CN111879942A - Full-range C-reactive protein latex enhanced immunoturbidimetric assay kit - Google Patents
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- 108010074051 C-Reactive Protein Proteins 0.000 title claims abstract description 63
- 102100032752 C-reactive protein Human genes 0.000 title claims abstract description 62
- 239000004816 latex Substances 0.000 title claims abstract description 39
- 229920000126 latex Polymers 0.000 title claims abstract description 39
- 238000003149 assay kit Methods 0.000 title claims abstract description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 93
- 239000004094 surface-active agent Substances 0.000 claims abstract description 22
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 19
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 19
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 239000007853 buffer solution Substances 0.000 claims abstract description 12
- 239000000701 coagulant Substances 0.000 claims abstract description 9
- 239000003755 preservative agent Substances 0.000 claims abstract description 8
- 230000002335 preservative effect Effects 0.000 claims abstract description 8
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 claims abstract description 3
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- 239000004005 microsphere Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 239000002245 particle Substances 0.000 claims description 10
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 7
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 239000008118 PEG 6000 Substances 0.000 claims description 5
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
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- 239000012190 activator Substances 0.000 claims description 4
- 239000002981 blocking agent Substances 0.000 claims description 4
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- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 3
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 claims description 3
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims description 3
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- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
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- 235000010234 sodium benzoate Nutrition 0.000 claims description 3
- 239000004299 sodium benzoate Substances 0.000 claims description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 3
- 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 2
- 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 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- 239000007832 Na2SO4 Substances 0.000 claims description 2
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 239000013504 Triton X-100 Substances 0.000 claims description 2
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- -1 DTT Chemical compound 0.000 claims 1
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- 239000002202 Polyethylene glycol Substances 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
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- 230000008733 trauma Effects 0.000 description 1
- 238000004879 turbidimetry Methods 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N33/531—Production of immunochemical test materials
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- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
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- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Nanotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit, which comprises an R1 reagent and an R2 reagent, wherein the R2 reagent is a latex reagent coupled with a C-reactive protein antibody, the R1 reagent comprises a buffer solution, a coagulant, an inorganic salt, a surfactant and a preservative, and the pH value of the R1 reagent is 5.0; the content of inorganic salt in the R1 reagent is 0.65-1.0mol/L, and the content of surfactant is 5-15 g/L; the R1 reagent also comprises 0.1-0.5g/L protein reducing agent. According to the invention, the pH value of the R1 reagent is reduced, and the high-concentration inorganic salt, the high-concentration surfactant and the low-concentration protein reducing agent are added into the R1 reagent, so that the sensitivity of the reagent is obviously enhanced, and meanwhile, the protein reducing agent can avoid the phenomenon that the linear range of the reagent is reduced due to the change of the pH, so that the full-range C-reactive protein latex enhanced immunoturbidimetry detection kit with the obviously enhanced sensitivity and linearity is obtained.
Description
Technical Field
The invention belongs to the technical field of clinical chemical detection, and particularly relates to a full-range C-reactive protein latex enhanced immunoturbidimetric assay kit.
Background
C-reactive protein (CRP) is a protein which rises sharply in plasma when a body is infected or damaged, and is a pentamer formed by non-covalent association of 5 identical non-glycosylated subunits, and disulfide bonds are formed between cysteines at positions 36 and 97 in each subunit; the CRP concentration in the plasma is extremely low in normal people, the concentration is obviously increased by ten times or even hundred times when cells are infected and tissues are damaged, and the CRP concentration is rapidly increased and decreased; the CRP rising speed, amplitude and duration are closely related to the severity of the disease condition and tissue damage; therefore, the method has better clinical significance for diagnosis, auxiliary diagnosis, curative effect observation and prognosis judgment of various diseases such as infection, inflammatory diseases, malignant tumors, connective tissue diseases, cardiovascular diseases, trauma and the like.
The clinical detection method of CRP concentration mainly includes latex agglutination method, immunochromatography, chemiluminescence method, radioimmunoassay, enzyme-linked immunosorbent assay, latex-enhanced turbidimetry, etc. CRP detection is roughly divided into two types according to the concentration detection range; the first is the common CRP assay, usually 3-200mg/L, but its sensitivity is relatively low and the linear range cannot cover trace CRP. The second type is the detection of hypersensitive CRP (high-sensitivity CRP, hs-CRP), the sensitivity can be lower than 0.3mg/L, CRP with the concentration range of 0.1-10mg/L can be accurately quantified, and the kit is clinically used for the assessment of the heart disease onset risk, but high-value samples cannot be detected.
At present, when a CRP latex enhanced immunoturbidimetry assay kit is used, two microspheres with different particle sizes are mostly adopted to be respectively coupled with two antibodies with different affinities and then mixed to serve as a reaction reagent, for example, patent CN110702908A discloses a full-scale C-reactive protein detection kit, wherein reagent R1 is a phosphate buffer solution, and reagent R2 is a conjugate formed by respectively crosslinking two kinds of latex with two kinds of particle sizes with two kinds of anti-human C-reactive protein antibodies. Although this method will result in a better reaction curve, there is a problem in that the ratio of large-sized microspheres is increased for enhanced sensitivity, resulting in poor linearity of the reagent; or the use of monoclonal antibodies to enhance linearity, leading to the problem of insufficient sensitivity of the reagent to trace CRP samples. And the monoclonal antibody is expensive and is not suitable for popularization and use in primary hospitals. In latex immunoturbidimetry, conventional methods for increasing the sensitivity of reagents to trace amounts of sample include: increasing the concentration of a sensitizer (such as polyethylene glycol series) or increasing the proportion of large-particle-size latex, and the like. But the non-specific reaction can be influenced by the high content of the sensitizer; increasing the proportion of large particle size latex leads to poor reagent linearity. Moreover, the above methods can only enhance the sensitivity of the reagent within a small range, and can also affect the linear range of the reagent and generate the back-band effect. Therefore, to obtain a full-scale C-reactive protein latex enhanced turbidimetric immunoassay reagent, the sensitivity of the reagent needs to be significantly enhanced without affecting the linear range.
Disclosure of Invention
The invention aims to provide a full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit, which is mainly used for optimizing an R1 reagent, and remarkably enhances the sensitivity of the reagent by reducing the pH value of the R1 reagent and adding a high-concentration inorganic salt, a high-concentration surfactant and a low-concentration protein reducing agent into an R1 reagent without influencing the linear range of the reagent.
In order to achieve the purpose, the invention adopts the technical scheme that:
a full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit, comprising a R1 reagent and a R2 reagent, wherein the R2 reagent is a latex reagent coupled with a C-reactive protein antibody, the R1 reagent comprises a buffer solution, a coagulant, an inorganic salt, a surfactant and a preservative, wherein the pH of the R1 reagent is 5.0; the content of inorganic salt in the R1 reagent is 0.65-1.0mol/L, and the content of surfactant is 5-15 g/L; the R1 reagent also comprises 0.1-0.5g/L protein reducing agent.
The specific principle of the invention is as follows: the conventional R1 reagent for treating serum samples mainly comprises a buffer and a coagulant, and appropriate amounts of inorganic salts and surfactants are added to maintain the activity of CRP. In order to avoid protein denaturation caused by pH change and too high content of inorganic salt and surfactant, the pH value of the conventional R1 reagent is 7.0-8.0, and the content of inorganic salt and surfactant is low. The invention is based on the special structure of CRP (namely CRP is a pentasphere formed by non-covalently linking 5 identical non-glycosylated subunits, and also has 1 inter-bond disulfide bond, the concave surface of the pentasphere contains a ligand binding site, and CRP exists in a pentamer form under a normal state), the content and the pH value of inorganic salt and surfactant in R1 reagent are adjusted, on the premise of not influencing the secondary structure of CRP, the non-covalent bond among CRP pentamers is destroyed by using high-concentration salt, high-concentration surfactant and low pH value, the disulfide bond is exposed, and then the disulfide bond is broken by using low-concentration protein reducing agent, so that CRP protein is decomposed into monomers. Then adding an R2 reagent coupled with CRP antibody, wherein the CRP monomer is easier to be immune combined with the active site of the antibody and form a compound net-shaped precipitate, thereby obviously enhancing the sensitivity of the detection reagent, and simultaneously, the addition of the protein reducing agent can avoid the phenomenon of the linear range reduction of the reagent caused by the pH change, thereby obtaining the full-range C-reactive protein latex enhanced immunoturbidimetry detection kit with obviously enhanced sensitivity and unaffected linearity.
Preferably, the content of the inorganic salt in the R1 reagent is 0.8mol/L, the content of the surfactant is 10g/L, and the content of the protein reducing agent is 0.3 g/L.
Preferably, the protein reducing agent is at least one of TCEP, DTT, β -mercaptoethanol, and SDS.
Preferably, the surfactant is at least one of Tween-20, Tween-80, EMULGEN A-60, EMULGEN A-90, EMULGEN B-66, EMULGEN 709, Brij-35, Triton X-405, Triton X-114, Triton X-100, and GENAPOLX-080.
Preferably, the inorganic salt is NaCl, KCl, CaCl2、MgCl2、Na2SO4、K2SO4At least one of (1).
Preferably, the content of the buffer solution in the R1 reagent is 50-100mmol/L, the content of the coagulant is 5-15g/L, and the content of the preservative is 0.5-1 g/L.
Preferably, the buffer solution is any one of MES buffer solution, PBS buffer solution, phosphate buffer solution, HEPES buffer solution and Tris-HCl buffer solution.
Preferably, the coagulant is at least one of PEG4000, PEG6000, PEG8000, PEG 20000.
Preferably, the preservative is at least one of sodium azide, potassium sorbate, sodium benzoate, Proclin series.
Preferably, the preparation method of the R2 reagent comprises the following steps:
s1, activating the latex microspheres with small particle size of 60-90nm by using an activating agent, adding a C-reactive protein antibody to couple with the latex microspheres with small particle size, adding a sealing agent to centrifuge, and carrying out ultrasonic resuspension by using R2 storage liquid to obtain A liquid;
s2, activating the large-particle-size latex microspheres with the particle sizes of 200-250nm by using an activator, adding a C-reactive protein antibody to couple with the large-particle-size latex microspheres, adding a sealing agent to centrifuge, and carrying out ultrasonic resuspension by using R2 storage liquid to obtain B liquid;
s3, mixing the solution A and the solution B according to the concentration ratio of 3:1-8:1 to obtain the R2 reagent;
wherein the activator is 1% EDC; the blocking agent is a mixture of 10% BSA and 10% glycine in equal proportion; the R2 stock solution is any one of MES buffer solution, phosphate buffer solution and Tris-HCl buffer solution.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the concentration of inorganic salt and surfactant in the R1 reagent is properly increased and the pH value of the R1 reagent is reduced, so that the noncovalent bond between CRP pentamers is destroyed, the disulfide bond is exposed, and then the disulfide bond is broken by using a low-concentration protein reducing agent, so that CRP protein is decomposed into monomers, and is easier to be subjected to immunological combination with the active site of an antibody and form complex reticular precipitation, thereby the sensitivity of the detection reagent is obviously enhanced, and meanwhile, the phenomenon that the linear range of the reagent is reduced due to the change of pH can be avoided by adding the protein reducing agent, so that the full-range C-reactive protein latex enhanced immunoturbidimetry detection kit with obviously enhanced sensitivity and linearity is obtained.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the examples and comparative examples of the invention, only one preparation method of the R2 reagent is listed, and the preparation method specifically comprises the following steps:
s1, taking 2mL of polystyrene carboxyl microspheres (from JSR Life Sciences) with the particle size of 83nm into an EP tube, adding 8mL of MES buffer (50mmol/L, pH5.0), adding 0.5mL of 1% EDC (from Thermo Fisher scientific), and stirring at 37 ℃ for 10min for activation; then adding 1mL of goat anti-human C reactive protein antibody with the concentration of 12mg/mL, and stirring for 2h at 37 ℃ to couple the antibody and the microspheres; adding 1mL of mixed solution of 10% BSA (from Roche) and 10% glycine (from national drug group) at equal ratio as blocking agent, centrifuging at 15000rpm for 35min, and ultrasonic-resuspending with 30mL of buffer solution (50mmol/L, pH7.5) to obtain solution A;
s2, adding 0.25mL of 240nm polystyrene carboxyl microspheres (from JSR Life Sciences) into an EP tube, adding 2mL of MES buffer (50mmol/L, pH5.0), adding 0.1mL of 1% EDC (from ThermoFisher Scientific), and stirring at 37 deg.C for 10min to activate; then adding 0.3mL of rabbit anti-human C-reactive protein antibody with the concentration of 12.3mg/mL, and stirring for 2h at 37 ℃ to couple the antibody and the microspheres; adding 1mL of mixed solution of 10% BSA (purchased from Roche) and 10% glycine (purchased from national drug group) at equal ratio as blocking agent, centrifuging at 10000rpm for 20min, and ultrasonic-resuspending with 30mLHEPES buffer solution (50mmol/L, pH7.5) to obtain solution B;
and S3, mixing the solution A and the solution B according to the concentration ratio of 5:1 to obtain the R2 reagent.
Example 1
The embodiment provides a full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit, which comprises an R1 reagent and an R2 reagent. The preparation method of the R2 reagent is shown above. The R1 reagent is: 60mmol/L MES buffer (pH5.0), 0.8mol/L NaCl, 10g/L Tween-20, 10g/L PEG6000, 0.8g/L sodium azide and 0.3g/L TCEP (tris (2-carboxyethyl) phosphine) mixed solution, and the R1 reagent pH is 5.0.
Example 2
The present embodiment is different from embodiment 1 in that: the content of NaCl in the R1 reagent is 0.65mol/L, the content of Tween-20 is 5g/L, and the content of TCEP is 0.1 g/L.
Example 3
The present embodiment is different from embodiment 1 in that: the content of NaCl in the R1 reagent is 1.0mol/L, the content of Tween-20 is 15g/L, and the content of TCEP is 0.5 g/L.
Example 4
The present embodiment is different from embodiment 1 in that: the R1 reagent includes: 100mmol/L Tris-HCl buffer (pH5.0), 0.8mol/L of an equal proportion mixture of NaCl and KCl, 10g/L of an equal proportion mixture of Triton X-114 and GENAPOLX-080, 15g/L of an equal proportion mixture of PEG6000 and PEG8000, 1.0g/L of an equal proportion mixture of sodium azide and Proclin300, and 0.3g/L DTT (dithiothreitol).
Example 5
The present embodiment is different from embodiment 1 in that: the R1 reagent includes: 50mmol/L PBS buffer (pH5.0), 0.8mol/L K2SO4And KCl, 10g/L of Tween-80 and EMULGEN A-60, 5g/L of PEG6000 and PEG20000, 0.5g/L of potassium sorbate and sodium benzoate, and 0.3g/L of beta-mercaptoethanol.
Comparative example 1
This comparative example differs from example 1 in that: the NaCl content in the R1 reagent was 0.5 mol/L.
Comparative example 2
This comparative example differs from example 1 in that: the NaCl content in the R1 reagent was 1.2 mol/L.
Comparative example 3
This comparative example differs from example 1 in that: the content of Tween-20 in the R1 reagent is 3 g/L.
Comparative example 4
This comparative example differs from example 1 in that: the content of Tween-20 in the R1 reagent is 18 g/L.
Comparative example 5
This comparative example differs from example 1 in that: there was no TCEP in the R1 reagent.
Comparative example 6
This comparative example differs from example 1 in that: the TCEP content in the R1 reagent was 0.6 g/L.
Comparative example 7
This comparative example differs from example 1 in that: the pH of the R1 reagent was 4.0.
Comparative example 8
This comparative example differs from example 1 in that: the pH of the R1 reagent was 7.0.
Application example
The reagents prepared in the above examples and comparative examples were used according to the conventional methods of use, and the analytical sensitivity and linear range of the different reagents were evaluated according to the following methods:
(1) evaluation of analytical sensitivity: repeatedly measuring 5% human serum albumin as a blank sample for 20 times, calculating a mean value X (mg/L) and a standard deviation SD, wherein the calculated mean value +2SD is an analysis sensitivity value, and the measurement result is shown in table 1;
(2) evaluation of Linear Range: measuring a sample with the C-reactive protein concentration of 0-350mg/L, repeatedly measuring each concentration for three times, calculating a measured mean value, obtaining a linear relation according to the theoretical concentration and the measured mean value, substituting the theoretical concentration into the linear relation to calculate a corresponding yi estimated value and the relative deviation between the mean value and the yi estimated value ((mean-yi value)/yi value); the correlation coefficient is more than or equal to 0.99, the relative deviation of each point is within 10%, the use requirement can be met, and the determination result is shown in table 2.
TABLE 1 analytical sensitivity evaluation data
TABLE 2 Linear evaluation data
Wherein, table 1 is the determination result of the analysis sensitivity, and the analysis sensitivity value is the detection limit, which is the lowest concentration of the reagent. In examples 1-3, the pH value, the contents of inorganic salt, surfactant and protein reducing agent are all within the protection range of the application, and the analysis sensitivity is less than 0.2 mg/L. And compared with example 1, when the kinds and contents of the buffer, coagulant, preservative and inorganic salt, surfactant and protein reducing agent were changed (examples 4 to 5), the influence on the analysis sensitivity was small. When the contents of the inorganic salt, the surfactant and the protein reducing agent and the pH value are changed (comparative examples 1 to 8), the detection value of the analysis sensitivity is obviously improved, namely the lowest concentration which can be detected by the reagent is obviously improved, and the sensitivity of the reagent is obviously reduced.
Table 2 shows the results of the linear detection, and it can be seen from the results that the reagents obtained in examples 1-5 all have linear correlation coefficients greater than 0.99 and relative deviations less than 10%, which indicates that the reagents prepared in examples 1-5 have good linear correlation, can meet the use requirements, and have linear high values as high as 350 mg/L. Whereas in the absence of protein reducing agent (comparative example 5), the pH value results in a significant effect on the linearity of the agent; when the pH value of the reagent is changed (comparative examples 7-8), the influence of the pH value on linearity cannot be compensated even if a protein reducing agent exists in the system, so that the linear correlation coefficients of the reagents in comparative example 5 and comparative examples 7-8 are both obviously lower than 0.99, and the relative deviation is both obviously greater than 10%, namely the linear correlation is obviously poor, and the linear high value cannot reach 350 mg/L. Comparative examples 2 and 4 have a low measurement value but have a small influence on the linear correlation because the inorganic salt or surfactant content is too high, which causes denaturation of part of the C-reactive protein.
Combining the measurement results of the above tables 1 and 2, wherein the sensitivity of the reagents is poor although the comparative examples 1 to 4 and the comparative example 6 have good linear correlation, the lowest detectable concentration is higher than 0.4mg/L, and the low-concentration C-reactive protein in the sample cannot be detected; and comparative example 5 and comparative examples 7 to 8 were poor in both sensitivity and linear correlation. Only the reagents of the embodiments 1 to 5 have significantly enhanced sensitivity, the analysis sensitivity can reach about 0.1mg/L, the linearity is not affected, and the high linear value can reach 350mg/L, so that the reagent can be used for detecting the content of common CRP and the content of hypersensitive CRP, thereby fully meeting the clinical use requirements.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit, comprising a R1 reagent and a R2 reagent, wherein the R2 reagent is a latex reagent coupled with a C-reactive protein antibody, the R1 reagent comprises a buffer solution, a coagulant, an inorganic salt, a surfactant and a preservative, and the pH value of the R1 reagent is 5.0; the content of inorganic salt in the R1 reagent is 0.65-1.0mol/L, and the content of surfactant is 5-15 g/L; the R1 reagent also comprises 0.1-0.5g/L protein reducing agent.
2. The full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit according to claim 1, wherein the content of inorganic salts in the R1 reagent is 0.8mol/L, the content of surfactant is 10g/L, and the content of protein reducing agent is 0.3 g/L.
3. The full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit according to claim 1 or 2, wherein the protein reducing agent is at least one of TCEP, DTT, beta-mercaptoethanol and SDS.
4. The full-scale C-reactive protein latex-enhanced immunoturbidimetric assay kit according to claim 1 or 2, wherein the surfactant is at least one of Tween-20, Tween-80, EMULGEN A-60, EMULGEN A-90, EMULGEN B-66, EMULGEN 709, Brij-35, Triton X-405, Triton X-114, Triton X-100 and GENAPOLX-080.
5. The full-scale C-reactive protein latex-enhanced immunoturbidimetric assay kit according to claim 1 or 2, wherein the inorganic salt is NaCl, KCl, CaCl2、MgCl2、Na2SO4、K2SO4At least one of (1).
6. The full-scale range C-reactive protein latex enhanced immunoturbidimetric assay kit according to claim 1, wherein the buffer solution content in the R1 reagent is 50-100mmol/L, the coagulant content is 5-15g/L, and the preservative content is 0.5-1 g/L.
7. The full-scale C-reactive protein latex-enhanced immunoturbidimetric assay kit according to claim 1 or 6, wherein the buffer is any one of MES buffer, PBS buffer, phosphate buffer, HEPES buffer, Tris-HCl buffer.
8. The full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit according to claim 1 or 6, wherein the coagulant is at least one of PEG4000, PEG6000, PEG8000 and PEG 20000.
9. The full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit according to claim 1 or 6, wherein the preservative is at least one of sodium azide, potassium sorbate, sodium benzoate and Proclin series.
10. The full-scale C-reactive protein latex enhanced immunoturbidimetric assay kit according to claim 1, wherein the preparation method of the R2 reagent comprises the following steps:
s1, activating the latex microspheres with small particle size of 60-90nm by using an activating agent, adding a C-reactive protein antibody to couple with the latex microspheres with small particle size, adding a sealing agent to centrifuge, and carrying out ultrasonic resuspension by using R2 storage liquid to obtain A liquid;
s2, activating the large-particle-size latex microspheres with the particle sizes of 200-250nm by using an activator, adding a C-reactive protein antibody to couple with the large-particle-size latex microspheres, adding a sealing agent to centrifuge, and carrying out ultrasonic resuspension by using R2 storage liquid to obtain B liquid;
s3, mixing the solution A and the solution B according to the concentration ratio of 3:1-8:1 to obtain the R2 reagent;
wherein the activator is 1% EDC; the blocking agent is a mixture of 10% BSA and 10% glycine in equal proportion; the R2 stock solution is at least one of MES buffer solution, phosphate buffer solution and Tris-HCl buffer solution.
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