CN108663516B - Preparation method and kit of sensitized latex - Google Patents
Preparation method and kit of sensitized latex Download PDFInfo
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- CN108663516B CN108663516B CN201810239797.9A CN201810239797A CN108663516B CN 108663516 B CN108663516 B CN 108663516B CN 201810239797 A CN201810239797 A CN 201810239797A CN 108663516 B CN108663516 B CN 108663516B
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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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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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
- G01N33/531—Production of immunochemical test materials
Abstract
A preparation method of sensitized latex and a kit, belonging to the technical field of medical reagents. The invention aims to provide a method and a kit for preparing sensitized latex by using a tangential flow filtration system concentration liquid change method. The method comprises the following steps: preparing an EDC solution; obtaining activated latex microspheres; mixing the activated latex microspheres with a coupling buffer; coupling latex microspheres with antibodies; adding bovine serum albumin mother liquor; and filtering the mixed solution of the solution and the storage buffer solution to obtain the sensitized latex. The invention overcomes the defects of low coupling efficiency, unstable reagent, black precipitate and large blank absorbance caused by using a centrifugal machine and a cell ultrasonic crusher, and is more beneficial to batch production.
Description
Technical Field
The invention belongs to the technical field of medical reagents.
Background
Latex-enhanced turbidimetric immunoassay (LETIA) is a stable and accurate method for detecting humoral protein homogeneous phase immunoturbidimetric assay. The principle is that through the monoclonal antibody crosslinked on the surface of the polymer latex microsphere, when the antigen is combined with the microsphere crosslinked with the antibody, the antigen and the microsphere can be rapidly gathered together in a short time, and the absorbance of a reaction solution is changed. Furthermore, the change in the absorbance of the reaction solution has a linear relationship with the concentration of the antigen to be measured within a certain range, and can be used to reflect the concentration of the antigen to be measured.
The LETIA method needs to adopt a physical or chemical method to couple the antibody on the surface of the microsphere, and the physical adsorption method is a weak acting force because the protein and the latex particles are combined together by electrostatic interaction or hydrophobic interaction, so that the protein is easy to be dissociated from the latex microsphere, the instability of a reagent and the low coupling efficiency are caused, and the practical application is limited to a certain extent. The chemical coupling method is that functional groups on the surface of the latex microsphere are combined with amino groups on an immune active molecule in a covalent bond mode, so that the stability and the anti-interference capability of the reagent are enhanced. Therefore, in the current domestic research and development, a latex enhanced turbidimetric reagent is mostly prepared by a chemical coupling method, namely, carbodiimide is used for activating carboxyl, and then antibody is added for coupling.
The chemical coupling method comprises the following steps: the one-step method (not separating the excessive activating agent) and the two-step method (removing the excessive activating agent after activation) are mainly used for separating the activating agent by adopting a centrifugal machine high-speed centrifugation method, and the smaller the particle size of the latex microspheres, the larger the centrifugal force required and the longer the centrifugation time. After the microspheres are coupled with the antibody, the latex precipitate is separated by a centrifuge, the latex precipitate is dispersed in a liquid change way, a small amount of production is dispersed by low-power ultrasonic cleaning equipment, the batch production is separated by an ultrasonic cell disruptor, due to repeated ultrasound, a black object falls into the solution due to the variable depression at the lower end of an amplitude transformer of the ultrasonic cell disruptor, and the reagent becomes unstable along with the prolonging of time. In addition, since there is a possibility that denatured IgG is generated due to uncertainty in the power of ultrasound and the influence of ultrasound temperature, RF interference is easily caused.
Disclosure of Invention
The invention aims to provide a method and a kit for preparing sensitized latex by using a tangential flow filtration system concentration liquid change method.
The method comprises the following steps:
(1) preparing: preparing 60-300nm polystyrene latex microspheres, and diluting the latex microspheres to a final concentration of 1-3% w/v by using 50 mM/L2- (N-morpholine) ethanesulfonic acid with the pH of 6.0 to obtain a latex microsphere solution; dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 50 mM/L2- (N-morpholine) ethanesulfonic acid (EDC) at pH6.0 to obtain an EDC solution with a concentration of 0.01 g/mL;
(2) and (3) activation: dripping each mg of microspheres into the latex microsphere solution according to the proportion of 10-100 mu L of EDC solution by taking the weight of the latex microspheres as a reference, oscillating and uniformly mixing, and oscillating and activating for 20-30min at the temperature of 10-37 ℃ and at the oscillating speed of 50-200rpm/min to obtain activated latex microspheres;
(3) liquid changing: pouring the activated latex microspheres into a sample container of a tangential flow filtration system, pouring a coupling buffer solution into a cleaning solution container, and setting a target pump speed of 30-60mL/min, a transmembrane pressure of 2.5Psi and a shear force of 2000-4000s on an instrument-1Pressing the start key after the parameter setting is completedFiltering, wherein the volume of the coupling buffer solution is 3-6 times of the volume of the latex microspheres to be filtered, the filtering is stopped by pressing an end key, and the whole process is controlled within 1 h;
(4) coupling: based on the weight of the latex microspheres, the weight ratio of microspheres: adding the antibody into the solution (3) according to the proportion of the antibody =0.1-0.5%, oscillating and mixing uniformly, and oscillating and coupling for 2-3h at the temperature of 10-37 ℃, wherein the oscillating speed is 50-200 rpm/min;
(5) and (3) sealing: adding 5% bovine serum albumin mother liquor into the solution (4) according to the proportion that the final volume of the solution is 0.5-2%, uniformly mixing by shaking, sealing by shaking at 10-37 ℃ for 1-2h, and the shaking speed is 50-200 rpm/min;
(6) cleaning and liquid changing: pouring the solution (5) into a sample container of a tangential flow filtration system, pouring the storage buffer solution into a cleaning solution container, and setting the target pump speed on the instrument to be 30-60mL/min, the transmembrane pressure to be 2.5Psi and the shearing force to be 2000-4000s-1And after the parameter setting is finished, pressing a start key to filter, wherein the volume of the storage liquid is 3-6 times of the volume of the latex microspheres to be filtered, and pressing an end key to stop filtering to obtain the sensitized latex.
The coupling buffer solution is one of 50-200mM/L phosphate buffer solution, carbonate buffer solution, borate buffer solution and MES buffer solution, and the pH value is 6-10.
The antibody of the present invention is one of a monoclonal antibody and a polyclonal antibody.
The storage buffer solution is one of 20-100mM/L phosphate buffer solution, HEPES buffer solution, glycine buffer solution, Tris buffer solution, PIPES buffer solution and citric acid buffer solution, and the pH value is 6-10; to increase the stability of the reagents, stabilizers are generally added to the stock solutions.
The stabilizer of the invention is one or more of 1-5% of glycerol, sucrose, mannitol and trehalose.
The detection kit containing the sensitization latex comprises R1 and R2 reagents, wherein R1 comprises buffer solution, monovalent or divalent salt, surfactant, PEG and sodium azide; r2 is a sensitized latex.
The buffer solution is one of Tris, phosphate and carbonate buffer solution, and the concentration is 20-500 mM/L.
The monovalent or divalent salt is one or more of sodium chloride, potassium chloride, calcium chloride and magnesium chloride, and the concentration is 0.1-15 g/L.
The surfactant is one of Tween series and Triton series, and the concentration is 1-10 mL/L.
The invention overcomes the defects of low coupling efficiency, unstable reagent, black precipitate and large blank absorbance caused by using a centrifugal machine and a cell ultrasonic crusher, and is more beneficial to batch production.
Drawings
FIG. 1 is a graph showing the comparison results in example 1 of the present invention;
FIG. 2 is a graph showing comparative results of example 2 of the present invention;
FIG. 3 is a graph showing the comparison results in example 3 of the present invention.
Detailed Description
The method comprises the following steps:
(1) preparing: preparing 60-300nm polystyrene latex microspheres, and diluting the latex microspheres to a final concentration of 1-3% w/v by using 50 mM/L2- (N-morpholine) ethanesulfonic acid with the pH of 6.0 to obtain a latex microsphere solution; dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 50 mM/L2- (N-morpholine) ethanesulfonic acid (EDC) at pH6.0 to obtain an EDC solution with a concentration of 0.01 g/mL;
(2) and (3) activation: dripping each mg of microspheres into the latex microsphere solution according to the proportion of 10-100 mu L of EDC solution by taking the weight of the latex microspheres as a reference, oscillating and uniformly mixing, and oscillating and activating for 20-30min at the temperature of 10-37 ℃ and at the oscillating speed of 50-200rpm/min to obtain activated latex microspheres;
(3) liquid changing: pouring the activated latex microspheres into a sample container of a tangential flow filtration system, pouring a coupling buffer solution into a cleaning solution container, and setting a target pump speed of 30-60mL/min, a transmembrane pressure of 2.5Psi and a shear force of 2000-4000s on an instrument-1After the parameter setting is finished, the starting key is pressed to filter, the volume of the coupling buffer solution is 3-6 times of the volume of the latex microspheres to be filtered, the filtering is stopped by pressing the ending key, and the whole process is controlled inWithin 1 h;
(4) coupling: based on the weight of the latex microspheres, the weight ratio of microspheres: adding the antibody into the solution (3) according to the proportion of the antibody =0.1-0.5%, oscillating and mixing uniformly, and oscillating and coupling for 2-3h at the temperature of 10-37 ℃, wherein the oscillating speed is 50-200 rpm/min;
(5) and (3) sealing: adding 5% bovine serum albumin mother liquor into the solution (4) according to the proportion that the final volume of the solution is 0.5-2%, uniformly mixing by shaking, sealing by shaking at 10-37 ℃ for 1-2h, and the shaking speed is 50-200 rpm/min;
(6) cleaning and liquid changing: pouring the solution (5) into a sample container of a tangential flow filtration system, pouring the storage buffer solution into a cleaning solution container, and setting the target pump speed on the instrument to be 30-60mL/min, the transmembrane pressure to be 2.5Psi and the shearing force to be 2000-4000s-1And after the parameter setting is finished, pressing a start key to filter, wherein the volume of the storage liquid is 3-6 times of the volume of the latex microspheres to be filtered, and pressing an end key to stop filtering to obtain the sensitized latex.
The coupling buffer solution is one of 50-200mM/L phosphate buffer solution, carbonate buffer solution, borate buffer solution and MES buffer solution, and the pH value is 6-10.
The antibody of the present invention is one of a monoclonal antibody and a polyclonal antibody.
The storage buffer solution is one of 20-100mM/L phosphate buffer solution, HEPES buffer solution, glycine buffer solution, Tris buffer solution, PIPES buffer solution and citric acid buffer solution, and the pH value is 6-10; to increase the stability of the reagents, stabilizers are generally added to the stock solutions.
The stabilizer of the invention is one or more of 1-5% of glycerol, sucrose, mannitol and trehalose.
The detection kit containing the sensitization latex comprises R1 and R2 reagents, wherein R1 comprises buffer solution, monovalent or divalent salt, surfactant, PEG and sodium azide; r2 is a sensitized latex.
The buffer solution is one of Tris, phosphate and carbonate buffer solution, and the concentration is 20-500 mM/L.
The monovalent or divalent salt is one or more of sodium chloride, potassium chloride, calcium chloride and magnesium chloride, and the concentration is 0.1-15 g/L.
The surfactant is one of Tween series and Triton series, and the concentration is 1-10 mL/L.
The present invention will be described in further detail with reference to the following examples:
example 1
The cystine protease inhibitor C sensitized latex prepared according to the preferred embodiment of the present invention comprises the following steps:
(1) preparing: preparing 30mL of 150nm polystyrene latex microspheres, and diluting the latex microspheres to 100mL by using 50 mM/L2- (N-morpholine) ethanesulfonic acid with the pH of 6.0 to obtain a latex microsphere solution; weighing 0.05g 1-Ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) dissolved in 5mL 50 mM/L2- (N-morpholino) ethanesulfonic acid pH6.0 to give a 0.01g/mL EDC solution;
(2) and (3) activation: dropwise adding 5mL of EDC solution into the latex microsphere solution, uniformly mixing by shaking, and carrying out shaking activation at 25 ℃ for 25min at a shaking speed of 150rpm/min to obtain activated latex microspheres;
(3) liquid changing: pouring the activated latex microspheres into a sample container of a tangential flow filtration system, pouring a coupling buffer solution into a cleaning solution container, and setting a target pump speed of 30mL/min, a transmembrane pressure of 2.5Psi and a shear force of 2000s on an instrument-1After the parameter setting is finished, the starting key is pressed for filtering, the volume of the coupling buffer solution is 6 times of the volume of the latex microspheres to be filtered, and the ending key is pressed for stopping filtering;
(4) coupling: measuring 265mg of antibody, adding the antibody into the solution (3), uniformly mixing by shaking, coupling by shaking at 25 ℃ for 3 hours, and controlling the shaking speed to be 150 rpm/min;
(5) and (3) sealing: measuring 25mL of 5% bovine serum albumin mother liquor, adding the 5% bovine serum albumin mother liquor into the solution (4), uniformly mixing by shaking, sealing by shaking at 25 ℃ for 1h, and controlling the shaking speed to be 150 rpm/min;
(6) cleaning and liquid changing: pouring the solution (5) into a sample container of a tangential flow filtration system, pouring the storage buffer into a cleaning solution container, and setting the target pump speed at 30mL/min and the transmembrane pressure at 30mL/min on the instrument2.5Psi, shear force 2000s-1And after the parameter setting is finished, pressing a start key to filter, wherein the volume of the storage liquid is 6 times of the volume of the latex microspheres to be filtered, and pressing an end key to stop filtering to obtain the sensitized latex.
Preferably, the coupling buffer in (3) is 50mM/L phosphate buffer solution, and the pH is 7.5;
preferably, the antibody in (4) is a polyclonal antibody;
preferably, the storage buffer in (6) is 100mM/LTris buffer, pH 8.5; in order to improve the stability of the reagent, a stabilizer is generally added into the storage solution;
still further, the stabilizer is 1% mannitol and 2% trehalose;
the cystine protease inhibitor C detection kit containing the prepared sensitized latex is characterized in that: including R1 and R2 reagents, wherein R1 includes buffers, mono or divalent salts, surfactants, PEG, and sodium azide; r2 is sensitizing latex;
the specific operation of preparing R1 is as follows: weighing 1L of purified water, adding 2.385g of anhydrous disodium hydrogen phosphate, 0.4992g of sodium dihydrogen phosphate, 9g of sodium chloride, 10g of PEG6000 and 1g of sodium azide into a beaker respectively, and stirring for dissolving; then adding 2mL of Tween 20, and uniformly stirring to obtain an R1 reagent;
application of kit in biochemical analyzer
The test kit comprises: the kit of example 1, the cystine protease inhibitor C kit of comparative example 1 (manufactured by Beijing Baishanglide biological Co., Ltd.)
Testing an instrument: hitachi 7100 biochemical analyzer
Specifically, the parameters are set as follows: the test method comprises the following steps: a two-point end-point method; dominant wavelength: 546 nm; light spot measurement: 18-34; sample size: 6; reagent amount R1: r2= 180: 45, a first step of; the calibration method comprises the following steps: spline
Setting of standard curve: the following data were obtained by placing standards of known concentrations, i.e., 0.50, 1.00, 2.00, 4.00, 8.00g/L, in the calibration plate, respectively, and setting the locations and concentrations of the standards:
TABLE 1 (example 1)
TABLE 2 (comparative example 1)
In parallel with the comparative example, 25 clinical samples of different concentrations were tested to obtain the following data (table 3):
the results in table 3 show that the kit prepared by the present invention has better correlation with comparative example 1, and the measured values are consistent.
After calibration, the same serum sample was measured 10 times, and the average value and SD value were obtained from the results of concentration measurement 10 times, and the CV value was calculated. The results are shown in Table 4:
as can be seen from the data in Table 4, the measurement results of example 1 on the same sample are not much different from those of comparative example 1 in repeatability, which shows that the reagent has good accuracy in measuring the sample.
The self-matched linear samples were assayed and 8mg/L of caspase inhibitor C antigen was diluted to 5 concentrations, each concentration assay result is shown in Table 5.
As can be seen from the data in Table 5 and the results in FIG. 1, the linear measurements of the examples are slightly higher than those of the comparative examples.
Example 2
The retinol binding protein sensitized latex prepared according to the preferred embodiment of the present invention comprises the following steps:
(1) preparing: preparing 15mL of 123nm polystyrene latex microspheres, and diluting the latex microspheres to 100mL by using 50 mM/L2- (N-morpholine) ethanesulfonic acid with the pH of 6.0 to obtain a latex microsphere solution; weighing 0.03g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) dissolved in 3mL of 50 mM/L2- (N-morpholino) ethanesulfonic acid pH6.0 to give a 0.01g/mL EDC solution;
(2) and (3) activation: dropwise adding 3mL of EDC solution into the latex microsphere solution, uniformly mixing by shaking, and carrying out shaking activation at 37 ℃ for 25min at a shaking speed of 220rpm/min to obtain activated latex microspheres;
(3) liquid changing: pouring the activated latex microspheres into a sample container of a tangential flow filtration system, pouring a coupling buffer solution into a cleaning solution container, and setting a target pump speed of 50mL/min, a transmembrane pressure of 2.5Psi and a shear force of 2000s on an instrument-1After the parameter setting is finished, the starting key is pressed for filtering, the volume of the coupling buffer solution is 5 times of the volume of the latex microspheres to be filtered, and the ending key is pressed for stopping filtering;
(4) coupling: measuring 22.5mg of antibody, adding the antibody into the solution (3), uniformly mixing by shaking, coupling by shaking at 37 ℃ for 2.5h, and the shaking speed is 220 rpm/min;
(5) and (3) sealing: measuring 10mL of 5% bovine serum albumin mother liquor, adding the solution into the solution (4), uniformly mixing by shaking, sealing by shaking at 37 ℃ for 1h, and controlling the shaking speed to be 220 rpm/min;
(6) cleaning and liquid changing: pouring the solution (5) into a sample container of a tangential flow filtration system, pouring the storage buffer into a washing solution container, setting the target pump speed on the apparatus at 50mL/min, the transmembrane pressure at 2.5Psi, and the shear force at 2000s-1And after the parameter setting is finished, pressing a start key to filter, wherein the volume of the storage liquid is 5 times of the volume of the latex microspheres to be filtered, and pressing an end key to stop filtering to obtain the sensitized latex.
Preferably, the coupling buffer in (3) is 50mM/L borate buffer, and the pH is 8.4;
preferably, the antibody in (4) is a polyclonal antibody;
preferably, the storage buffer in (6) is 20mM/L phosphate buffer, and the pH is 7.5; in order to improve the stability of the reagent, a stabilizer is generally added into the storage solution;
still further, the stabilizer is 3% glycerin;
the retinol binding protein detection kit containing the preparation method is characterized in that: including R1 and R2 reagents, wherein R1 includes buffers, mono or divalent salts, surfactants, PEG, and sodium azide; r2 is sensitizing latex;
the specific operation of preparing R1 is as follows: weighing 1L of purified water, adding 2.385g of anhydrous disodium hydrogen phosphate, 0.4992g of sodium dihydrogen phosphate, 7g of sodium chloride, 7g of PEG6000 and 1g of sodium azide into a beaker respectively, and stirring for dissolving; then adding 5mL of Tween 80, and uniformly stirring to obtain an R1 reagent;
application of kit in biochemical analyzer
The test kit comprises: the kit of example 2, the retinol binding protein kit of comparative example 2 (manufactured by Ningboruiyuan biological Co., Ltd.)
Testing an instrument: hitachi 7100 biochemical analyzer
Specifically, the parameters are set as follows: the test method comprises the following steps: a two-point end-point method; dominant wavelength: 600 nm; secondary wavelength: 700 nm; light spot measurement: 18-34; sample size: 3; reagent amount R1: r2= 210: 70; the calibration method comprises the following steps: spline
Setting of standard curve: the following data were obtained by placing standards of known concentrations, i.e., 0, 15.12, 30.25, 60.5, 121mg/L, respectively, in the calibration plate, and setting the locations and concentrations of the standards:
TABLE 6 (example 2)
TABLE 7 (comparative example 2)
In parallel with the comparative example, 25 clinical samples of different concentrations were tested to obtain the following data (table 8):
the results in table 8 show that the kit prepared by the present invention has better correlation with comparative example 2, and the measured values are consistent.
After calibration, the same serum sample was measured 10 times, and the average value and SD value were obtained from the results of concentration measurement 10 times, and the CV value was calculated. The results are shown in Table 9:
as can be seen from the data in Table 9, the measurement results of example 2 on the same sample are not much different from those of comparative example 2, which shows that the reagent has good accuracy in measuring the sample.
Self-prepared linear samples were assayed by diluting the retinol binding protein antigen 130mg/L to 5 concentrations, and the results of each concentration assay are shown in Table 10
As can be seen from the data in Table 10 and the results in FIG. 2, the values measured on the linear lines of the examples are less deviated and more accurate than those measured in comparative example 2.
Example 3
The rheumatoid factor sensitized latex prepared according to the preferred embodiment of the invention comprises the following steps:
(1) preparing: preparing 20mL of 160nm polystyrene latex microspheres, and diluting the latex microspheres to 100mL by using 50 mM/L2- (N-morpholine) ethanesulfonic acid with the pH of 6.0 to obtain a latex microsphere solution; weighing 0.03g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) dissolved in 4mL of 50 mM/L2- (N-morpholino) ethanesulfonic acid pH6.0 to give a 0.01g/mL EDC solution;
(2) and (3) activation: dropwise adding 4mL of EDC solution into the latex microsphere solution, uniformly mixing by shaking, and activating by shaking at 37 ℃ for 30min at a shaking speed of 220rpm/min to obtain activated latex microspheres;
(3) liquid changing: pouring the activated latex microspheres into a sample container of a tangential flow filtration system, pouring a coupling buffer solution into a cleaning solution container, and setting a target pump speed of 45mL/min, a transmembrane pressure of 2.5Psi and a shear force of 2000s on an instrument-1After the parameter setting is finished, the starting key is pressed for filtering, the volume of the coupling buffer solution is 6 times of the volume of the latex microspheres to be filtered, and the ending key is pressed for stopping filtering;
(4) coupling: measuring 30mg of antibody, adding the antibody into the solution (3), uniformly mixing by shaking, coupling by shaking at 37 ℃ for 3 hours, and shaking at the speed of 220 rpm/min;
(5) and (3) sealing: measuring 16mL of 5% bovine serum albumin mother solution, adding the 5% bovine serum albumin mother solution into the solution (4), uniformly mixing by shaking, sealing by shaking at 37 ℃ for 1h, and controlling the shaking speed to be 220 rpm/min;
(6) cleaning and liquid changing: pouring the solution (5) into a sample container of a tangential flow filtration system, pouring the storage buffer into a washing solution container, setting the target pump speed on the apparatus at 45mL/min, the transmembrane pressure at 2.5Psi, and the shear force at 2000s-1And after the parameter setting is finished, pressing a start key to filter, wherein the volume of the storage liquid is 6 times of the volume of the latex microspheres to be filtered, and pressing an end key to stop filtering to obtain the sensitized latex.
Preferably, the coupling buffer in (3) is 20mM/L carbonate buffer, and the pH is 9.21;
preferably, the antibody in (4) is a monoclonal antibody;
preferably, the storage buffer in (6) is 20mM/L glycine buffer, and the pH is 9.1; in order to improve the stability of the reagent, a stabilizer is generally added into the storage solution;
still further, the stabilizer is 1% sucrose;
the rheumatoid factor detection kit containing the preparation method is characterized in that: including R1 and R2 reagents, wherein R1 includes buffers, mono or divalent salts, surfactants, PEG, and sodium azide; r2 is sensitizing latex;
the specific operation of preparing R1 is as follows: measuring 1L of purified water in a beaker, respectively adding 0.0311g of Tris, 3.114g of Tris-HCL, 9g of sodium chloride, 10g of PEG6000 and 1g of sodium azide, and stirring for dissolving; then adding 2mL of triton 100, and uniformly stirring to obtain an R1 reagent;
application of kit in biochemical analyzer
The test kit comprises: the kit of example 3, the rheumatoid factor detection kit of comparative example 3 (manufactured by Ningbo Meikang Bio Inc.)
Testing an instrument: hitachi 7100 biochemical analyzer
Specifically, the parameters are set as follows: the test method comprises the following steps: a two-point end-point method; dominant wavelength: 600 nm; light spot measurement: 18-34; sample size: 3; reagent amount R1: r2= 180: 45, a first step of; the calibration method comprises the following steps: spline
Setting of standard curve: the following data were obtained by placing standards of known concentrations, i.e., 0, 15.13, 30.25, 60.5, 121IU/mL, respectively, in the calibration disk, setting the locations and concentrations of the calibrators:
table 11 (example 3)
TABLE 12 (comparative example 3)
In parallel with the comparative example, 25 clinical samples of different concentrations were tested to obtain the following data (table 13):
the results in table 13 show that the kit prepared by the present invention has better correlation with comparative example 3, and the measured values are consistent.
After calibration, the same serum sample was measured 10 times, and the average value and SD value were obtained from the results of concentration measurement 10 times, and the CV value was calculated. The results are shown in Table 14:
as can be seen from the data in Table 14, the measurement results of example 2 on the same sample are not much different from those of comparative example 3, which shows that the reagent has good accuracy in measuring the sample.
Self-prepared linear samples were assayed by diluting 150IU/mL rheumatoid factor antigen to 5 concentrations, each concentration determined as shown in Table 15
As can be seen from the data in Table 10 and the results in FIG. 3, the values measured on the linear lines of the examples are less deviated and more accurate than those measured in comparative example 2.
Claims (9)
1. A preparation method of sensitized latex comprises the following steps:
(1) preparing: preparing 60-300nm polystyrene latex microspheres, and diluting the latex microspheres to a final concentration of 1-3% w/v by using 50mM 2- (N-morpholine) ethanesulfonic acid with the pH of 6.0 to obtain a latex microsphere solution; dissolving EDC with 50mM 2- (N-morpholine) ethanesulfonic acid (pH6.0) to obtain EDC solution with concentration of 0.01 g/mL;
(2) and (3) activation: dropwise adding each mg of microspheres into the latex microsphere solution according to the proportion of 10-100 mu L of EDC solution by weight of the latex microspheres, uniformly mixing by oscillation, and performing oscillation activation at 10-37 ℃ for 20-30min at the oscillation speed of 50-200rpm to obtain activated latex microspheres;
the method is characterized in that:
(3) liquid changing: pouring the activated latex microspheres into a sample container of a tangential flow filtration system, pouring a coupling buffer solution into a cleaning solution container, and setting a target pump speed of 30-60mL, a transmembrane pressure of 2.5Psi and a shear force of 2000-4000s on an instrument-1After the parameter is set, the filter is carried out by pressing a start key, and buffer solution is coupledThe volume of the latex microsphere is 3-6 times of the volume of the latex microsphere to be filtered, the filtration is stopped by pressing an end key, and the whole process is controlled within 1 h;
(4) coupling: based on the weight of the latex microspheres, the weight ratio of microspheres: adding the antibody into the solution obtained in the step (3) according to the proportion that the antibody =0.1-0.5%, oscillating and uniformly mixing, and oscillating and coupling for 2-3h at the temperature of 10-37 ℃, wherein the oscillating speed is 50-200 rpm;
(5) and (3) sealing: adding 5% bovine serum albumin mother liquor into the solution obtained in the step (4) according to the proportion that the final volume of the solution is 0.5-2%, uniformly mixing by shaking, sealing by shaking for 1-2h at 10-37 ℃, and the shaking speed is 50-200 rpm;
(6) cleaning and liquid changing: pouring the solution obtained in the step (5) into a sample container of a tangential flow filtration system, pouring a storage buffer solution into a cleaning solution container, and setting a target pump speed of 30-60mL/min, a transmembrane pressure of 2.5Psi and a shear force of 2000-4000s on an instrument-1And after the parameter setting is finished, pressing a start key to filter, wherein the volume of the storage buffer solution is 3-6 times of the volume of the latex microspheres to be filtered, and pressing an end key to stop filtering to obtain the sensitized latex.
2. The method for producing the sensitized latex according to claim 1, characterized in that: the coupling buffer is one of 50-200mM phosphate buffer, carbonate buffer, borate buffer and MES buffer, and has a pH of 6-10.
3. The method for producing the sensitized latex according to claim 1, characterized in that: the antibody is one of a monoclonal antibody and a polyclonal antibody.
4. The method for producing the sensitized latex according to claim 1, characterized in that: the storage buffer solution is one of 20-100mM phosphate buffer solution, HEPES buffer solution, glycine buffer solution, Tris buffer solution, PIPES buffer solution and citric acid buffer solution, and the pH value is 6-10; a stabilizer is added to the storage buffer.
5. The method for producing the sensitized latex according to claim 4, characterized in that: the stabilizer is one or more of 1-5% glycerol, sucrose, mannitol, and trehalose.
6. A detection kit containing the sensitized latex according to claim 1, characterized in that:
comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a buffer solution, a monovalent or divalent salt, a surfactant, PEG and sodium azide; the R2 reagent is a sensitizing latex.
7. The detection kit according to claim 6, characterized in that: the buffer solution in the R1 reagent is one of Tris, phosphate and carbonate buffer solution, and the concentration is 20-500 mM.
8. The detection kit according to claim 6, characterized in that: the monovalent or divalent salt is one or more of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride, and has a concentration of 0.1-15 g/L.
9. The detection kit according to claim 6, characterized in that: the surfactant is one of Tween series and Triton series, and the concentration is 1-10 mL/L.
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| CN111707817A (en) * | 2020-05-29 | 2020-09-25 | 吉林基蛋生物科技有限公司 | Preparation and detection method of hepatobiliary acid determination kit |
| CN111999501A (en) * | 2020-08-20 | 2020-11-27 | 安徽伊普诺康生物技术股份有限公司 | Kit for measuring human serum lipoprotein phospholipase A2 and preparation and use methods thereof |
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