CN115236323A - Kit for detecting rabies antibody in human plasma by latex enhanced immunoturbidimetry - Google Patents
Kit for detecting rabies antibody in human plasma by latex enhanced immunoturbidimetry Download PDFInfo
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- CN115236323A CN115236323A CN202210692305.8A CN202210692305A CN115236323A CN 115236323 A CN115236323 A CN 115236323A CN 202210692305 A CN202210692305 A CN 202210692305A CN 115236323 A CN115236323 A CN 115236323A
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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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
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- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
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- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a kit for detecting the content of a rabies antibody in human plasma by a latex enhanced immunoturbidimetry method, which comprises a reagent R1, a reagent R2 and a rabies antibody calibrator, wherein: the reagent R1 comprises a first buffer solution; the reagent R2 comprises polystyrene latex particles coated with rabies antigens and a second buffer solution; the rabies antibody calibrator comprises a third buffer and a series of rabies antibodies with different concentrations. The kit has the advantages of high accuracy, good repeatability, strong specificity, high sensitivity, simple operation and the like.
Description
Technical Field
The invention relates to a latex-enhanced immunoturbidimetry kit, in particular to a kit for detecting rabies antibodies in human plasma by a latex-enhanced immunoturbidimetry method.
Background
The vast majority of rabies cases do not receive a standard treatment after exposure. Timely post-exposure treatment is the only effective means for preventing rabies after exposure. The world health organization believes that timely, scientific and thorough post-exposure prophylactic treatment can avoid the occurrence of rabies.
After injection of rabies vaccine or rabies patient immunoglobulin, whether the in vivo antibody reaches the protection level can be detected. It is generally considered that the neutralizing antibody titer reaches 0.5IU/ml, and the protective power is obtained. WHO recommends only mouse intracerebral neutralization and foci inhibition assays. In actual production enterprises, the ELISA method is most widely used for detecting the titer of the anti-rabies antibody in human plasma. The ELISA method is relatively complex in operation, and samples need to be diluted before detection, so that the problem of large difference between batch difference plates exists, the rabies antibody titer value is inaccurate, and great influence is brought to actual production. Therefore, a rabies antibody detection method with high accuracy, good repeatability, strong specificity, high sensitivity and simple operation is urgently needed at present.
Disclosure of Invention
In order to solve the technical problems, the invention provides a rabies antibody content detection kit.
In a first aspect, the invention relates to a kit for detecting the content of a rabies antibody in human plasma by a latex enhanced immunoturbidimetry method, which comprises a reagent R1, a reagent R2 and a rabies antibody calibrator, wherein:
the reagent R1 comprises a first buffer solution;
the reagent R2 comprises polystyrene latex particles coated with rabies antigens and a second buffer solution;
the rabies antibody calibrator comprises a third buffer solution and a series of rabies antibodies with different concentrations;
wherein, the polystyrene latex particles coated with the rabies antigen comprise a combination of activated polystyrene latex particles and rabies antigen;
activating polystyrene latex particles by using N-hydroxysuccinimide and carbodiimide, wherein in the activation process, the polystyrene latex particles are uniformly mixed in a fourth buffer solution, and the N-hydroxysuccinimide and the carbodiimide are added, wherein the mass ratio of the polystyrene to the N-hydroxysuccinimide to the carbodiimide is 500-2000.
Preferably, the mass ratio of the polystyrene to the N-hydroxysuccinimide to the carbodiimide is 600-800;
preferably, the fourth buffer is one or a combination of two or more of glycine buffer, tris buffer, HEPES buffer, MES buffer, MOPS buffer and MOPSO buffer.
Preferably, the reagent R1 further comprises one or more than two substances selected from a first protective agent, a first preservative, a first sensitizer and a first electrolyte; more preferably, the reagent R1 comprises a first protective agent, a first preservative, a first electrolyte, a first sensitizer and a first buffer.
Preferably, the reagent R2 further comprises one or more than two substances selected from a second protective agent, a second preservative and a second surfactant; more preferably, the reagent R2 comprises a second protective agent, a second preservative, a second surfactant, a second buffer, and polystyrene latex particles coated with rabies antigen.
Preferably, the rabies antibody calibrator comprises one or more than two substances selected from a third protective agent, a third preservative agent and a third buffer solution; more preferably, a third protective agent, a third preservative agent, a third buffer and a series of rabies antibodies with different concentrations are included.
Specifically, the invention relates to a kit for detecting the content of a rabies antibody in human plasma by a latex-enhanced immunoturbidimetry method, which comprises a reagent R1, a reagent R2 and a rabies antibody calibrator, wherein:
the reagent R1 comprises a first protective agent, a first preservative, a first electrolyte, a first sensitizer and a first buffer solution;
the reagent R2 comprises a second protective agent, a second preservative, a second surfactant, a second buffer solution and polystyrene latex particles coated with rabies antigens;
the rabies antibody calibrator comprises a third protective agent, a third preservative agent, a third buffer solution and a series of rabies antibodies with different concentrations.
Preferably, the reagent R2 independently further contains a reaction enhancer;
preferably, the reaction enhancer is selected from one or a combination of more than two of mannitol, glycerol and trehalose;
preferably, the mass percentage of the reaction enhancer is 1-10%, preferably 1.8-5%.
Preferably, the size of the rabies antigen-coated polystyrene latex particles in the reagent R2 is in the range of 150 to 300nm, for example, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm and a range between any two of the above values, and more preferably, the size of the polystyrene latex particles is in the range of 150nm to 250nm. It should be emphasized here and in the following that "192nm" in the present application does not mean that the particle size of each latex particle in the reagent is exactly 192nm, and that a 192nm sized latex particle means a particle size with a median diameter of 192nm, for example in the range of 170-210nm, with a median diameter of 192nm, due to errors in the manufacturing process of the latex particles. The particle size of the microsphere is closely related to the degree of reaction and the width of linear range, and the general rule is that the larger the particle size is, the higher the degree of reaction is, and the narrower the linearity is; the smaller the particle size, the lower the reactivity and the wider the linearity.
Preferably, the mass percentage of the rabies antigen-coated polystyrene latex particles in the reagent R2 is 0.05-1%, more preferably, the mass percentage of the latex particles in the reagent R2 is 0.05% -0.2%, for example, 0.05%, 0.1%, 0.15%, 0.2% and a range between any two of the above values. Within a certain range, the lower the mass percentage of the latex particles in the reagent R2, the lower the background of the reagent and the lower the reactivity, otherwise, the higher the background of the reagent and the higher the reactivity, and because a biochemical analyzer has a certain detection range, the background and the reactivity of the reagent need to reach and do not exceed a range, the mass percentage range of the R2 is 0.05-1 percent, the requirement can be met, and below the range, the reactivity is too low, and above the range, the background is too high, and the reactivity also exceeds the upper limit of the biochemical analyzer.
Preferably, the latex particles in the reagent R2 are polystyrene latex particles.
Preferably, the carboxyl content of the rabies antigen-coated polystyrene latex particles is 50-200. Mu. Mol/g, such as 50. Mu. Mol/g, 60. Mu. Mol/g, 70. Mu. Mol/g, 80. Mu. Mol/g, 90. Mu. Mol/g, 100. Mu. Mol/g, 110. Mu. Mol/g, 120. Mu. Mol/g, 130. Mu. Mol/g, 140. Mu. Mol/g, 150. Mu. Mol/g, 160. Mu. Mol/g, 170. Mu. Mol/g, 180. Mu. Mol/g, 190. Mol/g, 200. Mu. Mol/g, and ranges between any two of the above, and more preferably, the carboxyl content of the polystyrene latex particles is 94. Mu. Mol/g.
The rabies antigen is inactivated natural rabies virus or recombinant rabies antigen, and preferably, the rabies antigen is recombinant rabies antigen.
The combination of the latex particles and the rabies antigens in the reagent R2 can be prepared by adopting a physical adsorption method or a chemical crosslinking method, and preferably, the rabies antigens are combined with the latex particles by adopting the chemical crosslinking method.
Preferably, the mass ratio of the latex particles to the rabies antigen is 10 to 120, and more preferably, the mass ratio of the latex particles to the rabies antigen is 24.
The first protective agent, the second protective agent or the third protective agent in the reagent R1, the reagent R2 and the rabies antibody calibrator respectively and independently comprise one or more of protein, amino acid, polyhydroxy alcohol, inorganic salt, metal complexing agent, suspending agent and antioxidant.
The protein is selected from one or the combination of more than two of bovine serum albumin and chicken egg albumin.
The amino acid is selected from one or the combination of more than two of glycine, alanine and polylysine.
The selected polyhydric alcohol is one or more selected from mannitol and glycerol.
The inorganic salt is selected from one or the combination of more than two of sodium chloride, potassium chloride or magnesium chloride.
The metal complex is one or the combination of more than two of EDTA, EDTP and DTPA.
The suspending agent is one or the combination of more than two of sucrose, trehalose and lactose.
The antioxidant is selected from one or the combination of more than two of tert-butyl p-hydroxyanisole and propyl gallate.
Preferably, the concentrations of the first protective agent, the second protective agent or the third protective agent in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently selected from 0.01-1%, 0.1-10% and 0.1-5% by mass, preferably 0.05-0.5%,0.1-5% and 0.1-4%, the protective agent is added to protect the target protein in the solution system, and different protective agents and different mass percentage ranges are selected according to the purpose to be achieved and different protein types.
The first preservative, the second preservative or the third preservative in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently selected from one or a combination of more than two of potassium sorbate, gentamicin, PC300, PC910 and sodium nitrite, and preferably, the first preservative, the second preservative or the third preservative in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently selected from one or a combination of more than two of PC300 and gentamicin.
Preferably, the concentrations of the first preservative, the second preservative or the third preservative in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently 0.01-0.5%, 0.01-1% by mass, and more preferably, the concentrations of the first preservative, the second preservative or the third preservative are respectively and independently 0.05-0.1%, 0.1-0.5% by mass. The preservative mainly has the function of preventing the reagent from growing bacteria and deteriorating in the storage process, and if the preservative is not added, the effective period of the reagent is only 1 week. The selection of the mass fraction range of the preservative is not limited to the above range, and for example, increasing the mass fraction may also achieve the technical solution of the present invention, but may increase the cost and waste materials, and the selection range of the preservative is designed based on this consideration.
The first electrolyte in the reagent R1 is selected from one or a combination of two or more of inorganic salts, preferably the first electrolyte is selected from one or a combination of two or more of monovalent metal ion inorganic salts and divalent metal ion inorganic salts, more preferably the first electrolyte is selected from one or a combination of two or more of sodium ion salts and magnesium ion salts, particularly preferably the first electrolyte is selected from one or a combination of two or more of sodium ion salts.
Preferably, the concentration of the first electrolyte in the reagent R1 and the rabies antibody calibrator is 1-20.0% by mass, more preferably 1-10% by mass, and more preferably 1-5.8% by mass. The electrolyte is used for providing a reaction environment, and the reaction degree is generally reduced and the effect of non-specific reaction is reduced in an experiment.
The first buffer solution, the second buffer solution or the third buffer solution in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently selected from one or more than two combinations of glycine buffer solution, tris buffer solution, HEPES buffer solution, MES buffer solution, MOPS buffer solution and MOPSO buffer solution, preferably, the first buffer solution, the second buffer solution or the third buffer solution is selected from one or more than two combinations of MES buffer solution, tris buffer solution and glycine buffer solution.
Preferably, the concentration of the buffer solution in the reagent R1, the reagent R2 and the rabies antibody calibrator is independently selected from 10 to 100mmol/L, and preferably, the pH value of the buffer solution is independently selected from 6.0 to 8.5.
Preferably, the reagent R2 independently contains one or two of the surfactants.
The surfactant in the reagent R2 is selected from one or the combination of more than two of Tween-20, tween-60, tween-80, span-40, span-80 and TtitonX-100, preferably, the surfactant in the reagent R2 is Tween-20.
The concentration of the surfactant in the reagent R2 is independently 0.01 to 1% by mass, and more preferably, the concentration of the surfactant is 0.02 to 0.05% by mass, for example, 0.02%,0.03%,0.04%,0.05%, and a range between any two of the above values.
Preferably, the rabies antibodies with series concentrations in the rabies antibody calibration product are calibration products with different concentrations prepared by a dilution of the calibration product, and the rabies antibody calibration product contains 5 rabies antibodies with different concentrations, such as 0.0IU/mL, 2.5IU/mL, 5.0IU/mL, 10.0IU/mL and 20.0IU/mL.
The rabies antibody in the rabies antibody calibrator is derived from human rabies immunoglobulin or anti-rabies serum, and preferably, the rabies antibody used in the rabies antibody calibrator is derived from human rabies human immunoglobulin.
In a second aspect, the invention provides application of the kit for determining the content of the rabies antibody in detection of the rabies antibody in human plasma.
In a third aspect, the invention provides a method for detecting the content of a rabies antibody, which is to detect a sample by using the kit provided by the invention.
Specifically, the rabies antibody detection method comprises the following steps:
incubating a sample and a reagent R1 together, adding a reagent R2 to generate turbidity, detecting the turbidity by using a biochemical analyzer, and calculating an antibody concentration value corresponding to the sample according to a standard curve, wherein the standard curve is obtained by drawing by using a 5-point calibration method and a spline function as a calculation mode.
More specifically, the rabies antibody detection method comprises the following steps:
the method comprises the steps of mixing a sample and a reagent R1 uniformly, incubating for 5 minutes, adding a reagent R2, continuing to react for 5 minutes, measuring absorbance values (A1 and A2) at the 5.5 th and 10 th minutes of reaction, calculating an absorbance difference (delta A), and calculating the content of the rabies antibody in the sample according to a standard curve, wherein the selected detection wavelength is 340-800nm, preferably the detection wavelength is 500-700nm, particularly preferably the detection wavelength is 546 nm.
In a fourth aspect, the invention also provides application of the rabies antibody detection kit in human plasma rabies antibody titer screening.
In the fifth aspect, the invention also provides application of the rabies antibody detection method in human plasma rabies antibody titer screening.
In a sixth aspect, the invention also provides a rabies antigen coated polystyrene latex particle, which comprises a combination of the activated polystyrene latex particle and a rabies antigen.
The preparation method of the rabies antigen coated polystyrene latex particles comprises the following steps:
(1) Activating polystyrene latex particles;
(2) Coupling rabies antigens with activated polystyrene latex particles, and adding a blocking agent to obtain the rabies antigen-coated polystyrene latex particles.
Preferably, the polystyrene latex particles are activated by using N-hydroxysuccinimide and carbodiimide in the step (1), and more preferably, the polystyrene latex particles are mixed in a fourth buffer solution, the N-hydroxysuccinimide and the carbodiimide are added, mixed, and stirred to activate the polystyrene latex particles.
Preferably, the mass ratio of the polystyrene to the N-hydroxysuccinimide to the carbodiimide is (500-2000): 5, more preferably, the mass ratio of the polystyrene to the N-hydroxysuccinimide to the carbodiimide is (600-800): 5, and particularly preferably, the mass ratio of the polystyrene to the N-hydroxysuccinimide to the carbodiimide is 800.
More preferably, in the step (1), the polystyrene latex particles are uniformly mixed in the fourth buffer solution, so that the final concentration of the polystyrene latex particles in the activation system is 0.5-2%. Adding N-hydroxysuccinimide to mix to a final concentration of 0.05mg/ml, preferably mixing for 1-2 minutes, adding carbodiimide to mix to a final concentration of 0.06mg/ml, preferably mixing for 1-2 minutes, stirring in a stirrer, preferably stirring at room temperature for 20-50 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes and ranges between any two of the above values, removing impurities from the activated polystyrene latex particles, preferably removing impurities by desalting column or centrifugation.
The fourth buffer solution is one or the combination of more than two of glycine buffer solution, tris buffer solution, HEPES buffer solution, MES buffer solution, MOPS buffer solution and MOPSO buffer solution.
Preferably, the rabies antigen in the step (2) is diluted by a fifth buffer solution, the activated polystyrene latex particles prepared in the step (1) are added, the stirring is carried out for 3 to 20 hours, and a blocking agent is added to obtain the rabies antigen coated polystyrene latex particles.
More preferably, the rabies antigen is diluted to 0.01-0.1mg/mL by using a fifth buffer solution in the step (2), the diluted rabies antigen is placed on a stirrer for uniformly mixing, preferably, the uniformly mixing time is 2-10 minutes, and the activated polystyrene latex particles prepared in the step (1) are continuously and rapidly added to enable the mass fraction of the microspheres to reach 0.05-0.2%.
Preferably, in step (2), the mixture is stirred at room temperature for 3 to 20 hours, for example, 3h,4h,5h,6h, 7h,8h,9h,10h,1h, 12h,13h,14h,15h, 116h, 17h,18h,19h, and 20h, and ranges between any two of the above values.
Preferably, the blocking agent is added to perform the reaction in step (2) at a final concentration of 35-50. Mu.L/mL, such as 35. Mu.L/mL, 36. Mu.L/mL, 37. Mu.L/mL, 38. Mu.L/mL, 39. Mu.L/mL, 40. Mu.L/mL, 41. Mu.L/mL, 42. Mu.L/mL, 43. Mu.L/mL, 44. Mu.L/mL, 45. Mu.L/mL, 46. Mu.L/mL, 47. Mu.L/mL, 48. Mu.L/mL, 49. Mu.L/mL, 50. Mu.L/mL, preferably at a final concentration of 40. Mu.L/mL, preferably at a reaction time of 0.5-3h after addition of the blocking agent, such as 0.5h,1h,1.5h,2h,2.5h,3h, and ranges between any two of the above values, more preferably at a reaction time of 1 hour.
Preferably, in the step (2), after the blocking agent is added to complete the reaction, centrifuging for 10-50min at 11000rmp-15000rmp to remove the supernatant, and more preferably centrifuging for 30min at 13000rpm to remove the supernatant to obtain the rabies antigen coated polystyrene latex particles.
In a specific embodiment of the invention, after centrifuging to remove the supernatant, the polystyrene latex particles coated with the rabies antigens are dispersed under ultrasonic conditions, specifically, the polystyrene latex particles coated with the rabies antigens are dispersed under 1% power conditions, and the ultrasonic is started for 2 seconds and stopped for 3 seconds and stopped for 1 minute.
Preferably, the fifth buffer is one or a combination of two or more selected from the group consisting of Tris buffer, HEPES buffer, MES buffer, MOPS buffer and MOPSO buffer.
Preferably, the blocking agent is one or a combination of more than two of glycine and bovine serum albumin, and preferably, the blocking agent is glycine.
The kit for determining the content of the rabies antibody in human plasma provided by the invention adopts a method that the rabies antigen coats the latex particles, can be specifically combined with the rabies antibody, has the characteristics of high accuracy, good repeatability, strong specificity, high sensitivity, simple operation and the like, can be used for reagent detection, can be applied to a full-automatic biochemical analyzer and a transmission immunoturbidimeter, and has high detection speed. The kit can quickly, accurately, stably and high-flux test the content of the rabies antibody in 0-20IU/mL linearity in human plasma, and meets the requirement of blood product enterprises on the determination of the content of the rabies antibody in the immunized human plasma.
Drawings
FIG. 1 is a graph showing the results of a linear correlation coefficient test of the particle size of rabies antigen-coated polystyrene latex particles with reactivity;
FIG. 2 is a graph showing the results of the measurement of linear correlation coefficients of different buffers and the reactivity in the reagent R1;
FIG. 3 is a graph showing the results of the measurement of linear correlation coefficients of different buffers in the reagent R2 and the reactivity;
FIG. 4 is a correlation between the results of the assay with the test kit of the present invention obtained in example 5 and the results of the assay with the imported kit;
FIG. 5 shows the results of the linear measurement of the kit of the present invention obtained in example 5.
Detailed Description
The reaction principle of the latex enhanced immunoturbidimetry provided by the invention is as follows: the latex particles coated with the rabies antigens can be specifically combined with rabies antibodies in a sample to form an antibody-antigen-latex particle immune complex, when light with a certain wavelength passes through the immune complex, a part of light signals can be absorbed by the immune complex, the quantity of the immune complex is in direct proportion to the intensity of the absorbed light signals, so that the quantity of the antibodies in the sample is in direct proportion to the intensity of the absorbed light signals, and the content of the rabies antibodies in the sample can be calculated according to a calibration curve and the absorbance of the sample.
The kit of the present invention will be described below with reference to specific examples.
The following examples use the following sources of reagents, all reagents or equipment or procedures not described herein being routinely determinable by one of ordinary skill in the art:
TABLE 1 reagents used in the examples
Example 1 preparation of latex particles coated with rabies antigen
Polystyrene latex particles used by the kit are self-produced, persulfate (potassium persulfate, sodium persulfate, ammonium persulfate and the like) is used as an initiator to initiate styrene and vinyl carboxylic acid functional monomers (methacrylic acid, acrylic acid, maleic acid and the like) to be copolymerized to prepare the polystyrene microspheres with surfaces rich in carboxyl groups by a polymerization method of soap-free emulsion (no emulsifier is added or a small amount of emulsifier is added). By adjusting parameters such as water-oil ratio, functional monomer ratio, reaction temperature and the like, the microspheres with adjustable particle size of 50-350 nm and controllable carboxyl density of 0-400 mu mol/g can be prepared.
Respectively taking 3.25mL of polystyrene latex particle solutions with the particle sizes of 150nm, 192nm, 200nm, 250nm and 300nm, wherein the solid content of the polystyrene latex particles is 9.23g/100mL of the solution (the solid content is gram number of latex microsphere solid contained in each 100mL of the polystyrene latex particle solution), and uniformly mixing the polystyrene latex particles with different particle sizes with 41.2mL of purified water and 5mL of MES buffer solution with the concentration of 500mM and the pH value of 6.10 for later use.
Dissolving carbodiimide with purified water to a concentration of 10mg/mL, dissolving N-hydroxysuccinimide to a concentration of 10mg/mL, adding 0.3mL of carbodiimide solution and 0.25mL of N-hydroxysuccinimide solution into the mixed solutions respectively, uniformly mixing to obtain a final concentration of 0.06mg/mL of carbodiimide and a final concentration of 0.05mg/mL of N-hydroxysuccinimide, and continuously stirring at room temperature for 30 minutes.
Taking a Sephadex G25 desalting column, and balancing the Sephadex G25 desalting column by using MES buffer solution with the concentration of 10mM and the pH value of 7.2, wherein the using amount of the MES buffer solution is 2 times of the column volume.
And (3) passing the activated polystyrene latex particles through a Sephadex G25 desalting column, and collecting eluent with the volume 1.1 times that of the activated solution.
5 portions of 187.5mL of 10mM MES buffer (pH7.2) were added with 12.5mL of rabies antigen (1 mg/mL) and mixed uniformly, 50mL of each of the activated polystyrene latex particles were added continuously and rapidly, and the mixture was stirred at room temperature for 10 hours.
Blocked with 10mL of 1M glycine solution at pH11.0 and stirred at room temperature for 1 hour.
After centrifugation at 4 ℃ and 13000rpm for 30 minutes and supernatant removal, the mixture was washed repeatedly with a wash solution (0.5 g/LSDS,0.1MGly buffer, pH 8.0) for 2 times to give the rabies antigen-coated latex particles.
Example 2 determination of Linear correlation coefficient of particle size and reactivity of rabies antigen-coated latex particles
The rabies antigen-coated latex particles with the particle diameters of 150nm, 192nm, 200nm, 250nm and 300nm prepared in example 1 are taken to respectively test the 5-point reactivity of the calibrator and fit a straight line.
As shown in FIG. 1, the linear correlation coefficient between the particle size and the reactivity of the rabies antigen-coated latex particles was 0.91 or more. However, the reactivity was significantly low under the condition of a particle size of 150nm, high under the condition of a particle size of 300nm, but the 0 point was also high and the linear high-end reactivity was not high, and the linear correlation coefficient was 0.91454, so that the detection performance for rabies antibodies was not excellent when the particle sizes of the rabies antigen-coated latex particles were 150nm and 300 nm. When the particle size of the latex particles coated with the rabies antigen is 192nm-250nm, the reactivity and the linear correlation coefficient are in a better range, and the latex particles can be used for detecting the rabies antibody, particularly when the particle size of the latex particles coated with the rabies antigen is 192nm, the linear correlation coefficient reaches 0.99267, and the reactivity is at the highest level.
Example 3 Effect of different activator amounts on reagent reactivity and Linear correlation coefficient
Selecting latex particles with the particle size of 192nm for activation, wherein the mass ratio of microspheres to an activating agent is (1/2/3); 800; 500. The obtained reactivity and linear correlation coefficient are shown in Table 2 below
TABLE 2 relationship between different activator dosages and the degree of reaction and correlation coefficient
Concentration of calibrator IU/mL | Ratio 1 | Ratio 2 | Ratio 3 |
0 | -99 | -0.5 | -185 |
2.5 | 22 | 426 | 106 |
5.0 | 462 | 873 | 340.5 |
10.0 | 1250 | 1941 | 705 |
20.0 | 2576 | 3344 | 1534 |
R 2 | 0.9937 | 0.9927 | 0.9963 |
As can be seen from Table 2 above, the amount of the activator affects the reactivity and the relation with the reactivity is not linear but has an optimum amount, and the ratio 2 is the condition of the highest reactivity, and when the amount of the activator is lower or higher than the optimum amount for a certain amount of the latex particles, the reactivity is also decreased accordingly, and in this example, the effect of decreasing the amount of the activator on the reactivity is smaller than that of increasing the amount of the activator, so the ratio 2 is selected as the activation condition of the latex particles.
Example 4 determination of Linear correlation coefficient of different buffers and reactivity in reagent R1
Tris buffer (50mM, pH8.0), MOPSO buffer (50mM, pH7.2), MES buffer (50mM, pH6.1) were selected, and 1g/LBSA, 20g/l PEG6000 and 58g/LNaCl were added, respectively, as reagents R1 for the test. Latex particles with a particle size of 192nm coated with rabies antigen were taken to test the 5-point reactivity of the calibrator and fitted with a straight line.
The experimental result is shown in fig. 2, the linear correlation coefficients all reach more than 0.90, and only the MES system in the three buffer solution conditions has good reactivity and linearity, so the MES system is selected as the R1 buffer solution.
Example 5 determination of Linear correlation coefficients of different buffers and reactivity in reagent R2
Selecting Tris buffer (50mM, pH8.0), HEPES buffer (50mM, pH7.8), MOPS buffer (50mM, pH7.0), and MES buffer (50mM, pH6.1), and adding bovine serum albumin 0.1wt% respectively; mannitol 1.82wt%; TWEEN-20.02wt%; 0.05wt% of PC is used as a reagent R2 storage solution, 192nm latex particles coated with rabies antigens are respectively added, the concentration of the latex particles reaches 0.12%, then 5-point reactivity of a calibrator is tested, and a straight line is fitted.
As shown in FIG. 3, the linear correlation coefficients are all above 0.90, and the Tris buffer and MES buffer are relatively better and respectively reach 0.9880 and 0.9985. Comparing the two reactivities, the Tris buffer has higher reactivity, more space is reserved for subsequent adjustment, and therefore, a Tris system is selected as an R2 buffer.
Example 6 implementation of the kits of the invention with a buffer alone for reagents R1, R2 and calibrator
A kit for determining concentration of rabies antibody, wherein reagents R1 and R2 and a calibrator diluent in the kit are prepared and diluted by using buffer solution respectively, so as to realize the functions of the kit disclosed by the invention.
Wherein the reagent R1: MES buffer pH6.1 mmol/L; and (3) reagent R2: tris buffer pH8.5 mmol/L is used for diluting rabies antigen coated polystyrene latex particles, the particle size of the latex is 192nm, and the carboxyl density: 94 mu mol/g, and the final concentration of latex is 0.12%; rabies antibody calibrator: the concentration of rabies antibody was 0IU/mL, 2.5IU/mL, 5.0IU/mL, 10.0IU/mL, 20.0IU/mL by diluting with glycine buffer solution pH7.8 mmol/L.
The reaction degree, linear correlation coefficient, accuracy, sensitivity and precision of the kit are tested for testing. As can be seen from table 3, the kit of this embodiment has a high reactivity point of 1288, although the correlation coefficient meets the requirement and the accuracy of the two standards also meets the requirement, the reactivity is low and the low measurement value is inaccurate, so that a simple solution system cannot meet the test requirement of the kit, and a sensitizer, a protective agent and a preservative are required to be added.
Table 3 example 6 reactivity, linear correlation, accuracy
Calibrator concentration IU/mL | Degree of reaction |
0 | -139 |
2.5 | 11 |
5.0 | 231 |
10.0 | 625 |
20.0 | 1288 |
R 2 | 0.9977 |
Standard 1 deviation | 13% |
Standard 2 deviation | 7% |
Example 7 kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 8A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 9 kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 10A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 11A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 12A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 13A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 14 kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 15A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 16A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 17 kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 18 kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 19A kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 20 kit for determining concentration of rabies antibody
A kit for determining concentration of rabies antibody, which comprises the following components:
example 21 method for detecting rabies antibody content by using the kit
A Hitachi 7170 full-automatic biochemical analyzer is used for detecting a sample under the conditions that the wavelength is 546nm and the temperature is 37 ℃, the adopted analysis method is a two-point end point method, the reaction direction is ascending, and the calibration mode adopts a spline fitting method.
And (3) preparing a standard curve: add 150. Mu.L of reagent R1 to 2. Mu.L of calibrator at the respective concentrations: 0.0IU/mL, 2.5IU/mL, 5.0IU/mL, 10.0IU/mL, 20.0IU/mL, after incubating for 5min at 37 ℃,50 muL of reagent R2 is added, the first point (A1) is read after the reaction starts for 30s, the second point (A2) is read after the reaction starts for 5min, the absorbance difference Delta A = A2-A1 is calculated, the measurement is repeated for 2 times in each tube, the absorbance difference Delta A measured for 2 times in each calibrator tube is taken as the ordinate, the corresponding concentration is taken as the abscissa, and a standard curve of concentration-absorbance difference is prepared.
And (3) detecting the content of rabies antibodies: and (3) taking a sample to be detected, determining the absorbance difference of the sample by a method for making a standard curve, and substituting the absorbance difference into the standard curve to calculate the content of the rabies antibody in the sample to be detected.
Comparing the above examples 7-19, calculating curve correlation coefficients according to the reactivity of 5 points of each combined test calibrator, and performing tests on standard 1-GBW (E) 090279, standard 2-GBW (E) 090280 and standard 3-GBW (E) 090281, which are purchased from the chinese institute for food and drug assay, wherein the concentration of standard 1 is 2IU/mL, the concentration of standard 2 is 4IU/mL, and the concentration of standard 3 is 8IU/mL, calculating accuracy deviations, as can be seen from the results: the high point reactivity of the 12 examples, with the exception of examples 8/10/11/12/15/19/20, was essentially a 3000-fold fluctuation with linear correlations >0.99, with standard deviations controlled to within 15%. Among them, the standard product of example 8/16/20 has a large deviation. Example 8 reduced microsphere concentration compared to example 7, resulting in reduced overall reactivity, which was the lowest of 12 groups and only met the minimum requirements of the experiment. In example 9, the concentration of microspheres in R2 is increased compared with that in example 7, the background is increased due to the increase of the concentration, the high-point reactivity reaches the upper detection limit of a biochemical analyzer, and the integral linear discrimination is poor. Example 13 increased the sensitizer content in R1, the overall reactivity was improved, while point 0, 1270, was a non-specific reaction, and could be missed during sample detection. In example 15, the surfactant concentration in R2 was increased as compared with example 7, and the reactivity was lowered, and the discrimination was inferior to that in example 7. In example 19/20, the reagent components were replaced compared with example 7, but the effect was not as good as in example 7, and it is assumed that ovalbumin in example 19 had a strong inhibitory effect on the reactivity, PEG20000 sensitization effect was significant in example 20, non-specific reaction occurred at 0 value, and the linear correlation coefficient was not as good as in example 7. The above examples 8/9/10/11/12/13/15/16/19/20 can achieve the technical index of the kit of the invention, but the performance is worse than that of other examples. The performance of the remaining examples was essentially at the same level. The specific comparative data are shown in Table 5.
TABLE 5 examples 7-19 reactivity, linear correlation, deviation from accuracy
The 7/14/17/18 examples were compared and tested for sensitivity, precision, and thermal stability at 37 ℃ for 7 days. The sensitivity test method is that 5% bovine serum albumin solution is used as a blank sample, the measurement is repeated for 20 times according to the biochemical analyzer measurement method, and the lowest detection limit is reported by adding two times of standard deviation to the blank mean value, namely the sensitivity. The precision testing method comprises the following steps: and selecting one sample of the low-value sample and one sample of the high-value plasma sample, continuously measuring the same sample for 10 times by using the kit, and calculating the variation coefficient of the detection sample of the kit. Thermal stability at 37 ℃ for 7 days is generally used as a preliminary measure of reagent stability, and is determined by: after 9 groups of examples were placed in an oven at 37 ℃ and stored for 7 days, 2 concentration points of 2.5IU/mL and 10IU/mL were taken out and measured 3 times each according to the biochemical analyzer measurement method, and the relative deviation between the detected value and the indicated value was calculated. The results show that: the sensitivity results of the 4 groups of embodiments all meet the requirements, and the levels are basically consistent; the precision results of the 4 groups of examples all meet the minimum requirement of +/-15%, the precision of the high point is better than that of the low point, and the precision of the 2 points is integrated, so that the result is better in example 18, and the precision is 2.7% and 1.5%; the results of the thermal stability at 37 ℃ of the 4 groups of examples all meet the minimum requirement of +/-15%, the deviation is the minimum of 0.57% and 1.23% of the example 7, and the protective agent component in the R2 reagent has a larger influence on the stability. The best results of example 5 were obtained with respect to sensitivity, precision and thermal stability at 37 ℃ for 7 days. The specific data are shown in tables 6, 7 and 8.
TABLE 6 sensitivity of the set of examples
Sensitivity of the probe | Example 7 | Example 14 | Example 17 | Example 18 |
1 | 0.00 | 0.00 | 0.01 | 0.00 |
2 | 0.00 | 0.00 | 0.02 | 0.00 |
3 | 0.00 | 0.01 | 0.00 | 0.00 |
4 | 0.00 | 0.00 | 0.00 | 0.00 |
5 | 0.00 | 0.00 | 0.00 | 0.00 |
6 | 0.00 | 0.00 | 0.01 | 0.01 |
7 | 0.00 | 0.00 | 0.01 | 0.00 |
8 | 0.00 | 0.00 | 0.00 | 0.00 |
9 | 0.01 | 0.00 | 0.00 | 0.00 |
10 | 0.00 | 0.01 | 0.00 | 0.00 |
11 | 0.00 | 0.00 | 0.00 | 0.00 |
12 | 0.00 | 0.00 | 0.00 | 0.00 |
13 | 0.00 | 0.00 | 0.00 | 0.00 |
14 | 0.00 | 0.00 | 0.00 | 0.00 |
15 | 0.00 | 0.02 | 0.00 | 0.00 |
16 | 0.00 | 0.00 | 0.00 | 0.00 |
17 | 0.02 | 0.00 | 0.00 | 0.00 |
18 | 0.00 | 0.00 | 0.01 | 0.00 |
19 | 0.00 | 0.00 | 0.01 | 0.01 |
20 | 0.00 | 0.00 | 0.00 | 0.00 |
Mean value | 0.00 | 0.00 | 0.00 | 0.00 |
SD | 0.01 | 0.00 | 0.01 | 0.00 |
Mean +2 sd | 0.01 | 0.01 | 0.02 | 0.01 |
Precision of set of examples in Table 7
TABLE 8 7-day stability at 37 ℃ for the examples in group 9
Example 22 evaluation of the Performance of rabies antibody detection kit
Test materials: the kit of example 5, a commercially available control kit using enzyme-linked immunosorbent assay (ELISA).
The reagent composition of the control kit was as follows:
96-hole enzyme label plate pre-coated with rabies antigen
Enzyme-labeled secondary antibody, substrate, stop solution, sample diluent, calibrator, negative quality control material, positive quality control material and concentrated washing solution.
I. Correlation
The kit of the present invention prepared in example 7 was subjected to a correlation test with a commercially available control kit using enzyme-linked immunosorbent assay (ELISA), 50 samples of fresh human plasma (including negative and positive samples) were detected according to the detection method of the present invention and the method described in the specifications of the commercially available kit, and the measurement values were subjected to a correlation analysis according to the measurement methods described in the specifications of the commercially available kit, and the results are shown in FIG. 3, where the X and Y axes are both measurement values and the correlation coefficient R is a correlation coefficient 2 =0.9636, regression equation: y =0.81863x +1.0653, and the result shows that the kit has good correlation with the commercially available kit, and the correlation data is shown in Table 9.
TABLE 9 correlation test data of the kit of the present invention and commercially available kits
Linearity
The test sample was diluted with a calibrator diluent at a high rabies antibody concentration point to prepare a 20IU/mL concentration point, and diluted at a ratio of 1/2,1/4,1/8,1/16,1/32 by a double dilution with physiological saline. Preparing into 6 concentration point diluents, repeatedly measuring for 3 times according to the respective method of the kit, calculating the average value, performing correlation comparison between the measured value of the sample and the multiple proportion, obtaining linear regression equation, and calculating the correlation coefficient r of the equation 2 The linear experimental data are shown in fig. 4. The results show that the kit of the invention has a linear equation: y =0.96965x-0.24055Coefficient of correlation r 2 =0.99917, the kit of the invention has good linear correlation in the range of 0.63IU/mL-20.0 IU/mL.
Accuracy of
The accuracy test is carried out by using quality control products with target values of 2.5IU/mL (2.13-2.88 IU/mL) and 10IU/mL (8.5-11.5 IU/mL), the test is carried out for 3 times continuously according to the test method of the kit, the test deviation calculation is carried out on the mean value of the test results and the target values, the result shows that the test value of the kit is tested at the low value of-4 percent, the test value at the high value of 4.3 percent, and the accuracy test data are shown in a table 10:
TABLE 10 accuracy test data sheet of the kit of the present invention
Precision IV
The method comprises the following steps of selecting each sample of a low-value sample and a high-value plasma sample, continuously measuring the same sample for 10 times by using the kit, and calculating the variation coefficient of a detection sample of the kit, wherein the result shows that the precision of the low-value sample measured by the kit is 6.5%, the precision of the high-value sample measured by the kit is 5.3%, and the precision experimental data are shown in a table 11:
TABLE 11 precision experimental data table of the kit of the present invention
Plasma sample one | Plasma sample 2 | |
1 | 2.5 | 10.5 |
2 | 2.6 | 10.9 |
3 | 2.2 | 10.2 |
4 | 2.3 | 10.8 |
5 | 2.5 | 10.8 |
6 | 2.4 | 10.8 |
7 | 2.4 | 10.5 |
8 | 2.3 | 10.6 |
9 | 2.6 | 10.5 |
10 | 2.7 | 10.5 |
Ave | 2.45 | 10.55 |
STD | 0.16 | 0.55 |
CV | 6.5% | 5.3% |
Sensitivity v. degree of sensitivity
The 5% bovine serum albumin solution is used as a blank sample, the measurement is repeated for 20 times according to the measurement method of the biochemical analyzer, the blank mean value plus two times of standard deviation reports the lowest detection limit, the result shows that the sensitivity of the kit is 0.02IU/mL, and the sensitivity data is shown in Table 12:
TABLE 12 Experimental data sheet for sensitivity of the kit of the invention
VI, difference between batches
3 batches of the kit are prepared according to the method of the embodiment of the invention, the same serum sample is tested by using the 3 batches of the kit, each batch number is repeatedly tested for 3 times, the mean value of the 3 tests of each batch is respectively calculated, and the relative deviation is calculated, and the result shows that the relative deviation of the kit is 2.3%. The results are shown in Table 13.
TABLE 13 Experimental data sheet of the kit of the invention
VII interference test
Taking a rabies antibody calibrator with the concentration of 5.0IU/mL, respectively adding each interference substance solution with the same volume, enabling the concentration of each added interference substance to reach 5g/L of hemoglobin, 400 mu mol/L of bilirubin and 3g/L of chyle, repeatedly measuring each prepared sample for 3 times by using the kit, taking the mean value, comparing the mean value with a sample added with distilled water with the same volume, observing the relative deviation between the sample added with the interference substance and the sample added with the distilled water, and displaying the result that the deviation between the measured value of the sample added with the interference substance and the sample of the distilled water with the same volume is not more than 10 percent, wherein when the interference substances with certain concentrations in a detected sample comprise the hemoglobin, the bilirubin and the chyle, the influence on the kit is small. Specific data are shown in Table 14.
TABLE 14 influence of interfering substances on the kit of the invention
VIII. Stability
The kit of the invention is examined for bottle opening stability and long-term stability.
Bottle opening stability: after the kit is unpacked and placed at the temperature of 2-8 ℃ for 30 days, the kit is taken out to be measured for 2.5IU/mL and 10IU/mL according to the measuring method of the biochemical analyzer, each concentration point is measured for 3 times, the relative deviation between the detected value and the marked value is calculated, and the result shows that after the kit is unpacked for 30 days, the deviation between the measured value and the marked value of the kit is-6.7 percent and 7.3 percent, and the stability of the unpacked bottle is good.
Long-term stability: after the kit is stored at 2-8 ℃ for 12 months, the kit is taken out to measure 2 concentration points of 2.5IU/mL and 10IU/mL according to the biochemical analyzer measuring method, each concentration point is measured for 3 times, and the relative deviation between the detection value and the marked value is calculated, so that the result shows that the deviation between the measurement value and the marked value of the kit is-8.8 percent and-1.7 percent, the kit is relatively stable when being stored at 2-8 ℃, and the specific data are shown in a table 15.
TABLE 15 rabies kit open bottle and long term stability test data sheet
Claims (21)
1. A kit for detecting the content of rabies antibodies in human plasma by a latex-enhanced immunoturbidimetry method comprises a reagent R1, a reagent R2 and a rabies antibody calibrator, wherein:
the reagent R1 comprises a first buffer solution;
the reagent R2 comprises polystyrene latex particles coated with rabies antigen and a second buffer solution;
the rabies antibody calibrator comprises a third buffer solution and a series of rabies antibodies with different concentrations;
wherein, the polystyrene latex particles coated with the rabies antigen comprise a combination of activated polystyrene latex particles and rabies antigen;
activating polystyrene latex particles by using N-hydroxysuccinimide and carbodiimide, wherein in the activation process, the polystyrene latex particles are uniformly mixed in a fourth buffer solution, and the N-hydroxysuccinimide and the carbodiimide are added, wherein the mass ratio of the polystyrene to the N-hydroxysuccinimide to the carbodiimide is 500-2000.
2. The kit according to claim 1, wherein the mass ratio of polystyrene, N-hydroxysuccinimide and carbodiimide is from 600 to 800;
preferably, the fourth buffer is one or a combination of two or more of glycine buffer, tris buffer, HEPES buffer, MES buffer, MOPS buffer and MOPSO buffer.
3. The kit according to claim 1 or 2, characterized in that the reagent R1 further comprises one or more substances selected from the group consisting of a first protective agent, a first preservative, a first sensitizer and a first electrolyte;
preferably, the reagent R1 comprises a first protective agent, a first preservative, a first electrolyte, a first sensitizer and a first buffer.
4. The kit according to any one of claims 1 to 3, wherein the reagent R2 further comprises one or more substances selected from the group consisting of a second protective agent, a second preservative, a second surfactant, and a second buffer;
preferably, the reagent R2 comprises a second protective agent, a second preservative, a second surfactant, a second buffer, and polystyrene latex particles coated with rabies antigen.
5. The kit according to any one of claims 1 to 4, wherein the rabies antibody calibrator further comprises one or more substances selected from the group consisting of a third protective agent, a third preservative agent and a third buffer;
preferably, the rabies antibody calibrator comprises a third protective agent, a third preservative agent, a third buffer solution and a series of rabies antibodies with different concentrations.
6. The kit according to claim 1,
the reagent R1 comprises a first protective agent, a first preservative, a first electrolyte, a first sensitizer and a first buffer solution;
the reagent R2 comprises a second protective agent, a second preservative, a second surfactant, a second buffer solution and polystyrene latex particles coated with rabies antigens;
the rabies antibody calibrator comprises a third protective agent, a third preservative agent, a third buffer solution and a series of rabies antibodies with different concentrations.
7. The kit according to any one of claims 1 to 6, wherein reagent R2 independently further comprises a reaction enhancer;
preferably, the reaction enhancer is selected from one or a combination of more than two of mannitol, glycerol and trehalose;
preferably, the mass percentage of the reaction enhancer is 1-10%, preferably 1.8-5%.
8. The kit according to any one of claims 1 to 7, wherein the size of the rabies antigen coated polystyrene latex particles in the reagent R2 is in the range of 150 to 300nm,
preferably, the mass percent of the polystyrene latex particles coated with the rabies antigen in the reagent R2 is 0.05-1%,
more preferably, the mass percentage of the rabies antigen-coated polystyrene latex particles in the reagent R2 is 0.05% -0.2%.
9. The kit according to any one of claims 1 to 8, wherein the carboxyl content of the rabies antigen coated polystyrene latex particles is 50 to 200 μmol/g, preferably 94 μmol/g.
10. The kit of any one of claims 1-9, wherein the rabies antigen is an inactivated native rabies virus or a recombinant rabies antigen.
11. The kit according to any one of claims 1 to 10, wherein the binding of the latex particles to the rabies antigens in the reagent R2 can be prepared by a physical adsorption method or a chemical crosslinking method, and preferably, the rabies antigens are bound to the latex particles by the chemical crosslinking method;
preferably, the mass ratio of the latex particles to the rabies antigens is 10-120, and more preferably, the mass ratio of the latex particles to the rabies antigens is 24.
12. The kit of any one of claims 1-11, wherein the first protective agent, the second protective agent, or the third protective agent in the reagent R1, the reagent R2, and the rabies antibody calibrator are each independently selected from one or a combination of two or more of a protein, an amino acid, a polyhydroxy alcohol, an inorganic salt, a metal complexing agent, a suspending agent, and an antioxidant;
preferably, the concentrations of the first protective agent, the second protective agent or the third protective agent in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently 0.01-1%, 0.1-10%, 0.1-5% in terms of mass percentage,
more preferably 0.05-0.5%,0.1-5%,0.1-4%.
13. The kit according to any one of claims 1 to 12, wherein the first preservative, the second preservative or the third preservative in the reagent R1, the reagent R2 and the rabies antibody calibrator are each independently selected from one or a combination of two or more of potassium sorbate, gentamicin, PC300, PC910 and sodium nitrite;
preferably, the concentrations of the first preservative, the second preservative or the third preservative in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently 0.01-0.5%, 0.01-1% by mass,
more preferably, the concentration of the preservative is 0.05-0.1%, 0.1-0.5% by mass.
14. The kit according to any one of claims 1 to 13, wherein the first electrolyte in the reagent R1 is selected from one or a combination of two or more of inorganic salts;
preferably, the first electrolyte concentration of the reagent R1 is 1-20.0% by mass,
more preferably, the first electrolyte concentrations are each independently 1-10% by mass,
most preferably, the first electrolyte concentration is 1-5.8% by mass.
15. The kit according to any one of claims 1 to 14, wherein the first buffer, the second buffer or the third buffer in the reagent R1, the reagent R2 and the rabies antibody calibrator are each independently selected from one or a combination of two or more of glycine buffer, tris buffer, HEPES buffer, MES buffer, MOPS buffer and MOPSO buffer;
preferably, the concentrations of the first buffer solution, the second buffer solution or the third buffer solution in the reagent R1, the reagent R2 and the rabies antibody calibrator are respectively and independently selected from 10 to 100mmol/L,
preferably, the pH of the first buffer, the second buffer, or the third buffer is each independently selected from 6.0-8.5.
16. The kit according to any one of claims 1 to 15, wherein the sensitizer in the reagent R1 is selected from one or a combination of two or more of PEG6000, PEG8000 and PEG 20000;
preferably, the concentration of the sensitizer in the reagent R1 is 0.5-10% by mass percent,
more preferably, the concentration of the sensitizer in the reagent R1 is 1-4% by mass.
17. The kit according to any one of claims 1 to 16, wherein the surfactant in the reagent R2 is selected from one or a combination of two or more of Tween-20, tween-60, tween-80, span-40, span-80 and ttiton x-100;
preferably, the concentration of the surfactant in the reagent R2 is 0.01-0.1 percent by mass,
more preferably, the concentration of the surfactant is 0.02 to 0.05 mass%.
18. The kit of any one of claims 1-17, wherein the calibration sample of rabies antibodies comprises 5 rabies antibodies at different concentrations,
preferably, the concentration of the 5 different concentrations of the rabies antibody is 0IU/mL, 2.5IU/mL, 5.0IU/mL, 10.0IU/mL and 20.0IU/mL respectively.
19. Use of the kit of any one of claims 1-18 for the detection of rabies antibodies in human plasma.
20. A method for detecting the content of rabies antibodies, wherein the method for detecting the content of the rabies antibodies is to detect a sample by using the kit of any one of claims 1 to 18.
21. Use of the kit according to any one of claims 1 to 18 or the method for detecting the content of rabies antibodies according to claim 19 in the screening of human plasma rabies antibody titer.
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