Detection sensitivity-improving method, and kit for improving detection sensitivity

Abstract

Provided is a detection sensitivity-improving method for improving the sensitivity of detection of a protein. The detection sensitivity-improving method, which is used in a method for detecting, by an antigen-antibody reaction, a protein supported by a matrix, includes performing, before the antigen-antibody reaction, a boiling treatment in which the matrix on which the protein is supported is boiled in a sodium sulfate solution to thereby improve the sensitivity of detection of the protein by the antigen-antibody reaction. The protein is an etiological protein of dementia, such as Aβ and Tau. The method can also be applied to the improvement of the sensitivity of detection of a tag protein such as a His tag, an myc tag and a FLAG tag.

Description

Method for improving detection sensitivity and kit for improving detection sensitivity

[0001] The present invention relates to a method for improving detection sensitivity in a method for detecting a protein by an antigen-antibody reaction, and a kit for improving detection sensitivity.

[0002] Western blotting, which utilizes antigen-antibody reactions by specific antibodies, is an inexpensive and simple method that is essential for detecting proteins in life science research. Improving the detection sensitivity of Western blotting directly promotes basic research and is expected to be beneficial in clinical research, such as detecting biomarkers. Increasing the detection sensitivity of Western blotting requires obtaining antibodies with high specificity and affinity for the target, but obtaining high-performance antibodies is not always possible.

[0003] To achieve high sensitivity, commercially available reagents include those that enhance the affinity between antigens and primary antibodies and those that enhance the chemiluminescence produced by oxidoreductases bound to secondary antibodies (Patent Documents 1 and 2). Patent Document 1 discloses a method for enhancing the sensitivity of Western blotting by adding an antibody-binding domain such as protein A to a self-assembling protein, specifically HBsAg (hepatitis B virus surface antigen) protein, to produce enzyme-labeled virus-like particles. Patent Document 2 discloses a composition for promoting immune responses, containing a blocking agent at a weight percent concentration of 0.01% or more and polyethylene glycol with an average molecular weight of 2,000 to 26,000 at a weight percent concentration of 1 to 10%.

[0004] However, these methods often fail to increase detection sensitivity, and while sensitivity may increase, noise may also increase, limiting their effectiveness. Therefore, there is a great need for a technique that can reliably increase detection sensitivity in Western blotting.

[0005] Immunohistochemical staining using antibodies is also widely used in pathological examinations and pharmacological studies. This method uses antibodies that specifically bind to antigens in tissues and utilizes the reaction between an antibody-labeled enzyme and a substrate to stain specific sites, making the antigens visible under a microscope. Antigen retrieval methods to improve the detection sensitivity of hard-to-see proteins include enzyme treatment, heat treatment, and pH change treatment. Enzyme treatment uses enzymes such as pepsin and trypsin, but the enzymes may completely digest the antigen, resulting in loss of antigenicity. Heat treatments include microwave treatment and autoclave treatment. However, microwave treatment involves placing slides in a staining basket in a beaker containing buffer, which may result in protein detachment from the block. Autoclave treatment also involves treating slides immersed in buffer, but has the disadvantage of being longer than microwave treatment. pH change treatment involves incubation in citrate buffer at 37°C, but the disadvantage is that it does not sufficiently improve detection sensitivity.

[0006] International Publication No. 2016 / 017037 Japanese Patent Application Laid-Open No. 2006-126166

[0007] The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for improving the detection sensitivity of proteins in an assay method using an antigen-antibody reaction.

[0008] The method for improving detection sensitivity according to the present invention is a method for improving the detection sensitivity of a protein in an assay in which a protein supported on a matrix is ​​detected by an antigen-antibody reaction, and is characterized by comprising a boiling treatment step in which the matrix on which the protein is supported is boiled in a sodium sulfate solution, and a reaction step in which an antigen-antibody reaction is carried out using an antibody after the boiling treatment step.

[0009] According to the present invention, the detection sensitivity of proteins can be improved in assay methods using antigen-antibody reactions.

[0010]

[0023] Figure 1 is a diagram outlining the method for improving detection sensitivity of the present invention, which includes an acid treatment step. It is a photograph showing band intensities in Western blotting, comparing band intensities after boiling with sodium sulfate with those after untreated, boiled with water, and boiled with PBS. It is a diagram comparing band intensities in Western blotting relatively, comparing band intensities after boiling with sodium sulfate with those after untreated, boiled with water, and boiled with PBS. It is a photograph and graph showing band intensities when the sodium sulfate solution concentrations are 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, and 0.7M. It is a photograph and graph showing band intensities when the sodium sulfate solution concentrations are 0.01M, 0.05M, 0.1M, 0.2M, 0.5M, and 1.0M.

[0023] Figure 1 shows a photograph and graph comparing the effects of boiling in 0.4 M sodium sulfate solution on Myc-tagged proteins, using nitrocellulose membranes and PVDF membranes as transfer membranes for Western blotting. Figure 2 shows a photograph and graph comparing the effects of boiling in 0.4 M sodium sulfate solution on His-tagged proteins, using nitrocellulose membranes and PVDF membranes as transfer membranes for Western blotting. Figure 3 shows a photograph and graph comparing the effects of boiling in 0.4 M sodium sulfate solution on MAP2 and Tau proteins, using nitrocellulose membranes and PVDF membranes as transfer membranes for Western blotting. Figure 4 shows a photograph and graph comparing the band intensities remaining after overnight shaking in TBSt in the cases of untreated, PBS-boiled, and Na2SO4-boiled samples after transfer to nitrocellulose or PVDF membranes. Figure 5 shows a graph numerically illustrating the band intensities remaining after overnight shaking in TBSt in the cases of untreated, PBS-boiled, and Na2SO4-boiled samples after transfer to nitrocellulose or PVDF membranes. Fig. 1 is a photograph of Aβ senile plaques in an AD model mouse, comparing an untreated case with a case where Na2SO4 boiling was treated. Fig. 2 is a diagram showing the improvement in detection sensitivity when an acid treatment step is included. Fig. 3 is a diagram showing the improvement in detection sensitivity when a different antibody is used when an acid treatment step is included.1 shows the results of improving detection sensitivity by performing acid treatment even when boiling treatment is not performed. 2 shows the results of improving detection sensitivity when boiling treatment and acid treatment are performed on the cerebral cortex of a 3-month-old human APP knock-in mouse (APPNLGF mouse). 3 shows the results of improving detection sensitivity when boiling treatment and acid treatment are performed on the cerebral cortex of a 4-month-old human APP knock-in mouse (APPNLGF mouse).

[0011] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.

[0012] The method for improving detection sensitivity according to the present invention is a method for improving the detection sensitivity of proteins in an assay in which proteins supported on a matrix are detected by antigen-antibody reaction, and is characterized by comprising a boiling treatment step in which the matrix on which the proteins are supported is boiled in a sodium sulfate solution, and a reaction step in which an antigen-antibody reaction is carried out using antibodies after the boiling treatment step.

[0013] The present inventors have discovered that in a method for detecting proteins by antigen-antibody reaction, the protein detection sensitivity can be dramatically improved by boiling in a sodium sulfate solution, and have completed the present invention based on this discovery.

[0014] The present invention can be applied to, for example, improving the protein detection sensitivity in Western blotting. That is, the method for improving detection sensitivity in Western blotting involves boiling a nitrocellulose membrane carrying a protein in a sodium sulfate solution prior to a primary antibody reaction, thereby improving the protein detection sensitivity in an antigen-antibody reaction. After the boiling treatment, the nitrocellulose membrane is washed with distilled water or the like, and then the steps of normal Western blotting are carried out.

[0015] Proteins for which the detection sensitivity can be improved are dementia-causing proteins.

[0016] The dementia-causing protein is not particularly limited, but may be, for example, any of Aβ, Tau, MAP2, α-synuclein, PQBP1, huntingtin, or prion.

[0017] Aβ (amyloid beta) is a 36-43 amino acid peptide that is crucially involved in Alzheimer's disease as it is the main component of amyloid plaques found in the brains of patients with Alzheimer's disease. This peptide is derived from the amyloid precursor protein and is produced by cleavage by β-secretase and γ-secretase.

[0018] Tau (tau protein) is a protein that stabilizes microtubules and is abundant in neurons of the central nervous system. The neurofibrillary tangles seen in Alzheimer's disease are caused by hyperphosphorylation of tau.

[0019] MAP2 is a microtubule-associated protein abundant in vertebrate neurons. The C-terminus of MAP2 is highly homologous to tau, and it causes neurodegenerative diseases similar to those caused by tau.

[0020] Alpha-synuclein is a 14.5 kDa protein consisting of 140 amino acids encoded by the SNCA gene, and is abundant in presynaptic terminals in the central nervous system. Highly phosphorylated and abnormally aggregated alpha-synuclein is the major component of Lewy bodies found in neurons in Parkinson's disease and dementia with Lewy bodies, and of glial inclusions found in oligodendrocytes in multiple system atrophy.

[0021] PQBP1 (polyglutamine-binding protein 1) is involved in many synaptic functional proteins, and its reduction leads to abnormalities in synaptic function.

[0022] Huntingtin is a protein encoded by the HTT gene in humans. Mutations in the HTT gene cause Huntington's disease, and huntingtin plays an important role in nerve cells.

[0023] Prions are infectious agents composed of proteins that spread by transferring their structure to normal proteins. Unlike other infectious agents, prions do not contain nucleic acids such as DNA or RNA.

[0024] Furthermore, a protein that can improve detection sensitivity is a tag protein.

[0025] The tag protein is not particularly limited, but may be, for example, a His (registered trademark) tag, a myc tag, a FLAG (registered trademark) tag, a glutathione-S-transferase (GST) tag, a maltose-binding protein (MBP) tag, or a PA tag.

[0026] His (registered trademark) tag is a type of tag peptide consisting of approximately six consecutive histidine His residues.

[0027] The myc tag is an epitope tag derived from the c-myc gene, containing 10 amino acids and a molecular weight of 1.2 kDa. Using recombinant DNA technology, the Myc tag can be fused to the N- or C-terminus of a protein of interest.

[0028] A FLAG® tag is a polypeptide protein tag that can be added to proteins using recombinant DNA technology.

[0029] The glutathione-S-transferase (GST) tag is a protein with a molecular weight of 26 kDa, and utilizes a gene derived from Schistosoma japonicum.

[0030] The maltose-binding protein (MBP) tag is a protein that has binding affinity for maltose and maltodextrin. It has the property of improving the solubility of expressed proteins when fused to a target protein, and is used for the purification and detection of recombinant proteins.

[0031] The PA tag is a peptide tag consisting of 14 amino acids that utilizes the PLAG sequence of human podoplanin.

[0032] The concentration of the sodium sulfate solution used in the boiling treatment is not particularly limited as long as it is applicable to antigen activation, and can be, for example, 0.01 M to 1.0 M, preferably 0.1 M to 0.7 M, more preferably 0.3 M to 0.6 M, and optimally 0.5 M.

[0033] The boiling treatment with sodium sulfate is preferably carried out by immersing the material in a boiling sodium sulfate solution. The boiling treatment time is not particularly limited, but is, for example, 3 to 10 minutes, preferably 4 to 8 minutes, and more preferably 5 to 6 minutes.

[0034] The present invention can further improve the detection sensitivity of proteins by comprising a boiling step in which a matrix supporting the protein is boiled in a sodium sulfate solution, a reaction step in which an antigen-antibody reaction is carried out using an antibody after the boiling step, an acid treatment step in which an acidic buffer solution is applied after the reaction step, and a re-reaction step in which an antigen-antibody reaction is carried out again using the same antibody after the acid treatment step.

[0035] The acidic buffer solution is not particularly limited, but may have a pH of, for example, 1 to 6, preferably 2 to 5, and more preferably 2 to 4. The acidic buffer solution contains, for example, at least one anion species selected from the group consisting of acetate ions, citrate ions, glycine, succinate ions, phosphate ions, and formate ions, preferably glycine, and more preferably a glycine-hydrochloride buffer solution.

[0036] The detection sensitivity improvement method of the present invention, which includes an acid treatment step, is described below. Here, the case where the matrix is ​​a nitrocellulose membrane used in Western blotting is described. Figure 1 is a diagram outlining the detection sensitivity improvement method of the present invention, which includes an acid treatment step. As shown in Figure 1, for example, a membrane is attached to a gel after SDS-PAGE, and a voltage is applied to transfer the separated proteins from the gel to the membrane. The membrane to which the proteins are transferred is preferably a nitrocellulose membrane. Next, the membrane to which the proteins are transferred is boiled in a sodium sulfate solution (boiling step). Next, after the boiling step, an antigen-antibody reaction is carried out with a primary antibody and a secondary antibody (reaction step). The primary antibody is an antibody against the target protein, and the secondary antibody is an enzyme such as HRP (horseradish peroxidase). Next, after the reaction step, an acidic buffer solution is applied (acid treatment step). The acidic buffer solution is preferably a glycine hydrochloride buffer solution. Next, after the acid treatment step, a second antigen-antibody reaction is carried out with the same primary and secondary antibodies (re-reaction step). This further improves protein detection sensitivity.

[0037] Furthermore, unlike the above, the method for improving protein detection sensitivity of the present invention also encompasses methods that do not include a boiling treatment step. That is, the method for improving protein detection sensitivity of the present invention is a method for improving protein detection sensitivity in an assay in which a protein supported on a matrix is ​​detected by an antigen-antibody reaction, and is characterized by comprising a reaction step in which an antigen-antibody reaction is carried out using an antibody on the matrix on which the protein is supported, an acid treatment step in which an acidic buffer solution is applied after the reaction step, and a re-reaction step in which an antigen-antibody reaction is carried out again using the same antibody after the acid treatment step.

[0038] Furthermore, the present invention can be applied to, for example, improving the protein detection sensitivity in paraffin-embedded sections. That is, in a method for improving the detection sensitivity in paraffin-embedded sections, the paraffin-embedded sections on which proteins are supported are subjected to a boiling treatment in a sodium sulfate solution prior to an immune reaction, thereby improving the protein detection sensitivity by an immune reaction.

[0039] The protein that can improve the detection sensitivity, the concentration of the sodium sulfate solution used in the boiling treatment, and the boiling treatment time are the same as those described above.

[0040] In addition, the kit for improving the detection sensitivity of a protein according to the present invention is characterized by comprising a sodium sulfate solution and an instruction manual containing instructions for performing a boiling treatment in which a matrix on which a protein is supported is boiled in the sodium sulfate solution prior to an antigen-antibody reaction.

[0041] (1) Example 1: Effect of Boiling with Sodium Sulfate. Aβ42 samples were prepared by dissolving Aβ42 (Sigma-Aldrich) in DMSO (180 μl) and further diluting with PBS to a final concentration of 10 μM. Buffer was added to the Aβ42 samples, and the solubilized fraction was separated by SDS-polyacrylamide gel (17%) electrophoresis. After separation of 0.88 pg to 450 pg of synthetic Aβ42 on a 12% acrylamide gel, the resulting solution was transferred to a nitrocellulose membrane (Cleartrans Nitrocellulose Membrane, 0.2 μm, Fujifilm Wako Co., Ltd.). The nitrocellulose membrane was then boiled in 0.4 M sodium sulfate for 5 minutes. The membrane was then blocked with 5% skim milk for 30 minutes and reacted with anti-Aβ antibody (82E1, Immuno-Biological Laboratories Co., Ltd.) at room temperature for 2 hours. After washing the nitrocellulose membrane with TBSt, it was reacted with HRP-labeled anti-mouse IgG antibody as a secondary antibody for 1 hour, treated with a chemiluminescence reagent (Immunostar, Fujifilm Wako Co., Ltd.), and chemiluminescence was detected using a chemiluminescence detector (LAS4000, Fujifilm Wako Co., Ltd.).

[0042] As controls, 450 pg (100 fmol) of Aβ42 was simply transferred onto a nitrocellulose membrane (untreated), or transferred and then boiled in ion-exchanged water (water), or transferred and then boiled in PBS (PBS). These were then blocked with 5% skim milk for 30 minutes, reacted with anti-Aβ antibodies and HRP-labeled anti-mouse IgG antibodies, and then subjected to chemiluminescence treatment in the same way as the nitrocellulose membrane boiled in 0.4 M sodium sulfate. Images were then taken simultaneously with the nitrocellulose membrane boiled in 0.4 M sodium sulfate.

[0043] Figure 2 is a photograph showing the band intensity of Western blotting, comparing the band intensity after boiling with sodium sulfate with that after untreated, boiled in water, and boiled in PBS. As shown in Figure 2, the band intensity after boiling with sodium sulfate was significantly improved in sensitivity compared to untreated, boiled in water, and boiled in PBS.

[0044] Figure 3 shows a comparative comparison of band intensities in Western blotting, comparing the band intensities of untreated, boiled in water, and boiled in PBS. As shown in Figure 3, the band intensity of 450 pg of Aβ on untreated nitrocellulose membrane was approximately 150-fold higher than that of Aβ on a nitrocellulose membrane boiled in 0.4 M sodium sulfate.

[0045] The results of Example 1 demonstrate that the detection sensitivity of proteins can be significantly improved by boiling the transfer membrane in a sodium sulfate solution after electrophoresis in the Western blotting method.

[0046] (2) Example 2: Investigation of the optimal concentration of sodium sulfate. Synthetic Aβ42 (400 pg) was separated on a 12% acrylamide gel, transferred to a nitrocellulose membrane (Cleartrans nitrocellulose membrane, 0.2 μm, Fujifilm Wako Co., Ltd.), and boiled for 5 minutes in sodium sulfate at concentrations of 0.01 to 0.7 M. The membrane was then blocked with 5% skim milk for 30 minutes and reacted with anti-Aβ antibody (82E1, Immuno-Biological Laboratories Co., Ltd.) at room temperature for 2 hours. The nitrocellulose membrane was washed with TBSt and then reacted with HRP-labeled anti-mouse IgG antibody as a secondary antibody for 1 hour. The membrane was then treated with a chemiluminescence reagent (Immunostar, Fujifilm Wako Co., Ltd.), and chemiluminescence was detected using a chemiluminescence detector (LAS4000, Fujifilm Wako Co., Ltd.).

[0047] Figure 4 is a photograph and graph showing the band intensity when the sodium sulfate solution concentrations were 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, and 0.7 M. As shown in Figure 4, boiling in sodium sulfate solution improved the detection sensitivity in all cases, but excellent detection sensitivity was observed when boiling in 0.3 M to 0.6 M sodium sulfate, with the maximum sensitivity enhancement being observed when boiling in 0.5 M sodium sulfate.

[0048] The results of Example 2 revealed that the detection sensitivity could be improved favorably when the concentration of the sodium sulfate solution used for boiling treatment was between 0.1 M and 0.7 M, with 0.3 M to 0.6 M being more preferable, and 0.5 M being optimal.

[0049] Synthetic Aβ42 (400 pg) was then separated on a 12% acrylamide gel and transferred to a nitrocellulose membrane (Cleartrans Nitrocellulose Membrane 0.45 μm, Fujifilm Wako Co., Ltd.). The membrane was then boiled in 0.01–1.0 M sodium sulfate for 5 min. After blocking with 5% skim milk for 30 min, the membrane was incubated with anti-Aβ antibody (82E1, Immuno-Biological Laboratories Co., Ltd.) for 2 h at room temperature. After washing with TBSt, the membrane was incubated with HRP-conjugated anti-mouse IgG as a secondary antibody for 1 h. The membrane was then treated with a chemiluminescent reagent (Immunostar, Fujifilm Wako Co., Ltd.) and detected using a chemiluminescence detector (LAS4000, Fujifilm Wako Co., Ltd.).

[0050] Figure 5 is a photograph and graph showing the band intensity when the sodium sulfate solution concentrations were 0.01 M, 0.05 M, 0.1 M, 0.2 M, 0.5 M, and 1.0 M. As shown in Figure 5, boiling in sodium sulfate solution significantly improved the detection sensitivity, with boiling in 0.5 M sodium sulfate showing the greatest increase in sensitivity.

[0051] (3) Example 3 Verification of the Versatility of Boiling Treatment with Sodium Sulfate When a Myc-Tagged Protein is Used Cell lysates expressing a Myc-tagged Notch fragment (75 kDa) were subjected to SDS-polyacrylamide gel (17%) electrophoresis and transferred to a nitrocellulose membrane (Cleartrans Nitrocellulose Membrane, 0.2 μm, Fujifilm Wako Co., Ltd.) or a PVDF membrane (AS ONE 64-3966-43, 2322505 / WSE-4060, 0.2 μm). The membrane was then subjected to the respective treatments in the same manner as in Example 1, and the protein was detected.

[0052] Figure 6 is a photograph and graph comparing the effect of boiling in 0.4 M sodium sulfate on Myc-tagged proteins using nitrocellulose membranes and PVDF membranes as transfer membranes for Western blotting. As shown in Figure 6, when a PVDF membrane was used as the transfer membrane, boiling in sodium sulfate did not significantly improve detection sensitivity. However, when a nitrocellulose membrane was used as the transfer membrane, boiling in sodium sulfate significantly improved detection sensitivity.

[0053] The results of Example 3 revealed that the protein that can improve the detection sensitivity in Western blotting is not only Aβ but also Myc tag.

[0054] Furthermore, the results of Example 3 revealed that the detection sensitivity can be improved when a nitrocellulose membrane is used as the transfer membrane after electrophoresis in the Western blotting method.

[0055] (4) Example 4 Verification of the Versatility of Boiling Treatment with Sodium Sulfate When a His®-Tagged Protein is Used Cell lysates expressing His®-tagged APP fragments (12 kDa) were subjected to SDS-polyacrylamide gel (17%) electrophoresis and transferred to a nitrocellulose membrane (Cleartrans Nitrocellulose Membrane, 0.2 μm, Fujifilm Wako Co., Ltd.) or a PVDF membrane (AS ONE 64-3966-43, 2322505 / WSE-4060, 0.2 μm). The membrane was then subjected to the respective treatments in the same manner as in Example 1, and the protein was detected.

[0056] Figure 7 is a photograph and graph comparing the effect of boiling in 0.4 M sodium sulfate solution on His®-tagged proteins using a nitrocellulose membrane and a PVDF membrane as transfer membranes for Western blotting. As shown in Figure 7, when a PVDF membrane was used as the transfer membrane, boiling in sodium sulfate solution did not significantly improve detection sensitivity. However, when a nitrocellulose membrane was used as the transfer membrane, boiling in sodium sulfate solution significantly improved detection sensitivity.

[0057] The results of Example 4 demonstrated that the detection sensitivity of Western blotting can be improved not only for Aβ but also for His (registered trademark) tags, demonstrating that even peptide tags can improve detection sensitivity.

[0058] Furthermore, the results of Example 4 also revealed that the detection sensitivity can be improved when a nitrocellulose membrane is used as the transfer membrane after electrophoresis in the Western blotting method.

[0059] (5) Example 5 Verification of the versatility of boiling treatment with sodium sulfate when MAP2 and tau proteins were used. Brain lysates from dementia model mice were developed by SDS-polyacrylamide gel (10%) electrophoresis, transferred to a nitrocellulose membrane (Cleartrans nitrocellulose membrane 0.2 μm, Fujifilm Wako Co., Ltd.) or a PVDF membrane (AS ONE 64-3966-43, 2322505 / WSE-4060, 0.2 μm), boiled in 0.4 M NaSO solution, and detected with antibodies specific for MAP2 and tau, respectively.

[0060] Figure 8 is a photograph and graph comparing the effect of boiling in 0.4 M sodium sulfate solution on MAP2 and tau proteins, using nitrocellulose membranes and PVDF membranes as transfer membranes for Western blotting. As shown in Figure 8, when a PVDF membrane was used as the transfer membrane, boiling in sodium sulfate solution did not significantly improve detection sensitivity. However, when a nitrocellulose membrane was used as the transfer membrane, boiling in sodium sulfate solution significantly improved detection sensitivity for both MAP2 and tau.

[0061] The results of Example 5 demonstrated that the detection sensitivity of proteins capable of improving in Western blotting is not limited to Aβ, but can also be improved for MAP2 and tau. Neurofibrillary tangles observed in Alzheimer's disease are caused by the hyperphosphorylation of tau protein, and tau is a dementia-causing protein. MAP2 is a microtubule-associated protein abundant in vertebrate neurons. The C-terminus of MAP2 is highly homologous to tau, causing neurodegenerative diseases similar to those of tau, making MAP2 a dementia-causing protein. Thus, it was demonstrated that boiling in sodium sulfate solution can improve the detection sensitivity of dementia-causing proteins such as Aβ, tau, and MAP2.

[0062] Furthermore, the results of Example 5 also revealed that the detection sensitivity can be improved when a nitrocellulose membrane is used as the transfer membrane after electrophoresis in the Western blotting method.

[0063] (6) Example 6 Investigation of the mechanism of sensitization by boiling treatment with sodium sulfate A size marker (Bio-Rad) was developed by SDS-polyacrylamide gel (12%) electrophoresis (Tris-lysine buffer) and transferred to a nitrocellulose membrane (Cleartrans nitrocellulose membrane 0.2 μm, Fujifilm Wako Co., Ltd.). After that, the band intensity remaining on the nitrocellulose membrane was measured for the following treatments: simply transferred to the nitrocellulose membrane (untreated), boiled in PBS after transfer (PBS boiling), or boiled in 0.4 M NaSO solution (NaSO boiling). After each treatment, the membrane was shaken overnight in TBSt at room temperature. As a comparative example, a size marker (Bio-Rad) was electrophoresed on a 12% SDS-polyacrylamide gel (Tris-lysine buffer) and transferred to a PVDF membrane (AS ONE 64-3966-43, 2322505 / WSE-4060, 0.2 μm). The membrane was then boiled in PBS after transfer (PBS-boiling), or in 0.4 M NaSO solution (NaSO-boiling). After each treatment, the membrane was shaken overnight in TBSt at room temperature, and the band intensity remaining on the PVDF membrane was measured.

[0064] Figure 9 is a photograph of the band intensity remaining after overnight shaking in TBSt after transfer to a nitrocellulose or PVDF membrane, in the untreated, PBS-boiled, or Na2SO4-boiled cases. As shown in Figure 9, when a nitrocellulose membrane was used as the transfer membrane, almost no band intensity remained in the untreated case. However, the band intensity remained at the same level in both the PBS-boiled and Na2SO4-boiled cases. On the other hand, when a PVDF membrane was used as the transfer membrane, the band intensity remained at the same level in both the PBS-boiled and Na2SO4-boiled cases.

[0065] Figure 10 is a graph showing the numerical values ​​of band intensity remaining after overnight shaking in TBSt after transfer to a nitrocellulose or PVDF membrane, in the untreated, PBS-boiled, or Na2SO4-boiled cases. As shown in Figure 10, when a nitrocellulose membrane was used as the transfer membrane, almost no band intensity remained in the untreated case. However, the band intensity remained at the same level in both the PBS-boiled and Na2SO4-boiled cases. On the other hand, when a PVDF membrane was used as the transfer membrane, the band intensity remained at the same level in both the PBS-boiled and Na2SO4-boiled cases.

[0066] The results of Example 6 show that nitrocellulose membranes are superior to PVDF membranes in promoting protein fixation to the matrix. Furthermore, the improvement in protein detection sensitivity by boiling in sodium sulfate solution is thought to be due to antigen retrieval rather than promotion of protein fixation to the matrix.

[0067] (7) Example 7 Application to Immunohistochemical Staining A 3-month-old AD model mouse (APP KI NLGF) was euthanized, and the cerebral mass was excised and fixed overnight at 4°C in 10% neutral buffered formalin. The sections were dehydrated with ethanol, cleared with xylene, and then paraffin-substituted and embedded. The paraffin blocks were sliced ​​to 5 μm using a sliding microtome to prepare paraffin-embedded tissue sections. After deparaffinization, the sections were boiled in 0.4 M sodium sulfate solution for 5 minutes. Senile plaques were detected by reaction with an anti-Aβ antibody (82E1, Immuno-Biological Laboratories, Inc.).

[0068] Figure 11 shows photographs of Aβ senile plaques in AD model mice, comparing the untreated and the Na2SO4 boiled treatment. As shown in Figure 11, the detection sensitivity of senile plaques was significantly improved in the Na2SO4 boiled treatment compared to the untreated case.

[0069] The results of Example 7 demonstrated that the improvement in protein detection sensitivity achieved by boiling in a sodium sulfate solution can also be applied to immunohistochemical staining. In other words, the improvement in protein detection sensitivity achieved by boiling in a sodium sulfate solution is thought to be due to antigen activation, suggesting that the present invention is not limited to Western blotting and immunohistochemical staining, but can be applied to improving the sensitivity of any detection method that utilizes an antigen-antibody reaction, such as ELISA.

[0070] (8) Example 8: Further Improvement in Detection Sensitivity with an Acid Treatment Step (Part 1) In Example 8, the detection sensitivity was improved by further adding an acid treatment step. Specifically, synthetic Aβ42 (400 pg) was separated on a 12% acrylamide gel, transferred to a nitrocellulose membrane (Cleartrans nitrocellulose membrane, 0.2 μm, Fujifilm Wako Co., Ltd.), and boiled in 0.4 M sodium sulfate for 5 minutes. The membrane was then blocked with 5% skim milk for 30 minutes. After detection with a mouse monoclonal antibody (6E10, 1 / 1000 dilution) recognizing the amino-terminal end of amyloid beta as the primary antibody and a secondary antibody (HRP-labeled anti-mouse IgG antibody), the nitrocellulose membrane was immersed for 1 hour in 0.2 M glycine (pH 2.0), which is used to elute antigen proteins from the antibody. The membrane was then thoroughly washed with TBSt or similar and again reacted with the same primary antibody (6E10, 1 / 1000 dilution) and secondary antibody (HRP-conjugated anti-mouse IgG antibody). Chemiluminescent substrate reagent (Immunostar, Fujifilm Wako Co., Ltd.) was added, and chemiluminescence was detected using a chemiluminescence detector (LAS4000, Fujifilm Wako Co., Ltd.). Figure 12 shows the improved detection sensitivity achieved by including an acid treatment step. As shown in Figure 12, under the same imaging conditions, the band intensity increased approximately 20-fold compared to the band intensity before 0.2 M glycine treatment.

[0071] (9) Example 9: Further Improvement in Detection Sensitivity with Acid Treatment Step (Part 2) In Example 9, the improvement in detection sensitivity with an acid treatment step was demonstrated using a different antibody. Specifically, synthetic Aβ42 (400 pg) was separated on a 12% acrylamide gel, transferred to a nitrocellulose membrane (Cleartrans nitrocellulose membrane, 0.2 μm, Fujifilm Wako Co., Ltd.), and boiled in 0.4 M sodium sulfate for 5 minutes. The membrane was then blocked with 5% skim milk for 30 minutes. After detection with a mouse monoclonal antibody (82E1, 1 / 100 dilution) recognizing the amino-terminal end of amyloid beta as the primary antibody and a secondary antibody (HRP-labeled anti-mouse IgG antibody), the nitrocellulose membrane was immersed in 0.2 M glycine (pH 2.0), the same solution used to elute antigen proteins from the antibody, for 1 hour. The membrane was then thoroughly washed with TBSt or similar and again reacted with the same primary antibody (82E1, 1 / 100 dilution) and secondary antibody (HRP-conjugated anti-mouse IgG antibody). Chemiluminescent substrate reagent (Immunostar, Fujifilm Wako Co., Ltd.) was added, and chemiluminescence was detected using a chemiluminescence detector (LAS4000, Fujifilm Wako Co., Ltd.). Figure 13 shows the improved detection sensitivity achieved using a different antibody in the acid treatment step. As shown in Figure 13, under the same imaging conditions, the band intensity increased approximately 2.5-fold compared to the band intensity before 0.2 M glycine treatment.

[0072] (10) Example 10: Improvement in detection sensitivity without boiling treatment. In Example 10, we demonstrated that acid treatment improved detection sensitivity even without boiling treatment. Specifically, synthetic Aβ42 (400 pg) was separated on a 12% acrylamide gel and transferred to a nitrocellulose membrane (Cleartrans nitrocellulose membrane, 0.2 μm, Fujifilm Wako Co., Ltd.). Subsequently, a mouse monoclonal antibody (82E1, diluted 1 / 100) recognizing the amino terminal end of amyloid β was used as the primary antibody, and detection was performed with a secondary antibody (HRP-labeled anti-mouse IgG antibody). The nitrocellulose membrane was then immersed for 1 hour in 0.2 M glycine (pH 2.0), which is used to elute antigen proteins from the antibody. The membrane was then thoroughly washed with TBSt or similar and again reacted with the same primary antibody (82E1, 1 / 100 dilution) and secondary antibody (HRP-conjugated anti-mouse IgG antibody). Chemiluminescent substrate reagent (Immunostar, Fujifilm Wako Co., Ltd.) was added, and chemiluminescence was detected using a chemiluminescence detector (LAS4000, Fujifilm Wako Co., Ltd.). Figure 14 shows the improved detection sensitivity achieved by acid treatment, even without boiling. As shown in Figure 14, under the same imaging conditions, the band intensity increased approximately threefold compared to the band intensity before 0.2 M glycine treatment.

[0073] (11) Example 11 Application to Immunohistochemical Staining In Example 11, we demonstrated the improved detection sensitivity of area histochemical staining when acid treatment was performed. Specifically, the cerebral cortex of a 3-month-old human knock-in mouse (APPNLGF mouse) was excised and fixed overnight at 4°C in 10% neutral buffered formalin. The sections were dehydrated with ethanol, cleared with xylene, and paraffin-substituted and then embedded. The paraffin blocks were sliced ​​to 6 μm using a sliding microtome to prepare paraffin-embedded tissue sections. After deparaffinization, anti-Aβ antibody (82E1, Immuno-Biological Laboratories, Inc.) was used to detect senile plaques (untreated).

[0074] Next, paraffin-embedded tissue sections were prepared as described above, deparaffinized, and then boiled in 0.4 M sodium sulfate solution in an autoclave at 110°C for 10 minutes. Senile plaques were detected by reacting with anti-Aβ antibody (82E1, Immuno-Biological Laboratories, Inc.) (boiling treatment).

[0075] Next, paraffin-embedded tissue sections were prepared as described above and immersed in 0.2 M glycine solution (pH 2.0) at room temperature for 1 hour. Senile plaques were detected by reaction with anti-Aβ antibody (82E1, Immuno-Biological Laboratories, Inc.) (acid treatment).

[0076] Next, paraffin-embedded tissue sections were prepared as described above, deparaffinized, and then boiled in 0.4 M sodium sulfate solution for 10 minutes in an autoclave at 110°C. The boiled sections were then immersed in 0.2 M glycine solution (pH 2.0) at room temperature for 1 hour. Senile plaques were detected by reacting with an anti-Aβ antibody (82E1, Immuno-Biological Laboratories, Inc.) (boiling + acid treatment).

[0077] Figure 15 shows the improvement in detection sensitivity of the cerebral cortex of 3-month-old human APP knock-in mice (APPNLGF mice) after boiling and acid treatment. As shown in Figure 15, in 3-month-old sections, only a small amount of intracerebral Aβ aggregation was detected without treatment, but Aβ aggregation was significantly detected after sodium sulfate boiling. Aβ aggregation was also detected with acid treatment compared to untreated sections. When sodium sulfate boiling and low pH treatment were used in combination, Aβ aggregation was detected more intensely.

[0078] Next, we investigated the improvement in detection sensitivity of the cerebral cortex of 4-month-old human APP knock-in mice (APPNLGF mice) by the same method as described above: untreated, boiled, acid-treated, and boiled + acid-treated. Figure 16 shows the improvement in detection sensitivity of the cerebral cortex of 4-month-old human APP knock-in mice (APPNLGF mice) by boiling and acid treatment. As shown in Figure 16, in 4-month-old sections, only a small amount of Aβ aggregation was detected in the untreated brain, but Aβ aggregation was significantly detected after sodium sulfate boiling. Acid treatment also detected Aβ aggregation compared to the untreated brain. The combined use of sodium sulfate boiling and low pH treatment resulted in a stronger detection of Aβ aggregation.

[0079] It can be used for protein detection tests such as Western blotting, immunohistochemical staining, and ELISA.

Claims

1. A method for improving the detection sensitivity of a protein in an assay in which a protein supported on a matrix is ​​detected by an antigen-antibody reaction, the method comprising: a boiling step in which the matrix on which the protein is supported is boiled in a sodium sulfate solution; and a reaction step in which an antigen-antibody reaction is carried out using an antibody after the boiling step.

2. The method for improving detection sensitivity according to claim 1, characterized in that it comprises: an acid treatment step in which an acidic buffer solution is applied after the reaction step; and a re-reaction step in which an antigen-antibody reaction is carried out again with the antibody after the acid treatment step.

3. The method for improving detection sensitivity according to claim 2, wherein the acidic buffer solution is a glycine hydrochloride buffer solution.

4. The method for improving detection sensitivity according to claim 1, characterized in that the matrix is ​​a nitrocellulose membrane used in Western blotting, and the antigen-antibody reaction is a primary antibody reaction by a primary antibody and a secondary antibody reaction by a secondary antibody for detecting proteins transferred to the nitrocellulose membrane after electrophoresis in Western blotting.

5. The method for improving detection sensitivity according to claim 4, wherein the protein is a dementia-causing protein.

6. The method for improving detection sensitivity according to claim 5, characterized in that the dementia-causing protein is any of Aβ, Tau, MAP2, α-synuclein, PQBP1, huntingtin, and prion.

7. The method for improving detection sensitivity according to claim 4, wherein the protein is a tag protein.

8. The method for improving detection sensitivity according to claim 7, wherein the tag protein is any one of a His tag, a myc tag, a FLAG tag, a glutathione-S-transferase (GST) tag, a maltose-binding protein (MBP) tag, and a PA tag.

9. The method for improving detection sensitivity according to claim 1, wherein the concentration of the sodium sulfate solution is 0.3M to 0.6M.

10. The method for improving detection sensitivity described in claim 1, characterized in that the matrix is ​​a paraffin section used in immunohistochemical staining, and the antigen-antibody reaction is an immune reaction for detecting the protein.

11. The method for improving detection sensitivity according to claim 10, wherein the protein is a dementia-causing protein.

12. The method for improving detection sensitivity described in claim 11, characterized in that the dementia-causing protein is any of Aβ, Tau, MAP2, α-synuclein, PQBP1, huntingtin, and prion.

13. The method for improving detection sensitivity according to claim 10, wherein the protein is a tag protein.

14. The method for improving detection sensitivity according to claim 13, wherein the tag protein is any one of a His tag, a myc tag, a FLAG tag, a glutathione-S-transferase (GST) tag, a maltose-binding protein (MBP) tag, and a PA tag.

15. The method for improving detection sensitivity according to claim 10, wherein the concentration of the sodium sulfate solution is 0.3M to 0.6M.

16. A kit for improving the detection sensitivity of a protein in an assay in which a protein supported on a matrix is ​​detected by an antigen-antibody reaction, the kit comprising: a sodium sulfate solution; and instructions for performing a boiling treatment in which the matrix on which the protein is supported is boiled in the sodium sulfate solution prior to the antigen-antibody reaction.

17. The kit described in claim 16, characterized in that the matrix is ​​a nitrocellulose membrane used in Western blotting, and the antigen-antibody reaction is a primary antibody reaction by a primary antibody and a secondary antibody reaction by a secondary antibody for detecting proteins transferred to the nitrocellulose membrane after electrophoresis in Western blotting.

18. The kit according to claim 16, wherein the matrix is ​​a paraffin section used in immunohistochemical staining, and the antigen-antibody reaction is an immune reaction for detecting the protein.

19. A method for improving the detection sensitivity of a protein in an assay in which a protein supported on a matrix is ​​detected by an antigen-antibody reaction, the method comprising: a reaction step in which an antigen-antibody reaction is carried out using an antibody on the matrix on which the protein is supported; an acid treatment step in which an acidic buffer solution is applied after the reaction step; and a re-reaction step in which an antigen-antibody reaction is carried out again using the antibody after the acid treatment step.

20. The method for improving detection sensitivity according to claim 19, wherein the acidic buffer solution is a glycine hydrochloride buffer solution.

Images