CN114791495A - Protein chip for detecting pathogenic antibody of nervous system autoimmune disease - Google Patents

Abstract

A protein chip for detecting the pathogenic antibodies of autoimmune diseases of the nervous system, the chip is a microarray with different areas distributed on a glass substrate, and specific antibodies capable of detecting the antibodies of autoimmune diseases of the nervous system are respectively fixed in each microarray area. Sexual Antigen Probes. The present invention combines NDMA1 protein, IgLON5 protein, Ma2 protein, CASPR2 protein, MOG protein, Amphiphysin protein, GAD65 protein, GAD67 protein, AMPAR1 protein, AMPAR2 protein, GABABR B1 protein, ZIC4 protein, Recoverin protein, DNER protein, MAG protein, NOVA protein ‑1 protein, AChR (1‑210aa) protein, GQ1b glycosphingolipids, GT1b glycosphingolipids, GD1b glycosphingolipids, GD1a glycosphingolipids, GM3 glycosphingolipids, GM2 glycosphingolipids, and GM1 glycosphingolipids , Develop protein chips to detect autoimmune disease-causing antibodies, and achieve the advantages of high throughput, strong specificity, high sensitivity, parallel processing and low cost.

Description

A protein chip for the detection of causative antibodies in autoimmune diseases of the nervous system

technical field

The invention relates to the technical field of medical in vitro diagnosis, in particular to a protein chip for detecting the pathogenic antibodies of autoimmune diseases of the nervous system, a preparation method and application thereof.

Background technique

Autoimmune diseases of the nervous system are a major group of important diseases in the field of neurology. They are characterized by the complexity of immune diseases and the high lethality and disability of neurological diseases, so they are highly concerned by clinicians and researchers. At present, the commonly used antibody detection methods at home and abroad are mostly based on ELISA detection method, which can only detect one antibody at a time. If all antibodies are to be detected, the workload is large, the time consumption is long, and the cost is high.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a protein chip for detecting the pathogenic antibodies of autoimmune diseases of the nervous system in view of the problems of low efficiency and long time in the detection of autoimmune diseases of the nervous system.

The technical scheme of the present invention is as follows: a protein chip for detecting the pathogenic antibodies of autoimmune diseases of the nervous system, the chip is a microarray with different areas distributed on a glass substrate, and each microarray area is respectively fixed to detect the nervous system Antigen-specific probes for autoimmune disease antibodies.

The probes can be specifically combined with pathogenic antibodies, and 24 probes are designed. The sources of the designed probes are as follows:

A method for preparing a protein chip for detecting pathogenic antibodies of autoimmune diseases of the nervous system, comprising the following steps:

1. Design antigen-specific probes:

The target gene is amplified by PCR and inserted into the corresponding expression vector, and the sequence of the constructed vector is verified by sequencing, and the correct plasmid is amplified and transmitted to the downstream;

2. Expression and purification:

(1) HEK293 system

Plasmids are mixed with transfection reagents in optimal ratios, then added to flasks or bioreactors containing HEK293, cells are cultured in serum-free medium and incubated at 37°C with appropriate stirring speed on an orbital shaker or bioreactor The cell culture medium was centrifuged for 6 days in the Erlenmeyer flask on the device; the cell culture supernatant was loaded onto the affinity purification column at a suitable flow rate, and the molecular weight and purity of the purified protein were analyzed by SDS-PAGE;

(2)insect cell

Insert the cDNA of interest into the baculovirus vector, then generate recombinant baculovirus according to the manufacturer's manual, amplify the recombinant baculovirus in cells to prepare high-titer viral libraries, and for protein expression, infect with the recombinant baculovirus according to standard protocols cells, and express the target protein under optimal conditions, the cell pellet is collected by centrifugation and resuspended in homogenization buffer, the pellet is homogenized and centrifuged to remove cellular debris, the lysate supernatant is collected and loaded into the and purification column, the target protein is eluted from the column using elution buffer, the fractions containing the desired protein are pooled and exchanged into the formulation buffer, the protein concentration of the final product is determined by UV or BCA analysis, The purity of the final product was analyzed by SDS-PAGE;

(3)E.colic

The vector containing the gene of interest (GOI) was transferred into E. coli, and then high-expressing transformants were selected and established. An expression strain was selected for fermentation to express the target protein, and a single colony was cultured overnight at 37°C in MDG medium (Sino Biologic). Seed cultures were inoculated into TB medium (Sino Biologic) and grown at 37°C to an OD600 of 0.8, and the cultures were induced by using 0.4 mM isopropyl β-D-1-thiogalactoside (IPTG). To initiate protein expression, after IPTG induction, the culture temperature was moved to 18°C and allowed to ferment overnight for target protein expression. The cells were harvested by centrifugation, lysed by sonication, and the inclusion bodies were extracted by centrifugation. Before the denaturation process, they were washed with Buffer The particles were washed 3 times, inclusion bodies were dissolved in 8M urea, any precipitate in the process was removed by centrifugation, and the protein solution was loaded onto a metal ion affinity chromatography column using elution buffer containing 8M urea and 250mM imidazole The target protein was eluted with the buffer solution, the fractions containing the protein of interest were pooled and refolded, the purified target protein was refolded by dialysis in refolding buffer at 4°C for 2 days, any precipitate in the process was removed by centrifugation, The refolded buffer was exchanged for protein storage buffer using a Sephadex 25 column, the protein purity was determined by Coomassie blue-stained SDS-PAGE gel under reducing conditions, and the protein concentration was quantified using the BCA quantification method;

3. Preparation of chips:

(1) Dilute with probe diluent to the required concentration, distribute in 384-well plates, and store at 4°C for future use (if it is ordered within 1 day, it can be placed at 4 degrees, and if it exceeds, it needs to be stored at -80 degrees);

(2) Set the software parameters of the spotter, the array is as follows:

1 2 3 4 5 6 7 8 9 10 11 12 A POS1 POS1 POS1 POS2 POS2 POS2 NEG NEG NEG 1 1 1 B 2 2 2 3 3 3 4 4 4 5 5 5 C 6 6 6 7 7 7 8 8 8 9 9 9 D 10 10 10 11 11 11 12 12 12 13 13 13 E 14 14 14 15 15 15 16 16 16 17 17 17 F 18 18 18 19 19 19 20 20 20 twenty one twenty one twenty one G twenty two twenty two twenty two twenty three twenty three twenty three twenty four twenty four twenty four 25 25 25 H 26 26 26 27 27 27 NEG NEG NEG POS1 POS1 POS1

(3) Input the output parameters of the array design into the machine software and save and record;

The sample is placed on the sample stage (the temperature can be set in advance on the sample stage to protect the sample), and the machine is run; the machine will spot the sample on the corresponding carrier; after the chip spotting is completed, run the quality control program to check the spot system Whether the sample is placed at the set position, that is, whether the array is neat and regular, the chips that do not meet the standard are picked out and discarded; the quality control pictures and parameters of each available and unavailable slide are saved; the spotting is completed;

(4) After the chip point-making is completed, it needs to be dried for 3-5 hours; after the drying is completed, seal the package and place it at 4 degrees; select one piece, carry out experimental measurement, and evaluate the time by comparing with the signal value of the quality control. Whether the ordered chips meet the standard; the chips that meet the standard are packaged and output.

The application of protein chip for detecting autoimmune pathogenic antibodies of nervous system includes the following steps:

(1) Processing samples:

Dilute the sample with the sample diluent to obtain 100 μL of each sample loaded;

(2) Dry chips:

Take the slide chip out of the box, and after equilibrating at room temperature for 20-30min, open the packaging bag, peel off the sealing strip, and then place the chip in a vacuum desiccator or dry at room temperature for 1-2 hours;

(3) Sample incubation:

Add 100 μL of sample diluent to each well, incubate for 1 h on a shaker at room temperature, block the quantitative antibody chip, remove the buffer from each well, add 100 μL of sample to the well, and incubate at 4°C overnight;

(4) Cleaning the chip:

Use the Thermo Scientific Wellwash Versa chip washer to wash the slides, which is divided into two steps. First, wash with Wash Solution I, wash 10 times with 250 μL of Wash Solution I per well, and shake for 10 s each time, and choose a high vibration intensity. Then use the washing solution II channel for cleaning, wash 6 times with 250 μL of washing solution II per well, shake for 10 s each time, and choose a high vibration intensity;

(5) Biotin antibody incubation:

Centrifuge the biotin antibody vial, dilute the biotin antibody of all species by 6000 times, mix well and centrifuge again quickly. Add 100 μL of detection antibody to each well and incubate for 2 hours on RT shaker;

(6) Cleaning the chip:

Use the Thermo Scientific Wellwash Versa chip washer to wash the slides, which is divided into two steps. First, wash with Wash Solution I, wash 10 times with 250 μL of Wash Solution I per well, and shake for 10 s each time, and choose a high vibration intensity. Then use the washing solution II channel for cleaning, wash 6 times with 250 μL of washing solution II per well, shake for 10 s each time, and choose a high vibration intensity;

(7) Incubation of Cy3-streptavidin:

Centrifuge the Cy3-streptavidin vial, then add 1.4 ml of sample diluent, mix well, and centrifuge again quickly. Add 80 μL of Cy3-streptavidin to each well, wrap the slides with aluminum foil and incubate in the dark for 1 hour on a RT shaker;

(8) Cleaning the chip:

Use the Thermo Scientific Wellwash Versa chip washer to wash the slides. It is divided into two steps. First, wash with Wash Solution I, 250 μL of Wash Solution I in each well, wash 10 times, and shake for 10 s each time. Wash with the washing solution II channel, wash 6 times with 250 μL of washing solution II per well, shake for 10 s each time, and choose a high vibration intensity;

(9) Fluorescence detection:

Scan the signal with a laser scanner such as InnoScan 300, using Cy3 or green channel (excitation frequency = 532 nm);

(10) Data analysis:

Data analysis was performed using the data analysis software of QAH-CUST.

The antibodies detected by the protein chip of the present invention include NDMA1 protein, IgLON5 protein, Ma2 protein, CASPR2 protein, MOG protein, Amphiphysin protein, GAD65 protein, GAD67 protein, AMPAR1 protein, AMPAR2 protein, GABABRB1 protein, ZIC4 protein, Recoverin protein, DNER protein , MAG protein, NOVA-1 protein, AChR(1-210aa) protein, GQ1b glycosphingolipid, GT1b glycosphingolipid, GD1b glycosphingolipid, GD1a glycosphingolipid, GM3 glycosphingolipid, GM2 glycosphingolipid, and GM1 glycosphingolipid A total of 24 antigens such as lipids.

The significant effect of the present invention is: the present invention, by means of biochip technology, converts such known NDMA1 protein, IgLON5 protein, Ma2 protein, CASPR2 protein, MOG protein, Amphiphysin protein, GAD65 protein, GAD67 protein, AMPAR1 protein, AMPAR2 protein, GABABR B1 protein, ZIC4 protein, Recoverin protein, DNER protein, MAG protein, NOVA-1 protein, AChR(1-210aa) protein, GQ1b Glycosphingolipid, GT1b Glycosphingolipid, GD1b Glycosphingolipid, GD1a Glycosphingolipid, GM3 A total of 24 antigens, including glycosphingolipids, GM2 glycosphingolipids and GM1 glycosphingolipids, have been developed, and protein chips have been developed to detect pathogenic antibodies in blood pressure or cerebrospinal fluid of patients with autoimmune diseases, achieving high-throughput, strong specificity, and sensitivity for detection. It has the advantages of high performance, parallel processing and low cost, which is superior to other detection technologies and has important reference value for the diagnosis, treatment and prognosis of diseases.

Description of drawings

Fig. 1 is the ROC curve analysis schematic diagram of the chip of the present invention detecting the expression of NMDA1 antibody in patient's plasma;

Figure 2 is a schematic diagram of the fluorescence intensity of NMDA1 antibody in the plasma of NMDAR group (NMDA1 antibody positive patients) and CON group (NMDA1 antibody negative patients) patients detected by the chip of the present invention;

3 is a schematic diagram of a protein chip scanning picture of the plasma of patients in the NMDAR group and the CON group in the chip detection of the present invention;

4 is a schematic diagram of the fluorescence intensity of NMDA1 antibody in the plasma of four groups of mice in the CON group (control group), NDMA1 group (model group), GLYX-13 group (treatment group) and BE group (treatment group) in the chip detection of the present invention;

Figure 5 is a schematic diagram of protein chip scanning pictures of mouse plasma in CON group (control group), NDMA1 group (model group), GLYX-13 group (treatment group) and BE group (treatment group) in the chip detection of the present invention.

Detailed ways

The specific implementations of the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments.

A protein chip for detecting pathogenic antibodies of autoimmune diseases of the nervous system, the method comprises the following steps:

1. A protein chip for detecting pathogenic antibodies of autoimmune diseases of the nervous system and its preparation process and application method, the chip is a microarray with different regions distributed on a glass substrate, and each microarray region is fixed to detect Antigen-specific probes for antibodies to autoimmune diseases of the nervous system.

2. The probes can be specifically combined with pathogenic antibodies, and 24 probes are designed. The sources of the designed probes are as follows:

The protein chip designed and synthesized above was used to detect the expression of anti-NMDAR antibody in the plasma of patients with autoimmune encephalitis and the cerebrospinal fluid of the mouse model of autoimmune encephalitis, respectively. Data analysis was performed using the data analysis software of QAH-CUST. Compared with the clinical test results, the sensitivity of detecting anti-NMDAR antibody encephalitis patients' plasma was 100%, and the specificity was 50% (see Figure 1). The results of plasma detection of anti-NMDAR mice were consistent with those of Elisa (see Figure 2).

Claims (3)

1. A protein chip for detecting pathogenic antibodies of autoimmune diseases of a nervous system is characterized in that microarrays with different areas are distributed on a glass substrate, and specific antigen probes capable of detecting the antibodies of the autoimmune diseases of the nervous system are respectively fixed in each microarray area;

the probes can be specifically combined with pathogenic antibodies, and 24 probes are designed, wherein the sources of the designed probes are as follows:

probe needle Expression System/Item number Concentration (mg/mL) IgLON5 HEK293 0.325 nmda1 HEK293 0.365 ZIC4 insect cell 0.1 GQIb 1516 0.2 GT1b 1063 0.2 GD1b 1501 0.2 GD1a 1062 0.2 GM3 1503 0.2 GM2 1502 0.2 GM1 MD-19-0006P 0.2 Ma2 230-00883-10 0.24 Recoverin 230-00699-10 0.2 DNER 230-30104-10 0.2 CASPR2 230-30105-10 0.2 MOG 10364-H08E 0.2 NOVA-1 230-30081 0.2 Amphiphysin 230-30073-10 0.2 GAD65 17802-H09B 0.2 GAD67 12570-H08B 0.2 MAG 13186-H08H 0.2 ampar1 HEK293 0.2775 ampar2 HEK293 0.26 GABABR B1 insect cell 0.345 AChR E.colic 0.25

2. A preparation method of a protein chip for detecting pathogenic antibodies of autoimmune diseases of a nervous system is characterized by comprising the following steps:

(1) designing an antigen-specific probe:

after PCR amplification, the target gene is inserted into a corresponding expression vector, the sequence of the constructed vector is verified through sequencing, and the correct plasmid is amplified and transmitted to the downstream;

(2) expression and purification:

phi HEK293 System

Mixing the plasmid with transfection reagent in an optimal ratio, adding into a flask or a bioreactor containing HEK293, culturing the cells in serum-free medium, maintaining in an orbital shaker or a conical flask on the bioreactor at 37 ℃ and a proper stirring speed for 6 days, and centrifuging the cell culture solution; loading the cell culture supernatant onto an affinity purification column at a suitable flow rate, and analyzing the molecular weight and purity of the purified protein by SDS-PAGE;

②insect cell

inserting the target cDNA into a baculovirus vector, then generating a recombinant baculovirus according to manufacturer's manual, amplifying the recombinant baculovirus in cells to prepare a high titer viral pool, for protein expression, infecting the cells with the recombinant baculovirus according to standard protocols and expressing the target protein under optimal conditions, collecting the cell particles by centrifugation and resuspending in a homogenization buffer, homogenizing and centrifuging the particles to remove cell debris, collecting the lysate supernatant and loading it onto an affinity purification column, eluting the target protein from the column using an elution buffer, pooling the fractions containing the desired protein and exchanging it into the formulation buffer, the protein concentration of the final product being determined by UV or BCA analysis, and the purity of the final product being analyzed by SDS-PAGE;

③E.colic

the vector containing the gene of interest (GOI) was transferred into E.coli, and then a high-expression transformant was selected and established. One expression strain was selected for fermentation to express the protein of interest, and individual colonies were cultured overnight at 37 ℃ in MDG medium (Sino Biologic). Inoculating the seed culture into TB medium (Sino Biologic) and culturing at 37 ℃ to OD600 of 0.8, initiating protein expression by inducing the culture with 0.4mM isopropyl beta-D-1-thiogalactoside (IPTG), after IPTG induction, moving the culture temperature to 18 ℃ and allowing fermentation overnight for target protein expression, collecting the cells by centrifugation, sonicating the cells, centrifuging the inclusion bodies for extraction, washing the particles 3 times with wash buffer before the denaturation process, dissolving the inclusion bodies in 8M urea, removing any precipitate during the process by centrifugation and loading the protein solution onto a metal ion affinity chromatography column, eluting the target protein with elution buffer containing 8M urea and 250mM imidazole, combining and refolding the fractions containing the protein of interest, refolding the purified target protein by dialysis in refolding buffer at 4 ℃ for 2 days, removing any precipitate in the process by centrifugation and exchanging refolded buffer for protein storage buffer using a Sephadex 25 column, determining protein purity by Coomassie blue staining SDS-PAGE gel under reducing conditions, and quantifying protein concentration using BCA quantification method;

(3) preparing a chip:

diluting with a probe diluent to a required concentration, subpackaging in a 384-well plate, and storing at 4 ℃ for later use (if the concentration is 4 ℃ in 1 day, storing at-80 ℃ if the concentration exceeds the preset concentration);

setting the software parameters of the sample applicator, and making an array diagram as follows:

1 2 3 4 5 6 7 8 9 10 11 12 A POS1 POS1 POS1 POS2 POS2 POS2 NEG NEG NEG 1 1 1 B 2 2 2 3 3 3 4 4 4 5 5 5 C 6 6 6 7 7 7 8 8 8 9 9 9 D 10 10 10 11 11 11 12 12 12 13 13 13 E 14 14 14 15 15 15 16 16 16 17 17 17 F 18 18 18 19 19 19 20 20 20 21 21 21 G 22 22 22 23 23 23 24 24 24 25 25 25 H 26 26 26 27 27 27 NEG NEG NEG POS1 POS1 POS1

inputting the output parameters of the array design into the machine software, storing and recording;

placing the sample on a sample table (the temperature can be set in advance on the sample table to protect the sample), and operating the machine; the machine spots the samples on respective carriers; after the chip pointing is finished, running a quality control program to check whether the pointed sample is pointed at a set position, namely whether the array is regular or not, and picking out and discarding chips which do not meet the standard; storing the quality control pictures and parameters of each usable slide and each unusable slide; finishing the ordering;

fourthly, drying the chips for 3 to 5 hours after the chips are ordered; after drying, sealing, packaging and placing at 4 ℃; selecting one, carrying out experimental determination, and evaluating whether the clicked chip meets the standard or not by comparing with the quality control signal value; and packaging and outputting the chips meeting the standard.

3. The application of the protein chip for detecting the autoimmune pathogenic antibody of the nervous system is characterized by comprising the following steps:

(1) treating a sample:

diluting the sample with a sample diluent to obtain 100 mu L of each sample;

(2) and (3) drying the chip:

taking out the slide chip from the box, balancing at room temperature for 20-30min, opening the packaging bag, uncovering the sealing strip, and then drying the chip in a vacuum drier or at room temperature for 1-2 hours;

(3) sample incubation:

adding 100 mu L of sample diluent into each well, incubating for 1h on a shaking table at room temperature, sealing the quantitative antibody chip, removing buffer solution from each well, adding 100 mu L of sample into each well, and incubating overnight at 4 ℃;

(4) cleaning the chip:

the slide is cleaned by using a Thermo Scientific Wellwash Versa chip plate cleaning machine in two steps, cleaning is carried out by using a cleaning solution I, 250 mu L of the cleaning solution I is used for cleaning 10 times in each hole, each time, the vibration is carried out for 10s, and the vibration intensity is selected to be high. Then, cleaning by using a washing liquid II channel, wherein 250 mu L of washing liquid II is washed for 6 times, each time is oscillated for 10s, and the oscillation intensity is selected to be high;

(5) biotin antibody incubation:

the biotin antibody vials were centrifuged and the biotin antibodies of all species were diluted 6000 times, mixed well and then centrifuged rapidly again. Add 100. mu.L of detection antibody to each well and incubate for 2 hours on RT shaker;

(6) cleaning the chip:

the slide is cleaned by using a Thermo Scientific Wellwash Versa chip plate cleaning machine in two steps, cleaning is carried out by using a cleaning solution I, 250 mu L of the cleaning solution I is used for cleaning 10 times in each hole, each time, the vibration is carried out for 10s, and the vibration intensity is selected to be high. Then, cleaning by using a washing liquid II channel, wherein each hole is cleaned by 250 mu L of washing liquid II for 6 times, each time is vibrated for 10s, and the vibration intensity is selected to be high;

(7) incubation of Cy 3-streptavidin:

the Cy 3-streptavidin vial was centrifuged, then 1.4ml of sample diluent was added, mixed well and centrifuged quickly again. Adding 80 mu L of Cy 3-streptavidin into each well, wrapping the slide with aluminum foil paper, incubating in a dark place, and incubating on an RT shaker for 1 hour;

(8) cleaning the chip:

cleaning the glass slide by using a Thermo Scientific Wellwash Versa chip plate cleaning machine, wherein the method comprises two steps of cleaning by using a cleaning solution I, cleaning by using a cleaning solution II channel for cleaning, wherein 250 mu L of the cleaning solution I is used for each hole for 10 times, each time of vibration is 10s, the vibration intensity is selected to be high, and cleaning by using a cleaning solution II channel for cleaning, each 250 mu L of the cleaning solution II is used for 6 times, each time of vibration is 10s, and the vibration intensity is selected to be high;

(9) fluorescence detection:

scanning the signal with a laser scanner such as InnoScan 300, using Cy3 or green channel (excitation frequency 532 nm);

(10) and (3) data analysis:

data analysis was performed using data analysis software of QAH-CUST.

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