WO2016009971A1 - Mesure simplifiée d'un anticorps anti-récepteur de phospholipase a2 - Google Patents

Mesure simplifiée d'un anticorps anti-récepteur de phospholipase a2 Download PDF

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WO2016009971A1
WO2016009971A1 PCT/JP2015/069961 JP2015069961W WO2016009971A1 WO 2016009971 A1 WO2016009971 A1 WO 2016009971A1 JP 2015069961 W JP2015069961 W JP 2015069961W WO 2016009971 A1 WO2016009971 A1 WO 2016009971A1
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pla2r
antibody
phospholipase
membrane
trehalose
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Japanese (ja)
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秋山　真一
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国立大学法人名古屋大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • the present invention relates to a technique for measuring an anti-phospholipase A2 receptor antibody.
  • Phospholipase A2 receptor is a type I transmembrane protein with a molecular weight of about 180 kDa that is expressed in human glomerular podocytes.
  • Anti-PLA2R antibodies in blood are considered promising as the only biomarkers for pathologic differentiation and disease prediction of idiopathic membranous nephropathy, which is the main causative disease of nephrotic syndrome (Non-patent Document 1). Idiopathic membranous nephropathy was confirmed by exclusion diagnosis until anti-PLA2R antibody was discovered.
  • membranous nephropathy there are two types of membranous nephropathy: secondary membranous nephropathy with known causative agent and idiopathic membranous nephropathy with unknown causative agent. Diagnosed as secondary membranous nephropathy if the patient has a causative agent of secondary membranous nephropathy (eg, taking antirheumatic drugs, autoimmune disease, tumor, etc.), otherwise idiopathic The patient was diagnosed with membranous nephropathy.
  • a causative agent of secondary membranous nephropathy eg, taking antirheumatic drugs, autoimmune disease, tumor, etc.
  • anti-PLA2R antibodies can be used as a differential biomarker for idiopathic membranous nephropathy, if the anti-PLA2R antibody is present in the patient's blood (possibly without renal biopsy), the idiopathic membrane In addition to being diagnosed as having nephropathy, the treatment method is completely different between secondary membranous nephropathy and idiopathic membranous nephropathy. It is very important to confirm the presence or absence of PLA2R antibody (qualitative test).
  • the patient serum sample as primary antibody and chemically labeled anti-human IgG antibody as secondary antibody are sequentially reacted to PLA2R protein presented as antigen, and anti-PLA2R contained in the sample Detect antibodies. Since the anti-PLA2R antibody recognizes the three-dimensional epitope of PLA2R, which is an antigen, it is necessary to keep the three-dimensional structure of the PLA2R protein, which is an antigen, in a natural form (native form). It has been found that when the three-dimensional structure of PLA2R is broken, the anti-PLA2R antibody cannot bind to PLA2R (Non-patent Document 1), but detailed studies such as crystal structure analysis are not yet available.
  • Non-patent Document 1 an expensive imager (GE) is used to perform chemiluminescence using HRP labeled on the secondary antibody in the signal acquisition step which is the final step of the assay step. LAS4000 made) and complicated darkroom operation are necessary.
  • the indirect fluorescent antibody method genetically modified mammalian cells (for example, HEK293 cells) prepared so that recombinant PLA2R is expressed on the cell membrane are adherently cultured on a 96-well microplate or a glass slide with a chamber, and then the cells. Recombinant PLA2R that is solid-phased on the cell surface by fixing with formalin is used as an antigen (Non-patent Document 2).
  • the indirect fluorescent antibody method can also be carried out at a low cost, and is technically simpler than the Western blot method. On the other hand, the qualitative properties are remarkably inferior to the Western blot method, and the measurement sensitivity is not so good. In addition, an expensive fluorescence microscope is required in the microscope, which is the final stage of the assay process.
  • ELISA is characterized by simple procedures and excellent quantification, but requires high-purity recombinant PLA2R, and therefore the assay cost is extremely high. Moreover, it is inferior in qualitativeness and measurement sensitivity compared with the Western blot method, and an expensive plate reader is required in the signal acquisition process.
  • patient serum is diluted to an optimal concentration.
  • the anti-PLA2R antibody concentration can be increased to a measurable range by reducing the dilution rate of the serum sample during measurement. Need to be measured.
  • the antibody and protein concentrations other than the anti-PLA2R antibody in the serum also increase, and as a result, a non-specific signal is amplified.
  • anti-PLA2R antibody signals are obtained at the PLA2R band position, so it is possible to selectively measure anti-PLA2R antibody signals even if non-specific signal amplification occurs, but indirect fluorescent antibodies In the method and ELISA, non-specific signals and anti-PLA2R antibody signals cannot be measured separately in principle. Therefore, when it is desired to measure a serum sample having a low concentration of the original anti-PLA2R antibody, the anti-PLA2R antibody cannot be measured under a low dilution rate condition that causes a non-specific signal.
  • anti-PLA2R autoantibodies possessed by patients with idiopathic membranous nephropathy, etc. recognize the PLA2R three-dimensional epitope, so if the PVDF membrane after transferring the PLA2R protein is preserved, the three-dimensional structure of PLA2R will be preserved during storage. It is empirically known that the antigenicity is reduced or lost depending on the collapse and the change in the three-dimensional structure. Even if it is stored in a solution in which glycerol, which is widely used as a protein protective agent, coexists, it cannot be stored for more than one week while retaining the antigenicity of PLA2R.
  • an object of the present invention is to provide a means for measuring an anti-PLA2R antibody that is easy to operate and can be operated at a low cost.
  • the membrane immersed in a storage buffer containing trehalose did not deteriorate the storage stability even when dried (that is, the storage period was greatly extended), and could be stored at room temperature for a long time. .
  • the ability to store PLA2R-transferred (solid-phase) membranes in a dry state significantly improves the processability of the membrane.
  • the dried membrane onto which PLA2R has been transferred is cut into strips, If each piece is bonded and fixed to a carrier such as a glass slide, a simple anti-PLA2R antibody measuring instrument that has excellent operability and can be stored for a long period of time can be realized.
  • PLA2R antigenicity enhancement effect is also obtained by trehalose treatment, so there is no need to use ultra-sensitive chemiluminescence method for signal measurement, which is the final step of Western blotting. ) Can be applied (visual signal measurement is possible). This means that no special equipment or power supply is required for measurement, and measurement at a very low cost is realized.
  • the following invention is mainly based on the above results and considerations.
  • [1] comprising a step of treating a phospholipase A2 receptor with a trehalose solution, Preparation method of phospholipase A2 receptor maintaining antigenicity.
  • [2] The preparation method according to [1], wherein the trehalose concentration of the trehalose solution is 2% (w / v) to 40% (w / v).
  • [3] The preparation method according to [1], wherein the trehalose concentration of the trehalose solution is 2% (w / v) to 40% (w / v).
  • [4] The preparation method according to [1], wherein the trehalose solution has a trehalose concentration of 5% (w / v) to 20% (w / v).
  • [5] The preparation method according to any one of [1] to [4], wherein a drying treatment is performed after the step.
  • [6] The preparation method according to [5], wherein the drying treatment is air drying or freeze drying.
  • [7] The preparation method according to any one of [1] to [6], wherein the phospholipase A2 receptor is a recombinant protein.
  • [8] The preparation method according to any one of [1] to [7], wherein the phospholipase A2 receptor is in a state of being immobilized on a PVDF or nitrocellulose membrane.
  • the measurement method according to [15] comprising the following steps (1) and (2): (1) a step of bringing a specimen into contact with the membrane; and (2) a step of detecting a produced antigen-antibody complex.
  • the secondary antibody was changed to an IgG subclass-specific antibody, and anti-PLA2R antibody was detected in patient serum for each subclass.
  • Phospholipase A2 Receptor Maintaining Antigenicity
  • treatment with a trehalose solution is effective as a means for enhancing the preservation while maintaining the antigenicity of phospholipase A2 receptor (PLA2R).
  • trehalose treatment step a step of treating PLA2R with a trehalose solution
  • PLA2R is a single-transmembrane type I transmembrane protein expressed on the surface of human glomerular podocytes. It consists of a cysteine-rich domain, a fibronectin domain, 8 C-type lectin domains, and a transmembrane domain from the N-terminus, with two N-type sugar chain modifications and 17 disulfide bonds inside.
  • the amino acid sequence of PLA2R (NCBI Reference Sequence: NP_031392.3, DEFINITION secretory phospholipase A2 receptor, isoform 1, precursor [Homo sapiens]) is shown in SEQ ID NO: 1.
  • Non-patent Document 1 Non-patent Document 1
  • PLA2R maintaining amino acids 21 to 633 of the amino acid sequence of PLA2R (SEQ ID NO: 1) are important for antigenicity.
  • the treatment with the trehalose solution can prevent the three-dimensional structure from being collapsed during storage, and can maintain the antigenicity of PLA2R over a long period of time. That is, it is possible to provide an antigen for measuring anti-PLA2R antibody having excellent storage stability. Whether PLA2R maintains antigenicity can be confirmed by Western blotting using an anti-PLA2R antibody derived from a patient with idiopathic membranous nephritis as a primary antibody.
  • Recombinant PLA2R As PLA2R used in the trehalose treatment step, recombinant (recombinant) PLA2R or PLA2R separated and purified from human glomerular lysate can be used.
  • Recombinant PLA2R uses cDNA (an example is shown in SEQ ID NO: 2, in which MycTag sequence, HisTag sequence and stop codon are linked to a sequence encoding hPLA2R1), for example, mammals. It can be obtained by a production method using cells (such as HEK293 cells) as a host.
  • a trehalose solution with a predetermined concentration is used.
  • the trehalose concentration in the trehalose solution is preferably 2% (w / v) to 40% (w / v), more preferably 2 to 20%, and even more preferably 5 to 20%. If the trehalose concentration is too low, the desired effect (that is, preventing the collapse of the conformation of PLA2R) will not be obtained sufficiently, and if the trehalose concentration is too high, the effect on the measurement due to the remaining trehalose will be reduced. Increase is a problem.
  • Trehalose (substance name, generic name) is a compound represented by ⁇ -D-Glucopyranosyl (1,1) - ⁇ -D-Glucopyranoside. Trehalose is provided by Hayashibara Co., Ltd.
  • a trehalose solution can be prepared by dissolving trehalose in a physiological buffer (eg, PBS), physiological saline, distilled water, or the like.
  • a surfactant for example, 0.2% Tween
  • antibiotics for example, ascorbic acid
  • antioxidant for example, ascorbic acid
  • PLA2R is brought into contact with the trehalose solution.
  • PLA2R in a state of being immobilized on a membrane suitable for Western blotting, dot blotting, or the like, that is, a membrane made of PVDF or nitrocellulose is used.
  • a membrane suitable for Western blotting, dot blotting, or the like that is, a membrane made of PVDF or nitrocellulose is used.
  • Such PLA2R can be prepared by separation of a sample containing PLA2R by electrophoresis and subsequent transfer to a membrane (Western blotting).
  • PLA2R can be prepared by dropping and infiltrating PLA2R onto a membrane (for example, PVDF membrane or nitrocellulose membrane). In this case, it is preferable to use PLA2R having high purity.
  • blocking treatment is performed as follows. That is, blocking treatment is performed by immersing the carrier on which PLA2R is solid-phased at a room temperature or 4 ° C for a predetermined time (for example, 30 minutes to 16 hours) in a blocking agent (for example, Blocking® One® solution, 5% skim milk aqueous solution, etc. manufactured by Nacalai). I do.
  • a blocking agent for example, Blocking® One® solution, 5% skim milk aqueous solution, etc. manufactured by Nacalai.
  • the trehalose treatment may be performed before or after the blocking treatment.
  • PLA2R labeled in advance.
  • a PLA2R solid-phase membrane suitable for immunochromatography can be obtained (details will be described later).
  • the labeling substance a coloring substance, an enzyme, a radioisotope, or the like is used.
  • a color substance because the measurement result can be observed with the naked eye and a quick and simple determination can be made.
  • the color developing material are metal colloids (gold colloid, platinum colloid, etc.), synthetic latex colored with pigments, and natural latex.
  • the treatment time in the trehalose treatment step is, for example, 1 second to 14 days, preferably 1 second to 1 minute.
  • the temperature condition during the treatment is, for example, 4 ° C. to 40 ° C., preferably 20 ° C. to 25 ° C.
  • Optimal processing conditions may be set through preliminary experiments.
  • the PLA2R and the trehalose solution are typically contacted by immersing the membrane in a trehalose solution.
  • a drying treatment may be performed after the trehalose treatment step. This process can improve storage stability. Moreover, handling becomes easy.
  • general drying treatment methods such as air drying, vacuum drying, reduced pressure drying, and freeze drying can be employed.
  • samples that are frozen and solidified in a low-pressure environment generally having a boiling point of about -20 ° C (107 Pa, 0.8 Torr) to about -50 ° C (4 Pa, 0.03 Torr) (for example, The water is removed by sublimation from the one frozen at about -40 ° C.
  • the freeze-drying treatment it can be uniformly dehydrated from the inside, and since a high degree of dryness is realized, it can be dried while maintaining its original function and form at a high level.
  • the freeze-drying process is as follows: 1. Less degradation during processing 2. Can be sterilized easily. 3. A dry product excellent in resilience is obtained. It has the characteristics that a dried product excellent in storage stability can be obtained.
  • the freeze-drying process can be performed by a freeze-dryer equipped with a vacuum chamber, a cooling / heating device, and an exhaust device (cold trap and vacuum pump). Numerous freeze-drying apparatuses are commercially available, and the drying process can be carried out using any one selected from these.
  • the processing conditions can be set based on the instruction manual attached to the device to be used. In that case, the size of the sample to be subjected to the drying treatment, the degree of dryness, and the like can be considered.
  • the membrane is formed (eg, cut into a predetermined shape such as a strip shape) before or after the trehalose treatment step.
  • the above drying treatment is performed after the trehalose treatment step, such molding can be performed either before the trehalose treatment step, between the trehalose treatment step and the drying treatment, or after the drying treatment step.
  • PLA2R solid-phased on the membrane can be stored for a long time while maintaining its antigenicity. For example, it is stored at 4 ° C to 28 ° C. In order to prevent a decrease in antigenicity, it is preferably stored at a low temperature (eg, 4 ° C. to 25 ° C., preferably 4 ° C. to 15 ° C.).
  • a low temperature eg, 4 ° C. to 25 ° C., preferably 4 ° C. to 15 ° C.
  • PLA2R maintaining antigenicity can be obtained.
  • the PLA2R is an antigen suitable for measurement of anti-PLA2R antibodies.
  • a membrane (PLA2R solid-phased membrane) obtained by solidifying PLA2R with improved storage stability is obtained.
  • the membrane is suitable for measurement of anti-PLA2R antibody using Western blotting or dot blotting.
  • a labeled PLA2R solid-phase membrane obtained when the above preparation method is performed using labeled PLA2R is suitable for measurement of an anti-PLA2R antibody using immunochromatography.
  • a “PLA2R solid-phase membrane” suitable for measurement of anti-PLA2R antibodies can be obtained.
  • the present invention provides, as another aspect, a measurement method using a membrane obtained by the above preparation method (hereinafter referred to as “PLA2R solid phase membrane”).
  • the membrane obtained by the above preparation method is a flat or rod-like insoluble support (polystyrene resin, polycarbonate resin, silicon resin, nylon resin, etc., or a substance such as glass.
  • a slide glass can be used as a specific example
  • the measurement method of the present invention may be carried out by using the one immobilized on (an embodiment of an anti-PLA2R antibody measurement instrument).
  • an antigen-antibody reaction between PLA2R and anti-PLA2R antibody is used.
  • the specific operation procedure varies depending on the measurement principle employed, the following two steps (1) and (2) are performed roughly in the measurement method of the present invention.
  • a blood sample (serum, plasma, etc.) is used as the sample in step (1).
  • the origin of the specimen that is, the subject is not particularly limited. However, since the information (measurement results) obtained by the measurement method of the present invention is useful for determination / diagnosis of idiopathic membranous nephropathy, in a preferred embodiment, determination / diagnosis of idiopathic membranous nephropathy is necessary.
  • a typical subject is a patient with nephrotic syndrome, a history of idiopathic membranous nephropathy. The measurement results for these persons help to determine a more appropriate treatment policy, and promote the improvement of the treatment effect and improvement of the patient's QOL (Quality of Life).
  • a healthy person may be the subject, and the measurement result in this case is useful for early detection and prevention of idiopathic membranous nephropathy.
  • the “healthy person” as used herein refers to a person who has not been determined to have idiopathic membranous nephropathy at the time of applying the measurement method of the present invention.
  • the specimen is subjected to pretreatment as necessary. As pretreatment, for example, dilution of a specimen, removal of contaminants by filter filtration or centrifugation can be performed.
  • the contact between the specimen and the PLA2R solid-phase membrane is performed by dropping or spreading the specimen on the PLA2R solid-phase membrane, immersing the PLA2R membrane in the specimen, or the like.
  • Specific modes of contact, contact conditions, and the like can be appropriately set according to the measurement principle employed.
  • step (2) the antigen-antibody complex produced in step (1) is detected.
  • the detection operation depends on the measurement principle employed. For example, when the principle of enzyme immunoassay (EIA method) is used, an antigen-antibody complex is detected using the color of the enzyme substrate as an index.
  • EIA method enzyme immunoassay
  • various detection means using antigen-antibody reaction can be employed.
  • enzyme immunoassay EIA method
  • immunochromatography fluorescence immunoassay
  • FIA method fluorescence immunoassay
  • RIA method radioimmunoassay
  • the EIA method and immunochromatography are preferable from the viewpoint of simplicity.
  • an enzyme-labeled secondary antibody usually an anti-human IgG antibody
  • an immune complex is detected using color development or luminescence of a substrate based on an enzyme reaction as a signal.
  • enzymes used for labeling are peroxidase, ⁇ -D-galactosidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, glucose-6-phosphate dehydrogenase.
  • substrates are 3,3 ′, 5,5′-tetramethylbenzidine (TMB), diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and luminol.
  • Immunochromatography is a particularly excellent technique in terms of simplicity.
  • a test strip is usually used for the measurement using immunochromatography.
  • a specimen addition part (sample pad) is formed on one end of a support with an antigen-impregnated member impregnated with labeled PLA2R (antigen) sandwiched between them, and a specimen-absorbing member (absorption) is formed on the other end.
  • Pad is formed.
  • a determination unit in which a capture antibody (anti-human IgG antibody) is immobilized is provided between the sample addition unit and the sample absorption unit.
  • a control line an antibody that recognizes the antigen is immobilized is provided at the tip of the determination unit to confirm the development of the antigen.
  • the test strip having the above configuration when the specimen is added to the specimen addition portion, contact with the antigen occurs in the antigen-impregnated member.
  • the detection target that is, anti-PLA2R antibody
  • the detection target that is, anti-PLA2R antibody
  • the determination unit develops color.
  • the control line develops color. If there is no detection target in the sample, only the control line will be colored. In this way, the presence or absence of the detection target in the sample can be determined by examining the color of the determination unit and the control line.
  • a measuring instrument in which a PLA2R solid-phased membrane is fixed to a flat insoluble support (for example, a slide glass) is prepared.
  • a predetermined amount for example, 5 to 100 ⁇ l
  • a specimen patient serum
  • a predetermined dilution rate for example, ⁇ 1 to ⁇ 1/1000
  • PBS containing a predetermined concentration of a surfactant for example, 0.2% Tween
  • a surfactant for example 0.2% Tween
  • an HRP-labeled anti-human IgG antibody is dropped on the PLA2R solid phased membrane, and allowed to stand at 37 ° C. for a predetermined time (for example, 5 minutes to 8 hours).
  • an HRP substrate eg, TMB
  • an anti-PLA2R antibody signal is observed (see FIG. 4).
  • TMB an anti-PLA2R antibody signal is observed (see FIG. 4).
  • an anti-PLA2R antibody measuring instrument and kit using the PLA2R solid phase membrane obtained by the above preparation method includes a PLA2R solid-phase membrane as a component.
  • the PLA2R solid-phase membrane is fixed to an insoluble support for easy handling.
  • Another embodiment is a test strip using a PLA2R solid phase membrane. The configuration of the test strip is as described above.
  • the measuring instrument of the present invention can be stored for a long period of time while maintaining the antigenicity of its main component PLA2R.
  • a low temperature eg, 4 ° C. to 25 ° C., preferably 4 ° C. to 15 ° C.
  • it can be stored for a long period of time (typically 5 months or more) even at room temperature.
  • the kit of the present invention includes the measuring instrument of the present invention as a main component.
  • Reagents enzyme substrate, coloring reagent, buffer solution, etc.
  • other instruments and devices such as containers, reaction devices, etc.
  • a control reagent positive control
  • a control reagent negative control
  • an instruction manual is attached to the kit of the present invention.
  • PLA2R cDNA was introduced into HEK293 cells to express PLA2R protein
  • PLA2R protein was prepared from HEK293 cell lysate.
  • a CMV promoter, human PLA2R cDNA, and protein purification tag cDNA are sequentially ligated and inserted into a mammalian cell gene expression plasmid vector having a pcDNA vector (Lifetechnologies) as the backbone, and a human PLA2R expression plasmid vector.
  • a histidine tag is used as a protein purification tag, and the recombinant Pla2r protein is expressed as a fusion protein with the histidine tag.
  • This vector was transformed by transfecting HEK293 cells using transfection reagent (Promega), G418 sulfate (SIGMA aldrich), 10% fetal calf serum (Biowest), penicillin streptomycin mixture (SIGMA aldrich) ) Containing DMEM (SIGMA aldrich) was used to obtain HEK293 cells that constantly express recombinant PLA2R.
  • transfection reagent Promega
  • G418 sulfate SIGMA aldrich
  • 10% fetal calf serum Biowest
  • penicillin streptomycin mixture SIGMA aldrich
  • Containing DMEM SIGMA aldrich
  • the recombinant HEK293 cells after culturing were detached from the petri dish, washed with a phosphate buffer (SIGMA aldrich), and then dissolved in RIPA buffer (Wako Pure Chemical Industries) to prepare a lysate solution.
  • This lysate solution can be used as an antigen for detecting anti-PLA2R antibody by Western blotting.
  • affinity column purification using the affinity between the histidine tag and Ni resin is performed.
  • a fusion protein of recombinant PLA2R and histidine tag was prepared.
  • the prepared recombinant PLA2R was separated by polyacrylamide gel electrophoresis, and then transferred to a PVDF membrane (Lifetechnologies).
  • the transferred PDVF membrane was immersed in a blocking agent (Nalocai Blocing One Solution) stock solution and subjected to a blocking treatment at 37 ° C. for 30 minutes, and then treated and stored under the following predetermined conditions.
  • a blocking agent Nalocai Blocing One Solution
  • Condition 1 Store at 4 ° C in 0.2% Tween20 / PBS Condition 2: Store at 4 ° C after air drying Condition 3: Store at 4 ° C in 0.2% Tween20 / PBS after fixation with 4% paraformaldehyde Condition 4: 25% glycerol / 0.2% Storage at 4 ° C in Tween20 / PBS Condition 5: Storage at 4 ° C in 50% glycerol / 0.2% Tween20 / PBS Condition 6: Storage at 4 ° C in 10% trehalose / 0.2% Tween20 / PBS Condition 7: 10% trehalose / 0.2 After soaking in% Tween20 / PBS, air-dry and store at room temperature
  • FIG. 2 shows an example of a method for producing an anti-PLA2R antibody simple measuring instrument, which consists of the following procedures.
  • FIG. 3 shows an example of the measurement method using the anti-PLA2R antibody simple measurement instrument prepared by the method of FIG. 2.
  • the following operations are performed.
  • (i) Prepare a simple measuring instrument for anti-PLA2R antibody and add patient serum (10 ⁇ l) dropwise
  • ii Let stand at 37 ° C for 60 minutes
  • (iii) Wash (1 minute)
  • (iv) Add secondary antibody dilution and leave at 37 ° C for 45 minutes
  • Wash (1 minute) (vi) Add HRP substrate (eg TMB) and let stand at room temperature until color develops
  • (vii) Visually check the results
  • the anti-PAL2R antibody in clinical samples was measured by the above measurement method. Serum dilution was 1/10. Moreover, the serum of a healthy subject was used as a negative control (negative control), and the serum of an idiopathic membranous nephropathy patient known to contain anti-PLA2R2IgG as a positive control (positive control). The measurement results are shown in FIG. In patient sera # 3 and # 4, two distinct bands indicating the presence of anti-PLA2R antibodies were observed. The secondary antibody was changed from an anti-human IgG-Fc antibody to an IgG subclass-specific antibody, and anti-PLA2R antibody in patient serum was detected for each subclass (FIG. 5).
  • the measurement method using the anti-PLA2R antibody simple measurement instrument was compared with the conventional Western blot method and the measurement method using a commercially available ELISA kit (manufactured by Euroimmun). Specifically, anti-PLA2R antibody was measured by each method using patient serum as a sample, and the detection sensitivity and specificity were compared. As a result, the sensitivity (52.7%) and specificity (95.5%) equivalent to the Western blot method were obtained by the measurement method using the anti-PLA2R antibody simple measurement instrument. The sensitivity was superior to the commercially available ELISA kit (the sensitivity with the commercially available ELISA kit was 39.7%).
  • the anti-PLA2R antibody in the sample could be detected with high sensitivity.
  • the trehalose treatment significantly increased the storage stability of the membrane with PLA2R immobilized, which proved that a simple measuring instrument could be realized.
  • the ELISA plate prepared as described above was allowed to stand overnight at room temperature, it was immobilized on the bottom of the well using patient serum as the primary antibody and HRP-labeled anti-human IgG mouse monoclonal antibody as the secondary antibody.
  • a signal based on the antigenicity of the recombinant PLA2R immobilized on the trehalose-treated group was observed, whereas no signal was observed in the untreated group (FIG. 6). That is, even when immobilized on an ELISA plate, the antigenic protective effect of PLA2R against dry storage was observed.
  • a PLA2R antigen having excellent storage stability can be obtained.
  • the PLA2R antigen enables measurement of an anti-PLA2R antibody simply and at low cost. Further, by utilizing the principle of Western blotting, measurement with excellent qualitative properties can be achieved.
  • Anti-PLA2R antibody in blood is considered promising as the only biomarker for idiopathic membranous nephropathy and for pathologic differentiation and disease prediction, and the present invention contributes to the development and establishment of diagnostic technology for idiopathic membranous nephropathy To do.

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Abstract

　La présente invention traite le problème consistant à fournir un nouveau moyen permettant de mesurer des anticorps anti-récepteur de phospholipase A2. Selon la présente invention, des anticorps anti-récepteur de phospholipase A2 dans un échantillon sont détectés en utilisant, comme antigène, les récepteurs de phospholipase A2 dans lesquels l'antigénicité est maintenue par un traitement dans une solution de tréhalose.
PCT/JP2015/069961 2014-07-14 2015-07-10 Mesure simplifiée d'un anticorps anti-récepteur de phospholipase a2 WO2016009971A1 (fr)

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