CN117517647A - Latex-enhanced immunoturbidimetry detection kit and preparation method thereof - Google Patents
Latex-enhanced immunoturbidimetry detection kit and preparation method thereof Download PDFInfo
- Publication number
- CN117517647A CN117517647A CN202311387064.7A CN202311387064A CN117517647A CN 117517647 A CN117517647 A CN 117517647A CN 202311387064 A CN202311387064 A CN 202311387064A CN 117517647 A CN117517647 A CN 117517647A
- Authority
- CN
- China
- Prior art keywords
- latex
- antibody
- reagent
- coated
- latex particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000004816 latex Substances 0.000 title claims abstract description 90
- 229920000126 latex Polymers 0.000 title claims abstract description 90
- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 239000002245 particle Substances 0.000 claims abstract description 70
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 62
- 229940045944 sodium lauroyl glutamate Drugs 0.000 claims abstract description 28
- IWIUXJGIDSGWDN-UQKRIMTDSA-M sodium;(2s)-2-(dodecanoylamino)pentanedioate;hydron Chemical compound [Na+].CCCCCCCCCCCC(=O)N[C@H](C([O-])=O)CCC(O)=O IWIUXJGIDSGWDN-UQKRIMTDSA-M 0.000 claims abstract description 28
- 239000000872 buffer Substances 0.000 claims abstract description 16
- 239000003381 stabilizer Substances 0.000 claims abstract description 15
- 239000003792 electrolyte Substances 0.000 claims abstract description 14
- 239000007853 buffer solution Substances 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- 238000003018 immunoassay Methods 0.000 claims description 11
- 239000003755 preservative agent Substances 0.000 claims description 11
- 230000002335 preservative effect Effects 0.000 claims description 11
- 239000007987 MES buffer Substances 0.000 claims description 10
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 claims description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- 239000007995 HEPES buffer Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 4
- 229930182566 Gentamicin Natural products 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 4
- 229960002518 gentamicin Drugs 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 229940033663 thimerosal Drugs 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 16
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 230000000052 comparative effect Effects 0.000 description 31
- 238000012360 testing method Methods 0.000 description 16
- 239000004005 microsphere Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 13
- 230000009021 linear effect Effects 0.000 description 13
- 239000004094 surface-active agent Substances 0.000 description 9
- 238000005859 coupling reaction Methods 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 230000004913 activation Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000013112 stability test Methods 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000005484 gravity Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000001516 effect on protein Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000007779 soft material Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid 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
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
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
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention discloses a latex-enhanced immunoturbidimetry detection kit and a preparation method thereof, wherein the latex-enhanced immunoturbidimetry detection kit comprises a reagent R2, and the reagent R2 comprises the following components in concentration: 25 mM-50 mM R2 buffer, 5wt.% to 10wt.% of antibody-coated latex particles, 0.05wt.% to 0.1wt.% sodium lauroyl glutamate, 1g/L-50g/L stabilizer, and 5g/L-40g/L R2 electrolyte. According to the invention, sodium lauroyl glutamate is added into the reagent R2, so that the storage stability of the reagent R2 system is improved, and the reaction sensitivity is prevented from being reduced.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a latex-enhanced immunoturbidimetry detection kit and a preparation method thereof.
Background
The latex enhanced immunoturbidimetry is a relatively stable and accurate humoral protein homogeneous phase immunoturbidimetry detection method which appears in recent years. The detection principle is that the surface of the macromolecule nanometer microsphere is coated with a specific antibody, and when the antigen in the sample is mixed with the nanometer microsphere coated with the specific antibody, the antigen can be quickly gathered together in a short time to form an antigen-antibody-nanometer microsphere compound, so that the change of absorbance is caused. The change value of the absorbance of the reaction liquid has stronger correlation with the concentration of the detected antigen, and the content of the detected antigen in the sample can be reacted in a certain range.
The existing latex enhanced immunoturbidimetry detection kit generally comprises a reagent R1, a reagent R2, a calibration reagent and a quality control reagent, wherein the main component in the reagent R2 is a nanoparticle coated with an antibody. In the antibody-latex system of the reagent R2, various interaction forces such as physical chemistry exist among the nano-microspheres, the antibodies and the antibodies, and the mutual balance of the forces is an important precondition for maintaining the stability of the antibody-latex system and ensuring that the product always has better immunoreactivity. However, during actual storage, the reagent R2 system tends to be unstable due to various factors, and its response sensitivity gradually decreases with the increase of storage time.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a latex-enhanced immunoturbidimetry detection kit and a preparation method thereof, wherein sodium lauroyl glutamate is added into a reagent R2 to improve the storage stability of the reagent R2 system, so that the reaction sensitivity is prevented from being reduced.
In order to achieve the above object, a first technical solution of the present invention is as follows:
a latex enhanced immunoturbidimetry detection kit comprises a reagent R2,
the reagent R2 comprises the following components in concentration:
25 mM-50 mM R2 buffer, 5wt.% to 10wt.% of antibody-coated latex particles, 0.05wt.% to 0.1wt.% sodium lauroyl glutamate, 1g/L-50g/L stabilizer, and 5g/L-40g/L R2 electrolyte.
The second technical scheme of the invention is as follows:
the preparation method of the latex enhanced turbidimetric immunoassay detection kit comprises the following steps:
preparing the antibody-coated latex particles;
mixing the R2 buffer solution, the latex particles coated by the antibody, the sodium lauroyl glutamate, the stabilizer and the R2 preservative to obtain the reagent R2.
The implementation of the embodiment of the invention has the following beneficial effects:
according to the embodiment of the invention, the sodium lauroyl glutamate is added into the reagent R2, the hydrophobic terminal contained in the sodium lauroyl glutamate seals the hydrophobic region on the surface of the latex particles, so that the hydrophilicity of the latex particles is enhanced, and the mutual adhesion among the latex particles is prevented, thereby improving the suspension stability of the antibody-coated latex particle compound of the reagent R1, helping the antibody-coated latex particle compound overcome the action of gravity, enabling the antibody-coated latex particle compound to be uniformly suspended in a buffer solution and keep a stable state, avoiding the mutual aggregation among the latex microsphere compounds to generate precipitation, and enabling the reagent R2 to have better stability and repeatability. Sodium lauroyl glutamate belongs to an amino acid, has high safety to environment and organisms, good biodegradability, good compatibility with other various surfactants, and good antibacterial and bactericidal properties.
The invention protects the antibody through the stabilizer, maintains the structure of the antibody, and improves the stability of the reagent R2.
The invention constructs a liquid environment similar to the cell internal environment through the sodium lauroyl glutamate cooperative stabilizer, the electrolyte and the like, and utilizes the principle that the cell internal environment has macromolecular crowding or limiting effect, namely, in crowding environment, other biological macromolecules occupy most of the volume, can form a size exclusion effect on protein, and the available space of the protein is obviously reduced, so that the stability of the protein can be improved, and the sodium lauroyl glutamate cooperative stabilizer, the electrolyte and the like form macromolecular crowding environment, thereby improving the long-term stability of the antibody and the latex particle compound coated by the antibody. The stability of the reagent R2 is increased, so that the latex microsphere compound included in the antibody can be uniformly dispersed at each corner in the solution, and the stability and the reaction sensitivity of the reagent can be improved.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention discloses a latex-enhanced immunoturbidimetry detection kit, which comprises a reagent R2, wherein the reagent R2 comprises the following components in concentration:
25 mM-50 mM R2 buffer, 5wt.% to 10wt.% of antibody-coated latex particles, 0.05wt.% to 0.1wt.% sodium lauroyl glutamate, 1g/L-50g/L stabilizer, and 5g/L-40g/L R2 electrolyte.
In the prior art, the reason for the instability of the reagent R2 is: first, the latex particles coated with the antibody gradually aggregate at the bottom of the buffer solution due to the gravity of the latex particles during long-term storage to form a precipitate, and second, the storage environment is unfavorable for the stability of the antibody, and the structure of the latex particles is destroyed, so that the stability of the reagent R2 is destroyed. This is related to the nature of the antibody itself, which is a proteinaceous soft material whose essential characteristics are that under minor external effects (including temperature and external forces, etc.), a pronounced macroscopic effect is produced, a so-called small-impact large effect being a fundamental characteristic of soft material systems.
According to the invention, sodium lauroyl glutamate is added into the reagent R2, the hydrophobic end of the sodium lauroyl glutamate is used for sealing the surface hydrophobic region of the latex particles, so that the hydrophilicity of the latex particles is enhanced, and the mutual adhesion among the latex particles is prevented, thereby improving the suspension stability of the antibody-coated latex particle compound of the reagent R1, helping the antibody-coated latex particle compound to overcome the action of gravity, enabling the antibody-coated latex particle compound to be uniformly suspended in a buffer solution and keep a stable state, avoiding the mutual aggregation among the latex microsphere compounds to generate precipitation, and enabling the reagent R2 to have better stability and repeatability. Sodium lauroyl glutamate belongs to an amino acid, has high safety to environment and organisms, good biodegradability, good compatibility with other various surfactants, and good antibacterial and bactericidal properties.
The invention protects the antibody through the stabilizer, maintains the structure of the antibody, and improves the stability of the reagent R2.
The invention constructs a liquid environment similar to the cell internal environment through the sodium lauroyl glutamate cooperative stabilizer, the electrolyte and the like, and utilizes the principle that the cell internal environment has macromolecular crowding or limiting effect, namely, in crowding environment, other biological macromolecules occupy most of the volume, can form a size exclusion effect on protein, and the available space of the protein is obviously reduced, so that the stability of the protein can be improved, and the sodium lauroyl glutamate cooperative stabilizer, the electrolyte and the like form macromolecular crowding environment, thereby improving the long-term stability of the antibody and the latex particle compound coated by the antibody. The stability of the reagent R2 is increased, so that the latex microsphere compound included in the antibody can be uniformly dispersed at each corner in the solution, and the stability and the reaction sensitivity of the reagent can be improved.
In a specific embodiment, the stabilizer comprises one or more of trehalose, sucrose, and mannitol; the R2 electrolyte comprises one or more of sodium chloride, potassium chloride and magnesium chloride; the R2 buffer solution comprises one or more than two of MOPSO, MES, HEPES, phosphate and Tris buffer solution; the pH of the reagent R2 is 7.0-7.5.
In a preferred embodiment, the antibodies are RBP antibodies, the RBP level in serum is closely related to human kidney function, and when kidney diseases occur, the RBP level in serum and urine can be obviously increased to more than 200mg/L due to reduced glomerular filtration rate and renal tubule reabsorption disorder. The conventional RBP measuring range is 3-140mg/L, so that the high-linearity RBP measuring reagent can better meet clinical requirements. The invention can improve the reaction sensitivity of the immunoturbidimetry detection by improving the long-term stability of the latex particle complex in the reagent R2.
Furthermore, the linear range and sensitivity of detection are further improved by arranging large-particle-size antibody-coated latex particles and small-particle-size antibody-coated latex particles, specifically, the large-particle-size antibody-coated latex particles have a particle size of 150nm to 300nm and the small-particle-size antibody-coated latex particles have a particle size of 60nm to 90nm.
Preferably, the volume ratio of the large particle size antibody-coated latex particles to the small particle size antibody-coated latex particles is 1:1 to 1:10.
in one embodiment, the RBP antibody is present in the reagent R2 at a concentration of 0.02mg/mL to 0.2mg/mL.
In a specific embodiment, the agent R2 further comprises 0.05% -0.5% preservative. Specifically, the R2 preservative comprises one or more than two of PC-300, gentamicin, merthiolate and sodium azide.
In a specific embodiment, the latex-enhanced turbidimetric immunoassay kit further comprises a reagent R1, wherein the reagent R1 comprises the following components in concentration:
50 mM-100 mM of R1 buffer solution, 5g/L-40g/L of R1 electrolyte and 0.05% -0.5% of R1 preservative.
Preferably, the R1 buffer comprises one or more than two of MOPSO, MES, HEPES, phosphate and Tris buffers; the R1 electrolyte comprises one or more of sodium chloride, potassium chloride and magnesium chloride; the R1 preservative comprises one or more than two of PC-300, gentamicin, merthiolate and sodium azide; the pH of the reagent R1 is 7.0 to 7.5.
The invention also discloses a preparation method of the latex enhanced immunoturbidimetry detection kit, which comprises the following steps:
preparing antibody coated latex particles;
mixing the R2 buffer solution, the latex particles coated by the antibody, sodium lauroyl glutamate, a surfactant, a blocking agent, a stabilizer and an R2 preservative to obtain the reagent R2.
The preparation method of the antibody coated latex particles comprises the following steps:
1) And mixing the latex particles with an activation buffer solution for activation to obtain an activated latex particle solution.
In this step, the pH of the activation buffer is 6.5-7.0 and the concentration is 50 mM-100 mM, and the activation buffer may include one or more than two of MOPSO, MES, HEPES buffers.
2) And diluting the antibody by using a coupling buffer solution, adding the diluted antibody into the activated latex particle solution, and carrying out a coupling reaction to obtain a reaction solution after the coupling reaction.
In this step, the pH of the coupling buffer is 7.5-8.0 and the concentration is 50 mM-100 mM, and the coupling buffer may include one or more of MOPSO, MES, HEPES buffers.
Preferably, the conditions of the coupling reaction are: and reacting for 1-2 h at the constant temperature of 35-40 ℃.
3) And adding a blocking solution into the reaction solution after the coupling reaction to carry out the blocking reaction, and obtaining the latex particles coated with the antibody after the reaction is completed.
In this step, the mass concentration of the blocking solution is 0.1% -0.5%.
In this step, antibody-coated latex particles are obtained by centrifugation after completion of the reaction. The centrifugal speed is 10000 rmp-20000 rmp, and the centrifugal time is 30 min-60 min.
Preferably, the conditions of the blocking reaction are: and reacting for 1-2 h at the constant temperature of 35-40 ℃.
The following are specific examples.
Example 1
A latex enhanced turbidimetric immunoassay kit comprises a reagent R1 and a reagent R2.
Reagent R1 comprises the following components in concentration:
50-100mM MOPSO buffer, 15g/L NaCl,0.1wt.% sodium azide, pH 7.2.
Reagent R2 comprises the following concentration components:
25-50mM MOPSO buffer, 0.01% solid content of 300nm large particle size antibody coated latex particles, 0.15% solid content of 60nm small particle size antibody coated latex particles, 10g/L NaCl,5wt.% trehalose, 0.05wt.% sodium lauroyl glutamate, 0.1wt.% PC-300, pH 7.0-7.5.
The preparation of reagent R2 comprises the following steps:
1) Mixing 1mL of latex microsphere with the particle size of 60nm with an activation buffer solution (30 mmol/L MES buffer solution, pH value of 6.5) according to a volume ratio of 1:8, and uniformly mixing;
2) 0.6mL of 10mg/mL activator (EDC is weighed according to the concentration of 10mg/mL and is used in preparation) is added into the step (1), evenly mixed and placed in a constant temperature shaking table for reaction for 20min, and the temperature is: 37 ℃;
3) Diluting the RBP antibody to 1mg/mL with coupling buffer (30 mmol/L MES buffer, pH 7.5), adding 6mL of the diluted RBP antibody to the step (2), mixing uniformly, and placing in a constant temperature shaking table for reaction for 2h, wherein the temperature is as follows: 37 ℃;
4) 1mL of blocking solution (0.5 wt.% BSA) was added to step (3), mixed well, and placed in a constant temperature shaker for 2h of reaction at the temperature: 37 ℃;
5) Centrifuging after the reaction is completed, removing supernatant, wherein the rotating speed is 15000rpm, and the centrifuging time is 45 minutes;
6) Removing the supernatant, re-dissolving with 60mL of R2 buffer solution, and re-suspending the latex microspheres, wherein a cell disruption instrument can be used for assisting in re-suspending in the re-suspending process;
7) Mixing 0.5mL of latex microsphere with the particle size of 300nm with an activation buffer solution (30 mmol/L MES buffer solution, pH value of 6.5) according to a volume ratio of 1:6, and uniformly mixing;
8) 0.4mL of 10mg/mL activator (EDC is weighed according to the concentration of 10mg/mL and is used in preparation) is added into the step (7), evenly mixed and placed in a constant temperature shaking table for reaction for 20min, and the temperature is: 37 ℃; the other steps are the same as the preparation steps of the small-particle-size antibody-coated latex particle reagent, and 300nm large-particle-size antibody-coated latex particles are prepared.
9) Mixing the latex microsphere coated by the large-particle-size antibody and the latex microsphere coated by the small-particle-size antibody according to the volume ratio of 1:6, and then adding the mixture into a mixed solution of MOPSO, naCl, trehalose, sodium lauroyl glutamate and PC-300 to obtain the reagent R2.
Example 2
Example 2 differs from example 1 in that: the 90nm small particle size antibody coated latex particles, the 200nm large particle size antibody coated latex particles, and 0.07wt.% sodium lauroyl glutamate were the same as in example 1.
Example 3
Example 4 differs from example 1 in that: the buffer was changed to 50mM HEPES buffer, and the other components and the process were the same as in example 1.
Example 4
Example 4 differs from example 1 in that: the buffer was changed to 35mM MES buffer, 0.1wt.% sodium lauroyl glutamate, and the other components and processes were the same as in example 1.
Comparative example 1
Comparative example 1 differs from example 1 only in that: latex particles coated with only one 60nm small particle size antibody.
Comparative example 2
Comparative example 2 differs from example 1 only in that: latex particles coated with only one 300nm small particle size antibody.
Comparative example 3
Comparative example 3 differs from example 3 in that: 0.05wt.% sodium lauroyl glutamate was replaced with 0.05% Triton X-100, and the other components and processes were the same as in example 3.
Comparative example 4
Comparative example 4 differs from example 4 in that: 0.1wt.% sodium lauroyl glutamate was replaced with 0.1wt.% Triton X-100, and the other components and processes were the same as in example 1.
Comparative example 5
Comparative example 5 differs from example 3 in that: the sodium lauroyl glutamate was removed and the other components and processes were the same as in example 1.
Test example 1 (Linear Range evaluation)
Experimental instrument selection: keman CP880, prepared with the reagents of comparative examples 1-2 and examples 1-2, was calibrated by selecting RBP calibrator, and then tested for linear range and stability, and evaluated as follows: linear range evaluation: measuring samples with different concentrations of 0.5-310mg/L, repeatedly measuring each concentration for three times, and calculating a mean theoretical value, a relative deviation and a linear correlation coefficient R; r is more than 0.9900, the relative deviation of each point is within 10 percent, and the use requirement can be met. The results are shown in Table 1.
Table 1: linear range data for examples 1-2 and comparative examples 1-2
As can be seen from the data in table 1, the linear properties of example 1 and example 2 measured 310mg/L, and the linear correlation coefficient R and the relative deviation of the respective concentrations were all satisfactory; comparative example 1 shows 310mg/L linear properties, but the lower-stage relative deviation is more than 10%, and the correlation coefficient R is less than 0.99; comparative example 2 was only about 38.75mg/L linear, indicating a wider linear range of latex microsphere size particle size combinations. From the broader linear range of comparative example 1 relative to comparative example 2, the better sensitivity of comparative example 2 relative to comparative example 1, it was demonstrated that the small particle size of the latex microspheres has a major effect on the linear range and the large particle size has a major effect on the sensitivity.
Test example 2 (precision evaluation)
Experimental instrument selection: the Keman CP880, using the reagents prepared in comparative examples 4-5 and examples 3-4, selects RBP calibrator for calibration, and then detects the precision, and the evaluation method is as follows: precision evaluation: samples were assayed at 40mg/L and 150mg/L concentrations, each concentration was repeated 10 times, and the mean was calculated. The results are shown in tables 2 and 3.
Table 2: examples 3-4, comparative examples 4-5 Low value sample precision data
Comparative example 4 | Comparative example 5 | Example 3 | Example 4 | |
Test 1 | 38.42 | 46.58 | 39.47 | 42.37 |
Test 2 | 41.33 | 31.69 | 38.99 | 40.25 |
Test 3 | 43.56 | 36.77 | 42.64 | 39.53 |
Test 4 | 35.33 | 32.27 | 41.93 | 38.24 |
Test 5 | 42.59 | 45.42 | 42.85 | 41.27 |
Test 6 | 44.44 | 42.66 | 41.34 | 39.96 |
Test 7 | 36.56 | 41.27 | 38.98 | 40.29 |
Test 8 | 37.39 | 37.38 | 39.64 | 41.04 |
Test 9 | 40.25 | 39.64 | 40.08 | 42.59 |
Test 10 | 41.38 | 32.33 | 41.19 | 43.33 |
AVE | 40.125 | 38.601 | 40.711 | 40.887 |
SD | 3.08 | 5.45 | 1.47 | 1.55 |
CV | 7.69% | 14.11% | 3.61% | 3.80% |
Table 3: examples 3-4, comparative examples 4-5 high value sample precision data
As can be seen from the data in tables 2 and 3: the data in examples 3 and 4 show that the coefficient of variation is smaller for both low and high value samples than for the comparative example set, and that no surfactant was added in comparative example 5, so that the precision CV for the two different levels of calibrator test was greater than 10%, indicating that the addition of surfactant had a greater impact on the precision of the reagents.
Test example 3 (stability evaluation)
Experimental instrument selection: the prepared reagents of comparative examples 4 to 5 and examples 3 to 4 were placed in a 37℃incubator and a 2 8 ℃refrigerator, respectively, in a Keman CP 880. After RBP calibrator is selected for calibration, stability is detected, and the evaluation method is as follows: stability evaluation: samples of 40mg/L and 150mg/L concentration were assayed at 2-8deg.C by removing the reagents from the Keman CP880 apparatus for testing at week 1, week 2, week 3, week 4, month 2, month 4, month 6, month 8, month 10, month 11, month 12; the reagents were removed on a Keman CP880 instrument at 37℃for testing on days 1, 2, 3, 4, 5, 6, 7, 10, 12, 14, 15. The results are shown in tables 4 to 7.
Table 4: stability test data for examples 3-4 and comparative examples 4-5 at 2-8deg.C
Table 5: stability test data for examples 3-4 and comparative examples 4-5 at 2-8deg.C
Table 6: stability test data at 37℃for examples 3-4 and comparative examples 4-5
Table 7: stability test data at 37℃for examples 3-4 and comparative examples 4-5
From the data in tables 4 to 7, it can be seen that: the examples 3 and 4 with the surfactant added have good performance in the accelerated stability test at 2-8 ℃ and 37 ℃, but the comparative example 5 without the surfactant has poor stability test results, the surfactants used in the examples 3 and 4 are amino acid type surfactant sodium lauroyl glutamate, and the stability effect is better than Triton X-100, so that the stability of the kit is improved by using sodium lauroyl glutamate.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A latex enhanced immunoturbidimetry detection kit is characterized by comprising a reagent R2,
the reagent R2 comprises the following components in concentration:
25 mM-50 mM R2 buffer, 5wt.% to 10wt.% of antibody-coated latex particles, 0.05wt.% to 0.1wt.% sodium lauroyl glutamate, 1g/L-50g/L stabilizer, and 5g/L-40g/L R2 electrolyte.
2. The latex-enhanced turbidimetric immunoassay kit of claim 1, wherein said stabilizer comprises one or more of trehalose, sucrose, mannitol;
the R2 electrolyte comprises one or more of sodium chloride, potassium chloride and magnesium chloride.
3. The latex-enhanced turbidimetric immunoassay kit of claim 1, wherein said R2 buffer comprises one or more of MOPSO, MES, HEPES, phosphate and Tris buffer;
the pH of the reagent R2 is 7.0-7.5.
4. The kit for detecting the immune turbidimetric latex of claim 1, wherein the antibody-coated latex particles comprise large-size antibody-coated latex particles and small-size antibody-coated latex particles, the large-size antibody-coated latex particles have a particle size of 150nm to 300nm, and the small-size antibody-coated latex particles have a particle size of 60nm to 90nm.
5. The latex-enhanced turbidimetric immunoassay kit of claim 4, wherein said antibody is an RBP antibody and the concentration of said RBP antibody in said reagent R2 is 0.02mg/mL to 0.2mg/mL.
6. The latex-enhanced turbidimetric immunoassay kit of claim 1, wherein said reagent R2 further comprises 0.05% to 0.5% of an R2 preservative.
7. The latex-enhanced turbidimetric immunoassay kit of claim 6, wherein said R2 preservative comprises one or more of PC-300, gentamicin, thimerosal and sodium azide.
8. The latex-enhanced turbidimetric immunoassay kit of any of claims 1 to 7, further comprising a reagent R1, said reagent R1 comprising the following concentrations of the components:
50 mM-100 mM of R1 buffer solution, 5g/L-40g/L of R1 electrolyte and 0.05% -0.5% of R1 preservative.
9. The latex-enhanced turbidimetric immunoassay kit of claim 8, wherein said R1 buffer comprises one or more of MOPSO, MES, HEPES, phosphate and Tris buffer;
the R1 electrolyte comprises one or more of sodium chloride, potassium chloride and magnesium chloride;
the R1 preservative comprises one or more than two of PC-300, gentamicin, merthiolate and sodium azide;
the pH of the reagent R1 is 7.0-7.5.
10. A method for preparing the latex-enhanced turbidimetric immunoassay kit of any one of claims 1 to 9, comprising the following steps:
preparing the antibody-coated latex particles;
mixing the R2 buffer solution, the latex particles coated by the antibody, the sodium lauroyl glutamate, the stabilizer and the R2 preservative to obtain the reagent R2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311387064.7A CN117517647A (en) | 2023-10-25 | 2023-10-25 | Latex-enhanced immunoturbidimetry detection kit and preparation method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311387064.7A CN117517647A (en) | 2023-10-25 | 2023-10-25 | Latex-enhanced immunoturbidimetry detection kit and preparation method thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117517647A true CN117517647A (en) | 2024-02-06 |
Family
ID=89755826
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311387064.7A Pending CN117517647A (en) | 2023-10-25 | 2023-10-25 | Latex-enhanced immunoturbidimetry detection kit and preparation method thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117517647A (en) |
-
2023
- 2023-10-25 CN CN202311387064.7A patent/CN117517647A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103048464B (en) | Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof | |
CN103123319B (en) | Heart-type fatty acid binding protein content detection kit and preparation method thereof | |
CN102590524A (en) | Assay kit for neutrophil gelatinase-associated lipocalin | |
CN112730839B (en) | Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method | |
CN103323596B (en) | Detection kit for myeloperoxidase content and preparation method thereof | |
CN103954766B (en) | Transferring receptor detection kit and preparation method thereof | |
CN111965342B (en) | Method, reagent and kit for improving linear range and stability of latex immunoturbidimetry | |
CN106932594A (en) | A kind of preferred cystatin C detection kit and preparation method thereof | |
CN109470872B (en) | Magnetic particle buffer | |
CN112698023B (en) | Preservation solution of alkaline phosphatase-labeled conjugate and preparation method thereof | |
CN117517647A (en) | Latex-enhanced immunoturbidimetry detection kit and preparation method thereof | |
CN112285345A (en) | Glycocholic acid detection kit | |
CN111320696A (en) | MMP-3 antibody compound based on streptavidin latex and kit thereof | |
JP5191291B2 (en) | Method and reagent kit for measuring hemoglobin in stool specimen | |
CN114594269B (en) | Serum amyloid A detection kit and preparation method thereof | |
CN114487446A (en) | Stable apolipoprotein B determination kit with strong anti-interference capability | |
CN115856317A (en) | Rheumatoid factor determination kit | |
CN110221081B (en) | Retinol binding protein latex reagent R2, preparation method and kit thereof | |
CN111766381A (en) | Determination kit based on latex immunoturbidimetry and application thereof | |
CN112782409A (en) | Kit for determining IV type collagen in human blood and detection method | |
CN112595845A (en) | Hyaluronic acid detection kit and detection system | |
CN115327137B (en) | Human immunoglobulin G4 subtype chemiluminescence immunoassay kit | |
JPH1151938A (en) | Immunological latex nephelometry quantifying reagent | |
CN111896757B (en) | D-dimer determination kit for improving whole blood sample measurement value, preparation method and application | |
CN107976537B (en) | Stabilizer for prealbumin determination kit and preparation method thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |