CN113391073A

CN113391073A - Magnetic particle chemiluminescence immunoassay kit for NPTX2

Info

Publication number: CN113391073A
Application number: CN202110646688.0A
Authority: CN
Inventors: 孙月鹏; 胡秀丽; 孙月满
Original assignee: Zhongxiu Technology Co ltd
Current assignee: Zhongxiu Technology Co ltd
Priority date: 2021-06-10
Filing date: 2021-06-10
Publication date: 2021-09-14

Abstract

The present invention discloses a magnetic particle chemiluminescence immunoassay kit for human neuron pentraxin II (hereinafter referred to as NPTX2), the kit includes calibrator, quality control substance, NPTX2 magnetic particle suspension, NPTX2 Antibody marker, concentrated washing solution, luminescent substrate; the calibrator is configured by NPTX2 antigen; the quality control substance is a certain concentration of NPTX2 antigen, which is configured by the dilution of quality control substance; the The NPTX2 magnetic particle suspension contains a solution of magnetic particle-labeled NPTX2 antibody at a certain concentration. The beneficial effects of the invention are as follows: the kit of the invention has the advantages of less pollution, high degree of automation, simple operation, good repeatability, high sensitivity and good specificity for rapid quantitative detection of NPTX2.

Description

Magnetic particle chemiluminescence immunoassay kit for NPTX2

Technical Field

The invention relates to the technical field of kit detection, in particular to a magnetic particle chemiluminescence immunoassay kit and a method thereof for detecting human neuron positive pentameric protein II (NPTX 2).

Background

Alzheimer's Disease (AD) is a degenerative dementia of the central nervous system, which is mainly caused by the degeneration of neurons and the reduction of neurites, seriously affecting the life of patients. The etiology and pathogenesis of AD are not clarified, and the characteristic pathogenesis is changed into extracellular senile plaques formed by deposition of beta amyloid protein, nerve intracellular neurofibrillary tangles formed by hyperphosphorylation of Tau protein, neuron loss accompanied with glia cell proliferation and the like. Early detection of proteins such as beta-amyloid, Tau and NPTX2 in cerebrospinal fluid or blood plays a very important role in early detection of AD patients. NPTX2 is one of the members of the neuronal pentraxin family, which currently includes NPTX1, NPTX2 and NPTR 3 members, and is involved in the remodeling of neuronal synapses. NPTX2 is expressed in the brain of alzheimer's disease patients. NPTX2 down-regulation can lead to the disruption of the pyramidal neuron-pv neuron-interneuron circuit, which is important for the homeostasis of rhythmicity and excitability of the brain. NPTX2 down-regulation, an important mechanism in the pathogenesis of AD, is closely associated with the deterioration of cognitive function. NPTX2 is a previously unrecognized protein that is important for the progression of human cognitive dysfunction and AD. NPTX2 is considered to be a better biomarker for predicting progression of alzheimer's disease.

The currently available detection methods for human neuron positive pentameric protein II (NPTX2) mainly include radioimmunoassay RIA, enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunochromatography and the like. The methods have certain defects, the RIA pollution is large, the ELISA method operation process is complex, the detection time is too long, the NPTX2 automatic detection is not easy to realize, and the immunochromatography detection has poor repeatability and low sensitivity. Therefore, a rapid quantitative detection kit for NPTX2, which has the advantages of small pollution, high automation degree, simple operation, good repeatability, high sensitivity and good specificity, and a use method thereof are still needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a magnetic particle chemiluminescence immunoassay kit for NPTX 2. The kit has the advantages of small pollution, high automation degree, simple operation, good repeatability, high sensitivity and good specificity of NPTX2 rapid quantitative detection.

The purpose of the invention is realized by the following technical scheme: the invention discloses a magnetic particle chemiluminescence immunoassay kit for NPTX2, which comprises a calibrator, a quality control product, an NPTX2 magnetic particle suspension, an NPTX2 antibody marker, concentrated lotion and a luminescent substrate;

the calibrator is configured by NPTX2 antigen;

the quality control product is NPTX2 antigen with a certain concentration and is prepared from a quality control product diluent;

the NPTX2 magnetic particle coated antibody contains a NPTX2 antibody solution labeled by magnetic particles with a certain concentration.

The preparation method of the NPTX2 magnetic particle suspension is as follows:

1) and (3) activation: adding 100 mul of 50mg/mLEDC and 50mg/mL NHS into 20 mul of magnetic particle stock solution, activating at 200r/min for 0.5-1 h; EDC solution is prepared by dissolving EDC reagent in 100mM MES buffer solution with pH5.0, NHS solution is prepared by dissolving NHS reagent in 100mM MES buffer solution with pH 5.0;

2) washing: separating the solution on a magnetic frame, discarding the supernatant, adding 300 μ L MES buffer solution, mixing, separating on the magnetic frame, discarding the supernatant, and washing for 2 times;

3) coating: adding MES buffer solution and pre-coated antibody, wherein the mass ratio of the magnetic particles to the antibody is 80:1, uniformly mixing, and reacting at room temperature for 8-12 h;

4) and (3) sealing: separating the solution on a magnetic frame, removing the supernatant, adding 100-300 mu L of magnetic particle sealing solution, shaking up, and reacting at room temperature for 8-12 h;

5) and (3) separating the solution on a magnetic frame, removing supernatant, and adding a certain magnetic bead sealing solution to obtain NPTX2 magnetic particle suspension.

The NPTX2 antibody marker is an NPTX2 antibody solution containing a certain concentration of alkaline phosphatase marker or an acridinium ester marker NPTX2 antibody solution.

The preparation method of the alkaline phosphatase NPTX2 antibody marker is as follows:

1) respectively desalting and concentrating an NPTX2 antibody and alkaline phosphatase, wherein the mass of the alkaline phosphatase is 1.5-2 times of that of the antibody, and then adding an activating agent, and respectively activating for 20min at room temperature; desalting using Sephadex G25 gel column to remove salt; the activator for NPTX2 antibody activation is 2IT (2-Iminothiolan hydrochloride), and the activator for alkaline phosphatase activation is SMCC (succinimidyl 4- (N-maleimidyl 1) cyclohexane-1-carboxylate);

2) the activated NPTX2 antibody and alkaline phosphatase can be mixed according to the mass ratio for reaction; then carrying out termination reaction and separation and purification, removing the unbound NPTX2 antibody and alkaline phosphatase, preparing an antibody marker concentrated solution, and storing at 2-8 ℃; in the process of terminating the reaction, 10mM maleimide is added firstly, and the reaction is carried out for 10min at room temperature; then adding 100mM of ethanolamine; the separation and purification column is Superdex 200.

The preparation method of the acridinium ester labeled antibody is as follows:

1) the NPTX2 antibody and acridinium ester with the mass 50 times of that of the antibody are fully and uniformly mixed, reacted at 37 ℃ for more than 2 hours, and added with lysine to terminate the reaction;

2) purifying the marked acridinium ester marked antibody by using a desalting chromatographic column, collecting the purified acridinium ester marked antibody, and storing at-20 ℃.

The detection method of the kit is as follows:

1) immune reaction: 10 mu L of sample/quality control material, 20 mu L of magnetic particles and 100 mu L of antibody marker, incubating for 30min at 37 ℃, and washing for 6 times;

2) magnetic separation: settling the magnetic particles in a magnetic field, removing the supernatant, adding 150 mu L of washing solution, settling the magnetic particles in the magnetic field, removing the supernatant, repeating the steps for 4 times, and removing the unbound antibody;

3) and (3) detection reading: adding a luminescent substrate, reacting at room temperature, and detecting the luminescent value within 10s-5 min;

4) and (3) calculating: the compound catalyzes a luminescent substrate to emit photons, and the luminous intensity is in direct proportion to the content of NPTX 2; selecting a proper curve fitting mode to establish a standard curve; the kit preferably adopts a four-parameter fitting mode, establishes a standard curve by taking the concentration value of the calibrator as an x axis and the log value of the luminous intensity value of the calibrator as a y axis, and calculates the corresponding concentration value according to the luminous intensity value of the sample to be detected.

The calibrator provided by the invention is a calibrator containing NPTX2 antigen;

the method for configuring the calibration product comprises the following steps: the Tris buffer containing 1% casein was used as the calibrator diluent, and NPTX2 antigen was diluted in a gradient with the calibrator diluent to working concentrations of 50, 100, 250, 500, 1000, 3000pg/mL, respectively.

The quality control product is a quality control product containing NPTX2 antigen;

the quality control product configuration method comprises the following steps: tris buffer containing 1% casein is used as quality control product diluent, and NPTX2 antigen is diluted to 500pg/mL and 1000pg/mL in quality control product diluent in a gradient manner.

The method for determining the performance index of the kit comprises the following steps:

1) accuracy: NPTX2 sample A with the concentration of about 3000pg/mL (the concentration deviation is allowed to be +/-20%) is added into NPTX2 sample B with the concentration ranging from 150 to 300pg/mL, and the volume ratio of the added sample A to the added sample B is 1: 9, detecting the mixed sample by using the kit, repeating the detection for 3 times, calculating the recovery rate R according to the following formula,

in the formula: r-recovery rate; v-volume of solution A added; v₀-volume of serum B; c-the detection concentration of the serum sample after the solution A is added; c0 — detection concentration of serum sample B; the concentration of the Cs-a solution;

2) blank limit: detecting by using a zero-concentration calibrator or sample diluent as a sample, repeatedly measuring for 20 times to obtain a light absorption value of 20 measurement results, calculating an average value M and a standard deviation SD of the light absorption value, obtaining a light absorption value corresponding to M +2SD, substituting the light absorption value corresponding to M +2SD into an equation according to a calibration curve of the calibrator used by the kit, and calculating a corresponding concentration value, namely a blank limit;

3) repeatability: detecting quality control serum or fresh human serum with different concentrations for 10 times, respectively, calculating average value M and standard deviation SD of 10 detection results, and obtaining coefficient of variation CV according to formula CV (SD/M) 100%, wherein CV value should not be greater than 10%;

4) batch-to-batch repeatability: respectively detecting quality control serum or fresh human serum with high and low concentrations by using three batches of reagents, repeating the detection 10 times each time, respectively calculating the average value M and standard deviation SD of 30 detection results, and obtaining a coefficient of variation CV according to a formula CV (SD/M100%), wherein the CV value is not more than 15%;

5) linearity: diluting the NPTX2 high-value sample close to the upper limit of the linear interval to at least 5 concentrations by negative serum according to a certain proportion, wherein the sample of the low-value concentration needs to be close to the lower limit of the linear interval; repeatedly detecting each concentration for 2 times by using the kit, and respectively calculating the average value yi of the detection result; calculating a linear regression equation by taking the dilution ratio (xi) as an independent variable and the mean value yi of the detection result as a dependent variable; and calculating a correlation coefficient r of the linear regression, wherein r is more than or equal to 0.9900.

The evaluation result parameters of the kit are defined as follows:

1) accuracy: the accuracy is evaluated by dilution and recovery, and the result is between 85 and 115 percent;

2) blank limit: not greater than 20 pg/mL;

3) repeatability: the repeatability is less than or equal to 10 percent, and the batch repeatability is less than or equal to 15 percent;

4) dose-response curve linearity: within the range of 20-3000pg/mL, the correlation coefficient r of the dose-response curve is more than or equal to 0.99.

5) And (3) sample determination: compared with Western blot assay, NPTX2 clinical compliance rate is more than 85% when 50 AD patient samples are compared and determined.

The kit for quantitatively detecting the human neuron positive pentameric protein II (NPTX2) has the following advantages: 1. the quantitative detection of human neuron positive pentameric protein II (NPTX2) is realized; 2. by combining chemiluminescence and magnetic particle immune separation technologies, the method is convenient to operate, can realize automation, and improves the sensitivity and accuracy of human neuron ortho-pentameric protein II (NPTX2) detection; 3. the alkaline phosphatase labeling detection technology is adopted, so that the sensitivity is high and the stability is good; 4. by adopting the acridinium ester labeling detection technology, the luminescent system is simple, a catalyst is not needed, the cost is saved, the detection time is faster, and the method is the main development direction of the magnetic particle chemiluminescence immunoassay method in the future; 5. the kit provided by the invention can meet the social and commercial requirements as a product.

Drawings

FIG. 1 is a schematic diagram of the principle steps of the kit of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. Example (b): a magnetic particle chemiluminescence immunoassay kit for NPTX2, the kit comprises: calibrator, quality control material, NPTX2 magnetic particle suspension, NPTX2 antibody marker, concentrated lotion, luminescent substrate,

wherein the calibrator is configured from NPTX2 antigen and is used to establish a calibration curve;

the quality control product is NPTX2 antigen with a certain concentration and is prepared from a quality control product diluent.

The NPTX2 magnetic particle suspension described above contained a concentration of magnetic particle labeled NPTX2 antibody solution.

The NPTX2 magnetic microparticle suspension described above was prepared as follows.

1) And (3) activation: 20 mu L of magnetic particle stock solution is added with 100 mu L of 50mg/mLEDC and 50mg/mL NHS, 200r/min and activated for 0.5-1 h. EDC solution was prepared by dissolving EDC reagent in 100mM MES buffer at pH5.0, and NHS solution was prepared by dissolving NHS reagent in 100mM MES buffer at pH 5.0.

2) Washing: separating the above solution on magnetic frame, discarding supernatant, adding 300 μ L MES buffer solution, mixing, separating on magnetic frame, discarding supernatant, and washing for 2 times.

3) Coating: adding MES buffer solution and pre-coated antibody (the mass ratio of the magnetic particles to the antibody is 80:1), mixing uniformly, and reacting at room temperature for 8-12 h.

4) And (3) sealing: separating the solution on a magnetic frame, discarding the supernatant, adding 100-300 mu L of magnetic particle sealing solution, shaking up, and reacting at room temperature for 8-12 h.

The preparation method of the alkaline phosphatase NPTX2 antibody marker is shown as follows.

1) The NPTX2 antibody and alkaline phosphatase (the mass of the alkaline phosphatase is 1.5-2 times of that of the antibody) are respectively desalted and concentrated, and then an activating agent is added to respectively activate for 20min at room temperature. Desalting the salt was removed using Sephadex G25 gel column. The activator for NPTX2 antibody activation is 2IT (2-Iminothiolan hydrochloride), and the activator for alkaline phosphatase activation is SMCC (succinimidyl 4- (N-maleimidyl 1) cyclohexane-1-carboxylate).

2) The activated NPTX2 antibody and alkaline phosphatase may be mixed in a mass ratio and reacted. Then, a termination reaction and separation and purification were carried out to remove unbound NPTX2 antibody and alkaline phosphatase, and an antibody marker concentrate was prepared and stored at 2-8 ℃. During the termination of the reaction, 10mM maleimide was added first, and the reaction was carried out at room temperature for 10 min. Further adding ethanolamine in an amount of 100 mM. The separation and purification column is Superdex 200.

The preparation method of the acridinium ester labeled antibody is shown below.

1) The NPTX2 antibody and acridinium ester (the mass of the acridinium ester is 50 times of that of the antibody) are fully mixed, reacted at 37 ℃ for more than 2 hours, and added with lysine to terminate the reaction.

The calibrator is a calibrator containing NPTX2 antigen.

The method for configuring the calibration material comprises the following steps: the Tris buffer containing 1% casein was used as the calibrator diluent, and NPTX2 antigen was diluted in a gradient with the calibrator diluent to working concentrations of 50, 100, 250, 500, 1000, 3000pg/mL, respectively.

The quality control product is a quality control product containing NPTX2 antigen.

The concentrated washing solution is phosphate buffer solution containing Tween-20.

The luminous substrate solution is acridinium ester luminous substrate solution.

The components are assembled into a kit and stored at 2-8 ℃.

The detection method of the kit is as follows:

1) immune reaction: 10 u L samples/quality control, 20 u L magnetic particles, 100 u L enzyme conjugate labeled antibody, 37 degrees C temperature incubation for 30min, washing 6 times.

3) and (3) detection reading: adding a luminescent substrate, reacting at room temperature, and detecting the luminescent value within 5 min.

4) And (3) calculating: the compound catalyzes a luminescent substrate to emit photons, and the luminous intensity is in direct proportion to the content of NPTX 2. And selecting an appropriate curve fitting mode to establish a standard curve. The kit preferably adopts a four-parameter fitting mode, establishes a standard curve by taking the concentration value of the calibrator as an x axis and taking the logarithm (log) value of the luminous intensity value of the calibrator as a y axis, and calculates the corresponding concentration value according to the luminous intensity value of the sample to be detected.

The NPTX2 antibody used in the preparation process is provided by Abcam, the NPTX2 antigen is provided by Abcam, alkaline phosphatase is purchased from BBI, 2-IT is purchased from thermo fisher scientific, SMCC is purchased from thermo fisher scientific, magnetic microparticles is purchased from thermo fisher scientific, EDC is purchased from sigma, NHS is purchased from sigma, and acridine ester is purchased from BBI, England.

Preparation of NPTX2 magnetic particle suspension

Preparation of NPTX2 antibody marker

Preparation of alkaline phosphatase-labeled antibody

1) Desalting: the buffer system of the antibody is changed into TSE buffer solution by using a desalting column; the buffer system of alkaline phosphatase was changed to ALP dialysis buffer pH 7.6 using desalting column and the mass of alkaline phosphatase was 1.5-2 times that of antibody). Desalting the salt was removed using Sephadex G25 gel column. TSE pH8.5 buffer (1000 g): purified water, 5.84g of sodium chloride, 14.92g of triethanolamine and 0.41g of EDTA.2Na.2H2O.

2) And (3) centrifugal concentration: the concentration of antibody was in the range of 2-4mg/mL and the concentration of alkaline phosphatase was in the range of 3-5 mg/mL.

3) And (3) activation: dissolving 2-IT in 2-IT diluent to obtain 13.5mg/ml solution, activating antibody at room temperature for 30 min. SMCC was dissolved in DMF to make 6.5mg/ml, and the antibody was activated for 30min at room temperature. The activator for NPTX2 antibody activation is 2-IT (2-Iminothiolan hydrochloride), and the activator for alkaline phosphatase activation is SMCC (succinimidyl 4- (N-maleimidyl 1) cyclohexane-1-carboxylate).

4) Reaction: the reaction between the antibody and alkaline phosphatase may be carried out by mixing them in a mass ratio. Reacting at 2-8 deg.c for over 12 hr.

5) And (3) terminating the reaction: first, 10mM maleimide was added and the reaction was carried out at room temperature for 10 min. The reaction was stopped by adding 100mM ethanolamine.

6) Separation and purification: the column Superdex 200 was used for separation and purification to remove unbound NPTX2 antibody and alkaline phosphatase. The antibody marker concentrate was stored at 2-8 ℃.

7) NPTX2 antibody markers: the NPTX2 antibody marker concentrate was mixed with 1% BSA

0.1% Tween-20, 0.1% P-300, and 5mM MgCl2 in Tris buffer as antibody marker diluent, wherein the dilution ratio is 1:500-1:3000.

Preparation of acridinium ester labeled antibody

1) Reaction: the concentration of the antibody is within the range of 1-5mg/mL, the concentration of the acridine ester is within the range of 5-10mmol/L, and the reaction of the antibody and the acridine ester can be mixed according to the mass ratio and then reacted. The mixture is placed at 37 ℃ for reaction for more than 2 h.

2) And (3) sealing: adding lysine solution with concentration of 5-15g/L, and sealing for 15-60 min.

3) And (3) purification: purification was performed using a desalting column to remove unbound NPTX2 antibody and acridinium ester. An equal volume of glycerol was added to the antibody label and stored at-20 ℃.

4) Antibody labeling substance: and (3) diluting the labeled antibody concentrated solution by using a Tris buffer solution containing 1% BSA, 0.1% Tween-20 and 0.1% P-300 as an antibody label diluent at a dilution ratio of 1:1000-1:5000.

The calibrator is a calibrator containing NPTX2 antigen.

The concentrated washing solution is phosphate buffer solution containing Tween-20, and the working concentration of the washing solution is as follows: diluted 10-fold with purified water for use.

The luminescent substrate solution is alkaline phosphatase luminescent substrate solution or acridinium ester luminescent substrate solution.

The detection method of the kit is as follows:

3) and (3) detection reading: and adding a luminescent substrate to detect the luminescence value.

Performance index of the kit of the invention

1) Accuracy: adding a human neuron n-penta-protein II sample A with a concentration of about 3000pg/mL (the concentration deviation is allowed to be +/-20%) into a human neuron n-penta-protein II sample B with a concentration ranging from 150-300 pg/mL, wherein the volume ratio of the added sample A to the added sample B is 1: 9, detecting the mixed sample by using the kit, repeating the detection for 3 times, calculating the recovery rate (R) according to the following formula,

in the formula: r-recovery rate; v-volume of solution A added; v0 — volume of serum B; c-the detection concentration of the serum sample after the solution A is added; c0 — detection concentration of serum sample B; concentration of Cs-A solution. The recovery rate is 85 to 115 percentIn the meantime.

2) Blank limit: detecting with zero concentration calibrator or sample diluent as sample, repeating the determination for 20 times to obtain light absorption value of 20 measurement results, calculating average value (M) and Standard Deviation (SD) to obtain light absorption value corresponding to M +2SD, substituting the light absorption value corresponding to M +2SD into equation according to calibration curve of calibrator used in kit, and calculating corresponding concentration value, which is blank limit.

3) Repeatability: detecting quality control serum or fresh human serum with different concentrations for 10 times, respectively, calculating average value M and standard deviation SD of 10 detection results, respectively, obtaining coefficient of variation CV according to formula CV (SD/M) 100%, wherein CV value should not be greater than 10%.

4) Batch-to-batch repeatability: three batches of reagents are respectively used for detecting quality control serum or fresh human serum with different concentrations, the detection is repeated for 10 times each time, the average value M and the standard deviation SD of 30 detection results are respectively calculated, the coefficient of variation CV is obtained according to the formula CV (SD/M) 100%, and the CV value is not more than 15%.

5) Linearity: the NPTX2 high-value sample, which is near the upper limit of the linear interval, is diluted with negative serum to a certain proportion of at least 5 concentrations, wherein the low-value concentration sample is near the lower limit of the linear interval. Each concentration was repeatedly measured 2 times using the kit, and the mean value (yi) of the measurement results was obtained. And (3) calculating a linear regression equation by taking the dilution ratio (xi) as an independent variable and the mean value (yi) of the detection results as a dependent variable. And calculating a correlation coefficient (r) of the linear regression, wherein r is more than or equal to 0.9900.

Evaluation results of the methodology

1) Accuracy: the accuracy is evaluated by dilution and recovery, and the result is between 85% and 115%.

2) Blank limit: not greater than 20 pg/mL.

3) Repeatability: the repeatability in analysis is less than or equal to 10 percent, and the repeatability between analyses is less than or equal to 15 percent.

5) And (3) sample determination: the clinical coincidence rate of human neuron orthopentaprotein II (NPTX2) is more than 85% when 50 AD patient samples are compared and compared with Western blot assay.

The invention combines the chemiluminescence detection technology with the magnetic particle immune separation technology, quantitatively detects the content of the human neuron positive pentameric protein II (NPTX2), realizes high-flux and automatic detection, has good detection repeatability, and improves the sensitivity and the accuracy of the detection of the human neuron positive pentameric protein II (NPTX 2).

The above detailed description of embodiments of the invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. In addition, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.

Claims

1. Magnetic particle chemiluminescence immunoassay kit for NPTX2, characterized in that: the kit comprises calibrator, quality control substance, NPTX2 magnetic particle suspension, NPTX2 antibody marker, concentrated washing solution, luminescent bottom thing;

The calibrator is configured by NPTX2 antigen;

The quality control product contains a certain concentration of NPTX2 antigen, and is configured from the quality control product diluent;

The NPTX2 magnetic particle suspension contains a certain concentration of magnetic particle-labeled NPTX2 antibody solution.

2. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 1, characterized in that: the preparation method of the NPTX2 magnetic particle suspension is as follows:

1) Activation: 20μL of magnetic particle stock solution, add 100μL of 50mg/mLEDC, 50mg/mL NHS, 200r/min, activate for 0.5-1h; EDC solution is obtained by dissolving EDC reagent in 100mM MES buffer with pH 5.0, NHS solution is obtained by dissolving NHS reagent in 100mM MES buffer at pH 5.0;

2) Washing: put the above solution on a magnetic rack to separate, discard the supernatant, add 300 μL of MES buffer, mix well, separate and discard the supernatant on a magnetic rack, and wash twice;

3) Coating: add MES buffer and pre-coated antibody, the mass ratio of magnetic particles and antibody is 80:1, after mixing, react at room temperature for 8-12 hours;

4) Blocking: put the above solution on a magnetic stand to separate, discard the supernatant, add 100-300 μL of magnetic particle blocking solution, shake well, and react at room temperature for 8-12 hours;

5) The above solution was placed on a magnetic rack for separation, the supernatant was discarded, and a certain amount of magnetic beads were added to seal the solution to obtain a suspension of NPTX2 magnetic particles.

3. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 1, wherein the NPTX2 antibody marker is an alkaline phosphatase-labeled NPTX2 antibody solution or acridine containing a certain concentration Ester-labeled NPTX2 antibody solution.

4. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 3, wherein the preparation method of the alkaline phosphatase NPTX2 antibody marker is as follows:

1) NPTX2 antibody and alkaline phosphatase, the quality of alkaline phosphatase is 1.5-2 times that of the antibody, respectively, desalted, concentrated, and then added with activator, at room temperature, and activated for 20min respectively; desalted by Sephadex G25 gel column Salt; the activator used for NPTX2 antibody activation is 2IT, and the activator used for alkaline phosphatase activation is SMCC;

2) The activated NPTX2 antibody and alkaline phosphatase can be mixed and reacted according to the mass ratio; then the termination reaction and separation and purification are performed to remove the unbound NPTX2 antibody and alkaline phosphatase, and the antibody marker concentrate is prepared and stored. At 2-8°C; in the process of terminating the reaction, firstly add 10mM maleimide, and react at room temperature for 10min; then add 100mM ethanolamine; the separation and purification column is SuperdexTM200.

5. the magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 3, is characterized in that: described acridine ester-labeled antibody preparation method is as follows:

1) NPTX2 antibody and acridine ester, the mass of acridine ester is 50 times that of the antibody, mix well, react at 37 degrees for more than 2 hours, and add lysine to stop the reaction;

2) Purify the labeled acridinium ester-labeled antibody with a desalting chromatography column, collect the purified acridinium ester-labeled antibody, and store at -20°C.

6. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 1, wherein the kit detection method is as follows:

1) Immune reaction: 10 μL sample/quality control, 20 μL magnetic particles, 100 μL antibody marker, incubate at 37°C for 30 min, and wash 6 times;

2) Magnetic separation: sediment the magnetic particles in a magnetic field, discard the supernatant, add 150 μL of washing solution, sediment the magnetic particles in the magnetic field, discard the supernatant, repeat 4 times to remove unbound antibodies;

3) Detection and reading: add a luminescent substrate, react at room temperature, and detect the luminescence value within 10s-5min;

4) Calculation: The complex catalyzes the emission of photons from the luminescent substrate, and the luminous intensity is proportional to the content of NPTX2; select an appropriate curve fitting method to establish a standard curve; this kit recommends a four-parameter fitting method, using the calibrator concentration value is the x-axis, the logarithm of the luminescence intensity value of the calibrator is used to establish a standard curve on the y-axis, and the corresponding concentration value is calculated back according to the luminescence intensity value of the sample to be tested.

7. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 1, wherein the calibrator is a calibrator containing NPTX2 antigen;

The calibrator preparation method: use Tris buffer containing 1% casein as the calibrator diluent, dilute the NPTX2 antigen with the calibrator diluent, and dilute to the working concentration, respectively 50, 100, 250, 500, 1000, 3000pg/mL.

8. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 1, wherein the quality control product is a quality control product containing NPTX2 antigen;

The quality control product configuration method: use Tris buffer containing 1% casein as the quality control product diluent, and the NPTX2 antigen is serially diluted to 500 pg/mL and 1000 pg/mL with the quality control product diluent.

9. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 1, characterized in that: the performance index determination method of the kit:

1) Accuracy: Add standard sample A with a concentration of about 3000 pg/mL to standard sample B with a concentration range of 150-300 pg/mL, and the volume ratio between the added sample A and sample B is 1:9. The kit detects the mixed samples, repeats the detection 3 times, and calculates the recovery rate R according to the following formula,

In the formula: R-recovery rate; V-volume of adding solution A; V0-volume of serum B; C-detection concentration of serum sample after adding solution A; _C0 -detection concentration of serum sample B; concentration of Cs-A solution ;

2) Blank limit: use zero-concentration calibrator or sample diluent as a sample for detection, repeat the measurement 20 times, obtain the absorbance value of the 20 measurement results, calculate the mean value M and standard deviation SD, and obtain the value of M+2SD. The corresponding absorbance value, according to the calibration curve of the calibrator used in the kit, the absorbance value corresponding to M+2SD is brought into the equation, and the corresponding concentration value is obtained, which is the blank limit;

3) Repeatability: high and low two different concentrations of quality control serum or fresh human serum were tested, and each test was repeated 10 times, and the mean M and standard deviation SD of the 10 test results were calculated respectively, according to the formula CV=SD/M *100% to get the coefficient of variation CV, the CV value should not be greater than 10%;

4) Repeatability between batches: use three batches of reagents to detect quality control serum or fresh human serum with two different concentrations of high and low, repeat the test 10 times each time, and calculate the average value M and standard of the 30 test results. Difference SD, the coefficient of variation CV is obtained according to the formula CV=SD/M*100%, and the CV value should not be greater than 15%;

5) Linearity: Dilute the high-value samples close to the upper limit of the linear range with negative serum in a certain proportion to at least 5 concentrations, of which the samples with low-value concentrations must be close to the lower limit of the linear range; use the kit to repeat the detection of each concentration twice , respectively, to obtain the mean value yi of the detection results; take the dilution ratio xi as the independent variable, and take the mean value yi of the detection results as the dependent variable to obtain the linear regression equation; calculate the correlation coefficient r of the linear regression, r≥0.9900.

10. The magnetic particle chemiluminescence immunoassay kit for NPTX2 according to claim 1, wherein the evaluation result parameters of the kit are limited:

1) Accuracy: The accuracy is evaluated by the recovery rate, and the result is between 85% and 115%;

2) Blank limit: not more than 20pg/mL;

3) Repeatability: repeatability ≤ 10%, repeatability between batches ≤ 15%;

4) Linearity of dose-response curve: in the range of 20-3000pg/mL, the correlation coefficient of dose-response curve r≥0.99;

5) Sample determination: 50 AD patient samples were compared and compared with Western blot determination, and the clinical coincidence rate of NPTX2 was over 85%.