CN112485419A - Zinc transporter 8 antibody detection kit - Google Patents
Zinc transporter 8 antibody detection kit Download PDFInfo
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- CN112485419A CN112485419A CN202011344497.0A CN202011344497A CN112485419A CN 112485419 A CN112485419 A CN 112485419A CN 202011344497 A CN202011344497 A CN 202011344497A CN 112485419 A CN112485419 A CN 112485419A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention relates to the field of biological detection, and particularly discloses a zinc transporter 8 antibody detection kit which comprises zinc transporter 8 antigen coated magnetic beads, biotin-labeled zinc transporter 8 antigen, alkaline phosphatase-labeled streptavidin and chemiluminescent substrate solution. The kit prepared by the invention has the advantages of simple components, convenient operation, quick and convenient detection, high sensitivity, strong specificity and stable quality, can obviously improve the discrimination of positive serum and negative serum, reduces the omission phenomenon of the positive serum caused by a single antigen, and further greatly reduces the omission phenomenon of related diseases during early diagnosis.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a zinc transporter 8 antibody detection kit.
Background
The zinc transport protein 8(ZnT8, also named zinc transporter 8) is encoded by a gene SLC30A8 (chromosome 8q24.11), belongs to a member of zinc transport protein (ZnT)/SLC30 family, is only highly expressed in islet beta cells, is distributed on a vesicle membrane containing insulin particles in the islet cells, and plays an important role in the maturation, storage and secretion processes of insulin. The main function of the medicine is to transport Zn2+ into the vesicle by utilizing the concentration difference of H + formed by a proton pump on the vesicle membrane, so that Zn2+ is accumulated in the vesicle to participate in the maturation and storage of insulin.
ZnT8 is involved in the pathophysiological processes of diabetic islet cell dysfunction and insulin hyposecretion. Zinc is a regulator of glucagon secretion by islet alpha and beta cells and insulin paracrine and autocrine, while ZnT8 may influence insulin synthesis and secretion by affecting zinc ion concentration. The ZnT8 protein as an autoantigen causes T lymphocyte-mediated autoimmune reaction which is characterized by beta cell damage, further triggers the specific damage of the autoimmune system to the beta cells of the pancreatic islets, leads to the absolute deficiency of insulin in vivo and finally triggers type 1 diabetes (T1 DM).
ZnT8A is internationally recognized as the fourth main islet autoantibody after IAA and IA-2A, GADA, and has important application in diagnosing T1DM and discussing pathogenesis. According to the research, the positive rate of the ZnT8A infant with the adolescent type 1 diabetes is the highest, and the positive rate is reduced along with the increase of the age. In addition, researches show that the positive rate of a newly-discovered T1DM patient is as high as 60-80%, and a 26% positive rate still exists in a T1DM patient with negative IA-2A, IAA and GADA, so that the positive detection rate of T1DM can be improved by combining ZnT8A with IA-2A, IAA and GADA antibodies, and people with more serious insulin deficiency can be distinguished from IA-2A, IAA and GADA positive patients, so that the diagnosis of patients is more accurate, and the treatment of the patients is more targeted.
At present, common methods for clinically detecting the zinc transporter 8 antibody include an enzyme-linked immunosorbent assay (double-antigen sandwich method), an indirect magnetic particle chemiluminescence assay and the like, and the detection methods have some defects in clinical application. The zinc transporter 8 antibody enzyme-linked immunosorbent assay (double antigen sandwich method) has some defects in clinical application. Firstly, the method fixes the antigen on a special microporous plate, and the antigen can only be used according to the specification of the plate hole when in use, and can not realize independent and single-person detection. Secondly, the kit has complex components and complicated operation steps, and is not favorable for clinical examination operators to accurately and quickly obtain examination results. In addition, the detection process of the method is in an open space, and cross contamination of reagents is easily caused to influence the accuracy of the detection result. The indirect magnetic particle chemiluminescence method adopts an enzyme-labeled or chemiluminescent substance-labeled anti-human antibody, and the antibody can be combined with immune complexes of zinc transporters 8 and 8 on magnetic beads, and can also be combined with antibodies of human IgG and other types adsorbed on the magnetic beads to cause false positive. In addition, the indirect magnetic particle chemical method for detecting the antibody type of the zinc transporter 8 antibody has limitations, cannot detect all types of zinc transporter 8 antibodies in blood, and is not beneficial to early screening, early diagnosis and early treatment of diseases.
Therefore, it is needed to provide a kit for detecting the zinc transporter 8 antibody in blood, which has the advantages of stable quality, rapid and convenient detection, high sensitivity and strong specificity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a zinc transporter 8 antibody detection kit, the kit has the advantages of simple components, convenient operation, quick and convenient detection, high sensitivity, strong specificity and stable quality, and the kit prepared by the invention can also obviously improve the discrimination of positive serum and negative serum and reduce the omission phenomenon of positive serum by a single antigen.
In order to achieve the purpose, the invention adopts the technical scheme that:
a zinc transporter 8 antibody detection kit comprises zinc transporter 8 antigen coated magnetic beads, biotin labeled zinc transporter 8 antigen, alkaline phosphatase labeled streptavidin and chemiluminescent substrate solution.
In the technical scheme of the invention, the inventor firstly adopts a magnetic bead double-antigen sandwich method to coat a zinc transporter 8 antigen with magnetic beads, a biotin-labeled zinc transporter 8 antigen and a zinc transporter 8 antibody in a blood sample to form an antigen-antibody-antigen complex, and detects a positive serum sample and a negative serum sample, wherein the positive serum sample and the negative serum sample have good discrimination, and the method can accurately detect various types of zinc transporter 8 antibodies in serum, simultaneously overcomes the problem of high false positive of the zinc transporter 8 antibody measured by an indirect method, greatly reduces the phenomena of missed detection and false positive in early diagnosis of related diseases, and is favorable for a clinician to more accurately make a clinical scheme.
As a preferred embodiment of the zinc transporter 8 antibody detection kit, the concentration of the zinc transporter 8 antigen coated magnetic beads is 50-200mg/L, the concentration of the biotin-labeled zinc transporter 8 antigen is 20-100. mu.g/L, and the concentration of the alkaline phosphatase-labeled streptavidin is 2-10. mu.g/L.
As a preferred embodiment of the zinc transporter 8 antibody detection kit of the present invention, the zinc transporter 8 includes a zinc transporter 8(R325) and a zinc transporter 8 (W325).
As a preferred embodiment of the zinc transporter 8 antibody detection kit of the present invention, the preparation method of the zinc transporter 8 antigen coated magnetic bead comprises the following steps: and (2) taking the carboxylated magnetic beads, removing the supernatant after magnetic separation, adding a buffer solution for washing, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution and a 2- (N-morphine-forest) ethanesulfonic acid aqueous solution containing N-hydroxysuccinimide, activating the carboxyl groups on the surfaces of the magnetic beads at room temperature, washing and resuspending, then adding a zinc transporter 8, removing the supernatant through magnetic separation, and adding a buffer solution containing bovine serum albumin to obtain the zinc transporter 8 antigen coated magnetic beads.
The zinc transporter 8 antigen coated magnetic bead prepared by the invention can be combined with all kinds of zinc transporter 8 antibodies, and the phenomenon of missed detection of the conventional reagent is avoided.
As a preferred embodiment of the zinc transporter 8 antibody detection kit, the particle size of the carboxylated nanometer magnetic bead is 0.5-1.5 μm.
As a preferred embodiment of the zinc transporter 8 antibody detection kit of the present invention, the preparation method of the biotin-labeled zinc transporter 8 antigen comprises the following steps: adding the water solution of the zinc transporter 8 into the buffer solution, mixing uniformly, adding the dimethylformamide solution containing the biotin label, mixing uniformly, adding ethanolamine to obtain a reaction solution, and purifying the reaction solution by using a G-25 desalting column to obtain the zinc transporter.
As a preferred embodiment of the zinc transporter 8 antibody detection kit, the preparation method of the alkaline phosphatase labeled streptavidin comprises the following steps:
s1, activation of alkaline phosphatase: mixing alkaline phosphatase with a buffer solution to prepare an alkaline phosphatase solution, adding a SATA solution for reaction, desalting, adding a hydroxylamine hydrochloride solution, reacting at room temperature, and desalting;
s2, activation of streptavidin: adding 4- (N-maleimide methyl) cyclohexane carboxylic acid-N-succinimide ester into a streptavidin solution, and reacting for 1-2h at room temperature;
s3, alkaline phosphatase labeling of streptavidin: and mixing the activated alkaline phosphatase with streptavidin, reacting at room temperature for 1-3h, taking out and purifying to obtain the product.
As a preferred embodiment of the zinc transporter 8 antibody detection kit of the present invention, the kit further comprises a treatment agent.
As a preferred embodiment of the zinc transporter 8 antibody detection kit, the processing agent comprises a surfactant, Proclin300, a chloride salt and a Tris-HCl buffer containing 2% BSA.
In addition, another object of the present invention is to provide a method for detecting a zinc transporter 8 antibody using the above-mentioned kit.
In order to realize the purpose, the invention adopts the technical scheme that: adopting zinc transporter 8 antigen coated magnetic beads, biotin-labeled zinc transporter 8 antigen and zinc transporter 8 antibody in a blood sample to incubate at room temperature, adding alkaline phosphatase-labeled streptavidin after magnetic separation and washing, performing magnetic separation and washing again, finally adding chemiluminescent substrate solution, and detecting a luminescence value.
By adopting the method to measure the luminous value and comparing the luminous value with a standard curve, the content of the zinc transporter 8 antibody in the sample can be calculated, and the positive correlation between the content of the zinc transporter 8 antibody and the luminous value is found; the invention adopts the 8 antigens of the composite zinc transporter, the composite antigen can be combined with most antibodies in the blood sample, the missing detection phenomenon of the past reagent is avoided, and simultaneously, the method adopts the double-antigen sandwich to detect all kinds of antibodies in the blood sample, and the development condition of the disease can be reflected more accurately.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention provides a zinc transporter 8 antibody detection kit, which has the advantages of simple components, convenient operation, quick and convenient detection, high sensitivity, strong specificity and stable quality;
2) the invention provides a zinc transporter 8 antibody detection kit, which is characterized in that zinc transporter 8 antigen coated magnetic beads, biotin labeled zinc transporter 8 antigen and zinc transporter 8 antibody in a blood sample form an antigen-antibody-antigen complex, and a positive serum sample and a negative serum sample are detected, so that the positive serum sample and the negative serum sample have good discrimination.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
In the following examples or comparative examples, the nanobeads are made of nanoscale Fe2O3Or Fe3O4The magnetic particles and the organic polymer material are compounded to form the nano-scale solid phase microsphere with superparamagnetism and extremely large protein adsorption capacity, and the nano-scale solid phase microsphere has the properties that the nano-scale solid phase microsphere can be quickly magnetized under the action of an external magnetic field and the remanence is zero after the magnetic field is removed.
Example 1 detection kit for Zinc transporter 8 antibody
A zinc transporter 8 antibody detection kit comprises zinc transporter 8 antigen coated magnetic beads, biotin labeled zinc transporter 8 antigen, alkaline phosphatase labeled streptavidin and chemiluminescent substrate solution.
Wherein the concentration of the zinc transporter 8 antigen coated magnetic beads is 50-200 mg/L;
the concentration of the biotin-labeled zinc transporter 8 antigen is 20-100 mug/L;
the concentration of the streptavidin labeled by alkaline phosphatase is 2-10 mug/L;
in the preparation method of the kit for detecting a zinc transporter 8 antibody of the present example, the other reagents except the following reagents are prepared according to a conventional method.
Preparation of zinc transporter 8 antigen coated magnetic beads
Taking 3mg of carboxylated nano magnetic beads (the particle size is 0.5 mu m), after magnetic separation, adding 100 mu L of 25mM 2- (N-morphine-forest) ethanesulfonic acid buffer solution (pH5.0) for washing for 5 times, and after magnetic separation, respectively adding 50 mu L of newly prepared and cold 50mg/mL EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) aqueous solution and 50 mu L of 25mM 2- (N-morphine-forest) ethanesulfonic acid (pH5.0) aqueous solution containing 50mg/mL N-hydroxysuccinimide; after 30min of activation at room temperature, the activated beads were washed twice with 100. mu.L of 25mM 2- (N-morphinan) ethanesulfonic acid buffer (pH5.0), and then 100. mu.L of 25mM 2- (N-morphinan) ethanesulfonic acid buffer (pH5.0) was added for resuspension; respectively taking 120 mu g of zinc transporter 8(R325) and zinc transporter 8(W325) and adding into the activated magnetic beads, and reacting for 2 hours at room temperature; after magnetic separation and removal of the supernatant, 200. mu.L of 100mM Tris-HCl buffer (pH8.0) containing 2% by mass of BSA was added to obtain a zinc transporter 8 antigen-coated magnetic bead stock solution.
The mother liquor of the zinc transporter 8 antigen coated magnetic beads is diluted to 50-200mg/L by 25mM Tris-HCl (pH8.0) containing 0.1% Tween-20, 0.05% Proclin300, 0.15M sodium chloride and 2% BSA, and the zinc transporter 8 antigen coated magnetic beads are prepared.
II, biotin labeling of zinc transporter 8 antigen
mu.L of 10mg/mL zinc transporter 8(R325) and 4. mu.L of 10mg/mL zinc transporter 8(W325) aqueous solutions were added to 200. mu.L of 50mM borate buffer (pH8.5), mixed, and 8-10. mu.L of 20mg/mL NHS-LC-Biotin dimethylformamide solution was added and mixed. After 1 hour of reaction at room temperature, 1.2. mu.L of ethanolamine was added, the reaction was terminated at room temperature for 30 minutes, and the reaction mixture was purified by using a G-25 desalting column and then diluted to 20-100. mu.g/L with 25mM Tris-HCl (pH8.0) containing 0.1% Tween-20, 0.05% Proclin300, 0.15M sodium chloride and 2% BSA, to obtain biotin-labeled zinc transporter 8 antigen.
Alkaline phosphatase labeled streptavidin
Activation of alkaline phosphatase: mixing 7 μ L of alkaline phosphatase and 133 μ L of 100mM PBS buffer solution (pH7.2) to prepare 1mg/mL alkaline phosphatase solution, adding 1 μ L of SATA (N-Succinimidyl-S-acetylthioacetate) (dissolved in DMSO) with a mass concentration of 10mg/mL, reacting for 30min, and desalting after 30min (centrifugal desalting column, 1000g 2 min); to the solution was added 7. mu.L of a 1M hydroxylamine hydrochloride solution (dissolved in 100mM PBS containing 25mM EDTA), and the mixture was reacted at room temperature for 2 hours and then desalted.
Activation of streptavidin: mu.L of a streptavidin solution (0.97mg/mL) was added to 2.2. mu.L of 10mg/mL SMCC (4- (N-maleimidomethyl) cyclohexanecarboxylic acid N-succinimidyl ester) and reacted at room temperature for 1 hour.
Alkaline phosphatase-labeled streptavidin: mixing the activated alkaline phosphatase with streptavidin, reacting at room temperature for 2h, taking out, purifying with 6mL sephadex G75 chromatographic column, and diluting to 2-10 μ G/L with 25mM Tris-HCl (pH8.0) containing 0.1% Tween-20, 0.05% Proclin300, 0.15M sodium chloride and 2% BSA.
In other embodiments, the particle size of the carboxylated nano-magnetic particles can be 1.0 μm or 1.2 μm or 1.5 μm, but is not limited to this embodiment.
Fourthly, the method for detecting the concentration of the zinc transporter 8 antibody by adopting the kit of the embodiment comprises the following steps:
and (3) taking 25 mu L of blood sample, 50 mu L of zinc transporter 8 antigen coated magnetic beads and 50 mu L of biotin labeled zinc transporter 8 antigen, incubating for 30min at 37 ℃, carrying out magnetic separation and washing for 3 times, adding 50 mu L of reagent alkaline phosphatase labeled streptavidin, incubating for 10min at 37 ℃, carrying out magnetic separation and washing for 3 times, adding 100 mu L of chemiluminescent substrate solution, and detecting the luminescence value. After the measurement, a standard curve is drawn by using the value of the concentration of the zinc transporter 8 antibody in the standard as an X coordinate and the signal value as a Y coordinate, and the concentration of the zinc transporter 8 antibody in the sample is calculated according to the standard curve.
Example 2 detection kit for zinc transporter 8 antibody
A zinc transporter 8 antibody detection kit, which is substantially the same as the detection kit of example 1 except that:
wherein the concentration of the zinc transporter 8 antigen coated magnetic beads is 50-200 mg/L;
the concentration of the biotin-labeled zinc transporter 8 antigen is 20-100 mug/L;
the concentration of the streptavidin labeled by alkaline phosphatase is 2-10 mug/L;
in the method for preparing the kit for detecting a zinc transporter 8 antibody of this example, the preparation method of example 1 is referred to, and the other steps are performed according to a conventional method except for the following steps.
Preparation of zinc transporter 8 antigen coated magnetic beads
Taking 3mg of carboxylated nano magnetic beads (the particle size is 0.5 mu m), after magnetic separation, adding 100 mu L of 25mM 2- (N-morphine-forest) ethanesulfonic acid buffer solution (pH5.0) for washing for 5 times, and after magnetic separation, respectively adding 50 mu L of newly prepared and cold 50mg/mL EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) aqueous solution and 50 mu L of 25mM 2- (N-morphine-forest) ethanesulfonic acid (pH5.0) aqueous solution containing 50mg/mL N-hydroxysuccinimide; after 30min of activation at room temperature, the activated beads were washed twice with 100. mu.L of 25mM 2- (N-morphinan) ethanesulfonic acid buffer (pH5.0), and then 100. mu.L of 25mM 2- (N-morphinan) ethanesulfonic acid buffer (pH5.0) was added for resuspension; adding 120 mu g of zinc transporter 8(R325) into the activated magnetic beads, and reacting for 2 hours at room temperature; after magnetic separation and removal of the supernatant, 200. mu.L of 100mM Tris-HCl buffer (pH8.0) containing 2% by mass of BSA was added to obtain a zinc transporter 8 antigen-coated magnetic bead stock solution.
The mother liquor of the zinc transporter 8 antigen coated magnetic beads is diluted to 50-200mg/L by 25mM Tris-HCl (pH8.0) containing 0.1% Tween-20, 0.05% Proclin300, 0.15M sodium chloride and 2% BSA, and the zinc transporter 8 antigen coated magnetic beads are prepared.
The first step of this example differs from the first step of example 1 in that the zinc transporter 8(R325) is coated separately, and the remaining steps are the same as in example 1.
Example 3 detection kit for zinc transporter 8 antibody
A zinc transporter 8 antibody detection kit, which is substantially the same as the detection kit of example 1 except that:
wherein the concentration of the zinc transporter 8 antigen coated magnetic beads is 50-200 mg/L;
the concentration of the biotin-labeled zinc transporter 8 antigen is 20-100 mug/L;
the concentration of the streptavidin labeled by alkaline phosphatase is 2-10 mug/L;
in the method for preparing the kit for detecting a zinc transporter 8 antibody of this example, the preparation method of example 1 is referred to, and the other steps are performed according to a conventional method except for the following steps.
Preparation of zinc transporter 8 antigen coated magnetic beads
Taking 3mg of carboxylated nano magnetic beads (the particle size is 0.5 mu m), after magnetic separation, adding 100 mu L of 25mM 2- (N-morphine-forest) ethanesulfonic acid buffer solution (pH5.0) for washing for 5 times, and after magnetic separation, respectively adding 50 mu L of newly prepared and cold 50mg/mL EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) aqueous solution and 50 mu L of 25mM 2- (N-morphine-forest) ethanesulfonic acid (pH5.0) aqueous solution containing 50mg/mL N-hydroxysuccinimide; after 30min of activation at room temperature, the activated beads were washed twice with 100. mu.L of 25mM 2- (N-morphinan) ethanesulfonic acid buffer (pH5.0), and then 100. mu.L of 25mM 2- (N-morphinan) ethanesulfonic acid buffer (pH5.0) was added for resuspension; adding 120 mu g of zinc transporter 8(W325) into the activated magnetic beads, and reacting for 2 hours at room temperature; after magnetic separation and removal of the supernatant, 200. mu.L of 100mM Tris-HCl buffer (pH8.0) containing 2% by mass of BSA was added to obtain a zinc transporter 8 antigen-coated magnetic bead stock solution.
The mother liquor of the zinc transporter 8 antigen coated magnetic beads is diluted to 50-200mg/L by 25mM Tris-HCl (pH8.0) containing 0.1% Tween-20, 0.05% Proclin300, 0.15M sodium chloride and 2% BSA, and the zinc transporter 8 antigen coated magnetic beads are prepared.
The first step of this example differs from the first step of example 1 in that the zinc transporter 8(W325) is coated alone, and the remaining steps are the same as in example 1.
Example 4A Zinc transporter 8 antibody detection kit (Indirect method)
A zinc transporter 8 antibody detection kit, which is substantially the same as the detection kit of example 1 except that:
wherein the concentration of the zinc transporter 8 antigen coated magnetic beads is 50-200 mg/L;
the concentration of the alkaline phosphatase labeled goat anti-human IgG antibody is 2-10 mug/L;
in the method for preparing the kit for detecting a zinc transporter 8 antibody of this example, the preparation method of example 1 is referred to, and the other steps are performed according to a conventional method except for the following steps.
Alkaline phosphatase labeled goat anti-human IgG antibody
Activation of alkaline phosphatase: mixing 7 μ L of alkaline phosphatase and 133 μ L of 100mM PBS buffer solution (pH7.2) to prepare 1mg/mL alkaline phosphatase solution, adding 1 μ L of SATA (N-Succinimidyl-S-acetylthioacetate) (dissolved in DMSO) with a mass concentration of 10mg/mL, reacting for 30min, and desalting after 30min (centrifugal desalting column, 1000g 2 min); to the solution was added 7uL of 1M hydroxylamine hydrochloride solution (dissolved in 100mM PBS containing 25mM EDTA), and the mixture was reacted at room temperature for 2 hours and then desalted.
Alkaline phosphatase-labeled goat anti-human IgG: mixing the activated alkaline phosphatase with goat anti-human IgG antibody, reacting at room temperature for 2h, taking out, purifying with 6mL sephadex G75 chromatographic column, and diluting to 50-100ng/mL with 25mM Tris-HCl (pH8.0) containing 0.1% Tween-20, 0.05% Proclin300, 0.15M sodium chloride and 2% BSA.
Step three of this example differs from step three of example 1 only in that streptavidin was replaced with goat anti-human IgG antibody.
Fourthly, the method for detecting the concentration of the zinc transporter 8 antibody by adopting the kit of the embodiment comprises the following steps:
after 25. mu.L of the blood sample was diluted 20 times, 25. mu.L of the diluted sample was incubated with 100. mu.L of zinc transporter 8 antigen-coated magnetic beads at 37 ℃ for 30 min. After 3 times of magnetic separation and washing, 50. mu.L of alkaline phosphatase-labeled goat anti-human IgG was added and incubated at 37 ℃ for 10 min. After 3 times of magnetic separation and washing, 100. mu.L of chemiluminescent substrate solution was added to detect the luminescence value. After the measurement, a standard curve is drawn by using the value of the concentration of the zinc transporter 8 antibody in the standard as an X coordinate and the signal value as a Y coordinate, and the concentration of the zinc transporter 8 antibody in the sample is calculated according to the standard curve.
The difference between the step four of the present example and the step four of the example 1 is that an indirect method is used to replace the magnetic bead double antigen sandwich method, and the rest steps are the same as the example 1.
Test example I, evaluation of Performance of Zinc Transporter 8 antibody detection kit
The kit for detecting the zinc transporter 8 antibody and the detection method thereof in the embodiment are adopted for experimental comparison.
1. Sensitivity of the probe
The functional sensitivity is also called clinical sensitivity (clinical sensitivity) and is used for distinguishing the analysis ability from the existence on the basis of low concentration, the value is determined by taking the standard as gradient dilution, the same instrument, the same batch number reagent and the same standard curve are adopted for measuring 1 time per day and 3 times per time, the data of more than 3 weeks are measured totally, and the maximum dilution tube concentration when the batch variation Coefficient (CV) is less than or equal to 20 percent is taken as the functional sensitivity.
(1) Preparation of standard sample: preparing zinc transporter 8 antibody standard samples with concentration values of 10U/mL, 20U/mL, 75U/mL, 500U/mL and 2000U/mL respectively, and performing functional sensitivity experiments, wherein the numbers of the zinc transporter 8 antibody standard samples are respectively No. 1-No. 5.
(2) The detection method comprises the following steps: the standard samples were measured on a full-automatic chemiluminescence analyzer SMART 6500 using the kits and the detection methods of examples 1-4, respectively, and the sensitivity of the kit obtained by the detection of the measurement results was 0.15U/mL.
2. Linear range
Selecting a high-value serum sample and a negative serum sample of the clinical zinc transporter 8 antibody, mixing to prepare a linear sample, wherein the concentrations are respectively 10U/mL, 20U/mL, 75U/mL, 500U/mL and 2000U/mL. The linear sample is detected by using the zinc transporter 8 antibody detection reagent, the correlation coefficient r of the concentration of the linear sample and the corresponding luminescence value is calculated, and the result shows that the correlation coefficient r is 0.996.
3. Interference test
Taking mixed high, medium and low value serum to be respectively added with interferents: bilirubin, hemoglobin, ascorbic acid, and glyceride were added at a ratio of 1:20, and the measurement values of the mixed serum and the mixed serum added with various interfering substances were measured, respectively, and the deviation between the two was calculated to be within an acceptable range of. + -. 10%. The result shows that the interference of each interfering substance on the detection reagent meets the relevant clinical standard.
Test example II comparison of magnetic beads coated with Zinc Transporter 8 antigen with magnetic beads coated with Zinc Transporter 8 antigen alone
8 zinc transporter 8 antibody serum samples are taken and respectively numbered 1-8, wherein 4 negative sera and 4 positive sera are respectively obtained from the serum samples which are determined to be negative and positive by a ZnT8 autoantibody (ZnT8Ab) ELISA detection kit produced by RSR company of volunteers, and the zinc transporter 8 antibody detection kit prepared in the embodiment 1-3 is adopted to respectively detect the zinc transporter 8 antibody clinical negative and positive samples, and the detection results are shown in the table 1.
TABLE 1
As can be seen from the data in Table 1, compared with the single zinc transporter 8 antigen coated magnetic bead, the zinc transporter 8 antigen coated magnetic bead has the advantages that the discrimination of positive serum and negative serum is obviously improved, and the omission phenomenon of the positive serum caused by the single antigen is reduced.
Test example III comparison of detection of negative and positive sera by magnetic bead double antigen Sandwich method and Indirect method
10 samples of zinc transporter 8 antibody serum are taken and numbered 1-10 respectively, the serum samples are obtained from negative and positive serum samples determined by a ZnT8 autoantibody (ZnT8Ab) ELISA detection kit produced by RSR company of volunteers, wherein the negative serum and the positive serum are 5 samples respectively, and the results are shown in Table 2 according to the method for detecting the concentration of the zinc transporter 8 antibody by the kit of example 1 and the kit of example 4.
TABLE 2
From the data in table 2, in the aspect of detecting clinical negative serum samples of the zinc transporter 8 antibody, the magnetic bead double-antigen sandwich method reagent detects 5 clinical negative samples of the zinc transporter 8 antibody, and the luminescence values of all the negative samples are within the control range around 1000; in example 4, 5 clinical negative samples of the zinc transporter 8 antibody are detected by adopting an indirect method reagent, and two false positive samples and one suspected positive sample appear;
in the aspect of detecting clinical positive samples of zinc transporter 8 antibodies, the magnetic bead double antigen reagent detects 5 clinical positive samples of zinc transporter 8 antibodies, 5 positive serum samples have good discrimination with negative serum samples, and the detection luminescence value of 3 positive samples is obviously improved compared with the detection luminescence value of an indirect method, which shows that the magnetic bead double antigen sandwich method reagent can detect various types of zinc transporter 8 antibodies in serum, and the omission phenomenon of indirect method positive samples can be avoided in actual clinical examination.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A zinc transporter 8 antibody detection kit is characterized by comprising zinc transporter 8 antigen coated magnetic beads, biotin-labeled zinc transporter 8 antigen, alkaline phosphatase-labeled streptavidin and chemiluminescent substrate solution.
2. The kit of claim 1, wherein the concentration of the zinc transporter 8 antigen coated magnetic beads is 50-200mg/L, the concentration of the biotin-labeled zinc transporter 8 antigen is 20-100 μ g/L, and the concentration of the alkaline phosphatase-labeled streptavidin is 2-10 μ g/L.
3. The kit of claim 1, wherein the zinc transporter 8 comprises a zinc transporter 8(R325) and a zinc transporter 8 (W325).
4. The kit of claim 1, wherein the zinc transporter 8 antigen coated magnetic bead is prepared by a method comprising the steps of: and (2) taking the carboxylated magnetic beads, removing the supernatant after magnetic separation, adding a buffer solution for washing, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution and a 2- (N-morphine-forest) ethanesulfonic acid aqueous solution containing N-hydroxysuccinimide, activating the carboxyl groups on the surfaces of the magnetic beads at room temperature, washing and resuspending, then adding a zinc transporter 8, removing the supernatant through magnetic separation, and adding a buffer solution containing bovine serum albumin to obtain the zinc transporter 8 antigen coated magnetic beads.
5. The kit of claim 4, wherein the carboxylated magnetic beads have a particle size of from 0.5 to 1.5 μm.
6. The kit of claim 1, wherein the biotin-labeled zinc transporter 8 antigen is prepared by a method comprising the steps of: adding the water solution of the zinc transporter 8 into the buffer solution, mixing uniformly, adding the dimethylformamide solution containing the biotin label, mixing uniformly, adding ethanolamine to obtain a reaction solution, and purifying the reaction solution by using a G-25 desalting column to obtain the zinc transporter.
7. The kit of claim 1, wherein the method for preparing streptavidin labeled with alkaline phosphatase comprises the steps of:
s1, activation of alkaline phosphatase: mixing alkaline phosphatase with a buffer solution to prepare an alkaline phosphatase solution, adding a SATA solution for reaction, desalting, adding a hydroxylamine hydrochloride solution, reacting at room temperature, and desalting;
s2, activation of streptavidin: adding 4- (N-maleimide methyl) cyclohexane carboxylic acid-N-succinimide ester into a streptavidin solution, and reacting for 1-2h at room temperature;
s3, alkaline phosphatase labeling of streptavidin: and mixing the activated alkaline phosphatase with streptavidin, reacting at room temperature for 1-3h, taking out and purifying to obtain the product.
8. The kit of any one of claims 1 to 7, wherein the kit further comprises a treatment agent.
9. The kit of claim 8, wherein the treatment agents comprise a surfactant, Proclin300, chloride salt, and Tris-HCl buffer with 2% BSA.
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| WO2023103464A1 (en) * | 2021-12-09 | 2023-06-15 | 深圳市亚辉龙生物科技股份有限公司 | Zinc transporter 8 antibody chemiluminescence immunoassay kit and preparation method thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103328532A (en) * | 2010-01-19 | 2013-09-25 | 赛里根Ii有限公司 | Novel reagents for directed biomarker signal amplification |
| WO2016193714A1 (en) * | 2015-06-03 | 2016-12-08 | The University Of Lincoln | Diagnostic target |
| CN106226526A (en) * | 2016-06-30 | 2016-12-14 | 深圳市亚辉龙生物科技股份有限公司 | A kind of Zinc transporter 8 antibody chemical luminescence immunity detection reagent and preparation method thereof |
| CN106771233A (en) * | 2016-12-05 | 2017-05-31 | 上海良润生物医药科技有限公司 | ZnT8A autoantibody detection kits |
| CN108414766A (en) * | 2018-01-29 | 2018-08-17 | 上海良润生物医药科技有限公司 | Kit for quantitatively detecting diabetes autoantibody and its application |
| CN108918870A (en) * | 2018-07-07 | 2018-11-30 | 广东省实验动物监测所 | The liquid-phase chip detection method of pig parvoviral |
| CN111316099A (en) * | 2017-07-12 | 2020-06-19 | 约翰霍普金斯大学 | Proteoliposome-based ZNT8 autoantigen for diagnosis of type 1 diabetes |
| CN111579791A (en) * | 2020-04-30 | 2020-08-25 | 江苏省人民医院（南京医科大学第一附属医院） | Electrochemical luminescence detection kit for zinc transporter 8 islet autoantibodies |
| CN111579781A (en) * | 2020-05-21 | 2020-08-25 | 深圳市宇诺生物技术有限公司 | Hepatitis C virus antibody detection kit, preparation method and detection method |
-
2020
- 2020-11-25 CN CN202011344497.0A patent/CN112485419A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103328532A (en) * | 2010-01-19 | 2013-09-25 | 赛里根Ii有限公司 | Novel reagents for directed biomarker signal amplification |
| WO2016193714A1 (en) * | 2015-06-03 | 2016-12-08 | The University Of Lincoln | Diagnostic target |
| CN106226526A (en) * | 2016-06-30 | 2016-12-14 | 深圳市亚辉龙生物科技股份有限公司 | A kind of Zinc transporter 8 antibody chemical luminescence immunity detection reagent and preparation method thereof |
| CN106771233A (en) * | 2016-12-05 | 2017-05-31 | 上海良润生物医药科技有限公司 | ZnT8A autoantibody detection kits |
| CN111316099A (en) * | 2017-07-12 | 2020-06-19 | 约翰霍普金斯大学 | Proteoliposome-based ZNT8 autoantigen for diagnosis of type 1 diabetes |
| CN108414766A (en) * | 2018-01-29 | 2018-08-17 | 上海良润生物医药科技有限公司 | Kit for quantitatively detecting diabetes autoantibody and its application |
| CN108918870A (en) * | 2018-07-07 | 2018-11-30 | 广东省实验动物监测所 | The liquid-phase chip detection method of pig parvoviral |
| CN111579791A (en) * | 2020-04-30 | 2020-08-25 | 江苏省人民医院（南京医科大学第一附属医院） | Electrochemical luminescence detection kit for zinc transporter 8 islet autoantibodies |
| CN111579781A (en) * | 2020-05-21 | 2020-08-25 | 深圳市宇诺生物技术有限公司 | Hepatitis C virus antibody detection kit, preparation method and detection method |
Non-Patent Citations (7)
| Title |
|---|
| BERNICE LO等: "Antibodies against insulin measured by electrochemiluminescence predicts insulitis severity and disease onset in non-obese diabetic mice and can distinguish human type 1 diabetes status", 《JOURNAL OF TRANSLATIONAL MEDICINE》 * |
| FARIBA VAZIRI-SANI等: "A novel triple mix radiobinding assay for the three ZnT8 (ZnT8-RWQ) autoantibody variants in children with newly diagnosed diabetes", 《J IMMUNOL METHODS》 * |
| 唐伟国等: "《医学检验诊断试剂的制备与应用》", 31 October 1996, 上海科学技术文献出版社 * |
| 张吉林等: "《医学分子生物学实验指导》", 30 September 2005, 中国医药科技出版社 * |
| 杨新建: "《动物细胞培养技术》", 31 August 2013, 中国农业大学出版社 * |
| 陈宏: "《基因工程原理与应用》", 31 January 2004, 中国农业出版社 * |
| 陈恒等: "锌转运体8自身抗体电化学发光法的建立与应用", 《中华糖尿病杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023103464A1 (en) * | 2021-12-09 | 2023-06-15 | 深圳市亚辉龙生物科技股份有限公司 | Zinc transporter 8 antibody chemiluminescence immunoassay kit and preparation method thereof |
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