CN114924079A - Anti-glutamate decarboxylase antibody determination kit and detection method - Google Patents
Anti-glutamate decarboxylase antibody determination kit and detection method Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N2800/00—Detection or diagnosis of diseases
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Abstract
The invention relates to the technical field of biological analysis, and provides an anti-glutamate decarboxylase antibody determination kit and a detection method. The kit comprises a reagent 1 and a reagent 2 which are independent from each other, wherein the reagent 1 consists of a stabilizing agent and a preservative which are placed in a proper buffer solution; the reagent 2 is composed of bovine serum albumin, a surfactant, a preservative and glutamic acid decarboxylase coated antigen latex particles which are placed in a proper buffer solution. The method for detecting the serum anti-glutamate decarboxylase antibody by using the latex immunoturbidimetry as the detection principle has the advantages of high sensitivity, strong specificity, simple and convenient operation and the like, can be applied to various full-automatic biochemical analyzers for large-batch detection, has low detection cost, and is suitable for clinical popularization and use.
Description
Technical Field
The invention relates to the technical field of biological analysis, in particular to an anti-glutamate decarboxylase antibody determination kit and a detection method.
Background
Glutamate decarboxylase (GAD) consists of two isomers, GAD65 and GAD 67. GAD65 is the main target antigen of Anti-glutamate decarboxylase antibody (Anti-GAD) for type 1 diabetes patients. The positive rate of Anti-GAD in patients with pre-diabetes and type 1 diabetes is 70% -90%, and the Anti-GAD is the most sensitive index of high risk group of diabetes. The positive rate of Anti-GAD is higher in older children and patients with late-onset type 1 diabetes. For the slowly progressing type 1 diabetes, i.e., latent autoimmune diabetes in adults, Anti-GAD can be used for differential diagnosis with type 2 diabetes. Anti-GAD is also associated with a rare neurological disease, stiff person syndrome, with a positive rate of 60% -100%.
At present, the glutamate decarboxylase antibody is mainly detected clinically, and common methods comprise an indirect immunofluorescence method and an enzyme-linked immunosorbent assay, but the methods have some defects. The indirect immunofluorescence method has more complex operation, needs an expensive fluorescence microscope, can only carry out qualitative detection, and has insufficient objectivity of an analysis result; the ELISA method cannot perform independent and single-person detection, the types of reagents used for quantitative determination are more, the operation of reagent filling is very complicated, the detection reagents are easy to cross-contaminate, and the accuracy and precision of detection results are poor.
Disclosure of Invention
The invention aims to overcome at least one of the defects in the prior art and provides an anti-glutamate decarboxylase antibody determination kit and a detection method. The purpose of the invention is realized based on the following technical scheme:
in one aspect of the object of the invention, an anti-glutamate decarboxylase antibody determination kit is provided, which comprises a reagent 1 and a reagent 2 which are independent of each other, wherein:
the reagent 1 consists of a stabilizing agent and a preservative which are put in a proper buffer solution;
the reagent 2 is composed of bovine serum albumin, a surfactant, a preservative and glutamic acid decarboxylase coated antigen latex particles which are placed in a proper buffer solution.
Preferably, the stabilizer comprises one or more of sodium chloride, potassium chloride, sucrose;
the preservative comprises one or more of sodium azide, Procline300, potassium sorbate, sodium benzoate and sodium nitrite;
the buffer solution comprises sodium citrate, 2-morpholine ethanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, tris (hydroxymethyl) aminomethane or PBS buffer solution;
the surfactant comprises one or more of Tween 20, Tween 40 and Triton X-100.
Preferably, the reagent 1 comprises 50-150 mmol/L of buffering agent, 0.05-0.3 mol/L of stabilizing agent and 0.01-0.06 wt% of preservative, and the pH value of the buffering agent is 6.5-7.5;
the reagent 2 comprises 50-150 mmol/L of a buffering agent, 0.5-2 wt% of bovine serum albumin, 0.5-2 wt% of a surfactant, 0.01-0.06 wt% of a preservative, 0.2-3 wt% of glutamic acid decarboxylase coated antigen latex particles, and the pH value of the buffering agent is 6.5-7.5.
Preferably, the reagent 1 comprises 100mmol/L of buffering agent, 0.15mol/L of stabilizing agent and 0.03 wt% of preservative;
the reagent 2 comprises 100mmol/L of buffer, 1 wt% of bovine serum albumin, 1 wt% of surfactant, 0.03 wt% of preservative and 1 wt% of glutamic acid decarboxylase-coated antigen latex particles.
Preferably, the kit for determining the anti-glutamate decarboxylase antibody further comprises a calibrator, wherein the calibrator comprises 50 wt% of human serum, 25-100 mmol/L of buffer, 2-10 wt% of protective agent, 0.05-0.3 mol/L of sodium chloride, 0.01-0.06 wt% of preservative and the anti-glutamate decarboxylase antibody with the target value concentration range of 270-400 IU/mL.
Preferably, the kit for determining the anti-glutamate decarboxylase antibody further comprises a quality control product, wherein the quality control product comprises 50 wt% of human serum, 25-100 mmol/L of buffering agent, 2-10 wt% of protective agent, 0.05-0.3 mol/L of sodium chloride, 0.01-0.06 wt% of preservative and the anti-glutamate decarboxylase antibody, the target concentration range of the level 1 of the anti-glutamate decarboxylase antibody is 3-50 IU/mL, and the target concentration range of the level 2 of the anti-glutamate decarboxylase antibody is 100-200 IU/mL.
Preferably, the buffer comprises sodium citrate, 2-morpholine ethanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, tris or PBS, and the preservative comprises one or more of sodium azide, Procline300, potassium sorbate, sodium benzoate, sodium nitrite.
In another aspect of the present invention, a method for detecting the content of an anti-glutamate decarboxylase antibody is provided, wherein the anti-glutamate decarboxylase antibody detection kit is adopted to perform detection by a latex immunoturbidimetry method: adding the reagent 1 and the human serum sample according to the proportion, mixing uniformly, reacting for 5 minutes at 37 ℃, adding the reagent 2, mixing uniformly, reading the absorbance value A1 at 600nm after 30 seconds, and reading the absorbance value A2 after 5 minutes. Δ a ═ a2-a1 was calculated.
Preferably, the volume ratio of the reagent 1 and the reagent 2 to the human serum sample for the immune agglutination reaction is 100-150: 30-60: 15.
preferably, the detection range of the concentration of the anti-glutamate decarboxylase antibody of the human serum sample is 0-300 IU/mL.
The invention can obtain at least one of the following beneficial effects:
the kit adopts a latex immunoturbidimetry method as a detection principle to measure the anti-glutamate decarboxylase antibody of serum, in a reaction system, the anti-glutamate decarboxylase antibody in a sample is combined with latex particles coated with glutamate decarboxylase antigen to form an insoluble immune complex, so that reaction liquid is turbid, and the activity of the anti-glutamate decarboxylase in the sample can be calculated according to the change of turbidity absorbance. Compared with the current clinical use kit, the kit has consistent specificity, sensitivity and accuracy, can be applied to various full-automatic biochemical analyzers for large-batch determination, does not need expensive detection instruments, has short detection time, convenient operation, low detection cost and no radioactive pollution, and is more suitable for clinical popularization and use.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
And (3) checking the principle:
in a reaction system, an anti-glutamate decarboxylase antibody in a sample is combined with the glutamic acid decarboxylase coated antigen latex particles to form an insoluble immune complex, so that reaction liquid is turbid, and the activity of the anti-glutamate decarboxylase in the sample can be calculated according to the change of turbidity absorbance.
EXAMPLE 1 preparation of the kit
Reagent 1 and reagent 2
1. Reagent main raw material
The main raw material of the anti-glutamate decarboxylase antibody determination kit (latex immunoturbidimetry) is latex particles coated with glutamate decarboxylase antigen, and products of two manufacturers, namely Beijing Baishanglide and Beijing Huayuan mountain water biotechnology limited company, are selected through investigation and screening of the manufacturers at home and abroad.
Buffer and pH:
| buffer system | pH value |
| Tris-HCl (Tris-hydroxymethyl aminomethane) | 7.5 |
| PBS(NaH 2 PO 4 ·Na 2 HPO 4 ·12H 2 O) | 7.5 |
| Citric acid sodium salt | 6.5 |
| HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) | 7.5 |
| MES (2-morpholine ethanesulfonic acid) | 6.5 |
2. Experimental apparatus and conditions
Full-automatic biochemical analyzer (model Olympus AU400)
The detection method comprises the following steps: temperature detection by end-point method: 37 deg.C
And (3) detecting the wavelength: optical path of 600nm cuvette: 1cm
Water as blank control
3. Experimental methods
60mL of each buffer was used as reagent 1, and 20mL of each buffer was added to 0.2mL of glutamic acid decarboxylase-coated antigen latex particles, and the mixture was thoroughly mixed and used as reagent 2.
Reagent 1: s: adding the reagent 1 and an anti-glutamate decarboxylase antibody sample of 75IU/mL according to the volume ratio of 135:15:45 of the reagent 2, uniformly mixing, reacting at 37 ℃ for 5 minutes, adding the reagent 2, uniformly mixing, reading an absorbance value A1 at 600nm after 30 seconds, and reading an absorbance value A2 after 5 minutes. Calculating the value of delta A which is A2-A1 and the value of delta A is more than or equal to 0.01; and (3) replacing a sample detection reagent blank with purified water, wherein A is less than or equal to 2.0.
1) Calibration data:
| buffer system | Citric acid sodium salt | PBS | MES | HEPES | Tris-HCl |
| 0 | -0.0118 | -0.0118 | -0.0100 | -0.0119 | -0.0118 |
| 15.5 | 0.0404 | 0.0401 | 0.0420 | 0.0410 | 0.0404 |
| 55 | 0.1951 | 0.1161 | 0.1964 | 0.1018 | 0.0802 |
| 104.5 | 0.4717 | 0.2422 | 0.4489 | 0.2025 | 0.2396 |
| 204.2 | 0.7193 | 0.3562 | 0.6785 | 0.3811 | 0.2953 |
| 265.4 | 0.9960 | 0.5441 | 0.9590 | 0.5632 | 0.4923 |
| ABS | 0.5877 | 0.6063 | 0.6111 | 0.5801 | 0.6042 |
2) Test data:
and analyzing test data, wherein in 5 buffer systems, when the buffer solutions are MES and sodium citrate, the CV values of measured values are relatively close, and the mean value is smaller than the CV values of other buffer solutions, which indicates that when the buffer solutions are MES and sodium citrate, the repeatability of the Anti-GAD sample is the best, and further research needs to be carried out to determine the reagent buffer system.
3) Accelerated test at 37 ℃:
reagents prepared under MES and sodium citrate buffer systems are respectively subjected to accelerated stability at 37 ℃. Detecting at 0 day, 4 days and 7 days, repeatedly measuring and comparing the quality control product of the manufacturer for 3 times, calculating the CV value and the deviation of the average value and 0 day, and observing the stability of the reagent under different buffer solution systems.
As can be seen from the data of 3 times of tests, the deviation of the reagent test sample prepared by MES buffer solution is gradually increased along with the change of time, while the deviation of the reagent test sample prepared by sodium citrate buffer solution is not greatly changed along with the change of time, and the stability of the reagent prepared by sodium citrate buffer solution is best by comparison, so that the Anti-GAD reagent selects a sodium citrate buffer system with the pH value of 6.5.
4. Preparation of reagents 1 and 2
(1) Reagent 1 was prepared as follows: 100mmol/L sodium citrate, 0.15mol/L sodium chloride and 0.03 wt% preservative; the raw materials are weighed according to the proportion and then added into pure water, the stirring speed is 100r/min, and the stirring time is 30 min.
(2) Reagent 2 was prepared as follows: 100mmol/L of sodium citrate, 1 wt% of bovine serum albumin, 201 wt% of tween-201, 0.03 wt% of preservative and 1 wt% of glutamic acid decarboxylase coated antigen latex particles; the raw materials are weighed according to the proportion and then added into pure water, the stirring speed is 100r/min, and the stirring time is 30 min.
The antiseptic is sodium azide.
Second, calibration material and quality control material
1. Selection of buffer and pH
The test method comprises the following steps:
10mL of each buffer solution is respectively added with 1500IU of anti-glutamate decarboxylase antibody and fully mixed.
1) Test data:
in 4 buffer systems, when the buffer solution is Tris-HCl, the measured CV values are relatively close to each other in 3 pH ranges and are all smaller than the CV values of other buffer solutions, which indicates that when the buffer solution is Tris-HCl, the Anti-GAD sample has the best repeatability, so Tris-HCl is selected as the Anti-GAD calibrator quality control buffer system.
2) Accelerated test at 37 ℃:
samples prepared by 3 pH gradients under a Tris-HCl buffer system are respectively subjected to accelerated stability at 37 ℃. And (3) detecting for 0 day, 4 days and 7 days respectively, repeatedly measuring the samples prepared under 3 pH values in a Tris-HCl buffer system for 3 times, calculating the deviation of the CV values and the mean value from 0 day, and observing the stability of the samples under different pH values.
As can be seen from the data of 3 times of tests, the change of the samples under 3 pH values shows a consistent trend, namely the deviation is gradually increased along with the change of time when the samples are placed at 37 ℃, and the comparison shows that the stability of the prepared Anti-GAD sample is the best when the pH value is 7.5, so that a Tris-HCl buffer system with the pH value of 7.5 is selected as the Anti-GAD calibrator and the quality control product.
2. Preparation of calibrator and quality control material
1) The calibrator comprises 50% human serum, 50mmol/L buffer, 5% protective agent, 0.15mol/L sodium chloride, 0.03% preservative, and anti-glutamate decarboxylase antibody with a target value concentration range of 270-400 IU/mL. Weighing the raw materials according to the above proportion, adding into pure water, stirring at 100r/min for 30min to obtain the final product.
2) The quality control product comprises 50% of human serum, 50mmol/L of buffering agent, 5% of protective agent, 0.15mol/L of sodium chloride, 0.03% of preservative and anti-glutamate decarboxylase antibody, wherein the target concentration range of the level 1 of the anti-glutamate decarboxylase antibody is 3-50 IU/mL, and the target concentration range of the level 2 of the anti-glutamate decarboxylase antibody is 100-200 IU/mL. Weighing the raw materials according to the proportion, adding the raw materials into pure water, and stirring at the speed of 100r/min for 30min to obtain the product.
The antiseptic is sodium azide.
The freeze-drying parameters of the calibrator and the quality control product are set as follows:
| set temperature (. degree. C.) | Setting time (min) | Duration (min) | |
| Prefreezing | -45 | 300 | |
| Sublimation drying stage 1 | -30 | 30 | 180 |
| Sublimation drying stage 2 | -20 | 30 | 180 |
| Sublimation drying stage 3 | -10 | 30 | 300 |
| Sublimation drying stage 4 | -5 | 30 | 180 |
| Sublimation drying stage 5 | 0 | 30 | 180 |
| Stage 1 of analytical drying | 5 | 30 | 60 |
| 2 nd stage of desorption drying | 15 | 30 | 60 |
| 3 rd stage of desorption drying | 30 | 30 | 180 |
In order to improve the appearance of the calibrator and the quality control product after freeze-drying, 5 percent of trehalose is added as a protective agent and an excipient. The calibrator and the quality control product are in compact cake shapes after being freeze-dried, and meet the technical requirements of products.
Third, the composition of the kit
EXAMPLE 2 detection method of kit
Detection method
1. Reagent preparation
The liquid double-reagent can be used after opening the bottle, and the reagent can be stored for 30 days at the temperature of 2-8 ℃ after opening the bottle. Before the calibrator and the quality control product are used, 1.00mL of purified water is used for redissolving, the temperature is kept between 10 and 30 ℃ for 30 minutes, and after the freeze-dried substances are completely dissolved, the original calibrator solution and the original quality control product solution can be used after being gently mixed. The original calibrator solution was diluted with purified water to six concentration gradients according to the following table (vol/vol):
| serial number | 1 | 2 | 3 | 4 | 5 | 6 |
| Purified water stock solution | 1:0 | 15:1 | 7:1 | 3:1 | 1:1 | 0:1 |
2. Parameter setting
The detection method comprises the following steps: temperature detection by end-point method: 37 deg.C
And (3) checking wavelength: optical path of 600nm cuvette: 1cm
Water as blank control
3. Inspection step
4. Calibration procedure
And multi-point calibration correction is adopted.
5. Quality control program
Before the sample is detected every day, quality control is required to ensure the stability of the test system. The quality control product measurement result is within the allowable range.
6. Computing
7. Reference interval
Serum: less than or equal to 10IU/mL
The reference interval is obtained by verifying 200 normal population samples by the company and adopting a normal distribution method under a 95% confidence interval, and each laboratory is recommended to establish the reference interval due to the differences of geography, race, gender, age and the like.
Second, product performance index
1. Blank limit: the blank limit of the kit is less than or equal to 2 IU/mL.
2. Accuracy: the international standard substance (number: WHO 97/550) was tested in duplicate for 3 times, and the relative deviation of the test results did not exceed. + -. 10%.
3. Linearity:
within the interval of [3, 300] IU/mL, the linear correlation coefficient r is more than or equal to 0.990;
within the interval of [3, 100] IU/mL, the absolute deviation of linearity is not more than +/-10 IU/mL;
within the interval of (100, 300] IU/mL, the relative deviation of linearity does not exceed +/-10%.
4. Precision:
the CV of the variation coefficient of the result is less than or equal to 10 percent when the samples with high and low concentrations are tested.
Three batches of the kit are randomly drawn, the same sample is tested, and the relative range (R) among the batches is less than or equal to 10 percent.
The coefficient of variation CV among the calibrator and the quality control bottles is less than or equal to 10 percent.
5. Quality control material assignment validity:
the quality control detection result is within the quality control range.
The quality control detection result is within the quality control range.
6. Evaluation of interference Capacity
If the specimen contains the following interferents in concentration, the detection result is not obviously influenced:
bilirubin is less than or equal to 20mg/dL, hemoglobin is less than or equal to 5g/L, ascorbic acid is less than or equal to 20mg/dL, triglyceride is less than or equal to 15mmol/L, fat emulsion is less than or equal to 750mg/dL, and rheumatoid factor is less than or equal to 500 IU/mL.
For samples outside the linear range of the kit, the results are reported multiplied by the dilution factor, but the sample dilution factor should not be more than 5.
Example 3 detection of the kit of example 1
The kit of example 1 is tested according to the testing method and the performance index of the product of example 2.
1. Margin limit
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Mean value of | SD | Margin limit |
| 0.53 | 0.76 | 0.75 | 0.62 | 0.72 | 0.56 | 0.63 | 0.61 | 0.69 | 0.80 | 0.70 | 0.10 | 0.90 |
The blank limit of the kit meets the requirement of less than or equal to 0.9 ng/mL.
2. Accuracy of
3. Linear range
Standard samples were prepared, and samples at each concentration were measured 3 times using the kit of example 1 and averaged to perform correlation analysis of the measured values. The experimental verification shows that the analysis and measurement range of the anti-glutamate decarboxylase antibody is 0-300U/mL, R is 1.0000, and the results are shown in the following table.
4. Precision degree
The reagents prepared in example 1 were used to determine the in-batch precision. Two levels of quality control serum, low and high, were selected as evaluation samples, precision within the batch: the measurement was repeated 10 times in a short period of time and under stable conditions, and the measurement results were recorded for each time and the mean, Standard Deviation (SD), and Coefficient of Variation (CV) were calculated.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Mean value of | SD | CV |
| 25.82 | 24.55 | 24.98 | 25.40 | 24.94 | 25.69 | 25.00 | 24.87 | 25.07 | 24.26 | 25.06 | 0.48 | 1.92％ |
| 143.41 | 147.78 | 152.25 | 152.09 | 155.05 | 146.08 | 150.90 | 152.88 | 150.10 | 152.55 | 150.31 | 3.55 | 2.36％ |
The CV in the kit is up to 2.36 percent and meets the requirement that the reagent is less than or equal to 10 percent.
The measurement method of the variation coefficient between the calibrator and the quality control vial was the same as above.
The variation coefficient between the calibrator and the quality control bottle meets the requirement that the reagent is less than or equal to 10 percent.
5. Quality control material assignment validity
| 1 | 2 | 3 | Range of quality control | Conclusion | |
| Level 1 | 24.73 | 25.66 | 24.96 | 25.04(20.03～30.05)IU/mL | Qualified |
| Level 2 | 146.38 | 153.63 | 151.49 | 149.9(119.92～179.88)IU/mL | Qualified |
6. Evaluation of shelf-Life stability
And (4) carrying out experimental monitoring on the 14 th month sample remained at the temperature of between 2 and 8 ℃, wherein all indexes of the sample are in accordance with requirements.
The specific data are as follows:
batch number: SA01810101 date of production: 2018.04.03 expiration date to: 2019.04.02 detection time: 2019.06.10
7. Evaluation of reconstitution stability
And carrying out redissolution stability detection on the 2-8 ℃ sample calibrator and the quality control product in the near-term effect, wherein all indexes of the sample calibrator and the quality control product meet the requirements. The specific data are as follows:
1) calibrator, quality control of a batch of product redissolving assay data on day 9 (control):
batch number: production date of SA 01810101: 2018.04.03 valid period to: 2019.04.02 reconstitution date: 2019.03.29 detection time: 2019.03.29
2) The initial time of the calibration material and the quality control material of a batch of products is redissolved, and the measurement data is stored for 9 days under the condition of 2-8 ℃ in a sealed way:
batch number: production date of SA 01810101: 2018.04.03 valid period to: 2019.04.02 reconstitution date: 2019.03.20 detection time: 2019.03.29
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still make modifications to the technical solutions described in the foregoing embodiments, or make equivalent substitutions and improvements to part of the technical features of the foregoing embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An anti-glutamate decarboxylase antibody assay kit, comprising a reagent 1 and a reagent 2 which are independent of each other, wherein:
the reagent 1 consists of a stabilizing agent and a preservative which are put in a proper buffer solution;
the reagent 2 is composed of bovine serum albumin, a surfactant, a preservative and latex particles coated with glutamic acid decarboxylase antigen in a proper buffer solution.
2. The anti-glutamate decarboxylase antibody assay kit as claimed in claim 1, wherein said stabilizing agent comprises one or more of sodium chloride, potassium chloride, sucrose;
the preservative comprises one or more of sodium azide, Procline300, potassium sorbate, sodium benzoate and sodium nitrite;
the buffer solution comprises sodium citrate, 2-morpholine ethanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, tris (hydroxymethyl) aminomethane or PBS buffer solution;
the surfactant comprises one or more of Tween 20, Tween 40 and Triton X-100.
3. The kit for determining an anti-glutamate decarboxylase antibody according to claim 1, wherein the reagent 1 comprises 50-150 mmol/L of a buffering agent, 0.05-0.3 mol/L of a stabilizing agent, 0.01-0.06 wt% of a preservative, and the pH value of the buffering agent is 6.5-7.5;
the reagent 2 comprises 50-150 mmol/L of a buffering agent, 0.5-2 wt% of bovine serum albumin, 0.5-2 wt% of a surfactant, 0.01-0.06 wt% of a preservative, 0.2-3 wt% of glutamic acid decarboxylase coated antigen latex particles, and the pH value of the buffering agent is 6.5-7.5.
4. The kit for the determination of an anti-glutamate decarboxylase antibody according to claim 3, wherein said reagent 1 comprises buffer 100mmol/L, stabilizer 0.15mol/L, preservative 0.03 wt%;
the reagent 2 comprises 100mmol/L of buffer, 1 wt% of bovine serum albumin, 1 wt% of surfactant, 0.03 wt% of preservative and 1 wt% of glutamic acid decarboxylase-coated antigen latex particles.
5. The kit for determining an anti-glutamate decarboxylase antibody according to claim 1, further comprising a calibrator comprising 50 wt% human serum, 25-100 mmol/L buffer, 2-10 wt% protective agent, 0.05-0.3 mol/L sodium chloride, 0.01-0.06 wt% preservative, and an anti-glutamate decarboxylase antibody having a target concentration ranging from 270-400 IU/mL.
6. The kit for determining an anti-glutamate decarboxylase antibody according to claim 1, further comprising a quality control substance, wherein the quality control substance comprises 50 wt% of human serum, 25-100 mmol/L of buffer, 2-10 wt% of protective agent, 0.05-0.3 mol/L of sodium chloride, 0.01-0.06 wt% of preservative, and an anti-glutamate decarboxylase antibody, wherein the target concentration range of the anti-glutamate decarboxylase antibody at level 1 is 3-50 IU/mL, and the target concentration range of the anti-glutamate decarboxylase antibody at level 2 is 100-200 IU/mL.
7. The anti-glutamate decarboxylase antibody assay kit according to claim 5 or 6, wherein the buffering agent comprises sodium citrate, 2-morpholine ethanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, tris (hydroxymethyl) aminomethane or PBS, and the preservative comprises one or more of sodium azide, Procline300, potassium sorbate, sodium benzoate and sodium nitrite.
8. A method for detecting the content of an anti-glutamate decarboxylase antibody is characterized in that the anti-glutamate decarboxylase antibody detection kit as claimed in any one of claims 1 to 7 is adopted, and the detection is carried out by a latex immunoturbidimetry method: adding the reagent 1 and the human serum sample according to the proportion, mixing uniformly, reacting for 5 minutes at 37 ℃, adding the reagent 2, mixing uniformly, reading the absorbance value A1 at 600nm after 30 seconds, and reading the absorbance value A2 after 5 minutes. Δ a ═ a2-a1 was calculated.
9. The kit for determining an anti-glutamate decarboxylase antibody according to claim 8, wherein the volume ratio of the reagent 1, the reagent 2 and the human serum sample for immunoagglutination reaction is 100-150: 30-60: 15.
10. the kit for determining an anti-glutamate decarboxylase antibody according to claim 8, wherein the concentration of the anti-glutamate decarboxylase antibody in the human serum sample is detected within the range of 0-300 IU/mL.
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