CN113447650B - Detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody - Google Patents
Detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides a detection kit for an anti-peptidyl prolyl cis-trans isomerase D-IgG antibody. An antigen-antibody complex is formed by contacting a test serum sample with a target antigen peptidyl-prolyl cis-trans isomerase D (peptidyl prolyl cis-trans isomerase D) polypeptide or binding fragment thereof to cause an immune reaction, and detecting anti-peptidyl prolyl cis-trans isomerase D-IgG autoantibodies in the formed antigen-antibody complex. The invention identifies an antibody aiming at the anti-peptidyl prolyl cis-trans isomerase D-IgG in the children nephrotic syndrome for the first time, and provides a detection kit aiming at the antibody. Provides a better detection method for identifying autoimmune nephrotic syndrome related to the anti-peptidyl prolyl cis-trans isomerase D-IgG antibody at home and abroad.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to a detection kit for an anti-peptidyl prolyl cis-trans isomerase D-IgG antibody.
Background
Primary renal syndrome (PNS) is a clinical syndrome in which there are many factors that cause increased permeability of glomerular basement membrane and increased plasma protein leaching, loss from the urine, and thus a series of pathological changes. PNS is a common glomerular disease of children, and the annual incidence rate is reported to be 1.15-16.9/10 and 0000. The pathological type, which is characterized by minimal change renal syndrome (MCNS), is the most common, and is classified into hormone sensitive type (SSNS), hormone dependent type (SDNS), and hormone resistant type (SRNS) according to the response effect of PNS to hormone therapy. Although most of the children patients are sensitive to the response of hormone therapy and have good prognosis, about 10-20% of the children patients still have the response of dependence or drug resistance to the hormone therapy, and if other drug intervention therapy is not carried out, the drug can progress to glomerulosclerosis and finally end-stage nephropathy, the health of the children patients is seriously affected, and the families and the society cannot bear heavy economic burden. And the pathogenesis of the disease is not yet clear. The clinical symptoms of PNS are usually manifested as edema, profuse proteinuria, hypoproteinemia, hyperlipidemia, etc., but the causes of these clinical manifestations are very complex, and it is difficult to clearly diagnose PNS. Therefore, if the exact cause of PNS can be found, the PNS can be diagnosed as soon as possible in the cause, so as to intervene and treat as soon as possible, which has important clinical significance.
At present, the disease of most of PNS patients clinically treated by hormones and immunosuppressants can be improved, and the fact that the PNS is closely related to autoimmunity of the patients is indirectly suggested. It is well known that B lymphocytes play the most important role in humoral immunity, however, the local part of the kidney of PNS infants lacks antibody deposition during the past years, thus neglecting the role of B lymphocyte-mediated humoral immunity in PNS disease. In recent years, it has been found that, in a patient with relapsed hormone-sensitive nephrotic syndrome, the number of B cells in peripheral blood is significantly increased, and that activated B cells are greatly increased in a patient with hormone-dependent nephrotic syndrome, while in a patient with remission-stage hormone-dependent nephrotic syndrome, the number of B cells is significantly decreased, suggesting that B lymphocyte dysfunction plays an important role in PNS. In recent years, much global research has found that a significant therapeutic effect is achieved in the treatment of children's refractory nephrotic syndrome with rituximab specific for CD20+ B cells. However, in the treatment of SDNS with rituximab, it was found that the B cell scavenging efficacy was maintained for about 5 months, but at 6 and 7 months, the disease was recurring due to the rise in B cell numbers. Thus, pathological B cell clones in the PNS patient are suggested to be identified and accurately eliminated, so that the PNS recovery is facilitated, and the risk of humoral immune deficiency caused by indiscriminate B cell elimination by rituximab and other methods is reduced.
However, to date, the target antigens to which pathological B cells are directed in PNS infants are still not well understood. Pathologically, minimal morbid nephropathy or focal segmental glomerulosclerosis is considered podocytosis resulting in massive proteinuria due to loss or alteration of podocyte function. Podocytes are renal glomerular epithelial cells that attach to the outside of the glomerular basement membrane. It is the last barrier to protein loss, and podocyte injury often causes massive proteinuria.
The mitochondrial matrix protein encoded by Peptidyl-prolyl cis-trans isomerase D, which is the switch in the mitochondrial permeability transition pore, is a regulatory switch. Animal experiments of mice show that when the peptidyl-prolyl cis-trans isomerase D gene is knocked down, the mice show resistance to myocardial damage induced by ischemia-reperfusion; over-expression, which is indicative of myocardial hypertrophy, decreased cardiomyocyte function, probably by mitochondrial pathway regulation of cellular necrosis or by antioxidant pathways. Peptidyl-prolyl cis-trans isocerase D knockout mice are resistant to high fat diet-induced obesity or hepatic steatosis (Wang X, et al. cyclophilin D deficience epitopes mitochondmy, hepatology,2018 Jul; 68(1): 62-77.). In addition, peptidyl-prolyl cis-trans isomerase D is specifically expressed in human tumor cells such as ovarian cancer, uterine cancer, breast cancer, liver cancer, renal cancer and the like.
The relationship between peptidyl-prolyl cis-trans isomerase D and kidney disease is currently mostly focused on studies on acute kidney injury. For example, in a study of cyclosporin A-induced acute kidney injury, peptidyl-prolyl cis-trans isomerase D exerts a Renal protective effect by resisting oxidative stress (Jelena Klawitter, et al. cyclophilin D knock out protects the mouse kit against acquired cyclosporine A-induced oxidative stress. am J Physiol Renal Physiol, 2019 Sep; 317 (3): F683-F694). In another cisplatin-induced acute kidney injury study, peptidyl-prolyl cis-trans isomerase D served to protect the kidney by modulating fatty acid beta-oxidation. Fatty acid oxidation is the primary energy source for the kidney and is inhibited during acute kidney injury. Peptidyl-prolyl cis-trans isomerase D may play a key role in regulating overall cellular metabolism, including glucose and fatty acid metabolism, as well as mitochondrial bioenergetics, including the tricarboxylic acid cycle, electron transporter chains and oxidative phosphorylation in cells in a tissue/organ specific manner (Hee-Seong Jang, et al. Proximal tissue cycle and D regulation of protein activity in protein-induced amino kinase Int,2020 Feb; 97(2): 327. quadrature. 339.).
However, no report has been made on the relationship between peptidyl-prolyl cis-trans isomerase D and nephrotic syndrome. In the prior art, the study for identifying autoimmune nephrotic syndrome by detecting serum anti-peptidyl-prolyl cis-trans isomerase D-IgG antibody is also blank at present.
Disclosure of Invention
The invention provides a detection kit for an anti-peptidyl prolyl cis-trans isomerase D-IgG antibody, namely a detection kit for detecting serum anti-peptidyl-prolyl cis-trans isomerase D-IgG autoantibody, wherein the kit detects the antibody in a detected sample through immunoreaction with peptidyl-prolyl cis-trans isomerase D (peptidyl prolyl cis-trans isomerase D) antigen protein (particularly shown in a sequence identification number SEQ ID NO. 1).
The kit of the invention comprises: the kit comprises peptidyl-prolyl cis-trans isomerase D antigen protein, labeled antibody fluid (enzyme-labeled or chemiluminescent labeled anti-human IgG solution), solid phase carrier coated with peptidyl-prolyl cis-trans isomerase D antigen, sample diluent, antibody diluent, antigen diluent, substrate chromogenic solution, washing solution, stop solution, standard substance, positive quality control substance and negative quality control substance.
The sequence of the peptidyl-prolyl cis-trans isomerase D antigen protein is shown in SEQ ID NO: 1, and the following components: FADIVPKTAENFRALCTGEKGIGHTTGKPLHFKGCPFHRIIKKFMIQGGDFSNQNGTGGESIYGEKFEDENFHYKHDREGLLSMANAGRNTNGSQFFITTVPTPHLDGKHVVFGQVIKGIGVARILENVEVKGEKPAKLCVIAECGELKEGDDGGIFPKDGSGDSHPDFPEDADIDLKDVDKILLITEDLKNIGNTFFKSQNWEMAIKKYAEVLRYVDSSKAVIETADRAKLQPIALSCVLNIGACKLKMSNWQGAIDSCLEALELDPSNTKALYRRAQGWQGLKEYDQALADLKKAQGIAPEDKAIQAELLKVKQKIKAQKDKEKAVYAKMFA are provided.
According to the invention, the peptidyl-prolyl cis-trans isomerase D antigen protein is purified by a molecular sieve, gel filtration chromatography, affinity chromatography, an ion exchange column and a hydrophobic column.
The peptidyl-prolyl cis-trans isomerase D antigen protein of the present invention may be a fusion protein using a tag having a biological or physical function, particularly an N-terminal or C-terminal tag, preferably a C-terminal tag. These tags facilitate purification, immobilization, and precipitation of antigenic proteins. 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 present invention, the antigenic protein peptidyl-prolyl cis-trans isomerase D can be expressed in bacteria such as Escherichia coli, fungal yeast, mammalian cells.
In a preferred embodiment, the peptidyl-prolyl cis-trans isomerase D antigen protein of the present invention is presented in an immobilized form, the term "immobilized" referring to binding to an insoluble solid support in an aqueous solution. Preferably, the binding is by electrostatic interaction, hydrophobic interaction, covalent bond. The solid phase carrier is preferably polystyrene, a material microporous plate, a nitrocellulose membrane and magnetic beads.
The immobilization mode of peptidyl-prolyl cis-trans isomerase D antigen protein comprises a reversible immobilization mode or an irreversible immobilization mode. For example, the molecules are bound by a cleavable covalent bond (e.g., a disulfide bond that can be cleaved by addition of a thiol-containing reagent), and this immobilization is reversible. In addition, if the molecule is immobilized to the support through a covalent bond that is not cleaved in aqueous solution (a bond formed by the reaction of an epoxide group with the amine group coupling the lysine side chain to the affinity column), the immobilization is irreversible. The immobilization may also be an indirect way of immobilizing the protein: such as immobilizing 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 method for fixing peptidyl-prolyl cis-trans isomerase D antigen protein is a direct coating method: (1) the antigen is combined on the nitrocellulose membrane or the polystyrene microporous plate in a physical adsorption mode or non-covalent bond; (2) the magnetic particle belt with carboxyl functional group is combined with protein amino, and the antigen is combined on the magnetic particle by means of chemical coupling.
The substrate color developing solution is TMB, luminol, hydrogen peroxide and acridinium ester; the antigen diluent is: 1xPBS, pH7.40; the antibody diluent is as follows: 0.15% BSA +0.01M PBS (pH7.40); the sample diluent is: 6% fetal bovine serum +0.01M PBS (pH7.40); the washing solution is as follows: 1xPBS (pH7.40) +0.1 Tween-20; the stop solution is: 2M sulfuric acid.
In one embodiment of the present invention, the standard and the positive quality control substance are preferably prepared from human homologous antigen, and are recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or antibodies against peptidyl-prolyl cis-trans isomerase D-IgG extracted from patient serum as the positive quality control substance and the standard, and the negative quality control substance is serum of a healthy examiner.
The labeled antibody fluid can be Horseradish Peroxidase (HRP) labeled anti-human IgG, biotin labeled anti-human IgG and acridinium ester labeled anti-human IgG.
According to the invention, the sample to be tested is a liquid sample containing antibodies, preferably pleural fluid, 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 method for expressing and purifying recombinant protein peptidyl-prolyl cis-trans isomerase D by using a gene recombination method as an antigen protein in a kit, then coating the mixture on a solid phase carrier, adding a positive quality control product or a standard product or serum to be detected for incubation, adding a labeled secondary antibody for reaction, binding with anti-peptidyl-prolyl cis-trans-isomerase D-IgG antibody in serum to form a coating antigen peptidyl-prolyl cis-trans-isomerase D-detecting serum anti-peptidyl-prolyl cis-trans-isomerase D-IgG antibody-labeled anti-human IgG antibody complex, the detection kit can be used for qualitatively or quantitatively analyzing the concentration of the antibody against peptidyl-prolyl cis-trans isomerase D-IgG in serum by detecting optical signals by optical methods such as a light color development method, a chemiluminescence method and a fluorescence method.
By applying the kit, an anti-peptidyl-prolyl cis-trans-isomerase D-IgG autoantibody is detected in a part of patients with autoimmune nephrotic syndrome for the first time, and the target antigen aimed by the autoantibody is peptidyl-prolyl cis-trans-isomerase D on glomerular podocytes. Therefore, the kit can be used for detecting the anti-peptidyl-prolyl cis-trans isomerase D-IgG autoantibody and provides a basis for researching the autoimmune nephrotic syndrome.
Compared with the prior art, the kit has the advantages that:
(1) the invention identifies the existence of the anti-peptidyl-prolyl cis-trans isomer D-IgG autoantibody in patients with autoimmune nephrotic syndrome for the first time, and invents a detection kit aiming at the antibody. At present, the research related to peptidyl-prolyl cis-trans-isomerase D-IgG antibody at home and abroad is limited to the molecular mechanism research, and the invention fills the blank of detection of the peptidyl-prolyl cis-trans-isomerase D-IgG antibody at home and abroad.
(2) The kit relates to qualitative detection of an anti-peptidyl-prolyl cis-trans isocerase D-IgG antibody in serum, wherein the solid-phase membrane immunoassay qualitative detection is simple and convenient to operate, the dosage of a reagent is small, a trace amount of antigen can be adsorbed by the extremely strong adsorption capacity of an NC membrane, and the adsorbed NC membrane can be stored for a long time (the NC membrane can be stored for half a year at the temperature of minus 20 ℃) and does not influence the activity of the NC membrane. Is suitable for large-scale screening.
(3) The invention relates to a magnetic particle chemiluminescence immune analysis quantitative detection kit, which utilizes magnetic particles as solid phase carriers, the diameter of the magnetic particles is only 1.0 mu m, thus greatly increasing the surface area of the coating, increasing the adsorption quantity of antigens, improving the reaction speed, and ensuring that the cleaning and the separation are simpler and more convenient, thereby reducing pollution and reducing the probability of cross infection. On the other hand, the acridine ester luminescent agent is adopted to directly mark the anti-human IgG, the chemical reaction is simple and quick, and no catalyst is needed; the acridinium ester chemiluminescence is of the scintillation type by initiating the luminescent reagent (H)2O2NaOH) can reach the maximum after 0.4s, the half-life period is 0.9s, the detection is basically finished within 2s, and the rapid detection is convenient.
(4) The invention discovers that peptidyl-prolyl cis-trans isomerase D-IgG autoantibodies exist in nephrotic syndrome for the first time, and defines the cause of autoimmunity. Compared with the existing kidney biopsy detection method, the application of the kit fills the blank, and the pain and the economic burden of a patient can be relieved.
Drawings
FIG. 1: the peptidyl-prolyl cis-trans isomerase D protein on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. FIG. 1A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of healthy people; FIG. 1B: the first antibody is a two-dimensional electrophoresis protein spot of serum of an autoimmune nephrotic syndrome patient; FIG. 1C: mass spectrometric identification of the target antigen peptidyl-prolyl cis-trans isomerase D protein.
FIG. 2: SDS-PAGE identification of the expressed recombinant peptidyl-prolyl cis-trans isomerase D antigen protein.
FIG. 3: a solid-phase membrane immunoassay kit is used for detecting antibody membrane bars of peptidyl-prolyl cis-trans isomerase D-IgG in serum of patients with autoimmune nephrotic syndrome.
FIG. 4: schematic diagram of magnetic particle chemiluminescence kit for detecting anti-peptidyl-prolyl cis-trans isomerase D-IgG antibody.
FIG. 5: schematic representation of the coated carboxyl magnetic particles of the antigenic protein peptidyl-prolyl cis-trans isomerase D.
FIG. 6: detection of anti-peptidyl-prolyl cis-trans isomerase D-IgG antibodies in patients with different renal diseases, wherein NC: healthy people; HP: allergic purpura; HPN: purpuric nephritis; IgAN: IgA nephropathy; and NS: autoimmune nephrotic syndrome.
FIG. 7 is a schematic view of: the application value of the antibody against peptidyl-prolyl cis-trans isomerase D-IgG for detecting autoimmune nephrotic syndrome is evaluated by the ROC curve.
Detailed Description
The invention is further described below with reference to the following figures and specific examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
Example 1 peptidyl-prolyl cis-trans isomerase D on podocytes is a target antigen for autoantibodies in patients with autoimmune nephrotic syndrome
(1) Extraction of glomerular podocyte total protein: the podocyte strain (MPC5) was cultured, washed 2-3 times with PBS, then sufficiently lysed on ice using a focused ultrasound machine (Covaris S220, Gene) in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitor (# ab 65621; Abcam, 1: 200 dilution), and then the samples were centrifuged at 12000g for 30min at 4 ℃. Collecting the supernatant, namely the total protein of the glomerular podocyte. The total protein concentration of the collected glomerular podocytes was determined using the BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: extracting total protein of glomerular podocyte, performing two-dimensional electrophoresis, transferring to nitrocellulose membrane, incubating with serum of autoimmune nephrotic syndrome patient and healthy person as primary antibody, and developing with secondary antibody as shown in fig. 1A and 1B. (3) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: differential analysis of positive spots was performed after visualization in step (2), protein spots were selected which were strongly positive for nephrotic syndrome patients on two-dimensional electrophoresis gel and negative or weakly positive for healthy persons, the selected protein spots were excised 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 then 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) mass spectrometer to obtain a peptide mass spectrum, which was identified as peptidyl-prolyl cis-trans isomerate D protein, as shown in FIG. 1C.
EXAMPLE 2 expression and purification of recombinant peptidyl-prolyl cis-trans isomerase D antigen protein
The gene of the encoded peptidyl-prolyl cis-trans isomerase D protein is used as a template by utilizing a genetic engineering method to carry out PCR amplification, and then an expression vector is constructed to carry out protein expression. The expressed antigen protein of the invention contains tag peptide of His tag. The expressed recombinant protein was purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc., and finally the molecular weight of the recombinant protein peptidyl-prolyl cis-trans isomerase D was identified by SDS-PAGE to be 41KDa, the results are shown in FIG. 2.
Example 3 optimization of reaction conditions of the kit according to the present invention using orthogonal assay design orthogonal tables were selected according to 4 factors such as the coating concentration of the antigen peptidyl-prolyl cis-trans isomerase D (four coating concentrations of 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, 200. mu.g/mL), each reaction time (30min, 45min) and temperature (25 ℃, 35 ℃), the optimal dilution of the enzyme-labeled secondary antibody (four dilutions of 1: 100, 1: 500, 1: 1000, 1: 1500), and each factor was repeatedly measured at 2 levels for standard positive serum and standard negative serum. The ratio (P/N) of the highest luminescence (P) of the positive sera to the lowest luminescence (N) of the negative sera was selected. The average P/N value of repeated measurement is statistically processed to determine the optimal coating condition and the optimal dilution of the secondary antibody to carry out orthogonal optimization, thereby obviously improving the positive detection rate of the standard positive serum. Through orthogonal design, the optimal antigen coating concentration of the kit is 100 mu g/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 labeled anti-human IgG antibody is 1: 500.
EXAMPLE 4 preparation of solid-phase Membrane immunoassay kit for detecting anti-peptidyl-prolyl cis-trans isomerase D-IgG antibody
4.1 composition of solid phase membrane immunoassay kit for detecting anti-peptidyl-prolyl cis-trans isomerase D-IgG:
1. a nitrocellulose membrane coated with peptidyl-prolyl cis-trans isomerase D antigen protein,
2. and (3) standard substance: human anti-His tag immunoglobulin G (purchased from invitro lake),
3. the dilution liquid of the antibody is used as the antibody,
4. an antigen diluent is added to the antigen-containing solution,
5. an anti-human IgG antibody marked by horseradish peroxidase,
6. the washing liquid is used for washing the glass fiber,
7. a TMB color-developing agent,
8. and (4) stopping the solution.
4.2 the detection steps are as follows:
4.2.1 coating, sealing: the antigen was diluted with 0.01M PBS (pH7.4) and 10. mu.l of the diluted antigen was spotted on a nitrocellulose membrane, which was placed in a 37 ℃ incubator for 30 minutes, the nitrocellulose membrane was placed in a plate tank, 150. mu.l of 3% BSA was added thereto, the resulting mixture was sealed in a 37 ℃ incubator for 15 minutes, and after the blocking solution was aspirated off, the membrane was washed 2 times with a washing solution.
4.2.2 serum incubation (first incubation): 100 microliter of the standard substance diluted by the antigen releasing solution and the detected serum sample are loaded in the reaction tank, a negative control and a positive control are simultaneously carried out, the nozzle of the sample loader does not touch the surface of the membrane, and one nozzle is replaced when each serum is loaded. The reaction vessel with the added sample was placed on a shaker and incubated at room temperature (20-25 ℃) for 45 minutes.
4.2.3 Wash (first wash): pouring out the liquid in the reaction tank, and washing the liquid for about 10 seconds by using diluted washing liquid; in washing, the washing solution is allowed to sufficiently flow through the reaction vessel. The cleaning is repeated for 5 times, and the liquid is poured and washed while the liquid flows down along the reaction tank, so that the cross contamination is avoided. And (5) drying the reaction tank after cleaning.
4.2.4 incubation with secondary antibody working solution (second incubation): the horseradish peroxidase-labeled anti-human IgG antibody was diluted with the antibody diluent, and then 6 drops (300. mu.l) of the secondary antibody working solution were added to the reaction tank, and incubated on a shaker at room temperature (20-25 ℃) for 45 minutes.
4.2.5 Wash (second wash): the process is the same as step 3.
4.2.6. Chromogenic incubation (third incubation): 6 drops (300. mu.l) of the chromogenic solution were added to the strips and incubated on a shaker at room temperature (20-25 ℃) for 20 minutes.
4.2.7. And (3) terminating the reaction: the reaction vessel was flushed with running water to terminate the reaction.
4.2.8. Interpretation results: the test strips were taken out and dried by air blow (about 5 minutes) or placed in an oven at 37-50 ℃ for over 20 minutes. And (3) carrying out naked eye qualitative judgment, wherein the positive is the one with obvious brown spots, (see figure 3) or placing the membrane strip on a developing instrument for scanning, and drawing a standard curve by using analysis software carried by the developing instrument to carry out semi-quantitative analysis on the level of the antipeptidyl-prolyl cis-trans isocerase D-IgG in the serum by taking the concentration of a reference standard substance as an ordinate and taking a gray value read by the instrument as an abscissa.
EXAMPLE 5 preparation of a chemiluminescent immunoassay kit for the detection of anti-peptidyl-prolyl cis-trans isomerase D-IgG antibodies
5.1 composition of chemiluminescence immunoassay kit for detecting anti-peptidyl-prolyl cis-trans isomerase D-IgG:
1. magnetic particle solution coated with peptidyl-prolyl cis-trans isomerase D antigen protein,
2. and (3) standard substance: human anti-His tag immunoglobulin G (purchased from invitro lake),
3. the dilution liquid of the sample is used for diluting the sample,
4. the acridinium ester is marked on the anti-human IgG solution,
5. the quality control material is prepared by the following steps of,
6. the pre-excitation liquid is mixed with the water,
7. the excitation liquid is used for exciting the liquid,
8. and (4) washing liquid.
5.2 detection principle: the kit adopts an indirect method to detect the anti-peptidyl-prolyl cis-trans isomerase D-IgG antibody in human serum, and the whole process comprises two steps of reactions: in the first step, a magnetic bead solution is mixed with a diluted sample, a specific anti-peptidyl-prolyl cis-trans isomer D-IgG antibody is bound to the magnetic beads, and the mixture is washed to remove a residual solution. Secondly, adding acridinium ester labeled anti-human IgG antibody to form a magnetic bead-antigen-anti-peptidyl-prolyl cis-trans isocerase D-IgG antibody-acridinium ester antibody compound, washing to remove residual liquid, and adding a pre-excitation liquid (H)2O2) And performing luminescence reaction with exciting solution (NaOH), recording luminescence value, wherein the antibody concentration is in direct proportion to the luminescence value, and calculating the concentration value 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 mode of the kit is that carboxyl magnetic particles are activated by EDC/Sulfo-NHS and 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, then carrying out magnet separation, and removing the supernatant;
b) adding 200 microliters of 10mg/mL sodium periodate into 200 microliters of 2% dextran solution to react;
c) after the reaction is finished, adding 10mg/mL EDC solution prepared by phosphate buffer solution and 10mg/mLSulfo-NHS solution, and uniformly mixing for 60 minutes;
d) magnet separation, abandoning the supernatant, taking 400 microliter 0.05M phosphate buffer solution to wash the magnetic beads, adding 400 microliter of sealing solution to fix the volume for preservation.
Removing supernatant of the activated magnetic beads, adding pre-cooled 1ml of 20mM MES, and continuously washing the magnetic beads for 2 times; adding 200 mu L of 2mg/mL peptidyl-prolyl cis-trans isomerase D antigen protein into activated magnetic beads, fully and uniformly mixing, and standing at room temperature for reaction for 16 hours; after the reaction is finished, adding a PBS buffer solution with the pH of 7.4 and containing 0.2 percent Tween20, and repeatedly washing the magnetic beads for 2 times; then adding a PBS buffer solution with the pH of 7.4 and containing 0.2 percent Tween20 and 0.2 percent BSA until the final concentration of the magnetic beads is 10mg/mL, fully and uniformly mixing, and standing at room temperature for reaction for 30 min; after the reaction was completed, the supernatant was discarded, and the magnetic beads were resuspended in PBS buffer pH7.4 containing 0.2% Tween20 and 0.2% BSA, and the activated magnetic beads were crosslinked with the antigenic protein peptidyl-prolyl cis-trans isocyanate D (see FIG. 5).
5.5 acridinium ester labeled anti-human IgG solution, comprising the following steps:
a) preparing 2mg/mL acridinium ester solution by using dimethylformamide;
b) preparing 1mg/mL anti-human IgG antibody by using 0.2M (PH8.0) carbonate buffer;
c) uniformly mixing and stirring acridinium ester and an anti-human IgG antibody in a molar ratio of 4:1, and reacting for 40 minutes;
d) the reaction was stopped by adding 20. mu.l of carbonate buffer containing 5% lysine for 30 minutes;
e) desalting to remove impurities to obtain 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 the diluted sample, adding the magnetic particle solution and the sample diluent, and reacting for 12min at 37 ℃;
5.6.3 washing with washing solution for 3 times;
5.6.4 adding acridinium ester labeled anti-human IgG solution, reacting at 37 deg.C for 12 min;
5.6.5 washing solution for 3 times;
5.6.6 adding pre-exciting liquid (H)2O2) Reacting with an excitation solution (NaOH), and collecting a luminescence measured value;
5.6.7 the concentration measurement is calculated from the calibration curve.
Example 6 detection of anti-peptidyl-prolyl cis-trans isomerase D-IgG antibodies in various renal disease patients
6.1 Subjects included patients diagnosed with autoimmune nephrotic syndrome from 6 months 2018 to 6 months 2020, and healthy controls were selected from healthy examiners who had a contemporaneous visit. Serum samples were taken from nephrotic syndrome patients and healthy controls. All subjects had a first serum sample collection prior to no immunosuppressive treatment.
6.2 detection of serum anti-peptidyl-prolyl cis-trans-isomerase D-IgG antibodies in various patients with nephropathy the kit of the present invention was used to detect the levels of anti-peptidyl-prolyl cis-trans-isomerase D-IgG antibodies in the serum of patients diagnosed with various nephropathy from 6 months 2018 to 6 months 2020, including 466 nephrotic syndrome, 168 Henoch-Schonlein purpura, 137 Henoch-Schonlein purpura, 133 IgA nephropathy and 195 healthy children at the same time, and it was shown that anti-peptidyl-prolyl cis-trans-isomerase D-IgG antibodies were positive in some patients with autoimmune nephrotic syndrome, while anti-peptidyl-prolyl-trans-isomerase D-IgG antibodies were negative in Henoch-Schonlein purpura, nephrotic patients and healthy children (see FIG. 6).
EXAMPLE 7 evaluation of value of anti-peptidyl-prolyl cis-trans isomerase D-IgG antibody as a serological marker for detection of autoimmune nephrotic syndrome in patients with nephrotic syndrome in example 6.2 the results of detection of anti-peptidyl-prolyl cis-trans isomerase D-IgG antibody in patients with nephrotic syndrome in example 6 were analyzed using the ROC curve to determine the value of the antibody in detection of autoimmune nephrotic syndrome. The results showed that the anti-peptidyl-prolyl cis-trans isomerase D-IgG antibody as a serum marker was of great value for the detection of patients with autoimmune nephrotic syndrome, and when the diagnostic threshold was set to be greater than 41.1, the diagnostic sensitivity was 63.6%, the specificity was 68.1%, and the area under the curve was 0.701 (see FIG. 7).
Sequence listing
<110> Zhejiang university college of medicine subsidiary children hospital
<120> detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 334
<212> PRT
<213> peptidyl-prolyl cis-trans isomerase D protein (Artificial sequence Unknow)
<400> 1
Phe Ala Asp Ile Val Pro Lys Thr Ala Glu Asn Phe Arg Ala Leu Cys
1 5 10 15
Thr Gly Glu Lys Gly Ile Gly His Thr Thr Gly Lys Pro Leu His Phe
20 25 30
Lys Gly Cys Pro Phe His Arg Ile Ile Lys Lys Phe Met Ile Gln Gly
35 40 45
Gly Asp Phe Ser Asn Gln Asn Gly Thr Gly Gly Glu Ser Ile Tyr Gly
50 55 60
Glu Lys Phe Glu Asp Glu Asn Phe His Tyr Lys His Asp Arg Glu Gly
65 70 75 80
Leu Leu Ser Met Ala Asn Ala Gly Arg Asn Thr Asn Gly Ser Gln Phe
85 90 95
Phe Ile Thr Thr Val Pro Thr Pro His Leu Asp Gly Lys His Val Val
100 105 110
Phe Gly Gln Val Ile Lys Gly Ile Gly Val Ala Arg Ile Leu Glu Asn
115 120 125
Val Glu Val Lys Gly Glu Lys Pro Ala Lys Leu Cys Val Ile Ala Glu
130 135 140
Cys Gly Glu Leu Lys Glu Gly Asp Asp Gly Gly Ile Phe Pro Lys Asp
145 150 155 160
Gly Ser Gly Asp Ser His Pro Asp Phe Pro Glu Asp Ala Asp Ile Asp
165 170 175
Leu Lys Asp Val Asp Lys Ile Leu Leu Ile Thr Glu Asp Leu Lys Asn
180 185 190
Ile Gly Asn Thr Phe Phe Lys Ser Gln Asn Trp Glu Met Ala Ile Lys
195 200 205
Lys Tyr Ala Glu Val Leu Arg Tyr Val Asp Ser Ser Lys Ala Val Ile
210 215 220
Glu Thr Ala Asp Arg Ala Lys Leu Gln Pro Ile Ala Leu Ser Cys Val
225 230 235 240
Leu Asn Ile Gly Ala Cys Lys Leu Lys Met Ser Asn Trp Gln Gly Ala
245 250 255
Ile Asp Ser Cys Leu Glu Ala Leu Glu Leu Asp Pro Ser Asn Thr Lys
260 265 270
Ala Leu Tyr Arg Arg Ala Gln Gly Trp Gln Gly Leu Lys Glu Tyr Asp
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Gln Ala Leu Ala Asp Leu Lys Lys Ala Gln Gly Ile Ala Pro Glu Asp
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Lys Ala Ile Gln Ala Glu Leu Leu Lys Val Lys Gln Lys Ile Lys Ala
305 310 315 320
Gln Lys Asp Lys Glu Lys Ala Val Tyr Ala Lys Met Phe Ala
325 330
Claims (6)
1. The application of peptidyl prolyl cis-trans isomerase D antigen protein in preparing a kit for detecting autoimmune nephrotic syndrome is characterized in that the kit consists of the peptidyl prolyl cis-trans isomerase D antigen protein, labeled antibody liquid, a solid phase carrier coating peptidyl prolyl cis-trans isomerase D antigen, sample diluent, antibody diluent, antigen diluent, substrate color former, washing solution, stopping solution, standard substance, positive quality control substance and negative quality control substance; the sequence of the peptidyl prolyl cis-trans isomerase D antigen protein is shown as SEQ ID: 1 is shown in the specification; the labeled antibody liquid is an enzyme-labeled, chemiluminescent or biotin-labeled anti-human IgG secondary antibody; the peptidyl prolyl cis-trans isomerase D antigen protein has a label with biological or physical function; the standard substance and the positive quality control substance are recombinant human anti-tag peptide immunoglobulin G, or anti-peptidyl prolyl cis-trans isomerase D-IgG antibody is extracted from serum, and the negative quality control substance is serum of a health physical examiner; autoimmune nephrotic syndrome is diagnosed by detecting anti-peptidyl prolyl cis-trans isomerase D-IgG antibodies in a serum sample from a patient.
2. The use of claim 1, wherein the tag is a sequence or domain capable of specifically binding to a ligand, and is selected from one or more of a His tag, a GST tag, a maltose binding protein, a thioredoxin, and a fluorescent tag.
3. The use of claim 1, wherein the peptidyl prolyl cis-trans isomerase D antigen protein is purified by molecular sieve, affinity chromatography, ion exchange column or hydrophobic column.
4. The use of claim 1, wherein the peptidyl prolyl cis-trans isomerase D antigen protein is immobilized on a solid support selected from nitrocellulose membranes, polystyrene, microwell plates, biochips, or magnetic beads.
5. The use according to claim 4, wherein the peptidyl prolyl cis-trans isomerase D antigen protein is immobilized on the solid support by direct means using physical adsorption or chemical binding.
6. The use according to claim 1, wherein the antigen dilution is: 1xPBS, pH7.40; the antibody diluent is: 0.15% BSA +0.01M PBS pH7.40; the sample diluent is: 6% fetal bovine serum +0.01M PBS pH7.40; the substrate color developing solution is TMB, luminol, hydrogen peroxide and acridinium ester; the stop solution is: 2M sulfuric acid.
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| CN105988011A (en) * | 2016-03-31 | 2016-10-05 | 刘以鹏 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for idiopathic membranous nephropathy and detection method thereof |
| CN106999547A (en) * | 2014-07-24 | 2017-08-01 | 欧蒙医学诊断技术有限公司 | Method and kit for monitoring membranous nephropathy |
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| CN1393557A (en) * | 2001-06-29 | 2003-01-29 | 上海博德基因开发有限公司 | Polypeptide-human protein-14.08 containing peptidyl-prolyl cis-trans isomerase characteristic sequence fragment and polynucleotide for coding it |
| US20140248297A1 (en) * | 2011-05-12 | 2014-09-04 | Novarx Corporation | Predictors of response to immunotherapy |
| KR101471272B1 (en) * | 2013-01-29 | 2014-12-11 | 인제대학교 산학협력단 | Biomarker composition for diagnosis of bortezomib resistance comprising Cyclophilin D and diagnostic kit using the same |
| CN109182322A (en) * | 2018-10-25 | 2019-01-11 | 天津市湖滨盘古基因科学发展有限公司 | The Peptidyl prolyl cis-trans isomerases mutain of people a kind of and its application |
| CN110358829A (en) * | 2019-07-09 | 2019-10-22 | 江苏医药职业学院 | Detect application and the kit of the reagent of recombined human peptidyl prolyl cis-trans isomerase-H expression |
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| CN105988011A (en) * | 2016-03-31 | 2016-10-05 | 刘以鹏 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for idiopathic membranous nephropathy and detection method thereof |
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Application publication date: 20210928 Assignee: Century Yishu (Hangzhou) Medical Diagnostic Technology Co.,Ltd. Assignor: ZHEJIANG University Contract record no.: X2023980035174 Denomination of invention: A detection kit for D-IgG antibody against peptidylprolyl cis trans isomerase Granted publication date: 20220513 License type: Exclusive License Record date: 20230505 |
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