An immunoglobulin G4 detection kit and preparation method thereof

Abstract

The present invention provides an immunoglobulin G4 detection kit and a preparation method thereof. The kit is composed of a first reagent R1 and a second reagent R2: the first reagent R1 comprises a zwitterionic buffer having a concentration of 25 to 200 mmol / L, 5 to 10 g / L inorganic salt, 0.5 w% to 5 w% surfactant 1, and 0.05 w% to 1 w% preservative; the second reagent R2 comprises a zwitterionic buffer having a concentration of 25 to 200 mmol / L, 5 to 10 g / L inorganic salt, 50 to 100 g / L sucrose, 50 to 100 g / L trehalose, 0.5 w% to 5 w% surfactant 2, 1 w% to 5 w% latex microspheres coupled with immunoglobulin G4 monoclonal antibodies labeled by carrier molecules, 1 w% to 5 w% blocking agent, and 0.05 w% to 1 w% preservative. The kit of the present invention has the advantages of high coupling efficiency, high sensitivity, anti-hook effect, and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to an immunoglobulin G4 detection kit and a preparation method thereof. Background Art

[0002] Human immunoglobulin G4 is a subtype of immunoglobulin G, accounting for 4%-5% of total immunoglobulin G. Compared with other immunoglobulin G subtypes, the concentration of immunoglobulin G4 is lower. However, elevated or decreased immunoglobulin G4 can be seen in a variety of immune system diseases. Immunoglobulin G4-related diseases are a type of chronic, progressive inflammatory disease with fibrosis and sclerosis of unknown etiology that has been newly discovered in recent years. The main feature is a significant increase in serum immunoglobulin G4 levels; affected organ tissues are enlarged or nodular / sclerotic lesions due to infiltration of a large number of lymphocytes and immunoglobulin G4-positive plasma cells with fibrosis. The Chinese Expert Consensus on the Diagnosis and Treatment of Immunoglobulin G4-Related Diseases (2021 Edition) clearly states that elevated serum immunoglobulin G4 is an important indicator for the diagnosis and assessment of immunoglobulin G4-RD. Therefore, the detection of serum immunoglobulin G4 concentration has certain clinical value.

[0003] At present, the methods for determining IgG4 include enzyme-linked immunosorbent assay, fluorescent immunochromatography, immunochromatography colloidal gold method, chemiluminescence, immunoturbidimetry, latex-enhanced immunoturbidimetry, etc. Since IgG4 is highly homologous to IgG1, IgG2, and IgG3 in structure, the kit needs to have high specificity. In addition, since the level of IgG4 in healthy people is low, and the high value of IgG4 has clinical significance, the kit needs to have the characteristics of high sensitivity, linearity, and anti-hook effect.

[0004] According to the existing technology, although the enzyme-immunoassay method is cheap, the quantitative determination result is unstable and time-consuming; although the fluorescent immunochromatography method is fast, it has a large sample size requirement and poor repeatability; the immunochromatography colloidal gold method is cheap, but the determination result is not accurate enough; the chemiluminescence method has a high sensitivity, but the stability of the reagent is poor; the common immunoturbidimetry method is prone to cross-reaction, and the reagent dosage is large; the latex-enhanced immunoturbidimetry method has better test stability and accuracy compared to the methodologies such as enzyme-linked immunosorbent assay and immunochromatography, and the detection flux is high. However, in the preparation process of latex-enhanced immunoturbidimetry reagent, there is a great randomness in the coupling of antibodies and microspheres. If the connected antibody is invalid, the sensitivity of the reagent is greatly reduced. In CN106771251A, a method of glycosylated modified antibodies is adopted. Although this method can improve the sensitivity of the reagent, the stability of the reagent box is poor, and the preparation process is more complicated and difficult to control. Summary of the invention

[0005] The purpose of the present invention is to provide an immunoglobulin detection kit and a preparation method thereof, which mainly solves the technical problems existing in the prior art such as slow detection speed, low sensitivity, poor repeatability, and the occurrence of HOOK effect.

[0006] The technical solution of the present invention is as follows:

[0007] An immunoglobulin G4 detection kit of the present invention is characterized by comprising a first reagent R1 and a second reagent R2:

[0008] The first reagent R1 includes a zwitterionic buffer with a concentration of 25 to 200 mmol / L, 5 to 10 g / L of inorganic salt, 0.5 w% to 5 w% of surfactant 1, and 0.05 w% to 1 w% of preservative;

[0009] The second reagent R2 comprises a zwitterionic buffer having a concentration of 25 to 200 mmol / L, 5 to 10 g / L inorganic salt, 50 to 100 g / L sucrose, 50 to 100 g / L trehalose, 0.5 w% to 5 w% surfactant 2, 1 w% to 5 w% latex microspheres coupled with immunoglobulin G4 monoclonal antibodies labeled with carrier molecules, 1 w% to 5 w% blocking agent, and 0.05 w% to 1 w% preservative;

[0010] The zwitterionic buffer is one or more of 2-(N-morpholino)ethanesulfonic acid (MES), 2[(2-amino-2-oxoethyl)amino]ethanesulfonic acid (ACES), 3-(N-morpholino)propanesulfonic acid (MOPS), and N-(2-hydroxyethyl)piperidin-N / -(2-ethanesulfonic acid) (HEPES);

[0011] The inorganic salt is sodium chloride and / or potassium chloride; the preservative is sodium azide or ProClin-300;

[0012] The surfactant 1 is one or more of Tween-20, Triton X-100, Triton X-405 or Span-20; the surfactant 2 is one or more of polyoxyethylene ether Brij58 (CAS No.: 9004-95-9), lauroyl polyfructose, and nonionic surfactant A60;

[0013] The latex microspheres are one or more of carboxyl polystyrene latex microspheres with a particle size of 50-100 nm or 100-300 nm, and the loading amount of the immunoglobulin G4 monoclonal antibody on the latex microspheres is (5-50):1; the carrier molecule is one or more of bovine serum albumin, polylysine or aspartic acid;

[0014] The blocking agent is selected from one or more of bovine serum albumin, casein, and Tween-20.

[0015] Preferably, the pH of the first reagent R1 and the second reagent R2 is 6.0-8.0.

[0016] The preparation method of the immunoglobulin G4 detection kit of the present invention is characterized in that carrier molecules are coupled to the surfaces of two latex microspheres with different particle sizes respectively, and then directionally coupled with the reduced antibodies to generate the latex microspheres labeled with the carrier molecules and coupled with the immunoglobulin G4 monoclonal antibodies.

[0017] More specifically, the method for preparing the immunoglobulin G4 detection kit of the present invention is characterized by comprising the following steps:

[0018] S1, taking a certain amount of the zwitterionic buffer and dissolving it in purified water, then adding the inorganic salt and the preservative; after stirring evenly, adjusting the pH value to 6.0-8.0, then adding the surfactant 1, stirring and dissolving evenly, and then fixing the volume to obtain the first reagent R1;

[0019] S2, (1) dissolving a certain amount of the buffer solution in purified water, and then adding the inorganic salt, sucrose, trehalose and preservative; after stirring evenly, adjusting the pH value to 6.0-8.0, and then adding the surfactant 2, stirring and dissolving evenly, and then fixing the volume to obtain a latex microsphere dilution solution; (2) taking the latex microspheres with a particle size of 50-100 nm, dissolving them in an activation buffer solution; then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, and shaking the reaction at room temperature for 0.5-1h; then centrifuging to remove the supernatant, and resuspending the precipitate with the cross-linking buffer solution to obtain activated latex microspheres; (3) adding the carrier molecules to the activated latex microspheres, shaking the reaction at room temperature for 1-4h; adding the blocking agent, and continuing the reaction for 0.5-1h; washing with the cross-linking buffer solution and centrifuging to remove the supernatant, resuspending the precipitate with the latex microsphere dilution solution to obtain a latex microsphere-carrier molecule complex, and storing it at 2-8°C for use;

[0020] S3, dissolving the latex microsphere-carrier molecule complex in the activation buffer, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and reacting at room temperature for 0.5 to 1 hour by shaking; removing the supernatant by centrifugation, and resuspending the precipitate with the cross-linking buffer to obtain an activated latex microsphere-carrier molecule complex;

[0021] The immunoglobulin G4 monoclonal antibody and the reducing agent are mixed in a phosphate buffer solution (PBS) with a pH value of 7.0 to 7.5, reduced at room temperature for 30 to 120 minutes, centrifuged to remove excess reducing agent, added with 0.1 w% to 0.3 w% iodoacetamide, and the thiol groups are blocked at room temperature for 30 to 60 minutes to obtain a reduced antibody;

[0022] S4, adding the reduced antibody to the activated latex microsphere-carrier molecule complex, shaking and reacting at room temperature for 1 to 4 hours, adding a blocking agent, and continuing the reaction for 0.5 to 1 hour; washing with a cross-linking buffer, centrifuging to remove the supernatant, and resuspending the precipitate with a latex microsphere diluent to obtain a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 50 to 100 nm;

[0023] S5, taking latex microspheres with a particle size of 200 to 300 nm, repeating steps S2 to S5, and obtaining a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 200 to 300 nm;

[0024] S6, mixing a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 50 to 100 nm and a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 200 to 300 nm in a ratio of (1.5 to 3.5):1 to form a second reagent R2;

[0025] S7, combining the first reagent R1 and the second reagent R2 to obtain the immunoglobulin G4 detection kit.

[0026] Preferably, in step S2, the mass ratio of the latex microspheres to the activation buffer is 1:(5-20); the ratio of the mass of the latex microspheres to the sum of the masses of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(0.01-0.2); the mass ratio of the carrier molecule to the latex microspheres is (5-50):1.

[0027] Preferably, in step S3, the mass ratio of the latex microsphere-carrier molecule complex to the activation buffer is 1:(5-20); the mass ratio of the latex microsphere-carrier molecule complex to the sum of the masses of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(0.01-0.2).

[0028] Preferably, in step S3, the mass ratio of the immunoglobulin G4 monoclonal antibody to the reducing agent is 1:(5-15); the reducing agent is one of β-mercaptoethanol (ME), dithiothreitol (DTT), and tris(2-carboxyethyl)phosphine (TCEP).

[0029] Preferably, in step S4, the mass ratio of the reduced antibody to the latex microspheres is (5-50):1.

[0030] Preferably, the activation buffer is one of 2-(N-morpholino)ethanesulfonic acid (MES), 2[(2-amino-2-oxoethyl)amino]ethanesulfonic acid (ACES), and 3-(N-morpholino)propanesulfonic acid (MOPS) at a concentration of 25 to 100 mmol / L; the cross-linking buffer is one of 2-(N-morpholino)ethanesulfonic acid (MES), 2[(2-amino-2-oxoethyl)amino]ethanesulfonic acid (ACES), and 3-(N-morpholino)propanesulfonic acid (MOPS) at a concentration of 25 to 100 mmol / L.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The kit of the present invention uses a method of selecting two latex microspheres with different particle sizes to couple with antibodies respectively, so that the reagent has both sensitivity and anti-HOOK effect ability; by optimizing the coupling process of latex microspheres and antibodies, that is, adding a carrier molecule between the microspheres and the reduced antibodies, the antibodies are extended from the surface of the microspheres, thereby reducing the steric hindrance of antigen-antibody binding, and at the same time, since the reduced antibodies have free thiol groups, they are easy to generate stable thioether bonds with the carboxyl groups on the latex particles, so as to realize the directional coupling of the F (c) end of the antibody and the latex microspheres, thereby greatly improving the sensitivity of the reagent. Compared with the prior art, the kit of the present invention has the advantages of high coupling efficiency, high sensitivity, and prevention of HOOK effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results show the detection of double-size latex microspheres against the anti-HOOK effect of reagents. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art should recognize that the embodiments are only used to illustrate the present invention, and are not used to limit the present invention, and any changes or modifications to the embodiments within the scope of the present invention are within the scope of the claims of the present invention.

[0035] Example 1

[0036] Example 1 is the preparation process and use method of the immunoglobulin G4 detection kit:

[0037] 1. Preparation of R1 reagent

[0038] Take 12g of 3-(N-morpholino)propanesulfonic acid (MOPS) in purified water, add 5g of NaCl and 5g of NaN3, stir evenly, adjust the pH to 7.0, then add 5g of Tween-20, stir and dissolve evenly, and adjust the volume to 1L as the first reagent R1;

[0039] 2. Preparation of R2 reagent

[0040] (1) Preparation of latex microsphere dilution:

[0041] Dissolve 12g of 3-(N-morpholino)propanesulfonic acid (MOPS) in purified water, add 10g of NaCl, 50g of sucrose, 50g of trehalose and 5g of NaN3, stir evenly, adjust the pH to 7.0, then add 10g of Triton X-405, stir evenly, and dilute to 1L;

[0042] (2) Preparation of latex microsphere-carrier molecule complex:

[0043] 1 ml of carboxyl polystyrene latex microspheres (Bangs) with a particle size of 60 nm were dissolved in 10 ml of 25 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) buffer with a pH value of 5.5, and then 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 mg of N-hydroxysuccinimide were added to the 2-(N-morpholino)ethanesulfonic acid (MES) buffer in sequence to obtain a latex microsphere activation reaction system, which was shaken at room temperature for 0.5 to 1 h; the latex microsphere activation reaction system was centrifuged to remove the supernatant, and 10 ml of Resuspend the precipitate with 50 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) buffer at a pH value of 6.0 to obtain activated latex microspheres; add 7 ml of 5 mg / ml aspartic acid to the activated latex microspheres, shake and react at room temperature for 1 hour, then add bovine serum albumin and continue to react for 1 hour; wash twice with 50 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) buffer at a pH value of 6.0, centrifuge to remove the supernatant, resuspend the precipitate with latex microsphere diluent to obtain a latex microsphere-aspartic acid complex with a particle size of 60 nm, and store it at 2-8° C. for use;

[0044] Take 1 ml of carboxyl polystyrene latex microspheres (Bangs) with a particle size of 288 nm, repeat this step (the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride added is 15 mg, the amount of N-hydroxysuccinimide added is 100 mg, the amount of 5 mg / ml aspartic acid added is 1.5 ml, and the other steps are the same) to obtain a latex microsphere-aspartic acid complex with a particle size of 288 nm, and store it at 2-8°C for use;

[0045] (3) Antibody reduction:

[0046] 20 ml of 2 mg / ml immunoglobulin G4 monoclonal antibody was mixed with 500 mg of β-mercaptoethanol (ME) in 150 mmol / L phosphate buffer (PBS) with a pH value of 7.4, and reduced at room temperature for 30 min. The excess reducing agent was removed by centrifugation, and 1 mg of iodoacetamide was added. The thiol groups were blocked at 37°C for 30 min to obtain the reduced antibody.

[0047] (4) Coupling of Antibodies to Latex Microsphere-Aspartic Acid Complexes:

[0048] 1 ml of latex microsphere-aspartic acid complex with a particle size of 60 nm was dissolved in 10 ml of 2-(N-morpholino)ethanesulfonic acid (MES) buffer with a pH value of 5.5 and a concentration of 25 mmol / L, and then 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 mg of N-hydroxysuccinimide were added to the 2-(N-morpholino)ethanesulfonic acid (MES) buffer in sequence, and the reaction was shaken at room temperature for 0.5 h; the supernatant was removed by centrifugation, and the precipitate was resuspended with 50 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) buffer with a pH value of 6.0 to obtain an activated latex microsphere-aspartic acid complex with a particle size of 60 nm;

[0049] Take 1 ml of latex microsphere-aspartic acid complex with a particle size of 288 nm, adopt the same steps (the addition amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 15 mg, the addition amount of N-hydroxysuccinimide is 100 mg, and the other steps are the same) to obtain an activated latex microsphere-aspartic acid complex with a particle size of 288 nm;

[0050] Add 17 ml of reduced immunoglobulin G4 monoclonal antibody to the activated latex microsphere-aspartic acid complex with a particle size of 60 nm, add 3 ml of reduced immunoglobulin G4 monoclonal antibody to the activated latex microsphere-aspartic acid complex with a particle size of 288 nm, oscillate and react at room temperature for 1 hour, add bovine serum albumin, and continue to react for 1 hour; wash twice with MOPS buffer with a pH value of 7.4 and a concentration of 50 mmol / L, centrifuge to remove the supernatant, resuspend the precipitate with latex microsphere diluent, and obtain latex reagents coupled to immunoglobulin G4 monoclonal antibodies of two particle sizes.

[0051] (5) Preparation of R2 reagent

[0052] A latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 60 nm and a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 288 nm are mixed in a ratio of 2:1 to form a second reagent R2;

[0053] 3. Combine the first reagent R1 and the second reagent R2 to obtain the immunoglobulin G4 detection kit.

[0054] 4. How to use the kit

[0055] In this embodiment, a fully automatic biochemical analyzer (Hitachi 7180) was used as the detection tool, and the instrument parameters were set according to Table 1.

[0056] Table 1 Setting parameters on the fully automatic biochemical analyzer (Hitachi 7180)

[0057] Machine parameters Hitachi 7180 method 2PE time 10min wavelength cancel / 570nm Metering point 18-34 Sample loading volume 1.5uL Reaction direction ↑ Absorbance limit 32000 The first reagent R1 amount 150uL Second reagent R2 amount 50uL Calibration Type SPLINE Calibration Points 5 Range point 3

[0058] After adding the first reagent R1 to the human serum sample, mix well and incubate at 37°C for 3 to 5 minutes; then add the second reagent R2 and mix well; incubate at 37°C for 30 seconds and read the absorbance A1, and then incubate for about 270 seconds and read the absorbance A2.

[0059] The immunoglobulin G4 in the human serum sample forms an insoluble immune complex with the antibody coated on the polystyrene particles in the reagent, and its turbidity is proportional to the immunoglobulin G4 content in the human serum sample. The complex turbidity ΔA is measured at 570nm, and the calculation method is ΔA=A2-A1. The immunoglobulin G4 content in the sample is automatically calculated by the automatic biochemical analyzer through the calibration curve.

[0060] Example 2

[0061] Example 2 is a verification of the carrier molecule improving the sensitivity of the reagent:

[0062] In order to verify that the introduction of carrier molecules into the kit of the present invention during the latex preparation process improves the sensitivity of the reagent, four groups of kits were set up for comparison.

[0063] Kit 1: the kit prepared in Example 1 of the present invention.

[0064] Kit 2: The difference from Kit 1 is that in Kit 2, the second reagent R2, during the coupling process between latex and antibody, the reduced antibody is directly coupled to the latex microspheres without adding a carrier molecule. The rest of the preparation method is the same as Kit 1.

[0065] Kit 3: Immunoglobulin G subtype 4 detection kit (latex enhanced immunoturbidimetric method - marketed product A)

[0066] Kit 4: Immunoglobulin G4 Assay Kit (latex enhanced immunoturbidimetric method - marketed product B)

[0067] In this embodiment, a fully automatic biochemical analyzer (Hitachi 7180) was used as the detection tool. Kit 1 and Kit 2 were tested using the method of using the kit in Example 1, and Kit 3 and Kit 4 were tested according to their respective product instructions.

[0068] (1) Analytical sensitivity

[0069] Four sets of kits were used to simultaneously test samples with an analytical sensitivity of 1 g / L (assigned by the Siemens Immunoglobulin G4 Assay Kit (nephelometry)) to compare analytical sensitivities.

[0070] Table 2 Analytical sensitivity test results

[0071]

[0072]

[0073] Note: ΔA is the absorbance change.

[0074] As can be seen from Table 2, the analytical sensitivity of the kit 1 of the present invention is significantly greater than that of the kit 2, indicating that the analytical sensitivity of the reagent is significantly improved after the introduction of the carrier molecule. Compared with other manufacturers, the analytical sensitivity of the kit 1 of the present invention is significantly better than that of the kit 3 and the kit 4.

[0075] (2) Repeatability of low-value samples

[0076] Four sets of kits were used to simultaneously test low-value samples of immunoglobulin G4 (assigned by Siemens immunoglobulin G4 assay kit (nephelometry)), and the CV of the low-value samples was calculated to compare the repeatability of the low-value samples.

[0077]

[0078]

[0079] Table 3 Repeatability test results of low value samples

[0080] As can be seen from Table 3, the repeatability data of the low-value samples show that after the introduction of the carrier molecule, the repeatability of the kit 1 of the present invention is significantly improved, and the kit 1 of the present invention is superior to the repeatability of the kits of other manufacturers.

[0081] (3) Comparison of clinical low-value samples

[0082] Four sets of kits were used to simultaneously test samples with low immunoglobulin G4 values ​​(assigned by Siemens immunoglobulin G4 assay kit (nephelometry)). Each sample was measured twice, and the deviation between the mean and the target value (Siemens assigned value) was calculated.

[0083] Table 4 Test results of clinical samples

[0084]

[0085] According to Table 4, it can be seen that after the carrier molecule is introduced into the kit 1 of the present invention, the deviation between the low-value sample and the Siemens test result is significantly reduced, and the deviation is smaller than the deviation between the kit 3 and the Siemens; the deviation between the kit 4 and the Siemens when detecting low-value samples is small, but its hook shape is serious (see Example 3). In summary, after the carrier molecule is introduced into the kit 1 of the present invention, the sensitivity of the reagent is significantly improved.

[0086] Example 3

[0087] Example 3 is a verification of the dual-size latex microspheres taking into account both reagent sensitivity and anti-HOOK ability:

[0088] In order to verify that the double-diameter latex microspheres used in the kit of the present invention can enable the reagent to have both sensitivity and anti-HOOK effect, five groups of kits were set up for comparison.

[0089] Physiological saline was used to perform gradient dilutions (100%, 80%, 40%, 20%, 10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08%, 0.04%) of immunoglobulin G4 high-value samples (nephelometry, Siemens). Three groups of test kits were used to test the gradient dilution samples simultaneously. Each sample was measured twice, and the mean was calculated for comparison.

[0090] Kit 1: the kit prepared in Example 1 of the present invention.

[0091] Kit 5: The difference from Kit 1 is that the second reagent R2 in Kit 2 uses latex with small particle size for coupling, and the rest of the preparation method is the same as Kit 1.

[0092] Kit 6: The difference from Kit 1 is that the second reagent R2 in Kit 3 uses latex with large particle size for coupling, and the rest of the preparation method is the same as Kit 1.

[0093] Kit 3: Immunoglobulin G subtype 4 detection kit (latex enhanced immunoturbidimetric method - marketed product A)

[0094] Kit 4: Immunoglobulin G 4 Assay Kit (latex enhanced immunoturbidimetric method - marketed product B)

[0095] In this embodiment, a fully automatic biochemical analyzer (Hitachi 7180) was used as the detection tool. Kit 1, Kit 5 and Kit 6 were tested using the method of using the kit in Example 1, and Kit 3 and Kit 4 were tested according to their respective product instructions.

[0096] Table 5 Test results of double-size latex microspheres on the anti-HOOK effect of reagents

[0097]

[0098] Depend on Figure 1 It can be seen that kit 5 has good linearity and anti-HOOK effect ability, but poor sensitivity (see Table 5); kit 6 and kit 5 have good sensitivity, but serious HOOK effect; kit 6 is less sensitive than kit 1 of the present invention. Therefore, kit 1 of the present invention has both sensitivity and anti-HOOK effect ability.

[0099] Example 4

[0100] Example 4 is the performance verification of the immunoglobulin immunoglobulin G4 detection kit:

[0101] The performance of the kit prepared in Example 1 was verified.

[0102] (1) Accuracy verification

[0103] The kit was used on a fully automatic biochemical analyzer (Hitachi 7180) to detect the reference material ERM-DA470K / IFCC. Each sample was tested 3 times, and the deviation between the measured mean and the target value was calculated.

[0104] Table 6 Sample test results

[0105] Test results 1 0.368 2 0.376 3 0.356 Mean(g / L) 0.367 Reference material (g / L) 0.373 Relative deviation -1.70％

[0106] According to the test results in Table 6, the deviation between the mean value of the reference material ERM-DA470K / IFCC detected by this kit three times and the target value is only -1.70%, which meets the requirement of accuracy ≤±15%.

[0107] (2) Precision verification

[0108] The quality control products were tested using this kit using a fully automatic biochemical analyzer (Hitachi 7180) according to the above method. Each quality control product was tested 10 times and the CV was calculated.

[0109] Table 7 Precision test results

[0110]

[0111]

[0112] Table 8 Batch difference test results

[0113]

[0114] It can be seen from Tables 7 and 8 that, according to the test results, the repeatability and batch difference of the kit 1 of the present invention at two concentration levels both meet the requirement of ≤±10%.

[0115] (3) Linear verification

[0116] Using a fully automatic biochemical analyzer (Hitachi 7180), the immunoglobulin G4 high-value samples were gradiently diluted (100%, 80%, 40%, 20%, 10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08%, 0.04%) using the calibration diluent as described above. The gradient dilution samples were tested using the kit, and each sample was measured 3 times.

[0117] Table 9 Linearity verification results

[0118]

[0119] As can be seen from Table 9, this kit has good linearity in the range of 0.04-4g / L, with a correlation coefficient >0.99; in the range of 0.04-1.5g / L, the absolute deviation can be controlled within ±0.1g / L; in the range of 1.5-4.0g / L, the relative deviation can be controlled within ±10g / L.

Claims

1. An immunoglobulin G4 detection kit, characterized in that: It consists of a first reagent R1 and a second reagent R2: The first reagent R1 includes a zwitterionic buffer having a concentration of 25 to 200 mmol / L, 5 to 10 g / L of an inorganic salt, 0.5 w% to 5 w% of a surfactant 1, and 0.05 w% to 1 w% of a preservative; The second reagent R2 comprises a zwitterionic buffer having a concentration of 25 to 200 mmol / L, 5 to 10 g / L inorganic salt, 50 to 100 g / L sucrose, 50 to 100 g / L trehalose, 0.5 w% to 5 w% surfactant 2, 1 w% to 5 w% latex microspheres coupled with immunoglobulin G4 monoclonal antibodies labeled with carrier molecules, 1 w% to 5 w% blocking agent, and 0.05 w% to 1 w% preservative; The latex microspheres are carboxyl polystyrene latex microspheres of two different particle sizes, 50-100 nm and 100-300 nm; the loading amount of the immunoglobulin G4 monoclonal antibody on the latex microspheres is (5-50):1; the two latex microspheres of different particle sizes are activated respectively, and then the carrier molecules are coupled to the surface, and then they are activated again respectively, and then the reduced antibodies are directionally coupled to generate two latex microspheres of different particle sizes that are labeled with the carrier molecules and coupled with the immunoglobulin G4 monoclonal antibody; Mixing latex microspheres with a particle size of 50 to 100 nm and labeled with carrier molecules and coupled with immunoglobulin G4 monoclonal antibodies and latex microspheres with a particle size of 200 to 300 nm and labeled with carrier molecules and coupled with immunoglobulin G4 monoclonal antibodies in a ratio of (1.5 to 3.5):1 to obtain latex microspheres with a particle size of 200 to 300 nm and labeled with carrier molecules and coupled with immunoglobulin G4 monoclonal antibodies; The zwitterionic buffer is one or more of 2-(N-morpholino)ethanesulfonic acid, 2[(2-amino-2-oxoethyl)amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and N-(2-hydroxyethyl)piperidin-N / -(2-ethanesulfonic acid); the inorganic salt is sodium chloride and / or potassium chloride; the preservative is sodium azide or ProClin-300; the blocking agent is one or more of bovine serum albumin, casein, and Tween-20; the surfactant 1 is one or more of Tween-20, Triton X-100, Triton X-405, or Span-20; the surfactant 2 is one or more of polyoxyethylene ether Brij58, lauroyl polyfructose, and nonionic surfactant A60; and the carrier molecule is aspartic acid.

2. The immunoglobulin G4 detection kit according to claim 1, characterized in that: The pH of the first reagent R1 and the second reagent R2 is 6.0-8.

0.

3. The method for preparing the immunoglobulin G4 detection kit according to claim 2, characterized in that: The following steps are involved: S1, taking a certain amount of the zwitterionic buffer and dissolving it in purified water, then adding the inorganic salt and the preservative; after stirring evenly, adjusting the pH value to 6.0-8.0, then adding the surfactant 1, stirring and dissolving evenly, and then fixing the volume to obtain the first reagent R1; S2, (1) dissolving a certain amount of the buffer solution in purified water, and then adding the inorganic salt, sucrose, trehalose and preservative; after stirring evenly, adjusting the pH value to 6.0-8.0, and then adding the surfactant 2, stirring and dissolving evenly, and then fixing the volume to obtain a latex microsphere diluent; (2) taking the latex microspheres with a particle size of 50-100 nm, dissolving them in an activation buffer solution; then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, and shaking the reaction at room temperature for 0.5-1h; then centrifuging to remove the supernatant, and resuspending the precipitate with the cross-linking buffer solution to obtain activated latex microspheres; (3) adding the carrier molecules to the activated latex microspheres, shaking the reaction at room temperature for 1-4h; adding the blocking agent, and continuing the reaction for 0.5-1h; washing with the cross-linking buffer solution and centrifuging to remove the supernatant, resuspending the precipitate with the latex microsphere diluent to obtain a latex microsphere-carrier molecule complex, and storing it at 2-8°C for use; S3, dissolving the latex microsphere-carrier molecule complex in the activation buffer, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and reacting at room temperature for 0.5 to 1 hour by shaking; removing the supernatant by centrifugation, and resuspending the precipitate with the cross-linking buffer to obtain an activated latex microsphere-carrier molecule complex; Immunoglobulin G4 monoclonal antibody and reducing agent are mixed in a phosphate buffer with a pH value of 7.0 to 7.5, reduced at room temperature for 30 to 120 minutes, centrifuged to remove excess reducing agent, added with 0.1w% to 0.3w% iodoacetamide, and the thiol group is blocked at room temperature for 30 to 60 minutes to obtain a reduced antibody; S4, adding the reduced antibody to the activated latex microsphere-carrier molecule complex, shaking and reacting at room temperature for 1 to 4 hours, adding a blocking agent, and continuing the reaction for 0.5 to 1 hour; washing with a cross-linking buffer, centrifuging to remove the supernatant, and resuspending the precipitate with a latex microsphere diluent to obtain a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 50 to 100 nm; S5, taking latex microspheres with a particle size of 200 to 300 nm, repeating steps S2 to S5, and obtaining a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 200 to 300 nm; S6, mixing a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 50 to 100 nm and a latex reagent coupled to immunoglobulin G4 monoclonal antibody with a particle size of 200 to 300 nm in a ratio of (1.5 to 3.5):1 to form a second reagent R2; S7, combining the first reagent R1 and the second reagent R2 to obtain the immunoglobulin G4 detection kit.

4. The method for preparing the immunoglobulin G4 detection kit according to claim 3, characterized in that: In step S2, the mass ratio of the latex microspheres to the activation buffer is 1:(5-20); the mass ratio of the latex microspheres to the mass sum of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(0.01-0.2); and the mass ratio of the carrier molecule to the latex microspheres is (5-50):

1.

5. The method for preparing the immunoglobulin G4 detection kit according to claim 3, characterized in that: In step S3, the mass ratio of the latex microsphere-carrier molecule complex to the activation buffer is 1:(5-20); the mass ratio of the latex microsphere-carrier molecule complex to the mass sum of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(0.01-0.2).

6. The method for preparing the immunoglobulin G4 detection kit according to claim 3, characterized in that: In the step S3, the mass ratio of the immunoglobulin G4 monoclonal antibody to the reducing agent is 1:(5-15); the reducing agent is one of β-mercaptoethanol, dithiothreitol, and tri(2-carboxyethyl)phosphine; in the step S4, the mass ratio of the reduced antibody to the latex microspheres is (5-50):

1.

7. The method for preparing the immunoglobulin G4 detection kit according to claim 3, characterized in that: The activation buffer is one of 2-(N-morpholino)ethanesulfonic acid, 2[(2-amino-2-oxoethyl)amino]ethanesulfonic acid, and 3-(N-morpholino)propanesulfonic acid at a concentration of 25 to 100 mmol / L; the cross-linking buffer is one of 2-(N-morpholino)ethanesulfonic acid, 2[(2-amino-2-oxoethyl)amino]ethanesulfonic acid, and 3-(N-morpholino)propanesulfonic acid at a concentration of 25 to 100 mmol / L.

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