The application relates to a split application of a detection kit for detecting an anti-aconitate hydratase-IgG antibody, which has the application number 202110742938.0 and the application date 2021, 7 and 1.
Disclosure of Invention
The invention aims to provide a detection kit for detecting serum anti-aconitate hydratase-IgG autoantibodies, which detects antibodies in a detected sample through an immune reaction with aconitate hydratase antigen protein (particularly shown according to a sequence identification number SEQ ID NO. 1).
The kit comprises aconitate hydratase antigen protein, labeled antibody liquid (enzyme labeled or chemiluminescent labeled anti-human IgG solution), a solid phase carrier coated with aconitate hydratase antigen, sample diluent, antibody diluent, antigen diluent, substrate developing solution, washing solution, stop solution, standard substance, positive quality control substance and positive quality control substance.
The sequence of aconitate hydratase antigen protein is shown as SEQ ID NO. 1:
MAPYSLLVTRLQKALGVRQYHVASVLCQRAKVAMSHFEPNEYIHYDLLEKNINIVRKRLNRPLTLSEKIVYGHLDDPASQEIERGKSYLRLRPDRVAMQDATAQMAMLQFISSGLSKVAVPSTIHCDHLIEAQVGGEKDLRRAKDINQEVYNFLATAGAKYGVGFWKPGSGIIHQIILENYAYPGVLLIGTDSHTPNGGGLGGICIGVGGADAVDVMAGIPWELKCPKVIGVKLTGSLSGWSSPKDVILKVAGILTVKGGTGAIVEYHGPGVDSISCTGMATICNMGAEIGATTSVFPYNHRMKKYLSKTGREDIANLADEF.
According to the invention, aconitate hydratase antigen proteins are purified by molecular sieves, gel filtration chromatography, affinity chromatography, ion exchange columns, hydrophobic columns.
The aconitate hydratase antigen proteins of the invention may be fusion proteins, using tags with biological or physical functions, in particular N-terminal or C-terminal tags, preferably C-terminal tags. These tags facilitate antigen protein purification, immobilization, and precipitation. In a preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand and the tag peptide is selected from the group consisting of His tag, GST tag, maltose binding protein, thioredoxin and fluorescent tag or biotin tag.
According to the invention, the antigenic protein aconitate hydratase may be expressed in bacteria such as E.coli, fungal yeasts, mammalian cells.
In a preferred embodiment, the aconitate hydratase antigen proteins of the invention are presented in immobilized form, the term "immobilized" referring to binding to an insoluble solid carrier in aqueous solution. Preferably, the binding is by electrostatic interactions, hydrophobic interactions, covalent bonds. The solid phase carrier is preferably polystyrene, a material micro-pore plate, a nitrocellulose membrane or magnetic beads.
The aconitate hydratase antigen protein fixing mode comprises a reversible fixing mode or an irreversible fixing mode. For example, the molecules are bound by cleavable covalent bonds (e.g., disulfide bonds that can be cleaved by addition of a thiol-containing reagent), and the immobilization is reversible. In addition, if the molecule is immobilized to the carrier by a covalent bond that does not cleave in aqueous solution (a bond formed by the reaction of an epoxide group with an amine group that couples a lysine side chain to the affinity column), the immobilization is irreversible. Immobilization may also be an indirect way of immobilizing a protein, such as an antibody having a specific affinity for the molecule, and then forming a complex to achieve the effect of immobilizing the molecule-antibody complex.
The aconitate hydratase antigen protein fixing method is a direct coating method, wherein (1) antigen is combined on a nitrocellulose membrane or a polystyrene micro-pore plate through a physical adsorption mode or a non-covalent bond, and (2) a magnetic particle belt with a carboxyl functional group is combined with protein amino, and the antigen is combined on the magnetic particle through a chemical coupling mode.
The substrate chromogenic solution is TMB, luminol, hydrogen peroxide and acridinium ester, the antigen diluent is 1xPBS, pH7.40, the antibody diluent is 0.15%BSA+0.01M PBS (pH 7.40), the sample diluent is 6% fetal bovine serum+0.01M PBS (pH 7.40), the washing solution is 1xPBS (pH 7.40) +0.1Tween-20, and the stopping solution is 2M sulfuric acid.
In one embodiment of the invention, the standard and the positive quality control are preferably prepared from antigens homologous to human, and are recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or anti-aconitate hydratase-IgG antibodies are extracted from serum of patients as positive quality control and standard, and the negative quality control is serum of healthy physical examination people.
The labeled antibody liquid of the invention can be horseradish peroxidase (Horseradish Peroxidase, HRP) labeled anti-human IgG, biotin labeled anti-human IgG, acridinium ester labeled anti-human IgG.
According to the invention, the sample to be tested is a liquid sample containing antibodies, preferably hydrothorax, ascites, urine, whole blood, plasma, most preferably serum. The serum is mammalian serum, preferably human serum. The sample to be tested may be further processed prior to testing, including fractionation, centrifugation or enrichment.
The invention relates to a detection kit which is used for carrying out qualitative or quantitative analysis on the concentration of anti-aconitate hydratase-IgG antibodies in serum by taking recombinant protein aconitate hydratase expressed and purified by a gene recombination method as antigen protein in a kit, coating the antigen protein on a solid phase carrier, adding a positive quality control product or standard product or serum to be detected for incubation, adding a labeled secondary antibody for reaction, combining the labeled secondary antibody with anti-aconitate hydratase-IgG antibodies in serum to form a coating antigen aconitate hydratase-serum to be detected anti-aconitate hydratase-IgG antibody-labeled anti-human IgG antibody complex, and detecting optical signals by an optical method such as a light color development method, a chemiluminescence method and a fluorescence luminescence method.
By applying the kit, an anti-aconitate hydratase-IgG autoantibody is detected in the body of a part of patients with autoimmune nephrotic syndrome for the first time, and the target antigen aimed by the autoantibody is determined to be aconitase on glomerular podocytes (aconitate hydratase). Therefore, the kit provided by the invention can be used for detecting the anti-aconitate hydratase-IgG autoantibody and provides basis for researching autoimmune nephrotic syndrome.
Compared with the prior art, the kit has the following beneficial effects:
(1) The invention identifies the existence of the anti-aconitate hydratase-IgG autoantibody in the body of the patient with autoimmune nephrotic syndrome for the first time, and invents a detection kit aiming at the antibody. At present, no research related to aconitate hydratase-IgG antibodies is seen at home and abroad, and the invention fills the gap of detecting aconitate hydratase-IgG antibodies at home and abroad.
(2) The kit disclosed by the invention relates to qualitative detection of anti-aconitate hydratase-IgG antibodies in serum, wherein the operation of solid-phase membrane immunoassay is simple and convenient, the dosage of reagents is small, trace antigen can be adsorbed by the NC membrane with extremely strong adsorption capacity, and the adsorbed NC membrane can be stored for a long time (-20 ℃ can be stored for half a year) without affecting the activity of the NC membrane. Is suitable for large-scale screening.
(3) The invention relates to a magnetic particle chemiluminescence immunoassay quantitative detection kit, which uses magnetic particles as a solid phase carrier, wherein the diameter of the magnetic particles is only 1.0 mu m, so that the coating surface area is greatly increased, the adsorption quantity of antigens is increased, the reaction speed is improved, and the cleaning and separation are simpler and more convenient, thereby reducing pollution and reducing the cross infection probability. On the other hand, the anti-human IgG is directly marked by adopting an acridine ester luminescent agent, the chemical reaction is simple and quick, a catalyst is not needed, the acridine ester chemiluminescence is in a flashing type, the emission intensity can reach the maximum after the luminescent agent (H2O2 and NaOH) is started for 0.4s, the half-life period is basically ended within 0.9s, and the quick detection is convenient.
(4) No report is presented on the relation between aconitate hydratase and nephrotic syndrome at present, the invention discovers that aconitate hydratase-IgG antibody exists in nephrotic syndrome for the first time, and the cause of autoimmune disease is clarified. In the prior art, the study of detecting serum anti-aconitate hydratase-IgG antibodies to identify autoimmune nephrotic syndrome is blank, and the invention provides a noninvasive detection method for the disease.
Detailed Description
The invention is further described below with reference to the drawings and specific examples. The following examples are only intended to illustrate the invention and are not intended to limit the scope of the invention.
EXAMPLE 1 aconitate hydratase on podocytes is the target antigen against which autoantibodies are directed in patients with autoimmune nephrotic syndrome
(1) Extraction of total glomerular podocyte proteins podocyte strain (MPC 5), washing 2-3 times with PBS, then performing sufficient lysis with focused ultrasound (Covaris S220, gene) on ice in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitor (#ab 65621; abcam,1:200 dilution), and then placing the sample in centrifuge, 12000g,4℃and centrifuging for 30min. Collecting the supernatant, namely the total protein of the collected glomerular podocytes. The total protein concentration of the collected glomerular podocytes was determined using the BCA protein concentration determination kit. (2) Two-dimensional electrophoresis, namely extracting total proteins of glomerular podocytes, performing two-dimensional electrophoresis, transferring to a nitrocellulose membrane, incubating with serum of autoimmune nephrotic syndrome patient and healthy person as primary antibodies, and developing with secondary antibodies, as shown in figures 1A and 1B. (3) Matrix assisted laser Desorption/ionization time of flight mass spectrometry (MALDI/MS) the difference analysis of positive spots was performed after development in step (2), the protein spots were selected for strong positive and negative or weak positive for kidney disease syndrome patients on two-dimensional electrophoresis gel, the selected protein spots were cut off from the gel, the dried gel was digested with trypsin (0.1. Mu.g/. Mu.L), 10. Mu.L of 25mM ammonium bicarbonate was added to the reaction mixture, incubated overnight at 37℃and peptides were extracted from the gel with trifluoroacetic acid (0.1%). The extracted peptides were analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) to obtain a peptide mass spectrum, identified as aconitate hydratase proteins, FIG. 1C.
EXAMPLE 2 expression and purification of recombinant aconitate hydratase antigen proteins
And (3) carrying out PCR amplification by using a gene encoding aconitate hydratase protein as a template by using a genetic engineering method, and then constructing an expression vector to carry out protein expression. The antigen protein expressed by the invention contains a tag peptide of His tag. The expressed recombinant protein is purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc., and finally SDS-PAGE is used to identify that the molecular weight of the recombinant protein aconitate hydratase is 37kDa, and the result is shown in figure 2.
Example 3 optimization of the reaction conditions of the kit Using orthogonal test design according to the invention
Orthogonal tables were selected based on 4 factors including antigen aconitate hydratase coating concentration (50. Mu.g/mL, 100. Mu.g/mL, 150. Mu.g/mL, 200. Mu.g/mL), reaction time (30 min, 45 min) and temperature (25 ℃, 35 ℃) and enzyme-labeled secondary antibody optimal dilutions (1:100, 1:500, 1:1000, 1:1500 four dilutions), each factor repeatedly measuring standard positive serum and standard negative serum at 2 levels. The ratio (P/N) of the highest luminescence value (P) of positive serum to the lowest luminescence value (N) of negative serum was selected. The average P/N value is repeatedly measured, and the optimal coating condition and the optimal dilution of the secondary antibody are determined through statistical treatment to carry out orthogonal optimization, so that the positive detection rate of standard positive serum is obviously improved. By orthogonal design, the optimal antigen coating concentration of the kit is 100 mug/mL, the optimal antigen-antibody reaction temperature is 25 ℃, the optimal antigen-antibody reaction time is 45 minutes, and the optimal working dilution of the optimal marked anti-human IgG antibody is 1:500.
Example 4 preparation of solid-phase Membrane immunoassay kit for detection of anti-aconitate hydratase-IgG antibody
4.1 Composition of solid-phase Membrane immunoassay kit for detection of anti aconitate hydratase-IgG:
1. nitrocellulose membrane coated with aconitate hydratase antigen protein;
2. standard human anti-His tag immunoglobulin G (purchased from english trauma in lakezhou),
3. The dilution of the antibody is performed by a liquid,
4. An antigen diluent, wherein the diluent comprises a diluent,
5. Horseradish peroxidase-labeled anti-human IgG antibody,
6. The washing liquid is used for washing the clothes,
7. The color-developing agent of TMB,
8. And (5) stopping liquid.
4.2 The detection steps are as follows:
4.2.1 coating and blocking 10. Mu.l of antigen was diluted with 0.01M PBS pH7.4 on nitrocellulose membrane, placed in a 37℃incubator for 30 minutes, nitrocellulose membrane was placed in a plate well, 150. Mu.l of 3% BSA was added and placed in a 37℃incubator for 15 minutes, and after the blocking solution was sucked off, the membrane was washed 2 times with washing solution.
4.2.2 Serum incubation (first incubation) 100. Mu.l of the antigen-releasing diluted standard and the serum sample to be tested were loaded into the reaction tank, negative and positive controls were simultaneously made, and the nozzle of the sample loading device was not touched to the surface of the membrane, taking care that one nozzle was replaced every time one serum was loaded. The reaction tank with the added sample is placed on a shaking table and incubated for 45 minutes at room temperature (20-25 ℃).
4.2.3 Washing (first washing) by pouring out the liquid in the reaction tank, rinsing with diluted washing liquid for 10 seconds, and allowing the washing liquid to sufficiently flow through the reaction tank during rinsing. Repeated washing is carried out for 5 times, and the liquid is poured down along the reaction tank when the liquid is washed, so that cross contamination is avoided. And spin-drying the reaction tank after cleaning.
4.2.4 Secondary antibody working solution incubation (second incubation) horseradish peroxidase-labeled anti-human IgG antibody was diluted with antibody dilution, followed by addition of 6 drops (300. Mu.L) of secondary antibody working solution to the reaction tank and incubation on a shaker at room temperature (20-25 ℃) for 45 min.
4.2.5 Cleaning (second cleaning) the procedure is the same as in step 3.
4.2.6. Incubation (third incubation) 6 drops (300. Mu.l) of the chromogenic solution were added to the strip and incubated on a shaker at room temperature (20-25 ℃) for 20 minutes.
4.2.7. Terminating reaction, namely flushing the reaction tank by running water to terminate the reaction.
4.2.8. And judging the result, namely taking out the test strip, drying the test strip by using an electric hair dryer (about 5 minutes) or placing the test strip in a 37-50 ℃ oven for drying for more than 20 minutes. And (3) carrying out naked eye qualitative judgment, wherein the person with obvious brown spots is positive (see figure 3) or the membrane strip is placed on a developing instrument for scanning, the analysis software carried by the developing instrument takes the concentration of a reference standard substance as an ordinate and the gray value read by the instrument as an abscissa, and a standard curve is drawn to carry out semi-quantitative analysis on the level of anti-aconitate hydratase-IgG in serum.
EXAMPLE 5 preparation of chemiluminescent immunoassay kit for detection of anti-aconitate hydratase-IgG antibodies
5.1 Composition of chemiluminescent immunoassay kit for detection of anti aconitate hydratase-IgG:
1. A magnetic particle solution coated with aconitate hydratase antigen proteins,
2. Standard human anti-His tag immunoglobulin G (purchased from english trauma in lakezhou),
3. The sample diluent is used for the preparation of a sample diluent,
4. The acridinium ester marks the anti-human IgG solution,
5. The quality control product is prepared from the components,
6. The pre-excitation liquid is used for pre-exciting the liquid,
7. The excitation liquid is used for exciting the liquid,
8. Washing liquid.
5.2 Principle of detection the anti-aconitate hydratase-IgG antibody in human serum by indirect method, the whole process comprises two steps of reaction, namely, firstly, mixing magnetic bead solution with diluted sample, combining specific anti-aconitate hydratase-IgG antibody to the magnetic beads, and washing to remove residual solution. In the second step, acridinium ester labeled anti-human IgG antibody is added to form magnetic bead-antigen-aconitate hydratase-IgG antibody-acridinium ester antibody complex, after washing to remove residual liquid, pre-excitation liquid (H2O2) and excitation liquid (NaOH) are added to carry out luminescence reaction, luminescence value is recorded, antibody concentration is in direct proportion to the luminescence value, and concentration measurement value is calculated through a calibration curve (see figure 4).
5.3 The solid phase carrier of the kit is magnetic particles containing carboxyl functional groups.
5.4 The antigen coating method of the kit is that carboxyl magnetic particles are activated by EDC/Sulfo-NHS and are covalently combined with antigen (amino residue) to form a magnetic particle solution. The coating step is as follows:
a) Adding 40 microliters of magnetic bead stock solution into 400 microliters of 0.05M phosphate buffer solution, uniformly mixing for 8 minutes, performing magnet separation, and discarding the supernatant;
b) 200 microliters of 2% glucan solution is taken, and 200 microliters of 10mg/mL sodium periodate is added for reaction;
c) Adding 10mg/mL EDC solution prepared by phosphate buffer solution and 10mg/mLSulfo-NHS solution after the reaction is finished, and uniformly mixing for 60 minutes;
d) Separating by magnet, discarding supernatant, taking 400 microliter of 0.05M phosphate buffer solution to wash the magnetic beads, and adding 400 microliter of sealing and preserving solution to a constant volume for preservation.
The activated beads were discarded, pre-chilled 1mL of 20mM MES was added to continue washing the beads 2 times, 200. Mu.L of 2mg/mL of antigen protein aconitate hydratase was added to the activated beads, the mixture was thoroughly mixed, left standing at room temperature for 16 hours, after the reaction was completed, pH7.4PBS buffer containing 0.2% Tween20 was added, washing the beads 2 times was repeated, then pH7.4PBS buffer containing 0.2% Tween20 and 0.2% BSA was added to the final concentration of the beads 10mg/mL, the mixture was thoroughly mixed, left standing at room temperature for 30 minutes, after the reaction was completed, the supernatant was discarded, and the beads were resuspended in PBS buffer containing 0.2% Tween20 and 0.2% BSA and crosslinked with antigen protein aconitate hydratase (see FIG. 5).
5.5 Acridinium ester-labeled anti-human IgG solution, comprising the steps of:
a) Preparing 2mg/mL of acridine ester solution by using dimethylformamide;
b) 1mg/mL of anti-human IgG antibody was formulated using 0.2M (pH 8.0) carbonate buffer;
c) Uniformly mixing acridinium ester with the molar ratio of 4:1 with the anti-human IgG antibody, and reacting for 40 minutes;
d) The reaction was stopped by adding 20. Mu.l of 5% lysine in carbonate buffer for 30 min;
e) The removal of impurities by desalting gave an acridinium ester-labeled anti-human IgG solution.
5.6 The detection steps are as follows:
5.6.1 diluting the sample according to a certain proportion;
5.6.2, taking a diluted sample, adding the magnetic particle liquid and the sample diluent, and reacting for 12min at 37 ℃;
5.6.3 washing with washing liquid for 3 times;
5.6.4 adding acridinium ester labeled anti-human IgG solution, reacting at 37 ℃ for 12min;
5.6.5 washing with washing liquid for 3 times;
5.6.6 adding pre-excitation liquid (H2O2) and excitation liquid (NaOH) for reaction, and collecting luminescence measurement values;
5.6.7 concentration measurements were calculated by means of a calibration curve.
Example 6 detection of anti-aconitate hydratase-IgG antibodies in patients with various types of kidney disease
6.1 Subjects were enrolled in patients diagnosed with nephrotic syndrome from 2018, month 6 to 2020, month 6, and the healthy control group was selected from healthy physical examination persons with contemporaneous visits. Serum samples were taken from patients with nephrotic syndrome and healthy controls. All subjects were subjected to a first serum sample collection prior to no immunosuppressive treatment.
6.2 Detection of serum anti-aconitate hydratase-IgG antibodies from patients with different renal diseases Using the kit of the present invention, the serum levels of anti-aconitate hydratase-IgG antibodies from patients diagnosed with various renal diseases from month 6 2018 to month 6 in 2020, including 466 cases of nephrotic syndrome, 168 cases of allergic purpura, 137 cases of purpura nephritis, 133 cases of IgA nephropathy and 195 healthy children in the same period, showed that the anti-aconitate hydratase-IgG antibodies were positive in some patients with autoimmune nephrotic syndrome, while the anti-aconitate hydratase-IgG antibodies were negative in patients with purpura nephritis, allergic purpura, igA nephropathy and healthy children (see FIG. 6).
Example 7ROC curve evaluation of the value of anti-aconitate hydratase-IgG antibody as a serological marker for detection of autoimmune nephrotic syndrome patient the detection of anti-aconitate hydratase-IgG antibody in autoimmune nephrotic syndrome patient of example 6.2 was analyzed using a ROC curve to determine the value of the antibody to detect autoimmune nephrotic syndrome. The results show that the anti-aconitate hydratase-IgG antibody as a serum marker has good value for diagnosis of patients with autoimmune nephrotic syndrome, and when the diagnosis is defined as being more than 53.4, the sensitivity of the diagnosis is 56.8%, the specificity is 87.2%, and the area under the curve is 0.773 (see FIG. 7).
SEQUENCE LISTING
<110> University of Zhejiang
<120> A test kit for detecting an anti-aconitate hydratase-IgG antibody
<130> 2022.5.10
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 322
<212> PRT
<213> Artificial sequence (Unknow)
<400> 1
Met Ala Pro Tyr Ser Leu Leu Val Thr Arg Leu Gln Lys Ala Leu Gly
1 5 10 15
Val Arg Gln Tyr His Val Ala Ser Val Leu Cys Gln Arg Ala Lys Val
20 25 30
Ala Met Ser His Phe Glu Pro Asn Glu Tyr Ile His Tyr Asp Leu Leu
35 40 45
Glu Lys Asn Ile Asn Ile Val Arg Lys Arg Leu Asn Arg Pro Leu Thr
50 55 60
Leu Ser Glu Lys Ile Val Tyr Gly His Leu Asp Asp Pro Ala Ser Gln
65 70 75 80
Glu Ile Glu Arg Gly Lys Ser Tyr Leu Arg Leu Arg Pro Asp Arg Val
85 90 95
Ala Met Gln Asp Ala Thr Ala Gln Met Ala Met Leu Gln Phe Ile Ser
100 105 110
Ser Gly Leu Ser Lys Val Ala Val Pro Ser Thr Ile His Cys Asp His
115 120 125
Leu Ile Glu Ala Gln Val Gly Gly Glu Lys Asp Leu Arg Arg Ala Lys
130 135 140
Asp Ile Asn Gln Glu Val Tyr Asn Phe Leu Ala Thr Ala Gly Ala Lys
145 150 155 160
Tyr Gly Val Gly Phe Trp Lys Pro Gly Ser Gly Ile Ile His Gln Ile
165 170 175
Ile Leu Glu Asn Tyr Ala Tyr Pro Gly Val Leu Leu Ile Gly Thr Asp
180 185 190
Ser His Thr Pro Asn Gly Gly Gly Leu Gly Gly Ile Cys Ile Gly Val
195 200 205
Gly Gly Ala Asp Ala Val Asp Val Met Ala Gly Ile Pro Trp Glu Leu
210 215 220
Lys Cys Pro Lys Val Ile Gly Val Lys Leu Thr Gly Ser Leu Ser Gly
225 230 235 240
Trp Ser Ser Pro Lys Asp Val Ile Leu Lys Val Ala Gly Ile Leu Thr
245 250 255
Val Lys Gly Gly Thr Gly Ala Ile Val Glu Tyr His Gly Pro Gly Val
260 265 270
Asp Ser Ile Ser Cys Thr Gly Met Ala Thr Ile Cys Asn Met Gly Ala
275 280 285
Glu Ile Gly Ala Thr Thr Ser Val Phe Pro Tyr Asn His Arg Met Lys
290 295 300
Lys Tyr Leu Ser Lys Thr Gly Arg Glu Asp Ile Ala Asn Leu Ala Asp
305 310 315 320
Glu Phe