An immunoglobulin G4 subtype detection kit and preparation method thereof

Abstract

The present invention discloses an immunoglobulin G4 subtype detection kit and a preparation method thereof. The detection kit comprises a first reagent, a sample pretreatment filter membrane, a second reagent and a third reagent. The sample pretreatment filter membrane consists of a first filter layer, a second filter layer and a third filter layer. The first filter layer is a polyimide fiber filter membrane; the second filter layer is a polyvinyl alcohol fiber filter membrane coupled with a hydroxy acid flexible arm and monoesterified citric acid; the third filter layer is a polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm and an Fc fragment tag. The present invention partially removes interferences such as chyle, bilirubin, hemoglobin, rheumatoid factor and the like in the sample to be detected by arranging a sample pretreatment filter membrane in the kit, thereby improving the anti-interference performance of the detection kit and increasing the detection accuracy and stability.

Description

Technical Field

[0001] The present invention relates to the field of clinical detection technology, and in particular to an immunoglobulin G4 subtype detection kit and a preparation method thereof. Background Art

[0002] Immunoglobulin G4 subtype (IgG4) is a globulin synthesized and secreted by plasma cells in lymphoid tissues. Its elevated level in the blood indicates that the body has immune diseases or antibody production disorders. It is an indicator of chronic, systemic, and autoinflammatory diseases. Such diseases mainly affect organs such as the pancreas, liver, gallbladder, and kidneys, such as interstitial pneumonia, systemic lupus erythematosus, or multiple myeloma. If its concentration decreases, it indicates the occurrence of type 1 diabetes, juvenile diabetes, nephrotic syndrome, and AIDS. Therefore, the detection of immunoglobulin G4 subtypes can provide an important basis for clinical diagnosis.

[0003] At present, the main methods for determining serum immunoglobulin G4 subtypes are enzyme-linked immunosorbent assay, one-way immunodiffusion method, and immunoturbidimetry. The enzyme-linked immunosorbent assay requires manual operation, and the detection time is more than 1 hour, which is extremely difficult to be widely used in clinical practice; the one-way immunodiffusion method is cumbersome and complicated to operate, and the detection time is longer, generally requiring more than 5 hours, and the test results cannot be obtained in time; the immunoturbidimetry method is simple to operate and takes a short time, but it is easily interfered by substances in the sample to be tested, such as chyle, bilirubin, etc.

[0004] Therefore, it is necessary to develop an immunoglobulin G4 subtype detection kit and a preparation method thereof that can eliminate the interference of chyle, bilirubin, hemoglobin, and rheumatoid factor. Summary of the invention

[0005] In view of this, the present invention provides an immunoglobulin G4 subtype detection kit and a preparation method thereof. By setting a sample pretreatment filter membrane in the kit, interfering substances such as chyle, bilirubin, hemoglobin, rheumatoid factor, etc. in the sample to be tested are partially removed, thereby improving the anti-interference performance of the detection kit, thereby increasing the detection accuracy and stability.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an immunoglobulin G4 subtype detection kit, the immunoglobulin G4 subtype detection kit comprising a first reagent, a sample pretreatment filter membrane, a second reagent and a third reagent;

[0008] The first reagent includes a first antiseptic, a first pH regulator, a reducing agent, a first protein stabilizer, and tris(hydroxymethyl)aminomethane;

[0009] The first preservative is sodium azide, with a mass concentration of 0.05%-0.11%; the first pH regulator is phosphate buffer, with a concentration of 50-70mmol / L; the reducing agent is one of cysteine, vitamin C, and reduced glutathione, with a concentration of 5-28mmol / L; the first protein stabilizer is mannitol, with a mass concentration of 0.9%-1.7%; the concentration of tris(hydroxymethyl)aminomethane is 30-90mmol / L;

[0010] The sample pretreatment filter membrane consists of a first filter layer, a second filter layer, and a third filter layer;

[0011] The first filter layer is a polyimide fiber filter membrane with a thickness of 5-20 microns; the second filter layer is a polyvinyl alcohol fiber filter membrane with a thickness of 10-30 microns, coupled with a hydroxy acid flexible arm, and the free end of the hydroxy acid flexible arm is coupled with monoesterified citric acid; the third filter layer is a polypropylene alcohol fiber filter membrane with a thickness of 5-10 microns, coupled with a short-chain dibasic acid flexible arm, and the free end of the short-chain dibasic acid flexible arm is coupled with an Fc fragment tag, and the amino acid sequence of the Fc fragment tag is gly-thr-ser-glu-lys-pro-leu-cys-cys-lys-ile-ile-leu-val-leu-glu-Arg; the first filter layer, the second filter layer, and the third filter layer of the sample pretreatment filter membrane are pressed in sequence, and the first filter layer first contacts the sample to be processed;

[0012] The second reagent includes a second preservative, a second pH buffer, and a desorbent;

[0013] The second preservative is Proclin300, with a mass concentration of 0.1%-0.3%; the second pH buffer is acetate buffer, with a concentration of 30-60mmol / L; the desorbent is 1,4-butanediol, with a mass concentration of 0.5%-1.1%;

[0014] The third reagent includes a third preservative, a third pH adjuster, latex microspheres loaded with immunoglobulin G4 subtype antibodies, and a second protein stabilizer;

[0015] The third preservative is sodium azide with a mass concentration of 0.1%-0.19%; the third pH regulator is sodium glycine buffer with a concentration of 70-120 mmol / L; the mass concentration of the latex microspheres loaded with immunoglobulin G4 subtype antibodies is 9-16 g / L; the second protein stabilizer is arginine and xylooligosaccharide, the mass concentration of arginine is 0.3%-0.5%, and the mass concentration of xylooligosaccharide is 0.9%-1.2%;

[0016] The pH of the first reagent is 6.1-6.3, the pH of the second reagent is 7.15-7.55, and the pH of the third reagent is 7.3-7.7, which are adjusted using sodium hydroxide or hydrochloric acid.

[0017] Preferably, the pore size of the sample pretreatment filter membrane is 4.5-16 microns.

[0018] Preferably, the hydroxy acid of the hydroxy acid flexible arm includes one of γ-hydroxybutyric acid and δ-hydroxyvaleric acid, the short-chain dibasic acid of the short-chain dibasic acid flexible arm includes one of succinic acid, glutaric acid, and adipic acid, and the short-chain dibasic acid is a dibasic acid with carboxyl groups at both ends of the molecule.

[0019] Preferably, the xylo-oligosaccharide of the second protein stabilizer comprises xylo-oligosaccharide containing 5-8 xylose residues.

[0020] Preferably, the latex microspheres loaded with immunoglobulin G4 subtype antibodies include carboxyl polystyrene latex microspheres.

[0021] In a second aspect, the present invention provides a method for preparing the immunoglobulin G4 subtype detection kit as described above, the preparation method comprising the following steps:

[0022] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of sodium azide is 0.05%-0.11%, the concentration of phosphate buffer is 50-70mmol / L, the concentration of cysteine, or vitamin C, or reduced glutathione is 5-28mmol / L, the mass concentration of mannitol is 0.9%-1.7%, and the concentration of tris(hydroxymethyl)aminomethane is 30-90mmol / L, and they are dissolved in a corresponding volume of deionized water, and the pH value is adjusted to 6.1-6.3 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent;

[0023] Prepare a sample pretreatment filter membrane: use an electrostatic spinning device to process polyimide into the polyimide fiber filter membrane, the nozzle of the electrostatic spinning device is connected to the positive electrode, the voltage between the positive electrode and the negative electrode of the electrostatic spinning device is 60-70kV, the diameter of the polyimide fiber is 2-5 microns, and the thickness of the polyimide fiber filter membrane is 5-20 microns, to obtain a first filter layer;

[0024] Using an electrospinning device to process polyvinyl alcohol into the polyvinyl alcohol fiber filter membrane, the nozzle of the electrospinning device is connected to the positive electrode, the voltage between the positive and negative electrodes of the electrospinning device is 65-80 kV, the diameter of the polyvinyl alcohol fiber is 0.5-1 micron, the thickness of the polyvinyl alcohol fiber filter membrane is 10-30 microns, the polyvinyl alcohol fiber filter membrane is mixed with a hydroxy acid solution, so that the carboxyl group of the hydroxy acid reacts with the hydroxyl group on the polyvinyl alcohol fiber filter membrane to form an ester bond, after the reaction is completed, filtering and washing to obtain a polyvinyl alcohol fiber filter membrane coupled with a hydroxy acid flexible arm, immersing the polyvinyl alcohol fiber filter membrane coupled with a hydroxy acid flexible arm in a phosphate buffer solution, adding citric acid, so that the hydroxyl group of the hydroxy acid flexible arm forms an ester bond with the carboxyl group of the citric acid, and obtaining the second filter layer;

[0025] The polypropylene alcohol is processed into the polypropylene alcohol fiber filter membrane by using an electrospinning device, the nozzle of the electrospinning device is connected to the positive electrode, the voltage between the positive electrode and the negative electrode of the electrospinning device is 75-90 kV, the diameter of the polypropylene alcohol fiber is 0.8-3 microns, the thickness of the polypropylene alcohol fiber filter membrane is 5-10 microns, the polypropylene alcohol fiber filter membrane is mixed with a short-chain dibasic acid solution, so that the carboxyl group at one end of the short-chain dibasic acid reacts with the hydroxyl group on the polypropylene alcohol fiber filter membrane to form an ester bond, after the reaction is completed, filtering and washing are performed to obtain a polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm, the polypropylene alcohol fiber filter membrane coupled with the short-chain dibasic acid flexible arm is immersed in a phosphate buffer, and an activator NHS-EDC is added, stirred evenly, a certain amount of morpholine is added, the Fc fragment tag is added, stirred evenly and incubated for a certain period of time, so that the other end of the carboxyl group of the short-chain dibasic acid flexible arm is connected to the Fc fragment tag, and the third filter layer is obtained;

[0026] The first filter layer, the second filter layer and the third filter layer are stacked in sequence, and an ultrasonic welding device is used to bond adjacent fibers under the action of ultrasonic vibration to obtain the sample pretreatment filter membrane;

[0027] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of Proclin 300 is 0.1%-0.3%, the concentration of acetate buffer is 30-60mmol / L, and the mass concentration of 1,4-butanediol is 0.5%-1.1%, dissolve them in the corresponding volume of deionized water, and adjust the pH value to 7.15-7.55 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent;

[0028] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of sodium azide is 0.1%-0.19%, the concentration of sodium glycine buffer is 70-120 mmol / L, the mass concentration of latex microspheres loaded with immunoglobulin G4 subtype antibodies is 9-16 g / L, the mass concentration of arginine is 0.3%-0.5%, and the mass concentration of xylooligosaccharide is 0.9%-1.2%. Dissolve them in the corresponding volume of deionized water, adjust the pH value to 7.3-7.7 with hydrochloric acid or sodium hydroxide solution to obtain the third reagent.

[0029] Preferably, the concentration of the hydroxy acid solution for preparing the second filter layer is 1.2-3.5 g / L, the concentration of the phosphate buffer for preparing the second filter layer is 50-90 mmol / L, and the concentration of the citric acid in the phosphate buffer is 8-16 g / L.

[0030] Preferably, the concentration of the phosphate buffer for preparing the third filter layer is 80-110 mmol / L, the concentration of NHS-EDC is 0.08-0.22 g / L, the concentration of the Fc fragment tag is 830-980 mg / L, and the concentration of the short-chain dibasic acid is 4.2-6.8 g / L.

[0031] Preferably, the latex microspheres have a diameter of 100-150 nm.

[0032] Preferably, the latex microspheres loaded with immunoglobulin G4 subtype antibodies are prepared by adding the latex microspheres to a buffer solution of 3-5.2 g / L MES·H2O, adjusting the pH to 6.9-7.1 using sodium hydroxide, and mixing evenly, adding 1.9-2.7 g / L boric acid and 0.5-1.4 g / L disodium hydrogen phosphate, adjusting the pH to 7.2-7.6 using sodium hydroxide, and mixing at 30-35° C. to obtain a latex microsphere suspension; then adding the immunoglobulin G4 subtype antibody to the latex microsphere suspension, wherein the concentration of the immunoglobulin G4 subtype antibody in the reaction system is 960-1270 mg / L, and then adding 0.1-0.18 g / L EDC·HCl solution and A 0.43-0.81 g / L NHS solution is stirred at 35-38°C for coupling reaction, and the reaction time is 5-7 hours; a blocking solution consisting of 110-150 mL / L Tween and 0.9-1.3 g / L BSA is added, and the mixture is stirred and dispersed at 26-30°C for 5-9 hours for blocking reaction; proclin300 is added to a concentration of 0.25-0.45 mL / L in the system, and the pH is adjusted to 6.8-7.3 using a sodium hydroxide solution or hydrochloric acid, and the mixture is stirred and soaked for 14-16 hours to obtain the latex microspheres loaded with immunoglobulin G4 subtype antibodies.

[0033] Compared with the prior art, the present invention has the following beneficial technical effects: ① A sample pretreatment filter membrane with three filter layers is arranged in the test kit, the first filter layer adsorbs and removes the chyle in the sample to be tested, and the chyle adsorbed to the first filter layer further dissolves and removes the bilirubin in the sample, the second filter layer can chelate the ferrous ions in the hemoglobin, thereby partially removing the hemoglobin, and the third filter layer is coupled with an Fc fragment label, which can specifically bind to the rheumatoid factor in the sample to be tested, thereby improving the anti-interference performance of the detection kit, thereby increasing the detection accuracy and stability; ② Due to the elimination of interference, the immunoglobulin G4 subtype and its corresponding antibody coupled to the latex microsphere react faster, saving more than 50% of the sample measurement time. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is described in detail below in conjunction with embodiments.

[0035] It should be noted that the following embodiments and features in the embodiments may be combined with each other in the absence of conflict; and, based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without making any creative work are within the scope of protection of the present disclosure.

[0036] It should be noted that various aspects of the embodiments within the scope of the appended claims are described below. It should be apparent that the aspects described herein may be embodied in a wide variety of forms, and any specific structures and / or functions described herein are merely illustrative. Based on the present disclosure, it should be understood by those skilled in the art that an aspect described herein may be implemented independently of any other aspect, and two or more of these aspects may be combined in various ways. For example, any number of aspects described herein may be used to implement the device and / or practice method. It should be noted that the serial numbers themselves, such as "first", "second", "third", etc., in the present application document only distinguish the objects described and do not have any order or technical meaning.

[0037] The present invention provides an immunoglobulin G4 subtype detection kit and a preparation method thereof, which partially removes interferences such as chyle, bilirubin, hemoglobin, rheumatoid factor, etc. in the sample to be tested by setting a sample pretreatment filter membrane in the kit, thereby improving the anti-interference performance of the detection kit and increasing the detection accuracy and stability.

[0038] The present invention provides an immunoglobulin G4 subtype detection kit and a preparation method thereof, wherein the detection kit comprises a first reagent, a sample pretreatment filter membrane, a second reagent and a third reagent;

[0039] The first reagent includes a first antiseptic, a first pH adjuster, a reducing agent, a first protein stabilizer, and tris(hydroxymethyl)aminomethane;

[0040] The first preservative is sodium azide, with a mass concentration of 0.05%-0.11%; the first pH adjuster is phosphate buffer, with a concentration of 50-70mmol / L; the reducing agent is one of cysteine, vitamin C, and reduced glutathione, with a concentration of 5-28mmol / L; the first protein stabilizer is mannitol, with a mass concentration of 0.9%-1.7%; the concentration of tris(hydroxymethyl)aminomethane is 30-90mmol / L;

[0041] The sample pretreatment filter membrane consists of a first filter layer, a second filter layer, and a third filter layer;

[0042] The first filter layer is a polyimide fiber filter membrane with a thickness of 5-20 microns; the second filter layer is a polyvinyl alcohol fiber filter membrane with a thickness of 10-30 microns, coupled with a hydroxy acid flexible arm, and the free end of the hydroxy acid flexible arm is coupled with monoesterified citric acid; the third filter layer is a polypropylene alcohol fiber filter membrane with a thickness of 5-10 microns, coupled with a short-chain dibasic acid flexible arm, and the free end of the short-chain dibasic acid flexible arm is coupled with an Fc fragment tag, and the amino acid sequence of the Fc fragment tag is gly-thr-ser-glu-lys-pro-leu-cys-cys-lys-ile-ile-leu-val-leu-glu-Arg; the first filter layer, the second filter layer, and the third filter layer of the sample pretreatment filter membrane are pressed in sequence, and the first filter layer first contacts the sample to be processed;

[0043] The second reagent includes a second preservative, a second pH buffer, and a desorbent;

[0044] The second preservative is Proclin300, with a mass concentration of 0.1%-0.3%; the second pH buffer is acetate buffer, with a concentration of 30-60mmol / L; the desorbent is 1,4-butanediol, with a mass concentration of 0.5%-1.1%;

[0045] The third reagent includes a third preservative, a third pH adjuster, latex microspheres loaded with immunoglobulin G4 subtype antibodies, and a second protein stabilizer;

[0046] The third preservative is sodium azide with a mass concentration of 0.1%-0.19%; the third pH adjuster is sodium glycine buffer with a concentration of 70-120mmol / L; the mass concentration of latex microspheres loaded with immunoglobulin G4 subtype antibodies is 9-16g / L; the second protein stabilizer is arginine and xylooligosaccharide, with a mass concentration of arginine of 0.3%-0.5% and a mass concentration of xylooligosaccharide of 0.9%-1.2%;

[0047] The pH of the first reagent is 6.1-6.3, the pH of the second reagent is 7.15-7.55, and the pH of the third reagent is 7.3-7.7, which are adjusted using sodium hydroxide or hydrochloric acid.

[0048] It should be noted that the purpose of setting the reducing agent in the first reagent is to keep the iron element in the sample to be tested in the state of ferrous ions, prevent it from being oxidized into ferric ions, increase the removal rate of hemoglobin in the sample to be tested by the second filter layer, and thereby reduce the impact of hemoglobin in the sample to be tested on the test results.

[0049] It should be noted that the function of the first filter layer of the sample pretreatment filter membrane is to adsorb and remove chylomicrons in the sample to be tested. The adsorbed chylomicrons form a chylomicron film on the surface of the polyimide fiber of the first filter layer, and the chylomicron film further dissolves the bilirubin in the sample to be tested, thereby achieving the purpose of removing bilirubin; the function of the second filter layer is to remove hemoglobin in the sample to be tested; the function of the third filter layer is to remove rheumatoid factor in the sample to be tested.

[0050] It should be noted that the purpose of setting the second reagent is to adjust the pH of the detection system to the optimal value for the reaction of IgG4 with its antibody, and to use the desorbent polyol therein to desorb the IgG4 non-specifically adsorbed onto the sample pretreatment filter membrane.

[0051] It should be noted that the function of the Fc fragment tag is to specifically affinity adsorb the interfering substance rheumatoid factor in the sample to be tested, remove it, and reduce its impact on the test results.

[0052] It should be noted that the first reagent adjusts the pH of the detection system to below 6.5, so that IgG4 has a positive charge and has the same electrostatic charge as the positively charged sample pretreatment filter membrane. The principle of like charges repel each other is used to avoid nonspecific adsorption of IgG4 by the filter membrane.

[0053] More preferably, the concentration of sodium hydroxide or hydrochloric acid used to adjust the pH is 2-5 mol / L.

[0054] Preferably, the pore size of the sample pretreatment filter membrane is 4.5-16 microns.

[0055] Preferably, the hydroxy acid of the hydroxy acid flexible arm includes one of γ-hydroxybutyric acid and δ-hydroxyvaleric acid, and the short-chain dibasic acid of the short-chain dibasic acid flexible arm includes one of succinic acid, glutaric acid, and adipic acid, and the short-chain dibasic acid is a dibasic acid with carboxyl groups at both ends of the molecule.

[0056] More preferably, the short-chain dibasic acid is a dibasic acid having carboxyl groups at both ends of the molecule.

[0057] Preferably, the xylo-oligosaccharide of the second protein stabilizer comprises xylo-oligosaccharide containing 5-8 xylose residues.

[0058] It should be noted that the role of xylooligosaccharide is to maintain the stability of the immunoglobulin G4 subtype antibody loaded on the latex microspheres, improve its storage stability, and extend the shelf life.

[0059] Preferably, the latex microspheres loaded with immunoglobulin G4 subtype antibodies include carboxyl polystyrene latex microspheres.

[0060] The present invention also provides a method for preparing the immunoglobulin G4 subtype detection kit described above, the preparation method comprising the following steps:

[0061] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of sodium azide in the first reagent is 0.05%-0.11%, the concentration of phosphate buffer is 50-70mmol / L, the concentration of cysteine, or vitamin C, or reduced glutathione is 5-28mmol / L, the mass concentration of mannitol is 0.9%-1.7%, and the concentration of tris(hydroxymethyl)aminomethane is 30-90mmol / L, which are dissolved in the corresponding volume of deionized water, and the pH value is adjusted to 6.1-6.3 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent;

[0062] Prepare sample pretreatment filter membrane: use electrostatic spinning equipment to process polyimide into polyimide fiber filter membrane, the nozzle of the electrostatic spinning equipment used is connected to the positive electrode, the voltage between the positive electrode and the negative electrode of the electrostatic spinning equipment is 60-70kV, the diameter of the polyimide fiber is 2-5 microns, and the thickness of the polyimide fiber filter membrane is 5-20 microns, to obtain the first filter layer;

[0063] Using an electrospinning device to process polyvinyl alcohol into a polyvinyl alcohol fiber filter membrane, the nozzle of the electrospinning device is connected to the positive electrode, the voltage between the positive and negative electrodes of the electrospinning device is 65-80 kV, the diameter of the polyvinyl alcohol fiber is 0.5-1 micron, the thickness of the polyvinyl alcohol fiber filter membrane is 10-30 microns, the polyvinyl alcohol fiber filter membrane is mixed with a hydroxy acid solution, the carboxyl group of the hydroxy acid reacts with the hydroxyl group on the polyvinyl alcohol fiber filter membrane to form an ester bond, after the reaction is completed, filtering and washing to obtain a polyvinyl alcohol fiber filter membrane coupled with a hydroxy acid flexible arm, immersing the polyvinyl alcohol fiber filter membrane coupled with a hydroxy acid flexible arm in a phosphate buffer solution, adding citric acid, so that the hydroxyl group of the hydroxy acid flexible arm and the carboxyl group of the citric acid form an ester bond, to obtain the second filter layer;

[0064] The polypropylene alcohol is processed into a polypropylene alcohol fiber filter membrane by using an electrospinning device, the nozzle of the electrospinning device is connected to the positive electrode, the voltage between the positive electrode and the negative electrode of the electrospinning device is 75-90 kV, the diameter of the polypropylene alcohol fiber is 0.8-3 microns, the thickness of the polypropylene alcohol fiber filter membrane is 5-10 microns, the polypropylene alcohol fiber filter membrane is mixed with a short-chain dibasic acid solution, and one end carboxyl group of the short-chain dibasic acid reacts with a hydroxyl group on the polypropylene alcohol fiber filter membrane to form an ester bond, after the reaction is completed, filtering and washing are performed to obtain a polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm, the polypropylene alcohol fiber filter membrane coupled with the short-chain dibasic acid flexible arm is immersed in a phosphate buffer, and an activator NHS-EDC is added, stirred evenly, a certain amount of morpholine is added, an Fc fragment tag is added, stirred evenly, and incubated for a certain period of time, so that the other end carboxyl group of the short-chain dibasic acid flexible arm is connected to the Fc fragment tag, and a third filter layer is obtained;

[0065] The first filter layer, the second filter layer and the third filter layer are stacked in sequence, and an ultrasonic welding device is used to bond adjacent fibers under the action of ultrasonic vibration to obtain the sample pretreatment filter membrane;

[0066] Preparation of the second reagent: prepare the volume of the second reagent as required, calculate and weigh the ingredients according to the following ratio: Proclin 300 mass concentration 0.1%-0.3%, acetate buffer concentration 30-60mmol / L, 1,4-butanediol mass concentration 0.5%-1.1%, dissolve in the corresponding volume of deionized water, adjust the pH value to 7.15-7.55 with hydrochloric acid or sodium hydroxide solution, and obtain the second reagent;

[0067] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: sodium azide mass concentration 0.1%-0.19%, sodium glycine buffer concentration 70-120 mmol / L, latex microspheres loaded with immunoglobulin G4 subtype antibody mass concentration 9-16 g / L, arginine mass concentration 0.3%-0.5%, xylooligosaccharide mass concentration 0.9%-1.2%, dissolve in the corresponding volume of deionized water, adjust the pH to 7.3-7.7 with hydrochloric acid or sodium hydroxide solution to obtain the third reagent.

[0068] More preferably, the molecular weight of polyimide is 60,000 to 100,000 g / mol, the molecular weight of polyvinyl alcohol is 90,000 to 140,000 g / mol, and the molecular weight of polypropylene alcohol is 10,000 to 50,000 g / mol.

[0069] More preferably, the immunoglobulin G4 subtype antibody comprises a rabbit anti-human immunoglobulin G4 subtype monoclonal antibody.

[0070] It should be noted that the electrospinning equipment is an electrospinning equipment in the prior art.

[0071] It should be noted that the ultrasonic welding device is an ultrasonic welding device in the prior art.

[0072] It should be noted that the method for preparing immunoglobulin G4 subtype monoclonal antibody is a preparation method in the prior art.

[0073] It should be noted that the preparation methods of Fc fragment tags include chemical synthesis, biological enzymatic reaction and other methods.

[0074] Preferably, the concentration of the hydroxy acid solution for preparing the second filter layer is 1.2-3.5 g / L, the concentration of the phosphate buffer for preparing the second filter layer is 50-90 mmol / L, and the concentration of citric acid in the phosphate buffer is 8-16 g / L.

[0075] Preferably, the concentration of phosphate buffer for preparing the third filter layer is 80-110 mmol / L, the concentration of NHS-EDC is 0.08-0.22 g / L, the concentration of Fc fragment tag is 830-980 mg / L, and the concentration of short-chain dibasic acid is 4.2-6.8 g / L.

[0076] Preferably, the latex microspheres have a diameter of 100-150 nm.

[0077] Preferably, latex microspheres loaded with immunoglobulin G4 subtype antibodies are prepared, the latex microspheres are added to a buffer solution of MES·H2O3-5.2 g / L, the pH of which is adjusted to 6.9-7.1 using sodium hydroxide, and after mixing evenly, 1.9-2.7 g / L of boric acid and 0.5-1.4 g / L of disodium hydrogen phosphate are added, the pH of which is adjusted to 7.2-7.6 using sodium hydroxide, and the mixture is mixed at 30-35° C. to obtain a latex microsphere suspension; then, the immunoglobulin G4 subtype antibody is added to the latex microsphere suspension, the concentration of the immunoglobulin G4 subtype antibody in the reaction system is 960-1270 mg / L, and 0.1- 0.18 g / L EDC·HCl solution and 0.43-0.81 g / L NHS solution are stirred at 35-38°C for coupling reaction, and the reaction time is 5-7 hours; a blocking solution composed of 110-150 mL / L Tween and 0.9-1.3 g / L BSA is added, and the mixture is stirred and dispersed at 26-30°C for 5-9 hours for blocking reaction; proclin300 is added to make its concentration in the system reach 0.25-0.45 mL / L, and the pH is adjusted to 6.8-7.3 with sodium hydroxide solution or hydrochloric acid, and the mixture is stirred and soaked for 14-16 hours to obtain latex microspheres loaded with immunoglobulin G4 subtype antibodies.

[0078] In one embodiment, the application process of the immunoglobulin G4 subtype detection kit is as follows:

[0079] (1) Take fresh blood and prepare a sample liquid;

[0080] (2) Add 4-8 μl of the sample liquid to 180-360 μl of the first reagent, mix well, and incubate at 36-39° C. for 5-8 minutes to obtain a mixed solution of the sample to be tested and the first reagent;

[0081] (3) Using a sample pretreatment filter membrane to filter the sample to be tested and the first reagent mixed solution obtained in the previous step, the mixed solution is passed through the first filter layer, the second filter layer and the third filter layer in sequence, and the time for the mixed solution to pass through the sample pretreatment filter membrane is controlled between 30-90 seconds to obtain a filtrate, and the sample pretreatment filter membrane is washed with 60-120 microliters of the second reagent, and the washing liquid is combined with the filtrate to obtain a mixed solution to be tested after the filter membrane treatment;

[0082] (4) Add 80-160 μl of the third reagent to the test mixture after the filter membrane treatment obtained in the previous step, incubate at 36-38° C. for 10-15 seconds, read the absorbance A1 at a wavelength of 570 nm, and read the absorbance A2 after 2.5-3 minutes;

[0083] (5) Calculate △A=A2-A1, and calculate the concentration of immunoglobulin G4 subtype in the sample to be tested based on the working curve.

[0084] In order to further understand the present invention, the technical solution of the present invention is described below in conjunction with embodiments, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0085] Example 1

[0086] The immunoglobulin G4 subtype detection kit of the present invention is prepared according to the following steps and conditions:

[0087] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: the sodium azide mass concentration of the first reagent is 0.05%, the phosphate buffer concentration is 62 mmol / L, the cysteine ​​concentration is 5 mmol / L, the mannitol mass concentration is 1.7%, and the tris(hydroxymethyl)aminomethane concentration is 55 mmol / L, and they are dissolved in a corresponding volume of deionized water, and the pH value is adjusted to 6.1 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent;

[0088] Preparation of sample pretreatment filter membrane: using the electrospinning equipment of the prior art to process polyimide into a polyimide fiber filter membrane, the molecular weight of the polyimide is 60000-70000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 70 kV, the diameter of the obtained polyimide fiber is 2 microns, the thickness of the polyimide fiber filter membrane is 5 microns, and the first filter layer is obtained;

[0089] The electrospinning equipment of the prior art is used to process polyvinyl alcohol into a polyvinyl alcohol fiber filter membrane, wherein the molecular weight of the polyvinyl alcohol is 110000-140000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 65 kV, the diameter of the polyvinyl alcohol fiber is 1.0 micron, the thickness of the polyvinyl alcohol fiber filter membrane is 10 microns, the polyvinyl alcohol fiber filter membrane is mixed with a hydroxy acid solution, the hydroxy acid is γ-hydroxybutyric acid, the concentration is 2.3 g / L, the mass ratio of the hydroxy acid solution to the polyvinyl alcohol fiber filter membrane is 5:1, and the reaction temperature is 400 ° C. The temperature is 100°C, the reaction time is 6 hours, so that the carboxyl group of the hydroxy acid reacts with the hydroxyl group on the polyvinyl alcohol fiber filter membrane to form an ester bond, after the reaction is completed, it is filtered and washed to obtain a polyvinyl alcohol fiber filter membrane coupled with a hydroxyl acid flexible arm, and the polyvinyl alcohol fiber filter membrane coupled with a hydroxyl acid flexible arm is immersed in a phosphate buffer solution with a concentration of 50 mmol / L, and citric acid is added with a citric acid concentration of 12 g / L, the reaction temperature is 65°C, and the reaction time is 9 hours, so that the hydroxyl group of the hydroxyl acid flexible arm forms an ester bond with the carboxyl group of the citric acid, thereby obtaining the second filter layer;

[0090] The electrospinning equipment of the prior art is used to process polypropylene alcohol into a polypropylene alcohol fiber filter membrane. The molecular weight of polypropylene alcohol is 10000-30000 g / mol. The nozzle of the electrospinning equipment is connected to the positive electrode. The voltage between the positive and negative electrodes is 90 kV. The diameter of the polypropylene alcohol fiber is 0.8 microns. The thickness of the polypropylene alcohol fiber filter membrane is 5 microns. The polypropylene alcohol fiber filter membrane is mixed with a short-chain dibasic acid solution. The short-chain dibasic acid is succinic acid. The concentration of the short-chain dibasic acid is 4.2 g / L. The carboxyl group at one end of the short-chain dibasic acid reacts with the hydroxyl group on the polypropylene alcohol fiber filter membrane to form an ester bond. After completion, the membrane was filtered and washed to obtain a polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm, and the polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm was immersed in a phosphate buffer solution, the concentration of the phosphate buffer solution was 110 mmol / L, and an activator NHS-EDC was added, the concentration of which was 0.08 g / L, and the membrane was stirred evenly, and a certain amount of morpholine was added, and an Fc fragment tag was added, and the concentration of the Fc fragment tag was 830 mg / L. After stirring evenly, the membrane was incubated at a temperature of 39° C. for 20 hours, so that the other end carboxyl group of the short-chain dibasic acid flexible arm was connected to the Fc fragment tag, and the third filter layer was obtained;

[0091] The first filter layer, the second filter layer and the third filter layer are stacked in sequence, and the adjacent fibers are bonded together under the action of ultrasonic vibration using an ultrasonic welding device of the prior art to obtain the sample pretreatment filter membrane with a pore size of 10-16 microns;

[0092] Preparation of the second reagent: prepare the volume of the second reagent as required, calculate and weigh the ingredients according to the following ratio: Proclin 300 with a mass concentration of 0.1%, acetate buffer with a concentration of 30 mmol / L, and 1,4-butanediol with a mass concentration of 0.8%, dissolve them in a corresponding volume of deionized water, and adjust the pH value to 7.15 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent;

[0093] Preparation of the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: sodium azide mass concentration 0.19%, sodium glycine buffer concentration 70 mmol / L, latex microspheres loaded with immunoglobulin G4 subtype antibody mass concentration 12 g / L, arginine mass concentration 0.3%, xylooligosaccharide mass concentration 0.9%, dissolve in the corresponding volume of deionized water, adjust the pH to 7.5 with hydrochloric acid or sodium hydroxide solution to obtain the third reagent.

[0094] The latex microspheres loaded with immunoglobulin G4 subtype antibodies are prepared. The latex microspheres are carboxyl polystyrene latex microspheres with a diameter of 100-120 nm. The latex microspheres are added to a MES·H2O solution with a concentration of 4.1 g / L. The solution is adjusted to a pH of 6.9 by using sodium hydroxide. After being mixed evenly, 1.9 g / L of boric acid and 1.0 g / L of disodium hydrogen phosphate are added. The solution is adjusted to a pH of 7.2 by using sodium hydroxide. The solution is mixed at 33° C. to obtain a latex microsphere suspension. Then, the immunoglobulin G4 subtype antibodies are added to the latex microsphere suspension. The immunoglobulin G4 subtype antibodies include rabbit anti-human immunoglobulin G4 subtype monoclonal antibodies. The concentration of immunoglobulin G4 subtype antibody in the reaction system is 960 mg / L, and then 0.15 g / L EDC·HCl solution and 0.43 g / L NHS solution are added, and the coupling reaction is stirred at 37.5°C for 5 hours; a blocking solution composed of 130 mL / L Tween and 0.9 g / L BSA is added, and the blocking reaction is stirred and dispersed at 26°C for 5 hours; proclin300 is added to make its concentration in the system reach 0.25 mL / L, and the pH is adjusted to 7.0 with sodium hydroxide solution or hydrochloric acid, and the mixture is stirred and soaked for 14 hours to obtain latex microspheres loaded with immunoglobulin G4 subtype antibody.

[0095] Example 2

[0096] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: the sodium azide mass concentration of the first reagent is 0.11%, the phosphate buffer concentration is 50 mmol / L, the vitamin C concentration is 16 mmol / L, the mannitol mass concentration is 0.9%, and the tris(hydroxymethyl)aminomethane concentration is 90 mmol / L, and the mixture is dissolved in a corresponding volume of deionized water, and the pH value is adjusted to 6.2 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent;

[0097] Preparation of sample pretreatment filter membrane: using the electrospinning equipment of the prior art to process polyimide into a polyimide fiber filter membrane, the molecular weight of the polyimide is 65000-90000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 64 kV, the obtained polyimide fiber has a diameter of 5 microns, and the thickness of the polyimide fiber filter membrane is 20 microns, to obtain the first filter layer;

[0098] The electrospinning equipment of the prior art is used to process polyvinyl alcohol into a polyvinyl alcohol fiber filter membrane, wherein the molecular weight of the polyvinyl alcohol is 90000-110000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 72 kV, the diameter of the polyvinyl alcohol fiber is 0.7 micron, the thickness of the polyvinyl alcohol fiber filter membrane is 20 micron, the polyvinyl alcohol fiber filter membrane is mixed with a hydroxy acid solution, the hydroxy acid is δ-hydroxyvaleric acid, the concentration is 1.2 g / L, the mass ratio of the hydroxy acid solution to the polyvinyl alcohol fiber filter membrane is 4:1, and the reaction temperature is 400 °C. The temperature is 95°C, the reaction time is 7h, so that the carboxyl group of the hydroxy acid reacts with the hydroxyl group on the polyvinyl alcohol fiber filter membrane to form an ester bond, after the reaction is completed, it is filtered and washed to obtain a polyvinyl alcohol fiber filter membrane coupled with a hydroxyl acid flexible arm, and the polyvinyl alcohol fiber filter membrane coupled with a hydroxyl acid flexible arm is immersed in a phosphate buffer solution with a concentration of 70mmol / L, and citric acid is added with a citric acid concentration of 16g / L, the reaction temperature is 58°C, and the reaction time is 10h, so that the hydroxyl group of the hydroxyl acid flexible arm forms an ester bond with the carboxyl group of the citric acid, thereby obtaining the second filter layer;

[0099] The electrospinning equipment of the prior art is used to process polypropylene alcohol into a polypropylene alcohol fiber filter membrane, wherein the molecular weight of polypropylene alcohol is 20000-35000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 75 kV, the diameter of the polypropylene alcohol fiber is 3 microns, and the thickness of the polypropylene alcohol fiber filter membrane is 8 microns. The polypropylene alcohol fiber filter membrane is mixed with a short-chain dibasic acid solution, wherein the short-chain dibasic acid is glutaric acid, and the concentration of the short-chain dibasic acid is 5.5 g / L, so that the carboxyl group at one end of the short-chain dibasic acid reacts with the hydroxyl group on the polypropylene alcohol fiber filter membrane to form an ester bond, and the reaction is completed. After the mixture is formed, it is filtered and washed to obtain a polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm, and the polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm is immersed in a phosphate buffer solution, the concentration of the phosphate buffer solution is 95mmol / L, and an activator NHS-EDC is added, the concentration of which is 0.22g / L, and stirred evenly, and a certain amount of morpholine is added, and an Fc fragment tag is added, and the concentration of the Fc fragment tag is 980mg / L. After stirring evenly, it is incubated at a temperature of 41°C for 18h, so that the other end carboxyl group of the short-chain dibasic acid flexible arm is connected to the Fc fragment tag, and a third filter layer is obtained;

[0100] The first filter layer, the second filter layer and the third filter layer are stacked in sequence, and the adjacent fibers are bonded together under the action of ultrasonic vibration using an ultrasonic welding device of the prior art to obtain the sample pretreatment filter membrane with a pore size of 4.5-10 microns;

[0101] Preparation of the second reagent: prepare the volume of the second reagent as required, calculate and weigh the ingredients according to the following ratio: Proclin 300 with a mass concentration of 0.2%, acetate buffer with a concentration of 60 mmol / L, and 1,4-butanediol with a mass concentration of 0.5%, dissolve them in a corresponding volume of deionized water, and adjust the pH value to 7.55 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent;

[0102] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: sodium azide mass concentration 0.10%, sodium glycine buffer concentration 120 mmol / L, latex microspheres loaded with immunoglobulin G4 subtype antibody mass concentration 9 g / L, arginine mass concentration 0.4%, xylooligosaccharide mass concentration 1.2%, dissolve in the corresponding volume of deionized water, adjust the pH to 7.3 with hydrochloric acid or sodium hydroxide solution to obtain the third reagent.

[0103] The latex microspheres loaded with immunoglobulin G4 subtype antibodies are prepared. The latex microspheres are carboxyl polystyrene latex microspheres with a diameter of 110-130 nm. The latex microspheres are added to a MES·H2O solution with a concentration of 3 g / L. The solution is adjusted to a pH of 7.0 using sodium hydroxide. After mixing evenly, 2.7 g / L of boric acid and 0.5 g / L of disodium hydrogen phosphate are added. The solution is adjusted to a pH of 7.4 using sodium hydroxide. The solution is mixed at 30° C. to obtain a latex microsphere suspension. Then, the immunoglobulin G4 subtype antibodies are added to the latex microsphere suspension. The immunoglobulin G4 subtype antibodies include rabbit anti-human immunoglobulin G4 subtype monoclonal antibodies. The concentration of immunoglobulin G4 subtype antibody in the reaction system is 1270 mg / L, and then 0.1 g / L EDC·HCl solution and 0.81 g / L NHS solution are added, and the coupling reaction is stirred at 35°C for 6 hours; a blocking solution composed of 110 mL / L Tween and 1.3 g / L BSA is added, and the blocking reaction is stirred and dispersed at 28°C for 9 hours; proclin300 is added to make its concentration in the system reach 0.38 mL / L, and the pH is adjusted to 6.8 with sodium hydroxide solution or hydrochloric acid, and the mixture is stirred and soaked for 16 hours to obtain latex microspheres loaded with immunoglobulin G4 subtype antibody.

[0104] Example 3

[0105] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of sodium azide of the first reagent is 0.08%, the concentration of phosphate buffer is 70 mmol / L, the concentration of reduced glutathione is 5 mmol / L, the mass concentration of mannitol is 1.1%, and the concentration of tris(hydroxymethyl)aminomethane is 30 mmol / L, and they are dissolved in the corresponding volume of deionized water, and the pH value is adjusted to 6.3 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent;

[0106] Preparation of sample pretreatment filter membrane: using the electrospinning equipment of the prior art to process polyimide into a polyimide fiber filter membrane, the molecular weight of the polyimide is 80000-100000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 60 kV, the diameter of the obtained polyimide fiber is 3.5 microns, the thickness of the polyimide fiber filter membrane is 12 microns, and the first filter layer is obtained;

[0107] The electrospinning equipment of the prior art is used to process polyvinyl alcohol into a polyvinyl alcohol fiber filter membrane, wherein the molecular weight of the polyvinyl alcohol is 100000-120000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 80 kV, the diameter of the polyvinyl alcohol fiber is 0.5 micron, the thickness of the polyvinyl alcohol fiber filter membrane is 30 microns, the polyvinyl alcohol fiber filter membrane is mixed with a hydroxy acid solution, the hydroxy acid is δ-hydroxyvaleric acid, the concentration is 3.5 g / L, the mass ratio of the hydroxy acid solution to the polyvinyl alcohol fiber filter membrane is 6:1, and the reaction is carried out. The temperature is 102°C, the reaction time is 5 hours, the carboxyl group of the hydroxy acid reacts with the hydroxyl group on the polyvinyl alcohol fiber filter membrane to form an ester bond, after the reaction is completed, it is filtered and washed to obtain a polyvinyl alcohol fiber filter membrane coupled with a hydroxyl acid flexible arm, the polyvinyl alcohol fiber filter membrane coupled with a hydroxyl acid flexible arm is immersed in a phosphate buffer solution, the concentration of the phosphate buffer solution is 90 mmol / L, citric acid is added, the concentration of the citric acid is 8 g / L, the reaction temperature is 60°C, the reaction time is 8 hours, the hydroxyl group of the hydroxyl acid flexible arm forms an ester bond with the carboxyl group of the citric acid, and the second filter layer is obtained;

[0108] The electrospinning equipment of the prior art is used to process polypropylene alcohol into a polypropylene alcohol fiber filter membrane, wherein the molecular weight of polypropylene alcohol is 30000-50000 g / mol, the nozzle of the electrospinning equipment is connected to the positive electrode, the voltage between the positive and negative electrodes is 80 kV, the diameter of the polypropylene alcohol fiber is 2 microns, and the thickness of the polypropylene alcohol fiber filter membrane is 10 microns. The polypropylene alcohol fiber filter membrane is mixed with a short-chain dibasic acid solution, wherein the short-chain dibasic acid is adipic acid, and the concentration of the short-chain dibasic acid is 6.8 g / L, so that the carboxyl group at one end of the short-chain dibasic acid reacts with the hydroxyl group on the polypropylene alcohol fiber filter membrane to form an ester bond, and the reaction After completion, the membrane was filtered and washed to obtain a polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm, and the polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm was immersed in a phosphate buffer solution, the concentration of which was 80 mmol / L, and an activator NHS-EDC was added, the concentration of which was 0.15 g / L, and the membrane was stirred evenly, and a certain amount of morpholine was added, and an Fc fragment tag was added, the concentration of which was 900 mg / L, and the membrane was stirred evenly and incubated at a temperature of 40°C for 23 hours, so that the other end carboxyl group of the short-chain dibasic acid flexible arm was connected to the Fc fragment tag, and the third filter layer was obtained;

[0109] The first filter layer, the second filter layer and the third filter layer are stacked in sequence, and the adjacent fibers are bonded together under the action of ultrasonic vibration using an ultrasonic welding device of the prior art to obtain the sample pretreatment filter membrane with a pore size of 7-13 microns;

[0110] Preparation of the second reagent: prepare the volume of the second reagent as required, calculate and weigh the ingredients according to the following ratio: Proclin 300 with a mass concentration of 0.3%, acetate buffer with a concentration of 45 mmol / L, and 1,4-butanediol with a mass concentration of 1.1%, dissolve them in a corresponding volume of deionized water, and adjust the pH value to 7.35 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent;

[0111] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: sodium azide mass concentration 0.14%, sodium glycine buffer concentration 100 mmol / L, latex microspheres loaded with immunoglobulin G4 subtype antibody mass concentration 16 g / L, arginine mass concentration 0.5%, xylooligosaccharide mass concentration 1.0%, dissolve in the corresponding volume of deionized water, adjust the pH to 7.7 with hydrochloric acid or sodium hydroxide solution to obtain the third reagent.

[0112] The latex microspheres loaded with immunoglobulin G4 subtype antibodies are prepared. The latex microspheres are carboxyl polystyrene latex microspheres with a diameter of 120-150 nm. The latex microspheres are added to a MES·H2O solution with a concentration of 5.2 g / L. The solution is adjusted to a pH of 7.1 by using sodium hydroxide. After being mixed evenly, 2.1 g / L of boric acid and 1.4 g / L of disodium hydrogen phosphate are added. The solution is adjusted to a pH of 7.6 by using sodium hydroxide. The solution is mixed at 35° C. to obtain a latex microsphere suspension. Then, the immunoglobulin G4 subtype antibodies are added to the latex microsphere suspension. The immunoglobulin G4 subtype antibodies include rabbit anti-human immunoglobulin G4 subtype monoclonal Antibody, the concentration of immunoglobulin G4 subtype antibody in the reaction system is 1130 mg / L, and then 0.18 g / L EDC·HCl solution and 0.62 g / L NHS solution are added, and the coupling reaction is stirred at 38°C for 7 hours; a blocking solution composed of 150 mL / L Tween and 1.1 g / L BSA is added, and the blocking reaction is stirred and dispersed at 30°C for 7 hours; proclin300 is added to make its concentration in the system reach 0.45 mL / L, and the pH is adjusted to 7.3 with sodium hydroxide solution or hydrochloric acid, and stirred and soaked for 15 hours to obtain latex microspheres loaded with immunoglobulin G4 subtype antibody.

[0113] Comparative Example 1: No sample pretreatment filter membrane and second reagent

[0114] The preparation method of this comparative example does not have the steps of preparing the sample pretreatment filter membrane and the second reagent;

[0115] Other parameters are the same as those in Example 2;

[0116] The detection kit of this comparative example is obtained.

[0117] The immunoglobulin G4 subtype detection kit of Example 1-3 was tested and compared using the following detection method:

[0118] (1) Take fresh blood and prepare a sample liquid;

[0119] (2) Add 4 μl of the sample liquid to 180 μl of the first reagent, mix well, and incubate at 37° C. for 5 minutes to obtain a mixed solution of the sample to be tested and the first reagent;

[0120] (3) Using the sample pretreatment filter membrane to filter the sample to be tested and the first reagent mixed solution obtained in the previous step, the mixed solution is passed through the first filter layer, the second filter layer and the third filter layer in sequence, and the time for the mixed solution to pass through the sample pretreatment filter membrane is controlled between 45 seconds to obtain a filtrate, and the sample pretreatment filter membrane is washed with 90 microliters of the second reagent, and the washing liquid is combined with the filtrate to obtain the mixed solution to be tested after the filter membrane treatment;

[0121] (4) Add 100 μl of the third reagent to the test mixture after the filter membrane treatment obtained in the previous step, incubate at 38° C. for 12 seconds, read the absorbance A1 at a wavelength of 570 nm, and read the absorbance A2 after 2.5 minutes;

[0122] (5) Calculate △A=A2-A1, and calculate the concentration of immunoglobulin G4 subtype in the sample to be tested based on the working curve.

[0123] The test steps (1)-(2), (4) and (5) of the detection kit of Comparative Example 1 are the same as the above method, but the time for reading A2 in step (4) is 6 minutes after the reading of A1, and there is no step (3).

[0124] A batch of samples to be tested was tested 15 times using the detection kit corresponding to each embodiment and comparative example.

[0125] The standard deviation of the test results of each embodiment and comparative example was statistically calculated, and the results showed that the standard deviation of the test results of embodiments 1-3 was between 4.5% and 6.2%, and the total measurement time was 45 minutes; the standard deviation of comparative example 1 was 16.4%, and the total measurement time was 100 minutes. This shows that the technical solution of setting the sample pretreatment filter membrane and the second reagent of the present invention can significantly improve the anti-interference ability of the immunoglobulin G4 subtype detection kit, thereby improving its detection accuracy, and can also greatly shorten the detection time.

Claims

1. An immunoglobulin G4 subtype detection kit, characterized in that: The immunoglobulin G4 subtype detection kit comprises a first reagent, a sample pretreatment filter membrane, a second reagent and a third reagent; The first reagent includes an anti-first preservative, a first pH adjuster, a reducing agent, a first protein stabilizer and tris(hydroxymethyl)aminomethane; The first preservative is sodium azide, with a mass concentration of 0.05%-0.11%; the first pH adjuster is phosphate buffer, with a concentration of 50-70mmol / L; the reducing agent is one of cysteine, vitamin C, and reduced glutathione, with a concentration of 5-28mmol / L; the first protein stabilizer is mannitol, with a mass concentration of 0.9%-1.7%; the concentration of tris(hydroxymethyl)aminomethane is 30-90mmol / L; The sample pretreatment filter membrane consists of a first filter layer, a second filter layer and a third filter layer; The first filter layer is a polyimide fiber filter membrane with a thickness of 5-20 microns; The second filter layer is a polyvinyl alcohol fiber filter membrane with a thickness of 10-30 microns, coupled with a hydroxy acid flexible arm, and the free end of the hydroxy acid flexible arm is coupled with monoesterified citric acid; the third filter layer is a polypropylene alcohol fiber filter membrane with a thickness of 5-10 microns, coupled with a short-chain dibasic acid flexible arm, and the free end of the short-chain dibasic acid flexible arm is coupled with an Fc fragment tag, and the amino acid sequence of the Fc fragment tag is gly-thr-ser-glu-lys-pro-leu-cys-cys-lys-ile-ile-leu-val-leu-glu-Arg; the first filter layer, the second filter layer, and the third filter layer of the sample pretreatment filter membrane are pressed in sequence, and the first filter layer first contacts the sample to be treated; The hydroxy acid of the hydroxy acid flexible arm includes one of γ-hydroxybutyric acid and δ-hydroxyvaleric acid; The short-chain dibasic acid of the short-chain dibasic acid flexible arm includes one of succinic acid, glutaric acid, and adipic acid, and the short-chain dibasic acid is a dibasic acid with carboxyl groups at both ends of the molecule; The second reagent includes a second preservative, a second pH buffer and a desorbent; The second preservative is Proclin300, with a mass concentration of 0.1%-0.3%; the second pH buffer is acetate buffer, with a concentration of 30-60mmol / L; the desorbent is 1,4-butanediol, with a mass concentration of 0.5%-1.1%; The third reagent includes a third preservative, a third pH adjuster, latex microspheres loaded with immunoglobulin G4 subtype antibodies, and a second protein stabilizer; The third preservative is sodium azide with a mass concentration of 0.1%-0.19%; the third pH regulator is sodium glycine buffer with a concentration of 70-120 mmol / L; the mass concentration of the latex microspheres loaded with immunoglobulin G4 subtype antibodies is 9-16 g / L; the second protein stabilizer is arginine and xylooligosaccharide, the mass concentration of arginine is 0.3%-0.5%, and the mass concentration of xylooligosaccharide is 0.9%-1.2%; The pH of the first reagent is 6.1-6.3, the pH of the second reagent is 7.15-7.55, and the pH of the third reagent is 7.3-7.7, which are adjusted using sodium hydroxide or hydrochloric acid.

2. The immunoglobulin G4 subtype detection kit according to claim 1, characterized in that: The pore size of the sample pretreatment filter membrane is 4.5-16 microns.

3. The immunoglobulin G4 subtype detection kit according to claim 1, characterized in that: The xylo-oligosaccharide of the second protein stabilizer includes xylo-oligosaccharide containing 5-8 xylose residues.

4. The immunoglobulin G4 subtype detection kit according to claim 1, characterized in that: The latex microspheres loaded with immunoglobulin G4 subtype antibodies include carboxyl polystyrene latex microspheres.

5. A method for preparing the immunoglobulin G4 subtype detection kit according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of sodium azide is 0.05%-0.11%, the concentration of phosphate buffer is 50-70mmol / L, the concentration of reducing agent is 5-28mmol / L, the mass concentration of mannitol is 0.9%-1.7%, and the concentration of tris(hydroxymethyl)aminomethane is 30-90mmol / L, and dissolve them in the corresponding volume of deionized water, and adjust the pH value to 6.1-6.3 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent; The reducing agent is one of cysteine, vitamin C, and reduced glutathione; Prepare a sample pretreatment filter membrane: use an electrostatic spinning device to process polyimide into the polyimide fiber filter membrane, the nozzle of the electrostatic spinning device is connected to the positive electrode, the voltage between the positive electrode and the negative electrode of the electrostatic spinning device is 60-70kV, the diameter of the polyimide fiber is 2-5 microns, and the thickness of the polyimide fiber filter membrane is 5-20 microns, to obtain a first filter layer; Using an electrospinning device to process polyvinyl alcohol into the polyvinyl alcohol fiber filter membrane, the nozzle of the electrospinning device is connected to the positive electrode, the voltage between the positive and negative electrodes of the electrospinning device is 65-80 kV, the diameter of the polyvinyl alcohol fiber is 0.5-1 micron, the thickness of the polyvinyl alcohol fiber filter membrane is 10-30 microns, the polyvinyl alcohol fiber filter membrane is mixed with a hydroxy acid solution, so that the carboxyl group of the hydroxy acid reacts with the hydroxyl group on the polyvinyl alcohol fiber filter membrane to form an ester bond, after the reaction is completed, filtering and washing to obtain a polyvinyl alcohol fiber filter membrane coupled with a hydroxy acid flexible arm, immersing the polyvinyl alcohol fiber filter membrane coupled with a hydroxy acid flexible arm in a phosphate buffer solution, adding citric acid, so that the hydroxyl group of the hydroxy acid flexible arm forms an ester bond with the carboxyl group of the citric acid, and obtaining the second filter layer; The polypropylene alcohol is processed into the polypropylene alcohol fiber filter membrane by using an electrospinning device, the nozzle of the electrospinning device is connected to the positive electrode, the voltage between the positive electrode and the negative electrode of the electrospinning device is 75-90 kV, the diameter of the polypropylene alcohol fiber is 0.8-3 microns, the thickness of the polypropylene alcohol fiber filter membrane is 5-10 microns, the polypropylene alcohol fiber filter membrane is mixed with a short-chain dibasic acid solution, and one end carboxyl group of the short-chain dibasic acid reacts with the hydroxyl group on the polypropylene alcohol fiber filter membrane to form an ester bond, after the reaction is completed, filtering and washing are performed to obtain a polypropylene alcohol fiber filter membrane coupled with a short-chain dibasic acid flexible arm, the polypropylene alcohol fiber filter membrane coupled with the short-chain dibasic acid flexible arm is immersed in a phosphate buffer, and an activator NHS-EDC is added, stirred evenly, a certain amount of morpholine is added, the Fc fragment tag is added, stirred evenly, and incubated for a certain period of time, so that the other end carboxyl group of the short-chain dibasic acid flexible arm is connected to the Fc fragment tag, and the third filter layer is obtained; The first filter layer, the second filter layer and the third filter layer are stacked in sequence, and an ultrasonic welding device is used to bond adjacent fibers under the action of ultrasonic vibration to obtain the sample pretreatment filter membrane; Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of Proclin 300 is 0.1%-0.3%, the concentration of acetate buffer is 30-60mmol / L, and the mass concentration of 1,4-butanediol is 0.5%-1.1%, dissolve them in the corresponding volume of deionized water, and adjust the pH value to 7.15-7.55 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent; Preparation of the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: the mass concentration of sodium azide is 0.1%-0.19%, the concentration of sodium glycine buffer is 70-120 mmol / L, the mass concentration of latex microspheres loaded with immunoglobulin G4 subtype antibodies is 9-16 g / L, the mass concentration of arginine is 0.3%-0.5%, and the mass concentration of xylooligosaccharide is 0.9%-1.2%. Dissolve them in the corresponding volume of deionized water, adjust the pH value to 7.3-7.7 with hydrochloric acid or sodium hydroxide solution, and obtain the third reagent.

6. The method for preparing the immunoglobulin G4 subtype detection kit according to claim 5, characterized in that: The concentration of the hydroxy acid solution is 1.2-3.5 g / L, the concentration of the phosphate buffer is 50-90 mmol / L, and the concentration of the citric acid in the phosphate buffer is 8-16 g / L.

7. The method for preparing the immunoglobulin G4 subtype detection kit according to claim 5, characterized in that: The concentration of the phosphate buffer is 80-110 mmol / L, the concentration of NHS-EDC is 0.08-0.22 g / L, the concentration of the Fc fragment tag is 830-980 mg / L, and the concentration of the short-chain dibasic acid is 4.2-6.8 g / L.

8. The method for preparing the immunoglobulin G4 subtype detection kit according to claim 5, characterized in that: The diameter of the latex microspheres is 100-150 nm.

9. The method for preparing the immunoglobulin G4 subtype detection kit according to claim 5, characterized in that: The latex microspheres loaded with immunoglobulin G4 subtype antibodies are prepared by adding the latex microspheres to a buffer solution of 3-5.2 g / L MES·H2O, adjusting the pH to 6.9-7.1 using sodium hydroxide, and mixing evenly, adding 1.9-2.7 g / L boric acid and 0.5-1.4 g / L disodium hydrogen phosphate, adjusting the pH to 7.2-7.6 using sodium hydroxide, and mixing at 30-35° C. to obtain a latex microsphere suspension; then adding the immunoglobulin G4 subtype antibody to the latex microsphere suspension, wherein the concentration of the immunoglobulin G4 subtype antibody in the reaction system is 960-1270 mg / L, and then adding 0.1-0.18 g / L EDC·HCl solution and A 0.43-0.81 g / L NHS solution is stirred at 35-38°C for coupling reaction, and the reaction time is 5-7 hours; a blocking solution consisting of 110-150 mL / L Tween and 0.9-1.3 g / L BSA is added, and the mixture is stirred and dispersed at 26-30°C for 5-9 hours for blocking reaction; proclin300 is added to a concentration of 0.25-0.45 mL / L in the system, and the pH is adjusted to 6.8-7.3 using a sodium hydroxide solution or hydrochloric acid, and the mixture is stirred and soaked for 14-16 hours to obtain the latex microspheres loaded with immunoglobulin G4 subtype antibodies.