CN110907639A - Serum amyloid protein A detection kit and preparation method thereof - Google Patents
Serum amyloid protein A detection kit and preparation method thereof Download PDFInfo
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- CN110907639A CN110907639A CN201911233959.9A CN201911233959A CN110907639A CN 110907639 A CN110907639 A CN 110907639A CN 201911233959 A CN201911233959 A CN 201911233959A CN 110907639 A CN110907639 A CN 110907639A
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- ethanesulfonic acid
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- 102000054727 Serum Amyloid A Human genes 0.000 title claims abstract description 51
- 108700028909 Serum Amyloid A Proteins 0.000 title claims abstract description 51
- 238000001514 detection method Methods 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 51
- 239000004816 latex Substances 0.000 claims abstract description 42
- 229920000126 latex Polymers 0.000 claims abstract description 42
- 239000004005 microsphere Substances 0.000 claims abstract description 31
- 239000002245 particle Substances 0.000 claims abstract description 23
- 239000012279 sodium borohydride Substances 0.000 claims abstract description 23
- 229910000033 sodium borohydride Inorganic materials 0.000 claims abstract description 23
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 20
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 20
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 56
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 29
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 28
- 239000005018 casein Substances 0.000 claims description 18
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 18
- 235000021240 caseins Nutrition 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 11
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 11
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 11
- 239000002202 Polyethylene glycol Substances 0.000 claims description 9
- 229920001223 polyethylene glycol Polymers 0.000 claims description 9
- 229920000136 polysorbate Polymers 0.000 claims description 9
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims description 5
- 239000004793 Polystyrene Substances 0.000 claims description 5
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims description 5
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims description 5
- 229920002223 polystyrene Polymers 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 3
- 230000002335 preservative effect Effects 0.000 claims description 3
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- 238000003556 assay Methods 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 238000003149 assay kit Methods 0.000 claims 2
- 238000000034 method Methods 0.000 abstract description 8
- 125000003172 aldehyde group Chemical group 0.000 abstract description 6
- 238000000576 coating method Methods 0.000 abstract description 5
- 238000012412 chemical coupling Methods 0.000 abstract description 4
- 239000011248 coating agent Substances 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 21
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 8
- 239000007987 MES buffer Substances 0.000 description 7
- 239000002262 Schiff base Substances 0.000 description 7
- 150000004753 Schiff bases Chemical class 0.000 description 7
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 150000001299 aldehydes Chemical class 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 4
- 150000003335 secondary amines Chemical class 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000008878 coupling Effects 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000006722 reduction reaction Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
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- 238000005728 strengthening Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 206010028470 Mycoplasma infections Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 150000003973 alkyl amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- -1 diamine compounds Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
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- 208000015181 infectious disease Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
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- 244000052769 pathogen Species 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention discloses a serum amyloid A detection kit, which comprises a reagent 1 and a reagent 2, wherein the reagent 2 comprises latex particles coated with a mouse anti-human SAA monoclonal antibody, the mouse anti-human SAA monoclonal antibody, sodium borohydride, bovine serum albumin and bovine serum albumin. The kit adopts aldehyde group microspheres on the basis of a chemical coupling method, so that the binding force of the antibody and latex is improved, and the antibody coating efficiency is further improved.
Description
Technical Field
The invention relates to the field of medical detection, in particular to a serum amyloid A detection kit and a preparation method thereof.
Background
Serum Amyloid A (SAA) is an acute phase-reactive protein produced by hepatocytes and secreted into the serum, a heterogeneous class of proteins in the apolipoprotein family that binds to plasma High Density Lipoprotein (HDL). In the acute time-limited response, SAA is synthesized in the liver by activated macrophages and fibroblasts by stimulation with IL-1, IL-6 and TNF, and can rise to 1000 times of the initial concentration, but has a very short half-life of only about 50 minutes, and rapidly decreases to normal level after the body antigen is cleared. SAA has one of the most important differences compared to the most widely used CRP in the clinic at present: the SAA is increased and is found in virus, mycoplasma and bacterial infection, and the sensitivity is higher than CRP; CRP elevation is seen in bacterial infections, and infections with pathogens such as viruses and mycoplasma are not elevated or only slightly elevated.
In latex enhanced immunoturbidimetry, the general preparation process is to coat serum amyloid a antibody on latex by two coating methods, physical adsorption and chemical coupling. As taught by patent CN 105372434A: (1) the antibody of the sensitized latex particles obtained by the physical adsorption method is easy to fall off from the latex particles, and the latex obtained by the physical adsorption method is easy to be interfered, so that the false positive or false negative of the detection result is increased. (2) The chemical coupling method is mostly a random coupling method, and the antibody loses binding force due to the fact that the antibody is randomly coupled to different parts of latex. Therefore, complete random coupling will lose most of the binding capacity of the antibody, increasing the antibody dosage and production cost.
At present, the prior art scheme which is closer to the present invention is as follows: preparing 50Mm MES buffer solution with the pH value of 6.0; diluting the carboxyl latex microspheres with MES buffer solution, wherein the concentration is 1%; adding EDAC into the latex microsphere suspension, adding the EDAC into the latex microsphere suspension at a concentration of 40mg/ml, and incubating for 40 minutes at room temperature; washing the latex microspheres with MES buffer solution with the same volume, centrifuging for 30 minutes at 13000g/min, washing the latex microspheres with the same MES buffer solution again, centrifuging for 30 minutes, and then adding the latex microspheres into the MES buffer solution; dissolving rabbit anti-human serum amyloid A in MES buffer solution at a concentration of 1 mg/ml; mixing the antibody solution and the latex microsphere suspension, and stirring and reacting for 3 hours at 37 ℃; adding ethanolamine into the mixed solution, wherein the adding amount is 2.5ul/ml, and stirring for reaction for 10 minutes; unbound antibody was removed and the coated antibody latex particles were stored in MES buffer. The method has the defects that: the preparation process is complex, the centrifugation time is long, the dosage of the antibody is large, and the production cost is increased.
Disclosure of Invention
In order to solve the technical problem, the invention discloses a serum amyloid A detection kit, which adopts aldehyde microspheres on the basis of a chemical coupling method to improve the binding force of an antibody and latex, further improve the antibody coating efficiency and reduce the antibody dosage.
The invention is realized by the following technical scheme:
the serum amyloid A detection kit comprises a reagent 1 and a reagent 2, wherein the reagent 2 comprises latex particles coated with a mouse anti-human SAA monoclonal antibody, the mouse anti-human SAA monoclonal antibody, sodium borohydride, bovine serum albumin and casein.
Wherein the reagent 1 comprises: 4-hydroxyethyl piperazine ethanesulfonic acid, Tween-20, Brij-35, sodium chloride, polyethylene glycol 8000 and sodium azide, and the reagent 2 further comprises: 4-hydroxyethyl piperazine ethanesulfonic acid, Tween-20, glucose and sodium azide.
Further, the concentration of each component of the reagent 1 is as follows: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 355 g/L-10g/L of Brij, 10g/L of sodium chloride, 800010g/L of polyethylene glycol, 1g/L of sodium azide and 7.0 of pH value;
the concentration of each component of the reagent 2 is as follows: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 10g/L of latex particles coated with mouse anti-human SAA monoclonal antibodies, 250mg/L of mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 2g/L of casein, 5g/L, tween-202 ml/L of bovine serum albumin, 80g/L of glucose, 1g/L of sodium azide and 7.0 of pH value.
Further, the latex particles coated with the mouse anti-human SAA monoclonal antibody are aldehyde group modified polystyrene microspheres.
Aldehydes can be reacted with primary and secondary amines to form Schiff bases.
However, Schiff base formation is a relatively unstable, reversible interaction that is easily broken down by hydrolysis in aqueous solution, and is enhanced at alkaline pH but remains unstable.
Relatively unstable Schiff bases can be chemically stabilized by reduction, using reducing agents to convert the Schiff base interaction specifically to alkylamine linkages, which upon reduction are highly stable and do not readily hydrolyze in an aqueous environment.
According to the invention, the latex adopts aldehyde group microspheres, sodium borohydride is added into a reaction medium containing the aldehyde group microspheres and amine-containing antibodies, so that Schiff base intermediate reduction and covalent bond formation are caused, secondary amine connection is generated between the two molecules, the connection is highly stable, and the Schiff base intermediate cannot be easily hydrolyzed in a water environment, so that the aldehyde group microspheres and the antibodies are combined more stably, and the antibody coating efficiency is further improved.
Reductive amination of ammonia or diamine compounds with aldehydes:
in the invention, the key point is that the antibody and the microsphere are activated by using sodium borohydride, so that the binding force of the antibody and the microsphere can be improved, and meanwhile, casein and bovine serum albumin have the function of strengthening the antibody, so that the binding position of the antibody and the microsphere is more accurate, the coupling efficiency is improved, meanwhile, groups which do not participate in reaction on the microsphere are sealed, and the anti-interference capability of the reagent is improved by adding Brij-35 to the reality.
The addition of Tween-20 can protect the structure of the protein from being influenced.
The preparation method of the serum amyloid A detection kit comprises the following steps:
reagent 1:
(1) adding 4-hydroxyethyl piperazine ethanesulfonic acid, Tween-20, Brij-35, sodium chloride, polyethylene glycol 8000 and sodium azide into a reactor in sequence while stirring, and stirring until the components are completely dissolved to ensure that the concentrations of the components respectively reach the following concentrations: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 355 g/L-10g/L of Brij, 10g/L of sodium chloride, 800010g/L of polyethylene glycol, 1g/L of sodium azide and 7.0 of pH value;
reagent 2:
(1) preparing 4-hydroxyethyl piperazine ethanesulfonic acid solution;
(2) diluting the latex particles coated with the mouse anti-human SAA monoclonal antibody to 1% by using the solution (1), and then preheating the solution to 34-37 ℃;
(3) dripping all mouse anti-human SAA monoclonal antibodies into the solution (2), uniformly mixing, and reacting for 30 minutes;
(4) fully dissolving sodium borohydride by using the solution in the step (1), and then completely adding the sodium borohydride into the latex solution in the step (2) to react for 60 minutes;
(5) after 60 minutes, adding casein and bovine serum albumin to react for 30 minutes;
(6) after 30 minutes, adding Tween-20;
(7) adding glucose as a protective agent after 10 minutes, and adding sodium azide as a preservative;
the concentrations of the components are respectively as follows: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 10g/L of latex particles coated with mouse anti-human SAA monoclonal antibodies, 250mg/L of mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 2g/L of casein, 5g/L, tween-202 ml/L of bovine serum albumin, 80g/L of glucose, 1g/L of sodium azide and 7.0 of pH value.
The application of the serum amyloid A detection kit in serum amyloid A detection.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. according to the serum amyloid A detection kit, the latex adopts aldehyde microspheres, sodium borohydride is added into a reaction medium containing the aldehyde microspheres and the amine-containing antibody, so that the Schiff base intermediate is reduced and a covalent bond is formed, secondary amine connection is generated between the two molecules, the connection is highly stable, the secondary amine connection cannot be easily hydrolyzed in a water environment, the combination of the aldehyde microspheres and the antibody is more stable, the antibody coating efficiency is improved, and the antibody dosage is reduced;
2. according to the serum amyloid A detection kit, the key point is that the antibody and the microsphere are activated by using sodium borohydride, the binding force of the antibody and the microsphere can be improved, casein and bovine serum albumin have the function of strengthening the antibody, so that the binding position of the antibody and the microsphere is more accurate, the coupling efficiency is improved, groups which do not participate in reaction on the microsphere are sealed, and the anti-interference capability of the reagent is improved by adding Brij-35 to the actual detection kit.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.
Example 1
The serum amyloid A detection kit comprises the following reagents:
reagent 1: 50mMol/L of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 10g/L of sodium chloride, 800010g/L of polyethylene glycol and 1g/L of sodium azide, wherein the pH value is 7.0;
reagent 2: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, latex particles coated with mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 2g/L of casein, 5g/L of bovine serum albumin, 80g/L of glucose and 1g/L of sodium azide.
The latex particles coated with the mouse anti-human SAA monoclonal antibody are as follows: aldehyde group modified polystyrene microspheres.
Example 2
The serum amyloid A detection kit is prepared by the following steps:
reagent 1:
(1) adding 4-hydroxyethyl piperazine ethanesulfonic acid, Tween-20, Brij-35, sodium chloride, polyethylene glycol 8000 and sodium azide into a reactor in sequence while stirring, and stirring until the components are completely dissolved to ensure that the concentrations of the components respectively reach the following concentrations: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 355 g/L-10g/L of Brij, 10g/L of sodium chloride, 800010g/L of polyethylene glycol, 1g/L of sodium azide and 7.0 of pH value;
reagent 2:
(1) preparing 4-hydroxyethyl piperazine ethanesulfonic acid solution;
(2) diluting the latex particles coated with the mouse anti-human SAA monoclonal antibody to 1% by using the solution (1), and then preheating the solution to 34-37 ℃;
(3) dripping all mouse anti-human SAA monoclonal antibodies into the solution (2), uniformly mixing, and reacting for 30 minutes;
(4) fully dissolving sodium borohydride by using the solution in the step (1), and then completely adding the sodium borohydride into the latex solution in the step (2) to react for 60 minutes;
(5) after 60 minutes, adding casein and bovine serum albumin to react for 30 minutes;
(6) after 30 minutes, adding Tween-20;
(7) adding glucose as a protective agent after 10 minutes, and adding sodium azide as a preservative;
the concentrations of the components are respectively as follows: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 10g/L of latex particles coated with mouse anti-human SAA monoclonal antibodies, 250mg/L of mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 2g/L of casein, 5g/L, tween-202 ml/L of bovine serum albumin, 80g/L of glucose, 1g/L of sodium azide and 7.0 of pH value.
Example 3
The serum amyloid A detection kit comprises the following reagents:
reagent 1: 50mMol/L of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 10g/L of sodium chloride, 800010g/L of polyethylene glycol and 1g/L of sodium azide, wherein the pH value is 7.0;
reagent 2: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, latex particles coated with mouse anti-human SAA monoclonal antibodies, 0.1g/L of EDAC, 2g/L of casein, 5g/L of bovine serum albumin, 80g/L of glucose and 1g/L of sodium azide.
This example differs from example 1 in that: the latex particles coated with the mouse anti-human SAA monoclonal antibody adopt conventional microspheres, the reagent 2 does not contain sodium borohydride as a component, and EDAC is used as an activating agent.
Example 4
The serum amyloid A detection kit comprises the following reagents:
reagent 1: 50mMol/L of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 10g/L of sodium chloride, 800010g/L of polyethylene glycol and 1g/L of sodium azide, wherein the pH value is 7.0;
reagent 2: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, latex particles coated with mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 2g/L of casein, 5g/L of bovine serum albumin, 80g/L of glucose and 1g/L of sodium azide.
This example differs from example 1 in that: reagent 2 contained no components casein and bovine serum albumin.
Example 5
The serum amyloid A detection kit comprises the following reagents:
reagent 1: 50mMol/L of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 10g/L of sodium chloride, 800010g/L of polyethylene glycol and 1g/L of sodium azide, and the pH value is 7.0;
reagent 2: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, latex particles coated with mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 80g/L of glucose and 1g/L of sodium azide.
The latex particles coated with the mouse anti-human SAA monoclonal antibody are as follows:
this example differs from example 1 in that: reagent 2 does not contain the components casein and bovine serum albumin, and reagent 1 does not contain the component Brij-35.
The kit in the embodiments 1, 3-5 is used for detecting serum amyloid A, the detection method is latex enhanced immunoturbidimetry, the operation method of the detection is the same, the detection instruments are all Hitachi 7180 full-automatic biochemical analyzers, and the two-point end point method is utilized for determination: reagent 1 and reagent 2 were mixed as per 4: the ratio of 1 is placed on the corresponding reagent position, and the calibrator/sample is placed on the corresponding position of the sample plate, and the following specific operations are performed as shown in the following table 1:
tables 1 to 1: C1-C6: antigen concentration
C1：0.0mg/L；C2：5.0mg/L；C3：10.0mg/L；
C4：25.0mg/L；C5：50.0mg/L；C6：100.0mg/L.
Tables 1 to 2:
the following test results were obtained:
(1) in example 1, the reagent was stored at 237 ℃ and the absorbance on days 1 to 7 was as shown in Table 2 below:
| calibrating absorbance | C1 | C2 | C3 | C4 | C5 | C6 |
| Day 1 | -0.0045 | 0.0225 | 0.0540 | 0.1890 | 0.3230 | 0.4380 |
| 7 days at 37 DEG C | -0.0035 | 0.0225 | 0.0565 | 0.1915 | 0.3145 | 0.4120 |
| Deviation of | / | 0.00％ | 4.63％ | 1.32％ | -2.63％ | -5.94％ |
As can be seen from Table 2, the maximum deviation in absorbance between day 1 and day 7 at 37 ℃ was < 6%. Therefore, the stability of the binding of the antibody to the microsphere is high.
(2) The example 3 and the example 1 are calibrated and tested by the instrument, and the absorbance is shown in the following table 3:
| calibrating absorbance | C1 | C2 | C3 | C4 | C5 | C6 |
| Example 3 | -0.0010 | 0.0067 | 0.0120 | 0.0182 | 0.0245 | 0.0306 |
| Example 1 | -0.0045 | 0.0225 | 0.0540 | 0.1890 | 0.3230 | 0.4380 |
As can be seen from Table 3, in example 3, the reaction absorbance of example 1 is higher than that of example 1, which indicates that the binding rate of the aldehyde-modified polystyrene microsphere and the antibody is higher than that of the conventional microsphere, and the sodium borohydride catalyzes the aldehyde-modified polystyrene microsphere to make the binding with the antibody more stable.
(3) The retroactive high-value control substance (target value 50.5) and low-value control substance (target value 14.5) are respectively taken and detected by the embodiment 4 and the embodiment 1, each control substance is detected for 10 times, the average value is calculated and compared with the target value, and the result is shown in the following table 4.
| High value quality control material | Example 4 | Example 1 |
| Mean value of | 24.8 | 46.6 |
| Relative deviation of | -50.89％ | -7.72％ |
| Low value quality control material | Example 4 | Example 1 |
| Mean value of | 6.9 | 13.1 |
| Relative deviation of | -52.41％ | -9.66％ |
As can be seen from Table 4, the detection value of the reagent in the formula of example 1 is closer to the target value of the quality control substance, and the deviation between the detection value of the reagent in the formula of example 4 and the target value is larger, which shows that the reagent reaction system is optimized by adding casein and bovine serum albumin.
(4) Comparison experiment of anti-interference capability of reagent: taking a retrospective calibrator with the concentration of about 12mg/L, averagely dividing into 4 parts, which are A, B, C, D groups, each group is 900ul, then adding 100ul normal saline into the group A, adding 100ul 1140 ul bilirubin into the group B, adding 100ul 2280 ul bilirubin into the group C, adding 100ul3420 ul bilirubin into the group D, and then carrying out A, B, C, D four groups of samples simultaneously with the reagent prepared in the example 5 and the reagent prepared in the example 1. The results are given in table 5 below:
| example 5 | Deviation of | Example 1 | Deviation of | |
| A | 10.7 | / | 12.3 | / |
| B | 12.4 | 15.89％ | 12.4 | 0.81％ |
| C | 15.3 | 42.99％ | 12.8 | 4.07％ |
| D | 16.9 | 57.94％ | 13.3 | 8.13％ |
Group a served as control samples, and B, C, D was compared to each group a for one deviation. As can be seen from Table 5, the anti-bilirubin interference ability of the formulation of example 1 is superior to the assay results of example 5.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. The serum amyloid A detection kit comprises a reagent 1 and a reagent 2, and is characterized in that the reagent 2 comprises latex particles coated with a mouse anti-human SAA monoclonal antibody, the mouse anti-human SAA monoclonal antibody, sodium borohydride, bovine serum albumin and casein.
2. The serum amyloid a detection kit according to claim 1, wherein the reagent 1 comprises: 4-hydroxyethyl piperazine ethanesulfonic acid, Tween-20, Brij-35, sodium chloride, polyethylene glycol 8000 and sodium azide, and the reagent 2 further comprises: 4-hydroxyethyl piperazine ethanesulfonic acid, Tween-20, glucose and sodium azide.
3. The serum amyloid a detection kit according to claim 2, wherein the concentration of each component of reagent 1 is: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 355 g/L-10g/L of Brij, 10g/L of sodium chloride, 800010g/L of polyethylene glycol, 1g/L of sodium azide and 7.0 of pH value;
the concentration of each component of the reagent 2 is as follows: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 10g/L of latex particles coated with mouse anti-human SAA monoclonal antibodies, 250mg/L of mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 2g/L of casein, 5g/L, tween-202 ml/L of bovine serum albumin, 80g/L of glucose, 1g/L of sodium azide and 7.0 of pH value.
4. The serum amyloid A detection kit and the preparation method thereof according to claim 1, wherein the latex particles coated with the mouse anti-human SAA monoclonal antibody are aldehyde-modified polystyrene microspheres.
5. The method of making a serum amyloid a assay kit according to any one of claims 1-4, comprising the steps of:
reagent 1:
(1) adding 4-hydroxyethyl piperazine ethanesulfonic acid, Tween-20, Brij-35, sodium chloride, polyethylene glycol 8000 and sodium azide into a reactor in sequence while stirring, and stirring until the components are completely dissolved to ensure that the concentrations of the components respectively reach the following concentrations: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 205-15 ml/L of Tween, 355 g/L-10g/L of Brij, 10g/L of sodium chloride, 800010g/L of polyethylene glycol, 1g/L of sodium azide and 7.0 of pH value;
reagent 2:
(1) preparing 4-hydroxyethyl piperazine ethanesulfonic acid solution;
(2) diluting the latex particles coated with the mouse anti-human SAA monoclonal antibody to 1% by using the solution (1), and then preheating the solution to 34-37 ℃;
(3) dripping all mouse anti-human SAA monoclonal antibodies into the solution (2), uniformly mixing, and reacting for 30 minutes;
(4) fully dissolving sodium borohydride by using the solution in the step (1), and then completely adding the sodium borohydride into the latex solution in the step (2) to react for 60 minutes;
(5) after 60 minutes, adding casein and bovine serum albumin to react for 30 minutes;
(6) after 30 minutes, adding Tween-20;
(7) adding glucose as a protective agent after 10 minutes, and adding sodium azide as a preservative;
the concentrations of the components are respectively as follows: 50mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 10g/L of latex particles coated with mouse anti-human SAA monoclonal antibodies, 250mg/L of mouse anti-human SAA monoclonal antibodies, 0.05g/L of sodium borohydride, 2g/L of casein, 5g/L, tween-202 ml/L of bovine serum albumin, 80g/L of glucose, 1g/L of sodium azide and 7.0 of pH value.
6. Use of the serum amyloid a assay kit according to any one of claims 1-4 in serum amyloid a assays.
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