CN114441769A - Neuron antigen spectrum antibody detection kit and detection method thereof - Google Patents
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
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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
- G01N33/531—Production of immunochemical test materials
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a neuron antigen spectrum antibody detection kit and a detection method thereof. The detection membrane strip is coated with antigens Amphihysin, CV2, PNMA2(Ma2/Ta), Ri, Yo and Hu in parallel. The enzyme conjugate is one or more of alkaline phosphatase-labeled anti-human immunoglobulin G (IgG), IgA and IgM. The detection method is immunoblotting. The detection kit provided by the invention can conveniently and rapidly diagnose diseases in vitro, and has simple operation and high sensitivity; provides more diagnostic means for the medical research field of nervous system pathological changes.
Description
Technical Field
The invention relates to the field of kits, in particular to a neuron antigen spectrum antibody detection kit and a detection method thereof.
Background
Amphihysin, CV2, PNMA2(Ma2/Ta), Ri, Yo or Hu are neuron-related antigens, in a range of neurological diseases, the body can produce autoantibodies against these antigens, leading to a range of neurological diseases or disorders such as sensitive neuritis, brainstem encephalitis, cerebellar encephalitis and limbic encephalitis, and PNS.
The anti-Amphiphysin (128kDa) antibody takes Amphiphysin as a target antigen and participates in the endocytosis process of the vesicle. Therefore, the detection of the anti-Amphihyssin antibody is very important for diagnosing Eaton-Lambert myasthenia syndrome, autonomic, susceptible, sensory motor polyneuropathy, ankylosing encephalomyelitis, cerebellar syndrome, sensory neuropathy, ocular myoclonic dyskinesia, stiff person syndrome and other nervous system syndromes.
The detection of the anti-CV 2 antibody is very important for diagnosing limbic encephalitis, focal epilepsy, ocular myoclonic movement disorder, chabazitis, extrapyramidal motor syndrome, cerebellar degeneration, autonomy, susceptibility and sensory neuropathy.
The detection of anti-PNMA 2(Ma2/Ta) antibody is related to the diagnosis of limbic encephalitis, focal epilepsy, ocular myoclonic movement disorder, chabazin encephalitis, extrapyramidal motor syndrome, and cerebellar degeneration.
The detection of the anti-Ri antibody is related to the diagnosis of ocular myoclonic dyskinesia and rhombohedral encephalitis.
The detection of the anti-Yo antibody has important significance for diagnosing the ocular myoclonus dyskinesia and cerebellar degeneration of the patient with the positive antibody of the Pythagorean cell.
The detection of anti-Hu antibodies is relevant to the diagnosis of neurological syndromes such as retinopathy, encephalitis focusing on the brainstem, cerebellum and limbic system, epilepsy, ocular myoclonic dyskinesia, chabazitis, extrapyramidal motor syndrome, cerebellar degeneration, myelitis, autonomy, susceptibility, sensory neuropathy, mononeuropathy, motor syndrome, Eaton-Lambert myasthenia syndrome and the like.
Antibody-positive results for the above antigens may occur before the tumor is diagnosed. For patients positive for such antibodies, tumor detection must be performed immediately.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a neuron antigen spectrum antibody detection kit and a detection method thereof, the kit can carry out in-vitro diagnosis on antibody negativity or antibody positivity in a human body through an immunoblotting method, and effectively and quickly solves the health examination problem of the human body in nervous system diseases.
The technical scheme is as follows: the neuron antigen profile antibody detection kit comprises an antigen-coated detection membrane strip, an enzyme conjugate, a sample buffer solution and a substrate solution, wherein the antigen coated on the detection membrane strip comprises Amphipsin, PNMA2(Ma2/Ta), Ri, Yo and Hu.
The antigen coated on the detection membrane strip also comprises CV 2.
The enzyme conjugate comprises one or more of AP enzyme sheep antihuman, namely alkaline phosphatase labeled antihuman immunoglobulin G (IgG), IgA and IgM.
Wherein the pH value of the enzyme conjugate is 7-7.1, and the enzyme conjugate also comprises Tris (hydroxymethyl) aminomethane (Tris), sodium chloride, BSA (bovine serum albumin), sucrose, preservative (Proclin 300), Brij 35 (polyoxyethylene lauryl ether), acetaminophen, gentamicin sulfate and Tween 20.
The content of each liter of enzyme conjugate is as follows: 5.451-6.663g of Tris (hydroxymethyl) aminomethane (Tris), 8.1-9.9g of sodium chloride, 22.5-27.5g of BSA (bovine serum albumin), 13.5-16.5g of sucrose, 0.9-1.1ml of preservative (Proclin 300), 0.9-1.1g of Brij 35 (polyoxyethylene lauryl ether), 0.45-0.55g of acetaminophen, 0.036-0.044g of gentamycin sulfate, 200.45-0.55 ml of Tween and 0.72-0.88mg/L of AP enzyme goat antibody.
The pH value of the enzyme conjugate is preferably 7.05, and the content of each component in each liter of the enzyme conjugate is preferably as follows: 6.057g of Tris (hydroxymethyl) aminomethane (Tris), 9g of sodium chloride, 25g of BSA (bovine serum albumin), 15g of sucrose, 1ml of preservative (Proclin 300), 1g of Brij 35 (polyoxyethylene lauryl ether), 0.5g of acetaminophen, 0.04g of gentamicin sulfate, 200.5 ml of Tween and 0.8mg/L of AP enzyme goat antibody.
The enzyme conjugate was concentrated 10-fold, diluted with a sample buffer at the time of use, and used within one day after dilution.
The substrate solution comprises tetrazole nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate (NBT/BCIP). Wherein the pH value of the substrate solution is 9-10, and the substrate solution also comprises Tris, NaCl, Proclin 300, N-dimethylformamide and MgCl2。
The content of each liter of substrate liquid is as follows: tris 10.903-13.325g, NaCl 5.256-6.424g, Proclin 3000.45-0.55 ml, NBT 0.149-0.182g, BCIP 0.0747-0.0913g, N-dimethylformamide 4.95-6.05ml, MgCl2 0.086-0.105g。
The pH value of the substrate solution is preferably 9.6, and the content of each component in each liter of the substrate solution is preferably Tris 12.114g, NaCl 5.84g, Proclin 3000.5 ml, NBT 0.165g, BCIP 0.083g, N-dimethylformamide 5.5ml, MgCl20.095g。
The substrate liquid can be directly used, but the bottle cap is covered immediately after use due to the sensitivity of the substrate liquid to light.
The sample buffer comprises TBST buffer solution/skimmed milk powder (Tris-sodium chloride-Tween 20/skimmed milk powder), and can be directly used. Wherein the pH value of the sample buffer solution is 7-8, and the sample buffer solution also comprises EDTA and Proclin 300.
The content of each component in each liter of sample buffer solution is as follows: tris 5.451-6.663g, sodium chloride 7.893-9.647g, EDTA 0.9-1.1g, Proclin 3000.9-1.1 ml, Tween 204.5-5.5 ml and skimmed milk powder 9-11 g.
The pH value of the sample buffer is preferably 7.4, and the content of each component in each liter of the sample buffer is preferably as follows: tris 6.057g, sodium chloride 8.77g, EDTA 1g, Proclin 3001 ml, Tween 205 ml and skimmed milk powder 10 g.
The detection kit also comprises a washing buffer solution, wherein the washing buffer solution comprises TBST buffer solution (Tris-sodium chloride-Tween 20). Wherein the pH value of the washing buffer solution is 7-8, and the washing buffer solution also comprises Proclin 300.
The content of each component in each liter of washing buffer solution is as follows: tris 54.513-66.627g, sodium chloride 81-99g, Proclin 3000.45-0.55 ml and Tween 2018-22 ml.
The pH value of the washing buffer is preferably 7.4, and the content of each component in each liter of the washing buffer is preferably as follows: tris 60.57g, sodium chloride 90g, Proclin 3000.5 ml and Tween 2020 ml.
The washing buffer was concentrated 10 times, and diluted with distilled water before use, and used within one day after dilution.
The width of the detection membrane strip is more than or equal to 2.0mm, and a quality control band capable of indicating whether the experimental result is reliable or not is arranged in the functional area at the bottom of the detection membrane strip.
The detection method and the principle for detecting the antibody by using the kit of the invention are as follows:
in vitro diagnosis using immunoblotting: purified antigens are coated on the detection membrane strips in the kit in parallel. In the first incubation step, the diluted serum is reacted with a test membrane strip. If the sample is positive, specific IgG (also including IgA and IgM) binds to the corresponding antigen.
To detect bound antibody, a second incubation step is performed with the addition of an enzyme conjugate, followed by the addition of an enzyme substrate and a third incubation step to produce an observable color reaction. The detection result may be determined based on the generated color.
Has the advantages that: 1. the detection kit provided by the invention can conveniently and rapidly diagnose diseases in vitro, and has simple operation and high sensitivity; 2. provides more diagnostic means for the medical research field of nervous system pathological changes.
Drawings
FIG. 1 is a schematic view of the structure of a detection membrane strip in the detection kit of the present invention;
FIG. 2 is a schematic view showing the detection procedure when the detection kit of the present invention is used.
Detailed Description
Referring to fig. 1, the neuron antigen profile antibody detection kit according to an embodiment of the present invention includes an antigen-coated detection membrane strip, an enzyme conjugate, a sample buffer, a washing buffer, and a substrate solution.
The antigens coated on the detection membrane strip in parallel comprise Amphipsin, CV2, PNMA2(Ma2/Ta), Ri, Yo and Hu. The width of the detection membrane strip is 2.5mm, and a functional area at the bottom of the strip is provided with a quality control band which can indicate whether the experimental result is reliable or not.
The enzyme conjugate is concentrated by 10 times, the pH value of the enzyme conjugate is 7.05, and each liter of the enzyme conjugate comprises 6.057g of Tris, 9g of sodium chloride, 25g of BSA, 15g of sucrose, 3001 ml of Proclin, 351 g of Brij, 0.5g of acetaminophen, 0.04g of gentamycin sulfate, 200.5 ml of Tween and 0.8mg/L of AP enzyme goat anti-human.
The enzyme conjugate is used by diluting with a sample buffer, and is used within one day after dilution.
The pH value of the sample buffer solution is 7.4, and each liter of the sample buffer solution comprises 6.057g of Tris, 8.77g of sodium chloride, 1g of EDTA, 3001 ml of Proclin, 205 ml of Tween and 10g of skimmed milk powder. The sample buffer can be used directly.
The washing buffer was 10-fold concentrated at pH 7.4 and contained, per liter, Tris 60.57g, sodium chloride 90g, Proclin 3000.5 ml, Tween 2020 ml.
The washing buffer solution is diluted with distilled water and used within one day.
The pH value of the substrate solution is 9.6, and each liter of the substrate solution comprises Tris 12.114g, NaCl 5.84g, Proclin 3000.5 ml, NBT 0.165g, BCIP 0.083g, N-dimethylformamide 5.5ml, MgCl2 0.095g。
The substrate liquid can be directly used, but the bottle cap is covered immediately after use due to the sensitivity of the substrate liquid to light.
Incubation trays are required to complete the detection assays of the present invention. Meanwhile, the detection kit is also suitable for a full-automatic immunoblotting instrument.
Referring to fig. 2, the kit of the present invention is used for antibody detection by immunoblotting, and the principle and specific steps are as follows:
first, sample requirement
Sample preparation: human serum or plasma anticoagulated with ethylenediaminetetraacetic acid (EDTA), heparin and citrate.
Stability: the sample to be tested can be stored for 14 days at 2-8 ℃. The diluted samples should be tested within the same working day.
Sample dilution: patient samples were diluted 1:101 with sample buffer. For example: mu.L of serum was diluted with 1.5mL of sample buffer and mixed with a mixer, a non-available sample injector.
Second, inspection method
1. Reagent preparation and stability
Note that: all reagents should be equilibrated at room temperature (18-25 ℃) for about 30 minutes before use. From the first use, the reagent can be stabilized to the indicated expiration date without contamination when stored at 2-8 ℃.
(1) Antigen-coated test membrane strip: can be used directly. To prevent condensation of the film strip, the package can be opened only after the film strip has equilibrated to room temperature. The strips should be removed and the original package sealed immediately and stored at 2-8 ℃.
(2) Enzyme conjugate: concentrating by 10 times. In use, the required amount of sample buffer is diluted 1:10 by sucking from the bottle with a clean pipette. For example: one strip of test membrane was incubated and 0.15mL of enzyme conjugate was diluted with 1.35mL of sample buffer and the diluted enzyme conjugate was used up on the same day of the work.
(3) Sample buffer: can be used directly.
(4) Washing buffer solution: concentrating by 10 times. When in use, a clean suction pipe is used for sucking required amount from the bottle and diluting the bottle with distilled water at a ratio of 1: 10. Such as: one strip was incubated and 1mL of concentrated buffer was diluted with 9mL of distilled water. The diluted buffer should be used up on the same working day.
(5) Substrate solution: the product can be directly used; because of the sensitivity to light, the bottle cap should be closed immediately after use.
2. Operation process
(1) Pretreatment: the desired test membrane strip is removed and placed in the incubation well of the incubation tray. The side of the test membrane strip with the number faces upward. 1.5mL of each sample buffer was added to each of the incubation tanks, and after incubation for 5 minutes on a rocking shaker at room temperature, the liquid in the incubation tanks was aspirated.
(2) Incubation with serum:
s1: 1.5mL of each diluted serum sample was added to the incubation tank and incubated for 30 minutes at room temperature (18-25 ℃) on a rocking shaker.
Cleaning: the tank was aspirated and the membrane strips were washed 3 times for 5 minutes each with 1.5mL of wash buffer on a rocking shaker.
(3) Incubation of enzyme conjugate:
s2: 1.5mL of diluted enzyme conjugate (alkaline phosphatase-labeled anti-human IgG) was added to the incubation tank and incubated on a rocking shaker at room temperature for 30 minutes.
Cleaning: the tank was aspirated and the membrane strips were washed 3 times for 5 minutes each with 1.5mL of wash buffer on a rocking bed.
(4) Substrate incubation:
s3: 1.5mL of the substrate solution was added to each of the incubation tanks, and incubated for 10 minutes at room temperature (18-25 ℃) on a rocking shaker.
(5) And (4) terminating: the liquid in the tank was aspirated off, and the membrane strip was washed with distilled water 3 times for 1 minute each time.
3. And (5) judging a result: and (5) placing the detection membrane strip in a result judgment template, and judging a result after air drying.
Third, explanation of test results
The method comprises the following steps: the wet test membrane strip after incubation was placed on a plastic film in the results determination template and aligned with the markers. Carefully remove the water with absorbent paper (after complete drying, the film strip will adhere to the plastic film). Clearly visible bands appearing on the dry test membrane strip corresponding to the markings on the reference membrane strip are recorded on the outcome determination template. The appearance of a white band at the coated antigen should be judged negative.
The test result is reliable when the functional zone at the bottom of the detection membrane strip has a quality control band and only the quality control band has strong color reaction.
The antigens coated on the test strips and their arrangement are shown in table 1 below:
table 1: detecting coating antigens on membrane strips
The results were classified as negative, suspicious and positive according to the shade of the staining of the antigen, as shown in the experimental result control standard in Table 2 below.
TABLE 2 comparison of the results
Claims (10)
1. A neuron antigen profile antibody detection kit is characterized in that: the kit comprises an antigen-coated detection membrane strip, an enzyme conjugate, a sample buffer solution and a substrate solution, wherein the antigen coated on the detection membrane strip comprises Amphipsin, PNMA2(Ma2/Ta), Ri, Yo and Hu.
2. The neuronal antigen profiling antibody detection kit of claim 1, characterized in that: the antigen coated on the detection membrane strip also comprises CV 2.
3. The neuronal antigen profiling antibody detection kit of claim 1, characterized in that: the enzyme conjugate comprises one or more of alkali phosphatase-labeled anti-human immunoglobulin G, IgA and IgM.
4. The neuronal antigen profiling antibody detection kit according to claim 1 or 3, characterized in that: the enzyme conjugate was 10-fold concentrated and used diluted with sample buffer.
5. The neuronal antigen profiling antibody detection kit of claim 1, characterized in that: the substrate solution comprises tetrazole nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate.
6. The neuronal antigen profiling antibody detection kit of claim 1, characterized in that: the sample buffer comprises TBST buffer/skimmed milk powder.
7. The neuronal antigen profiling antibody detection kit according to claim 1 or 2, characterized in that: the washing buffer solution is 10 times concentrated and needs to be diluted by distilled water when in use.
8. The neuronal antigen profiling antibody detection kit of claim 1, characterized in that: the width of the detection membrane strip is more than or equal to 2.0 mm.
9. The neuronal antigen profiling antibody detection kit of claim 1, characterized in that: and the functional area at the bottom of the detection membrane strip is provided with a quality control band which can indicate whether the experimental result is reliable or not.
10. The method for detecting the antibody by using the neuron antigen profile antibody detection kit according to any one of claims 1 to 9, wherein the detection is performed by immunoblotting, comprising the steps of:
(1) combining and reacting the diluted serum sample with a detection membrane strip, and carrying out first-step incubation, wherein if the sample is positive, specific IgG, IgA or IgM is combined with corresponding antigen;
(2) adding diluted enzyme conjugate for second incubation in order to detect the antibody bound in step (1);
(3) in order to enable the detection result in the step (2) to be visually observed through color reaction, adding substrate solution to carry out third-step incubation;
(4) and (4) placing the detection membrane strip in the result judgment template and judging the detection result, namely finishing the antibody detection.
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| CN115097125A (en) * | 2022-06-01 | 2022-09-23 | 苏州邦器生物技术有限公司 | Antinuclear antibody spectrum detection kit and detection method thereof |
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| CN113373156A (en) * | 2021-06-30 | 2021-09-10 | 四川携光生物技术有限公司 | NMDAR recombinant protein related to autoimmune encephalitis, and coding sequence, preparation method and application thereof |
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| CN101490085A (en) * | 2006-06-12 | 2009-07-22 | 特鲁比昂药品公司 | Single-chain multivalent binding proteins with effector function |
| CN113373156A (en) * | 2021-06-30 | 2021-09-10 | 四川携光生物技术有限公司 | NMDAR recombinant protein related to autoimmune encephalitis, and coding sequence, preparation method and application thereof |
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| CN115097125A (en) * | 2022-06-01 | 2022-09-23 | 苏州邦器生物技术有限公司 | Antinuclear antibody spectrum detection kit and detection method thereof |
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