CN112326971A - Novel method and kit for NMDAR antibody quantitative detection - Google Patents
Novel method and kit for NMDAR antibody quantitative detection Download PDFInfo
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Abstract
The invention discloses a novel method and a kit for NMDAR antibody quantitative detection, which comprise a magnetic particle suspension coupled with streptavidin, NMDAR protein marked by biotin, NMDAR protein marked by chemiluminescent marker, NMDAR protein calibrator, NMDAR antibody quality control product, chemiluminescent liquid A, chemiluminescent liquid B and concentrated cleaning solution. The invention adopts a mode of combining chemiluminescence immunoassay and a magnetic particle technology, has simple and convenient operation, improves the sensitivity and the accuracy of detection, is easy to popularize and improve the detection efficiency, has lower cost and is easy to carry out batch detection.
Description
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to a novel method and a kit for quantitative detection of an NMDAR antibody.
Background
anti-NMDAR encephalitis is the most common autoimmune encephalitis, which occurs well in young women and can be associated with ovarian teratomas. Studies have shown that anti-NMDAR antibodies reversibly inhibit the accumulation of NMDAR on cell membranes; inhibition of pre-synaptic GABAergic neurons NMDAR may reduce GABA release, a significant increase in extracellular glutamate and alterations in GABAergic signaling may lead to a series of anti-NMDAR encephalitis manifestations: including psychobehavioral disorders, cognitive disorders, seizures, and the like, often accompanied by a variety of movement disorders, and may have both hyperkinetic and hypokinetic syndromes, including orofacial movement disorder (OFLD), dystonic posture of limbs, chorea, parkinsonism, and catatonic syndromes.
At present, cellular immunofluorescence is a main mode for detecting an NMDAR antibody, and the immunofluorescence technology is a process of labeling fluorescent pigments which do not influence the activity of antigen antibodies on antigens, combining the fluorescent pigments with corresponding antibodies, and presenting a specific fluorescence reaction under a fluorescence microscope. The immunofluorescence can accurately identify the correct expression site, but the immunofluorescence method has the problems of different plasmid vectors, different proportions of plasmid concentrations, different transfection efficiencies and different detection rates; meanwhile, the method is imported abroad, and the detection cost is high.
Disclosure of Invention
The invention aims to provide a novel method and a kit for NMDAR antibody quantitative detection, which adopt a mode of combining chemiluminescence immunoassay and a magnetic particle technology, are simple and convenient to operate, improve the sensitivity and accuracy of detection, are easy to popularize and improve the detection efficiency, have lower cost and are easy to detect in batches.
The invention is realized by the following technical scheme:
an NMDAR antibody quantitative detection kit comprises a streptavidin-coupled magnetic particle suspension, biotin-labeled NMDAR protein and chemiluminescent marker-labeled NMDAR protein.
Also comprises an NMDAR protein calibrator, an NMDAR antibody quality control product, a chemiluminescence solution A, a chemiluminescence solution B and concentrated cleaning solution.
The chemiluminescent marker is horseradish peroxidase.
The NMDAR protein calibrator is a calibrator solution with a series of concentration gradients prepared by adding NMDAR protein into 30% bovine serum solution serving as diluent.
The NMDAR antibody quality control product is a low-value quality control product and a high-value quality control product which are prepared by adding an NMDAR antibody into a diluent containing 30% of bovine serum.
The chemiluminescence solution A is a mixed solution of luminol, p-iodophenol and Tris-HC 1, and the chemiluminescence solution B is a carbamide peroxide solution.
The preparation method of the NMDAR antibody quantitative detection kit comprises the following steps:
step S1, coupling the magnetic particle chain enzyme avidin to prepare magnetic particle suspension;
s2, taking NMDAR purified protein for permeation, and adding biotin and NMDAR protein marked by biotin;
step S3, coupling the NMDAR protein with horseradish peroxidase to prepare the NMDAR protein marked by a chemiluminescent marker;
and step S4, assembling the reagents into a kit.
A novel method for the quantitative detection of NMDAR antibodies, said kit being used for the detection of NMDAR antibodies in serum and cerebrospinal fluid.
A novel method for quantitative detection of NMDAR antibodies, the detection method comprising the steps of: 1) adding biotin-labeled NMDAR protein into a streptavidin-coupled magnetic particle suspension for reaction; 2) adding a sample to be detected and NMDAR protein marked by a chemiluminescent marker for reaction to obtain a coupled compound; 3) adsorbing the compound at the bottom of the test tube by using a magnetic field, washing off free components, adding a mixed solution of a chemiluminescence solution A and a chemiluminescence solution B, and measuring a luminescence value RLU of a sample to be measured at the 5 th minute; 4) and establishing a Log (X) -Log (Y) mathematical model by the concentration of the calibrator NMDAR and the corresponding luminescence value RLU, and calculating the content of the NMDAR antibody of the sample to be detected.
The principle of the invention is as follows: the NMDAR antibody in serum or cerebrospinal fluid is measured by adopting a double-antigen sandwich method principle in a chemiluminescence method, a biotin-NMDAR conjugate is added into an avidin-magnetic particle suspension, a magnetic particle-avidin-biotin-NMDAR compound is formed through an affinity reaction of avidin and biotin, after a sample and enzyme are added, a magnetic particle-avidin-biotin-NMDAR-anti-NMDAR antibody-NMDAR-HRP compound is formed through an antigen-antibody reaction, under the action of an oxidizing agent, the HRP catalyzes luminol to generate aminophthalic acid ions in an excited state, and when the luminol returns to a ground state, photons of 425nm are released, so that the measurement of a luminescence value is realized.
The kit, namely the detection method, takes the magnetic particles as the solid phase carrier, greatly increases the effective coating amount of the antibody, saves the using amount of the antibody, increases the contact area and the luminous area of the antigen-antibody and improves the sensitivity of the reaction; the reaction is carried out in a liquid phase, so that the reaction time is greatly shortened; meanwhile, the introduced biotin-avidin system has the multi-stage signal amplification effect, does not increase non-specific interference, and has the characteristics of high sensitivity, good specificity, high stability, strong applicability, low experiment cost and the like.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the method adopts a mode of combining chemiluminescence immunoassay and a magnetic particle technology, is simple and convenient to operate, improves the sensitivity and accuracy of detection, is easy to popularize and improve the detection efficiency, has lower cost and is easy to detect in batches;
2. compared with a cell immunofluorescence method, the method is simple and convenient to operate, high in automation degree, low in human error, safe and free of environmental pollution. In addition, the concentration range of the detected sample is wide, the cost is low, and the stability is good.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.
Example 1
Preparation of magnetic particle-streptavidin suspension:
1.1 taking L00mL 0.1.1 mol/L2-alunite HE ethane sulfonic acid solution, sequentially adding 10mg of magnetic particles and 3mg of streptavidin, stirring for 30min, then adding 3.5mL of 10mg/mL carbodiimide hydrochloride solution, and reacting for lh;
and 1.2, adsorbing by using a magnetic frame, standing for 10min, removing liquid, adding 10mL of 0.0lmol/L PBS, repeating the process, washing for 3 times, and finally performing constant volume to 1L by using 0.01mol/L PBS to obtain the magnetic particle-streptavidin suspension.
Example 2
Preparation of Biotin-labeled NMDAR proteins
2.1 taking 0.5mg of NMDAR purified protein, and dialyzing for 2 hours at the temperature of 2-8 ℃ by using borate buffer solution;
2.2 adding 25mg biotin and dimethyl sulfoxide to obtain a final concentration of 10%, reacting in dark for 3h, and slowly oscillating;
2.3 adding 250ml mol/L ammonium chloride solution into the 2.2 solution, and reacting for 50min at normal temperature in a dark place;
2.4 dialyzing with 0.0L mol/L PBS solution at 2-8 deg.C for 2 days, and changing the solution 5 times to obtain biotin-labeled NMDAR protein.
Example 3
Preparation of NMDAR protein-enzyme conjugates
3.1 coupling the NMDAR with horseradish peroxidase by adopting a sodium periodate oxidation method, wherein the purity RZ of the horseradish peroxidase is more than or equal to 3.0, and the activity is more than or equal to 250U/mL;
3.2 dilution to working concentration 1: 5000, then 15% enzyme stabilizer is added and stored at 2-8 ℃, wherein the enzyme stabilizer is protein stabilizer product of Surmodics In Vitro Technologies.
Example 4
4.1 preparation of NMDAR protein calibrator:
diluting the pure NMDAR protein product with 30% bovine serum solution to form a series of gradients, wherein the concentrations are 0, 5, 10, 25, 50 and 100KU/mL respectively;
4.2 preparation of NMDAR antibody quality control product:
diluting the NMDAR antibody with a solution containing 30% of bovine serum to prepare a low-value quality control product and a high-value quality control product, wherein the concentration of the low-value quality control product is 10KU/mL, and the concentration of the high-value quality control product is 60 KU/mL.
Example 5
5.1 preparation of chemiluminescent solutions A and B
The solution A is 0.7g/L of luminol, 0.165g/L of p-iodophenol, the buffer solution is 5mmol/L of Tris-HC 1 with the pH value of 8.6, and the solution is stored away from light;
the liquid B is 0.675g/L carbamide peroxide, and is prepared by using process water; mixing solution A and solution B5 min before use.
Preparation of 5.220 times concentrated cleaning solution
The 20-fold concentrated washing solution comprises 58g/L of disodium hydrogen phosphate, 5.92g/L of sodium dihydrogen phosphate, 180g/L of NaCL 10mL/L of Tween-20 and L% of Proclin 300.
Example 6
Specificity and sensitivity assays
By performing experiments on 20 parts of normal healthy human serum and 20 parts of positive serum, as shown in tables 1 and 2, the results are in line with expectations, the consistency is high, and the specificity and the sensitivity of the NMDAR are both 100% and the results are good.
TABLE 1
TABLE 2
Example 7
Comparison of cellular immunofluorescence and chemiluminescence
The experimental method is as follows
7.1 cell fluorescence method is the dilution reporting method of results: 1:1 represents serum and diluent 1:1 mixing, 1:10 represents serum and dilution 1:10, mixing, and repeating the steps until a positive reaction occurs, namely reporting the dilution ratio at the moment.
7.2 samples of 5 dilutions were selected, 5 samples for each dilution, and 25 samples were obtained, and the serum was directly verified by magnetic particle chemiluminescence at each gradient, and the results are shown in Table 3.
TABLE 3
As can be seen from table 3, 1: mean 2.72, 1:10 mean 11.6, 1:30 mean 28.5, 1: 100 mean 52.2, 1: 300 is 75.8 ku/ml. The linear reaction is realized, and the correlation coefficient is 0.99.
And (4) conclusion: the chemiluminescence method results increase along with the positive result gradient in a linear manner, and accord with the expected linear increase of experiments.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. An NMDAR antibody quantitative detection kit is characterized by comprising a streptavidin-coupled magnetic particle suspension, biotin-labeled NMDAR protein and chemiluminescent label-labeled NMDAR protein.
2. The quantitative NMDAR antibody detection kit according to claim 1, characterized by further comprising an NMDAR protein calibrator, an NMDAR antibody quality control, a chemiluminescent solution a, a chemiluminescent solution B and a concentrated cleaning solution.
3. The NMDAR antibody quantitative detection kit according to claim 1, characterized in that the chemiluminescent label is horseradish peroxidase.
4. The NMDAR antibody quantitative detection kit according to claim 2, wherein the NMDAR protein calibrator is a calibrator solution with a series of concentration gradients prepared by adding NMDAR protein into a 30% bovine serum solution as a diluent.
5. The NMDAR antibody quantitative detection kit according to claim 2, wherein the NMDAR antibody quality control is a low-value quality control and a high-value quality control prepared by adding NMDAR antibody into a diluent containing 30% bovine serum.
6. The NMDAR antibody quantitative detection kit according to claim 2, wherein the chemiluminescence solution A is a mixed solution of luminol, p-iodophenol and Tris-HC 1, and the chemiluminescence solution B is a carbamide peroxide solution.
7. The method of making a quantitative NMDAR antibody detection kit according to any of claims 1 to 6, comprising the steps of:
step S1, coupling the magnetic particle chain enzyme avidin to prepare magnetic particle suspension;
s2, taking NMDAR purified protein for permeation, and adding biotin and NMDAR protein marked by biotin;
step S3, coupling the NMDAR protein with horseradish peroxidase to prepare the NMDAR protein marked by a chemiluminescent marker;
and step S4, assembling the reagents into a kit.
8. A novel method for quantitative detection of NMDAR antibodies, characterized in that the kit according to any of claims 1 to 6 is used for the detection of NMDAR antibodies in serum and cerebrospinal fluid.
9. The method of claim 8, wherein the detection method comprises the following steps: 1) adding biotin-labeled NMDAR protein into a streptavidin-coupled magnetic particle suspension for reaction; 2) adding a sample to be detected and NMDAR protein marked by a chemiluminescent marker for reaction to obtain a coupled compound; 3) adsorbing the compound at the bottom of the test tube by using a magnetic field, washing off free components, adding a mixed solution of a chemiluminescence solution A and a chemiluminescence solution B, and measuring a luminescence value RLU of a sample to be measured at the 5 th minute; 4) and establishing a Log (X) -Log (Y) mathematical model by the concentration of the calibrator NMDAR and the corresponding luminescence value RLU, and calculating the content of the NMDAR antibody of the sample to be detected.
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Cited By (1)
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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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