CN112526121B

CN112526121B - Serum amyloid A detection kit and detection system thereof

Info

Publication number: CN112526121B
Application number: CN202011511617.1A
Authority: CN
Inventors: 刘先成; 李永志; 皮小武; 徐良; 曾映; 王铮
Original assignee: Lifotronic Technology Co ltd
Current assignee: Lifotronic Technology Co ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2022-11-18
Anticipated expiration: 2040-12-18
Also published as: CN112526121A

Abstract

The invention relates to a serum amyloid A detection kit and a detection system thereof. The serum amyloid A detection kit comprises a first antibody liquid, a second antibody liquid and magnetic particle working liquid, wherein the first antibody liquid comprises a first marker labeled serum amyloid A antibody, bovine serum albumin, inorganic salt, sugar, surfactant and HEPES buffer solution, and the second antibody liquid comprises a second marker labeled serum amyloid A antibody, bovine serum albumin, inorganic salt, sugar, surfactant and HEPES buffer solution. The serum amyloid A detection kit can be used for quantitatively detecting serum amyloid A and is high in sensitivity.

Description

Serum amyloid A detection kit and detection system thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to a serum amyloid A detection kit and a detection system thereof.

Background

Serum Amyloid A (SAA), a precursor of tissue amyloid a, was isolated and identified from serum in 1976 as a highly conserved acute phase protein family member. SAA, like C-reactive protein (CRP), increases rapidly in levels after an organism is injured by infection, trauma, inflammation, etc. In recent years, with the intensive research on gene regulation, protein structure and biological function of SAA, it has been found that SAA is also involved in the pathogenesis of chronic inflammatory diseases.

The human SAA-encoding gene is clustered in the p15.1 region of chromosome 11 and is 150kb in size. The SAA family includes acute phase SAA (A-SAA) encoded by SAA1 and SAA2, constitutive SAA (C-SAA) encoded by SAA4, and epigenoid encoded by SAA 3. The nucleotides of SAA1 and SAA2 are 90% homologous, while SAA3 is about 60% homologous to the nucleotides of A-SAA. Except that the SAA3 gene has a3 exon and 2 intron structure, all other genes contain 4 exons and 3 introns. SAA1 and SAA2 genes are 15-20 kb in size, the promoter regions of the SAA1 and SAA2 genes contain recognition sequences of transcription factors of kernel factor-kB (NF-kB) and interleukin-6 (IL-6), are binding sites of RAS activating factors and can be induced and activated by interleukin-1 beta (IL-1 beta), IL-6 and Tumor Necrosis Factor (TNF); SAA3 is located at the downstream of SAA4 gene and has the size of 110kb; the SAA4 gene encodes C-SAA, is located only 9kb downstream of the SAA2 gene, and its promoter region includes only a truncated NF-. Kappa.B recognition sequence.

Human A-SAA is synthesized primarily in the liver, induced by cytokines IL-1 β, IL-6 and tumor necrosis factor- α (TNF- α), which act synergistically with glucocorticoids to enhance A-SAA production.

A particularly important property of SAA is that its degradation products can be deposited in different organs in the manner of amyloid a fibrils, which is a serious complication in chronic inflammatory diseases; serum amyloid A elevation is also seen in atherosclerosis, diabetic nephropathy, acute myocardial infarction, coronary heart disease, chronic kidney disease and other diseases.

At present, the detection method of SAA mainly comprises a colloidal gold method and an immunoturbidimetry method. The colloidal gold method mainly uses physical properties of colloidal gold, such as high electron density, particle size, shape and color reaction, and immunological and biological properties of the conjugate. Although the colloidal gold method is convenient and fast, the colloidal gold method can only be used for qualitative immunodetection at present. The detection principle of the immunoturbidimetry is that antigen and antibody quickly form an antigen-antibody complex in a special buffer solution, so that the reaction solution has turbidity. When the antibody in the reaction liquid is excessive, the formed complex increases along with the increase of the antigen amount, the turbidity of the reaction liquid also increases, and then the content of the detected object can be calculated by comparing with a series of standard products. The immune turbidimetry is used for detecting the SAA, and the sensitivity is lower.

Disclosure of Invention

Accordingly, it is necessary to provide a serum amyloid a detection kit which can quantitatively detect serum amyloid a and has high sensitivity.

A serum amyloid A detection kit comprises a first antibody liquid, a second antibody liquid and a magnetic particle working solution, wherein:

the first antibody liquid comprises a first marker labeled serum amyloid A antibody, bovine serum albumin, inorganic salt, sugar, a surfactant and a HEPES buffer solution, wherein in the first antibody liquid, the inorganic salt is selected from at least one of sodium chloride and potassium chloride, the concentration of the first marker labeled serum amyloid A antibody is 0.5-1.5 mg/L, the mass percentage of the bovine serum albumin is 0.5-5%, the mass percentage of the inorganic salt is 0.5-5%, the mass percentage of the sugar is 1-5%, the mass percentage of the surfactant is 0.02-1%, and the pH value of the first antibody liquid is 6.0-7.6;

the second antibody liquid comprises a second marker labeled serum amyloid A antibody, bovine serum albumin, inorganic salt, sugar, a surfactant and a HEPES buffer solution, wherein in the second antibody liquid, the inorganic salt is selected from at least one of sodium chloride and potassium chloride, the concentration of the second marker labeled serum amyloid A antibody is 0.5 mg/L-1.5 mg/L, the mass percentage of the bovine serum albumin is 0.5-5%, the mass percentage of the inorganic salt is 0.5-5%, the mass percentage of the sugar is 1-5%, the mass percentage of the surfactant is 0.02-1%, the pH of the second antibody liquid is 6.0-7.6, and the second marker is a chemiluminescent marker;

the magnetic particle working solution comprises magnetic particles coupled with avidin and a buffer solution, and the third marker can be specifically combined with the first marker.

The buffer solutions in the first antibody solution and the second antibody solution in the serum amyloid A detection kit are HEPES buffer solutions. Through the mutual matching of the HEPES buffer solution, bovine serum albumin, inorganic salt, sugar and surfactant, the serum amyloid A detection kit can quantify serum amyloid A, and is high in sensitivity and wide in detection range.

In one embodiment, the surfactant in the first antibody fluid and the surfactant in the second antibody fluid are each independently selected from at least one of dodecyl polyethylene glycol, polyethylene glycol p-isooctyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, and polyoxyethylene lauryl ether.

In one embodiment, the sugar in the first antibody fluid and the sugar in the second antibody fluid are each independently selected from at least one of sucrose and trehalose;

in one embodiment, the concentration of HEPES in the HEPES buffer in the first antibody solution is 0.005mol/L to 0.2mol/L.

In one embodiment, the concentration of HEPES in the HEPES buffer solution in the second antibody solution is 0.005 mol/L-0.2mol/L.

In one embodiment, the surfactant in the first antibody solution is polyoxyethylene sorbitan monolaurate, and the mass ratio of the HEPES to the polyoxyethylene sorbitan monolaurate in the first antibody solution is (0.24-95.2): 1, the mass ratio of HEPES to the inorganic salt is (0.048-4.76): 1, the mass ratio of bovine serum albumin to sugar is (0.5-1): 1.

in one embodiment, the surfactant in the second antibody liquid is polyoxyethylene sorbitan monolaurate, and the mass ratio of the HEPES to the polyoxyethylene sorbitan monolaurate in the second antibody liquid is (0.24-95.2): 1, the mass ratio of HEPES to inorganic salt is (0.048-4.76): 1, the mass ratio of bovine serum albumin to sugar is (0.5-1): 1.

in one embodiment, the magnetic particle working solution further comprises a buffer selected from at least one of phosphate buffer, tris buffer, HEPES buffer, and MOPSO buffer.

In one embodiment, the first label is biotin and the third label is avidin; and/or the second marker is ruthenium terpyridyl, acridine ester, alkaline phosphatase or horseradish peroxidase.

In one embodiment, the test kit further comprises at least one of a sample diluent, a quality control material and a calibrator.

In one embodiment, the sample diluent comprises an inorganic salt, sugar, a surfactant and a buffer, wherein in the sample diluent, the mass percentage of the inorganic salt is 0.5-5%, the mass percentage of the sugar is 1-5%, and the mass percentage of the surfactant is 0.02-1%.

In one embodiment, the surfactant in the sample diluent is selected from at least one of dodecyl polyethylene glycol, polyethylene glycol p-isooctyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, and polyoxyethylene lauryl ether.

In one embodiment, the first antibody liquid further contains a preservative, and the preservative in the first antibody liquid is 0.05-0.5% by mass;

in one embodiment, the second antibody liquid further contains a preservative, and the preservative in the second antibody liquid is 0.05-0.5% by mass;

in one embodiment, the magnetic particle working solution further contains a preservative, and the preservative in the magnetic particle working solution is 0.05-0.5% by mass.

A serum amyloid a detection system comprising:

a detection device; and

the serum amyloid A detection kit.

Drawings

FIG. 1 is a graph showing the relationship between the concentration of serum amyloid A and its signal value in a positive clinical specimen detected by the serum amyloid A detection kit of example 1;

FIG. 2 is a graph showing the relationship between the concentration of serum amyloid A and its signal value in a positive clinical specimen detected by the serum amyloid A detection kit of comparative example 1;

FIG. 3 is a graph showing the relationship between the concentration of serum amyloid A and the signal value measured by the serum amyloid A detection kit according to example 1, when the concentration of serum amyloid A is 0.20. Mu.g/mL-452.10. Mu.g/mL.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It should be noted that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Unless otherwise specified, the polyoxyethylene octylphenyl ether is TritonX-100, the polyethylene glycol is PEG40000, the polyoxyethylene sorbitan monolaurate is Tween-20, the polyoxyethylene sorbitan monooleate is Tween-80, and the cinnamyl alcohol polyoxyethylene ether is Brij35.

One embodiment of the present invention provides a serum amyloid a detection kit, which detects the content of serum amyloid a in a sample to be detected by detecting a formed serum amyloid a antibody-serum amyloid a complex. The serum of the serum amyloid A detection kit comprises a first antibody liquid, a second antibody liquid and a magnetic particle working solution.

Specifically, the first antibody solution includes a first marker-labeled serum amyloid a antibody, bovine serum albumin, an inorganic salt, a sugar, a surfactant, and a HEPES buffer. The serum amyloid A antibody marked by the first marker can be combined with the serum amyloid A antibody and can also be combined with the magnetic particles coupled with the third marker in the magnetic particle working solution. In the first antibody solution, the surfactant and the HEPES buffer solution have a synergistic effect, and the sensitivity of the serum amyloid A detection kit can be remarkably improved. In addition, the bovine serum albumin and the sugar have the function of maintaining the stability of the structure of the serum amyloid A antibody marked by the first marker, and are beneficial to prolonging the effective period of the serum amyloid A detection kit.

In one embodiment, the first label is biotin, i.e., the first label-labeled serum amyloid a antibody is a biotin-labeled serum amyloid a antibody. In this case, the method for producing a serum amyloid a antibody labeled with a first marker in a first antibody solution comprises the steps of: reacting N-hydroxysuccinimide activated biotin with serum amyloid A antibody according to the following formula (1-20): 1, reacting for 0.5-2 hours at 2-8 ℃ or room temperature (20-26 ℃), and dialyzing or passing through a column to remove redundant biotin to obtain the biotin-labeled serum amyloid A antibody. Of course, biotin-labeled serum amyloid a antibodies can also be prepared by other methods known in the art.

In the first antibody liquid, the inorganic salt is selected from at least one of sodium chloride and potassium chloride, the sugar is selected from at least one of sucrose and trehalose, the concentration of the serum amyloid A antibody marked by the first marker is 0.5-1.5 mg/L, the mass percentage of bovine serum albumin is 0.5-5%, the mass percentage of the inorganic salt is 0.5-5%, the mass percentage of the sugar is 1-5%, the mass percentage of the surfactant is 0.02-1%, and the pH of the first antibody liquid is 6.0-7.6. When the pH of the first antibody liquid is 6.0-7.6, the reactivity of the serum amyloid A antibody marked by the first marker is optimal, which is beneficial to improving the sensitivity of the reagent.

Optionally, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) in the HEPES buffer solution is 0.005mol/L to 0.2mol/L in the first antibody solution, and further the concentration of HEPES in the HEPES buffer solution is 0.01mol/L to 0.2mol/L in the first antibody solution. Further, in the first antibody solution, the concentration of HEPES in the HEPES buffer solution is 0.01mol/L to 0.1mol/L.

More specifically, the surfactant is selected from at least one of dodecyl polyethylene glycol, polyethylene glycol octyl phenyl ether (TritonX-100), polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene sorbitan monooleate (Tween-80), and polyoxyethylene lauryl ether. Further, the surfactant is polyoxyethylene sorbitan monolaurate. The synergistic effect of the polyoxyethylene sorbitan monolaurate and the HEPES buffer solution is better and obvious, and the sensitivity of the detection kit can be obviously improved.

In one embodiment, in the first antibody solution, the concentration of the first marker-labeled serum amyloid A antibody is 0.5mg/L to 1.5mg/L, the mass percentage of bovine serum albumin is 0.5% to 2%, the mass percentage of inorganic salt is 0.5% to 3%, the mass percentage of sugar is 1% to 5%, the mass percentage of surfactant is 0.1% to 1%, and the concentration of HEPES in the HEPES buffer solution is 0.01mol/L to 0.05mol/L; the pH of the first antibody liquid is 6.0 to 7.6. Further, in the first antibody liquid, the concentration of the serum amyloid A antibody marked by the first marker is 0.8-1.2 mg/L, the mass percentage of bovine serum albumin is 0.8-1.5%, the mass percentage of inorganic salt is 0.5-2%, the mass percentage of sugar is 2-4%, the mass percentage of surfactant is 0.02-0.1%, and the concentration of HEPES in a HEPES buffer solution is 0.01-0.1 mol/L; the pH of the first antibody liquid is 6.5 to 7.4.

In one embodiment, in the first antibody solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-95.2): 1, the mass ratio of HEPES to the inorganic salt is (0.048-4.76): 1, the mass ratio of bovine serum albumin to sugar is (0.5-1): 1. the proportion of HEPES, polyoxyethylene sorbitan monolaurate, inorganic salt, bovine serum albumin and sugar is set according to the above, so that the reactivity of the antibody working solution can reach a better state, and the sensitivity and stability of the reagent are improved. Further, in the first antibody solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-47.6): 1, the mass ratio of HEPES to inorganic salt is (0.067-2.38): 1, the mass ratio of bovine serum albumin to sugar is (0.6-0.9): 1.

Further, in the first antibody solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-4.8): 1, the mass ratio of HEPES to inorganic salt is (0.1-2): 1, the mass ratio of bovine serum albumin to sugar is (0.7-0.8): 1.

in one embodiment, the first antibody solution consists of first marker-labeled serum amyloid a antibody, bovine serum albumin, an inorganic salt, a sugar, a surfactant, and a HEPES buffer, except for inevitable impurities, wherein the first marker-labeled serum amyloid a antibody, bovine serum albumin, an inorganic salt, a sugar, a surfactant, and a HEPES buffer are each as described above in composition and proportion.

Specifically, the second antibody solution includes a second marker-labeled serum amyloid a antibody, bovine serum albumin, an inorganic salt, a sugar, a surfactant, and a HEPES buffer. The serum amyloid A antibody labeled with the second label can be combined with the serum amyloid A antibody, and the second label carried by the antibody is a label of chemiluminescence reaction and can participate in chemiluminescence reaction (for example, the second label is used as a substrate of the chemiluminescence reaction or catalyzes the substrate to generate chemiluminescence reaction) to be detected. In the second antibody solution, the surfactant and the HEPES buffer solution have a synergistic effect, and the sensitivity of the serum amyloid A detection kit can be remarkably improved. In addition, the bovine serum albumin and the sugar are used for stabilizing the structure of the labeled serum amyloid A antibody, so that the validity period of the serum amyloid A detection kit can be prolonged.

In one embodiment, the second marker is an acridine compound-NHS or Ru-NHS ester. In this case, the method for producing a serum amyloid a antibody labeled with a second marker in a second antibody solution comprises the steps of: mixing a serum amyloid A antibody with the pH of 6.5-8.0 and an acridine compound-NHS or Ru-NHS ester solution according to the mass ratio of (1-30): 1, reacting for 1-3 hours at 37 ℃ in a dark place, then adding glycine solution to terminate the reaction, dialyzing the reaction solution to remove redundant acridine compound-NHS or Ru-NHS ester, and obtaining the acridine compound or ruthenium terpyridyl labeled serum amyloid protein A antibody. Of course, labeled serum amyloid A antibodies can also be prepared by other methods.

In the second antibody liquid, the inorganic salt is selected from at least one of sodium chloride and potassium chloride, the sugar is selected from at least one of sucrose and trehalose, the concentration of the serum amyloid A antibody marked by the second marker is 0.5-1.5 mg/L, the mass percentage of bovine serum albumin is 0.5-5%, the mass percentage of the inorganic salt is 0.5-5%, the mass percentage of the sugar is 1-5%, the mass percentage of the surfactant is 0.02-1%, and the pH of the second antibody liquid is 6.0-7.6. When the pH value of the second antibody liquid is 6.0-7.6, the reactivity of the serum amyloid A antibody marked by the second marker is optimal, which is beneficial to improving the sensitivity of the reagent.

Optionally, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) in the HEPES buffer solution is 0.005mol/L to 0.2mol/L in the second antibody solution, and further, the concentration of HEPES in the HEPES buffer solution is 0.01mol/L to 0.2mol/L in the second antibody solution. Further, in the second antibody solution, the concentration of HEPES in the HEPES buffer solution is 0.01mol/L to 0.1mol/L.

Specifically, the surfactant is selected from at least one of dodecyl polyethylene glycol, polyethylene glycol octyl phenyl ether (TritonX-100), polyethylene glycol (such as PEG 40000), polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene sorbitan monooleate (Tween-80) and polyoxyethylene lauryl ether (Brij 35). Further, the surfactant is polyoxyethylene sorbitan monolaurate. The synergistic effect of the polyoxyethylene sorbitan monolaurate and the HEPES buffer solution is better and obvious, and the sensitivity of the detection kit can be obviously improved.

In one embodiment, in the second antibody solution, the concentration of the serum amyloid A antibody marked by the second marker is 0.5-1.5 mg/L, the mass percentage of bovine serum albumin is 0.5-2%, the mass percentage of inorganic salt is 0.5-3%, the mass percentage of sugar is 1-5%, the mass percentage of surfactant is 0.1-1%, and the concentration of HEPES in the HEPES buffer solution is 0.01-0.05 mol/L; the pH of the second antibody liquid is 6.0 to 7.6. Further, in the second antibody solution, the concentration of the serum amyloid A antibody marked by the second marker is 0.8-1.2 mg/L, the mass percentage of bovine serum albumin is 0.8-1.5%, the mass percentage of inorganic salt is 0.5-2%, the mass percentage of sugar is 2-4%, the mass percentage of surfactant is 0.02-0.1%, and the concentration of HEPES in the HEPES buffer solution is 0.01-0.1 mol/L; the pH of the second antibody solution is 6.5 to 7.4.

In one embodiment, in the second antibody solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-95.2): 1, the mass ratio of HEPES to inorganic salt is (0.048-4.76): 1, the mass ratio of bovine serum albumin to sugar is (0.5-1): 1. according to the proportion of HEPES, polyoxyethylene sorbitan monolaurate, inorganic salt, bovine serum albumin and sugar, the reaction activity of the serum amyloid A antibody working solution marked by the second marker can reach a better state, and the sensitivity and stability of the reagent are improved. Further, in the second antibody solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-47.6): 1, the mass ratio of HEPES to the inorganic salt is (0.067-2.38): 1, the mass ratio of bovine serum albumin to sugar is (0.6-0.9): 1.

Further, in the second antibody solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24 to 4.8): 1, the mass ratio of HEPES to inorganic salt is (0.1-2): 1, the mass ratio of bovine serum albumin to sugar is (0.7-0.8): 1.

in one embodiment, the second antibody solution consists of, in addition to unavoidable impurities, a second marker-labeled serum amyloid a antibody, bovine serum albumin, an inorganic salt, a sugar, a surfactant, and a HEPES buffer, wherein the second marker-labeled serum amyloid a antibody, bovine serum albumin, an inorganic salt, a sugar, a surfactant, and a HEPES buffer are each as described above in composition and proportion.

In one embodiment, the composition and content of the components other than the antibody in the first antibody liquid and the second antibody solution are the same. Further, in the first antibody solution, the inorganic salt is sodium chloride, the sugar is sucrose, and the surfactant is polyoxyethylene sorbitan monolaurate; in the second antibody fluid, the inorganic salt is also sodium chloride, the sugar is also sucrose, and the surfactant is also polyoxyethylene sorbitan monolaurate.

In the present embodiment, the serum amyloid a detection kit is a detection kit applied to an electrochemiluminescence platform, and the detection of serum amyloid a is performed by using the principle of a double antibody sandwich method (that is, when used, serum amyloid a in a sample to be detected is simultaneously bound to a serum amyloid a antibody labeled with a first marker in a first antibody liquid and a serum amyloid a antibody labeled with a second marker in a second antibody liquid). Thus, the epitope of the serum amyloid a antibody in the first antibody fluid and the serum amyloid a antibody in the second antibody fluid are different. Of course, in other embodiments, if a competitive method is used to detect serum amyloid a, the epitope of the serum amyloid a antibody in the first antibody fluid is the same as the epitope of the serum amyloid a antibody in the second antibody fluid. In an alternative embodiment, the serum amyloid a antibody is a monoclonal antibody, wherein the monoclonal antibody Fab fragment is preferably from mouse, rabbit, sheep.

In addition, the serum amyloid A detection kit is suitable for an electrochemical luminescence platform. Therefore, in the present embodiment, the second marker is ruthenium terpyridyl. It is understood that the labeling substance of the antibody in the second antibody solution is not limited to ruthenium terpyridyl, and may be other substances as long as it is suitable for the electrochemiluminescence platform. Of course, in other embodiments, the second marker may be selected based on the platform for which the test kit is specifically adapted. For example, when the serum amyloid A detection kit is suitable for a direct luminescence immunoassay platform, the second label may be an acridinium ester. When the serum amyloid a detection kit is suitable for a chemiluminescent enzyme immunoassay platform, the second label may be selected from horseradish peroxidase (HRP) or alkaline phosphatase (ALP).

Specifically, the magnetic particle working solution includes a magnetic particle to which a third label is coupled and a buffer. The magnetic particles coupled with the third marker are used for combining with the serum amyloid A antibody marked by the first marker in the first antibody liquid so as to fix an antibody-antigen-antibody complex formed after the reaction of the first antibody liquid, the second antibody liquid and the sample to be tested. Thus, the third label is capable of specifically binding to the first label. In an alternative specific example, the first label is biotin and the third label is avidin. Further, the third label is streptavidin. That is, the magnetic particles to which the third label is coupled are magnetic particles to which streptavidin is coupled. Of course, in other embodiments, the first label and the third label are not limited to the above, as long as the first label and the third label can specifically bind to each other, so that the serum amyloid a antibody labeled with the first label can be bound to the magnetic particles and immobilized.

Optionally, the buffer in the magnetic particle working solution is selected from at least one of phosphate buffer, tris buffer, HEPES buffer and MOPSO buffer. Of course, it is understood that, in other embodiments, the buffer solution in the magnetic particle working solution is not limited to the above, and may be other buffer solutions.

Optionally, the pH of the magnetic particle working solution is 6.5 to 7.6. In the pH range, the binding capacity of the magnetic particles and the antigen-antibody complex is stronger, which is beneficial to improving the correlation between the concentration of the test sample and the signal value. Further, the pH of the magnetic particle working solution is 6.8 to 7.4.

In some embodiments, the magnetic particle working solution comprises magnetic particles coupled with a third label, bovine serum albumin, casein, sugar, a surfactant, and HEPES buffer.

Specifically, in the magnetic particle working solution, the inorganic salt is at least one selected from sodium chloride and potassium chloride; the sugar is at least one selected from sucrose and trehalose; the surfactant is at least one selected from dodecyl polyethylene glycol, polyethylene glycol octyl phenyl ether (TritonX-100), polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene sorbitan monooleate (Tween-80) and polyoxyethylene lauryl ether. Further, the surfactant is polyoxyethylene sorbitan monolaurate. The synergistic effect of the polyoxyethylene sorbitan monolaurate and the HEPES buffer solution is better and obvious, and the sensitivity of the detection kit can be obviously improved.

Specifically, in the magnetic particle working solution, the mass percentage content of bovine serum albumin is 0.5% -5%; the mass percentage content of the casein is 0.02-1%; the mass percentage of the sugar is 0.5-5%, and the mass percentage of the surface active agent is 0.02-1%.

Optionally, the HEPES buffer solution has a HEPES concentration of 0.005mol/L to 0.2mol/L, and further, the HEPES buffer solution has a HEPES concentration of 0.01mol/L to 0.2mol/L in the magnetic fine particle working solution. Furthermore, the concentration of HEPES in the HEPES buffer solution is 0.01mol/L to 0.1mol/L.

In one embodiment, in the magnetic particle working solution, the mass ratio of the HEPES to the polyoxyethylene sorbitan monolaurate is (0.24-95.2): 1, the mass ratio of HEPES to inorganic salt is (0.048-4.76): 1, the mass ratio of bovine serum albumin to sugar is (0.5-1): 1. furthermore, in the magnetic particle working solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-47.6): 1, the mass ratio of HEPES to the inorganic salt is (0.067-2.38): 1, the mass ratio of bovine serum albumin to sugar is (0.6-0.9): 1.

Furthermore, in the magnetic particle working solution, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-4.8): 1, the mass ratio of HEPES to inorganic salt is (0.1-2): 1, the mass ratio of bovine serum albumin to sugar is (0.7-0.8): 1.

in some embodiments, the magnetic particle working solution further comprises a preservative. Furthermore, the preservative in the working solution of the magnetic particles is 0.05-0.5% by mass. Optionally, the preservative in the magnetic particle working solution is at least one of proclin 300 and sodium azide. In an alternative specific example, the preservative in the magnetic particle working solution is proclin 300. The proclin 300 is selected as the preservative of the magnetic particle working solution, so that the preservative effect can be achieved, and the sensitivity influence on the serum amyloid A detection kit is small.

In some embodiments, the serum amyloid a detection kit further comprises at least one of a sample diluent, a quality control material and a calibrator.

Specifically, the sample diluent is used to dilute the sample to be tested. The concentration of serum amyloid A in a human body is high, and the HOOK effect is easy to appear by direct test, so that a false low-point result is caused. The sample to be detected is diluted by the sample diluent for multiple times, the HOOK effect of serum amyloid A detection can be greatly eliminated under a certain dilution multiple (25-200 times), and the linearity of the test result is good in a large range, and the accuracy is good. Tests prove that the detection range of the serum amyloid A detection kit is superior to that of other serum amyloid A detection kits in the current market.

More specifically, the sample diluent includes inorganic salts, sugars, surfactants, and buffers. In the sample diluent, the inorganic salt is selected from at least one of sodium chloride and potassium chloride, the sugar is selected from at least one of sucrose and trehalose, the mass percentage of the inorganic salt is 0.5-5%, the mass percentage of the sugar is 1-5%, the mass percentage of the surfactant is 0.02-1%, and the pH value of the sample diluent is 6.0-7.6.

Optionally, the buffer in the sample dilution is selected from at least one of phosphate buffer, tris buffer, HEPES buffer, MES buffer, and citrate buffer. In an alternative specific example, the solute concentration in the buffer of the sample diluent is between 0.01mol/L and 0.2mol/L. Furthermore, the buffer solution in the sample diluent is HEPES buffer solution, and the concentration of HEPES in the HEPES buffer solution is 0.01mol/L-0.2mol/L. When the buffer solution in the sample diluent is also HEPES buffer solution, the sensitivity of the detection kit is improved. Of course, in other embodiments, the buffer in the sample diluent is not limited to the above, and may be another buffer.

Optionally, the surfactant in the sample diluent is selected from at least one of dodecyl polyethylene glycol, polyethylene glycol p-isooctyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, and polyoxyethylene lauryl ether. Further, the surfactant is polyoxyethylene sorbitan monolaurate. Of course, in other embodiments, the surfactant in the sample diluent is not limited to the above, and may be other substances that can be used as a surfactant.

It is understood that in some embodiments, the sample diluent may be free of inorganic salts, sugars, surfactants. In particular, the sample diluent is a common buffer. For example, phosphate buffer, tris buffer, HEPES buffer, MES buffer or citric acid buffer.

The quality control product comprises negative control product and positive control product.

Optionally, the negative control is a buffer or normal human serum. In some embodiments, the negative control further comprises 10wt% to 50wt% calf serum, 0.5wt% to 10wt% ethylene glycol, 0.05wt% to 0.5wt% proclin 300, and 0.02wt% to 1wt% surfactant, wherein the surfactant is selected from at least one of Tween-20, triton X-100, and Brij35.

The positive control contains serum amyloid A with the concentration of 160 mg/L-240 mg/L. In this embodiment, the serum amyloid a in the positive control is a recombinant protein produced by escherichia coli. Furthermore, the serum amyloid A in the positive control was a recombinant protein produced by commercially available E.coli. Specifically, the positive control includes serum amyloid a and buffer. The buffer solution in the positive control is at least one selected from phosphate buffer solution, tris buffer solution, HEPES buffer solution, MES buffer solution and citric acid buffer solution. Further, the pH value of the positive control is 6.0-7.6; the concentration of solute in the buffer solution of the negative control is 0.01mol/L-0.2mol/L. It is understood that in other embodiments, the buffer of the positive control is not limited to the above, and may be other buffers or normal human serum. In some embodiments, the positive control further comprises 10wt% to 50wt% calf serum, 0.5wt% to 10wt% ethylene glycol, 1wt% to 8wt% polysaccharide, 0.05wt% to 0.5wt% proclin 300, and 0.02wt% to 1wt% surfactant, wherein the surfactant is selected from at least one of Tween-20, triton X-100, and Brij 35; the polysaccharide is at least one selected from trehalose and sucrose. Wherein wt% represents mass percentage content.

The composition of the calibrator was about the same as that of the positive control, except that the concentration of serum amyloid A in the calibrator was 10. Mu.g/mL-300. Mu.g/mL.

In some embodiments, the first antibody fluid further comprises a preservative. Specifically, the mass percentage of the preservative in the first antibody solution is 0.05-0.5%. Further, the preservative in the first antibody fluid is selected from at least one of proclin 300 and sodium azide. In an alternative specific example, the preservative in the first antibody fluid is proclin 300. The proclin 300 is selected as the preservative of the first antibody liquid, so that the preservative effect can be achieved, and the sensitivity influence on the serum amyloid A detection kit is small.

In some embodiments, the second antibody fluid further comprises a preservative. Specifically, the preservative in the second antibody solution is 0.05-0.5% by mass. Further, the preservative in the second antibody solution is at least one selected from proclin 300 and sodium azide. In an alternative specific example, the preservative in the second antibody fluid is proclin 300. Proclin 300 is selected as a preservative of the second antibody solution, so that the preservative effect can be achieved, and the influence on the sensitivity of the serum amyloid A detection kit is small.

The serum amyloid A detection kit at least has the following advantages:

(1) Compared with the traditional chemiluminescence technology such as acridinium ester, isoluminol, spiral adamantane and the like, the method has the advantages that the starting is not uniform, the luminous signal is unstable, the electrochemical luminescence system is glow, the reaction is started through voltage, the problem of nonuniform reaction caused by reagent addition or mixing can be effectively solved, the reaction is ensured to be carried out stably and controllably, the continuous and stable optical signal is provided, the sensitivity and the accuracy of the SAA test are greatly improved through the cyclic amplification of the photomultiplier, and the method can be applied to the early detection of inflammatory infection.

(2) The quantitative serum amyloid protein A detection kit is suitable for a full-automatic chemical luminescence system, can be used for experimental automation of steps such as sample adding, incubation, cleaning and detection, avoids result deviation caused by manual operation, improves working efficiency, can test serum or plasma of a patient only by internally arranging a calibration curve to test software, can finish quantitative detection of serum amyloid protein A in 9 minutes, and is quicker, more reliable and more stable in detection.

(3) The streptavidin-biotin signal amplification system is utilized, so that the detection sensitivity is high, the yin-yang coincidence rate is high, and the repeatability is good.

In addition, the embodiment of the invention also provides a serum amyloid A detection system, which comprises detection equipment and the serum amyloid A detection kit.

Specifically, the detection device is an electrochemical analyzer. In one specific example, the electrochemical analyzer is an electrochemical analyzer of Shenzhen Pumen science and technology, inc.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and equipment used in the examples, unless otherwise specified, are all conventional in the art. The experimental procedures without specifying the specific conditions in the examples were carried out under the conventional conditions such as those described in the literature, in books, or as recommended by the manufacturer.

In the following examples, the serum amyloid a antibody in the biotin-labeled serum amyloid a antibody is a product of Medix, and the serum amyloid a antibody in the terpyridyl ruthenium-labeled serum amyloid a antibody is a product of wuhan huamei bioengineering ltd, unless otherwise specified.

Example 1

The serum amyloid a detection kit of embodiment 1 includes a magnetic particle working solution, a first antibody solution, a sample diluent, a second antibody solution, a calibrator, and a quality control. Wherein:

the magnetic particle working solution consists of streptavidin coupled magnetic particles, PBS buffer solution, bovine serum albumin, triton X-100 and proclin 300, and the pH value of the magnetic particle working solution is 7.0. In the magnetic particle working solution, the concentration of the streptavidin coupled magnetic particle is 0.5mg/mL, the concentration of PBS is 0.1mol/L, the mass percentage of bovine serum albumin is 0.5%, the mass percentage of Triton X-100 is 0.05%, and the mass percentage of proclin 300 is 0.05%.

The first antibody liquid is formed by mixing a biotin-labeled serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300, and the pH value of the first antibody liquid is 7.4; in the first antibody liquid, the concentration of the biotin-labeled serum amyloid A antibody is 1.0mg/L, the concentration of the HEPES buffer solution is 0.01mol/L (namely the concentration of the HEPES in the HEPES buffer solution is 0.01 mol/L), the mass percentage of the bovine serum albumin is 1.5%, the mass percentage of the sodium chloride is 1%, the mass percentage of the sucrose is 2%, the mass percentage of the Tween-20 is 0.05%, and the mass percentage of the proclin 300 is 0.05%; the mass ratio of HEPES to Tween-20 is 4.8:1, the mass ratio of HEPES to sodium chloride is 0.24:1, the mass ratio of bovine serum albumin to sucrose is 1.5:2;

the second antibody liquid is formed by mixing terpyridyl ruthenium labeled serum amyloid A antibody (with different epitope from biotin labeled serum amyloid A antibody), HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300, and the pH value of the second antibody liquid is 7.4; in the second antibody liquid, the concentration of the terpyridyl ruthenium labeled serum amyloid A antibody is 1.0mg/L, the concentration of the HEPES buffer solution is 0.01mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sodium chloride is 1%, the mass percentage of sucrose is 2%, the mass percentage of Tween-20 is 0.05%, and the mass percentage of proclin 300 is 0.05%; the mass ratio of HEPES to Tween-20 is 4.8:1, the mass ratio of HEPES to sodium chloride is 0.24:1, the mass ratio of bovine serum albumin to sucrose is 1.5:2;

the sample dilution was HEPES buffer solution at pH 7.4 and a concentration of 0.01 mol/L.

The calibrator comprises high-point working fluid, middle-point working fluid and low-point working fluid. The high-point working solution, the middle-point working solution and the low-point working solution of the calibrator respectively consist of premixed liquid and serum amyloid A, the premixed liquid of the calibrator consists of Tris buffer solution, trehalose, bovine serum albumin, ethylene diamine tetraacetic acid disodium dihydrate and proclin 300, and in the premixed liquid, the concentration of the Tris buffer solution is 0.05mol/L, the mass percentage of the trehalose is 6.22%, the mass percentage of the bovine serum albumin is 2.48%, the mass percentage of the ethylene diamine tetraacetic acid disodium dihydrate is 0.05%, the mass percentage of Tween-20 is 0.05% and the mass percentage of the proclin 300 is 0.1%. The concentrations of serum amyloid A in the high-point working solution, the medium-point working solution and the low-point working solution of the calibrator are 300 mug/mL, 100 mug/mL and 10 mug/mL respectively.

The quality control product comprises high-point working fluid and low-point working fluid. The compositions of the high-point working solution and the low-point working solution of the quality control product are approximately the same as those of the high-point working solution and the place working solution of the standard product, and the difference is that the concentrations of serum amyloid A in the high-point working solution and the low-point working solution of the quality control product are 200 mug/mL and 5 mug/mL respectively.

The preparation method of the serum amyloid A detection kit of the embodiment 1 includes but is not limited to the following steps:

(1) Preparation of streptavidin-coupled magnetic particle working solution

Magnetically separating a streptavidin-coupled magnetic particle suspension to remove a supernatant, and preparing a streptavidin-coupled magnetic particle working solution with the concentration of 0.5mg/mL by using a magnetic particle premix with the pH of 7.0 to prepare the streptavidin-coupled magnetic particle working solution, wherein the magnetic particle premix is prepared by mixing PBS buffer solution, bovine serum albumin, triton X-100 and proclin 300, the pH is 7.0, the concentration of PBS in the first antibody premix is 0.1mol/L, the mass percentage of the bovine serum albumin is 0.5%, the mass percentage of Triton X-100 is 0.05%, and the mass percentage of the proclin 300 is 0.05%.

(2) Preparing a first antibody solution and a second antibody solution

1) Preparing an antibody premix with pH of 7.4, wherein the antibody premix is prepared by mixing HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300. In the antibody premix, the concentration of the HEPES buffer solution is 0.01mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sodium chloride is 1%, the mass percentage of sucrose is 2%, the mass percentage of Tween-20 is 0.05%, and the mass percentage of proclin 300 is 0.05%.

2) Using the antibody premix prepared in the step 1) to respectively dilute the biotin-labeled serum amyloid A antibody (Medix corporation) and the terpyridyl ruthenium-labeled serum amyloid A antibody (Wuhan Huamei corporation) to 1.0mg/L to prepare a first antibody solution and a second antibody solution. Wherein the epitope of serum amyloid A antibody in the biotin-labeled serum amyloid A antibody is different from the epitope of serum amyloid A antibody in the ruthenium terpyridyl-labeled serum amyloid A antibody.

(3) Sample dilution preparation

HEPES buffer solution having a pH of 7.4 and a concentration of 0.01mol/L was prepared as a sample diluent.

(4) Preparation of calibrator and quality control product

1) Preparing a premixed solution with the pH value of 7.4, wherein the premixed solution comprises 0.05mol/L of Tris buffer solution, 6.22% of trehalose, 2.48% of bovine serum albumin, 0.05% of disodium ethylenediamine tetraacetate dihydrate, 0.05% of Tween-20 and 0.1% of proclin 300.

2) Preparing a serum amyloid A calibrator and a quality control product by using the premixed solution prepared in the step 1). The calibrator comprises a high-point working solution, a middle-point working solution and a low-point working solution, and the concentrations of serum amyloid A are 300 mug/mL, 100 mug/mL and 10 mug/mL respectively. The quality control product has a high point and a low point, and the concentration of serum amyloid A is 200 mug/mL and 20 mug/mL respectively.

(5) Reagent subpackaging and assembling

The streptavidin-coupled magnetic particle working solution is subpackaged by 5.0 mL/bottle, the first antibody liquid containing the biotin-labeled serum amyloid A antibody is subpackaged by 11 mL/bottle, the second antibody liquid containing the terpyridyl ruthenium-labeled serum amyloid A antibody is subpackaged by 11 mL/bottle, the sample diluent is subpackaged by 13 mL/bottle, the calibrator and the quality control material are subpackaged by 1.0 mL/bottle, and the components are assembled together and stored at 2-8 ℃.

Example 2

The reagents in the serum amyloid a detection kit of example 2 are substantially the same as those in the serum amyloid a detection kit of example 1, except that the concentration of HEPES in the HEPES buffer of the first antibody liquid of example 2 is 0.2mol/L, the concentration of HEPES in the HEPES buffer of the second antibody liquid of example 2 is 0.2mol/L, and the sample buffer is a HEPES buffer having a pH of 7.4 and a concentration of 0.2mol/L. In both the primary antibody fluid and the secondary antibody fluid of example 2, the mass ratio of HEPES to Tween-20 was 95.2:1, the mass ratio of HEPES to sodium chloride is 4.76:1.

example 3

The reagent in the serum amyloid a detection kit of example 3 is substantially the same as the reagent in the serum amyloid a detection kit of example 1, except that the concentration of HEPES in the HEPES buffer of the first antibody liquid of example 3 is 0.005mol/L, the concentration of HEPES in the HEPES buffer of the second antibody liquid of example 3 is 0.005mol/L, and the sample buffer of example 3 is a HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol/L. In both the primary antibody fluid and the secondary antibody fluid of example 3, the mass ratio of HEPES to Tween-20 was 2.4:1, the mass ratio of HEPES to sodium chloride is 0.12:1.

example 4

The reagents in the serum amyloid a detection kit of example 4 are substantially the same as those in the serum amyloid a detection kit of example 1, except that in the first antibody solution of example 4, the concentration of the terpyridyl ruthenium-labeled serum amyloid a antibody is 1.0mg/L, the mass percentage of bovine serum albumin is 0.5%, the mass percentage of sodium chloride is 0.5%, the mass percentage of sucrose is 1%, the mass percentage of Tween-20 is 0.02%, and the mass percentage of proclin 300 is 0.05%. In the second antibody solution of example 4, the concentration of the terpyridyl ruthenium labeled serum amyloid a antibody was 1.0mg/L, the mass percentage of bovine serum albumin was 0.5%, the mass percentage of sodium chloride was 0.5%, the mass percentage of sucrose was 1%, the mass percentage of Tween-20 was 0.02%, and the mass percentage of proclin 300 was 0.05%. In the primary antibody fluid and the secondary antibody fluid of example 4, the mass ratio of HEPES to Tween-20 was 12:1, the mass ratio of HEPES to sodium chloride is 0.48:1, the mass ratio of bovine serum albumin to sucrose is 0.5:1.

example 5

The reagents in the serum amyloid a detection kit according to example 5 are substantially the same as those in the serum amyloid a detection kit according to example 1, except that in the first antibody solution according to example 5, the concentration of the terpyridyl ruthenium labeled serum amyloid a antibody is 1.0mg/L, the mass percentage of bovine serum albumin is 5%, the mass percentage of sodium chloride is 5%, the mass percentage of sucrose is 5%, the mass percentage of Tween-20 is 1%, and the mass percentage of proclin 300 is 0.05%. In the second antibody solution of example 5, the concentration of the terpyridyl ruthenium labeled serum amyloid a antibody was 1.0mg/L, the mass percentage of bovine serum albumin was 5%, the mass percentage of sodium chloride was 5%, the mass percentage of sucrose was 5%, the mass percentage of Tween-20 was 1%, and the mass percentage of proclin 300 was 0.05%. In the first antibody liquid and the second antibody liquid of example 5, the mass ratio of HEPES to Tween-20 was 0.24:1, the mass ratio of HEPES to sodium chloride is 0.048:1, the mass ratio of bovine serum albumin to sucrose is 1:1.

example 6

The reagents in the serum amyloid a detection kit of example 6 were substantially the same as those in the serum amyloid a detection kit of example 5, except that the concentrations of the HEPES buffer in the first antibody liquid, the second antibody liquid, and the sample diluent in the serum amyloid a detection kit of example 6 were 0.002mol/L.

Example 7

The reagents in the serum amyloid a detection kit according to example 7 are substantially the same as those in the serum amyloid a detection kit according to example 4, except that the concentrations of the HEPES buffer in the first antibody liquid, the second antibody liquid, and the sample diluent in the serum amyloid a detection kit according to example 7 are 0.3mol/L.

Example 8

The reagents in the serum amyloid a assay kit of example 8 are substantially the same as those in the serum amyloid a assay kit of example 1, except that the surfactants in the first antibody solution, the second antibody solution and the sample diluent in the serum amyloid a assay kit of example 8 are each polyethylene glycol octylphenyl ether (triton x-100).

Example 9

The reagents in the serum amyloid a detection kit according to example 9 are substantially the same as those in the serum amyloid a detection kit according to example 1, except that the surfactants in the first antibody solution, the second antibody solution, and the sample diluent in the serum amyloid a detection kit according to example 9 are each polyoxyethylene sorbitan monooleate (Tween-80).

Example 10

The reagents in the serum amyloid a detection kit of example 10 are substantially the same as those in the serum amyloid a detection kit of example 5, except that the concentration of HEPES buffer in the first antibody solution in the serum amyloid a detection kit of example 10 is 0.005mol/L; in the second antibody solution of the serum amyloid A detection kit of example 10, the concentration of HEPES buffer was 0.005mol/L. In the first antibody liquid and the second antibody liquid of example 10, the mass ratio of HEPES to Tween-20 was 0.12:1; the mass ratio of HEPES to sodium chloride is 0.024:1.

example 11

The reagent in the serum amyloid a detection kit of example 11 is substantially the same as the reagent in the serum amyloid a detection kit of example 2, except that the first antibody solution in the serum amyloid a detection kit of example 11 contains Tween-20 in an amount of 0.025% by mass; in the second antibody solution of the serum amyloid A detection kit of the embodiment 11, the mass percentage content of the Tween-20 is 0.025%; in the primary antibody liquid and the secondary antibody liquid of example 11, the mass ratio of HEPES to Tween-20 is 190.4:1.

example 12

The reagent in the serum amyloid a detection kit of example 12 is substantially the same as the reagent in the serum amyloid a detection kit of example 4, except that the first antibody solution in the serum amyloid a detection kit of example 12 contains sucrose in an amount of 2% by mass; in the second antibody solution of the serum amyloid a detection kit of embodiment 12, the mass percentage of sucrose is 2%; in the first antibody liquid and the second antibody liquid of example 12, the mass ratio of bovine serum albumin to sucrose was 0.25:1.

example 13

The reagent in the serum amyloid a detection kit according to example 13 is substantially the same as the reagent in the serum amyloid a detection kit according to example 5, except that the first antibody solution in the serum amyloid a detection kit according to example 13 contains sucrose in an amount of 2.5% by mass; in the second antibody solution of the serum amyloid a detection kit of embodiment 13, the mass percentage content of sucrose is 2.5%; in the first antibody liquid and the second antibody liquid of example 13, the mass ratio of bovine serum albumin to sucrose was 2:1.

example 14

The reagent in the serum amyloid a detection kit of example 14 is substantially the same as the reagent in the serum amyloid a detection kit of example 2, except that the first antibody solution in the serum amyloid a detection kit of example 14 contains 0.5% by mass of sodium chloride; in the second antibody solution of the serum amyloid a detection kit according to example 14, the content of sodium chloride is 0.5% by mass. In the first antibody liquid and the second antibody liquid of example 14, the mass ratio of HEPES to sodium chloride was 9.52:1.

comparative example 1

The reagents in the serum amyloid a assay kit of comparative example 1 were approximately the same as those in the serum amyloid a assay kit of example 1, except that the buffers in the first antibody solution, the second antibody solution and the sample diluent of the serum amyloid a assay kit of comparative example 1 were all 0.01mol/L PBS buffer.

And (3) testing:

test example 1:

1. test of serum amyloid A detection kit of examples 1 to 14

The serum amyloid a detection kit of each example was used to test the sensitivity and test range at different dilution times and different buffer systems, respectively. Specifically, the test procedures of the serum amyloid a detection kit of each embodiment are as follows:

clinical samples of healthy people (the concentration of serum amyloid A contained in the clinical samples is 5.32 mug/mL) are respectively diluted by 0 time, 2 times, 4 times, 8 times, 16 times, 64 times, 128 times and 256 times and then are respectively used as samples to be detected by adopting the detection kit corresponding to each embodiment, and meanwhile, positive clinical samples with known serum amyloid A concentration are used as samples to be detected by adopting the detection kit corresponding to each embodiment. The detection method of each sample to be detected is as follows: diluting the samples to be detected by 100 times to obtain corresponding diluted samples; mu.L of diluted sample, 85. Mu.L of first antibody liquid and 85. Mu.L of second antibody liquid are added into a reaction cup to form biotin-labeled serum amyloid A antibody-serum amyloid A-terpyridyl ruthenium-labeled serum amyloid A antibody complex solution, 25. Mu.L of streptavidin-coupled magnetic particle working solution is added, incubation is carried out for 9min at 37 ℃ to form magnetic complex suspension, the magnetic complex suspension is placed in a magnetic field to be adsorbed on the surface of an electrode, washing liquid flows through the magnetic field to remove unbound substances, chemiluminescence substrate liquid (tripropylamine) is injected, and the luminescence intensity (namely, detection signal value) is detected, and the results are shown in Table 1.

In table 1, a sample 8 is a clinical sample of a healthy person, and the concentration of serum amyloid a contained in the sample 8 is 5.32 μ g/mL through clinical detection, a sample 7 is obtained by diluting the sample 8 by 2 times with a sample diluent, a sample 6 is obtained by diluting the sample 8 by 4 times with the sample diluent, a sample 5 is obtained by diluting the sample 8 by 8 times with the sample diluent, a sample 4 is obtained by diluting the sample 8 by 16 times with the sample diluent, a sample is obtained by diluting the sample 8 by 64 times with the sample diluent, a sample is obtained by diluting the sample 8 by 128 times with the sample diluent, and a sample 1 is obtained by diluting the sample 8 by 256 times with the sample diluent; the samples 10 to 16 are positive clinical samples; the concentration of the serum amyloid A in the samples 1 to 16 is obtained through clinical detection; r is a correlation between the concentration of serum amyloid A and the detection signal value thereof in samples 1 to 15 in Table 1.

As is clear from Table 1, the serum amyloid A detection kits of examples 1 to 14 can accurately detect a sample containing 0.166. Mu.g/mL serum amyloid A, and have high detection sensitivity. Furthermore, the serum amyloid a detection kits of embodiments 1 to 14 can also detect positive clinical samples (600 μ g/mL) diluted by 100, and the correlations between the serum amyloid a concentrations of samples 1 to 15 and the detection signal values thereof are good, which indicates that the serum amyloid a detection kit of the present study has a wide linear detection range. Wherein, in the first antibody liquid and/or the second antibody liquid, the mass ratio of HEPES to polyoxyethylene sorbitan monolaurate is (0.24-95.2): 1, the mass ratio of HEPES to the inorganic salt is (0.048-4.76): 1, the mass ratio of bovine serum albumin to sugar is (0.5-1): 1, the detected signal value is higher, the background of the sample is lower, and the correlation between the sample concentration and the signal value is better, which indicates that the reactivity of the antibody fluid reaches a better state under the proportion, and the sensitivity and the accuracy of the serum amyloid A detection kit can be improved.

2. Test of the detection kit of comparative example 1

(1) The sample to be tested comprises: respectively diluting a healthy human clinical sample (the concentration of serum amyloid A contained in the healthy human clinical sample is 5.32 mug/mL through clinical detection) by 0 time, 2 times, 4 times, 8 times, 16 times, 64 times, 128 times and 256 times to be used as samples to be detected; positive clinical samples of serum amyloid a concentrations are known. See table 2 for details. In Table 2, the 2-fold dilution means that a clinical sample of a healthy person (which is clinically detected to contain serum amyloid A at a concentration of 5.32. Mu.g/mL) is diluted 2-fold, and so on; positive clinical samples (10.67. Mu.g/mL) represent positive clinical samples containing 10.67. Mu.g/mL serum amyloid A, and so on.

(2) And (3) respectively detecting each sample to be detected by adopting the detection kit of the comparative example 1 and two detection methods. The first detection method was substantially the same as that of example 1, except that the detection kit used was the detection kit of comparative example 1; in the detection method, 6 μ L of the sample to be detected is directly reacted with 85 μ L of the first antibody liquid and 85 μ L of the second antibody liquid in the reaction cup without dilution. The second detection method was substantially the same as the detection method of example 1, except that the detection kit used was the detection kit of comparative example 1. The results of the test sample using the test kit of comparative example 1 and using the two test methods are shown in table 2.

TABLE 2

As can be seen from table 2, from the results of the first and second detection methods on each positive clinical sample, the second detection method was able to detect positive clinical samples with serum amyloid a concentration of 600 μ g/mL, and the correlation between the serum amyloid a concentration and the detection signal value of the second detection method was superior to the correlation between the serum amyloid a concentration and the detection signal value of the first detection method, indicating that it is advantageous to expand the detection range of serum amyloid a by diluting the sample 100 times.

Furthermore, the detection kit of comparative example 1 can detect a sample diluted 100 times after a clinical sample (5.32. Mu.g/mL) of a healthy person is diluted 16 times, which shows that the detection kit of comparative example 1 can detect a sample containing 0.333. Mu.g/mL of serum amyloid A, and the detection sensitivity is lower than that of the serum amyloid A detection kits of examples 1 to 14. Further, the relationship curve between the serum amyloid A concentration and the signal value thereof in the positive clinical sample diluted 100 times by using the serum amyloid A detection kit of example 1 is shown in FIG. 1. The relationship curve between the serum amyloid A concentration and the signal value thereof in the positive clinical sample diluted 100 times by using the serum amyloid A detection kit of comparative example 1 is shown in FIG. 2. As can be seen from FIGS. 1 and 2, the positive clinical sample of example 1 had a higher signal value, a lower background, and a correlation coefficient (R) between the serum amyloid A concentration and the signal value thereof ² ) And more preferably.

Test example 2:

linear range of serum amyloid a detection kit of example 1:

the linear range of the serum amyloid a detection kit of example 1 was calculated by separately testing the signal values of positive clinical samples of known serum amyloid a concentrations. The specific test steps are as follows:

diluting a positive clinical sample with known serum amyloid A concentration by 100 times to obtain a diluted sample, adding 6 mu L of the diluted sample, 85 mu L of first antibody liquid and 85 mu L of second antibody liquid into a reaction cup to form a biotin-labeled serum amyloid A antibody-serum amyloid A-ruthenium terpyridyl-labeled serum amyloid A antibody complex solution, adding 25 mu L of streptavidin-coupled magnetic particle working solution, incubating at 37 ℃ for 9min to form a magnetic complex suspension, placing the magnetic complex suspension on the surface of an electrode in a magnetic field, allowing a cleaning solution to flow through the magnetic field to remove unbound substances, injecting a chemiluminescent substrate solution (tripropylamine), and detecting the luminous intensity of the chemiluminescent substrate solution, wherein the results are shown in Table 3 and FIG. 3. Wherein, the concentration of serum amyloid A in a positive clinical sample with known serum amyloid A concentration is obtained by clinical detection.

TABLE 3

As can be seen from table 3 and fig. 3, when the serum amyloid a detection kit of the present application performs detection, the concentration of serum amyloid a has good linearity with the detection signal value within the range of 0.20 μ g/mL to 452.10 μ g/mL, which indicates that the detection linearity range of the serum amyloid a detection kit of the present application is 0.20 μ g/mL to 452.10 μ g/mL, which is wider than the detection linearity range of the conventional kit for detecting serum amyloid a (i.e., 0.5 μ g/mL to 200 μ g/mL), and the detection linearity range is wide. Furthermore, the serum amyloid A detection kit of the application detects the correlation coefficient R of a sample with the serum amyloid A concentration of 0.20-452.10 mu g/mL ² Is 0.9968 (i.e. the correlation r is 0.9983), which indicates that the serum amyloid A detection kit has higher accuracy in a wider linear range.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, without departing from the concept of the present invention, a person skilled in the art may make several modifications and improvements, for example, the content of the main effective component in the first antibody liquid, the second antibody liquid or the magnetic particle working solution is a multiple of the corresponding component in the present invention, and the person skilled in the art can obtain the technical solution of the present invention by adding water to make a multiple dilution when using, and these all fall into the protection scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. A serum amyloid A detection kit is characterized by comprising a first antibody liquid, a second antibody liquid and a magnetic particle working solution, wherein:

the first antibody liquid comprises a first marker labeled serum amyloid A antibody, bovine serum albumin, sodium chloride, sucrose, a surfactant and an HEPES buffer solution, wherein in the first antibody liquid, the surfactant is TritonX-100 or Tween-20, the concentration of the first marker labeled serum amyloid A antibody is 1mg/L, the mass percentage of the bovine serum albumin is 1.5-5%, the mass percentage of the sodium chloride is 1-5%, the mass percentage of the sucrose is 2-5%, the mass percentage of the surfactant is 0.05-1%, and the mass ratio of the HEPES to the surfactant is (0.24-95.2): 1, the mass ratio of HEPES to sodium chloride is (0.048-4.76): 1, the mass ratio of bovine serum albumin to sucrose is (0.75 to 1): 1, the concentration of HEPES in the HEPES buffer solution is 0.005 mol/L-0.2mol/L, and the pH value of the first antibody solution is 6.0-7.6;

the second antibody liquid comprises a second marker labeled serum amyloid A antibody, bovine serum albumin, sodium chloride, sucrose, a surfactant and an HEPES buffer solution, wherein in the second antibody liquid, the surfactant is TritonX-100 or Tween-20, the concentration of the labeled serum amyloid A antibody is 1mg/L, the mass percentage of the bovine serum albumin is 1.5-5%, the mass percentage of the sodium chloride is 1-5%, the mass percentage of the sucrose is 2-5%, the mass percentage of the surfactant is 0.02-1%, and the mass ratio of the HEPES to the surfactant is (0.24-95.2): 1, the mass ratio of HEPES to sodium chloride is (0.048 to 4.76): 1, the mass ratio of bovine serum albumin to sucrose is (0.75 to 1): 1, the concentration of HEPES in the HEPES buffer solution is 0.005 mol/L-0.2mol/L, the pH value of the second antibody solution is 6.0-7.6, and the second marker is a chemiluminescence marker;

the magnetic particle working solution includes magnetic particles coupled with a third label capable of specifically binding to the first label.

2. The serum amyloid A detection kit according to claim 1, wherein the magnetic particle working solution further comprises a buffer selected from at least one of phosphate buffer, tris buffer, HEPES buffer and MOPSO buffer.

3. The serum amyloid A detection kit according to any one of claims 1 to 2, wherein the first marker is biotin and the third marker is avidin; and/or the second marker is ruthenium terpyridyl, acridinium ester, alkaline phosphatase or horseradish peroxidase.

4. The serum amyloid a detection kit according to claim 1, wherein the detection kit further comprises at least one of a sample diluent, a quality control substance and a calibrator.

5. The serum amyloid A detection kit according to claim 4, wherein the sample diluent comprises 0.5-5% by mass of inorganic salts, 1-5% by mass of sugars and 0.02-1% by mass of surfactants.

6. The serum amyloid A assay kit according to claim 5, wherein the surfactant in the sample diluent is at least one selected from the group consisting of dodecyl polyethylene glycol, polyethylene glycol p-isooctyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, and polyoxyethylene lauryl ether.

7. The serum amyloid A detection kit according to claim 1, wherein the first antibody solution further contains a preservative, and the preservative in the first antibody solution is 0.05% -0.5% by mass;

and/or the second antibody liquid also contains a preservative, wherein the preservative in the second antibody liquid accounts for 0.05-0.5% by mass;

and/or the magnetic particle working solution also contains a preservative, and the preservative in the magnetic particle working solution accounts for 0.05-0.5% by mass.

8. The serum amyloid a detection kit according to claim 1, comprising a first antibody fluid, a second antibody fluid, a magnetic microparticle working fluid and a sample diluent, wherein:

the first antibody liquid is formed by mixing a biotin-labeled serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300; in the first antibody liquid, the concentration of a biotin-labeled serum amyloid A antibody is 1.0mg/L, the concentration of a HEPES buffer solution is 0.01mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sodium chloride is 1%, the mass percentage of sucrose is 2%, the mass percentage of Tween-20 is 0.05%, and the mass percentage of proclin 300 is 0.05%;

the second antibody liquid is formed by mixing a serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300, wherein the serum amyloid A antibody is marked by terpyridyl ruthenium and has different epitopes from the serum amyloid A antibody in the first antibody liquid; in the second antibody liquid, the concentration of the terpyridyl ruthenium labeled serum amyloid A antibody is 1.0mg/L, the concentration of the HEPES buffer solution is 0.01mol/L, the mass percentage content of bovine serum albumin is 1.5%, the mass percentage content of sodium chloride is 1%, the mass percentage content of sucrose is 2%, the mass percentage content of Tween-20 is 0.05%, and the mass percentage content of proclin 300 is 0.05%;

the magnetic particle working solution comprises magnetic particle working solution, wherein the magnetic particle working solution consists of streptavidin-coupled magnetic particles, PBS buffer solution, bovine serum albumin, triton X-100 and proclin 300, the concentration of the streptavidin-coupled magnetic particles in the magnetic particle working solution is 0.5mg/mL, the concentration of PBS is 0.1mol/L, the mass percentage of bovine serum albumin is 0.5%, the mass percentage of Triton X-100 is 0.05%, and the mass percentage of proclin 300 is 0.05%;

the sample diluent is HEPES buffer solution with pH of 7.4 and concentration of 0.01 mol/L.

9. The serum amyloid a detection kit according to claim 1, comprising a first antibody fluid, a second antibody fluid, a magnetic microparticle working fluid and a sample diluent, wherein:

the first antibody liquid is formed by mixing a biotin-labeled serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300; in the first antibody liquid, the concentration of a biotin-labeled serum amyloid A antibody is 1.0mg/L, the concentration of a HEPES buffer solution is 0.2mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sucrose is 2%, and the mass ratio of HEPES to Tween-20 is 95.2:1, the mass ratio of HEPES to sodium chloride is 4.76:1, proclin 300 with the mass percentage of 0.05 percent;

the second antibody liquid is formed by mixing a serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300, wherein the serum amyloid A antibody is marked by terpyridyl ruthenium and has different epitopes from the serum amyloid A antibody in the first antibody liquid; in the second antibody liquid, the concentration of the ruthenium terpyridyl labeled serum amyloid A antibody is 1.0mg/L, the concentration of the HEPES buffer solution is 0.2mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sucrose is 2%, and the mass ratio of HEPES to Tween-20 is 95.2:1, the mass ratio of HEPES to sodium chloride is 4.76:1, the mass percentage content of proclin 300 is 0.05 percent;

the magnetic particle working solution comprises a magnetic particle working solution, wherein the magnetic particle working solution consists of streptavidin coupled magnetic particles, PBS buffer solution, bovine serum albumin, triton X-100 and proclin 300, the concentration of the streptavidin coupled magnetic particles in the magnetic particle working solution is 0.5mg/mL, the concentration of PBS is 0.1mol/L, the mass percentage content of bovine serum albumin is 0.5%, the mass percentage content of Triton X-100 is 0.05%, and the mass percentage content of proclin 300 is 0.05%;

the sample diluent is HEPES buffer solution with pH 7.4 and concentration of 0.2mol/L.

10. The serum amyloid a detection kit according to claim 1, comprising a first antibody fluid, a second antibody fluid, a magnetic microparticle working fluid and a sample diluent, wherein:

the first antibody liquid is formed by mixing a biotin-labeled serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300; in the first antibody liquid, the concentration of a biotin-labeled serum amyloid A antibody is 1.0mg/L, the concentration of a HEPES buffer solution is 0.005mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sucrose is 2%, and the mass ratio of HEPES to Tween-20 is 2.4:1, the mass ratio of HEPES to sodium chloride is 0.12:1, the mass percentage content of proclin 300 is 0.05 percent;

the second antibody liquid is formed by mixing a serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300, wherein the serum amyloid A antibody is marked by terpyridyl ruthenium and has different epitopes from the serum amyloid A antibody in the first antibody liquid; in the second antibody liquid, the concentration of the terpyridyl ruthenium labeled serum amyloid A antibody is 1.0mg/L, the concentration of the HEPES buffer solution is 0.005mol/L, the mass percentage of the bovine serum albumin is 1.5%, the mass percentage of the sucrose is 2%, and the mass ratio of the HEPES to the Tween-20 is 2.4:1, the mass ratio of HEPES to sodium chloride is 0.12:1, the mass percentage content of proclin 300 is 0.05 percent;

the sample diluent is HEPES buffer solution with pH 7.4 and concentration of 0.05 mol/L.

11. The serum amyloid a detection kit according to claim 1, comprising a first antibody fluid, a second antibody fluid, a magnetic microparticle working fluid and a sample diluent, wherein:

the first antibody liquid is formed by mixing a biotin-labeled serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300; in the first antibody liquid, the concentration of a biotin-labeled serum amyloid A antibody is 1.0mg/L, the concentration of a HEPES buffer solution is 0.01mol/L, the mass percentage of bovine serum albumin is 5%, the mass percentage of sodium chloride is 5%, the mass percentage of sucrose is 5%, the mass percentage of Tween-20 is 1%, and the mass percentage of proclin 300 is 0.05%;

the second antibody liquid is formed by mixing a serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tween-20 and proclin 300, wherein the serum amyloid A antibody is marked by terpyridyl ruthenium and has different epitopes from the serum amyloid A antibody in the first antibody liquid; in the second antibody liquid, the concentration of the terpyridyl ruthenium labeled serum amyloid A antibody is 1.0mg/L, the concentration of the HEPES buffer solution is 0.01mol/L, the mass percentage of bovine serum albumin is 5%, the mass percentage of sodium chloride is 5%, the mass percentage of sucrose is 5%, the mass percentage of Tween-20 is 1%, and the mass percentage of proclin 300 is 0.05%;

the sample diluent is HEPES buffer solution with pH 7.4 and concentration of 0.01 mol/L.

12. The serum amyloid a detection kit according to claim 1, comprising a first antibody fluid, a second antibody fluid, a magnetic microparticle working fluid and a sample diluent, wherein:

the first antibody liquid is formed by mixing a biotin-labeled serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tritonX-100 and proclin 300; in the first antibody liquid, the concentration of a biotin-labeled serum amyloid A antibody is 1.0mg/L, the concentration of a HEPES buffer solution is 0.01mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sodium chloride is 1%, the mass percentage of sucrose is 2%, the mass percentage of TritonX-100 is 0.05%, and the mass percentage of proclin 300 is 0.05%;

the second antibody liquid is formed by mixing a serum amyloid A antibody, a HEPES buffer solution, bovine serum albumin, sodium chloride, sucrose, tritonX-100 and proclin 300, wherein the serum amyloid A antibody is marked by terpyridyl ruthenium and has different epitopes from the serum amyloid A antibody in the first antibody liquid; in the second antibody liquid, the concentration of the terpyridyl ruthenium labeled serum amyloid A antibody is 1.0mg/L, the concentration of the HEPES buffer solution is 0.01mol/L, the mass percentage of bovine serum albumin is 1.5%, the mass percentage of sodium chloride is 1%, the mass percentage of sucrose is 2%, the mass percentage of TritonX-100 is 0.05%, and the mass percentage of proclin 300 is 0.05%;

13. A serum amyloid a detection system, comprising:

a detection device; and

the serum amyloid A detection kit according to any one of claims 1 to 12.