CN111693692B

CN111693692B - Detection kit and detection method for PNS or AE autoantibody in human body fluid

Info

Publication number: CN111693692B
Application number: CN202010607670.5A
Authority: CN
Inventors: 闫亚平; 裴元元; 冯昆; 封雪; 李怡婷; 李科
Original assignee: Shaanxi Mybiotech Co ltd
Current assignee: Shaanxi Mybiotech Co ltd
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2021-12-10
Anticipated expiration: 2040-06-29
Also published as: CN111693692A

Abstract

The invention discloses a detection kit and a detection method for a PNS or AE autoantibody in human body fluid, wherein the detection kit comprises a test strip, a working solution, a confining protein solution, a secondary antibody and a color developing agent; the detection method comprises the steps of mixing working solution and blocking protein solution to form sample diluent, and adding a sample to be detected into the sample diluent; dripping a sample to be detected on a sample pad of the test strip; taking down the water absorption pad and the sample pad on the test strip, washing the carrier membrane, and fixing the water absorption pad and the sample pad at two ends of the carrier membrane after washing; immersing the sample pad end of the test strip into a second antibody diluent, and standing for 5-10 min at room temperature; washing the carrier film again; and immersing the sample pad end of the test strip into a color developing agent, and observing the color developing condition. In the detection process, the sample chromatography method is an inverted chromatography method, and the carrier membrane washing method is ultrasonic washing, so that the detection sensitivity and specificity are effectively increased. Antibodies can still be detected by diluting strong positive serum by more than ten thousand times.

Description

Detection kit and detection method for PNS or AE autoantibody in human body fluid

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a detection kit and a detection method for a PNS or AE autoantibody in human body fluid.

Background

The syndrome of Paraneoplastic Neurological Syndromes (PNS) refers to a disease associated with cancer that affects distant organs of the body before the tumor metastasizes, resulting in organ dysfunction. The pathogenesis of PNS is the immune response of tumor cells to neuronal surface proteins. Although the incidence of PNS is only 1% in cancer patients, it has important clinical implications, and the onset of PNS is often in early or limited stages of cancer, and thus PNS contributes to the discovery of potentially malignant primary tumors. According to the PNS diagnostic criteria published by Graus et al in 2004, neuronal autoimmune antibodies are usually detected in the serum or cerebrospinal fluid of PNS patients. There are nearly 20 types of PNS-related autoantibodies currently discovered.

Autoimmune Encephalitis (AE) refers to a class of encephalitis caused by an Autoimmune mechanism, which is clinically manifested as limbic encephalitis, and the intrinsic mechanism is that the immune system in the human body generates autoantibodies to intracellular proteins, synaptic receptors, ion channels and surface proteins of neurons. The latest diagnostic criteria for the diagnosis of autoimmune encephalitis are described in the literature published in 2018 by Divyanshu Dubey et al, and AE can be preliminarily determined by conventional medical imaging means, since AE is a heterogeneous group of diseases requiring the homogeneous grouping of patients by different autoantibodies. It follows that the identification of autoantibodies is of great importance for the diagnostic treatment of AE.

At present, methods for detecting autoantibodies in blood and cerebrospinal fluid of suspected PNS and AE patients mainly comprise an indirect immunofluorescence method, a flow cytometry method, an enzyme-linked immunosorbent assay, a protein immunoblotting method and an immunospot method, wherein both the indirect immunofluorescence method and the protein immunoblotting method are complex in operation, strong in operation skill (needing to be operated by professionals or experienced persons), and long in detection time (needing to be operated by two days); the enzyme-linked immunosorbent assay and the flow cytometry have high detection cost, and simultaneously need special equipment and an experienced person for operation; although the immuno-spot method improves the detection sensitivity, the detection cycle also requires 1 hour. In addition, the accumulated sensitivity of the methods in clinical detection is estimated to be 60-80%, and the methods have the defects of complicated operation, high detection cost, large antibody demand, time-consuming detection process and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a detection kit and a detection method for a PNS or AE autoantibody in human body fluid, and solves the problems of high detection cost, large antibody demand, low detection sensitivity, time-consuming detection process and the like of the existing detection method or detection kit.

In order to achieve the purpose, the invention adopts the following technical scheme:

the detection kit for the PNS or AE autoantibody in the human body fluid comprises a test strip, a sample diluent, a secondary antibody, a washing solution and a color developing agent;

the test strip comprises a backing plate, a carrier film provided with a control point and a detection point, a water absorption pad and a sample pad; a target antigen Ag is solidified on the detection point of the carrier film, and the Ag represents one of the target antigens corresponding to the PNS or AE disease autoantibody; negative control antigen is solidified on the control point; the water absorption pad, the carrier film and the sample pad are all laid on the backing plate, the water absorption pad is arranged at the contrast point end of the carrier film, and the sample pad is arranged at the detection point end of the carrier film;

the working solution is a mixed solution of 1 XPBS or 1 XPTBS or 100mM sodium phosphate buffer solution, 0.5-1% Triton X-100 or 0.5-1% Tween-20 and 0.02-0.05% EDTA, and the pH value of the working solution is 6.8-7.2;

the blocking protein solution is prepared by the following method:

transforming pET28a-GST or pET28a plasmid vector into Escherichia coli to obtain thallus containing pET28a-GST or pET28a plasmid; and (2) centrifuging the bacterial liquid containing pET28a-GST or pET28a plasmid thallus, adding a bacteria breaking liquid into the obtained supernatant, carrying out ultrasonic breaking for 15-35 min, centrifuging, taking the supernatant, and adding 5-10% of BSA, 2-5% of PEG12000 and 1 Xof protease inhibitor to obtain the protein blocking liquid.

Furthermore, the detection kit also comprises a washing solution, wherein the washing solution is a mixed solution of 50-80 mM Tris-HCl, 0.8-1% NaCl and 0.5-1% Triton X-100 or a mixed solution of 50-80 mM Tris-HCl, 0.8-1% NaCl and 0.5-1% Tween-20, and the pH of the washing solution is 8.2-8.5.

The invention also discloses a method for detecting the PNS or AE autoantibody in the human body fluid, which adopts the detection kit to detect, and the method comprises the following steps:

step 1, mixing a working solution and a blocking protein solution according to a volume ratio of 5: 1-15: 1 to form a sample diluent, and adding a sample to be detected into the sample diluent;

wherein, the sample diluent: the volume ratio of the sample to be detected is 10: 1;

step 2, dripping a sample to be detected added with the sample diluent on a sample pad of the test strip, inclining the test strip to enable the sample to be detected to uniformly flow through a carrier membrane containing target protein Ag, and standing for 2-3 min at room temperature;

step 3, taking down the water absorption pad and the sample pad on the test strip processed in the step 2, washing the carrier membrane, and fixing the water absorption pad and the sample pad at two ends of the carrier membrane after washing;

step 4, immersing the sample pad end of the test strip processed in the step 3 into a second antibody diluent, and standing for 5-10 min at room temperature;

step 5, taking down the water absorption pad and the sample pad on the test strip processed in the step 4, washing the carrier membrane, and fixing the water absorption pad and the sample pad at two ends of the carrier membrane after washing;

and 6, immersing the sample pad end of the test strip treated in the step 5 into a color developing agent, and observing the color developing condition.

Specifically, in the step 3, the carrier membrane is washed by the working solution;

in the step 5, the carrier membrane is washed by a washing solution, the washing solution is a mixed solution of 50-80 mM Tris-HCl, 0.8-1% NaCl and 0.5-1% Triton X-100 or a mixed solution of 50-80 mM Tris-HCl, 0.8-1% NaCl and 0.5-1% Tween-20, and the pH value of the washing solution is 8.2-8.5.

The invention also discloses a preparation method of the material for detecting the PNS or AE autoantibody in the human body fluid, which is used for obtaining the target antigen Ag and comprises the following steps:

step 1, obtaining a CDS sequence of a target antigen Ag as a target gene, and inserting the target gene with a restriction enzyme cutting site into a pET28a plasmid vector to obtain a recombinant plasmid vector pET28 a-Ag; ag represents one of target antigens corresponding to the autoantibodies of PNS or AE diseases;

step 2, transforming the recombinant vector pET28a-Ag into escherichia coli to obtain thalli containing a target antigen Ag;

step 3, purifying the thalli containing the target antigen Ag obtained in the step 2 by adopting an affinity chromatography;

and 4, verifying the product purified in the step 3, and solidifying the target antigen Ag which is verified to be error-free on a carrier membrane to obtain the PNS or AE autoantibody detection material in the human body fluid.

Specifically, the affinity chromatography purification process in step 3 comprises:

step 3.1, adding a bacteria-breaking liquid into the thalli containing the target antigen Ag obtained in the step 2, oscillating, whirling, ultrasonically crushing for 15-35 min, and centrifuging at 4-8 ℃ for 20-30min to obtain a supernatant, namely an initial sample liquid;

wherein, bacterial liquid: the volume ratio of the bacteria-breaking liquid is 20: 1; the bacteria breaking liquid corresponding to the His label antigen is a mixed liquid of 20mM Tris-HCl, 10-200 mM NaCl and 10-100 mM imidazole, and the pH of the bacteria breaking liquid is 7.4-9.0; the bacteria breaking solution corresponding to the GST tag antigen is 1 multiplied by PBS;

step 3.2, washing the affinity chromatography column by using deionized water, balancing 10-30 times of column volume by using a bacteria breaking liquid, adding the initial sample liquid obtained in the step 3.1 into the well-balanced affinity chromatography column, and combining for 1-2 hours at 4 ℃;

step 3.3, washing the affinity chromatography column obtained in the step 3.2 by 10-30 times of column volume with a first buffer solution, performing gradient elution on the washed pre-packed column with a first eluent, collecting the eluent, and desalting the eluent to obtain a purified target antigen Ag;

the first buffer solution corresponding to the His label antigen is a mixed solution of 20mM Tris-HCl and 100-1000 mM NaCl, and the pH value of the first buffer solution is 7.4-9.0; the first buffer solution corresponding to the GST tag antigen is 1 XPBS;

the first eluent corresponding to the His label antigen is a mixed solution of 20mM Tris-HCl and 10-1000 mM imidazole or a mixed solution of 20mM Tris-HCl and 10-300 mM Glu; the first eluent corresponding to the GST tag antigen is a mixed solution of 50mM Tris-HCl and 10-100 mM reduced glutathione, and the pH of the first eluent is 8.0.

Further, based on the purification in step 3, further purification can be carried out by ion exchange chromatography, hydrophobic chromatography or gel filtration chromatography.

Specifically, the purification process of the ion exchange chromatography comprises the following steps:

firstly, dialyzing an eluted sample after affinity chromatography by using a second buffer solution, replacing the second buffer solution for 3-4 times in the dialysis process, wherein the dialysis time is 2-8 hours each time, and taking out a sample from a dialysis bag to obtain an initial sample solution;

wherein the second buffer solution in the cation exchange chromatography is 20mM PB with the pH value of 5.0-7.0, and the second buffer solution in the anion exchange chromatography is 20mM Tris with the pH value of 7.0-9.0;

then, washing the ion exchange chromatography column by 10-30 times of column volume with deionized water, treating the column by 10-30 times of column volume with a second buffer solution, and loading the initial sample solution at the flow rate of 1 ml/min;

finally, washing the loaded ion exchange chromatographic column by using a second buffer solution, performing gradient elution on the washed ion exchange chromatographic column by using a second eluent, and collecting the eluent to obtain a target antigen Ag after further purification;

wherein the second eluent in the cation chromatography is a mixed solution of 20mM PB with pH 5.0-7.0 and 50-1000 mM NaCl; the second eluent in the anion chromatography is a mixture of 20mM Tris and 50-1000 mM NaCl, the pH value of which is 7.0-9.0.

Specifically, the hydrophobic chromatography purification process comprises:

first, NaCl or (NH) is added to an eluted sample of affinity chromatography₄)₂SO₄The final concentration of NaCl in the eluted sample is 0.6-2.0M and (NH)₄)₂SO₄The final concentration is 0.4-2.0M, and an initial sample solution is obtained;

then, washing the hydrophobic chromatography column by using deionized water, treating the column with 10-30 times of column volume by using a third buffer solution, and loading an initial sample solution at the flow rate of 1 ml/min;

wherein the third buffer solution has a pH of 6.0-9.0, 20mM PB or 0.4-2.0M (NH)₄)₂SO₄Or a mixture of 20mM Tris and 0.6-2.0M NaCl at a pH of 6.0-9.0;

finally, washing the column with a third buffer solution by 10-30 times of the volume of the column, performing gradient elution on the washed hydrophobic chromatographic column by using a third eluent, and collecting the eluent to obtain a target antigen Ag after further purification;

wherein the third eluent has pH of 6.0-9.0, 20mM PB or 2.0-0.4M (NH)₄)₂SO₄Or a mixture of 20mM Tris and 2.0-0.6M NaCl at a pH of 6.0-9.0.

Specifically, the purification process of the gel filtration chromatography comprises the following steps:

firstly, washing a gel filtration chromatography column by using deionized water, treating 10-30 times of column volume by using a fourth buffer solution, and loading an elution sample after affinity chromatography at a flow rate of 1 ml/min;

then eluting with a fourth buffer solution, and collecting eluent to obtain a target antigen Ag after further purification;

wherein the fourth buffer solution is pH 6.0-9.0, 20mM PB or mixture of pH 6.0-9.0, 20mM Tris and 50-500 mM NaCl.

Compared with the prior art, the invention has the beneficial effects that:

(2) in the detection process, the sample chromatography method is an inverted chromatography method, and the carrier membrane washing method is ultrasonic washing, so that the detection sensitivity and specificity are effectively increased. Antibodies can still be detected by diluting strong positive serum by more than ten thousand times.

(2) The detection method has the advantages of simple operation, no need of professional equipment assistance, short detection period and only 20min or so in the whole detection process.

(3) According to the invention, the blocking protein is prepared independently in the detection process, the blocking material is derived from the control cell which is homologous with the detection material, and the blocking protein is applied to the detection of the autoantibody, so that the pollution caused by the impurity protein can be effectively reduced, and the detection sensitivity is increased.

(4) The detection of the invention adopts a prokaryotic expression mode to obtain the antigen, the obtaining period is shorter, the cost is lower, the one-time obtaining amount is larger, and the kit with stable quality is obtained. Meanwhile, the purity of the target antigen is greatly improved by combining a plurality of purification methods. Therefore, the detection kit has higher sensitivity and specificity.

Drawings

FIG. 1 is a front view of the test strip of the present invention.

FIG. 2 is a side view of the structure of the test strip of the present invention.

FIG. 3 is a schematic flow chart of the detection method of the present invention.

FIG. 4 is the result obtained in example 3 of the present invention, in which panel A shows the positive and negative coloration results and panel B shows the positive detection rate in the serum sample and the cerebrospinal fluid sample.

FIG. 5 is the result obtained in comparative example 1 of the present invention, in which A is the positive and negative coloration results and B is the positive detection rate in the serum sample and the cerebrospinal fluid sample in comparative example 1 and example 3.

FIG. 6 is the result obtained in comparative example 2 of the present invention, in which A is the positive and negative coloration results and B is the positive detection rate in the serum sample and the cerebrospinal fluid sample in comparative example 2 and example 3.

FIG. 7 is the result obtained in comparative example 3 of the present invention, in which A is the positive and negative coloration results and B is the positive detection rate in the serum sample and the cerebrospinal fluid sample in comparative example 3 and example 3.

FIG. 8 is the result obtained in comparative example 4 of the present invention, in which A is the positive and negative coloration results and B is the positive detection rate in the serum sample and the cerebrospinal fluid sample in comparative example 4 and example 3.

Detailed Description

The invention discloses a detection kit for PNS or AE autoantibodies in human body fluid, which can detect the AE and twenty kinds of autoantibodies related to the PNS.

The detection kit specifically comprises a test strip, a working solution, a blocking protein solution, a secondary antibody and a color developing agent;

wherein, the test strip comprises a backing plate, a carrier film provided with a contrast point and a detection point, a water absorption pad and a sample pad; target antigens Ag are solidified on the detection points of the carrier film, wherein the Ag represents one of the target antigens corresponding to the autoantibodies of the PNS or AE diseases, namely different target antigens in the table 1; negative control antigen was immobilized on the control spot. The absorbent pad, the carrier film and the sample pad are laid on the backing plate, the absorbent pad is arranged at the control point end of the carrier film, and the sample pad is arranged at the detection point end of the carrier film, as shown in fig. 1 and fig. 2.

The negative control antigen in the present invention may be GAPDH or actin.

Wherein the working solution is a mixed solution of 1 XPBS or 1 XPTBS or 100mM sodium phosphate buffer solution, 0.5-1% Triton X-100 or 0.5-1% Tween-20 and 0.02-0.05% EDTA, and the pH value of the working solution is 6.8-7.2;

the blocking protein solution is prepared by the following method:

pET28a-GST or pET28a plasmid vectors were transformed into E.coli, and pET28a-GST or pET28a plasmids were specifically expressed in E.coli using an inducible approach for autoantibody target antigen expression. Obtaining thallus containing pET28a-GST or pET28as plasmid; and (2) centrifuging the bacterial liquid containing pET28a-GST or pET28a plasmid thallus, adding a bacteria breaking liquid into the obtained supernatant, carrying out ultrasonic disruption for 15-35 min, centrifuging, taking the supernatant, and adding 5-10% of BSA, 2-5% of PEG12000 and 1 Xprotease inhibitor to obtain the protein blocking liquid.

The disrupted solution of the plasmid vector pET28a-GST or pET28a in the present invention was 1 XPBS.

Specifically, the color developing agent of the invention is prepared by taking color developing solution and color developing buffer solution as the color developing solution according to the volume ratio: the developing buffer solution is mixed at a ratio of 1: 50-1: 80; wherein, the developing solution is a mixture of 70-80% DMSO, 2-3% NBT and 1-1.5% BCIP or a mixture of 70-80% DMF, 2-3% NBT and 1-1.5% BCIP; the color buffer solution is 0.1M Tris-HCl, 0.1-0.2M NaCl, 0.05M MgCl₂The mixed solution of (1).

The carrier film of the present invention may specifically be an NC film or a PVDF film.

The information of the detected autoantibody and the target antigen related to the kit of the present invention is shown in table 1:

TABLE 1 PNS and AE autoantibodies and information relating to target antigens

The invention also discloses a method for detecting the PNS or AE autoantibody in the human body fluid, the detection method is based on the detection kit of the invention for detection, the flow chart of the method is shown in figure 3, and the method comprises the following steps:

step 1, sample dilution and blocking:

mixing the working solution and the blocking protein solution according to the volume ratio of 5: 1-15: 1 to form a sample diluent, adding a sample to be detected into the sample diluent, diluting and blocking the sample to be detected, reducing the background and improving the detection sensitivity. Wherein, the sample diluent: the volume ratio of the sample to be detected is 10:1, and the sample to be detected in the invention is generally serum or cerebrospinal fluid.

Step 2, sample reverse chromatography:

and dripping the sample to be detected added with the sample diluent on a sample pad of the test strip, inclining the test strip to enable the sample to be detected to uniformly flow through a carrier membrane containing the target protein Ag, and standing for 2-3 min at room temperature.

And 3, washing the carrier membrane:

and (3) taking down the water absorption pad and the sample pad on the test strip treated in the step (2), and washing the carrier membrane to remove impurities. Specifically, ultrasonic washing is carried out for 10-30 s, and washing is carried out for 2-3 times. After washing, the absorbent pad and the sample pad are fixed at two ends of the carrier membrane.

Preferably, in this step, the carrier membrane is washed with a working solution which is a mixture of 1 XPBS or 1 XPTBS or 100mM sodium phosphate buffer, 0.5% to 1% Triton X-100 or 0.5% to 1% Tween-20, and 0.02% to 0.05% EDTA, and the pH of the working solution is 6.8 to 7.2.

Step 4, secondary antibody positive chromatography:

immersing the sample pad end of the test strip treated in the step 3 into a second antibody diluent, and standing for 5-10 min at room temperature;

and 5, washing the carrier membrane:

and (4) taking down the water absorption pad and the sample pad on the test strip treated in the step (4), and washing the carrier membrane to remove impurities. Specifically, ultrasonic washing is carried out for 10-30 s, and washing is carried out for 2-3 times. After washing, the absorbent pad and the sample pad are fixed at two ends of the carrier membrane.

Preferably, in this step, the carrier membrane is washed with the washing solution of the present invention described above.

Step 6, color development:

and (3) immersing the sample pad end of the test strip treated in the step (5) into a color developing agent, and observing the color developing condition, wherein the specific steps are as follows: standing for 2-5 min at room temperature in dark place, and observing the color development after about 5-10 min.

The target antigen Ag in the detection kit of the present invention can be obtained by the prior art, or can be obtained by the following method of the present invention, and the preparation method of the present invention is preferred. Specifically, the preparation method of the material for detecting the PNS or AE autoantibody in the human body fluid comprises the following steps:

step 1.1, obtaining a CDS sequence of an autoantibody target antigen Ag as a target gene by artificial synthesis or a PCR method, and adding NheI/NotI enzyme cutting sites at two ends of the target gene;

step 1.2, inserting a target gene with a restriction enzyme cutting site into a pET28a vector, wherein the insertion site is NheI/NotI to obtain a recombinant vector, and naming the recombinant vector as pET28 a-Ag;

and step 1.3, sequencing the recombinant plasmid pET28a-Ag, carrying out amplification culture on strains with target genes and vector strains which are correctly sequenced, and then carrying out plasmid extraction to obtain the recombinant vector pET28 a-Ag. Specifically, the vector strain of the present invention may be selected from DH5 α, TOP10 or JM 109.

And 2, transforming the recombinant vector pET28a-Ag into escherichia coli to obtain thalli containing the target antigen Ag.

Specifically, recombinant vector pET28a-Ag was subjected to expression of the autoantibody target antigen in E.coli using an induction method to obtain a sufficient amount of the autoantibody target antigen Ag. Specifically, the inducer IPTG may be used. Preferred E.coli strains of choice in the present invention are BL21(DE3), Arctic express, and Arctic RIL express.

Step 3, purifying the thalli containing the target antigen Ag obtained in the step 2 by adopting an affinity chromatography; specifically, the affinity chromatography purification process comprises:

wherein, bacterial liquid: the volume ratio of the bacteria-breaking liquid is 20: 1;

in the specific embodiment of the invention, the lysis solution in the affinity chromatography corresponding to the His-tag antigen is a mixed solution of 20mM Tris-HCl, 10-200 mM NaCl and 10-100 mM imidazole, and the pH of the lysis solution is 7.4-9.0; the lysis solution in the affinity chromatography corresponding to the GST-tag antigen was 1 XPBS.

Step 3.2, washing the affinity chromatography column by using deionized water, balancing 10-30 times of column volume by using a bacteria breaking solution, adding the initial sample solution obtained in the step 3.1 into the well-balanced affinity chromatography column, and combining for 1-2 hours at 4 ℃ to ensure that the target protein in the bacteria solution is fully combined on the column;

step 3.3, washing the affinity chromatography column obtained in step 3.2 with a first buffer solution for 10-30 times of the column volume to remove proteins not bound to the column; then, carrying out gradient elution on the washed pre-packed column by using a first eluent, eluting the target protein, collecting the eluent, and desalting the eluent to obtain a purified target antigen Ag;

in the specific embodiment of the invention, the first buffer solution in the affinity chromatography corresponding to the His label antigen is a mixed solution of 20mM Tris-HCl and 100-1000 mM NaCl, and the pH value of the first buffer solution is 7.4-9.0; the first eluent is a mixed solution of 20mM Tris-HCl and 10-1000 mM imidazole or a mixed solution of 20mM Tris-HCl and 10-300 mM Glu.

The first buffer solution in the affinity chromatography corresponding to the GST tag antigen is 1 XPBS; the first eluent is a mixed solution of 50mM Tris-HCl and 10-100 mM reduced glutathione, and the pH value of the first eluent is 8.0.

And 4, verifying the product purified in the step 3 to ensure that the purified target antigen Ag is correct, and solidifying the target antigen Ag without errors on a carrier membrane to obtain the PNS or AE autoantibody detection material in the human body fluid.

In the present invention, if the part target antigen does not satisfy the experimental requirements after affinity chromatography, ion exchange chromatography, hydrophobic chromatography or gel filtration chromatography may be used for further purification based on the purification in step 3.

Wherein, the purification process of the ion exchange chromatography comprises the following steps:

firstly, dialyzing an eluted sample after affinity chromatography by using a second buffer solution, replacing the second buffer solution for 3-4 times in the dialysis process, wherein the dialysis time is 2-8 hours each time, and taking out a sample from a dialysis bag to obtain an initial sample solution; in the embodiment of the present invention, the second buffer solution in the cation chromatography is 20mM PB with pH 5.0 to 7.0, and the second buffer solution in the anion chromatography is 20mM Tris with pH 7.0 to 9.0.

and finally, washing the loaded ion exchange chromatographic column by using a second buffer solution, performing gradient elution on the washed ion exchange chromatographic column by using a second eluent, and collecting the eluent to obtain the target antigen Ag after further purification.

Wherein, in the following examples of the present invention, the second eluent in the cation chromatography is a mixture of 20mM PB with pH 5.0-7.0 and 50-1000 mM NaCl; the second eluent in the anion chromatography is a mixture of 20mM Tris and 50-1000 mM NaCl, the pH value of which is 7.0-9.0.

The purification process of the hydrophobic chromatography comprises the following steps:

then, washing the hydrophobic chromatography column by using deionized water, treating the column with 10-30 times of column volume by using a third buffer solution, and loading an initial sample solution at the flow rate of 1 ml/min; wherein the third buffer solution has a pH of 6.0-9.0, 20mM PB or 0.4-2.0M (NH)₄)₂SO₄Or a mixture of 20mM Tris and 0.6-2.0M NaCl at a pH of 6.0-9.0;

finally, washing the column with a third buffer solution by 10-30 times of the volume of the column, performing gradient elution on the washed hydrophobic chromatographic column by using a third eluent, and collecting the eluent to obtain a target antigen Ag after further purification; wherein the third eluent has pH of 6.0-9.0, 20mM PB or 2.0-0.4M (NH)₄)₂SO₄Or a mixture of 20mM Tris and 2.0-0.6M NaCl at a pH of 6.0-9.0.

The purification process of the gel filtration chromatography comprises the following steps:

then eluting with a fourth buffer solution, and collecting eluent to obtain a target antigen Ag after further purification; wherein the fourth buffer solution is pH 6.0-9.0, 20mM PB or mixture of pH 6.0-9.0, 20mM Tris and 50-500 mM NaCl.

The negative control antigen in the detection kit of the present invention can be obtained by the prior art, or can be obtained by the method of the present invention described below, and the method of the present invention is preferred. Specifically, the process of preparing the negative control antigen by using the method of the present invention also includes four steps, wherein step 1 and step 2 are the same as step 1 and step 2 in the method for obtaining the target antigen Ag of the present invention, and only the target antigen Ag is replaced by the negative control antigen, specifically:

step 1, obtaining a CDS sequence of a negative control antigen as a target gene, and inserting the target gene with a restriction enzyme cutting site into a pET28a plasmid vector to obtain a recombinant plasmid vector.

And 2, transforming the recombinant vector into escherichia coli to obtain thalli containing the negative control antigen.

And 3, adding PBS into the thalli containing the negative control antigen obtained in the step 2, wherein the bacterial liquid is as follows: the volume ratio of PBS is 20:1, the PBS is vibrated, vortexed, ultrasonically crushed for 15-35 min and centrifuged for 20-30min at 4-8 ℃, and the obtained supernatant is a negative control antigen sample;

and 4, solidifying the negative control antigen sample obtained in the step 3 on the carrier membrane.

The following embodiments of the present invention are provided, and it should be noted that the following embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention fall into the protection scope of the present invention.

The affinity chromatography column, ion exchange chromatography column, hydrophobic chromatography column and gel filtration chromatography column used in the following examples of the present invention are all commercially available from GE corporation. pET28a vector was purchased from Shanghai enzyme research Biotech, Inc., vector strains DH5 alpha, TOP10, JM109, Escherichia coli BL21(DE3), Arctic express, and Arctic RIL express were purchased from Beijing Solebao scientific, Inc.; the carrier film was purchased from GE.

Example 1

The embodiment discloses a preparation method of a PNS or AE autoantibody detection material in human body fluid, wherein a target antigen in the embodiment is an antigen HuB of PNS diseases, and a negative control antigen is GAPDH, and the preparation method specifically comprises the following steps:

1. plasmid construction:

CDS sequences of autoantibody target antigens HuB and GAPDH are obtained by artificial synthesis or a PCR method to be used as target genes, and NheI/NotI enzyme cutting sites are added at two ends of the target genes;

inserting a target gene with an enzyme cutting site into a pET28a vector, wherein the insertion site is NheI/NotI to obtain a recombinant vector, and the recombinant vector is named as pET28a-HuB and pET28a-GAPDH, and specifically comprises the following steps:

the glycerol strain of pET28a plasmid was inoculated into 3mL LB liquid medium resistant to kanamycin, and the medium was incubated at 37 ℃ for 220r/min overnight with shaking. On the second day, 500. mu.L of the bacterial liquid is sucked for storing strains, and the remaining 2.5mL of the bacterial liquid is extracted by a plasmid miniextraction kit and the concentration is measured.

pET28a vector restriction: mu.L of the digestion system, 3. mu.g of the vector, 1. mu.L of each of NheI and NotI enzymes, 5. mu.L of 10 Xdigestion Buffer, and completion of ddH2O to 50. mu.L, and digestion in water bath at 37 ℃ for 2 h.

And (3) recovering the enzyme digestion product, separating the enzyme digestion product by 0.8% agarose gel electrophoresis, and recovering the vector fragment according to the instruction of the gel recovery kit.

Connecting the target gene with a vector fragment: 20 μ L ligation system, 50ng of pET28a vector, 200ng of target antigen gene fragment, 1 μ L of T4 ligase, and 1 μ L of ddH2O were supplemented to 20 μ L and ligated overnight at 16 ℃.

The above ligation product plasmids were transformed into E.coli Top10 competent cells, spread on a kanamycin solid LB plate, overnight at 37 ℃, and 5 strains were picked the next day, inoculated into a kanamycin-resistant liquid LB medium, and cultured overnight at 37 ℃. The next day, each tube sucks 500 μ L of bacterial liquid, the bacterial liquid is stored at the temperature of minus 20 ℃, the plasmid of the residual bacterial liquid is extracted slightly, and the bacterial strain with correct enzyme digestion identification is sent to sequencing for re-identification. The strains with correct sequencing and identification are reserved and stored at-80 ℃.

And (3) carrying out amplification culture on the strains with the target genes and the vector strain DH5 alpha with correct sequencing, carrying out plasmid large-scale extraction, and measuring the concentration to obtain recombinant vectors pET28a-Ag and pET28 a-GAPDH.

2. Protein induced expression

Expressing autoantibody target antigen and negative control antigen in Escherichia coli BL21(DE3) by using recombinant vectors pET28a-HuB and pET28a-GAPDH in an induction mode to obtain thallus containing the target antigen and thallus containing the negative control antigen;

3. purification of target antigen HuB

The obtained thallus containing the target antigen HuB is purified by adopting an affinity chromatography method, wherein the purification method comprises the steps of firstly carrying out affinity chromatography and then carrying out ion exchange chromatography.

4. Obtaining negative control antigen

Adding PBS into the thallus containing the negative control antigen obtained in the step 2, and adding bacterial liquid: and the volume ratio of PBS is 20:1, carrying out ultrasonic crushing for 15-35 min after oscillating vortex, and centrifuging for 20-30min at 4-8 ℃, wherein the obtained supernatant is the negative control antigen sample.

5. Immobilization of target antigen HuB and negative control antigen

And solidifying the purified target antigen HuB at the end close to the sample pad of the carrier membrane, and solidifying the negative control antigen at the end close to the water absorption pad of the carrier membrane to obtain the PNS or AE autoantibody detection test strip in the human body fluid.

The preparation method of the test strip corresponding to other target antigens in table 1 of the present invention is the same as that in example 1, except that the labels and purification methods corresponding to different target antigens are different, which is specifically shown in table 2.

TABLE 2 PNS and AE autoantibodies information on purification of target antigen expression

Example 2

The embodiment discloses a detection kit, which comprises a test strip, a sample diluent, a secondary antibody (specifically an AP-labeled goat anti-human IgG antibody), a washing solution and a color developing agent;

the test strip is a test strip corresponding to the HuB target antigen in embodiment 1, and specifically, the test strip comprises a backing plate, a carrier film provided with a control point and a detection point, a water absorption pad and a sample pad; a target antigen Ag is solidified on the detection point of the carrier film, and the Ag represents one of the target antigens corresponding to the PNS or AE disease autoantibody; negative control antigen was immobilized on the control spot. The absorbent pad, the carrier film and the sample pad are laid on the backing plate, the absorbent pad is arranged at the control point end of the carrier film, and the sample pad is arranged at the detection point end of the carrier film, as shown in fig. 1 and fig. 2.

And mixing the sample diluent with the working solution and the sealing protein solution according to the volume ratio of 10:1, then vortex and shake for a plurality of seconds, and standing for 5min at room temperature. The working solution was a mixture of 1 × PBS, 0.5% Triton X-100, and 0.04% EDTA, and the pH of the working solution was 6.8.

The blocking protein solution is prepared by the following method:

transforming pET28a-GST plasmid vector into Escherichia coli BL21(DE3) by using an inducer IPTG to obtain thallus containing pET28a-GST plasmid; centrifuging 5000-7500 g of the bacterial liquid for 5min, adding 1 XPBS of the bacterial liquid, carrying out ultrasonic crushing for 15-35 min and 6000-7500 g at the power of 10-30%, centrifuging for more than 25min, adding 25% of BSA (bovine serum albumin), 2% of PEG12000 and a protease inhibitor by the volume of 1/4 into supernatant to obtain a closed protein liquid, and storing at 4 ℃ for later use.

The washing solution of this example was 50mM Tris-HCl, 0.85% NaCl, 0.5% Triton X-100, pH 8.5.

The color developing agent of the embodiment is prepared by taking a color developing solution and a color developing buffer solution as the color developing solution according to the volume ratio: the developing buffer solution is mixed at a ratio of 1: 50-1: 80; wherein the developing solution is a mixed solution of 70% DMSO, 2% NBT and 1% BCIP; the color buffer solution is 0.1M Tris-HCl, 0.1M NaCl, 0.05M MgCl₂And (4) mixing the solution.

Example 3

The embodiment discloses a method for detecting a PNS or AE autoantibody in human body fluid, which adopts the detection kit described in embodiment 2 to detect, and specifically comprises the following steps:

sample dilution and blocking: according to the sample diluent: adding the sample to be detected into the sample diluent at a volume ratio of 10: 1.

Sample reverse chromatography: and adding the diluted and sealed sample to be detected into a 2mL EP tube with a small hole, contacting the bottom of the small hole with a sample pad, keeping a certain inclination angle, enabling the sample to be detected to uniformly flow through an NC membrane containing the target antigen at a constant speed, and standing for 2-3 min at room temperature.

Washing the carrier film: and (3) taking down the water absorption pad and the sample pad on the test strip, putting the test strip into a 10mL centrifuge tube added with 3mL working solution, and ultrasonically washing for 10-30 s for 2-3 times. After washing, the absorbent pad and the sample pad are fixed at two ends of the carrier membrane.

Secondary antibody positive chromatography: adding a 1:4000 diluted AP-labeled goat anti-human IgG antibody into a 2mL EP tube, 200 mu L per tube, immersing the sample pad end of the assembled test strip into a secondary antibody diluent, and carrying out room-temperature chromatography for 5-10 min;

washing the carrier film: and after the secondary antibody positive chromatography is finished, taking down the water absorption pad and the sample pad on the test strip, putting the test strip into a 10mL centrifuge tube added with 3mL of washing liquid, and carrying out ultrasonic washing for 10-30 s for 2-3 times. After washing, the absorbent pad and the sample pad are fixed at two ends of the carrier membrane.

Color development: and (3) immersing the sample pad end of the assembled test strip into a color developing agent, standing for 2-5 min at room temperature in a dark place, and observing the color developing condition after about 5-10 min.

The results are shown in FIG. 4, and the positive and negative results are shown in FIG. 4A, where the color of the detection point in the detection result of the positive sample is darker than that of the control point. Meanwhile, the detection results of 100 serum samples which are confirmed to be positive for the HuB and 100 cerebrospinal fluid samples which are confirmed to be positive for the HuB are respectively shown in FIG. 4B, and the positive detection rate of 100 HuB positive sera is 95%; the positive detection rate of the cerebrospinal fluid sample is 98 percent respectively.

Test kits for 34 target antigens such as HuB are prepared according to the methods of example 1 and example 2, 100 positive serum samples and 100 positive cerebrospinal fluid samples which are determined are tested according to the method provided in example 3, and the positive rate is counted, and the results are shown in Table 3.

TABLE 3, 34 autoantibody detection positive rate

Comparative example 1

This comparative example is the same as example 1 and example 2, differing from example 3 in that: the normal positive chromatography method is used in the sample reverse chromatography step.

100 serum samples that have been determined to be positive for HuB and 100 cerebrospinal fluid samples that have been determined to be positive for HuB were tested separately. The results are shown in FIG. 5, the positive and negative results are shown in FIG. 5A, and the color of the detection point in the detection result of the positive sample is darker than that of the control point; meanwhile, as shown in fig. 5B, in 100 cases of HuB positive serum, the positive detection rate of the method of the present invention is 95%, which is much higher than 39% of the method of comparative example 1; the positive detection rates of the cerebrospinal fluid sample are respectively 98% and 51%. The reverse chromatography detection method can obviously improve the positive detection rate of the serum and the cerebrospinal fluid.

Comparative example 2

This comparative example is the same as example 1 and example 2, differing from example 3 in that: the carrier membrane washing step was carried out by placing the membrane in a shaker (frequency about 40 times/min), and washing was repeated 3 times at room temperature for 3 min.

100 serum samples that have been determined to be positive for HuB and 100 cerebrospinal fluid samples that have been determined to be positive for HuB were tested separately. The results are shown in FIG. 6, the positive and negative results are shown in FIG. 6A, and the color of the detection point in the detection result of the positive sample is darker than that of the control point; meanwhile, as shown in fig. 6B, in 100 cases of HuB positive serum, the positive detection rate of the method of the present invention is 95%, which is much higher than 16% of the method of comparative example 2; the positive detection rates of the cerebrospinal fluid samples are 98 percent and 28 percent respectively. The positive detection rate of serum and cerebrospinal fluid can be obviously improved by using ultrasonic washing.

Comparative example 3

This comparative example is the same as example 1 and example 3, differing from example 2 in that: the sample diluent is a working solution.

The results of the tests were shown in FIG. 7 for 100 serum samples and 100 cerebrospinal fluid samples, respectively, which were confirmed to be positive for HuB. The positive and negative results are shown in FIG. 7A, the color of the detection point in the detection result of the positive sample is darker than that of the control point; meanwhile, as shown in fig. 7B, in 100 cases of HuB positive serum, the positive detection rate of the method of the present invention is 95%, which is much higher than 7% of the method of comparative example 3; the positive detection rates of the cerebrospinal fluid samples are 98 percent and 18 percent respectively. The blocking protein of the invention can obviously improve the positive detection rate of serum and cerebrospinal fluid.

Comparative example 4

This comparative example is the same as example 1 and example 3, differing from example 2 in that: the sample dilution was 1% BSA.

The results of the tests were shown in FIG. 8 for 100 serum samples and 100 cerebrospinal fluid samples, respectively, which were confirmed to be positive for HuB. The positive and negative results are shown in FIG. 8A, the color of the detection point in the detection result of the positive sample is darker than that of the control point; meanwhile, as shown in fig. 8B, in 100 cases of HuB positive serum, the positive detection rate of the method of the present invention is 95%, which is much higher than 59% of the method of comparative example 4; the positive detection rates of the cerebrospinal fluid samples are 98% and 74%, respectively. The blocking protein of the invention can obviously improve the positive detection rate of serum and cerebrospinal fluid.

Claims

1. The detection kit for the PNS or AE autoantibody in the human body fluid is characterized by comprising a test strip, a sample diluent, a secondary antibody, a washing solution and a color developing agent;

the sample diluent is formed by mixing working solution and enclosed protein solution according to the volume ratio of 5: 1-15: 1;

the blocking protein solution is prepared by the following method:

2. The kit for detecting an autoantibody in PNS or AE in human body fluid according to claim 1, further comprising a washing solution, wherein the washing solution is a mixture of 50 to 80mM Tris-HCl, 0.8 to 1% NaCl, 0.5 to 1% Triton X-100, or a mixture of 50 to 80mM Tris-HCl, 0.8 to 1% NaCl, 0.5 to 1% Tween-20, and the pH of the washing solution is 8.2 to 8.5.

3. A method for preparing a material for detecting PNS or AE autoantibodies in human body fluids for obtaining the target antigen Ag of claim 1 or 2, comprising the steps of:

4. The method of claim 3, wherein the step 3 of purifying by affinity chromatography comprises:

5. The method of claim 3, wherein the material for detecting PNS or AE autoantibodies in human body fluid is further purified by ion exchange chromatography, hydrophobic chromatography or gel filtration chromatography on the basis of the purification in step 3.

6. The method of claim 5, wherein the ion exchange chromatography purification process comprises:

7. The method of claim 5, wherein the hydrophobic chromatography purification process comprises:

8. The method of claim 5, wherein the gel filtration chromatography purification process comprises: