CN111896752A - C-reactive protein kit suitable for various POCT instruments - Google Patents
C-reactive protein kit suitable for various POCT instruments Download PDFInfo
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- CN111896752A CN111896752A CN202010800285.2A CN202010800285A CN111896752A CN 111896752 A CN111896752 A CN 111896752A CN 202010800285 A CN202010800285 A CN 202010800285A CN 111896752 A CN111896752 A CN 111896752A
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- 108010074051 C-Reactive Protein Proteins 0.000 title claims abstract description 134
- 102100032752 C-reactive protein Human genes 0.000 title claims abstract description 134
- 238000012123 point-of-care testing Methods 0.000 title claims abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 89
- 239000002245 particle Substances 0.000 claims abstract description 85
- 239000004816 latex Substances 0.000 claims abstract description 80
- 229920000126 latex Polymers 0.000 claims abstract description 80
- 239000003223 protective agent Substances 0.000 claims abstract description 25
- 239000000427 antigen Substances 0.000 claims abstract description 9
- 102000036639 antigens Human genes 0.000 claims abstract description 9
- 108091007433 antigens Proteins 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 239000003792 electrolyte Substances 0.000 claims abstract description 9
- 239000003755 preservative agent Substances 0.000 claims abstract description 9
- 230000002335 preservative effect Effects 0.000 claims abstract description 9
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- 239000000872 buffer Substances 0.000 claims abstract description 6
- 230000001376 precipitating effect Effects 0.000 claims abstract description 4
- 239000002562 thickening agent Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 94
- 239000004005 microsphere Substances 0.000 claims description 41
- 239000012190 activator Substances 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 239000002244 precipitate Substances 0.000 claims description 22
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000035484 reaction time Effects 0.000 abstract description 4
- 239000006228 supernatant Substances 0.000 description 36
- 238000002360 preparation method Methods 0.000 description 26
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 22
- 229940098773 bovine serum albumin Drugs 0.000 description 22
- 238000000034 method Methods 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- 238000009210 therapy by ultrasound Methods 0.000 description 18
- 239000008213 purified water Substances 0.000 description 13
- 239000007987 MES buffer Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 238000004132 cross linking Methods 0.000 description 9
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical group [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 238000002604 ultrasonography Methods 0.000 description 6
- 238000007865 diluting Methods 0.000 description 5
- 108091006905 Human Serum Albumin Proteins 0.000 description 4
- 102000008100 Human Serum Albumin Human genes 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 3
- 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 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 230000001808 coupling effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006749 inflammatory damage Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940093429 polyethylene glycol 6000 Drugs 0.000 description 1
- 239000012716 precipitator Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
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- 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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- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
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- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention provides a C-reactive protein kit suitable for various POCT instruments. The C-reactive protein kit comprises a reagent R1, a reagent R2, and a C-reactive protein calibrator, wherein the reagent R1 comprises a buffer, an electrolyte, a protective agent, a thickening agent, and a preservative; the reagent R2 comprises C-reactive protein polyclonal antibody latex particles, a buffer solution, a protective agent, an electrolyte, a precipitating agent and a preservative; the C-reactive protein calibrator comprises a C-reactive protein antigen, a buffer and a protective agent. The C-reactive protein kit has high sensitivity, good stability and good repeatability, can improve the production efficiency and shorten the reaction time, and is suitable for various POCT instruments.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a C-reactive protein kit suitable for various POCT instruments.
Background
C-reactive protein (CRP) is an acute phase-reactive protein and is present in very low concentrations in healthy people. Elevation of CRP concentration is observed in serum of patients with invasive bacterial infection, inflammation or tissue damage, and elevation of CRP can be detected 6-12 hours after inflammation begins, and the peak value is accelerated more rapidly than other inflammation indexes, so that the CRP can be used as an early diagnosis index for diagnosing related diseases.
There are also many methods for detecting CRP, and most of the current detection methods require a fully-automatic biochemical analyzer or a fully-automatic immunoassay analyzer, and although the test results of the instruments are accurate, the price of the instruments is often too high. Along with the development of the medical industry, the rapid detection instrument is applied, has low manufacturing cost and simple and rapid operation, is suitable for various medical places and families, and has wide market coverage rate.
At present, the chemical coupling in the method for connecting the microsphere and the object to be marked is to connect the object to be marked and the microsphere together through chemical reaction by using an activated cross-linking agent, and the stability after connection is better than that of physical adsorption. And among them, the carboxyl microspheres are more widely applied. In the existing method for preparing carboxyl microspheres by using an activating agent, more problems exist, such as the need of centrifugation after the antibody is coupled to the microspheres to remove free activating agent and free unbound antibody, which makes the operation process become inefficient and time-consuming, and is not beneficial to the industrial preparation of carboxyl microspheres; after the microspheres are activated by a cross-linking agent for a period of time, the cross-linking reaction can be carried out after an additional solution is added to adjust the pH value, so that the preparation time of the reaction is prolonged, and the complexity of the operation is increased; the requirement on the pH value is strict, and the requirements on the dissolving solution and the microspheres are higher, so that the coupling efficiency is low, and the operability of crosslinking is limited to a certain extent; in addition, the coupling effect obtained by the existing preparation method is usually limited to a certain similar instrument, at least more than two-step centrifugation or multi-step microsphere and antibody treatment is required, and the latex reagent has a narrow application range and is not suitable for various POCT instruments in the current market. Therefore, in summary, the most important problems of the cross-linking methods commonly used in the prior art are long coupling time, poor reagent compatibility and strict requirements on cross-linking conditions.
Disclosure of Invention
The invention aims to provide a C-reactive protein kit which is suitable for various POCT instruments, high in production efficiency and good in reagent compatibility.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a C-reactive protein kit applicable to a plurality of POCT instruments, which comprises a reagent R1 and a reagent R2;
the reagent R1 comprises a buffer solution, an electrolyte, a protective agent, a thickening agent and a preservative;
the reagent R2 comprises C-reactive protein polyclonal antibody latex particles, a buffer solution, a protective agent, an electrolyte, a precipitating agent and a preservative.
Further, the volume ratio of the reagent R1 to the reagent R2 is 5-10: 1.
Further, the volume ratio of the reagent R1 to the reagent R2 is 10: 1.
Further, the content of each component of the reagent R1 is as follows:
the pH value is 6.8-7.3.
Further, the content of each component of the reagent R1 is as follows:
the pH was 7.1.
Further, the optimal content of each component of the reagent R1 is as follows:
the pH was 7.1.
Further, the reagent R1 is formulated as: dissolving the components in water according to their respective concentrations or contents, and mixing.
Further, the content of each component of the reagent R2 is as follows:
the pH was 7.1.
Further, the content of each component of the reagent R2 is as follows:
the pH was 7.1.
Further, the optimal content of each component of the reagent R2 is as follows:
further, the preparation steps of the reagent R2 are as follows: and (3) uniformly mixing the protective agent, the buffer solution, the electrolyte, the precipitator and the preservative in the reagent R2 in water, adding the mixed C-reactive protein polyclonal antibody latex particles, and fully and uniformly mixing.
Further, in the reagent, the protective agent is bovine serum albumin.
Further, in the reagent, the buffer is a phosphate buffer.
Further, in the reagent, the electrolyte is sodium chloride.
Further, in the reagent, the preservative is sodium azide.
Further, in the reagent, the thickening agent is polyethylene glycol-6000.
Further, in the reagent, the precipitating agent is sucrose.
Further, the preparation steps of the mixed C-reactive protein polyclonal antibody latex particles are as follows:
(1) adding EDC activator solution into the C-reactive protein polyclonal antibody for reaction, and then respectively adding microsphere solutions with different particle sizes for reaction;
(2) centrifuging the reaction solution, collecting precipitate, adding a protective agent for ultrasonic dispersion, centrifuging again, and collecting precipitate to obtain C-reactive protein antibody polyclonal antibody latex particles with different particle sizes;
(3) and mixing the C-reactive protein antibody polyclonal antibody latex particles with different particle sizes to obtain the mixed C-reactive protein polyclonal antibody latex particles.
Further, the concentration of the C-reactive protein polyclonal antibody is 10 g/L.
Further, in the EDC activator solution, the mass-volume ratio of the EDC activator to water is 30-60:1, wherein the unit of mass is g, and the unit of volume is ml.
Further, in the EDC activator solution, the mass-to-volume ratio of the EDC activator to the water is 30: 1.
Further, in the EDC activator solution, the mass-to-volume ratio of the EDC activator to the water is 40: 1.
Further, in the EDC activator solution, the mass-to-volume ratio of the EDC activator to the water is 60: 1.
Further, the volume ratio of the C-reactive polyclonal protein antibody to the EDC activator solution is 5: 1-3.
Further, the volume ratio of the C-reactive polyclonal protein antibody to the EDC activator solution was 5: 3.
Further, the volume ratio of the C-reactive polyclonal protein antibody to the EDC activator solution was 20: 9.
Further, the volume ratio of the C-reactive polyclonal protein antibody to the EDC activator solution was 10: 3.
Further, the mixing mass ratio of the C-reactive protein antibody polyclonal antibody latex particles with different particle sizes is 1-9: 9-1.
Further, the optimal mixing mass ratio of the C-reactive protein antibody polyclonal antibody latex particles with different particle sizes is 1: 1.
Further, the preparation method of the microsphere solution comprises the following steps: 30ml of latex microspheres were diluted with 0.1mol/L MES buffer to a final reaction concentration of 5% in microsphere solution.
Further, the particle size of the latex microsphere is 40-100nm and 150-350nm respectively.
Further, the particle size of the latex microspheres is optimally 70nm and 300 nm.
Further, the concentration of the protective agent in the preparation process of the mixed C-reactive protein polyclonal antibody latex particles is 1-5%.
Further, the concentration of the protective agent during the preparation of the mixed C-reactive protein polyclonal antibody latex particles is optimally 4%.
Furthermore, the C-reactive protein kit also comprises a C-reactive protein calibrator.
Further, the C-reactive protein calibrator comprises a C-reactive protein antigen, a buffer and a protective agent.
Further, in the C-reactive protein calibrator, the buffer is a phosphate buffer.
Further, in the C-reactive protein calibrator, the protective agent is human serum albumin.
Further, the contents of the components of the C-reactive protein calibrator are as follows:
10-50mmol/L buffer solution;
0.1-10 per mill (volume ratio) of protective agent;
2.4mg/mL of C-reactive protein antigen;
the pH was 7.4.
Most preferably, the optimal content of each component of the C-reactive protein calibrator is as follows:
phosphate buffer solution 50 mmol/L;
1 per mill (volume ratio) of human serum albumin;
2.4mg/mL of C-reactive protein antigen;
the pH was 7.4.
The invention also provides application of the C-reactive protein kit to detection of C-reactive protein content.
The invention also provides a preparation method of the C-reactive protein kit, which comprises the following steps:
(1) dissolving each component in the reagent R1 in water according to the respective concentration or content, and uniformly mixing;
(2) and (3) uniformly mixing the protective agent, the buffering agent, the electrolyte and the preservative in the reagent R2 in water, adding the mixed C-reactive protein polyclonal antibody latex particles, and fully and uniformly mixing to obtain the reagent R2.
(3) And uniformly mixing the C-reactive protein antigen of the C-reactive protein calibrator, the buffer solution and the protective agent, diluting to a constant volume, and preparing the calibrator with the calibrated concentration.
Further, the calibration concentrations of the C-reactive protein calibrator are: 0mg/L, 5.3mg/L, 20mg/L, 40mg/L, 160mg/L and 320 mg/L.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the C-reactive protein kit is prepared by adopting a one-step crosslinking method, so that the crosslinking step is simplified, and the crosslinking mode is correspondingly shortened, so that the reagent production efficiency is improved, and the reaction time is shortened;
2. the invention mixes the antibody and the activator, and directly connects the antibody and the activator into the latex microsphere after fully combining, thereby improving the sensitivity of the reagent in the kit and solving the problem of poor compatibility of the reagent on different types of instruments.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples. Experimental procedures in the following examples, where specific conditions are not noted, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer; the reagents and materials are commercially available. Unless otherwise indicated, percentages and parts are by weight.
Example 1
The kit is a liquid double reagent, namely a reagent R1 and a reagent R2; wherein the volume ratio of the reagent R1 to the reagent R2 is 10: 1. in particular, the method comprises the following steps of,
the formulation of reagent R1 was as follows:
the pH was 7.1.
The reagent R2 contains the following components:
the pH was 7.1.
Preparation of a reagent:
1. preparation of the R1: dissolving the components in purified water according to the content, uniformly mixing, and fixing the volume.
2. The mixed C-reactive protein polyclonal antibody latex particles of 1.25g/L are prepared by a one-step method:
(1) respectively diluting 30ml of latex microspheres with the particle size of 70nm and 300nm with 0.1mol/L MES buffer solution to obtain a microsphere solution with the particle size of 70nm and a microsphere solution with the particle size of 300nm, wherein the final reaction concentration is 5%;
(2) preparing 5ml of 10g/L C-reactive polyclonal protein antibody, and standing at normal temperature for later use;
(3) weighing 0.1g EDC activator, fully dissolving in 3ml purified water, slowly pouring into the prepared C-reactive protein polyclonal antibody, and reacting at normal temperature for 15-20 min;
(4) adding 10ml of microsphere solution with the particle size of 70nm into the solution to react for 2 hours;
(5) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin as protective agent into the obtained precipitate, stopping for 3s after exceeding 3s, and performing ultrasonic treatment for 10min to obtain a latex solution, namely 1.25g/L of 120nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 ℃ for later use;
(6) centrifuging again at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 70nm C-reactive protein polyclonal antibody latex particles;
(7) repeating the steps (2) and (3), adding 10ml of microsphere solution with the particle size of 300nm into the prepared solution, and reacting for 2 hours;
(8) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin as protective agent into the obtained precipitate, stopping for 3s after super 3s, performing ultrasonic treatment for 10min to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C for later use;
(9) centrifuging again at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex particles;
(10) mixing the latex particles of the C-reactive protein polyclonal antibody of 70nm and 300nm according to the mass ratio of 1:1 to obtain the mixed latex particles of the C-reactive protein polyclonal antibody, and standing at the temperature of 2-8 ℃ for later use.
3. Preparation of the reagent R2:
dissolving components of the reagent R2, namely 4mmol/L bovine serum albumin, 10mmol/L phosphate buffer solution, 21.2g/L sodium chloride, 20g/L sucrose and 0.5g/L sodium azide in purified water, and uniformly mixing and fixing the volume; adding the prepared mixed C-reactive protein polyclonal antibody latex particles into 400ml of solution, and fully and uniformly mixing to obtain the reagent R2.
4. Preparation of a calibrator:
the formula of the C-reactive protein calibrator is as follows:
phosphate buffer solution 50 mmol/L;
1 per mill of human serum albumin;
2.4mg/mL of C-reactive protein antigen;
the pH was 7.4.
Dissolving 50mmol/L phosphate buffer solution, 2.4mg/mL C-reactive protein antigen and 1 thousandth of human serum albumin in purified water, uniformly mixing and fixing the volume, diluting the obtained mixed solution to prepare the calibrator with the calibration concentration, wherein the calibration concentration of the C-reactive protein calibrator is as follows: 0mg/L, 5.3mg/L, 20mg/L, 40mg/L, 160mg/L, 320 mg/L.
Example 2
The kit is a liquid double reagent, namely a reagent R1 and a reagent R2; wherein the volume ratio of the reagent R1 to the reagent R2 is 10: 1. in particular, the method comprises the following steps of,
the formulation of reagent R1 was as follows:
the pH was 7.1.
The reagent R2 contains the following components:
the pH was 7.1.
Preparation of a reagent:
1. preparation of the R1: dissolving the components in purified water according to the content, uniformly mixing, and fixing the volume.
2. Preparation of 1.25g/L of the Mixed C-reactive protein polyclonal antibody latex particles:
(1) respectively diluting 30ml of latex microspheres with the particle size of 70nm and 300nm with 0.1mol/L MES buffer solution to obtain a microsphere solution with the particle size of 70nm and a microsphere solution with the particle size of 300nm, wherein the final reaction concentration is 5%;
(2) preparing 5ml of 10g/L C-reactive protein polyclonal antibody, and standing at normal temperature for later use;
(3) weighing 0.075g EDC activator, fully dissolving in 3ml purified water, slowly pouring 2.25ml into the C-reactive protein polyclonal antibody, and reacting at normal temperature for 15-20 min;
(4) adding 10ml of microsphere solution with the particle size of 70nm into the solution, and reacting for 2 hours;
(5) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin as protective agent into the obtained precipitate, stopping for 3s after exceeding 3s, and performing ultrasonic treatment for 10min to obtain a latex solution, namely 1.25g/L of 120nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 ℃ for later use;
(6) centrifuging again at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 70nm C-reactive protein polyclonal antibody latex particles;
(7) repeating the steps (2) and (3), adding 10ml of microsphere solution with the particle size of 300nm into the prepared solution, and reacting for 2 hours;
(8) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin as protective agent into the obtained precipitate, stopping for 3s after super 3s, performing ultrasonic treatment for 10min to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C for later use;
(9) centrifuging again at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex particles;
(10) mixing the latex particles of the C-reactive protein polyclonal antibody of 70nm and 300nm according to the proportion of 1:1 to obtain the mixed latex particles of the C-reactive protein polyclonal antibody, and standing at the temperature of 2-8 ℃ for later use.
3. Preparation of the reagent R2:
dissolving the components of the reagent R2, namely 4mmol/L bovine serum albumin, 10mmol/L phosphate buffer solution, 21.2g/L sodium chloride, 20g/L sucrose and 10% sodium azide in purified water, uniformly mixing and fixing the volume, adding the prepared mixed C-reactive protein polyclonal antibody latex particles into 400ml solution, and fully and uniformly mixing to obtain the reagent R2.
4. Preparation of a calibrator: the procedure was the same as in step 4 of example 1.
Example 3
The kit is a liquid double reagent, namely a reagent R1 and a reagent R2, wherein the ratio of the reagent R1 to the reagent R2 is 10: 1. in particular, the method comprises the following steps of,
the formulation of reagent R1 was as follows:
the pH was 7.1.
The reagent R2 contains the following components:
preparation of a reagent:
1. preparation of the R1: dissolving the components in purified water according to the content, uniformly mixing, and fixing the volume.
2. Preparation of 1.25g/L of the Mixed C-reactive protein polyclonal antibody latex particles:
(1) respectively diluting 30ml of latex microspheres with the particle size of 70nm and 300nm with 0.1mol/L MES buffer solution to obtain a microsphere solution with the particle size of 70nm and a microsphere solution with the particle size of 300nm, wherein the final reaction concentration is 5%;
(2) preparing 5ml of 10g/L C-reactive protein polyclonal antibody, and standing at normal temperature for later use;
(3) weighing 0.025g EDC activator, fully dissolving in 1.5ml purified water, slowly pouring into the prepared C-reactive protein polyclonal antibody, and reacting at normal temperature for 15-20 min;
(4) adding 10ml of microsphere solution with the particle size of 70nm into the solution, and reacting for 2 hours;
(5) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin as protective agent into the obtained precipitate, stopping for 3s after super 3s, and performing ultrasonic treatment for 10min to obtain a latex solution which is 1.25g/L of 120nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 ℃ for later use;
(6) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25 g/L70 nm C-reactive protein polyclonal antibody latex particles;
(7) repeating the steps (2) and (3), adding 10ml of microsphere solution with the particle size of 300nm into the prepared solution, and reacting for 2 hours;
(8) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin as protective agent into the obtained precipitate, stopping for 3s after super 3s, and performing ultrasonic treatment for 10min to obtain a latex solution which is 1.25g/L of 300nm C-reactive protein polyclonal antibody latex particles, and standing at 2-8 ℃ for later use;
(9) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex particles;
(10) mixing the latex particles of the C-reactive protein polyclonal antibody of 70nm and 300nm according to the proportion of 1:1 to obtain the mixed latex particles of the C-reactive protein polyclonal antibody, and standing at the temperature of 2-8 ℃ for later use.
3. Preparation of the reagent R2:
dissolving 4mmol/L bovine serum albumin, 10mmol/L phosphate buffer solution, 21.2g/L sodium chloride, 20g/L sucrose and 20% sodium azide in purified water, uniformly mixing and fixing the volume, adding the prepared mixed C-reactive protein polyclonal antibody latex particles into 400ml of solution, and fully and uniformly mixing to obtain the reagent R2.
4. Preparation of a calibrator: the procedure was the same as in step 4 of example 1.
Comparative example 1
The preparation process of the C-reactive protein polyclonal antibody latex particles described in example 1 is changed from a one-step method to a two-step method, namely:
(1) preparing 5ml of 10g/L C-reactive protein antibody, and standing at normal temperature for later use;
(2) weighing 0.1g EDC activating agent, and fully dissolving in 3ml purified water;
(3) adding 10ml of microsphere solution with the particle size of 70nm into the EDC activator solution, and reacting for 15-20 min;
(4) centrifuging at 12000r for 15min, removing supernatant, adding 20ml MES solution with pH of 6.2 into the obtained precipitate, stopping for 3s after super 3s, and after 2min of ultrasound, slowly adding the prepared C-reactive protein antibody for reaction for 2 h;
(5) centrifuging at 12000r for 15min, removing supernatant, adding 20ml of 1% bovine serum albumin solution into the obtained precipitate, carrying out ultrasonic treatment for 1min at 1200w, 3s and 3s, and reacting for 2 h;
(6) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin solution, 1200w for 3s, and performing ultrasonic treatment for 10min to obtain a latex solution of 1.25g/L of 120nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C;
(7) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25 g/L70 nm C-reactive protein polyclonal antibody latex particles;
(8) repeating the steps (1) and (2);
(9) adding 10ml of microsphere solution with the particle size of 300nm into the EDC activator solution, and reacting for 15-20 min;
(10) centrifuging at 12000r for 15min, removing supernatant, adding 20ml MES solution with pH of 6.2 into the obtained precipitate, stopping for 3s after super 3s, and after 2min of ultrasound, slowly adding the prepared C-reactive protein antibody for reaction for 2 h;
(11) centrifuging at 12000r for 15min, removing supernatant, adding 20ml of 1% bovine serum albumin solution into the obtained precipitate, 1200w, stopping for 3s after super 3s, performing ultrasonic treatment for 1min, and reacting for 2 h;
(12) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin solution, 1200w, stopping for 3s after 3s, and performing ultrasonic treatment for 10min to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C;
(13) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex particles;
(14) mixing the latex particles of the C-reactive protein polyclonal antibody of 70nm and 300nm according to the proportion of 1:1 to obtain the mixed latex particles of the C-reactive protein polyclonal antibody, and standing at the temperature of 2-8 ℃ for later use.
The other reagents were prepared in the same manner as in example 1.
Comparative example 2
The preparation process of the C-reactive protein polyclonal antibody latex particles in the embodiment 2 is changed from a one-step method to a two-step method, namely:
(1) preparing 5ml of 10g/L C-reactive protein antibody, and standing at normal temperature for later use;
(2) 0.075g of EDC activator is weighed out and dissolved in 3ml of purified water;
(3) adding 10ml of microsphere solution with the particle size of 70nm into the EDC activator solution, and reacting for 15-20 min;
(4) centrifuging at 12000r for 15min, removing supernatant, adding 20ml MES solution with pH of 6.2 into the obtained precipitate, stopping for 3s after super 3s, and after 2min of ultrasound, slowly adding the prepared C-reactive protein antibody for reaction for 2 h;
(5) centrifuging at 12000r for 15min, removing supernatant, adding 20ml of 1% bovine serum albumin solution into the obtained precipitate, carrying out ultrasonic treatment for 1min at 1200w, 3s and 3s, and reacting for 2 h;
(6) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin solution, 1200w for 3s, and performing ultrasonic treatment for 10min to obtain a latex solution of 1.25g/L of 120nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C;
(7) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25 g/L700 nm C-reactive protein polyclonal antibody latex particles;
(8) repeating the steps (1) and (2);
(9) adding 10ml of microsphere solution with the particle size of 300nm into the EDC activator solution, and reacting for 15-20 min;
(10) centrifuging at 12000r for 15min, removing supernatant, adding 20ml MES solution with pH of 6.2 into the obtained precipitate, stopping for 3s after super 3s, and after 2min of ultrasound, slowly adding the prepared C-reactive protein antibody for reaction for 2 h;
(11) centrifuging at 12000r for 15min, removing supernatant, adding 20ml of 1% bovine serum albumin solution into the obtained precipitate, 1200w, stopping for 3s after super 3s, performing ultrasonic treatment for 1min, and reacting for 2 h;
(12) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin solution, 1200w, stopping for 3s after 3s, and performing ultrasonic treatment for 10min to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C;
(13) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex particles;
(14) mixing the latex particles of the C-reactive protein polyclonal antibody of 70nm and 300nm according to the proportion of 1:1 to obtain the mixed latex particles of the C-reactive protein polyclonal antibody, and standing at the temperature of 2-8 ℃ for later use.
The other reagents were prepared in the same manner as in example 2.
Comparative example 3
The preparation process of the C-reactive protein polyclonal antibody latex particles in the embodiment 3 is changed from a one-step method to a two-step method, namely:
(1) preparing 5ml of 10g/L C-reactive protein antibody, and standing at normal temperature for later use;
(2) 0.025g EDC activator was weighed and dissolved well in 1.5ml purified water;
(3) adding 10ml of microsphere solution with the particle size of 70nm into the EDC activator solution, and reacting for 15-20 min;
(4) centrifuging at 12000r for 15min, removing supernatant, adding 20ml MES solution with pH of 6.2 into the obtained precipitate, stopping for 3s after super 3s, and after 2min of ultrasound, slowly adding the prepared C-reactive protein antibody for reaction for 2 h;
(5) centrifuging at 12000r for 15min, removing supernatant, adding 20ml of 1% bovine serum albumin solution into the obtained precipitate, carrying out ultrasonic treatment for 1min at 1200w, 3s and 3s, and reacting for 2 h;
(6) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin solution, 1200w for 3s, and performing ultrasonic treatment for 10min to obtain a latex solution of 1.25g/L of 120nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C;
(7) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25 g/L70 nm C-reactive protein polyclonal antibody latex particles;
(8) repeating the steps (1) and (2);
(9) adding 10ml of microsphere solution with the particle size of 300nm into the EDC activator solution, and reacting for 15-20 min;
(10) centrifuging at 12000r for 15min, removing supernatant, adding 20ml MES solution with pH of 6.2 into the obtained precipitate, stopping for 3s after super 3s, and after 2min of ultrasound, slowly adding the prepared C-reactive protein antibody for reaction for 2 h;
(11) centrifuging at 12000r for 15min, removing supernatant, adding 20ml of 1% bovine serum albumin solution into the obtained precipitate, 1200w, stopping for 3s after super 3s, performing ultrasonic treatment for 1min, and reacting for 2 h;
(12) centrifuging at 12000r for 15min, removing supernatant, adding 40ml of 4% bovine serum albumin solution, 1200w, stopping for 3s after 3s, and performing ultrasonic treatment for 10min to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex solution, and standing at 2-8 deg.C;
(13) centrifuging at 12000r for 15min, and discarding the supernatant to obtain 1.25g/L of 300nm C-reactive protein polyclonal antibody latex particles;
(14) mixing the latex particles of the C-reactive protein polyclonal antibody of 70nm and 300nm according to the proportion of 1:1 to obtain the mixed latex particles of the C-reactive protein polyclonal antibody, and standing at the temperature of 2-8 ℃ for later use.
The other reagents were prepared in the same manner as in example 3.
Example 7
Four instruments of different models were used: PA-54 specific protein analyzer, PA-65 specific protein analyzer, PA-900 specific protein analyzer and PA-880 specific protein analyzer. The test basic parameters are shown in table 1.
TABLE 1 basic parameters
1. Kit sensitivity detection
Adding a reagent R1 and a calibrator into a blank reaction cup of a corresponding instrument according to the parameters in the table 1, uniformly mixing, adding a reagent R2, placing the mixture in a reaction space of the instrument, reading absorbance (A1) at 10S, reading corresponding absorbance (A2) according to the final reaction time of each instrument, and calculating absorbance delta A; wherein Δ a ═ a2-a 1.
TABLE 2 comparison of sensitivity of reagent kit in different types of instruments
From the results it can be seen that: the kit prepared by the invention has higher sensitivity.
2. Test of thermal stability of kit at 37 DEG C
The prepared reagent R2 was placed in a 37 ℃ incubator, and the absorbance of each spot was measured on each of the 0 th, 1 st, 3 th, 5 th and 7 th days using the unopened reagent.
TABLE 3 comparison of the thermal stability of the reagent kit in different types of instruments
From the results, the reaction amplitude of the reagent in the kit of the invention has no obvious change in the environment of 37 ℃ for 7 days, while the reaction amplitude of the reagent in the kit prepared by the two-step method gradually increases along with the lengthening of the time, thereby indicating that the kit prepared by the invention has better thermal stability.
3. Kit loss rate detection
The relative deviation between days 1, 3, 5 and 7 and day 0 and between day 7 and day 1 in table 3 was calculated, and it can be seen from the results that the loss rate of the kit prepared by the present invention was lower.
4. Kit repeatability detection
Selecting a sample with serum concentration of 1mg/L, repeatedly measuring 10 times by using a kit, calculating a mean value X and a standard deviation SD, and calculating an intra-batch precision CV value according to a formula, wherein CV is SD/X multiplied by 100 percent
TABLE 4 comparison of repeatability of reagent kit in different types of instruments
As can be seen from the results, the repeatability of the kit prepared by the invention is more stable.
In conclusion, the invention adopts a one-step crosslinking process, the antibody and the activator are mixed, the full reaction of the activator and the antibody is utilized to promote the antibody to be coated on the latex microsphere more completely, the sensitivity of the prepared kit is higher, the reaction time is short, the reagent is still stable and has good repeatability in instruments of different models, and the kit has better compatibility and low requirement on crosslinking; meanwhile, the kit is prepared by a one-step method, so that the production efficiency can be improved.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A C-reactive protein kit suitable for a plurality of POCT instruments is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises a buffer solution, an electrolyte, a protective agent, a thickening agent and a preservative;
the reagent R2 comprises C-reactive protein polyclonal antibody latex particles, a buffer solution, an electrolyte, a protective agent, a precipitating agent and a preservative.
2. The C-reactive protein kit of claim 1, wherein the volume ratio of the reagents R1 to R2 is 5-10: 1.
3. the C-reactive protein kit according to claim 1, wherein the contents of the components of the reagent R1 are as follows:
the pH value is 6.8-7.3.
4. The C-reactive protein kit according to claim 1, wherein the contents of the components of the reagent R2 are as follows:
the pH value is 6.8-7.3.
5. The C-reactive protein kit according to claim 1, wherein the mixed C-reactive protein polyclonal antibody latex particles are prepared by the following steps:
(1) adding EDC activator solution into the C-reactive protein polyclonal antibody for reaction, and then respectively adding microsphere solutions with different particle sizes for reaction;
(2) centrifuging the reaction solution, collecting precipitate, adding a protective agent for ultrasonic dispersion, centrifuging again, and collecting precipitate to obtain C-reactive protein antibody polyclonal antibody latex particles with different particle sizes;
(3) and mixing the C-reactive protein antibody polyclonal antibody latex particles with different particle sizes to obtain the mixed C-reactive protein polyclonal antibody latex particles.
6. The C-reactive protein kit as claimed in claim 5, wherein the particle size of the latex microspheres in the microsphere solution is 40-100nm and 150-350nm, respectively.
7. The C-reactive protein kit according to claim 5, wherein the mixing mass ratio of the C-reactive protein antibody polyclonal antibody latex particles with different particle sizes is 1-9: 9-1.
8. The C-reactive protein kit according to claim 1, wherein the C-reactive protein kit further comprises a C-reactive protein calibrator.
9. The C-reactive protein kit of claim 8, wherein the C-reactive protein calibrator comprises a C-reactive protein antigen, a buffer, and a protectant.
10. The C-reactive protein kit of claim 8, wherein the C-reactive protein calibrator comprises the following components:
buffer solution 10-50mmol/L
0.1-10 per mill of protective agent
2.4mg/mL of C-reactive protein antigen;
the pH was 7.4.
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Inventor after: Chen Xiao Inventor after: Wang Lujun Inventor after: Xu Ruiping Inventor before: Zhang Ranran Inventor before: Zhou Wei Inventor before: Mao Xiaobo |