WO2022134430A1 - 一种血清淀粉样蛋白 a 检测试剂盒及其制备与应用 - Google Patents

一种血清淀粉样蛋白 a 检测试剂盒及其制备与应用 Download PDF

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WO2022134430A1
WO2022134430A1 PCT/CN2021/092458 CN2021092458W WO2022134430A1 WO 2022134430 A1 WO2022134430 A1 WO 2022134430A1 CN 2021092458 W CN2021092458 W CN 2021092458W WO 2022134430 A1 WO2022134430 A1 WO 2022134430A1
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reagent
temperature
antibody
reagents
kit
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PCT/CN2021/092458
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English (en)
French (fr)
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王兴红
邹海涛
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深圳市科曼医疗设备有限公司
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Publication of WO2022134430A1 publication Critical patent/WO2022134430A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • 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

Definitions

  • the invention relates to the technical field of medical detection, in particular to a serum amyloid A detection kit and its preparation and application.
  • Serum amyloid A is a family of polymorphic proteins encoded by multiple genes.
  • SAA is synthesized in the liver by activated macrophages and fibroblasts, and the concentration rises rapidly within 48 to 72 hours, which can be increased to 100 to 1000 times the initial concentration, but its half-life is short, only 50 minutes or so, and declines rapidly during the recovery period of the disease.
  • SAA has high sensitivity and can provide better clinical reference value. As a new detection index, SAA has received more and more attention.
  • the serum amyloid A kit consists of two parts, reagent R1 and reagent R2, and reagent R1 and R2 are usually in liquid state.
  • the defects of the existing serum amyloid A kits include: the R1 reagent and the R2 reagent are easily frozen into a solid state when transported in a cold area in a liquid state, and become liquid again after the temperature is restored, so repeated freezing and thawing can easily lead to the detection of the reagents. Poor stability makes the requirements for transportation temperature too high. If constant temperature transportation is adopted, a temperature control device needs to be installed in the transportation tool, which increases the difficulty of transportation.
  • an embodiment provides a method for preparing a kit, the kit includes at least one of an R1 reagent and an R2 reagent, the R2 reagent contains a latex-antibody complex, and the antibody is selected from the group consisting of An antibody that can specifically bind to serum amyloid A, the preparation method comprises freeze-drying at least one of the R1 reagent and the R2 reagent, and the freeze-drying method comprises:
  • the first drying section under the condition of low air pressure, the freezing chamber storing the reagent pre-frozen to the first temperature is heated to the second temperature, and the holding time at the second temperature is 4-5h;
  • the second drying section after the first drying section, under the condition of low air pressure, the freezing chamber storing the reagent is heated from the second temperature to the third temperature, and the holding time at the third temperature is 5-10h;
  • the third drying section after the second drying section, under the condition of low air pressure, the freezing chamber storing the reagents is heated from the third temperature to the fourth temperature, and the holding time at the fourth temperature is 5-10h.
  • an embodiment provides a kit, characterized in that the kit includes at least one of an R1 reagent and an R2 reagent, the R2 reagent contains a latex-antibody complex, and the antibody is selected from the group consisting of R1 and R2. From an antibody that can specifically bind to serum amyloid A, the R1 reagent and the R2 reagent each independently contain at least one of a stabilizer and an excipient.
  • an embodiment provides the use of the kit of the second aspect in detecting serum amyloid A.
  • a serum amyloid A detection kit and preparation and application thereof according to the above embodiment, the reagent after freeze-drying treatment has high stability, is convenient for transportation in cold areas, and the rise and fall of ambient temperature has no significant impact on the detection performance of the detection reagent .
  • FIG. 4 is a graph showing the detection results of the freeze-dried reagents in Example 2.
  • FIG. 4 is a graph showing the detection results of the freeze-dried reagents in Example 2.
  • connection and “connection” mentioned in this application, unless otherwise specified, include both direct and indirect connections (connections).
  • a method for preparing a kit includes at least one of an R1 reagent and an R2 reagent, the R2 reagent contains a latex-antibody complex, and the antibody is selected from the group consisting of R1 and R2.
  • the preparation method includes lyophilizing at least one of the R1 reagent and the R2 reagent, and the lyophilization method includes:
  • the first drying section under the condition of low air pressure, the freezing chamber storing the reagent pre-frozen to the first temperature is heated to the second temperature, and the holding time at the second temperature is 4-5h;
  • the second drying section after the first drying section, under the condition of low air pressure, the freezing chamber storing the reagent is heated from the second temperature to the third temperature, and the holding time at the third temperature is 5-10h;
  • the third drying section after the second drying section, under the condition of low air pressure, the freezing chamber storing the reagents is heated from the third temperature to the fourth temperature, and the holding time at the fourth temperature is 5-10h.
  • the first temperature is -40°C.
  • the second temperature is -30°C.
  • the third temperature is 5°C.
  • the fourth temperature is 20°C.
  • the low pressure is 10 ⁇ 10Pa.
  • the freezing chamber containing the reagents is vented during the incubation period of the first drying stage.
  • the ventilation mode can be intermittent ventilation or continuous ventilation, and the purpose of ventilation is to discharge water molecules in the freezing chamber to promote the drying of reagents. Almost no water molecules are produced in the second drying section and the third drying section, so no ventilation is required.
  • the heating rates of the first drying section, the second drying section, and the third drying section are conventional technical means in the field. Those skilled in the art can select an appropriate heating rate as required, and gradually adjust the temperature in the refrigerated strong chamber where the reagents are stored. warming up.
  • the R1 reagent and the R2 reagent each independently contain at least one of a stabilizer and an excipient.
  • the R1 reagent and the R2 reagent each independently contain at least one of the following components at final concentrations: 1-10 g/L stabilizer, 1-10 g/L excipient.
  • the R1 reagent and the R2 reagent independently contain the following final concentrations of components: 1-10 g/L stabilizer, 1-10 g/L excipient.
  • the concentration of stabilizer includes, but is not limited to, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L and so on.
  • concentrations of excipients include, but are not limited to, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L and so on.
  • the stabilizer includes, but is not limited to, at least one of sucrose, trehalose, fructose, bovine serum albumin, casein, and gelatin.
  • the excipients include, but are not limited to, mannitol, polyethylene glycol (also known as PEG), polyvinylpyrrolidone (also known as PVP, CAS Reg. No: 9003-39-8), glycerol , at least one of ethylene glycol.
  • the polyethylene glycol includes, but is not limited to, at least one of PEG6000, PEG8000, PEG10000, and PEG20000.
  • the R1 reagent and the R2 reagent each independently further contain at least one of a buffer and a preservative.
  • the R1 reagent and the R2 reagent each independently further contain at least one of the following components at final concentrations: 5-50 mmol/L buffer, 0.5-5 g/L preservative.
  • the R1 reagent and the R2 reagent independently further contain the following components at final concentrations: 5-50 mmol/L buffer, 0.5-5 g/L preservative.
  • the concentration of the buffer includes, but is not limited to, 5 mmol/L, 10 mmol/L, 15 mmol/L, 20 mmol/L, 25 mmol/L mmol/L, 30 mmol/L, 35 mmol/L, 40 mmol/L, 45 mmol/L, 50 mmol/L, etc.
  • the concentration of preservative includes, but is not limited to, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L and so on.
  • the buffers include, but are not limited to, phosphate buffered saline (PBS), tris hydrochloride (Tris hydrochloride), glycine, borate, 4-hydroxyethyl Piperazineethanesulfonic acid (HEPES), 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO), morpholineethanesulfonic acid (MES, also known as 2-morpholineethanesulfonic acid, CAS number : 4432-31-9) at least one.
  • PBS phosphate buffered saline
  • Tris hydrochloride Tris hydrochloride
  • glycine borate
  • HEPES 4-hydroxyethyl Piperazineethanesulfonic acid
  • MOPSO 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid
  • MES morpholineethanesulfonic acid
  • the preservative includes, but is not limited to, at least one of sodium azide, ProClin-950, ProClin-300, KroVin100, KroVin300, KroVin500, KroVin750, thimerosal.
  • the latex-antibody complexes are antibody-conjugated latex microspheres.
  • the particle size of the latex microspheres is 50-300 nm.
  • the latex microspheres include, but are not limited to, polystyrene.
  • the latex-antibody complex is obtained by mixing and reacting latex microspheres and antibodies in a mass ratio of (10-50):1.
  • the mass ratio of latex microspheres to antibody includes, but is not limited to, 10:1, 20:1, 30:1, 40:1, 50:1, and the like.
  • the concentration of latex microspheres in the R2 reagent is 0.05-0.2 wt%.
  • the concentration of latex microspheres includes but is not limited to 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15 wt%, 0.16wt%, 0.17wt%, 0.18wt%, 0.19wt%, 0.2wt%, etc.
  • the pH of the R1 reagent, R2 reagent is independently 6.0-9.0.
  • the pH includes, but is not limited to, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0.
  • the serum amyloid A includes, but is not limited to, human serum amyloid A.
  • the antibodies include, but are not limited to, at least one of monoclonal antibodies, polyclonal antibodies.
  • Antibodies can be purchased from the market.
  • the antibodies include, but are not limited to, goat anti-human serum amyloid A polyclonal antibody, mouse anti-human serum amyloid A monoclonal antibody, and rabbit anti-human serum amyloid A polyclonal antibody. at least one.
  • a kit in some embodiments, includes at least one of an R1 reagent and an R2 reagent, the R2 reagent contains a latex-antibody complex, and the antibody is selected from the group consisting of An antibody that binds to serum amyloid A, the R1 reagent and the R2 reagent each independently contain at least one of a stabilizer and an excipient.
  • the R1 reagent and the R2 reagent each independently contain at least one of the following components at final concentrations: 1-10 g/L stabilizer, 1-10 g/L excipient.
  • the stabilizer is selected from at least one of sucrose, trehalose, fructose, bovine serum albumin, casein, and gelatin.
  • the excipient is selected from at least one of mannitol, polyethylene glycol, polyvinylpyrrolidone, glycerol, and ethylene glycol.
  • the polyethylene glycol is selected from at least one of PEG6000, PEG8000, PEG10000, and PEG20000.
  • the R1 reagent and the R2 reagent each independently further contain at least one of a buffer and a preservative.
  • the R1 reagent and the R2 reagent each independently further contain at least one of the following components at final concentrations: 5-50 mmol/L buffer, 0.5-5 g/L preservative.
  • the concentration of the buffer includes, but is not limited to, 5 mmol/L, 10 mmol/L, 15 mmol/L, 20 mmol/L, 25 mmol/L mmol/L, 30 mmol/L, 35 mmol/L, 40 mmol/L, 45 mmol/L, 50 mmol/L, etc.
  • the concentration of preservative includes, but is not limited to, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L and so on.
  • the buffer is selected from the group consisting of phosphate buffered saline, tris hydrochloride, glycine, borate, 4-hydroxyethylpiperazineethanesulfonic acid, 3-(N -At least one of morpholino)-2-hydroxypropanesulfonic acid and morpholineethanesulfonic acid.
  • the preservative is selected from at least one of sodium azide, ProClin-950, ProClin-300, KroVin100, KroVin300, KroVin500, KroVin750, thimerosal.
  • the R1 reagent and the R2 reagent are solid reagents obtained by lyophilization.
  • the kit is made by the method of the first aspect.
  • the solid R1 and R2 components in the kit need to be reconstituted by adding pure water.
  • the kit further includes a container for independently containing the R1 reagent and the R2 reagent.
  • the kit further includes an instruction manual for instructing the user to use the kit for sample detection.
  • kit of the second aspect in detecting serum amyloid A is provided.
  • the application includes use to detect the concentration of serum amyloid A in a sample.
  • the sample includes, but is not limited to, a body fluid sample.
  • the body fluid sample includes, but is not limited to, at least one of whole blood, serum, and plasma.
  • the sample is from a human body.
  • the serum amyloid A kit of the present embodiment contains two solid reagents, R1 and R2.
  • the R1 and R2 reagents are lyophilized from liquid reagents containing excipients and stabilizers to form solid R1 and R2 reagents.
  • the R2 reagent can be reconstituted with pure water to restore the R1 and R2 reagents to liquid state.
  • the R1 reagent contains the following final concentrations of components: 2g/L first stabilizer, 2g/L first excipient, 5mmol/L first buffer, 0.5g/L first preservative.
  • the first stabilizer is trehalose, bovine serum albumin, the final concentration of trehalose and bovine serum albumin in R1 reagent is 1g/L, the first excipient is mannitol, PEG6000, mannitol, PEG6000 in R1
  • the final concentrations in the reagents were all 1 g/L
  • the first buffer was Tris-HCl (tris-hydroxymethylaminomethane hydrochloride)
  • the first preservative was ProClin-300.
  • the R2 reagent contains the following final concentrations of components: latex microspheres 0.1wt%, 2g/L second stabilizer, 2g/L second excipient, 5mmol/L second buffer, 0.5g/L second preservative .
  • the second stabilizer is trehalose, bovine serum albumin
  • the final concentration of trehalose and bovine serum albumin in R2 reagent is 1g/L
  • the second excipient is mannitol, glycerol, mannitol, propylene glycol
  • the final concentration of triol in R2 reagent is 1g/L
  • the second buffer is Tris-HCl (tris-hydroxymethylaminomethane hydrochloride)
  • the second preservative is ProClin-300.
  • R1 reagent and R2 reagent The lyophilization method of R1 reagent and R2 reagent is as follows:
  • the first drying section vacuumize the air pressure in the refrigeration equipment to 30Pa, and increase the temperature in the refrigeration equipment. After the internal temperature of the refrigeration equipment rises to -30 °C, start to ventilate the refrigeration equipment intermittently, and at -30 Continue to run at °C for 5h, during this process, control the air pressure in the freezing chamber to 10 ⁇ 10Pa, until the product water line disappears.
  • the second drying section After the first drying section, the temperature inside the freezing equipment is gradually increased from -30°C to 5°C. During this process, the air pressure in the freezing equipment is controlled at 10 ⁇ 10pa, and the second drying section is carried out. , specifically at 5°C for 10h.
  • the third drying section After the second drying section, the temperature inside the refrigeration equipment is gradually increased from 5 °C to 20 °C, and the third drying section is carried out, specifically, the temperature is kept at 20 °C for 5 hours. The air pressure is controlled at 10 ⁇ 10pa. After the third drying section, solid R1 reagent and R2 reagent are prepared. The refrigeration equipment is opened, and the freeze-dried R1 reagent and R2 reagent are taken out and packaged separately. The lyophilized reagent of this example was obtained.
  • the lyophilized R1 and R2 reagents in this example were reconstituted with pure water. After reconstitution, the volumes of the R1 and R2 reagents were the same as the liquid reagents before lyophilization, and the concentration of serum amyloid A in the whole blood sample was detected.
  • the experimental group use the unlyophilized liquid R1 reagent and R2 reagent to detect the concentration of serum amyloid A in the whole blood sample, as the control group 1;
  • the obtained R1 reagent and R2 reagent were used to detect the concentration of serum amyloid A in the whole blood sample, and it was used as control group 2;
  • the concentration of serum amyloid A in the blood sample was taken as control group 3;
  • the lyophilized reagent in this example was lyophilized and reconstituted 3 times, and the obtained R1 reagent and R2 reagent were used to detect the serum amyloid A in the whole blood sample. concentration, as control group 4.
  • the lyophilization procedure for each lyophilization and reconstitution is the same as the steps A) to D) of this example.
  • reagents that only need to be lyophilized and reconstituted once they will be solid after lyophilization.
  • When testing samples use pure reagents. Reconstituted with water to the volume before lyophilization for subsequent sample testing; for reagents that need to be lyophilized for 2 or 3 times, after the last lyophilization, reconstitute with pure water to the volume before lyophilization, and then Carry out the next freeze-drying procedure according to steps A) to D) of this example. After the last freeze-drying, solid reagents are obtained.
  • Comparative experiment 1 Using the lyophilized reagent of this example, the R1 and R2 reagents that were liquid before lyophilization, the R1 and R2 reagents that were lyophilized and reconstituted once, and the lyophilized reagents that were lyophilized and reconstituted twice R1 and R2 reagents, freeze-dried reagents and R1 and R2 reagents that were lyophilized and reconstituted 3 times, independently detected whole blood samples, at a wavelength of 540 nm, through the same
  • the automatic specific protein analyzer can quantitatively detect the content of serum amyloid A in the whole blood sample by comparing the whole blood sample obtained by SAA reagent test assignment.
  • the R1 and R2 in the serum amyloid A kit are reconstituted, when used for detection, the R1 reagent and the R2 reagent are mixed in a volume ratio of 1:1.
  • the R1 reagent and the R2 reagent were mixed in a volume ratio of 1:1.
  • Figure 1 shows the results of detecting the content of serum-like amyloid A in whole blood samples using the R1 and R2 reagents in liquid state before freeze-drying and the R1 and R2 reagents after freeze-drying in this example.
  • the abscissa is the serum sample in whole blood.
  • the concentration of amyloid A (mg/L), the ordinate is the degree of reactivity.
  • the R1 and R2 reagents after freeze-drying in Example 1 were used to reconstitute and test whole blood samples of a certain concentration.
  • the reactivity of the lyophilized reagents is almost the same, indicating that the test values of the lyophilized and reconstituted reagents are not significantly different from those of the unlyophilized reagents, and the test is not affected by lyophilization, which significantly improves the stability of the reagents.
  • Figure 2 shows the lyophilized reagents of the present embodiment, the lyophilized reagents are lyophilized and reconstituted once, R1, R2, the lyophilized reagents are lyophilized and reconstituted twice, the lyophilized reagents are lyophilized again
  • the R1 and R2 reagents that have been reconstituted three times to detect the content of serum amyloid A in whole blood samples.
  • the abscissa is the concentration of serum amyloid A in whole blood (mg/L), and the ordinate is the degree of reactivity .
  • lyophilized R1 and R2 reagents for lyophilization and reconstitution 1 time, 2 times, and 3 times.
  • the procedure for each lyophilization and reconstitution is the same as the steps A) to D) of this example, and the lyophilization and reconstitution procedures are performed twice, For 3 times, after the last lyophilization and reconstitution, reconstitute with pure water to the volume before lyophilization, and then perform lyophilization according to steps A) to D) of this example.
  • the content of serum amyloid A in the whole blood sample was tested, and the result is shown in Figure 2.
  • the reactivity detected by the freeze-dried reagent of this embodiment is all highly similar, and are not affected by the lyophilized and reconstituted times, indicating that the lyophilized R1 and R2 reagents in this example are more stable, even in cold Regional transportation will not affect the detection accuracy of reagents due to changes in ambient temperature.
  • This comparative example provides common liquid reagents, wherein the R1 reagent does not contain the first stabilizer and the first excipient, and the R2 reagent does not contain the second stabilizer and the second excipient.
  • the other components and contents are the same as those in Example 1.
  • Three equal parts of ordinary liquid reagents were lyophilized and reconstituted for 1, 2, and 3 times respectively.
  • the method for each lyophilized and reconstituted was the same as that in Comparative Experiment 1. After obtaining the corresponding reagents, whole blood was detected.
  • Figure 3 shows the use of ordinary liquid reagents that have not been lyophilized, the R1 and R2 reagents that have been lyophilized and reconstituted once, the R1 and R2 reagents that have been lyophilized and reconstituted twice, and the common liquid reagents that have been lyophilized and reconstituted.
  • the serum amyloid A kit of the present embodiment contains two solid reagents, R1 and R2.
  • the R1 and R2 reagents are lyophilized from liquid reagents containing excipients and stabilizers to form solid R1 and R2 reagents.
  • the R2 reagent can be reconstituted with pure water to restore the R1 and R2 reagents to liquid state.
  • the R1 reagent contains the following components at final concentrations: 10 g/L primary stabilizer, 10 g/L g/L first excipient, 50 mmol/L first buffer, 5 g/L first preservative.
  • the first stabilizer is trehalose, bovine serum albumin, the final concentration of trehalose and bovine serum albumin in R1 reagent is 5g/L, the first excipient is mannitol, PEG6000, mannitol, PEG6000 in R1
  • the final concentrations in the reagents were all 5 g/L
  • the first buffer was Tris-HCl (tris-hydroxymethylaminomethane hydrochloride)
  • the first preservative was ProClin-300.
  • the R2 reagent contains the following final concentrations of components: latex microspheres 0.2wt%, 10 g/L secondary stabilizer, 10 g/L secondary excipient, 50 mmol/L second buffer, 5g/L second preservative.
  • the second stabilizer is trehalose, bovine serum albumin
  • the final concentration of trehalose and bovine serum albumin in R2 reagent is 5g/L
  • the second excipient is mannitol, glycerol, mannitol, propylene glycol
  • the final concentration of triol in R2 reagent was 5g/L
  • the second buffer was Tris-HCl (tris-hydroxymethylaminomethane hydrochloride)
  • the second preservative was ProClin-300.
  • R1 reagent and R2 reagent The lyophilization method of R1 reagent and R2 reagent is as follows:
  • the first drying section vacuumize the air pressure in the refrigeration equipment to 30Pa, and increase the temperature in the refrigeration equipment. After the internal temperature of the refrigeration equipment rises to -30 °C, start to ventilate the refrigeration equipment intermittently, and at -30 Keep running at °C for 5h, during this process, control the air pressure in the freezing chamber to 10 ⁇ 10Pa, until the product water line disappears.
  • the second drying section After the first drying section, the temperature inside the freezing equipment is gradually increased from -30°C to 5°C. During this process, the air pressure in the freezing equipment is controlled at 10 ⁇ 10pa, and the second drying section is carried out. , specifically at 5°C for 10h.
  • the third drying section After the second drying section, the temperature inside the refrigeration equipment is gradually increased from 5 °C to 20 °C, and the third drying section is carried out. Specifically, the operation is kept at 20 °C for 10 hours. The air pressure is controlled at 10 ⁇ 10pa. After the third drying section, solid R1 reagent and R2 reagent are prepared. The refrigeration equipment is opened, and the freeze-dried R1 reagent and R2 reagent are taken out and packaged separately.
  • the lyophilized R1 and R2 reagents in this example were reconstituted with pure water. After reconstitution, the volumes of the R1 and R2 reagents were the same as the liquid reagents before lyophilization, and the concentration of serum amyloid A in the whole blood sample was detected. , as the experimental group; non-lyophilized liquid R1 reagent and R2 reagent were used to detect the concentration of serum amyloid A in the whole blood sample, as the control group 5 .
  • the detection steps are as follows:
  • R1 and R2 in the serum amyloid A kit prepared in this example are reconstituted, when they are used to detect whole blood samples, the R1 reagent and the R2 reagent are mixed according to a volume ratio of 1:1. When the liquid reagent before lyophilization is used to detect whole blood samples, the R1 reagent and the R2 reagent are also mixed in a volume ratio of 1:1.
  • FIG. 4 is a graph of the detection results of the reagents reconstituted after lyophilization and the liquid reagents before lyophilization in this embodiment.
  • the present invention uses freeze-drying technology to freeze-dry the R1 and R2 reagents of the serum amyloid A kit, which can improve the convenience of transportation of the reagents in cold regions and the stability of repeated freeze-thaw of the reagents.
  • the present invention is easy to operate during detection, is in a solid state during transportation, is not affected by the temperature in cold regions, does not destroy the activity of the antibody after being reconstituted into a liquid during use, and is in a liquid state during detection, and its performance is not affected by influences. It ensures convenient storage in transportation in cold areas and enhances the stability of repeated freezing and thawing of reagents.

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Abstract

一种血清淀粉样蛋白A检测试剂盒及其制备与应用，所述试剂盒包括R1试剂、R2试剂中的至少一种，所述R2试剂含有胶乳抗体复合物，所述抗体选自可特异性结合至血清淀粉样蛋白A的抗体，所述制备方法包括对R1试剂、R2试剂中的至少一种进行冻干。冻干处理后的试剂稳定性高，便于在寒冷地区运输，环境温度的升降对检测试剂的检测性能无显著影响。

Description

一种血清淀粉样蛋白A检测试剂盒及其制备与应用 技术领域
本发明涉及医学检测技术领域，具体涉及一种血清淀粉样蛋白A检测试剂盒及其制备与应用。
 背景技术
血清淀粉样蛋白A(serumamyloidAprotein，SAA)是一类多基因编码的多形态蛋白家族，是组织淀粉样蛋白A的前体物质，属于急性时相反应蛋白，其相对分子量约12000。在急性时相反应中，如炎症或感染急性期，经IL-1、IL-6 和TNF刺激，SAA在肝脏中由被激活的巨噬细胞和纤维母细胞合成，在48～72 小时内浓度迅速升高，可升高到最初浓度的100～1000倍，但其半衰期短，只有50分钟左右，并在疾病恢复期迅速下降。某些疾病，如病毒感染、移植排斥反应、冠心病等，SAA的敏感性高，可为临床提供更好的参考价值，作为一个新的检测指标，SAA受到人们越来越多的关注。
通常血清淀粉样蛋白A试剂盒由两部分组成，试剂R1与试剂R2，R1试剂和R2试剂通常均为为液态状态。
现有的血清淀粉样蛋白A试剂盒的缺陷包括：R1试剂与R2试剂在液态状态下在寒冷地区运输容易被冻成固态，温度恢复后又变为液态，这样反复冻融容易导致试剂的检测稳定性差，使得对运输温度的要求过高，如果采用恒温运输，则需要在运输工具内安装温控装置，增加了运输的难度。
 技术解决方案
根据第一方面，一种实施例中提供一种试剂盒的制备方法，所述试剂盒包括R1试剂、R2试剂中的至少一种，所述R2试剂含有胶乳抗体复合物，所述抗体选自可特异性结合至血清淀粉样蛋白A的抗体，所述制备方法包括对R1试剂、R2试剂中的至少一种进行冻干，冻干方法包括：
第一干燥段：在低气压条件下，将存放有预冻至第一温度的试剂的冷冻腔室升温至第二温度，在第二温度下的保持时间为4-5h；
第二干燥段：第一干燥段结束后，在低气压条件下，将存放有试剂的冷冻腔室从第二温度升温至第三温度，在第三温度下的保持时间为5-10h；
第三干燥段：第二干燥段结束后，在低气压条件下，将存放有试剂的冷冻腔室从第三温度升温至第四温度，在第四温度下的保持时间为5-10h。
根据第二方面，一种实施例中提供一种试剂盒，其特征在于，所述试剂盒包括R1试剂、R2试剂中的至少一种，所述R2试剂含有胶乳抗体复合物，所述抗体选自可特异性结合至血清淀粉样蛋白A的抗体，所述R1试剂、R2试剂各自独立地含有稳定剂、赋形剂中的至少一种。
根据第三方面，一种实施例中提供第二方面所述试剂盒在检测血清淀粉样蛋白A中的应用。
 有益效果
依据上述实施例的一种血清淀粉样蛋白A检测试剂盒及其制备与应用，冻干处理后的试剂稳定性高，便于在寒冷地区运输，环境温度的升降对检测试剂的检测性能无显著影响。
 附图说明
图 1 为实施例 1 的冻干试剂与液态试剂检测结果图；
图 2 为实施例 1 制得的冻干试剂与该冻干试剂再冻干复溶 1 次、 2 次、 3 次后的检测结果图；
图 3 为普通液态试剂与分别冻干复溶 1 次、 2 次、 3 次的普通液态试剂的检测结果图；
图 4 为实施例 2 的冻干试剂检测结果图。
本发明的实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。其中不同实施方式中类似元件采用了相关联的类似的元件标号。在以下的实施方式中，很多细节描述是为了使得本申请能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。在某些情况下，本申请相关的一些操作并没有在说明书中显示或者描述，这是为了避免本申请的核心部分被过多的描述所淹没，而对于本领域技术人员而言，详细描述这些相关操作并不是必要的，他们根据说明书中的描述以及本领域的一般技术知识即可完整了解相关操作。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本文中为部件所编序号本身，例如“第一”、“第二”等，仅用于区分所描述的对象，不具有任何顺序或技术含义。而本申请所说“连接”、“联接”，如无特别说明，均包括直接和间接连接（联接）。
根据第一方面，在一些实施例中，提供一种试剂盒的制备方法，所述试剂盒包括R1试剂、R2试剂中的至少一种，所述R2试剂含有胶乳抗体复合物，所述抗体选自可特异性结合至血清淀粉样蛋白A的抗体，所述制备方法包括对R1试剂、R2试剂中的至少一种进行冻干，冻干方法包括：
第一干燥段：在低气压条件下，将存放有预冻至第一温度的试剂的冷冻腔室升温至第二温度，在第二温度下的保持时间为4-5h；
第二干燥段：第一干燥段结束后，在低气压条件下，将存放有试剂的冷冻腔室从第二温度升温至第三温度，在第三温度下的保持时间为5-10h；
第三干燥段：第二干燥段结束后，在低气压条件下，将存放有试剂的冷冻腔室从第三温度升温至第四温度，在第四温度下的保持时间为5-10h。
在一些实施例中，所述第一温度为-40℃。
在一些实施例中，所述第二温度为-30℃。
在一些实施例中，所述第三温度为5℃。
在一些实施例中，所述第四温度为20℃。
在一些实施例中，所述低气压为10±10Pa。
在一些实施例中，第一干燥段的保温期间对存放有试剂的冷冻腔室进行通气。通气的方式可以是间断性通气，也可以是连续通气，通气的目的是使得冷冻腔室内的水分子外排，促进试剂的干燥。第二干燥段、第三干燥段几乎没有水分子产生，因此无需通气。
第一干燥段、第二干燥段、第三干燥段的升温速率为本领域的常规技术手段，本领域技术人员可以根据需要选择合适的升温速率，对存放有试剂的冷冻强室内的温度进行逐步升温。
在一些实施例中，所述R1试剂、R2试剂各自独立地含有稳定剂、赋形剂中的至少一种。
在一些实施例中，所述R1试剂、R2试剂各自独立地含有如下终浓度的组分中的至少一种：1-10g/L稳定剂、1-10g/L赋形剂。
在一些实施例中，所述R1试剂、R2试剂独立地含有如下终浓度的组分：1-10g/L稳定剂、1-10g/L赋形剂。
在一些实施例中，稳定剂的浓度包括但不限于1 g/L、2 g/L、3 g/L、4 g/L、5 g/L、6 g/L、7 g/L、8 g/L、9 g/L、10 g/L等等。
在一些实施例中，赋形剂的浓度包括但不限于1 g/L、2 g/L、3 g/L、4 g/L、5 g/L、6 g/L、7 g/L、8 g/L、9 g/L、10 g/L等等。
在一些实施例中，所述稳定剂包括但不限于蔗糖、海藻糖、果糖、牛血清白蛋白、酪蛋白、明胶中的至少一种。
在一些实施例中，所述赋形剂包括但不限于甘露醇、聚乙二醇（亦称PEG）、聚乙烯吡咯烷酮（亦称PVP，CAS登录号：9003-39-8）、丙三醇、乙二醇中的至少一种。
在一些实施例中，所述聚乙二醇包括但不限于PEG6000、PEG8000、PEG10000、PEG20000中的至少一种。
在一些实施例中，所述R1试剂、R2试剂各自独立地还含有缓冲剂、防腐剂中的至少一种。
在一些实施例中，所述R1试剂、R2试剂各自独立地还含有如下终浓度的组分中的至少一种：5-50mmol/L缓冲剂、0.5-5g/L防腐剂。
在一些实施例中，所述R1试剂、R2试剂各自独立地还含有如下终浓度的组分：5-50 mmol/L缓冲剂、0.5-5g/L防腐剂。
在一些实施例中，缓冲剂的浓度包括但不限于5mmol/L、10mmol/L、15 mmol/L、20 mmol/L、25 mmol/L、30 mmol/L、35 mmol/L、40 mmol/L、45 mmol/L、50 mmol/L等等。
在一些实施例中，防腐剂的浓度包括但不限于0.5 g/L、0.6 g/L、0.7 g/L、0.8 g/L、0.9 g/L、1 g/L、2 g/L、3 g/L、4 g/L、5 g/L等等。
在一些实施例中，所述缓冲剂包括但不限于磷酸缓冲盐溶液（PBS）、三羟甲基氨基甲烷盐酸盐（Tris盐酸盐）溶液、甘氨酸、硼酸盐溶液、4-羟乙基哌嗪乙磺酸（HEPES）、3-(N-吗啉基)-2-羟基丙磺酸（MOPSO）、吗啉乙磺酸（MES，亦称2-吗啉乙磺酸，CAS号：4432-31-9）中的至少一种。
在一些实施例中，所述防腐剂包括但不限于叠氮化钠、ProClin-950、ProClin-300、KroVin100、KroVin300、KroVin500、KroVin750、硫柳汞中的至少一种。
在一些实施例中，所述胶乳抗体复合物为偶联有抗体的胶乳微球。
在一些实施例中，所述胶乳微球的粒径为50-300 nm。
在一些实施例中，所述胶乳微球包括但不限于聚苯乙烯。
在一些实施例中，所述胶乳抗体复合物是由胶乳微球与抗体按照（10-50）：1的质量比混合反应得到。
在一些实施例中，胶乳微球与抗体的质量比包括但不限于10：1、20：1、30：1、40：1、50：1等等。
在一些实施例中，所述R2试剂中胶乳微球的浓度为0.05-0.2wt%。胶乳微球的浓度包括但不限于0.05wt%、0.06wt%、0.07wt%、0.08wt%、0.09wt%、0.1wt%、0.11wt%、0.12wt%、0.13wt%、0.14wt%、0.15wt%、0.16wt%、0.17wt%、0.18wt%、0.19wt%、0.2wt%等等。
在一些实施例中，所述R1试剂、R2试剂的pH独立地为6.0-9.0。该pH包括但不限于6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0。
在一些实施例中，所述血清淀粉样蛋白A包括但不限于人源血清淀粉样蛋白A。
在一些实施例中，所述抗体包括但不限于单克隆抗体、多克隆抗体中的至少一种。抗体可以从市场上购买得到。
在一些实施例中，所述抗体包括但不限于羊抗人血清淀粉样蛋白A多克隆抗体、鼠抗人血清淀粉样蛋白A单克隆抗体、兔抗人血清淀粉样蛋白A多克隆抗体中的至少一种。
根据第二方面，在一些实施例中，提供一种试剂盒，所述试剂盒包括R1试剂、R2试剂中的至少一种，所述R2试剂含有胶乳抗体复合物，所述抗体选自可特异性结合至血清淀粉样蛋白A的抗体，所述R1试剂、R2试剂各自独立地含有稳定剂、赋形剂中的至少一种。
在一些实施例中，所述R1试剂、R2试剂各自独立地含有如下终浓度的组分中的至少一种：1-10g/L稳定剂、1-10g/L赋形剂。
在一些实施例中，所述稳定剂选自蔗糖、海藻糖、果糖、牛血清白蛋白、酪蛋白、明胶中的至少一种。
在一些实施例中，所述赋形剂选自甘露醇、聚乙二醇、聚乙烯吡咯烷酮、丙三醇、乙二醇中的至少一种。
在一些实施例中，所述聚乙二醇选自PEG6000、PEG8000、PEG10000、PEG20000中的至少一种。
在一些实施例中，所述R1试剂、R2试剂各自独立地还含有缓冲剂、防腐剂中的至少一种。
在一些实施例中，所述R1试剂、R2试剂各自独立地还含有如下终浓度的组分中的至少一种：5-50mmol/L缓冲剂、0.5-5g/L防腐剂。
在一些实施例中，缓冲剂的浓度包括但不限于5 mmol/L、10mmol/L、15 mmol/L、20 mmol/L、25 mmol/L、30 mmol/L、35 mmol/L、40 mmol/L、45 mmol/L、50 mmol/L等等。
在一些实施例中，防腐剂的浓度包括但不限于0.5 g/L、0.6 g/L、0.7 g/L、0.8 g/L、0.9 g/L、1 g/L、2 g/L、3 g/L、4 g/L、5 g/L等等。
在一些实施例中，所述缓冲剂选自磷酸缓冲盐溶液、三羟甲基氨基甲烷盐酸盐溶液、甘氨酸、硼酸盐溶液、4-羟乙基哌嗪乙磺酸、3-(N-吗啉基)-2-羟基丙磺酸、吗啉乙磺酸中的至少一种。
在一些实施例中，所述防腐剂选自叠氮化钠、ProClin-950、ProClin-300、KroVin100、KroVin300、KroVin500、KroVin750、硫柳汞中的至少一种。
在一些实施例中，所述R1试剂、R2试剂均为冻干所得的固态试剂。
在一些实施例中，所述试剂盒是由第一方面所述方法制得。
在一些实施例中，使用时，试剂盒中的固态R1和R2组分需要加入纯水使其复溶。
在一些实施例中，所述试剂盒还包括用于独立容纳R1试剂、R2试剂的容器。
在一些实施例中，所述试剂盒还包括使用说明书，用于指导用户使用该试剂盒进行样品检测。
根据第三方面，提供第二方面所述试剂盒在检测血清淀粉样蛋白A中的应用。
在一些实施例中，所述应用包括用于检测样本中血清淀粉样蛋白A的浓度。
在一些实施例中，所述样本包括但不限于体液样本。
在一些实施例中，所述体液样本包括但不限于全血、血清、血浆中的至少一种。
在一些实施例中，所述样本来自于人体。
实施例1
本实施例的血清淀粉样蛋白A试剂盒中含有固态的R1和R2两种试剂，所述R1和R2试剂是由含有赋形剂和稳定剂的液态试剂冻干而成，形成固态的R1和R2试剂，可用纯水复溶恢复成液态的R1和R2试剂。
本实施例的试剂盒制备方法如下：
1）R1的制备：
在调配罐中加入适量的水，调节搅拌器至合适转速，依次投入缓冲离子、稳定剂、赋形剂、防腐剂，15分钟至完全溶解，溶液澄清透明无沉淀后，调节pH至8.0，定容至最终体积。R1试剂含有如下终浓度的组分：2g/L第一稳定剂、2g/L第一赋形剂、5mmol/L第一缓冲剂、0.5g/L第一防腐剂。第一稳定剂为海藻糖、牛血清白蛋白，海藻糖、牛血清白蛋白在R1试剂中的终浓度均为1g/L，第一赋形剂为甘露醇、PEG6000，甘露醇、PEG6000在R1试剂中的终浓度均为1 g/L，第一缓冲剂为Tris-HCl（三羟甲基氨基甲烷盐酸盐），第一防腐剂为ProClin-300。
然后使用冻干装置将液态的R1进行冻干，后分装、标识。
2）R2的制备：
将粒径为100nm的胶乳微球稀释至一定浓度，并进行搅拌，同时边滴加兔抗人SAA抗体，每5秒加入一滴，胶乳微球与兔抗人SAA抗体按照20：1的质量比例进行混合，反应一定时间后加入10% BSA溶液反应一定时间，将混合液离心、超声重悬，将标记好的胶乳微球-SAA抗体复合物中加入适量的水，超声重悬后，依次投入第二稳定剂、第二赋形剂、第二缓冲剂、第二防腐剂，调节pH至8.0，定容至最终体积。
R2试剂含有如下终浓度的组分：胶乳微球 0.1wt%、2g/L第二稳定剂、2g/L第二赋形剂、5mmol/L第二缓冲剂、0.5g/L第二防腐剂。第二稳定剂为海藻糖、牛血清白蛋白，海藻糖、牛血清白蛋白在R2试剂中的终浓度均为1g/L，第二赋形剂为甘露醇、丙三醇，甘露醇、丙三醇在R2试剂中的终浓度均为1g/L，第二缓冲剂为Tris-HCl（三羟甲基氨基甲烷盐酸盐），第二防腐剂为ProClin-300。
然后使用冻干装置将液态的R2进行冻干，后分装、标识。
R1试剂、R2试剂的冻干方法如下：
A）将R1、R2试剂放入冷冻设备中进行产品预冻，设定冷冻设备内部温度为-40℃，冷冻设备开始降温，经4h后试剂温度稳定后，预冻结束。
B）第一干燥段：抽真空使冷冻设备内气压降至30Pa，并使冷冻设备内温度上升，待冷冻设备内部温度升至-30℃后，开始向冷冻设备内间断地通气，在-30℃下持续运行5h，在此过程中，将冷冻腔室内的气压控制在10±10Pa，直至产品水线消失。
C）第二干燥段：第一干燥段结束后，将冷冻设备内部温度从-30℃逐步升温至5℃，在此过程中，冷冻设备内的气压控制在10±10pa，进行第二干燥段，具体是在5℃下保持10h。
D）第三干燥段：第二干燥段结束后，冷冻设备内部温度从5℃逐步升温至20℃，进行第三干燥段，具体是在20℃下保持5h，在此过程中，冷冻设备内的气压控制在10±10pa，第三干燥段结束后，制得固态的R1试剂、R2试剂，将冷冻设备打开，将冻干后的R1试剂与R2试剂取出，独立包装。得到本实施例的冻干试剂。
用纯水将本实施例冻干的R1、R2试剂复溶，复溶后，R1试剂、R2试剂的体积与冻干前的液态试剂体积一致，检测全血样本中血清样淀粉蛋白A的浓度，作为实验组；采用未冻干的液态R1试剂、R2试剂检测全血样本中血清样淀粉蛋白A的浓度，作为对照组1；将本实施例的冻干试剂冻干复溶1次，使用所得的R1试剂、R2试剂检测全血样本中血清样淀粉蛋白A的浓度，作为对照组2；将本实施例的冻干试剂冻干复溶2次，使用所得的R1试剂、R2试剂检测全血样本中血清样淀粉蛋白A的浓度，作为对照组3；将本实施例的冻干试剂冻干复溶3次，使用所得的R1试剂、R2试剂检测全血样本中血清样淀粉蛋白A的浓度，作为对照组4。每次冻干复溶的冻干程序同本实施例的步骤A）至步骤D），对于只需冻干复溶1次的试剂，在冻干结束后为固态，在检测样品时，使用纯水复溶至冻干前的体积，用于后续样品检测；对于需要冻干复溶2次、3次的试剂，上一次冻干结束后，用纯水复溶至冻干前的体积，然后按照本实施例的步骤A）至步骤D）进行下一次冻干程序，最后一次冻干结束后，得到固态试剂，在检测样品时，使用纯水复溶至冻干前的体积，用于后续样品检测。
检测实验的具体步骤如下：
对比实验1：使用本实施例的冻干试剂，冻干前液态的R1、R2试剂，冻干试剂再冻干复溶1次的R1、R2试剂，冻干试剂再冻干复溶2次的R1、R2试剂，冻干试剂再冻干复溶3次的R1、R2试剂，独立检测全血样本，在540nm波长下，通过与已知浓度（使用深圳市国赛生物技术有限公司的Aristo全自动特定蛋白分析仪，进行SAA试剂测试赋值得到）的全血样本比较，可定量检测出全血样本中血清样淀粉蛋白A的含量。
血清淀粉样蛋白A试剂盒中的R1和R2复溶后，用于检测时，R1试剂与R2试剂按照1:1的体积比混合。各个对照组用于全血样本检测时，R1试剂与R2试剂均按照1:1的体积比混合。
结果如图1、图2所示。
图1为使用冻干前液态的R1、R2试剂，本实施例冻干后的R1、R2试剂，检测出全血样本中血清样淀粉蛋白A的含量结果图，横坐标为全血中血清样淀粉蛋白A的浓度（mg/L），纵坐标为反应度。
从图1可见，使用实施例1冻干后的R1、R2试剂复溶，测试一定浓度的全血样本，检测得到的相同浓度的样本的反应度与冻干前的液态R1、R2试剂检测所得的反应度几乎一致，说明冻干复溶后的试剂与未冻干的试剂测试值无显著差别，测试不受冻干的影响，显著提高了试剂的稳定性。
图2为使用本实施例的冻干试剂，冻干试剂再冻干复溶1次的R1、R2试剂，冻干试剂冻干再复溶2次的R1、R2试剂，冻干试剂再冻干复溶3次的R1、R2试剂，检测出全血样本中血清样淀粉蛋白A的含量结果图，横坐标为全血中血清样淀粉蛋白A的浓度（mg/L），纵坐标为反应度。
使用冻干后的R1、R2试剂冻干复溶1次、2次、3次，每次冻干复溶的程序同本实施例的步骤A）至步骤D），冻干复溶2次、3次时，上一次冻干复溶结束后，用纯水复溶至冻干前的体积，然后按照本实施例的步骤A）至步骤D）进行冻干。测试全血样本中的血清样淀粉蛋白A的含量，结果如图2所示，从其结果来看，用于检测相同浓度的全血样本时，本实施例的冻干试剂检测出的反应度与使用冻干复溶1次、2次、3次的试剂的检测结果均高度接近，不受冻干复溶次数的影响，说明本实施例冻干的R1、R2试剂更加稳定，即使在寒冷地区运输，也不会因环境温度的变化而影响试剂的检测准确性。
对比例1
本对比例提供普通液态试剂，其中的R1试剂不含第一稳定剂、第一赋形剂，R2试剂不含第二稳定剂、第二赋形剂，其他组分及含量同实施例1，将三等份的普通液态试剂，分别冻干复溶1次、2次、3次，每次冻干复溶的方法同对比实验1，得到相应的试剂后，进行全血检测。
图3为使用未经过冻干处理的普通液态试剂，普通液态试剂冻干复溶1次的R1、R2试剂，普通液态试剂冻干复溶2次的R1、R2试剂，普通液态试剂冻干复溶3次的R1、R2试剂，分别测试全血样本中的血清样淀粉蛋白A的含量的结果图。
从图3可知，用于检测相同浓度的全血样本时，冻干复溶次数越多，反应度越低，冻干复溶1次、2次、3次的R1、R2试剂模拟了试剂在寒冷地区运输时容易被冻成固态的情形，可见，普通液态试剂冻干复溶1次、2次、3次后，稳定性显著下降，说明稳定剂和赋形剂可显著提高R1试剂、R2试剂的稳定性。
实施例2
本实施例的血清淀粉样蛋白A试剂盒中含有固态的R1和R2两种试剂，所述R1和R2试剂是由含有赋形剂和稳定剂的液态试剂冻干而成，形成固态的R1和R2试剂，可用纯水复溶恢复成液态的R1和R2试剂。
本实施例的试剂盒制备方法如下：
1）R1的制备：
在调配罐中加入适量的水，调节搅拌器至合适转速，依次投入缓冲离子、稳定剂、赋形剂、防腐剂，15分钟至完全溶解，溶液澄清透明无沉淀后，调节pH至8.0，定容至最终体积。R1试剂含有如下终浓度的组分：10 g/L第一稳定剂、10 g/L第一赋形剂、50 mmol/L第一缓冲剂、5 g/L第一防腐剂。第一稳定剂为海藻糖、牛血清白蛋白，海藻糖、牛血清白蛋白在R1试剂中的终浓度均为5g/L，第一赋形剂为甘露醇、PEG6000，甘露醇、PEG6000在R1试剂中的终浓度均为5g/L，第一缓冲剂为Tris-HCl（三羟甲基氨基甲烷盐酸盐），第一防腐剂为ProClin-300。
然后使用冻干装置将液态的R1进行冻干，后分装、标识。
2）R2的制备：
将粒径为100nm的胶乳微球稀释至一定浓度，并进行搅拌，同时边滴加兔抗人SAA抗体，每5秒加入一滴，胶乳微球与兔抗人SAA抗体按照20：1的质量比例进行混合，反应一定时间后，再加入10% BSA溶液反应一定时间，将混合液离心、超声重悬，向标记好的胶乳微球-SAA抗体复合物中加入适量的水，超声重悬后，依次投入第二稳定剂、第二赋形剂、第二缓冲剂、第二防腐剂，调节pH至8.0，定容至最终体积。
R2试剂含有如下终浓度的组分：胶乳微球 0.2wt%、10 g/L第二稳定剂、10 g/L第二赋形剂、50 mmol/L第二缓冲剂、5g/L第二防腐剂。第二稳定剂为海藻糖、牛血清白蛋白，海藻糖、牛血清白蛋白在R2试剂中的终浓度均为5g/L，第二赋形剂为甘露醇、丙三醇，甘露醇、丙三醇在R2试剂中的终浓度均为5g/L，第二缓冲剂为Tris-HCl（三羟甲基氨基甲烷盐酸盐），第二防腐剂为ProClin-300。
然后使用冻干装置将液态的R2进行冻干，后分装、标识。
R1试剂、R2试剂的冻干方法如下：
A）将R1、R2试剂放入冷冻设备中进行产品预冻，设定冷冻设备内部温度为-40℃，冷冻设备开始降温，经4h后试剂温度稳定后，预冻结束。
B）第一干燥段：抽真空使冷冻设备内气压降至30Pa，并使冷冻设备内温度上升，待冷冻设备内部温度升至-30℃后，开始向冷冻设备内间断地通气，在-30℃下保持运行5h，在此过程中，将冷冻腔室内的气压控制在10±10Pa，直至产品水线消失。
C）第二干燥段：第一干燥段结束后，将冷冻设备内部温度从-30℃逐步升温至5℃，在此过程中，冷冻设备内的气压控制在10±10pa，进行第二干燥段，具体是在5℃保持运行10h。
D）第三干燥段：第二干燥段结束后，冷冻设备内部温度从5℃逐步升温至20℃，进行第三干燥段，具体是在20℃保持运行10h，在此过程中，冷冻设备内的气压控制在10±10pa，第三干燥段结束后，制得固态的R1试剂、R2试剂，将冷冻设备打开，将冻干后的R1试剂与R2试剂取出，独立包装。
用纯水将本实施例冻干的R1、R2试剂复溶，复溶后，R1试剂、R2试剂的体积与冻干前的液态试剂体积一致，检测全血样本中血清样淀粉蛋白A的浓度，作为实验组；采用未冻干的液态R1试剂、R2试剂检测全血样本中血清样淀粉蛋白A的浓度，作为对照组5。
检测步骤如下：
使用冻干前液态的R1、R2试剂，在540nm波长下，通过与已知浓度（使用深圳市国赛生物技术有限公司的Aristo全自动特定蛋白分析仪，进行SAA试剂测试赋值得到）的全血样本比较，可定量检测出全血样本中血清样淀粉蛋白A的含量。使用本实施例的方法制备的R1、R2冻干试剂进行复溶后测试已知浓度的全血样本，使用普通液态的R1、R2试剂测试同样的全血样本，将测试结果进行对比。
本实施例制得的血清淀粉样蛋白A试剂盒中的R1和R2复溶后，用于检测全血样本时， R1试剂与R2试剂按照1:1的体积比混合。冻干前的液态试剂用于检测全血样本时，R1试剂与R2试剂同样按照1:1的体积比混合。
图4为本实施例冻干后复溶的试剂与冻干前的液态试剂的检测结果图。
实验结果如下图4所示，图4的横坐标为全血中血清样淀粉蛋白A的浓度（mg/L），纵坐标为反应度。
从图4可知，使用本实施例2冻干后的R1、R2试剂复溶，测试一定浓度的全血样本，检测得到的相同浓度的样本的反应度与冻干前的液态R1、R2试剂检测所得的反应度几乎一致，说明冻干复溶后的试剂与未冻干的试剂测试值无显著差别，测试结果不受冻干的影响，显著提高了试剂的稳定性。
在一些实施例中，本发明使用冻干技术将血清淀粉样蛋白A试剂盒的R1和R2试剂进行冻干，可提高试剂在寒冷地区的运输方便性和试剂反复冻融的稳定性。
在一些实施例中，本发明检测时操作方便，运输时为固体状态，不受寒冷地区温度的影响，使用时复溶为液体后不会破坏抗体的活性，检测时为液体状态，性能不受影响。保证了寒冷地区运输方面方便储存、增强了试剂反复冻融的稳定性。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种试剂盒的制备方法，其特征在于，所述试剂盒包括R1试剂、R2试剂中的至少一种，所述R2试剂含有胶乳抗体复合物，所述抗体选自可特异性结合至血清淀粉样蛋白A的抗体，所述制备方法包括对R1试剂、R2试剂中的至少一种进行冻干，冻干方法包括：
    第一干燥段：在低气压条件下，将存放有预冻至第一温度的试剂的冷冻腔室升温至第二温度，在第二温度下的保持时间为4-5h；
    第二干燥段：第一干燥段结束后，在低气压条件下，将存放有试剂的冷冻腔室从第二温度升温至第三温度，在第三温度下的保持时间为5-10h；
    第三干燥段：第二干燥段结束后，在低气压条件下，将存放有试剂的冷冻腔室从第三温度升温至第四温度，在第四温度下的保持时间为5-10h。
  2. 如权利要求1所述的制备方法，其特征在于，所述第一温度为-40℃；
    和/或，所述第二温度为-30℃；
    和/或，所述第三温度为5℃；
    和/或，所述第四温度为20℃；
    和/或，所述低气压为10±10Pa；
    和/或，第一干燥段的保温期间对存放有试剂的冷冻腔室进行通气。
  3. 如权利要求1所述的制备方法，其特征在于，所述R1试剂、R2试剂各自独立地含有稳定剂、赋形剂中的至少一种；
    和/或，所述R1试剂、R2试剂各自独立地含有如下终浓度的组分中的至少一种：1-10g/L稳定剂、1-10g/L赋形剂；
    和/或，所述稳定剂选自蔗糖、海藻糖、果糖、牛血清白蛋白、酪蛋白、明胶中的至少一种；
    和/或，所述赋形剂选自甘露醇、聚乙二醇、聚乙烯吡咯烷酮、丙三醇、乙二醇中的至少一种；
    和/或，所述聚乙二醇选自PEG6000、PEG8000、PEG10000、PEG20000中的至少一种；
    和/或，所述R1试剂、R2试剂各自独立地还含有缓冲剂、防腐剂中的至少一种；
    和/或，所述R1试剂、R2试剂各自独立地还含有如下终浓度的组分中的至少一种：5-50mmol/L缓冲剂、0.5-5g/L防腐剂；
    和/或，所述缓冲剂选自磷酸缓冲盐溶液、三羟甲基氨基甲烷盐酸盐溶液、甘氨酸、硼酸盐溶液、4-羟乙基哌嗪乙磺酸、3-(N-吗啉基)-2-羟基丙磺酸、吗啉乙磺酸中的至少一种；
    和/或，所述防腐剂选自叠氮化钠、ProClin-950、 ProClin-300、KroVin100、KroVin300、KroVin500、KroVin750、硫柳汞中的至少一种。
  4. 如权利要求1所述的制备方法，其特征在于，所述胶乳抗体复合物为偶联有抗体的胶乳微球；
    和/或，所述胶乳微球的粒径为50-300 nm；
    和/或，所述胶乳微球选自聚苯乙烯；
    和/或，所述胶乳抗体复合物是由胶乳微球与抗体按照（10-50）：1的质量比混合反应得到；
    和/或，所述R2试剂中胶乳微球的浓度为0.05-0.2wt%；
    和/或，所述R1试剂、R2试剂的pH独立地为6.0-9.0。
  5. 如权利要求1所述的制备方法，其特征在于，所述血清淀粉样蛋白A选自人源血清淀粉样蛋白A；
    和/或，所述抗体选自单克隆抗体、多克隆抗体中的至少一种；
    和/或，所述抗体选自羊抗人血清淀粉样蛋白A多克隆抗体、鼠抗人血清淀粉样蛋白A单克隆抗体、兔抗人血清淀粉样蛋白A多克隆抗体中的至少一种。
  6. 一种试剂盒，其特征在于，所述试剂盒包括R1试剂、R2试剂中的至少一种，所述R2试剂含有胶乳抗体复合物，所述抗体选自可特异性结合至血清淀粉样蛋白A的抗体，所述R1试剂、R2试剂各自独立地含有稳定剂、赋形剂中的至少一种。
  7. 如权利要求6所述的试剂盒，其特征在于，所述R1试剂、R2试剂各自独立地含有如下终浓度的组分中的至少一种：1-10g/L稳定剂、1-10g/L赋形剂；
    和/或，所述稳定剂选自蔗糖、海藻糖、果糖、牛血清白蛋白、酪蛋白、明胶中的至少一种；
    和/或，所述赋形剂选自甘露醇、聚乙二醇、聚乙烯吡咯烷酮、丙三醇、乙二醇中的至少一种；
    和/或，所述聚乙二醇选自PEG6000、PEG8000、PEG10000、PEG20000中的至少一种。
    和/或，所述R1试剂、R2试剂各自独立地还含有缓冲剂、防腐剂中的至少一种；
    和/或，所述R1试剂、R2试剂各自独立地还含有如下终浓度的组分中的至少一种：5-50mmol/L缓冲剂、0.5-5g/L防腐剂；
    和/或，所述缓冲剂选自磷酸缓冲盐溶液、三羟甲基氨基甲烷盐酸盐溶液、甘氨酸、硼酸盐溶液、4-羟乙基哌嗪乙磺酸、3-(N-吗啉基)-2-羟基丙磺酸、吗啉乙磺酸中的至少一种；
    和/或，所述防腐剂选自叠氮化钠、ProClin-950、ProClin-300、KroVin100、KroVin300、KroVin500、KroVin750、硫柳汞中的至少一种。
  8. 如权利要求6所述的试剂盒，其特征在于，所述R1试剂、R2试剂均为冻干所得的固态试剂；
    和/或，所述试剂盒是由权利要求1-5任意一项所述制备方法制得；
    和/或，所述试剂盒还包括用于独立容纳R1试剂、R2试剂的容器；
    和/或，所述试剂盒还包括使用说明书。
  9. 如权利要求6-8任意一项所述试剂盒在检测血清淀粉样蛋白A中的应用。
  10. 如权利要求9所述的应用，所述应用包括用于检测样本中血清淀粉样蛋白A的浓度；
    和/或，所述样本选自体液样本；
    和/或，所述体液样本选自全血、血清、血浆中的至少一种；
    和/或，所述样本来自于人体。
PCT/CN2021/092458 2020-12-24 2021-05-08 一种血清淀粉样蛋白 a 检测试剂盒及其制备与应用 WO2022134430A1 (zh)

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