CN111856026A - Kit for determining III C based on latex enhanced immunoturbidimetry and preparation and use methods thereof - Google Patents
Kit for determining III C based on latex enhanced immunoturbidimetry and preparation and use methods thereof Download PDFInfo
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- G—PHYSICS
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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Abstract
The invention provides a kit for determining III C based on a latex enhanced immunoturbidimetry, which comprises a reaction buffer solution R1 and a latex reagent R2; the reaction buffer solution R1 comprises buffer solution, inorganic salt, turbidity-increasing agent, preservative, stabilizer and surfactant; the latex reagent R2 comprises at least two latex reagent component solutions formed by coupling the III C monoclonal antibody and latex microspheres, and the mixing ratio of each component solution is 1: 1; in addition, each latex reagent composition liquid also comprises a latex preservation liquid and latex microspheres preserved in the latex preservation liquid, wherein the latex preservation liquid comprises a buffer solution, inorganic salt, a stabilizer, a preservative and a surfactant. The invention also provides a preparation and use method of the kit for determining III C based on the latex enhanced immunoturbidimetry. The invention has the advantages that: the method has the advantages of simple operation, high detection speed, good selectivity, good specificity, high precision, good repeatability and consistency.
Description
Technical Field
The invention relates to the field of preparation of biological detection reagents, in particular to a kit for determining III C based on a latex enhanced immunoturbidimetry method and a preparation and use method thereof.
Background
The process of liver disease progression is characterized by the occurrence of hepatic fibrosis, with the progressive increase of hepatic fibrosis, a great amount of shedding and necrosis of liver cells occur, and meanwhile, the lobular structure of the liver gradually becomes twisted and weighted, and finally, liver cirrhosis occurs. Therefore, the symptoms of the patients can be greatly relieved by timely and effectively diagnosing and treating the patients with the liver diseases clinically, and the further deterioration of the disease condition can be avoided. Histopathological examination is the clinical diagnosis gold standard of hepatic fibrosis, and although the pathological examination has extremely high accuracy, the histopathological examination inevitably causes certain trauma to patients and is inconvenient for multiple diagnoses. Therefore, serology indexes are widely adopted clinically to judge the severity of hepatic fibrosis, wherein type III collagen (III C) is one of indexes with wide application and high accuracy.
Hepatic fibrosis is fibrosis caused by different degrees and different types of chronic liver diseases, and the basic problem of hepatic fibrosis such as chronic hepatitis B, chronic hepatitis C, fatty liver and the like is the increase of matrix such as inner collagen, non-collagen glycoprotein, protein diversity and the like of liver tissues. The type III collagen (III C) belongs to extracellular matrix components, and the content of the extracellular matrix components in serum can be obviously increased along with the more serious condition development, so the collagen III (III C) has obvious value for judging the severity of liver diseases and liver fibrosis.
As for the detection method of type III collagen, chemiluminescence is currently the main method on the market. Chemiluminescence is a sensitive and simple method, but it is poorly selective and reacts to a series of compounds rather than to a single compound. Therefore, a method for detecting type III collagen with good selectivity, good specificity and high precision is urgently needed.
Disclosure of Invention
The invention aims to provide a kit for determining III C based on a latex enhanced immunoturbidimetry, which has good selectivity, good specificity and high precision, and a preparation and use method thereof.
The invention adopts the following technical scheme to solve the technical problems:
a kit for determining III C based on latex enhanced immunoturbidimetry, comprising a reaction buffer R1 and a latex reagent R2;
the reaction buffer R1 comprises the following components in parts by weight: 5-12g/L of buffer solution, 2-15 g/L of inorganic salt, 10-30g/L of turbidity enhancer, 1-2mL/L of preservative, 3-14g/L of stabilizer, 0.5-2.5 mL/L of surfactant and purified water as solvent;
the latex reagent R2 comprises at least two latex reagent composition liquids formed by coupling a III C monoclonal antibody and latex microspheres, wherein each latex reagent composition liquid comprises a latex preservative solution and corresponding III C monoclonal antibody labeled latex microspheres which are preserved in the latex preservative solution and have the final concentration of 0.5-1.0 mg/mL; the latex preservation solution comprises the following components in percentage by weight: 2-7g/L of buffer solution, 5-12g/L of inorganic salt, 10-20g/L of stabilizing agent, 1-2mL/L of preservative, 1-7mL/L of surfactant and purified water as solvent; in the latex reagent R2, the mixing ratio of each latex reagent composition liquid formed by coupling the III C monoclonal antibody and the latex microspheres is 1: 1.
In a preferred embodiment of the present invention, in the reaction buffer R1:
the buffer solution is one of a tris buffer solution, a glycine buffer solution, a 2- (N-morpholine) ethanesulfonic acid buffer solution (namely, MES buffer solution) and a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution (namely, MOPSO buffer solution);
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
the turbidity increasing agent is one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000 and polyethylene glycol 8000;
the preservative is one of Proclin300 and thimerosal;
the stabilizer is one of BSA and sucrose;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
In a preferred embodiment of the present invention, the reaction buffer R1 has a pH of 7.1 to 7.9.
In a preferred embodiment of the present invention, in the latex preservative solution:
the buffer solution is one of a tris buffer solution, a glycine buffer solution, a 2- (N-morpholine) ethanesulfonic acid buffer solution and a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution;
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
The stabilizer is one or more of BSA, sucrose, mannitol and glycine;
the preservative is one or more of Proclin300 and thimerosal;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
In a preferred embodiment of the present invention, the latex preservative solution has a pH of 6.1 to 7.5.
In a preferred embodiment of the present invention, the diameter of the latex microspheres used in the III C monoclonal antibody-labeled latex microspheres is 100-300 nm.
In a preferred embodiment of the present invention, the latex reagent R2 is prepared by a method comprising:
firstly, taking latex microspheres with solid content of 10%, and adding the latex microspheres into a cleaning buffer solution; centrifuging, removing supernatant, carrying out resuspension ultrasonic treatment on the latex microsphere precipitate by using an activation buffer solution, and adding an activating agent for activation; centrifuging again, and carrying out heavy suspension ultrasonic treatment on the latex microsphere precipitate by using a coupling buffer solution to prepare activated latex microspheres;
uniformly mixing at least two III C monoclonal antibodies with the prepared activated latex microspheres according to a specific ratio, and performing coupling for a certain time to prepare coupled latex microspheres; wherein the v/w ratio of the activated latex microspheres to each III C monoclonal antibody is respectively 1 (3-5), and the coupling time is 1 h;
Adding a sealing buffer solution into the coupled latex microspheres for sealing; after the sealing is finished, centrifuging again, removing supernatant, adding latex preservation solution for resuspension and ultrasound, and preparing respective latex reagent composition solutions; then, the prepared latex reagent composition liquids are mixed to obtain the required latex reagent R2.
The preparation method of the reagent for determining III C based on the latex enhanced immunoturbidimetry comprises the following steps:
(1) preparing a reaction buffer R1:
according to the component content of the reaction buffer solution R1, mixing all the components in the same container, and uniformly mixing to obtain a reaction buffer solution R1;
(2) formulation of latex reagent R2:
firstly, taking latex microspheres with solid content of 10%, and adding the latex microspheres into a cleaning buffer solution; centrifuging, removing supernatant, carrying out resuspension ultrasonic treatment on the latex microsphere precipitate by using an activation buffer solution, and adding an activating agent for activation; centrifuging again, and carrying out heavy suspension ultrasonic treatment on the latex microsphere precipitate by using a coupling buffer solution to prepare activated latex microspheres;
uniformly mixing at least two III C monoclonal antibodies with the prepared activated latex microspheres according to a specific ratio, and performing coupling for a certain time to prepare coupled latex microspheres; wherein the v/w ratio of the activated latex microspheres to each III C monoclonal antibody is respectively 1 (3-5), and the coupling time is 1 h;
Adding a sealing buffer solution into the coupled latex microspheres for sealing; after the sealing is finished, centrifuging again, removing supernatant, adding latex preservation solution for resuspension and ultrasound, and preparing respective latex reagent composition solutions; then, the prepared latex reagent composition liquids are mixed to obtain the required latex reagent R2.
In a preferred embodiment of the present invention, in the first step, MES buffer is used as the washing buffer and the activation buffer; the coupling buffer solution adopts a phosphate buffer solution; the activator adopts EDAC/NHS; in the third step, the blocking buffer solution adopts one or more of BSA and glycine, and the blocking time is 1 h.
The use method of the kit for determining III C based on the latex enhanced immunoturbidimetry comprises the following specific steps:
(1) sucking 5 μ L of sample, adding 200 μ L of reagent R1, and incubating at 37 deg.C for 3-5 min;
(2) adding 50 μ L reagent R2, and incubating at 37 deg.C;
(3) reading absorbance a1 after 60s incubation; incubating for 5min, and reading the light absorption value A2; and finally, calculating delta A, wherein the delta A is A2-A1, and calculating the content of III C in the sample according to the delta A.
As one of the preferred embodiments of the present invention, each of the III C monoclonal antibodies used in the present invention is derived from a conventional III C monoclonal antibody which is commercially available in the art.
The reaction principle of the kit of the invention is as follows:
the type III collagen in the sample is combined with the specific anti-human type III collagen antibody latex particles in the reagent to form an antigen-antibody complex so as to generate turbidity, and the turbidity is in direct proportion to the type III collagen in the sample. The content of the type III collagen in the specimen can be calculated by measuring the absorbance value under a specific wavelength and referring to a calibration curve.
Compared with the prior art, the invention has the advantages that:
(1) the kit disclosed by the invention is used for measuring III C by using a latex enhanced immunoturbidimetry, and has the advantages of simplicity in operation, high detection speed, good selectivity, good specificity, high precision, good repeatability and good consistency;
(2) the invention can be used on a full-automatic biochemical analyzer, has low cost and high automation, can save the detection time and is convenient for clinical application;
(3) the kit prepared by the method can simultaneously improve the sensitivity and specificity of detection, so that the kit has wider linear range and higher accuracy and better meets the clinical requirements;
(4) in the application of the latex enhanced immunoturbidimetry, if the polyclonal antibody is adopted for marking, the sensitivity of the reagent can be effectively improved, but the specificity is not strong, so that the accuracy of the detection result is reduced; if a monoclonal antibody is used for marking latex, the specificity can be improved, but the sensitivity is influenced; aiming at the problems, in the kit, at least two III C monoclonal antibodies with stronger specificity are selected to be respectively marked on latex, and then the latex is mixed together, so that the specificity is stronger under the condition of keeping the sensitivity.
Drawings
FIG. 1 is a linear range linear regression plot of the kit for the latex enhanced immunoturbidimetry based assay of III C in example 6.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
A kit for the latex-enhanced immunoturbidimetry-based assay of III C of this example comprises reaction buffer R1 and latex reagent R2.
The reaction buffer R1 comprises the following components in parts by weight: 5g/L of tris buffer solution, 2g/L of magnesium chloride or potassium chloride, 1000 or 200010g/L of polyethylene glycol, 3001 mL/L of Proclin, 3 g/L of BSA, and 800.5mL/L of Tween, wherein the solvent is purified water, and the pH value is 7.1.
The latex reagent R2 comprises at least two latex reagent compositions formed by coupling the III C monoclonal antibody and latex microspheres, and each latex reagent composition comprises a latex preservative solution and corresponding III C monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have the final concentration of 0.5 mg/mL. Wherein, the latex preservative fluid comprises the following components in percentage by weight: 2g/L of tris buffer solution, 5g/L of sodium chloride or potassium chloride, 10g/L of BSA, 3001 mL/L of Proclin and 801mL/L of Tween, wherein the solvent is purified water and the pH value is 6.1.
Further, in the latex reagent R2, the mixing ratio of each latex reagent composition liquid formed by coupling the iiic mab and the latex microspheres was 1: 1.
Further, in the latex reagent R2, the latex microspheres used were 100nm in diameter.
Example 2
A kit for the latex-enhanced immunoturbidimetry-based assay of III C of this example comprises reaction buffer R1 and latex reagent R2.
The reaction buffer R1 comprises the following components in parts by weight: 12g/L of glycine buffer solution or 2- (N-morpholine) ethanesulfonic acid buffer solution, 15g/L of zinc chloride or calcium chloride, 800030g/L of polyethylene glycol, 2mL/L of thimerosal, 14g/L of sucrose, and 14g/L of triton X-1002.5mL/L, wherein the solvent is purified water, and the pH value is 7.9.
The latex reagent R2 comprises at least two latex reagent compositions formed by coupling the III C monoclonal antibody and latex microspheres, and each latex reagent composition comprises a latex preservative solution and corresponding III C monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have the final concentration of 1.0 mg/mL. Wherein, the latex preservative fluid comprises the following components in percentage by weight: 7 g/L of glycine buffer solution or 2- (N-morpholine) ethanesulfonic acid buffer solution, 12g/L of zinc chloride or calcium chloride, 20g/L of sucrose or mannitol, 2mL/L of thimerosal, X-1007 mL/L of triton, purified water as a solvent and 7.5 of pH.
Further, in the latex reagent R2, the mixing ratio of each latex reagent composition liquid formed by coupling the iiic mab and the latex microspheres was 1: 1.
Further, in the latex reagent R2, the latex microspheres used were 300nm in diameter.
Example 3
A kit for the latex-enhanced immunoturbidimetry-based assay of III C of this example comprises reaction buffer R1 and latex reagent R2.
The reaction buffer R1 comprises the following components in parts by weight: 8.06g/L MOPSO buffer solution, 5.4g/L sodium chloride, 600020g/L polyethylene glycol, 1.2 mL/L Proclin300, 6g/L BSA, and 201.5 mL/L Tween, wherein the solvent is purified water, and the pH value is 7.6.
The latex reagent R2 comprises at least two latex reagent compositions formed by coupling the III C monoclonal antibody and latex microspheres, and each latex reagent composition comprises a latex preservative solution and corresponding III C monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have the final concentration of 0.75 mg/mL. Wherein, the latex preservative fluid comprises the following components in percentage by weight: 4.3g/L of MOPSO buffer solution, 8.7g/L of magnesium chloride, 16g/L of glycine, 1.2 mL/L of Proclin 300and 204.5mL/L of Tween-L, wherein the solvent is purified water and the pH value is 6.6.
Further, in the latex reagent R2, the mixing ratio of each latex reagent composition liquid formed by coupling the iiic mab and the latex microspheres was 1: 1.
Further, in the latex reagent R2, the latex microspheres used were 200nm in diameter.
Example 4
This example is a method for preparing the kit for determining III C based on latex enhanced immunoturbidimetry as in examples 1-3 above, comprising the steps of:
(1) preparing a reaction buffer R1:
according to the component content of the reaction buffer solution R1, all the components are mixed in the same container, and after uniform mixing, the reaction buffer solution R1 is prepared.
(2) Formulation of latex reagent R2:
cleaning: taking 0.5mL of latex particles with the solid content of 10%, and adding the latex particles into 3.0mL of washing buffer solution; centrifuging at 20000r/min for 30min, and removing supernatant; the latex microparticle pellet was sonicated with 3.0mL of activation buffer to resuspend the pellet. In this step, MES buffer was used as the washing buffer and the activation buffer.
And (3) activation: weighing a certain amount of activating agent, adding the activating agent into the cleaned latex particles, uniformly mixing, and activating the latex particles; and centrifuging again, and carrying out resuspension ultrasonic treatment on the latex microsphere precipitate by using a coupling buffer solution to prepare the activated latex microspheres. In the step, phosphate buffer is adopted as coupling buffer; the activator adopts 0.1mL of 2 percent EDAC and 0.2mL of 4 percent NHS, and the activation time is 0.5 h; the ratio of the corresponding latex particulates to activator was 1: (15-25) (weight ratio).
Coupling: coupling at least two screened III C monoclonal antibodies with strong specificity with activated latex particles respectively to prepare coupled latex microspheres. In the step, the v/w ratio of the activated latex particles to each III C monoclonal antibody is respectively 1 (3-5), and the coupling time is 1 h.
And (3) sealing: after the coupling time is over, the centrifugation is started, the centrifugation is carried out for 30min at 20000r/min, and the supernatant is removed; resuspend the latex microparticle pellet with 5.0mL of blocking buffer, sonicate and then block for 1 h. In the step, blocking buffer adopts one or more of BSA and glycine, and preferably 1% Gly.
And (3) dissolving: centrifuging at 20000r/min for 30min, and removing supernatant; adding 40mL of latex preservation solution to carry out resuspension ultrasonic treatment on the latex particles, and then carrying out fixed dissolution to prepare respective latex reagent composition solutions; finally, mixing the prepared latex reagent composition liquid according to the proportion of 1: mixing at a ratio of 1 to obtain the required latex reagent R2.
Example 5
This example is a method of using the kit for the latex-enhanced immunoturbidimetry-based determination of III C in examples 1-3 above.
A detection instrument: hitachi 7180;
temperature: 37 ℃;
a cuvette: 1 cm;
The analysis method comprises the following steps: a two-point end-point method;
light spot measurement: 19 to 34;
primary and secondary wavelengths: 546/0, respectively;
sample size/R1/R2: 5uL/200uL/50 uL;
the reaction direction is as follows: (+).
The method comprises the following steps: see table 1.
TABLE 1 procedure for use of the kit of the invention
The calibration mode is a Spline function Spline. And establishing a working curve by adopting a multipoint calibration mode and using purified water as a zero point.
The calculation method comprises the following steps: and fitting a calibration curve to the corresponding delta A according to the concentration of the calibrator, and obtaining the concentration value of the sample through the calibration curve.
Example 6
This example is intended to evaluate the kit for the latex-enhanced immunoturbidimetry-based determination of III C in the above examples 1-3:
(1) linear correlation verification
Linear correlation coefficient: the high value specimens close to the upper limit of the linear interval were diluted with the low value specimens close to the lower limit of the linear interval in the range of [5.0ng/mL-90.0ng/mL ], mixed into specimens of at least 5 different concentrations (Xi), measured 3 times for each specimen, and the mean value (yi) of the measurement results was calculated, respectively, as shown in Table 2. The linear regression equation was calculated using the dilution concentration (Xi) as an independent variable and the measurement result mean (yi) as a dependent variable. And (3) calculating a correlation coefficient r of the linear regression according to the formula (1), wherein the obtained result is that r is more than or equal to 0.9900.
TABLE 2 comparison of the Linear correlation of the kit of the invention
FIG. 1 is a linear range linear regression plot of the kit of the present invention, as shown in FIG. 1, the straight line fitting curve of the kit is: y 1.0148x +0.0475, R21.0; wherein, R2>0.99, meeting the clinical requirements.
(2) Precision and repeatability verification
Repeatability: under the repeated condition, samples (quality control products, calibration products or other fixed value samples) with the concentrations of (15.0 +/-5.0) ng/mL and (40.0 +/-10.0) ng/mL are taken, the same batch of reagents are used for repeated measurement for 10 times, the average value and the standard deviation of the measured values are respectively calculated, the intra-batch Coefficient of Variation (CV) is calculated according to the formula (2), and the obtained result CV is less than or equal to 5.0 percent.
Coefficient of variation
In the formula: SD is standard deviation, and the calculation formula is
dn is the deviation of the measured values at the same level, and the calculation formula is
xn is measured at the same level for each time;
The results are shown in Table 3.
TABLE 3 summary table of the repeatability test results of the kit of the present invention
As can be seen from the detection results, the CV of the low-value sample is 3.49%, and the CV of the high-value sample is 3.22%, which are both less than 5%, and meet the clinical requirements.
(3) Accuracy verification
Taking a high-level quality control product and a low-level quality control product, measuring according to the operating steps of the specification, repeatedly measuring 3 times by using the same batch of reagent, recording the test result as (xi), calculating the relative deviation (Bi) according to the formula (3), wherein the 3 times of results all accord with the measured value and the corresponding deviation of the target value of the quality control product is less than or equal to 15.0 percent; if 2 times of results in 3 times of results meet the requirements and 1 time of results do not meet the requirements, continuously testing for 20 times again, and respectively calculating the relative deviation (Bi) according to the formula (3), if the results are more than or equal to 19 times, the results meet the requirements, and the accuracy is verified, namely, the requirements that the corresponding deviation of the measured value and the target value of the quality control product is less than or equal to 15.0 percent are met.
In the formula: xi is the result of the determination;
t is the target value.
The results are shown in Table 4.
TABLE 4 summary table of accuracy test results of the kit of the present invention
The detection result shows that the relative deviation of the low-value quality control is 5.7 percent, the relative deviation of the high-value quality control is 2.8 percent, and the relative deviation is less than 15 percent, so the clinical requirements are met.
In conclusion, the kit for determining III C based on the latex enhanced immunoturbidimetry and the preparation and use methods thereof provided by the invention can effectively improve the sensitivity and specificity of the III C detection reagent. The kit for detecting the III C is prepared by adopting an innovative technology of coupling at least two III C monoclonal antibodies with strong specificity through the marked latex particles and then mixing the coupling together, realizes the automatic detection on a full-automatic biochemical analyzer, effectively improves the sensitivity and the specificity of the reagent, and has extremely high application value and wide market prospect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A kit for measuring IIIC based on latex enhanced immunoturbidimetry is characterized by comprising a reaction buffer R1 and a latex reagent R2;
The reaction buffer R1 comprises the following components in parts by weight: 5-12g/L of buffer solution, 2-15g/L of inorganic salt, 10-30g/L of turbidity enhancer, 1-2mL/L of preservative, 3-14g/L of stabilizer, 0.5-2.5mL/L of surfactant and purified water as solvent;
the latex reagent R2 comprises at least two latex reagent constitutional solutions formed by coupling the IIIC monoclonal antibody and latex microspheres, and each latex reagent constitutional solution respectively comprises a latex preservative solution and corresponding IIIC monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have the final concentration of 0.5-1.0 mg/mL; the latex preservation solution comprises the following components in percentage by weight: 2-7g/L of buffer solution, 5-12g/L of inorganic salt, 10-20g/L of stabilizing agent, 1-2mL/L of preservative, 1-7mL/L of surfactant and purified water as solvent; in the latex reagent R2, the mixing ratio of each latex reagent composition liquid formed by coupling the IIIC monoclonal antibody and the latex microspheres is 1: 1.
2. The latex-enhanced immunoturbidimetry-based kit for the IIIC determination according to claim 1, characterized in that in the reaction buffer R1:
the buffer solution is one of a tris buffer solution, a glycine buffer solution, a 2- (N-morpholine) ethanesulfonic acid buffer solution and a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution;
The inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
the turbidity increasing agent is one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000 and polyethylene glycol 8000;
the preservative is one of Proclin300 and thimerosal;
the stabilizer is one of BSA and sucrose;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
3. The latex-enhanced immunoturbidimetry-based assay kit for IIIC according to claim 1, wherein the pH of the reaction buffer R1 is 7.1-7.9.
4. The latex-enhanced immunoturbidimetry-based assay kit for IIIC according to claim 1, wherein in the latex preservative solution:
the buffer solution is one of a tris buffer solution, a glycine buffer solution, a 2- (N-morpholine) ethanesulfonic acid buffer solution and a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution;
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
the stabilizer is one or more of BSA, sucrose, mannitol and glycine;
The preservative is one or more of Proclin300 and thimerosal;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
5. The latex-enhanced immunoturbidimetry-based IIIC assay kit according to claim 1, wherein the pH of the latex stock solution is 6.1-7.5.
6. The kit for determining IIIC based on latex-enhanced immunoturbidimetry according to claim 1, wherein the latex microspheres used in the IIIC monoclonal antibody-labeled latex microspheres have a diameter of 100-300 nm.
7. The latex-enhanced immunoturbidimetry-based assay kit for IIIC according to any of claims 1 to 6, wherein the latex reagent R2 is prepared by:
firstly, taking latex microspheres with solid content of 10%, and adding the latex microspheres into a cleaning buffer solution; centrifuging, removing supernatant, carrying out resuspension ultrasonic treatment on the latex microsphere precipitate by using an activation buffer solution, and adding an activating agent for activation; centrifuging again, and carrying out heavy suspension ultrasonic treatment on the latex microsphere precipitate by using a coupling buffer solution to prepare activated latex microspheres;
uniformly mixing at least two IIIC monoclonal antibodies with the prepared activated latex microspheres according to a specific ratio, and performing coupling for a certain time to prepare coupled latex microspheres; wherein the v/w ratio of the activated latex microspheres to each IIIC monoclonal antibody is 1 (3-5), and the coupling time is 1 h;
Adding a sealing buffer solution into the coupled latex microspheres for sealing; after the sealing is finished, centrifuging again, removing supernatant, adding latex preservation solution for resuspension and ultrasound, and preparing respective latex reagent composition solutions; then, the prepared latex reagent composition liquids are mixed to obtain the required latex reagent R2.
8. A method for preparing a reagent for latex-enhanced immunoturbidimetry-based IIIC determination according to any one of claims 1 to 7, comprising the steps of:
(1) preparing a reaction buffer R1:
according to the component content of the reaction buffer solution R1, mixing all the components in the same container, and uniformly mixing to obtain a reaction buffer solution R1;
(2) formulation of latex reagent R2:
firstly, taking latex microspheres with solid content of 10%, and adding the latex microspheres into a cleaning buffer solution; centrifuging, removing supernatant, carrying out resuspension ultrasonic treatment on the latex microsphere precipitate by using an activation buffer solution, and adding an activating agent for activation; centrifuging again, and carrying out heavy suspension ultrasonic treatment on the latex microsphere precipitate by using a coupling buffer solution to prepare activated latex microspheres;
uniformly mixing at least two IIIC monoclonal antibodies with the prepared activated latex microspheres according to a specific ratio, and performing coupling for a certain time to prepare coupled latex microspheres; wherein the v/w ratio of the activated latex microspheres to each IIIC monoclonal antibody is 1 (3-5), and the coupling time is 1 h;
Adding a sealing buffer solution into the coupled latex microspheres for sealing; after the sealing is finished, centrifuging again, removing supernatant, adding latex preservation solution for resuspension and ultrasound, and preparing respective latex reagent composition solutions; then, the prepared latex reagent composition liquids are mixed to obtain the required latex reagent R2.
9. The method for preparing the kit for the latex-enhanced immunoturbidimetry-based IIIC assay according to claim 8, wherein in the step (I), MES buffer is used as the washing buffer and the activation buffer; the coupling buffer solution adopts a phosphate buffer solution; the activator adopts EDAC/NHS; in the third step, the blocking buffer solution adopts one or more of BSA and glycine, and the blocking time is 1 h.
10. A method of using the kit for latex-enhanced immunoturbidimetry-based IIIC determination according to any one of claims 1 to 7, comprising the following specific steps:
(1) sucking 5 μ L of sample, adding 200 μ L of reagent R1, and incubating at 37 deg.C for 3-5 min;
(2) adding 50 μ L reagent R2, and incubating at 37 deg.C;
(3) reading absorbance a1 after 60s incubation; incubating for 5min, and reading the light absorption value A2; and finally, calculating delta A, wherein the delta A is A2-A1, and calculating the IIIC content in the sample according to the delta A.
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| CN112595853A (en) * | 2020-12-04 | 2021-04-02 | 安徽大千生物工程有限公司 | IL-6 immunoturbidimetry detection kit prepared based on recombinant monoclonal antibody and preparation method thereof |
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