Hexapeptin detection method and reagent strip

Abstract

The invention discloses a method and a reagent strip for detecting aglyptin, the reagent strip or kit comprises a magnetic particle working solution and an aglyptin monoclonal antibody 2 coupled with alkaline phosphatase, and the magnetic particle working solution is composed of streptavidin magnetic particles and an aglyptin monoclonal antibody 1 coupled with biotin. The kit provided by the invention is high in sensitivity and good in stability, can be used for rapid detection of stroke, heart failure and myocardial infarction, and provides a faster diagnosis technology for clinic.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a copeptin detection method and reagent strip. BACKGROUND

[0002] Copeptin (CPP) is a 39-amino acid residue polypeptide homologous to arginine vasopressin (AVP) and is the C-terminal part of pro-vasopressin (pro-VAP). Upon activation of the AVP system, CPP is stimulated to be secreted from the posterior pituitary into the circulation in equimolar amounts with AVP. Therefore, CPP directly reflects the AVP concentration and can be used as a surrogate biomarker for AVP secretion. A study shows that CPP is more sensitive than circulating cortisol in reflecting the body stress level. This new biomarker will be able to help make early decisions in clinical practice and show a degree of correlation with various acute disease states, such as cerebrovascular events, myocardial infarction, pneumonia, kidney disease and hypertension, osmotic changes, obesity, even bipolar disorder, major depressive disorder, and childhood abuse, etc.

[0003] The existing methods for determining copeptin include radioimmunoassay, immunochromatography and chemiluminescence, etc. The radioimmunoassay has the problems of long detection time and nuclear element pollution. The immunochromatography has the problems of low sensitivity, narrow linear range, complex operation, low degree of automation and great influence of human factors. The sensitivity of the current chemical detection of copeptin is still low and cannot meet the needs of cardiovascular diseases to be quickly and accurately detected. Therefore, there is an urgent need for a high-sensitivity chemiluminescence method for detecting copeptin in China to assist cardiovascular diseases to provide a fast and accurate clinical diagnosis scheme and reduce the probability of occurrence of various diseases caused by stroke, heart failure, myocardial infarction, etc. SUMMARY

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present application is to provide a copeptin detection kit or reagent strip with higher detection sensitivity.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a copeptin detection reagent strip or kit in the first aspect.

[0007] Further, the reagent strip or kit comprises a magnetic microparticle working solution and a copeptin monoclonal antibody 2 coupled with alkaline phosphatase.

[0008] Further, the magnetic microparticle working solution is composed of streptavidin magnetic microparticles and a copeptin monoclonal antibody 1 coupled with biotin.

[0009] In some embodiments, the kits can include a carrier means for closely related articles, such as vials, tubes, etc., each containing one of the separate components to be employed in the methods. One of the containers can include the components described above, other containers can include substances that are desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The containers can be unitary with the other components of the kit, or can be separate. The container can maintain the sterility of the component prior to use. One or more components can also include a label indicating that the component is used for a specific detection or diagnostic application. Further optional components of the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents (e.g., substrates that undergo a chemical change by enzymatic action), etc.

[0010] In some embodiments, the reagent strip further comprises a PVC base plate and a sample pad, an anti-and peptide hormone antibody tracer marker pad, a capture membrane, and a water absorption pad attached to the upper surface of the PVC base plate.

[0011] Specifically, the sample pad is generally glass fiber, filter paper or filter blood membrane; the marker pad can be glass fiber, filter paper or polyester film; when the sample pad and the marker pad are made of the same material, they can be integrated together, and a piece of glass fiber, filter paper or polyester film can complete the functions of the sample pad and the marker pad.

[0012] Specifically, the capture membrane can be a nitrocellulose membrane (NC membrane), a nylon membrane, etc., the T line of the capture membrane contains small molecule organic compound antigens, the C line is a positive control, and the C line can contain secondary antibodies that can bind to small molecule organic compound detection antibodies, or other proteins or antibodies that can be used as positive controls, such as biotin-labeled bovine serum albumin or ovalbumin (the marker pad contains streptavidin), or goat anti-rabbit antibody (the marker pad contains rabbit antibody).

[0013] Further, the and peptide hormone monoclonal antibody 2 conjugated with alkaline phosphatase and the and peptide hormone monoclonal antibody 1 conjugated with biotin bind to different epitopes of human and peptide hormone.

[0014] Further, the concentration of the magnetic particle working solution is 0.2 mg / mL to 1.0 mg / mL.

[0015] Further, the concentration of the magnetic particle working solution is 0.5 mg / mL.

[0016] Further, the preparation method of the magnetic particle working solution comprises the following steps:

[0017] After washing the streptavidin magnetic particles, the magnetic particles are configured into a magnetic particle suspension with a buffer solution;

[0018] reacting the andpepsin monoclone 1 and the biotin-N-hydroxysuccinimide activated lipid at a mass ratio of 50:1-10:1 at 4-30°C, pH 6.0-8.0 for 0.5 h-36 h; after the reaction, removing the excess biotin-N-hydroxysuccinimide activated lipid by dialysis to obtain the biotin-coupled andpepsin monoclone 1;

[0019] mixing the magnetic particle suspension and the biotin-coupled andpepsin monoclone 1 at a mass ratio of 1 mg:1 μg-1 mg:10 μg, washing and then using a buffer to prepare a magnetic particle working solution with a concentration of 0.2 mg / mL-1.0 mg / mL.

[0020] Further, the concentration of the magnetic particle suspension is 10 mg / mL.

[0021] Further, the andpepsin monoclone 1 and the biotin-N-hydroxysuccinimide activated lipid are reacted at a mass ratio of 20:1 at 25°C, pH 8.0 for 2 h.

[0022] Further, the concentration of the magnetic particle working solution is 0.5 mg / mL.

[0023] Further, the buffer is a PBS buffer containing 2% BSA, 0.1% ProClin300.

[0024] In some embodiments, the biotin-coupled andpepsin monoclone 1

[0025] Further, the preparation method of the andpepsin monoclone 2 coupled with alkaline phosphatase comprises the following steps:

[0026] mixing the activated andpepsin monoclone 2 and the activated alkaline phosphatase at a mass ratio of 1:1-1:10 for reaction, purifying using a gel filtration chromatography column to obtain the andpepsin monoclone 2 coupled with alkaline phosphatase.

[0027] Further, the activated andpepsin monoclone 2 and the activated alkaline phosphatase are mixed at a mass ratio of 1:2 for reaction for 30 min, purifying using a gel filtration chromatography column to obtain the andpepsin monoclone 2 coupled with alkaline phosphatase.

[0028] Further, the reagent strip or kit further comprises a chemiluminescent substrate solution.

[0029] In some embodiments, the chemiluminescent substrate solution comprises but is not limited to: AMPPD, APS-5 or CDP-Star type substrate. Different chemiluminescent substrate solutions can be selected by those skilled in the art according to the mechanism of action and the labeled enzyme.

[0030] In some embodiments, the high concentration of albumin in the chemiluminescent marker diluent prevents the alkaline phosphatase from denaturing due to adsorption and also prevents the aggregation of alkaline phosphatase molecules due to interaction between the molecules.

[0031] Further, the chemiluminescent marker diluent can contain magnesium chloride and calcium chloride, both of which contain Mg 2+ , Ca 2+ , which can improve the efficiency of the catalytic hydrolysis reaction of alkaline phosphatase and increase the rigidity of the protein by forming multiple points of contact with the protein, thereby improving the stability of the protein to temperature and the like.

[0032] Further, the chemiluminescent marker diluent can contain sodium chloride, the sodium ions of which can bind to the zinc binding center of alkaline phosphatase, causing the alkaline phosphatase to be in a lower energy level, thereby making the alkaline phosphatase more stable and more likely to maintain activity.

[0033] Further, the chemiluminescent marker diluent can contain a protective agent, preferably a polyhydroxy compound, which can form many hydrogen bonds with protein molecules, help form a solvent layer, and also increase the surface tension of solvent water, causing the alkaline phosphatase to preferentially hydrate, thereby increasing the stability of the alkaline phosphatase molecules.

[0034] Further, the chemiluminescent marker diluent can also contain a sugar substance, which has a polyol structure and can be connected to the surface molecules of alkaline phosphatase through hydrogen bonds and also effectively connected to external water molecules through hydrogen bonds, thereby stabilizing the alkaline phosphatase molecules.

[0035] Further, the chemiluminescent marker diluent can also contain a stabilizing agent, which can increase the surface tension of solvent water, causing the alkaline phosphatase to preferentially hydrate, thereby increasing the stability of the alkaline phosphatase molecules, and also can be connected to the surface molecules of alkaline phosphatase through hydrogen bonds, thereby stabilizing the alkaline phosphatase molecules.

[0036] Further, the kit can also include a procalcitonin standard.

[0037] Further, the procalcitonin standard is a diluent that dissolves and dilutes procalcitonin into 4 to 8 series of procalcitonin solutions with different concentrations.

[0038] Further, the kit can also include a procalcitonin quality control.

[0039] In some embodiments, the diluent for the andrographolide calibrator includes: 10-500 mM buffer, 1-10% protein, 1-5% saccharide, 10-20% protective agent, 1-5% stabilizer, 0.1-0.5% surfactant, 0.5-1.5% fish gelatin, 0.2-0.8% methyl cellulose, 0.1-0.6% preservative, pH 7.0-8.0.

[0040] In some embodiments, the protein is one or more of bovine serum albumin, casein, gamma globulin (bovine); the saccharide is one or more of galactose, lactose, fructose, sucrose, trehalose; the protective agent is one or more of glycerol, mannitol, sorbitol, inositol, xylitol; the stabilizer is one or more of polyethylene glycol 20000, polyethylene glycol 2000, polyethylene glycol 8000, polyvinylpyrrolidone; the surfactant is one or more of lauryl sulfosuccinate disodium, fatty alcohol polyoxyethylene (3) ether sulfosuccinate disodium, potassium lauryl ether phosphate, polyethylene glycol octylphenyl ether; the preservative is one or more of thiomersal, gentamicin, ProClin series preservatives, with ProClin 300 listed as an example of the ProClin series preservatives.

[0041] The high protein concentration in the diluent for the andrographolide calibrator prevents adsorption of andrographolide, and the saccharide and protective agent interfere with the refolding process of andrographolide, protecting the activity and interstitial structure of andrographolide antigen. The stabilizer substance can increase the surface tension of solvent water, causing andrographolide to preferentially hydrate, thereby increasing stability. Methyl cellulose and fish gelatin are very effective thickeners and stabilizers, enabling andrographolide solution to form a uniform suspension, maintaining an active state. The addition of a preservative greatly reduces the probability of long-term bacterial growth in the solution.

[0042] In some embodiments, the buffer in the diluent for the andrographolide calibrator is one or more of Tris buffer, HEPES buffer, boric acid buffer, phosphate buffer, acetate buffer, citric acid, (N-morpholine)ethanesulfonic acid (MES2) buffer, 3-(N-morpholine)propanesulfonic acid (MOPS) buffer, N-tris(hydroxymethyl)methyl-2-aminethanesulfonic acid monosodium salt (TAPSN) buffer, N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid buffer.

[0043] The second aspect of the present application provides a method for quantitatively detecting andrographolide.

[0044] Further, the method includes the step of detecting the sample to be tested using the components of the reagent strip or kit of the first aspect of the present application.

[0045] Further, the sample to be detected includes plasma, serum, cerebrospinal fluid, urine, saliva, tissue homogenate and / or cell culture supernatant.

[0046] Further, the method further comprises the steps of detecting gradient concentrations of the procalcitonin standard to prepare a standard curve, and calculating the concentration of the sample to be detected and the procalcitonin according to the standard curve.

[0047] Advantages and beneficial effects of the present application:

[0048] The kit or reagent strip of the present application comprises magnetic particle working solution and biotin-coupled alkaline phosphatase procalcitonin monoclonal antibody 2 reagent, and the whole kit system greatly improves the detection sensitivity; can be used for rapid detection of stroke, heart failure and myocardial infarction, and provides faster diagnosis technology for clinic. DETAILED DESCRIPTION

[0049] The technical solutions of the present application are further described and explained below by specific examples, and it should be understood that the specific examples described herein are only used to help understand the present application, and are not used to limit the specific embodiments of the present application. If not specifically stated, the raw materials used in the embodiments of the present application are all commonly used raw materials in the art, and the methods used in the embodiments are all conventional methods in the art.

[0050] Example 1

[0051] (1) Preparation of streptavidin magnetic bead suspension:

[0052] Take 2 mL of streptavidin magnetic particles with a concentration of 10 mg / mL (magnetic particles from Suzhou Fuduo Biotechnology Co., Ltd., product number CMP1003SA-2), place them on a magnetic stand for solid-liquid separation, and discard the supernatant. Add 2 mL of PBST cleaning solution, mix well, and place it on a magnetic stand for solid-liquid separation, and discard the supernatant. After 3 times of cleaning, configure it into a magnetic particle suspension with a concentration of 10 mg / mL using PBS buffer containing 2% BSA and 0.1% ProClin300, and store it at 2-8°C.

[0053] (2) Preparation of biotin-coupled mouse anti-human procalcitonin monoclonal antibody 1:

[0054] Take 20 mg of mouse anti-human and peptidyl monoclonal antibody 1 (from Beijing Shunlei Technology Co., Ltd., catalog number AB1005) and dissolve it in PBS to a concentration of 5 mg / mL, pH 8.0. Weigh 20 mg of biotin-N-hydroxysuccinimide activated lipid and dissolve it in dimethyl sulfoxide to a concentration of 5 mg / mL. Mix 3 mL of mouse anti-human and peptidyl monoclonal antibody 1 solution with 150 μL of biotin-N-hydroxysuccinimide activated lipid solution and mix well on a rotary mixer at 25°C for 2 hours. After the reaction, place the reaction solution in a 3K dialysis bag and dialyze with PBS buffer for 4 hours, repeating twice. After dialysis, biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1 is obtained. Its concentration is measured using the Lowry method and stored at 2–8°C.

[0055] (3) Preparation of magnetic bead working solution:

[0056] Take 1 mL of magnetic bead suspension and add 40 μg of biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1 at a mass ratio of 1 mg:4 μg. Mix well and react for 10 min. Separate the solid and liquid phases on a magnetic rack and discard the supernatant. Add 1 mL of PBST washing buffer, suspend and mix well, and separate the solid and liquid phases on a magnetic rack again, discarding the supernatant. After washing 3 times, prepare a 0.5 mg / mL magnetic bead working solution using PBS buffer containing 2% BSA and 0.1% ProClin300, and store at 2–8℃.

[0057] (4) Preparation of mouse anti-human and peptidyl monoclonal antibody 2 coupled with alkaline phosphatase:

[0058] 20 mg of mouse anti-human and peptidyl monoclonal antibody 2 (from Beijing Shunlei Technology Co., Ltd., catalog number AB1006) was prepared into a 5 mg / mL solution using 20 mM Tris, pH 8.0 buffer. 20 mg of alkaline phosphatase was also prepared into a 5 mg / mL solution using 20 mM Tris, pH 8.0 buffer. Activators (2-IT and Sulfo-SMCC) were added to the mouse anti-human and peptidyl monoclonal antibody 2 solution and the alkaline phosphatase solution, respectively, and the reactions were carried out for 30 min. The activated mouse anti-human and peptidyl monoclonal antibody 2 solution and the activated alkaline phosphatase solution were mixed at a mass ratio of 1:2 and reacted at room temperature for 30 min. The mixture was then purified using a Superdex 200 gel filtration chromatography column with PBS as the buffer to obtain the alkaline phosphatase-conjugated mouse anti-human and peptidyl monoclonal antibody 2. Glycerol was added to a final volume of 50% and the mixture was stored at -20℃.

[0059] Example 2

[0060] (1) Preparation of streptavidin magnetic bead suspension:

[0061] Take 3 mL of streptavidin magnetic particles with a concentration of 10 mg / mL, place them on a magnetic rack for solid-liquid separation, and discard the supernatant. Add 3 mL of PBST washing buffer, suspend and mix well, place on a magnetic rack for solid-liquid separation, and discard the supernatant. After washing 3 times, prepare a magnetic particle suspension with PBS buffer containing 2% BSA and 0.1% ProClin300 to a concentration of 10 mg / mL, and store at 2-8℃.

[0062] (2) Preparation of biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1:

[0063] Take 20 mg of mouse anti-human and peptidyl monoclonal antibody 1 and dissolve it in PBS to a concentration of 5 mg / mL, pH 8.5. Weigh 20 mg of biotin-N-hydroxysuccinimide activated lipid and dissolve it in dimethyl sulfoxide to a concentration of 5 mg / mL. Mix 3 mL of mouse anti-human and peptidyl monoclonal antibody 1 solution with 60 μL of biotin-N-hydroxysuccinimide activated lipid solution and mix well on a rotary mixer at 25°C for 4 hours. After the reaction, place the reaction solution in a 3K dialysis bag and dialyze with PBS buffer for 4 hours, repeating twice. After dialysis, biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1 is obtained. Its concentration is measured using the Lowry method and stored at 2–8°C.

[0064] (3) Preparation of magnetic bead working solution:

[0065] Take 2 mL of magnetic bead suspension and add 240 μg of biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1 at a mass ratio of 1 mg:12 μg. Mix well and react for 10 min. Separate the solids and liquids on a magnetic rack and discard the supernatant. Add 2 mL of PBST washing buffer, suspend and mix well, and separate the solids and liquids on a magnetic rack again, discarding the supernatant. After washing 3 times, prepare a 1.0 mg / mL magnetic bead working solution using PBS buffer containing 2% BSA and 0.1% ProClin300, and store at 2–8℃.

[0066] (4) Preparation of mouse anti-human and peptidyl monoclonal antibody 2 coupled with alkaline phosphatase:

[0067] Prepare a 5 mg / mL solution of mouse anti-human and peptidyl monoclonal antibody 2 using 20 mM Tris, pH 8.0 buffer. Prepare a 5 mg / mL solution of alkaline phosphatase using 20 mM Tris, pH 8.0 buffer. Add activator to the mouse anti-human and peptidyl monoclonal antibody 2 solution and the alkaline phosphatase solution, respectively, and react for 30 min. Mix the activated mouse anti-human and peptidyl monoclonal antibody 2 solution and the activated alkaline phosphatase solution at a mass ratio of 1:5 and react at room temperature for 30 min. Then purify using a Superdex 200 gel filtration chromatography column with PBS as the buffer to obtain the alkaline phosphatase-conjugated mouse anti-human and peptidyl monoclonal antibody 2. Add 50% glycerol to the final volume and store at -20℃.

[0068] Example 3

[0069] (1) Preparation of streptavidin magnetic bead suspension:

[0070] Take 1 mL of streptavidin magnetic particles with a concentration of 10 mg / mL, place them on a magnetic rack for solid-liquid separation, and discard the supernatant. Add 1 mL of PBST washing buffer, suspend and mix well, place on a magnetic rack for solid-liquid separation, and discard the supernatant. After washing 3 times, prepare a 10 mg / mL magnetic particle suspension with PBS buffer containing 2% BSA and 0.1% ProClin300, and store at 2-8℃.

[0071] (2) Preparation of biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1:

[0072] 20 mg of mouse anti-human and peptidyl monoclonal antibody 1 was dissolved in PBS to a concentration of 5 mg / mL at pH 5.5. 20 mg of biotin-N-hydroxysuccinimide activated lipid was also dissolved in dimethyl sulfoxide to a concentration of 5 mg / mL. 3 mL of mouse anti-human and peptidyl monoclonal antibody 1 solution was mixed with 300 μL of biotin-N-hydroxysuccinimide activated lipid solution and stirred on a rotary mixer at 4°C for 30 hours. After the reaction, the solution was placed in a 3K dialysis bag and dialyzed against PBS buffer for 4 hours, repeating twice. The resulting biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1 was obtained. Its concentration was measured using the Lowry method and stored at 2–8°C.

[0073] (3) Preparation of magnetic bead working solution:

[0074] Take 0.4 mL of the magnetic bead suspension and add 80 μg of biotin-conjugated mouse anti-human and peptidyl monoclonal antibody 1 at a mass ratio of 1 mg:20 μg. Mix well and react for 10 min. Separate the solid and liquid phases on a magnetic rack and discard the supernatant. Add 0.4 mL of PBST washing buffer, suspend and mix well, and separate the solid and liquid phases on a magnetic rack again, discarding the supernatant. After washing 3 times, prepare a 0.2 mg / mL magnetic bead working solution using PBS buffer containing 2% BSA and 0.1% ProClin300, and store at 2–8℃.

[0075] (4) Preparation of mouse anti-human and peptidyl monoclonal antibody 2 coupled with alkaline phosphatase:

[0076] Prepare a 5 mg / mL solution of mouse anti-human and peptidyl monoclonal antibody 2 using 20 mM Tris, pH 8.0 buffer. Prepare a 5 mg / mL solution of alkaline phosphatase using 20 mM Tris, pH 8.0 buffer. Add activator to both the mouse anti-human and peptidyl monoclonal antibody 2 solution and the alkaline phosphatase solution, and react for 30 min. Mix the activated mouse anti-human and peptidyl monoclonal antibody 2 solution and the activated alkaline phosphatase solution at a mass ratio of 1:10, and react at room temperature for 30 min. Then purify using a Superdex 200 gel filtration chromatography column with PBS as the buffer to obtain the alkaline phosphatase-conjugated mouse anti-human and peptidyl monoclonal antibody 2. Add 50% glycerol to the final volume and store at -20℃.

[0077] The prepared magnetic bead working solution, mouse anti-human and peptide monoclonal antibody 2 coupled with alkaline phosphatase, and purchased washing solution (TBST) and alkaline phosphatase luminescent substrate solution (AMPPD) are added to the corresponding reagent wells of the test strip to form the test strip. The test strip is then tested using a fully automated chemiluminescence immunoassay analyzer from Wuhan BGI Genomics Co., Ltd. During testing, the sample is added to the sample well, the test strip is placed on the instrument, and the test is automatically completed.

[0078] The kits obtained in steps 1, 2, and 3 were used to test the standard curve solutions at concentrations of 0, 5, 25, 50, 250, and 500 pg / mL, with each concentration tested three times and the average value taken. A standard curve was constructed using a four-parameter fitting method, with concentration values ​​on the x-axis and the average luminescence value on the y-axis. A standard solution at a concentration of 5 pg / mL was then tested 20 times. Based on the standard curve, the recovery rate of the 5 pg / mL standard solution was calculated. A recovery rate between 90% and 110% indicates that the sensitivity reaches 5 pg / mL; a recovery rate outside this range indicates that the sensitivity does not reach 5 pg / mL.

[0079] The results of the test standard curve solution and the test concentration of 5 pg / mL standard solution prepared in Example 1 are shown in Table 1 and Table 2, respectively. The four-parameter fitting correlation coefficient r of the test standard is 0.9973, and the recovery rate of the test concentration of 5 pg / mL standard is in the range of 90%-110%, indicating that the sensitivity of the kit prepared in Example 1 can reach 5 pg / mL.

[0080] The results of the test standard curve solution and the test concentration of 5 pg / mL standard solution prepared in Example 2 are shown in Tables 3 and 4, respectively. The correlation coefficient r of the four-parameter fitting of the test standard is 0.9961, and the recovery rate of the test concentration of 5 pg / mL standard is 82.2%, which is not in the range of 90%-110%, indicating that the sensitivity of the kit prepared in Example 2 does not reach 5 pg / mL.

[0081] The results of the test standard curve solution and the test concentration of 5 pg / mL standard solution prepared in Example 3 are shown in Tables 5 and 6, respectively. The correlation coefficient r of the four-parameter fitting of the test standard is 0.9990, and the recovery rate of the test concentration of 5 pg / mL standard is 85.6%, which is not in the range of 90%-110%, indicating that the sensitivity of the kit prepared in Example 3 does not reach 5 pg / mL.

[0082] Table 1 Results of test standard curve solution for kit in Example 1

[0083]

[0084] Table 2. Results of test using kit 1 with a standard solution concentration of 5 pg / mL

[0085]

[0086]

[0087] Table 3 Results of the standard curve solution test using the kit in Implementation 2

[0088]

[0089] Table 4. Results of test using kit 2 with a standard solution concentration of 5 pg / mL

[0090]

[0091]

[0092] Table 5 Results of reagent kit test standard curve solution in Implementation 3

[0093]

[0094] Table 6. Results of test using kit 3 with a standard solution concentration of 5 pg / mL

[0095]

[0096]

[0097] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A peptide detection strip or kit, characterized in that, The reagent strip or kit contains magnetic microparticle working solution and peptide monoclonal antibody 2 conjugated with alkaline phosphatase; The magnetic microparticle working solution consists of streptavidin magnetic microparticles and biotin-conjugated peptide monoclonal antibody 1.

2. The reagent strip or reagent kit according to claim 1, characterized in that, The conjugated alkaline phosphatase and peptidin monoclonal antibody 2 and the conjugated biotin and peptidin monoclonal antibody 1 bind to different epitopes of human peptidin. Preferably, the concentration of the magnetic microparticle working solution is 0.2 mg / mL to 1.0 mg / mL; Preferably, the concentration of the magnetic microparticle working solution is 0.5 mg / mL.

3. The reagent strip or reagent kit according to claim 1, characterized in that, The preparation method of the magnetic microparticle working fluid includes the following steps: After washing the streptavidin magnetic microparticles, they were prepared into a magnetic microparticle suspension using a buffer solution. Heptacin monoclonal antibody 1 was reacted with biotin-N-hydroxysuccinimide activated lipid at a mass ratio of 50:1 to 10:1 at 4℃ to 30℃ and pH 6.0 to 8.0 for 0.5 h to 36 h. After the reaction, excess biotin-N-hydroxysuccinimide activated lipid was removed by dialysis to obtain biotin-conjugated Heptacin monoclonal antibody 1. The magnetic microparticle suspension was mixed with biotin-conjugated and peptidyl monoclonal antibody 1 at a mass ratio of 1 mg:1 μg to 1 mg:10 μg. After washing, the mixture was prepared into a magnetic microparticle working solution with a concentration of 0.2 mg / mL to 1.0 mg / mL using buffer.

4. The reagent strip or reagent kit according to claim 3, characterized in that, The concentration of the magnetic particle suspension is 10 mg / mL; Preferably, the peptide monoclonal antibody 1 and biotin-N-hydroxysuccinimide activated lipid are reacted at a mass ratio of 20:1 at 25°C and pH 8.0 for 2 hours. Preferably, the concentration of the magnetic microparticle working solution is 0.5 mg / mL; Preferably, the buffer solution is a PBS buffer containing 2% BSA and 0.1% ProClin300.

5. The reagent strip or reagent kit according to claim 1, characterized in that, The preparation method of the conjugated alkaline phosphatase and peptidyl monoclonal antibody 2 includes the following steps: The activated glutathione 2 was mixed with activated alkaline phosphatase at a mass ratio of 1:1 to 1:10 and purified using a gel filtration chromatography column to obtain glutathione 2 conjugated with alkaline phosphatase.

6. The reagent strip or reagent kit according to claim 1, characterized in that, The activated glutathione 2 was mixed with activated alkaline phosphatase at a mass ratio of 1:2 and reacted for 30 min. The mixture was then purified using a gel filtration chromatography column to obtain glutathione 2 conjugated with alkaline phosphatase.

7. The reagent strip or reagent kit according to claim 1, characterized in that, The reagent strips or kits also include chemiluminescent substrate solutions; Preferably, the kit also includes peptide standards.

8. The reagent strip or reagent kit according to claim 7, characterized in that, The peptide standard is a diluent used to dissolve and dilute peptides into peptide solutions of 4 to 8 series concentrations. Preferably, the kit further includes peptide quality control products.

9. A method for quantitative detection of peptides, characterized in that, The method includes the step of detecting the sample to be tested using components in the reagent strip or kit of claim 1.

10. The method according to claim 9, characterized in that, The samples to be tested include plasma, serum, cerebrospinal fluid, urine, saliva, tissue homogenate and / or cell culture supernatant; Preferably, the method further includes the steps of detecting gradient concentrations and preparing a standard curve for peptide standards, and calculating the concentrations of the sample to be tested and peptides based on the standard curve.

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