Preparation method of dexamethasone detection reagent and dexamethasone kit

Abstract

The invention belongs to the field of dexamethasone detection, and provides a preparation method of a dexamethasone detection reagent and a dexamethasone kit.The preparation method comprises the following steps that IgG molecules and latex particles are coupled, and IgG sensitization latex is obtained; digesting and decomposing the obtained IgG sensitization latex with papain to obtain a mixture of two F (ab ') fragment sensitization latex and one F (c') fragment sensitization latex; separating the F (ab ') fragment sensitization latex from the F (c') fragment sensitization latex through centrifugation to obtain F (c ') fragment sensitization latex; the F (c ') fragment in the F (c') fragment sensitization latex is denatured to obtain denatured F (c ') fragment sensitization latex, and the dexamethasone detection reagent is prepared. Compared with the prior art, the IgG sensitization latex prepared by the invention not only can be used for producing a high-specificity dexamethasone detection reagent, but also has the advantages of low production cost and simple process, and is convenient for large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of dexamethasone detection, and particularly relates to a preparation method of a dexamethasone detection reagent and a dexamethasone kit. BACKGROUND

[0002] Dexamethasone is a glucocorticoid commonly used to treat inflammation and immune-related diseases. The determination of dexamethasone in serum is very important for evaluating the therapeutic effect and drug monitoring. At present, the detection methods of dexamethasone detection kits available for biochemical analyzers can be roughly divided into two types according to the determination principle: one is to detect the signal generated by the reaction of specific antibodies with dexamethasone, that is, the immunoassay method, for example, there are many companies selling dexamethasone detection kits using this method; the other is to use high performance liquid chromatography (HPLC) or liquid chromatography-tandem mass spectrometry (LC-MS / MS) for detection, which can provide higher sensitivity and specificity.

[0003] Although the dexamethasone detection kit obtained by the former method is simple to operate, it has the problem that it may be affected by the cross-reaction of other hormone drugs. Although the dexamethasone detection kit obtained by the latter method has high sensitivity and specificity, it has the problem of complex operation, which requires professional equipment and technology, and has high cost.

[0004] For the latter method, in order to improve the simplicity of operation and reduce the cost, the previous technology adopts solid phase extraction (SPE) and automatic sample pretreatment system to improve the sample processing throughput and reduce the operation cost. For example, there is a study on sample pretreatment of dexamethasone using solid phase extraction technology. However, this method requires specific solid phase extraction columns and organic solvents, and the operation process is relatively complex and the cost is high, which is difficult to be used for rapid detection in clinical laboratories.

[0005] At present, a rapid and simple dexamethasone detection method without high cost is needed to facilitate the wide application of clinical laboratories and on-site detection. Such a detection method should be able to provide sufficient sensitivity and specificity, while simplifying the operation steps and reducing the requirements for equipment and technical personnel. SUMMARY

[0006] To solve the problems in the prior art, the application provides a preparation method of a dexamethasone detection reagent, which can have the advantages of high reaction specificity of the dexamethasone detection reagent, low production cost and convenience for large-scale production.

[0007] To achieve the above-mentioned purpose, the specific technical scheme is as follows: One of the present application is to provide a preparation method of a dexamethasone detection reagent, comprising the following steps: The IgG molecule is coupled with latex particles to obtain IgG sensitized latex; The obtained IgG sensitized latex is digested and decomposed by papain to obtain a mixture of two F(ab') fragments and one F(c') fragment sensitized latex; The F(ab') fragment and F(c') fragment sensitized latex are separated by centrifugation to obtain F(c') fragment sensitized latex; The F(c') fragment in the F(c') fragment sensitized latex is denatured to obtain denatured F(c') fragment sensitized latex, and a dexamethasone detection reagent is prepared.

[0008] As a further scheme of the present application: the buffer range of the buffer solution is pH 4.5-7.4, the buffer concentration is 20 mmol / L, the stirring speed is 200-300 rpm, the coupling of the IgG molecule with the latex particles is carried out at a temperature of 20-30°C, the particle size of the latex particles is in the range of 60-80 nm, and the purity of the IgG molecule is greater than 98%.

[0009] As a further scheme of the present application: the digestion temperature of the IgG molecule sensitized latex is 37°C, and the digestion time of the IgG molecule sensitized latex is 3-6 hours.

[0010] The second aspect of the present application provides a dexamethasone detection kit, comprising: The detection kit comprises a box body, an enzyme-labeled plate arranged in the box body, and reagents arranged in the box body, and each well of the enzyme-labeled plate is coated with a coated antigen prepared by coupling an IgG molecule with latex particles. The reagents include: IgG sensitized latex antibody, IgG series standard solution, phosphate buffer, concentrated washing solution, chemiluminescent solution, 2M sulfuric acid termination solution.

[0011] As a further scheme of the present application: the enzyme-labeled plate is a milky white opaque polystyrene 96-well chemiluminescent enzyme-labeled plate.

[0012] As a further scheme of the present application: the concentrations of the IgG series standard solution are respectively: 0 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 2.1 ng / mL and 9.2 ng / mL.

[0013] As a further scheme of the present application: the phosphate buffer solution contains 6.34 g of NaH2PO4·2H2O and 44.3 g of Na2HPO4·12H2O per liter of aqueous solution.

[0014] As a further scheme of the present application: the concentrated washing solution contains 0.1% (volume fraction) Tween-20, pH 7.4, and 0.1 mol / L phosphate buffer solution.

[0015] As a further scheme of the present application: the chemiluminescence solution A is a tris(hydroxymethyl)aminomethane solution with a luminol content of 0.05M, a p-methylphenol content of 0.005M, and pH = 8; The B solution is a 200mL solution containing citric acid 3.5g, anhydrous Na2HPO66.32g, and a 0.75% hydrogen peroxide solution 0.64mL, the percentage being a mass percentage.

[0016] Compared with the prior art, the present application has at least the following beneficial effects: 1) The IgG sensitized latex prepared can be used to produce a dexamethasone detection reagent with high specificity, low production cost, simple process, and easy large-scale industrial production; 2) The dexamethasone detection method and kit of the present application have the characteristics of high sensitivity, simplicity, rapidness, and accuracy. Compared with the traditional colorimetric ELISA method, the sensitivity can be improved by one order of magnitude, and can play an important role in the detection of ofloxacin drug residues in animal food (such as aquatic products, animal tissues, and honey). BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a schematic diagram of the preparation method of a dexamethasone detection reagent. DETAILED DESCRIPTION

[0018] The present application will be described in detail below with reference to the specific embodiments shown in the accompanying drawings. However, these embodiments do not limit the present application, and any structural, method, or functional changes made by those of ordinary skill in the art based on these embodiments are included within the scope of protection of the present application.

[0019] EMBODIMENT

[0020] A preparation method of a dexamethasone detection reagent, comprising the following steps: S1: coupling IgG molecules with latex particles in a buffer solution at a temperature of 30-40℃ under stirring conditions, preferably at a temperature of 20-30℃, to obtain IgG sensitized latex, the buffer range of the buffer solution being pH 4.0-8.0, preferably pH 4.5-7.4, the buffer solution concentration being 15-30mmol / L, preferably 20mmol / L, the stirring speed being 100-300rpm, preferably 200-300rpm, the latex particles being physical adsorption microspheres, preferably polymethyl methacrylate, more preferably polystyrene latex, the latex particle size being in the range of 50-100nm, preferably 60-80nm, the purity of the IgG molecules being more than 90%, preferably more than 98%; S2: The resulting IgG sensitized latex is digested with papain to obtain a mixture of two F(ab') fragments and one F(c') fragment sensitized latex, the digestion temperature is 30-40°C, preferably 37°C, and the digestion time is 2-8 hours, preferably 3-6 hours; S3: The F(ab') fragments and F(c') fragments sensitized latex are separated by centrifugation to obtain F(c') fragment sensitized latex, the acceleration of the centrifugation is 5000-20000g, preferably 10000-15000g; S4: The F(c') fragments in the F(c') fragment sensitized latex are denatured to obtain denatured F(c') fragment sensitized latex, which is used as a dexamethasone detection reagent.

[0021] The above denaturation can be performed by conventional methods, not limited to thermal denaturation.

[0022] In addition, further purification can be performed after the F(ab') fragments and F(c') fragments sensitized latex are separated.

[0023] In the preparation method of the rheumatoid factor detection reagent of the present application, the amount of IgG molecules is calculated as 10 times the maximum amount of IgG molecules theoretically linked to the latex particles.

[0024] The particle size of the above latex particles is the average particle size measured using a Differential Mobility Analyzer (DMA), a transmission electron microscope, or an optical microscope, and the measurement method is a method commonly used in the art, such as a method specified by the National Institute of Technology and Science, Japan, the National Institute of Standards and Technology, USA, the International Bureau of Weights and Measures, etc.

[0025] In the above step S3, in order to further purify the F(c') fragments, repeated centrifugation and washing can be performed.

[0026] In the preparation method of the dexamethasone detection reagent, vortex shaking, ultrasonic resuspension, etc. can be used to ensure that the latex particles are fully and uniformly dispersed. The conditions for ultrasonic used in the latex dispersion can be, for example, 200-500w, 10-20min, ice water bath.

[0027] In the preparation method of the dexamethasone detection reagent, a highly specific F(c') fragment sensitized latex can be prepared by thermal denaturation, and the thermal denaturation conditions are 50°C for 60 minutes.

[0028] Experimental Example Experimental Materials Latex carrier (polystyrene latex microspheres manufactured by Sigma-Aldrich Co.); Papain (supplied by Thermo Fisher Scientific, product number CB5125); Human IgG (supplied by China Institute for Drug Control); Control 1 (Human IgG F(c') fragment, supplied by Sigma-Aldrich, product name Human IgG Fc Fragment); Control 2 (RF detection reagent obtained by coupling the F(c') fragment of Control 1 with latex particles and denaturation, manufactured by MP Biomedicals); Control 3 (RF detection reagent kit, manufactured by Bio-Rad, product name RHEUMATOID FACTOR Reagent kit, RF detection reagent kit using immunoturbidimetry, detection principle is to use anti-human rheumatoid factor antibody to react with RF for detection); Centrifuge (5424R high-speed refrigerated centrifuge provided by Eppendorf).

[0029] Preparation Example 1 S1: Coupling of IgG with latex particles 1) Weigh 8.2 mg of human IgG and dissolve it in 5 ml of 30 mM buffer to prepare solution 1; 2) Take 0.2 g of 10% (content of latex particles) polymethyl methacrylate latex with a particle size of 66 nm and dissolve it in 5 ml of 20 mM buffer to prepare solution 2; 3) Slowly add solution 2 to solution 1, water bath temperature 40°C, slowly stir (speed 300 rpm) for 8 hours; 4) After centrifugation at 20000 g for 30 minutes, wash with 10 ml of 30 mM buffer, then resuspend with pH 8.0, 200 mM glycine buffer by ultrasonic (500 w-20 min conditions) to prepare latex solution 1.

[0030] S2: Digestion of IgG sensitized latex 1) Add 2 KU / L of papain to latex solution 1 and react at 40°C for 2 hours; 2) Add 2 KU / L of papain and continue to react for 2 hours to prepare digestion solution 1.

[0031] S3: Separation of F(c') fragment sensitized latex and Fab fragment 1) Centrifuge digestion solution 1 at 4°C, 10000 g for 20 minutes, and discard the supernatant; 2) Wash twice with 10 ml of 30 mM buffer; 3) Resuspend with 30mM PBS buffer 5ml by ultrasonic (500w-20min) to prepare F(c') fragment sensitized latex emulsion 2.

[0032] S4: Heat denaturation of F(c') fragment sensitized latex emulsion 2 at 50℃ for 60min to prepare RF detection reagent of the present application.

[0033] Preparation examples 2-6, according to the data in the following table, according to the operation steps of preparation example 1, RF detection reagent was prepared: Preparation example table

[0034] Latex A: polymethyl methacrylate latex Latex B: polystyrene latex Experimental example 1 purity verification Using the latex emulsion 2 obtained in preparation example 1, F(c') fragment was separated from the microspheres, and then electrophoresis analysis of protein purity was carried out, as F(c') fragment of the present application; The supernatant obtained by centrifugation of control 1 was used as control F(c') fragment, and the supernatant was separated by Native-PAGE (non-denaturing polyacrylamide gel electrophoresis), and the gel electrophoresis separation result was shown by colloidal staining, and the image was analyzed by ImageMaster 2D software; The electrophoretic purity of the F(c') fragment of the present application and the control F(c') fragment sample with the same protein content was analyzed by Native-PAGE, and the results were shown by colloidal staining; From the experimental results, using ImageMaster 2D software to analyze the protein purity (colloidal staining) of the two samples in Native-PAGE, the protein purity of the two samples was calculated to be 97.63% (F(c') fragment of the present application) and 95.67% (control F(c') fragment) respectively in Rolling Disk mode, and the purity of the present application was better than that of the control.

[0035] Experimental examples 2-6 purity verification Using the RF detection reagent of preparation examples 2-6, according to the same operation as experimental example 1, experimental examples 2-6 were carried out, and the purity of F(c') fragment in the RF detection reagent of the present application was measured to be 98.33%, 97.87%, 97.59% and 99.36% respectively.

[0036] From the above experimental examples 1-6, it can be seen that the purity of F(c') fragment prepared by the method of the present application is high, which is better than that of the prior art control, and the separation effect of F(ab') fragment and F(c') fragment in the method of the present application is excellent.

[0037] A dexamethasone detection kit The detection kit comprises a box body, an enzyme-labeled plate coated with IgG molecules and latex particles in each well in the box body, and reagents in the box body, the reagents comprising: IgG sensitized latex antibody, IgG class series standard solution, phosphate buffer, concentrated washing solution, chemiluminescent solution, 2M sulfuric acid termination solution. The enzyme-labeled plate is a milky white opaque polystyrene 96-well chemiluminescent enzyme-labeled plate. The concentrations of the IgG class series standard solution are: 0 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 2.1 ng / mL and 9.2 ng / mL respectively. The phosphate buffer solution contains 6.34 g of NaH2PO4·2H2O and 44.3 g of Na2HPO4·12H2O in 1 L of aqueous solution. The concentrated washing solution contains 0.1% (volume fraction) Tween-20, pH 7.4, and 0.1 mol / L phosphate buffer solution. The chemiluminescent solution A contains 0.05 M of luminol and 0.005 M of p-cresol, pH 8, in tris(hydroxymethyl) aminomethane solution; the solution B contains 3.5 g of citric acid, 6.32 g of anhydrous Na2HPO6, and 0.64 mL of 0.75% hydrogen peroxide solution, the percentage being a mass percentage.

[0038] Sample pretreatment: Homogenize the tissue sample with a homogenizer, weigh 2.0±0.05 g of the homogenate into a 50 mL polystyrene centrifuge tube, add 0.6 mL of 0.1M hydrochloric acid solution, and then add 5.4 mL of anhydrous acetonitrile and mix well; Vibrate for 3 min, centrifuge at 3000 g or above for 10 min at room temperature (20-25°C); Take 1 mL of the upper organic phase into a 10 mL clean glass test tube, blow dry under nitrogen flow at 50-60°C water bath, add 1 mL of n-hexane, vortex for 30 s with a vortex instrument, then add 1 mL of phosphate buffer solution and vortex for 20 s, centrifuge at 3000 g or above for 5 min at room temperature (20-25°C), remove the upper n-hexane, and take 50 μL of the lower layer for analysis.

[0039] Detection step: add 50 μL of the IgG class series standard solution or sample solution to the micro-wells of the enzyme-labeled plate, then add 50 μL of the IgG sensitized latex antibody solution to each well, and then add 50 μL of the dexamethasone antibody solution, incubate at room temperature (20-25°C) for 15 min, pour out the liquid in the wells, add 250 μL of the washing solution to each well, wash for 5 times, pat dry, add 100 μL of the luminescent solution to each well, react for 3 min, and then measure the luminescent intensity of each well with a chemiluminescent immunoassay instrument.

[0040] The results are analyzed, the average of the luminescence intensity values of the obtained standard and sample is divided by the luminescence intensity value of the first standard (0 standard), and then multiplied by 100, taking the inhibition rate as the vertical coordinate and the logarithm of the dexamethasone concentration as the horizontal coordinate to draw a standard curve. The concentration of each sample can be read from the standard curve. Relative luminescence intensity (%) = RLU / RLU0, RLU is the luminescence intensity value measured by the standard or sample solution, and RLU0 is the luminescence intensity value of the blank (standard solution with a concentration of 0).

[0041] It should be understood that although the present specification is described in terms of examples, not every example contains only one independent technical solution, and the description of the specification is only for the sake of clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each example can also be appropriately combined to form other implementations that those skilled in the art can understand.

[0042] The above series of detailed descriptions are only specific descriptions of the feasible implementation of the present application, and are not intended to limit the protection scope of the present application. Any equivalent implementation or change made without departing from the spirit of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a dexamethasone detection reagent, characterized in that: The following steps are involved: S1: In a buffer solution at a temperature of 30-40° C. and under stirring, IgG molecules are coupled to latex particles to obtain IgG-sensitized latex, wherein the buffer solution has a pH range of 4.0-8.0, a buffer concentration of 15-30 mmol / L, and a stirring speed of 100-300 rpm. The latex particles are physical adsorption microspheres, preferably a latex of polymethyl methacrylate, butyl acrylate, polystyrene, or a copolymer thereof, the particle size of the latex particles is in the range of 50-100 nm, and the purity of the IgG molecules is above 90%; S2: digesting the obtained IgG sensitized latex with papain to obtain a mixture of two F(ab') fragments and one F(c') fragment sensitized latex at a temperature of 30-40°C for 2-8 hours; S3: separating the F(ab') fragment and the F(c') fragment sensitized latex by centrifugation to obtain the F(c') fragment sensitized latex, wherein the centrifugation acceleration is 5000-20000g; S4: denaturing the F(c') fragment in the F(c') fragment-sensitized latex to obtain a denatured F(c') fragment-sensitized latex, and preparing a dexamethasone detection reagent.

2. The method for preparing a dexamethasone detection reagent according to claim 1, wherein: In step S1, the buffer range of the buffer is pH 4.5-7.4, the buffer concentration is 20 mmol / L, the stirring speed is 200-300 rpm, the step of coupling the IgG molecules with the latex particles is carried out at a temperature of 20-30°C, the particle size of the latex particles is in the range of 60-80 nm, and the purity of the IgG molecules is greater than 98%.

3. The method for preparing a dexamethasone detection reagent according to claim 1, wherein: In step S2, the digestion temperature of the IgG molecule sensitized latex is 37° C., and the digestion time of the IgG molecule sensitized latex is 3-6 hours.

4. The method for preparing a dexamethasone detection reagent according to claim 1, wherein: In step S3, the centrifugal acceleration is 10000-15000 g.

5. A dexamethasone detection kit, comprising the IgG sensitized latex obtained by the preparation method of a dexamethasone detection reagent according to any one of claims 1 to 4, characterized in that: The detection kit comprises a box body, an ELISA plate and reagents arranged in the box body, wherein each well of the ELISA plate is coated with a coating antigen prepared by coupling IgG molecules with latex particles; The reagents include: IgG sensitized latex antibody, IgG series standard solution, phosphate buffer, concentrated washing solution, chemiluminescent solution, and 2M sulfuric acid stop solution.

6. A dexamethasone detection kit according to claim 5, characterized in that: The ELISA plate is a milky white opaque polystyrene 96-well chemiluminescence ELISA plate.

7. A dexamethasone detection kit according to claim 5, characterized in that: The concentrations of the IgG series standard solutions are: 0 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 2.1 ng / mL and 9.2 ng / mL respectively.

8. A dexamethasone detection kit according to claim 5, characterized in that: The phosphate buffer solution contains 6.34 g of NaH2PO4·2H2O and 44.3 g of Na2HPO4·12H2O per liter.

9. A dexamethasone detection kit according to claim 5, characterized in that: The concentrated washing solution contains 0.1% Tween-20 by volume, has a pH of 7.4, and a concentration of 0.1 mol / L phosphate buffer.

10. A dexamethasone detection kit according to claim 5, characterized in that: The chemiluminescent liquid A is a tris(hydroxymethyl)aminomethane solution with a luminol content of 0.05M, a p-cresol content of 0.005M, and a pH of 8; Solution B is a 200 mL solution containing 3.5 g of citric acid, 6.32 g of anhydrous Na2HPO6, and 0.64 mL of 0.75% urea hydrogen peroxide. The percentages are by mass.

Images