500 mL of ultrapure water in a beaker, take 1.15 g of Na2HPO4, 0.228 g of NaH2PO4, 1 g of Casein-Na, 7.5 g of PVP-K30, 2.5 g of Tween-20, adjust the pH of the solution to 7.4, and store at 2-8℃.

[0096] 8. Sample diluent preparation:

[0097] Add 1 L of ultrapure water in a beaker, take 1.15 g of Na2HPO4, 0.228 g of NaH2PO4, 8.7 g of NaCl, 10 g of BSA dissolved in 1 L of water, mix well, pH value is 7.4, and store at room temperature.

[0098] II. Quantum dot microsphere labeling

[0099] 1. Competitive inhibitor preparation:

[0100] (1) Take 11.915 g of 4-hydroxyethyl piperazine ethanesulfonic acid in 100 mL of ultrapure water, adjust the pH of the solution to 7.0 with 0.1 M NaOH, obtain 500 mM HEPES solution, and store at 2-8℃.

[0101] (2) Take 200 ul of 500 mM HEPES solution and add it to 1800 uL of pure water to obtain 50 mM HEPES;

[0102] (3) Take 0.02 g of tergitol NP9 and add it to 2 mL of 50 mM HEPES solution, mix well with a vortex instrument;

[0103] (4) Take 200 ug of biotin-BSA and add it to 2 mL of solution, and the competitive inhibitor is obtained.

[0104] 2. Quenching agent preparation:

[0105] (1) Take 1 g of PEG and put it in 100 mL of water, constantly stir during dissolution, and make the PEG fully dispersed in water;

[0106] (2) Heat by water bath, raise the water temperature to 60-70℃, fully dissolve and stand at room temperature;

[0107] (3) Add 0.18 g of ethanolamine, fully dissolve, and store at 2-8℃.

[0108] 3. Quantum dot microsphere labeling:

[0109] 1) 2 mL of 1 umol / L quantum dot microspheres were added to 20 mL of activation solution, mixed to form solution 1, and 500 ug of EDC and 5000 ug of NHS were added to 1 mL of activation solution to form solution 2 and solution 3. Solution 1, solution 2 and solution 3 were mixed, activated in a shaker for 30 min, and then centrifuged (2°C, 15000 rpm) for 20 min to discard the supernatant;

[0110] 2) 20 mL of coupling solution was added and resuspended by ultrasonic, 200 ug of SP10 monoclonal antibody 2, 200 ug of ACP monoclonal antibody 2, and 200 ug of PSA monoclonal antibody 2 were added to the solution, and the solution was coupled in a shaker (250 rpm, 25°C) for 30 min, then 2 mL of competitive inhibitor was added and mixed, and the solution was coupled in a shaker (250 rpm, 25°C) for 1.5 h;

[0111] 3) Microsphere blocking: 200 ul of quenching agent was added to the coupled microsphere solution, and the solution was reacted in a shaker (250 rpm, 25°C) for 60 min, then centrifuged (2°C, 15000 rpm) for 20 min to discard the supernatant, and then 20 mL of blocking solution was added and the solution was reacted in a shaker (250 rpm, 25°C) for 1 h;

[0112] 4) Microsphere preservation: the blocked microsphere solution was centrifuged (2°C, 15000 rpm) for 30 min to discard the supernatant, and 20 mL of preservation solution was added.

[0113] Three, sample pad processing:

[0114] The glass fiber membrane was soaked in the sample pad treatment solution at room temperature for 1 h, then taken out and dried in a 37°C oven overnight, taken out and placed in a sealed bag for dry storage.

[0115] Four, binding pad processing:

[0116] The glass fiber membrane was soaked in the binding pad treatment solution at room temperature for 1 h, then taken out and dried in a 37°C oven overnight, taken out and placed in a sealed bag for dry storage.

[0117] Five, binding pad gold spraying:

[0118] The glass fiber membrane was placed on the AUTOKUNHGS510 gold spraying platform, and the quantum dot immunofluorescent microspheres labeled with SP10, ACP and PSA antibodies were sprayed onto the binding pad at a spraying amount of 2 ul / cm, and dried at 37°C for 2 hours to obtain the product.

[0119] Six, nitrocellulose membrane marking:

[0120] The SP10, ACP, PSA three antibodies and avidin SA were respectively diluted to a concentration of 1 mg / ml with a line diluent, and were drawn on a nitrocellulose membrane on an AUTOKUNHGS510 gold mark platform with a line of 1 ul / cm to form SP10, ACP, PSA detection lines and avidin SA quality control lines, and the detection membrane was dried in an oven at 37s℃ overnight for standby.

[0121] Seven, test strip assembly:

[0122] The sample pad, binding pad, nitrocellulose membrane and water absorption membrane were sequentially and partially overlapped on the PVC base plate, and were cut into a paper strip with a length of 6-8 cm and a width of 3-4 mm, i.e. a quantum dot immunofluorescence method for detecting SP10 / ACP / PSA triple test paper strip.

[0123] Example 2:

[0124] A SP10 / ACP / PSA triple test paper strip, which is different from example 1 in that the competitive inhibitor is prepared by the following process: (1) weigh 11.915g of 4-hydroxyethyl piperazine ethanesulfonic acid in 100mL ultrapure water, adjust the solution PH value to 7.0 with 0.1M NaOH. Obtain 500mM HEPES solution, store at 2-8℃; (2) take 100ul solution of 500mM HEPES solution into 900uL pure water to obtain 50mM HEPES; (3) weigh 0.01g tergitol NP9 into 1mL of 50mM HEPES solution, mix well with vortex instrument; (4) weigh 100ug of biotin-BSA into 1mL of solution, i.e. competitive inhibitor.

[0125] Example 3:

[0126] A SP10 / ACP / PSA triple test paper strip, which is different from example 1 in that the competitive inhibitor is prepared by the following process: (1) weigh 23.83g of 4-hydroxyethyl piperazine ethanesulfonic acid in 200mL ultrapure water, adjust the solution PH value to 7.0 with 0.1M NaOH. Obtain 500mM HEPES solution, store at 2-8℃; (2) take 200ul solution of 500mM HEPES solution into 800uL pure water to obtain 100mM HEPES; (3) weigh 0.02g tergitol NP9 into 1mL of 100mM HEPES solution, mix well with vortex instrument; (4) weigh 200ug of biotin-BSA into 1mL of solution, i.e. competitive solution.

[0127] Example 4:

[0128] A SP10 / ACP / PSA triple test paper, the difference from example 1 is that the quenching agent is prepared by the following process: (1) take 1g of PVA into 100mL water, constantly stirring when dissolving, so that PVA is fully dispersed in water; (2) through water bath heating, water temperature is raised to 60 to 70 degrees Celsius after fully dissolving, and then standing to normal temperature; (3) add 0.18g of ethanolamine, fully dissolved. Store at 2-8℃.

[0129] Example 5:

[0130] A SP10 / ACP / PSA triple test paper, the difference from example 1 is that the quenching agent is prepared by the following process: (1) take 2g of PEG into 100mL water, constantly stirring when dissolving, so that PEG is fully dispersed in water; (2) through water bath heating, water temperature is raised to 60 to 70 degrees Celsius after fully dissolving, and then standing to normal temperature; (3) add 0.18g of ethanolamine, fully dissolved. Store at 2-8℃.

[0131] Example 6:

[0132] A SP10 / ACP / PSA triple test paper, the difference from example 1 is that the quenching agent is prepared by the following process: (1) take 4g of PVP into 100mL water, constantly stirring when dissolving, so that PEG is fully dispersed in water; (2) through water bath heating, water temperature is raised to 60 to 70 degrees Celsius after fully dissolving, and then standing to normal temperature; (3) add 0.36g of ethanolamine, fully dissolved. Store at 2-8℃.

[0133] Table 1 linear table of T / C and concentration relationship of SP10 in example 1

[0134]

[0135] Table 2 linear table of T / C and concentration relationship of ACP in example 1

[0136]

[0137] Table 3 linear table of T / C and concentration relationship of PSA in example 1

[0138]

[0139]

[0140] According to table 1, the T / C value of SP10 in example 1 is used to do linear relationship r 2 >0.99, the linear equation is y=65.079x 3 -144.6x 2+ 161.91 x - 2.0008; According to Table 2, the ACP of Example 1 has a linear relationship r of T / C value when T appears a hook 2 > 0.99, the linear equation is y = -120.9x 3 + 406.04x 2 + 132.13x + 20.547; According to Table 3, the PSA of Example 1 has a linear relationship r of T / C value when T appears a hook 2 > 0.99, the linear equation is y = 168.99x 3 - 1015.9x 2 + 4511.4x + 205.93, from the whole linear relationship, when the test concentration reaches a high concentration, the T / C value will be significantly improved when the hook-shaped effect of T value appears a hook-shaped effect after using an independent C line system.

[0141] Table 4 Linear table of T / C and concentration relationship of SP10 in Example 2

[0142]

[0143] Table 5 Linear table of T / C and concentration relationship of PSA in Example 2

[0144]

[0145]

[0146] Table 6 Linear table of T / C and concentration relationship of ACP in Example 2

[0147]

[0148] According to Table 4, the SP10 project has a linear relationship r of T / C value when T appears a hook 2 > 0.99, the linear equation is y = 13.2x 3 - 0.3524x 2 + 78.932x + 5.5669; According to Table 5, the PSA project has a linear relationship r of T / C value when T appears a hook 2 > 0.99, the linear equation is y = -120.98x 3 + 425.44x 2 + 94.502x + 22.959; According to Table 6, the ACP project has a linear relationship r of T / C value when T appears a hook 2 > 0.99, the linear equation is y = 210.66x 3 - 1235.6x 2+4745x+199.03; from the whole linear relationship, when the test concentration reaches high concentration, the T value out of the hook effect, after using the independent C line system, see T / C value will obviously improve this situation.

[0149] The linear relationship of examples 3-6 is similar to examples 1-2, and compared with examples 1-2, example 3 uses different concentrations of competitive inhibitors, examples 4-6 use different concentrations of quenching agents, and the hook effect is obviously improved.

[0150] Comparative example 1:

[0151] A SP10 / ACP / PSA triple test strip, compared with the examples, the marking process does not add competitive inhibitors, and there is no independent C line system;

[0152] I. Solution preparation:

[0153] 1. Preparation of activation solution:

[0154] Add 1L of ultrapure water to a beaker, weigh 0.286g of borax, 0.433g of boric acid, 0.1g of Tween-20 in water, add 0.1M HCL to adjust the solution pH to 8.0, and store at 2-8℃.

[0155] 2. Preparation of coupling solution:

[0156] Add 1L of ultrapure water to a beaker, weigh 11.915g of 40 hydroxyethyl piperazine ethanesulfonic acid and 0.1g of Tween-20 in water, add 0.1M NaOH to adjust the solution pH to 7.0, and store at 2-8℃.

[0157] 3. Preparation of blocking solution:

[0158] Add 1L of ultrapure water to a beaker, weigh 100g of BSA in water, mix well, and store at 2-8℃.

[0159] 4. Preparation of microsphere preservation solution:

[0160] Add 1L of ultrapure water to a beaker, weigh 0.433g of boric acid, 0.286g of borax, 10g of casein-Na, 100g of glucose in water, add 0.1M HCL to adjust the pH to 8.0, and store at 2-8℃.

[0161] 5. Preparation of line dilution solution:

[0162] Add 1L of ultrapure water in a beaker, take 0.24g KH2PO4, 1.44g Na2HPO4, 0.2g KCL, 8g NaCl, 20g trehalose, 50g NaCl dissolved in 1L water, stirring, pH value is 7.4, placed at 2-8℃ storage.

[0163] 6, the preparation of the pad processing liquid:

[0164] Add 1L of ultrapure water in a beaker, take 2.3g Na2HPO4, 0.456g NaH2PO4, 2g casein-Na, 15g PVP-K30, adjust the solution pH to 7.5, placed at 2-8℃ storage.

[0165] 7, sample pad processing liquid preparation:

[0166] Add 1L of ultrapure water in a beaker, take 2.3g Na2HPO4, 0.456g NaH2PO4, 2g Casein-Na, 15g PVP-K30, 10g Tween-20, adjust the solution pH to 7.4, placed at 2-8℃ storage.

[0167] II. Quantum dot microspheres label

[0168] 1, the preparation of the quencher:

[0169] (1) take 1g PVA into 100mL water, constantly stirring when dissolving, make PVA fully dispersed in water;

[0170] (2) through water bath heating, water temperature rises to 60-70℃ fully dissolved after standing to normal temperature;

[0171] (3) add 0.18g ethanolamine, fully dissolved, placed at 2-8℃ storage.

[0172] 2, quantum dot microspheres label:

[0173] 1) 1mL 1umol / L quantum dot microspheres are added to 10mL of activation solution, mixed to form solution 1, and 300ug EDC and 3000ug NHS are added to 1mL of activation solution to form solution 2 and solution 3. Mix solution 1, solution 2 and solution 3, and place in a shaker for activation for 30min, then centrifuge (2℃, 15000rpm) for 20min and discard the supernatant;

[0174] 2) add 10mL of coupling solution and resuspend by ultrasonic, add 100ug SP10 monoclonal antibody 2, 150ug ACP monoclonal antibody 2, 100ug PSA monoclonal antibody 2 in the solution, and couple in a shaker (250rpm, 25℃) for 1.5h;

[0175] 3) Microspheres blocking: add 100ul of quenching agent to the coupled microspheres solution, put into a shaker at 250rpm, 25℃, and react for 30min, then centrifuge (2℃, 15000rpm) for 20min, discard the supernatant, then add 10mL of blocking solution, and continue to put into a shaker (250rpm, 25℃) for 1h;

[0176] 4) Microspheres preservation: centrifuge (2℃, 15000rpm) the blocked microspheres solution for 15-30min, discard the supernatant, and add 10mL of preservation solution.

[0177] III. Sample pad processing:

[0178] Soak the glass fiber membrane at room temperature for 1h, then take out and dry in a 37℃ oven overnight, take out, and place in a sealed bag for dry storage.

[0179] IV. Binding pad processing:

[0180] Soak the glass fiber membrane at room temperature for 1h, then take out and dry in a 37℃ oven overnight, take out, and place in a sealed bag for dry storage.

[0181] V. Binding pad gold spraying:

[0182] Place the glass fiber membrane on the AUTOKUNHGS510 gold spraying platform, spray the quantum dot immunofluorescence microspheres labeled with SP10, ACP, and PSA antibodies onto the binding pad at a spraying amount of 1ul / cm, and dry at 37℃ for 2h to obtain.

[0183] VI. Nitrocellulose membrane scribing:

[0184] Dilute SP10, ACP, and PSA antibodies and avidin SA to a concentration of 1mg / ml with scribing diluent, scribe on the nitrocellulose membrane at a scribing amount of 1ul / cm on the AUTOKUNHGS510 gold spraying platform to form SP10, ACP, and PSA detection lines and goat anti-mouse control lines, respectively, to form a detection membrane, and dry in a 37s℃ oven overnight for standby.

[0185] VII. Test strip assembly:

[0186] Paste the sample pad, binding pad, nitrocellulose membrane, and water absorption membrane on the PVC bottom plate in sequence and partially overlap, cut into strips with a length of 6-8cm and a width of 3-4mm, and obtain the quantum dot immunofluorescence method for detecting SP10 / ACP / PSA triple test strips.

[0187] Table 7 Linear table of T / C and concentration relationship of SP10 in Comparative Example 1

[0188]

[0189] Table 8 Linear table of T / C vs concentration of PSA in Comparative Example 1

[0190]

[0191]

[0192] Table 9 Linear table of T / C vs concentration of ACP in Comparative Example 1

[0193]

[0194] According to Table 7, the T / C value of the SP10 project in the case of T appearing hook, without adding competitive inhibitor, the linear relationship r 2 <0.99, the linear equation is y = -1.2002x 2 +87.038x-19.827; according to Table 8, the T / C value of the PSA project in the case of T appearing hook, without adding competitive agent, the linear relationship r 2 <0.99, the linear equation is y = -1.2002x 2 +87.038x-19.827; according to Table 9, the T / C value of the ACP project in the case of T appearing hook, without adding competitive agent, the linear relationship r 2 <0.99, the linear equation is y = -23.828x 3 +310.05x 2 +325.9x+322.51; after not adding competitive agent, the C line sheep anti-mouse and the excess microspheres affinity is not good, when the high value appears hook-shaped effect, the T / C also does not have obvious improvement.

[0195] Comparative Example 2:

[0196] An SP10 / ACP / PSA triple test paper, the difference from the embodiment is that the marking process does not add quenching agent;

[0197] I. Solution preparation:

[0198] 1. Preparation of activation solution:

[0199] Add 500 mL of ultrapure water to a beaker, weigh 0.143 g of borax, 0.2165 g of boric acid, 0.2 g of Tween-20 in water, add 0.1 M HCL to adjust the solution pH to 8.0, and store at 2-8℃.

[0200] 2. Preparation of coupling solution:

[0201] Add 500 mL of ultrapure water in a beaker, weigh 5.9575 g of 4-hydroxyethyl piperazine ethanesulfonic acid and 0.32 g of Tween-20 in water, add 0.1M NaOH to adjust the solution pH to 7.0. Store at 2-8℃.

[0202] 3. Preparation of blocking solution:

[0203] Add 500 mL of ultrapure water in a beaker, weigh 5 g of BSA in water, stir well, and store at 2-8℃.

[0204] 4. Preparation of microsphere storage solution:

[0205] Add 500 mL of ultrapure water in a beaker, weigh 0.2165 g of boric acid, 0.143 g of borax, 5 g of casein-Na, 50 g of glucose in water, add 0.1M HCL to adjust the pH to 8.0, and store at 2-8℃.

[0206] 5. Preparation of streak dilution solution:

[0207] Add 500 mL of ultrapure water in a beaker, weigh 0.12 g of KH2PO4, 0.72 g of Na2HPO4, 0.1 g of KCL, 4 g of NaCL, 10 g of trehalose, 25 g of NaCL, dissolve in 500 mL of water, stir well, pH value is 7.4, and store at 2-8℃.

[0208] 6. Preparation of binding pad treatment solution:

[0209] Add 500 mL of ultrapure water in a beaker, weigh 1.15 g of Na2HPO4, 0.228 g of NaH2PO4, 1 g of Casein-Na, 7.5 g of PVP-K30, adjust the solution pH to 7.5, and store at 2-8℃.

[0210] 7. Preparation of sample pad treatment solution:

[0211] Add 500 mL of ultrapure water in a beaker, weigh 1.15 g of Na2HPO4, 0.228 g of NaH2PO4, 1 g of Casein-Na, 7.5 g of PVP-K30, 2.5 g of Tween-20, adjust the solution pH to 7.4, and store at 2-8℃.

[0212] 8. Preparation of sample dilution solution:

[0213] Add 1 L of ultrapure water in a beaker, weigh 1.15 g of Na2HPO4, 0.228 g of NaH2PO4, 8.7 g of NaCl, 10 g of BSA, dissolve in 1 L of water, stir well, pH value is 7.4, and store at room temperature.

[0214] II. Quantum dot microsphere labeling

[0215] 1. Competitive inhibitor preparation:

[0216] (1) Take 11.915 g of 4-hydroxyethylpiperazine ethanesulfonic acid in 100 mL of ultrapure water, and adjust the pH of the solution to 7.0 with 0.1 M NaOH. Obtain a 500 mM HEPES solution, and store at 2-8°C;

[0217] (2) Take 200 ul of the 500 mM HEPES solution and add it to 1800 uL of pure water to obtain a 50 mM HEPES solution;

[0218] (3) Take 0.02 g of tergitol NP9 and add it to 2 mL of the 50 mM HEPES solution, and mix it thoroughly with a vortex;

[0219] (4) Take 200 ug of biotin-BSA and add it to 2 mL of the solution to obtain the competitive inhibitor.

[0220] 2. Quantum dot microsphere labeling:

[0221] 1) Take 2 mL of 1 umol / L quantum dot microspheres and add them to 20 mL of activation solution to form solution 1, then take 500 ug of EDC and 5000 ug of NHS and add them to 1 mL of activation solution to form solutions 2 and 3. Mix solutions 1, 2, and 3, and place them in a shaker for activation for 30 min, then centrifuge (2°C, 15000 rpm) for 20 min and discard the supernatant;

[0222] 2) Add 20 mL of coupling solution and resuspend by ultrasonication, then add 200 ug of SP10 monoclonal antibody 2, 200 ug of ACP monoclonal antibody 2, and 200 ug of PSA monoclonal antibody 2 to the solution, and place it in a shaker (250 rpm, 25°C) for coupling for 30 min, then add 2 mL of competitive inhibitor and mix, and continue to place it in a shaker (250 rpm, 25°C) for coupling for 1.5 h;

[0223] 3) Microsphere blocking: add 20 mL of blocking solution to the coupled microsphere solution and continue to place it in a shaker (250 rpm, 25°C) for reaction for 1 h;

[0224] 4) Microsphere preservation: centrifuge (2°C, 15000 rpm) the blocked microsphere solution for 30 min and discard the supernatant, then add 20 mL of preservation solution.

[0225] Table 10 Linear table of T / C and concentration relationship of SP10 in Comparative Example 2

[0226]

[0227] Table 11 Linear table of T / C vs. concentration of PSA in Comparative Example 2

[0228]

[0229] Table 12 Linear table of T / C vs. concentration of ACP in Comparative Example 2

[0230]

[0231] Comparative Example 2, without adding a quenching agent, can cause non-specific binding with T2 line when testing SP10, non-specific binding with T1 and T3 when testing PSA concentration, and non-specific binding with T3 when testing ACP concentration.

[0232] The detection accuracy of the kit according to the present application is verified.

[0233] Ten clinical samples are taken for quantitative detection of each item. The test strip is taken out and balanced at room temperature for standby; 80ul of sample diluent is taken and mixed, two drops are added to the sample pad of the combined test strip, and after 10min, the test strip is placed under the same excitation light source, and the fluorescence intensity of T line and C line is detected respectively, according to the ratio of fluorescence intensity of T line and C line, the standard curve is substituted, and the concentration of SP10 / ACP / PSA in the sample is calculated.

[0234] Among them, SP10 / ACP / PSA are all self-produced antibodies of the company. The specific detection results are shown in Table 13.

[0235] Table 13 Detection results of SP10-ACP-PSA fluorescence kit in semen samples

[0236]

[0237]

Claims

1. A SP10 / ACP / PSA triple test paper strip comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent membrane, the sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent membrane being sequentially and adhesively attached to the base plate, characterized in that: The nitrocellulose membrane is provided with a first detection line T1, a second detection line T2, a third detection line T3 and a quality control line C; the first detection line T1 is marked with SP10 monoclonal antibody 1; the second detection line T2 is marked with ACP monoclonal antibody 1; the third detection line T3 is marked with PSA monoclonal antibody 1; the quality control line C is marked with avidin SA; and the conjugated pad is provided with quantum dot microspheres marked with SP10 monoclonal antibody 2, ACP monoclonal antibody 2 and PSA monoclonal antibody 2. A competitive inhibitor containing biotin Biotin-BSA is added in the coupling process of the quantum dot microspheres. The competitive inhibitor is biotin Biotin-BSA and tergitol NP9 added in a solution of hydroxyethylpiperazine ethanesulfonic acid. The preparation method of the competitive inhibitor comprises the following steps: (1) preparing an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid and adjusting the pH to neutral to obtain a HEPES solution; (2) adding tergitol NP9 into the HEPES solution and mixing to obtain a mixed solution; (3) adding biotin Biotin-BSA into the mixed solution to obtain the competitive inhibitor.

2. The preparation process of SP10 / ACP / PSA triple test paper strip according to claim 1, characterized in that: A quenching agent containing ethanolamine is added in the blocking process of the quantum dot microspheres.

3. The preparation process of SP10 / ACP / PSA triple test paper strip according to claim 1, wherein, The quantum dot microsphere marking comprises the following steps: (1) adding quantum dot microspheres into an activation solution to form a solution 1, then adding EDC and NHS into the activation solution to form a solution 2 and a solution 3, mixing the solution 1, the solution 2 and the solution 3 to obtain a mixed solution; (2) adding a coupling solution into the mixed solution and mixing, then adding SP10 monoclonal antibody 2, ACP monoclonal antibody 2 and PSA monoclonal antibody 2, coupling for 20-40 min, then adding the competitive inhibitor and mixing, continuing to couple for 1.5-2 h, and obtaining a microsphere solution after coupling; (3) adding a quenching agent into the microsphere solution and reacting for 30-60 min, centrifuging and discarding the supernatant, then adding a blocking solution and continuing to react for 1-1.5 h to obtain the marked quantum dot microspheres.

4. The preparation process of SP10 / ACP / PSA triple test paper according to claim 2, characterized in that: The quenching agent is ethanolamine dissolved in a PVA / PVP / PEG / Tween-20 solution, and the pH is adjusted to alkaline.

5. The preparation process of SP10 / ACP / PSA triple test paper according to claim 4, characterized in that, The quenching agent is prepared by the following method: (1) weighing PVA and putting it into water, continuously stirring during dissolving to make the PVA fully dispersed in the water; (2) heating by water bath, and when the water temperature is increased to 60-70℃, fully dissolving and then standing at room temperature; (3) adding ethanolamine, fully dissolving and storing at 2-8℃.

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