CN111999505A - Reagent for detecting antistreptolysin O and preparation method thereof - Google Patents
Reagent for detecting antistreptolysin O and preparation method thereof Download PDFInfo
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- CN111999505A CN111999505A CN202010806409.8A CN202010806409A CN111999505A CN 111999505 A CN111999505 A CN 111999505A CN 202010806409 A CN202010806409 A CN 202010806409A CN 111999505 A CN111999505 A CN 111999505A
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- 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
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
Abstract
The invention relates to the technical field of biology, in particular to a reagent for detecting antistreptolysin O and a preparation method thereof. The detection reagent provided by the invention comprises a reagent R1 and a reagent R2, wherein the reagent R1 adopts a double-surfactant system, and the reagent R2 adopts latex particles with two particle sizes. The detection reagent is suitable for latex particle enhanced immunoturbidimetry detection of the antistreptolysin O. Compared with the commercially available reagent A only containing single-particle-size latex particles, the reagent A has obvious advantages in the aspects of linearity, low-value precision and interference resistance. Experiments show that R in the linear detection of the reagent2The value can reach 0.9991, and the precision is very high, and the CV value is lower than 2%.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a reagent for detecting antistreptolysin O and a preparation method thereof.
Background
Streptolysin O (ASO for short) is one of metabolites of hemolytic streptococcus in human body, is a protein containing-SH group and having hemolytic property, can dissolve red blood cells of organism, is easy to be oxidized in air, can temporarily lose hemolytic ability after being oxidized, and can restore hemolytic ability after being reduced by reducing agent. After a human body is infected by streptococcus, streptolysin O can stimulate the immune system of the body to generate corresponding antibodies, so that the concentration of the antistreptolysin O in blood is increased, generally, 85-90% of patients infected by the streptolysin streptococcus exist in serum of months or a year from 15-20 days after infection to the time after recovery, if the antistreptolysin O is detected in the serum of the patients, the patients are infected by the streptolysin streptococcus recently, so the antistreptolysin O has important significance in clinical diagnosis, and the antistreptolysin O can be used for auxiliary diagnosis of streptococcal allergic diseases such as glomerulonephritis, rheumatic fever, scarlet fever, tonsillitis, pharyngitis, rheumatic heart disease and the like by detecting the antistreptolysin O in the serum.
The clinical detection method of the antistreptolysin O comprises a hemolysis inhibition method, a latex agglutination method, an enzyme-linked immunosorbent assay and a latex particle enhanced immunoturbidimetry. The hemolysis inhibition method has the disadvantages of complicated operation, easily affected measurement result and poor repeatability. The enzyme-linked immunosorbent assay has complex operation in the assay process, has higher professional requirements on testers, can only carry out qualitative or semi-quantitative detection, and cannot meet the clinical requirements. While latex agglutination is only a qualitative measure. Latex particle enhanced immunoturbidimetry (PETIA) is a detection method which is widely applied in recent years, and the PETIA method comprises a scattering turbidimetry detection method and a transmission turbidimetry detection method, wherein the detection principles of the scattering turbidimetry detection method and the transmission turbidimetry detection method are very similar and are based on antigen-antibody specific reaction. The antibody (antigen) is crosslinked on the surface of the polymer latex particles, and the antibody (antigen) crosslinked with the latex particles and the antigen (antibody) are combined and then rapidly aggregated together to form turbidity, so that the light transmittance or the light scattering of a reaction system is changed. The change in the light transmittance or light scattering of the reaction system is correlated with the antigen (antibody) concentration in the sample to be tested, and thus the detection of the change in the light transmittance or light scattering reflects the antigen (antibody) concentration in the sample to be tested. The method is time-saving and labor-saving, can be directly operated on a biochemical analyzer, is convenient and quick, has high accuracy of a measuring result, can carry out quantitative detection, and has few measured value influence factors, so the method is widely applied in recent years.
The conventional anti-streptolysin O detection kit on the market mostly adopts a latex particle enhanced immunoturbidimetry method, but in practical application, the linearity and precision of the commercially available reagent in detection are not compatible, so that the requirement of clinical detection cannot be met.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a reagent for detecting antistreptolysin O and a preparation method thereof, so that the linearity and precision of the detection are ensured.
The detection reagent for the antistreptolysin O provided by the invention comprises a reagent R1 and a reagent R2:
the reagent R1 comprises: 80-120 mmol/L phosphate buffer solution, gene 11 g/L-10 g/L, Tween-201 g/L-10 g/L, EDTA 2-4 g/L, NaN30.5-2 g/L, NaCl 4-6 g/L and bovine serum albumin 0.5-2 g/L;
the reagent R2 is prepared from the following raw materials: 80nm latex particles, 300nm latex particles, EDC, streptolysin O, MES buffer solution, Tris buffer solution, BSA and preservation solution;
the preservation solution comprises: 30mM TAPS, 1% BSA, 6% sucrose, 1g/L sodium azide and 0.9% sodium chloride.
In the reagent R1, a double-surfactant system is adopted, so that a good anti-interference effect is achieved in detection, and particularly chyle interference can be resisted. It was found in the study that neither of these surfactants used could be omitted or substituted, nor could the ratio be adjusted at will, and that any change in the above would lead to a decrease in the detection. In the reagent R2, latex particles with two particle sizes are adopted, the particle size selection is reasonable, and the reagent is properly matched with a buffer solution, so that the low-value precision, linearity and anti-interference performance of a measurement result are improved.
In the invention, the reagent R1 consists of 100mmol/L phosphate buffer solution, genape 15 g/L-7.5 g/L, Tween-202.5 g/L-5 g/L, EDTA 3g/L, NaN31g/L, NaCl 5g/L and bovine serum albumin 1 g/L; wherein the mass ratio of the gene 1 to the Tween-20 is 1: 1.
In the invention, the reagent R1 consists of 100mmol/L phosphate buffer solution, gene 15 g/L, Tween-205 g/L, EDTA 3g/L, NaN31g/L, NaCl 5g/L and bovine serum albumin 1 g/L;
the preparation method of the reagent R1 comprises the following steps: the components were added sequentially and the pH was then adjusted to 6.0. The preparation of R1 was carried out at room temperature. The room temperature is 18-30 ℃.
The preparation method of the reagent R2 comprises the following steps:
diluting streptolysin O with a Tris buffer solution to obtain an antigen diluent;
diluting 80nm latex particles with MES buffer solution, adding EDC for activation, mixing with antigen diluent for reaction, then adding BSA for blocking, centrifuging, collecting precipitate, and suspending with a preservation solution to obtain solution A;
diluting the latex particles with the size of 300nm by MES buffer solution, adding EDC for activation, mixing with antigen diluent for reaction overnight, then adding BSA for blocking, centrifuging, collecting precipitate, and suspending by a preservation solution to obtain solution B;
and mixing the solution A and the solution B to prepare a reagent R2.
The concentration of the Tris buffer solution is 50 mmol/L; in the antigen dilution, the concentration of streptolysin O is 0.3 mg/ml.
The concentration of the MES buffer solution is 10 mmol/L;
in the step of preparing the solution A, the 80nm latex particles are diluted to the concentration of 3.625g/L by MES buffer solution;
in the step of preparing the solution B, the 300nm latex particles are diluted to a concentration of 2.175g/L with MES buffer.
Adding EDC is adding MES buffer solution containing EDC, wherein the concentration of EDC is 1.4mg/ml, and the concentration of MES is 10 mmol/L; EDC is added in the step of preparing the solution A to the concentration of 0.15 mg/ml; EDC is added in the step of preparing the solution B to the concentration of 0.05 mg/ml; the activation condition is shaking for 20min at room temperature. The room temperature is 18-30 ℃.
Mixing the solution A and the antigen diluent in the step of preparing the solution A until the concentration of the antigen is 0.1 mg/ml; mixing the solution B with an antigen diluent in the step of preparing the solution B until the concentration of the antigen is 0.15 mg/ml; the mixing reaction with the antigen diluent is carried out for 8-12 h at room temperature. The room temperature is 18-30 ℃.
Adding BSA (bovine serum albumin) in the step of preparing the solution A until the concentration of the BSA is 1.2 mg/ml; and adding BSA (bovine serum albumin) in the step of preparing the solution B until the concentration of the BSA is 1.2mg/ml, and reacting at room temperature for 20-40 min under the closed reaction condition. The room temperature is 18-30 ℃.
The volume ratio of the liquid A to the liquid B is 1: 1.
In the reagent R2, the concentration of latex particles is 2.9g/L, wherein the mass of 80nm latex particles accounts for 37.5-62.5% of the total mass of the latex particles. In some embodiments, the mass of 80nm latex particles comprises 62.5% of the total latex particle mass.
The reagent is suitable for latex particle enhanced immunoturbidimetry detection, and a full-automatic biochemical analyzer is used for detection. The detection method is an end point method, the detection wavelength is 540nm, and the measurement temperature is 37 ℃.
The detection reagent provided by the invention comprises a reagent R1 and a reagent R2, wherein the reagent R1 adopts a double-surfactant system, and the reagent R2 adopts latex particles with two particle sizes. The detection reagent is suitable for latex particle enhanced immunoturbidimetry detection of the antistreptolysin O. Compared with the commercially available reagent A only containing single-particle-size latex particles, the reagent A has obvious advantages in the aspects of linearity, low-value precision and interference resistance. Experiments show that R in the linear detection of the reagent2The value can reach 0.9991, and the precision is very high, and the CV value is lower than 2%.
Drawings
FIG. 1 shows the results of linear investigation of each kit, with the theoretical concentration on the abscissa and the actual concentration on the ordinate.
Detailed Description
The invention provides a reagent for detecting antistreptolysin O and a preparation method thereof, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
An anti-streptolysin O double-particle-size latex enhanced immunoturbidimetry detection kit and a preparation method thereof are disclosed:
reagent R1 consisted of:
the components are added in sequence at room temperature to complete preparation, the pH is adjusted to 6.0, and the mixture is stored at 4 ℃ in a sealing way for later use.
Reagent R2 was prepared as follows:
(1) washing and purifying streptolysin O protein: and (3) putting the purchased streptolysin O protein solution into a dialysis bag, putting the dialysis bag into a physiological saline solution, and dialyzing overnight for later use.
(2) Activation of latex particles: latex particles having the same volume and particle diameters of 80nm and 300nm were diluted with 10mM MES buffer solution having a pH of 5.0, and the latex particle dilutions having two different particle diameters were referred to as solution a and solution B. EDC was accurately weighed and diluted with 10mM pH 5.0MES buffer to a concentration of 1.4 mg/ml. EDC was added to the solution A and the solution B so that the concentration of EDC in the solution A was 0.15mg/ml and the concentration of EDC in the solution B was 0.05mg/ml, respectively. Shaking solution A and solution B with EDC at room temperature for 20min on a shaking table.
(3) And (3) preparing an antigen diluent, namely adding the purified and washed streptolysin O into a 50mM Tris buffer solution with the pH value of 8.0 to prepare the antigen diluent, wherein the concentration of the streptolysin O in the antigen diluent is 0.3 mg/ml.
(4) Preparation of latex particles of different particle size coated with streptolysin O: respectively adding the prepared streptolysin O diluent into the activated latex particle A, B liquid to ensure that the antigen concentration in the A, B liquid is respectively as follows: 0.1mg/ml and 0.15mg/ml, and placing A, B solution on a mixing machine for reaction overnight under the condition of room temperature. After the reaction overnight, 10% BSA was added to A, B solution to make the BSA concentration in A, B solution 1.2mg/ml, and blocking treatment was performed for 30 min. And (3) centrifuging the sealed A, B at the rotating speed of 15000rpm for 5min, removing supernatant, and adding a certain volume of preservation solution, wherein the formula of the preservation solution is as follows: 30mM TAPS pH8.0+ 1% BSA + 6% sucrose +1g/L sodium azide + 0.9% sodium chloride, followed by ultrasonic dispersion.
(5) R2 preparation: mixing the prepared latex particle solutions coated with the streptolysin O with different particle diameters according to a certain volume proportion, and adding a corresponding volume of preservation solution according to the latex concentration (the preservation solution formula in the step 4 is the same), so that the concentration of the double-particle-diameter latex in the R2 is 2.9g/L, wherein the mass ratio of the small-particle-diameter latex particles to the total latex particles is 62.5%. The two solutions are mixed evenly according to a certain proportion to obtain the prepared R2 reagent.
Example 2
An anti-streptolysin O double-particle-size latex enhanced immunoturbidimetry detection kit and a preparation method thereof are disclosed:
reagent R1 the composition is the same as example 1
Reagent R2 was prepared as follows:
(1) streptolysin O protein purification by washing in the same manner as in example 1
(2) Activation of latex particles As in example 1
(3) Preparation of antigen dilutions
(4) Preparation of latex particles of different particle size coated with streptolysin O
(5) R2 preparation: mixing the prepared latex particle solutions coated with the streptolysin O with different particle diameters according to a certain volume proportion, and adding a corresponding volume of preservation solution according to the latex concentration (the preservation solution formula in the step 4 is the same), so that the concentration of the double-particle-diameter latex in the R2 is 2.9g/L, and the mass ratio of the small-particle-diameter latex particles to the total latex particles is 37.5%. The two solutions are mixed evenly according to a certain proportion to obtain the prepared R2 reagent.
Example 3
An anti-streptolysin O double-particle-size latex enhanced immunoturbidimetry detection kit and a preparation method thereof are disclosed:
the composition of the reagent R1 is the same as that in example 1.
Reagent R2 was prepared as follows:
(1) streptolysin O protein was purified by washing as in example 1.
(2) Activation of latex particles the same as in example 1.
(3) And (3) preparing an antigen diluent, namely adding the purified and washed streptolysin O into a 50mM Tris buffer solution with the pH value of 8.8 to prepare the antigen diluent, wherein the concentration of the streptolysin O in the antigen diluent is 0.3 mg/ml.
(4) Latex particles of different particle size coated with streptolysin O were prepared as in example 1.
(5) R2 preparation: same as example 1
Example 4
An anti-streptolysin O double-particle-size latex enhanced immunoturbidimetry detection kit and a preparation method thereof are disclosed:
reagent R1 consisted of:
the components are added in sequence at room temperature to complete preparation, the pH is adjusted to 6.0, and the mixture is stored at 4 ℃ in a sealing way for later use.
Reagent R2 was prepared as follows:
(1) streptolysin O protein purification by washing in the same manner as in example 1
(2) Activation of latex particles As in example 1
(3) Preparation of antigen dilutions
(4) Preparation of latex particles of different particle size coated with streptolysin O
(5) R2 preparation: same as example 1
Detection of antistreptolysin O
The detection is carried out by using a full-automatic biochemical analyzer according to the following detection parameters:
TABLE 1 detection parameters of the kit of the present invention on a fully automatic biochemical analyzer
(1) Detection of precision
The precision of the reagent according to the invention was determined 10 times with each of the two samples.
TABLE 2 precision investigation of the kit according to the invention (sample 1)
| Serial number | Example 1 | Example 2 | Example 3 | Commercial reagent A |
| 1 | 196.65 | 197.96 | 190.51 | 196.90 |
| 2 | 196.46 | 203.86 | 192.14 | 192.49 |
| 3 | 196.47 | 209.59 | 184.93 | 195.00 |
| 4 | 196.06 | 192.20 | 185.81 | 191.99 |
| 5 | 196.56 | 203.54 | 189.00 | 184.31 |
| 6 | 196.35 | 205.85 | 191.65 | 195.84 |
| 7 | 196.83 | 193.94 | 195.38 | 199.18 |
| 8 | 199.20 | 209.37 | 199.87 | 183.33 |
| 9 | 198.52 | 190.69 | 181.56 | 195.67 |
| 10 | 198.73 | 197.99 | 188.34 | 186.57 |
| AVE | 197.18 | 200.50 | 189.92 | 192.13 |
| SD | 1.16 | 6.92 | 5.29 | 5.54 |
| CV | 0.59％ | 3.45％ | 2.79％ | 2.89％ |
TABLE 3 precision investigation of the kit according to the invention (sample 2)
| Serial number | Example 1 | Example 2 | Example 3 | Commercial reagent A |
| 1 | 48.32 | 47.10 | 49.13 | 46.35 |
| 2 | 46.38 | 45.27 | 45.52 | 46.77 |
| 3 | 47.50 | 46.21 | 47.06 | 47.86 |
| 4 | 47.85 | 49.56 | 45.83 | 48.94 |
| 5 | 48.42 | 47.76 | 48.49 | 45.24 |
| 6 | 49.33 | 46.99 | 46.69 | 49.63 |
| 7 | 47.40 | 45.00 | 46.06 | 48.82 |
| 8 | 48.97 | 49.58 | 49.54 | 48.39 |
| 9 | 48.93 | 49.49 | 49.87 | 49.31 |
| 10 | 48.98 | 47.94 | 47.47 | 48.29 |
| AVE | 48.21 | 47.49 | 47.57 | 47.96 |
| SD | 0.92 | 1.70 | 1.60 | 1.41 |
| CV | 1.92％ | 3.58％ | 3.36％ | 2.95％ |
(2) Linear assay was carried out using the assay reagents prepared in examples 1 to 3 as subjects. Meanwhile, a commercially available reagent was used as a control. The results are shown in FIG. 1, and the results indicate that the detection reagent prepared in example 1 has the best detection linearity, R2The value may reach 0.9991.
(3) Anti-interference detection
TABLE 4. investigation of the anti-interference Performance of the kit of the invention
In summary, the results of comparing the precision, linearity and anti-interference performance of the kits of the present invention, examples 1, 2, 3, 4 and the commercial kit a, show: compared with the commercially available reagent A only containing single-particle-size latex particles, the reagent A has obvious advantages in the aspects of linearity, low-value precision and interference resistance. Among them, the effect of the detection reagent prepared in example 1 is more excellent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. An anti-streptolysin O detection reagent comprising a reagent R1 and a reagent R2:
the reagent R1 comprises: 80-120 mmol/L phosphate buffer solution, gene 11 g/L-10 g/L, Tween-201 g/L-10 g/L, EDTA 2-4 g/L, NaN30.5-2 g/L, NaCl 4-6 g/L and bovine serum albumin 0.5-2 g/L;
the reagent R2 is prepared from the following raw materials: 80nm latex particles, 300nm latex particles, EDC, streptolysin O, MES buffer solution, Tris buffer solution, BSA and preservation solution;
the preservation solution comprises: 30mM TAPS, 1% BSA, 6% sucrose, 1g/L sodium azide and 0.9% sodium chloride.
2. The detection reagent according to claim 1, wherein the reagent R1 is prepared from 100mmol/L phosphate buffer solution, gene 15 g/L-7.5 g/L, Tween-202.5 g/L-5 g/L, EDTA 3g/L, NaN31g/L, NaCl 5g/L and bovine serum albumin 1 g/L; wherein the mass ratio of the gene 1 to the Tween-20 is 1: 1.
3. The method for preparing the reagent R2 in the detection reagent according to claim 1, comprising:
diluting streptolysin O with a Tris buffer solution to obtain an antigen diluent;
diluting 80nm latex particles with MES buffer solution, adding EDC for activation, mixing with antigen diluent for reaction, then adding BSA for blocking, centrifuging, collecting precipitate, and suspending with a preservation solution to obtain solution A;
diluting the latex particles with the size of 300nm by MES buffer solution, adding EDC for activation, mixing with antigen diluent for reaction overnight, then adding BSA for blocking, centrifuging, collecting precipitate, and suspending by a preservation solution to obtain solution B;
and mixing the solution A and the solution B to prepare a reagent R2.
4. The method according to claim 3, wherein the concentration of the Tris buffer is 50 mmol/L; in the antigen dilution, the concentration of streptolysin O is 0.3 mg/ml.
5. The production method according to claim 3,
the concentration of the MES buffer solution is 10 mmol/L;
in the step of preparing the solution A, the 80nm latex particles are diluted to the concentration of 3.625g/L by MES buffer solution;
in the step of preparing the solution B, the 300nm latex particles are diluted to a concentration of 2.175g/L with MES buffer.
6. The method of claim 3, wherein the EDC is added as MES buffer solution containing EDC, wherein EDC concentration is 1.4mg/ml, MES concentration is 10 mmol/L; EDC is added in the step of preparing the solution A to the concentration of 0.15 mg/ml; EDC is added in the step of preparing the solution B to the concentration of 0.05 mg/ml; the activation condition is shaking for 20min at room temperature. .
7. The method according to claim 3, wherein the step of preparing solution A is carried out by mixing the solution A with an antigen diluent so that the antigen concentration is 0.1 mg/ml; mixing the solution B with an antigen diluent in the step of preparing the solution B until the concentration of the antigen is 0.15 mg/ml; the mixing reaction with the antigen diluent is carried out for 8-12 h at room temperature.
8. The method according to claim 3, wherein BSA is added to a concentration of 1.2mg/ml in the step of preparing solution A; and adding BSA (bovine serum albumin) in the step of preparing the solution B until the concentration of the BSA is 1.2mg/ml, and reacting at room temperature for 20-40 min under the closed reaction condition.
9. The method according to claim 3, wherein the volume ratio of the solution A to the solution B is 1: 1.
10. The method according to any one of claims 3 to 9, wherein the concentration of latex particles in the reagent R2 is 2.9g/L, and the mass of 80nm latex particles is 37.5% to 62.5% of the total mass of latex particles.
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| CN113433317A (en) * | 2021-06-23 | 2021-09-24 | 湖北民族大学附属民大医院 | Matrix metalloproteinase-3 determination kit and preparation method thereof |
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| CN113433317B (en) * | 2021-06-23 | 2024-02-02 | 湖北民族大学附属民大医院 | Matrix metalloproteinase-3 assay kit and preparation method thereof |
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