WO2019095403A1 - Aβ KIT, DETECTION METHOD AND USE - Google Patents

Aβ KIT, DETECTION METHOD AND USE Download PDF

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WO2019095403A1
WO2019095403A1 PCT/CN2017/112086 CN2017112086W WO2019095403A1 WO 2019095403 A1 WO2019095403 A1 WO 2019095403A1 CN 2017112086 W CN2017112086 W CN 2017112086W WO 2019095403 A1 WO2019095403 A1 WO 2019095403A1
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antibody
kit
sample
detection
tested
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PCT/CN2017/112086
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French (fr)
Chinese (zh)
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于顺
杨巍巍
李昕
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首都医科大学宣武医院
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the field of immunoassay, in particular to an A ⁇ detection kit, a detection method and an application thereof.
  • the ⁇ -amyloid peptide or protein (amyloid ⁇ -peptide, also known as amyloid ⁇ -protein A ⁇ or Abeta) has a molecular weight of about 4 kDa and is produced by the hydrolysis of ⁇ -amyloid precursor protein (APP).
  • APP ⁇ -amyloid precursor protein
  • the 43 amino acid polypeptide, its aberrant aggregate is the main component of the senile plaque, the main pathological structure in the brain of AD patients [1].
  • a ⁇ is a degradation product of amyloid precursor protein (APP) under pathological conditions. Under physiological conditions, APP is degraded by the secretory pathway (or non-amyloidogenic pathway).
  • APP first degraded into a soluble N-terminal fragment (sAPP ⁇ ) and a C-terminal fragment (C83) under the action of ⁇ -secretase, which was further decomposed by ⁇ -secretase. It is a smaller C-terminal fragment of 3 kDa.
  • APP degrades in the amyloidogenic pathway. APP is first degraded by ⁇ -secretase, releasing a smaller N-terminal fragment (sAPP ⁇ ) and a longer C-terminal fragment (C99) containing the entire amino acid sequence of A ⁇ , which is further in ⁇ - Producing a pathogenic A ⁇ peptide by the action of a secretase.
  • a ⁇ of different molecular sizes is present as a monomer when released, gradually aggregates into oligomers, fibrils and fibers, and deposits to form amyloid plaques (or senile plaques).
  • a ⁇ 42 is the easiest to aggregate And the most neurotoxic A ⁇ molecule.
  • a ⁇ oligomers exert neurotoxic effects through a variety of mechanisms, including activation of inflammatory responses, mitochondrial dysfunction, enhanced oxidative stress, impaired intracellular signaling pathways and synaptic plasticity, increased tau phosphorylation and GSK-3 ⁇ activity, Causes calcium metabolism disorders and induces apoptosis or death [2].
  • the above mechanism causes a positive feedback response: the production of A[beta] peptides results in a series of events that are detrimental to neurons, which in turn cause abnormalities in APP metabolism and more release of A[beta] peptides.
  • a ⁇ has been considered as the most suitable candidate for AD biomarkers.
  • the test focuses on A ⁇ in the cerebrospinal fluid and blood.
  • the levels of A ⁇ 42 (or A ⁇ 1-42) in AD patients decreased, the levels of t-tau and p-tau increased, and the changes in A ⁇ 42, t-tau (total tau) and p-tau (phosphorylated tau) levels Consistent.
  • t-tau, p-tau, and the ratio of A ⁇ 40 (A ⁇ 1-40) to A ⁇ 42 can effectively predict the deposition of A ⁇ in the brain.
  • cerebrospinal fluid A ⁇ as a diagnostic marker for AD has been validated in large populations and confirmed by neuropathological examination [3].
  • the detection of A ⁇ in cerebrospinal fluid has difficulty in sampling, complicated collection process, and large damage. Most patients are unwilling to accept it, especially for patients with early or possible AD, thus delaying treatment and missing the best treatment for AD. period.
  • SIMOA Single Molecular Array
  • IMR detects the magnetic susceptibility through a superconducting quantum interference device.
  • the SIMOA technique detects the presence of antigen molecules by fluorescence imaging of a single enzyme-labeled immune complex that reacts with a fluorescent substrate (Resorufin ⁇ -D-galactopyranoside).
  • IMR detected A ⁇ in plasma as follows: A ⁇ 42 level was increased, A ⁇ 40 level was decreased or unchanged [4,9-12]; and A ⁇ results in plasma detected by SIMOA technique were: A ⁇ 42 and A ⁇ 40 levels were decreased [ 4,13].
  • the present invention provides a novel kit for detecting A ⁇ in blood.
  • the detection of the kit can avoid the contamination of the sample by hemolysis, making the result more accurate and reliable.
  • the invention provides a kit for detecting A ⁇ in a sample to be tested, which comprises an anti-Hb antibody and an anti-A ⁇ antibody.
  • an anti-Hb antibody can be used as a capture antibody for capturing Hb in a sample to be tested; an anti-A ⁇ antibody can be used as The antibody is detected for detecting A ⁇ bound to Hb.
  • the kit for detecting A ⁇ in a sample to be tested of the present invention optionally further comprises an Hb-A ⁇ complex standard protein.
  • the Hb-A ⁇ complex standard protein can be combined with anti-Hb and A ⁇ antibodies to make a standard curve.
  • the anti-Hb antibody has a concentration of 0.5 to 4 ⁇ g/mL, preferably 1 to 2 ⁇ g/mL, more preferably 2 ⁇ g/mL.
  • the concentration of the anti-A ⁇ antibody is 0.1 to 2 ⁇ g/mL, preferably 1 ⁇ g/mL.
  • the anti-A ⁇ antibody is an enzyme-labeled anti-A ⁇ antibody.
  • the enzyme is Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP) or Glucose Oxidase (GO).
  • the anti-A ⁇ antibody is a fluorescent or chemiluminescent material or a colloidal gold-labeled anti-A ⁇ antibody, such as FITC or green fluorescent protein.
  • the anti-A ⁇ antibody is a biotinylated anti-A ⁇ antibody
  • the kit further comprises an avidin-labeled enzyme, such as an alkaline phosphatase. .
  • the anti-A ⁇ antibody is an anti-A ⁇ antibody not labeled with an enzyme
  • the kit further comprises an enzyme-labeled secondary antibody, or fluorescein, a chemiluminescent material, biotin, A secondary antibody labeled with other substances such as colloidal gold, which can specifically bind to an anti-A ⁇ antibody.
  • the sample to be tested in the present invention is derived from human, animal and/or cultured cells.
  • the sample to be tested is derived from a human, more preferably from an Alzheimer's (AD) patient or a population at risk of developing AD.
  • the sample is a blood sample, or a erythrocyte cytoplasmic protein from the blood, or any other tissue containing Hb.
  • the kit may further include a solid phase carrier, a buffer solution, a blocking solution, and the like.
  • the solid phase carrier is an ELISA plate, a microplate, a test tube or a microporous membrane.
  • the solid phase carrier is an ELISA plate; the material of the solid phase carrier is, for example, polystyrene or nitrocellulose. Plain, nylon, etc.
  • the buffer solution is selected from the group consisting of Tris-HCl buffer, PBS buffer, CBS buffer or buffered physiological saline;
  • the blocking solution is selected from 2% to 8% of newborn calf serum or gelatin-containing PBST.
  • the invention also provides the use of an anti-Hb antibody for the preparation of a kit or reagent for detecting A ⁇ content in a sample to be tested.
  • the invention provides the use of an anti-Hb antibody and an anti-A ⁇ antibody in the preparation of a kit or reagent for detecting A ⁇ content in a sample to be tested.
  • the invention provides the use of an anti-Hb antibody for the preparation of a kit or reagent for detecting whether a sample to be tested has AD or is at risk of AD.
  • the invention provides the use of an anti-Hb antibody and an anti-A ⁇ antibody in the manufacture of a kit or reagent for detecting whether a test sample has AD or is at risk of AD.
  • the present invention also provides a method for preparing an A ⁇ kit, comprising:
  • step 1) comprises formulating an anti-Hb antibody coating solution, preparing a blocking solution, and coating the ELISA plate.
  • Formulating an anti-Hb antibody coating solution diluting the anti-Hb antibody with a buffer solution;
  • Formulating a blocking solution adding 2% to 8% of newborn calf serum to a buffer solution, or dissolving gelatin in PBST;
  • Coated enzyme plate The prepared anti-Hb antibody coating solution is added to the well of the enzyme labeling plate, incubated overnight, and rinsed; the prepared blocking solution is added into the well of the enzyme labeling plate, incubated at constant temperature for 2-4 hours, and rinsed.
  • the buffer solution is preferably: Tris-HCl buffer, PBS buffer, CBS buffer or buffered physiological saline;
  • step 3 specifically includes the following steps:
  • the above step 2) is carried out by diluting the anti-A ⁇ antibody with a blocking solution.
  • the present invention also provides a method for detecting A ⁇ , which comprises the following steps:
  • the method for detecting A ⁇ mentioned above specifically includes:
  • biotinylated anti-A ⁇ antibody was added to the solid phase carrier, incubated, washed; avidin-labeled alkaline phosphatase was added, incubated, washed; and the p-nitrophenyl phosphate coloring solution was added for color development.
  • the detection of A[beta] in the present invention comprises qualitative and quantitative detection of A[beta].
  • the invention also provides the use of an A ⁇ kit for detecting A ⁇ .
  • the antibody of the present invention is a monoclonal antibody, for example, an antibody capable of specifically binding to Hb and an antibody capable of specifically binding to A ⁇ .
  • the antibody may be an intact antibody, a bispecific antibody, an antibody fragment, a genetically engineered antibody, or the like, and may be, for example, (Fab') 2 , Fab', Fab, scFv, diabody, minibody, murine antibody, and human Sourced antibodies and the like.
  • the enzyme coupled to the secondary antibody may be selected from Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP) or Glucose Oxidase (Glucose Oxidase).
  • HRP Horseradish Peroxidase
  • AP Alkaline Phosphatase
  • Glucose Oxidase Glucose Oxidase
  • the kit of the invention further contains instructions for use.
  • the A ⁇ kit provided by the present invention and the detection method thereof and related applications are an anti-Hb antibody as a capture antibody, which is used for capturing Hb, and an anti-A ⁇ antibody as a detection antibody to detect A ⁇ combined with the captured Hb.
  • the present invention has at least one of the following advantages:
  • the A ⁇ detection kit provided by the present invention is suitable for quantitative detection of all A ⁇ content bound to Hb, including quantitative detection of A ⁇ content bound to Hb in peripheral blood red blood cells;
  • FIG. 1 is a schematic view showing a process of separating erythrocyte cytoplasmic proteins in an embodiment of the present invention
  • Example 2 is a detection result of an A ⁇ kit in Example 5 of the present invention.
  • Fig. 3 is a graph showing the ROC curve of the detection result of the A ⁇ kit in Example 5 of the present invention.
  • the ⁇ -amyloid peptide or protein, amyloid, amyloid ⁇ -peptide, ⁇ -protein, Amyloid beta, A ⁇ , and Abeta are all the same substance, and the English name, short form, and the like are not case sensitive.
  • the preparation method of some reagents in the present invention is as follows, and other reagents are conventional preparation methods.
  • the concentration of PBS is 0.01 mol/L, and the preparation method is as follows:
  • the concentration of NaHCO 3 buffer was 200 mmol/L, pH 9.6, and the preparation method was as follows:
  • the concentration of the blocking solution is 2.5%, and the preparation method is:
  • PBST 0.01 mol/L PBS containing 0.05% Tween-20, and the preparation method is as follows:
  • the anti-Hb antibody is diluted to 0.2 ⁇ g/mL to 10 ⁇ g/mL with a buffer solution; the buffer solution is selected from the group consisting of Tris-HCl buffer, PBS buffer, CBS buffer or buffered saline. This example employed a NaHCO 3 buffer.
  • the anti-Hb antibody in the present embodiment is not limited, and may be any antibody capable of specifically recognizing hemoglobin.
  • the blocking solution is selected from 2% to 8% of newborn calf serum or PBST solution having a gelatin content of 2.5% (0.01 mol/L PBS containing 0.05% Tween-20). This example uses a PBST solution having a gelatin content of 2.5%.
  • the solid phase carrier can be an ELISA plate, a microplate, a test tube or a microporous membrane.
  • an ELISA plate is used, and the ELISA plate is modified, wherein the modification is performed by placing the ELISA plate on a medical purification operation table equipped with an ultraviolet lamp, and fixing the ultraviolet lamp and the microplate.
  • the microplate is UV treated by the vertical distance of the substrate.
  • the anti-A ⁇ antibody is diluted with an enzyme conjugate stabilizer.
  • the anti-A ⁇ antibody in the present embodiment is not limited, and may be any antibody capable of specifically recognizing A ⁇ .
  • the enzyme conjugate stabilizer is an agent capable of maintaining the stability between the antibody and the enzyme conjugate, and is capable of maintaining the activity of the antibody and the enzyme.
  • it may be an HRP enzyme conjugate stabilizer
  • the enzyme conjugate stabilizer in the present invention may be a commercially available product.
  • Horseradish peroxidase (HRP)-labeled anti-A ⁇ antibody was diluted with blocking solution.
  • the washing solution is a Tween-20-containing PBS solution (abbreviated as PBST solution), wherein the PBST solution may contain a biological liquid preservative such as ProClin300.
  • the substrate solution may be OPD (o-phenylenediamine), OT (o-toluidine), ABTS (2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid)), p- NPP (p-nitrophenyl phosphate) or tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB).
  • OPD o-phenylenediamine
  • OT o-toluidine
  • ABTS 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid)
  • p- NPP p-nitrophenyl phosphate
  • tetramethylbenzidine 3,3',5,5'-Tetramethylbenzidine, TMB.
  • Preferred is OPD.
  • the stop solution can be a 10% (v/v) sulfuric acid solution.
  • the sample diluent was a PBS solution.
  • the various reagents are respectively packaged, preferably using a packaging tube, and the amount of the reagents in each packaging tube is sufficient for one sample to be used, and can be expanded to 10, 100, 1000 samples. The amount of use.
  • Reaction 1 Add erythrocyte cytoplasmic sample or hemoglobin-A ⁇ complex standard protein to each well of the plate to incubate for 2 h. rinse.
  • Reaction 2 The mouse anti-A ⁇ antibody was diluted with blocking solution to a final concentration of 0.1-2 ⁇ g/mL. The antibody dilution was added to each well of the plate and incubated for 2 h. rinse.
  • Termination The color development was terminated with 10% H 2 SO 4 .
  • This example tested the effects of different anti-Hb antibody concentrations and sample concentrations on the kit test results.
  • the coating antibody anti-Hb antibody
  • the sample was diluted with PBS buffer, and the sample concentration was selected as follows, 1:1, 1 respectively. : 2, 1:4 four conditions. Its effect on the test results of the kit is shown in Table 1 below.
  • the OD values of the blood as it is and the dilution ratio of 1:1 are not much different. Considering the cost issue, choose a 1:1 dilution of blood for loading. When the coated antibody was 1-2 ⁇ g/mL, the measured OD value was in the linear range, and the coated antibody did not reach saturation, making the measured value more accurate. Considering the concentration of the sample, select a dilution ratio of 1:1, and coat the antibody to choose 2 ⁇ g/mL.
  • the detection antibody when the detection antibody was 0.5-2 ⁇ g/mL, the measured OD value was in the linear range. Taken together, the detection antibody was selected at 1 ⁇ g/mL.
  • Reaction 1 Add erythrocyte cytoplasmic sample or Hb-A ⁇ complex standard protein to each well of the plate to incubate for 2 h. rinse.
  • Reaction 2 The biotinylated murine anti-A ⁇ antibody was diluted with blocking solution to a final concentration of 0.5-4 ⁇ g/mL. The antibody dilution was added to each well of the plate and incubated for 2 h. rinse.
  • Termination The color development was terminated with 10% H 2 SO 4 .
  • this example tested the effects of different coating antibody (anti-Hb antibody) concentrations, sample concentrations, and detection antibody (anti-A ⁇ antibody) concentrations on the kit detection results.
  • the coating antibody (anti-Hb antibody) was selected under the conditions of 0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL and 4 ⁇ g/mL; the sample was diluted with PBS buffer, and the sample concentration was selected as follows, 1:1, 1 respectively. : 2, 1:4 conditions; detection antibody (anti-A ⁇ antibody) concentration was selected under conditions of 0.5 ⁇ g/mL, 1 ⁇ g/mL, and 2 ⁇ g/mL.
  • the concentration of the coated antibody was finally selected to be 2 ⁇ g/mL; the sample concentration was a dilution ratio of 1:1; and the detection antibody concentration was 1 ⁇ g/mL.
  • the A ⁇ kit was prepared under the optimal conditions selected in Example 2, and three batches were taken. Twenty kits were randomly selected from each batch for the following tests.
  • the A ⁇ kit was prepared according to the optimal conditions selected in Example 2 of the present invention, and the OD value of A ⁇ bound to Hb in the blood red blood cells of 50 normal patients and 50 AD patients was detected, and then plotted as a scatter plot, as shown in FIG. 2 Show.
  • the solid circle in Fig. 2 represents the OD value of each normal human Hb-A ⁇ .
  • Open circles represent the OD values of Hb-A ⁇ in each AD patient.
  • the horizontal line in the figure represents the mean level of normal people and AD.
  • the amount of Hb-A ⁇ in the blood red blood cells of AD patients is significantly higher than that of the normal population.
  • Figure 3 is a plot of ROC plotted against the OD value detected by the kit.
  • the sensitivity of the A ⁇ kit was initially determined to be 87.5%; the specificity was 81.25%; and the 95% confidence interval was 0.7615-0.9612.
  • the A ⁇ kit provided by the embodiments of the present invention has high sensitivity and specificity, and can be applied to the detection of Hb-A ⁇ in blood red blood cells. Moreover, by using the A ⁇ kit provided by the invention, the contamination of the sample by hemolysis can be avoided, and the detection result is more accurate and reliable, and the characteristics of the A ⁇ protein circulating in the blood, the cerebrospinal fluid and the interstitial fluid are determined according to the characteristics. The content of A ⁇ protein in other parts can be determined indirectly.
  • the sample containing hemoglobin is easy to collect and is easily accepted by the patient, so that the A ⁇ kit provided by the present invention can be applied to large-scale screening, so as to detect or determine whether it is an AD patient as early as possible, and also to facilitate neuroprotection.
  • the evaluation of the efficacy of drugs has a wide range of applications.

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Abstract

Provided is a detection kit for an amyloid-β (Aβ) peptide. The kit comprises an anti-hemoglobin (Hb) antibody and an anti-Aβ antibody, wherein the anti-Hb antibody is a capture antibody and is used for capturing Hb, and the anti-Aβ antibody is a detection antibody and is used for detecting the content of Aβ binding to Hb. The kit is suitable for the quantitative detection of the content of all Aβs binding to Hb, comprising the quantitative detection of the content of Aβ binding to Hb in peripheral blood erythrocytes; and the kit is suitable for screening large-scale populations, and can solve the problem of the contamination of samples caused by hemolysis that is present in the existing technique of detecting Aβ from cerebrospinal fluid, plasma and serum samples.

Description

Aβ试剂盒、检测方法及应用Aβ kit, detection method and application 技术领域Technical field
本发明涉及免疫测定领域，具体涉及Aβ检测试剂盒、检测方法及其应用。The invention relates to the field of immunoassay, in particular to an Aβ detection kit, a detection method and an application thereof.
背景技术Background technique
β-淀粉样肽或蛋白(amyloid β-peptide，又名amyloid β-protein Aβ或Abeta)分子量约4kDa，由β淀粉样前体蛋白(β-amyloid precursor protein，APP)水解而产生的含有39～43个氨基酸的多肽，其异常聚集体是构成AD病人脑中的主要病理结构——老年斑(senile plaque)的主要成分[1]。Aβ是淀粉样前体蛋白(amyloid precursor protein，APP)在病理情况下的降解产物。在生理情况下，APP以分泌途径(或非淀粉样生成途径)降解。APP首先在α-分泌酶(α-secretase)的作用下降解为可溶性的N-末端片段(sAPPα)和一个C-末端片段(C83)，后者被γ-分泌酶(γ-secretase)进一步分解为3kDa的较小的C-末端片段。在病理情况下，APP以淀粉样生成途径(amyloidogenic pathway)降解。APP首先被β-分泌酶(β-secretase)降解，释放出较小的N-末端片段(sAPPα)和一个较长的含有Aβ全部氨基酸序列的C-末端片段(C99)，后者进一步在γ-分泌酶的作用下产生具有致病性的Aβ肽。不同分子大小的Aβ在释放时以单体形式存在，逐渐聚集成寡聚体、原纤维和纤维，并沉积形成淀粉样斑块(或老年斑)。其中，Aβ42是最容易聚集 且神经毒性作用最强的Aβ分子。Aβ寡聚体通过多种机制发挥神经毒性作用，包括激活炎症反应，引起线粒体功能异常，增强氧化应激，损害细胞内信号通路和突触可塑性，增加tau蛋白的磷酸化和GSK-3β活性，引起钙代谢失调和诱发细胞凋亡或死亡[2]。上述机制引起一个正反馈反应：Aβ肽的产生导致一系列对神经元有害的事件，后者反过来引起APP代谢的异常和更多的Aβ肽的释放。The β-amyloid peptide or protein (amyloid β-peptide, also known as amyloid β-protein Aβ or Abeta) has a molecular weight of about 4 kDa and is produced by the hydrolysis of β-amyloid precursor protein (APP). The 43 amino acid polypeptide, its aberrant aggregate is the main component of the senile plaque, the main pathological structure in the brain of AD patients [1]. Aβ is a degradation product of amyloid precursor protein (APP) under pathological conditions. Under physiological conditions, APP is degraded by the secretory pathway (or non-amyloidogenic pathway). APP first degraded into a soluble N-terminal fragment (sAPPα) and a C-terminal fragment (C83) under the action of α-secretase, which was further decomposed by γ-secretase. It is a smaller C-terminal fragment of 3 kDa. In pathological conditions, APP degrades in the amyloidogenic pathway. APP is first degraded by β-secretase, releasing a smaller N-terminal fragment (sAPPα) and a longer C-terminal fragment (C99) containing the entire amino acid sequence of Aβ, which is further in γ - Producing a pathogenic Aβ peptide by the action of a secretase. Aβ of different molecular sizes is present as a monomer when released, gradually aggregates into oligomers, fibrils and fibers, and deposits to form amyloid plaques (or senile plaques). Among them, Aβ42 is the easiest to aggregate And the most neurotoxic Aβ molecule. Aβ oligomers exert neurotoxic effects through a variety of mechanisms, including activation of inflammatory responses, mitochondrial dysfunction, enhanced oxidative stress, impaired intracellular signaling pathways and synaptic plasticity, increased tau phosphorylation and GSK-3β activity, Causes calcium metabolism disorders and induces apoptosis or death [2]. The above mechanism causes a positive feedback response: the production of A[beta] peptides results in a series of events that are detrimental to neurons, which in turn cause abnormalities in APP metabolism and more release of A[beta] peptides.
鉴于Aβ在AD患者的大脑中积聚并且是AD发病机制的中心要素的事实，Aβ已经被认为是作为AD生物标志物的最合适候选物。目前，检测主要集中于脑脊液和血液中的Aβ。脑脊液中，AD病人的Aβ42(或Aβ1-42)含量降低，t-tau和p-tau含量增高，Aβ42、t-tau(总的tau)和p-tau(磷酸化tau)含量变化的检测结果一致。结合t-tau、p-tau，以及Aβ40(Aβ1-40)与Aβ42的比值，能有效地预测脑中Aβ的沉积。检测脑脊液中Aβ的变化可以将轻度认知功能障碍患者中呈现进行性认知功能下降或转为AD的与认知相对稳定的患者区分开来，且结果可靠。脑脊液Aβ作为AD诊断标志物的应用已经在大规模人群得到验证，并通过神经病理检测得到确认[3]。但是，检测脑脊液中的Aβ存在取样困难，采集过程复杂，损伤较大等问题，大多数患者不愿意接受，尤其是对于早期或可能患有AD的患者，从而延误治疗，错过AD治疗的最佳时期。In view of the fact that Aβ accumulates in the brain of AD patients and is a central element of the pathogenesis of AD, Aβ has been considered as the most suitable candidate for AD biomarkers. Currently, the test focuses on Aβ in the cerebrospinal fluid and blood. In cerebrospinal fluid, the levels of Aβ42 (or Aβ1-42) in AD patients decreased, the levels of t-tau and p-tau increased, and the changes in Aβ42, t-tau (total tau) and p-tau (phosphorylated tau) levels Consistent. Combining t-tau, p-tau, and the ratio of Aβ40 (Aβ1-40) to Aβ42 can effectively predict the deposition of Aβ in the brain. Detection of changes in Aβ in cerebrospinal fluid can distinguish patients with mild cognitive impairment from progressive cognitive decline or conversion to AD with patients with relatively stable cognition, and the results are reliable. The application of cerebrospinal fluid Aβ as a diagnostic marker for AD has been validated in large populations and confirmed by neuropathological examination [3]. However, the detection of Aβ in cerebrospinal fluid has difficulty in sampling, complicated collection process, and large damage. Most patients are unwilling to accept it, especially for patients with early or possible AD, thus delaying treatment and missing the best treatment for AD. period.
血液样本采集操作容易、创伤小，容易被大多数患者所接受。目前针对血液中Aβ的检测主要是测试血浆或血清中的Aβ。然而，针对血浆或血清中Aβ42的检测则呈现出矛盾的结果。传统的ELISA方法或Luminex检测法测试AD患者血浆的Aβ42含量的结果有的增高，有的降低，有的无变化。其主要原因之一被归结为 血浆Aβ42含量较低，传统方法的检测敏感性不够[4-8]。然而，检测结果不一致的问题并没有因为增加检测敏感性得到解决。例如新近推出的超敏感的检测技术。一个技术是免疫磁珠还原法(Immunomagnetic Reduction，IMR)，由MagQu Company,Ltd.公司(台湾新北市)开发。另一个技术是单分子阵列技术(Single molecular array，SIMOA)，由美国Quanterix公司(Lexington,MA)开发。这两个技术都是基于特异性抗体与待检物质或蛋白标准品之间的免疫反应。IMR是通过超导量子干扰装置检测交流磁化率(Magnetic susceptibility)。而SIMOA技术是通过与荧光底物(Resorufin β-D-galactopyranoside)反应的单个酶标免疫复合物的荧光成像检测抗原分子的存在。然而，IMR检测出血浆中的Aβ的结果是：Aβ42水平增高，Aβ40水平降低或不变[4,9-12]；而SIMOA技术检测出的血浆中Aβ的结果是：Aβ42和Aβ40水平降低[4,13]。Blood sample collection is easy to operate, minimally invasive, and easily accepted by most patients. At present, the detection of Aβ in blood is mainly to test Aβ in plasma or serum. However, detection of Aβ42 in plasma or serum presents contradictory results. The traditional ELISA method or Luminex test method has increased the plasma Aβ42 content of AD patients, and some of them have decreased, some have no change. One of the main reasons is attributed to The plasma Aβ42 content is low, and the detection sensitivity of the traditional method is not enough [4-8]. However, the problem of inconsistent detection results has not been solved by increasing the sensitivity of detection. For example, the recently introduced ultra-sensitive detection technology. One technique is Immunomagnetic Reduction (IMR), developed by MagQu Company, Ltd. (New Taipei, Taiwan). Another technology is Single Molecular Array (SIMOA), developed by Quantelix, USA (Lexington, MA). Both techniques are based on an immune response between a specific antibody and the substance to be tested or a protein standard. IMR detects the magnetic susceptibility through a superconducting quantum interference device. The SIMOA technique detects the presence of antigen molecules by fluorescence imaging of a single enzyme-labeled immune complex that reacts with a fluorescent substrate (Resorufin β-D-galactopyranoside). However, IMR detected Aβ in plasma as follows: Aβ42 level was increased, Aβ40 level was decreased or unchanged [4,9-12]; and Aβ results in plasma detected by SIMOA technique were: Aβ42 and Aβ40 levels were decreased [ 4,13].
迄今为止，不管使用传统的ELISA技术还是更先进的IMR或SIMOA技术，血液中Aβ的检测仍然没有一致性的结论。分析其原因，可能有以下几点：1.血液中的Aβ含量少，基本处于检测限以下，利用现有技术无法检测出Aβ，或检测结果不准确；2.利用现有技术检测血液中的Aβ时，无法避免溶血对其的影响。To date, the detection of Aβ in blood has not been consistent, regardless of whether traditional ELISA techniques or more advanced IMR or SIMOA techniques are used. Analysis of the reasons, there may be the following points: 1. The amount of Aβ in the blood is small, basically below the detection limit, the Aβ can not be detected by the prior art, or the detection result is inaccurate; 2. The prior art is used to detect the blood. At the time of Aβ, the effect of hemolysis on it cannot be avoided.
发明内容Summary of the invention
有鉴于此，本发明提供了一种新的检测血液中Aβ的试剂盒。该试剂盒的检测能够避免溶血对样本的污染，使得结果更加准确，可靠。In view of this, the present invention provides a novel kit for detecting Aβ in blood. The detection of the kit can avoid the contamination of the sample by hemolysis, making the result more accurate and reliable.
一方面，本发明提供了一种检测待测样品中Aβ的试剂盒，其包括抗Hb抗体和抗Aβ抗体。在本发明的试剂盒中，抗Hb抗体可作为捕获抗体，用于捕获待检样品中的Hb；抗Aβ抗体可作为 检测抗体，用于检测与Hb结合的Aβ。In one aspect, the invention provides a kit for detecting Aβ in a sample to be tested, which comprises an anti-Hb antibody and an anti-Aβ antibody. In the kit of the present invention, an anti-Hb antibody can be used as a capture antibody for capturing Hb in a sample to be tested; an anti-Aβ antibody can be used as The antibody is detected for detecting Aβ bound to Hb.
优选地，本发明的检测待测样品中Aβ的试剂盒还任选地包括Hb-Aβ复合体标准蛋白。该Hb-Aβ复合体标准蛋白可与抗Hb和Aβ抗体相结合，用于制作标准曲线。Preferably, the kit for detecting Aβ in a sample to be tested of the present invention optionally further comprises an Hb-Aβ complex standard protein. The Hb-Aβ complex standard protein can be combined with anti-Hb and Aβ antibodies to make a standard curve.
示例性地，所述抗Hb抗体的浓度为0.5～4μg/mL，优选的为1～2μg/mL，更优选的为2μg/mL。Illustratively, the anti-Hb antibody has a concentration of 0.5 to 4 μg/mL, preferably 1 to 2 μg/mL, more preferably 2 μg/mL.
示例性地，所述抗Aβ抗体的浓度为0.1～2μg/mL，优选的为1μg/mL。Illustratively, the concentration of the anti-Aβ antibody is 0.1 to 2 μg/mL, preferably 1 μg/mL.
在本发明提供的一具体实施例中，所述抗Aβ抗体为酶标记的抗Aβ抗体。所述酶为辣根过氧化物酶(Horseradish Peroxidase，HRP)、碱性磷酸酶(Alkaline Phosphatase，AP)或葡萄糖氧化酶(Glucose Oxidase，GO)。In a specific embodiment provided by the invention, the anti-Aβ antibody is an enzyme-labeled anti-Aβ antibody. The enzyme is Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP) or Glucose Oxidase (GO).
在本发明提供的另一具体实施例中，所述抗Aβ抗体为荧光或化学发光材料或胶体金标记的抗Aβ抗体，例如FITC或绿色荧光蛋白等。In another embodiment provided by the present invention, the anti-Aβ antibody is a fluorescent or chemiluminescent material or a colloidal gold-labeled anti-Aβ antibody, such as FITC or green fluorescent protein.
在本发明提供的另一具体实施例中，所述抗Aβ抗体为生物素化的抗Aβ抗体，以及，任选地，所述试剂盒还包括亲和素标记的酶，例如碱性磷酸酶。In another embodiment provided by the present invention, the anti-Aβ antibody is a biotinylated anti-Aβ antibody, and, optionally, the kit further comprises an avidin-labeled enzyme, such as an alkaline phosphatase. .
在本发明提供的又一具体实施例中，所述抗Aβ抗体为未被酶标记的抗Aβ抗体，所述试剂盒还包括酶标记的二抗，或荧光素、化学发光材料、生物素、胶体金等其他物质标记的二抗，所述二抗可特异性的与抗Aβ抗体相结合。In still another embodiment provided by the present invention, the anti-Aβ antibody is an anti-Aβ antibody not labeled with an enzyme, and the kit further comprises an enzyme-labeled secondary antibody, or fluorescein, a chemiluminescent material, biotin, A secondary antibody labeled with other substances such as colloidal gold, which can specifically bind to an anti-Aβ antibody.
示例性地，本发明中的待测样品是来源于人、动物和/或培养细胞。优选地，所述待测样品来源于人，更优选地，来源于阿尔茨海默(AD)患者或者具有患AD风险的人群。进一步优选地， 所述样品是血液样品，或来自于血液的红细胞胞浆蛋白，或其它任何含有Hb的组织。Illustratively, the sample to be tested in the present invention is derived from human, animal and/or cultured cells. Preferably, the sample to be tested is derived from a human, more preferably from an Alzheimer's (AD) patient or a population at risk of developing AD. Further preferably, The sample is a blood sample, or a erythrocyte cytoplasmic protein from the blood, or any other tissue containing Hb.
进一步的，所述试剂盒还可以包括固相载体、缓冲液、封闭液等。其中，所述固相载体为酶标板、微孔板、试管或微孔滤膜，优选的，所述固相载体为酶标板；固相载体的材质例如可为聚苯乙烯、硝酸纤维素、尼龙等。Further, the kit may further include a solid phase carrier, a buffer solution, a blocking solution, and the like. Wherein, the solid phase carrier is an ELISA plate, a microplate, a test tube or a microporous membrane. Preferably, the solid phase carrier is an ELISA plate; the material of the solid phase carrier is, for example, polystyrene or nitrocellulose. Plain, nylon, etc.
所述缓冲溶液选自Tris-HCl缓冲液、PBS缓冲液、CBS缓冲液或缓冲生理盐水；The buffer solution is selected from the group consisting of Tris-HCl buffer, PBS buffer, CBS buffer or buffered physiological saline;
所述封闭液选自2％-8％的新生牛血清或含明胶的PBST。The blocking solution is selected from 2% to 8% of newborn calf serum or gelatin-containing PBST.
另一方面，本发明还提供了抗Hb抗体在制备用于检测待测样本中Aβ含量的试剂盒或试剂中的用途。In another aspect, the invention also provides the use of an anti-Hb antibody for the preparation of a kit or reagent for detecting Aβ content in a sample to be tested.
在一具体的实施方式中，本发明提供了抗Hb抗体和抗Aβ抗体在制备用于检测待测样本中Aβ含量的试剂盒或试剂中的用途。In a specific embodiment, the invention provides the use of an anti-Hb antibody and an anti-Aβ antibody in the preparation of a kit or reagent for detecting Aβ content in a sample to be tested.
再一方面，本发明提供了抗Hb抗体在制备用于检测待测样品是否患有AD或患有AD风险的试剂盒或试剂中的用途。In a further aspect, the invention provides the use of an anti-Hb antibody for the preparation of a kit or reagent for detecting whether a sample to be tested has AD or is at risk of AD.
在一具体的实施方式中，本发明提供了抗Hb抗体和抗Aβ抗体在制备用于检测待测样本是否患有AD或患有AD风险的试剂盒或试剂中的用途。In a specific embodiment, the invention provides the use of an anti-Hb antibody and an anti-Aβ antibody in the manufacture of a kit or reagent for detecting whether a test sample has AD or is at risk of AD.
又一方面，本发明还提供了Aβ试剂盒的制备方法，其包括：In still another aspect, the present invention also provides a method for preparing an Aβ kit, comprising:
1)抗Hb抗体包被的固相载体的制备；和1) preparation of an anti-Hb antibody-coated solid phase carrier;
2)抗Aβ抗体的配制；以及任选地，2) formulation of an anti-Aβ antibody; and optionally,
3)Hb-Aβ复合物标准品的配制；3) Preparation of Hb-Aβ complex standard;
进一步的，上述步骤1)包括配制抗Hb抗体包被液、配制封闭液及包被酶标板。Further, the above step 1) comprises formulating an anti-Hb antibody coating solution, preparing a blocking solution, and coating the ELISA plate.
在本发明的一个具体实施方式中： In a specific embodiment of the invention:
配制抗Hb抗体包被液：采用缓冲溶液稀释抗Hb抗体；Formulating an anti-Hb antibody coating solution: diluting the anti-Hb antibody with a buffer solution;
配制封闭液：将2％-8％的新生牛血清加入缓冲溶液中，或将明胶溶于PBST中；以及，Formulating a blocking solution: adding 2% to 8% of newborn calf serum to a buffer solution, or dissolving gelatin in PBST;
包被酶标板：将配制的抗Hb抗体包被液加入酶标板孔中，孵育过夜，冲洗；将配制的封闭液加入酶标板孔中，恒温孵育2-4h，冲洗即得。Coated enzyme plate: The prepared anti-Hb antibody coating solution is added to the well of the enzyme labeling plate, incubated overnight, and rinsed; the prepared blocking solution is added into the well of the enzyme labeling plate, incubated at constant temperature for 2-4 hours, and rinsed.
在本发明中，所述缓冲溶液优选自：Tris-HCl缓冲液，PBS缓冲液，CBS缓冲液或缓冲生理盐水；In the present invention, the buffer solution is preferably: Tris-HCl buffer, PBS buffer, CBS buffer or buffered physiological saline;
进一步的，在本发明的一个具体实施方式中，上述步骤3)具体包括如下步骤：Further, in a specific implementation manner of the present invention, the foregoing step 3) specifically includes the following steps:
A、用缓冲液分别溶解磷酸化Aβ和Hb后，将其混合，振荡孵育；A. Dissolve phosphorylated Aβ and Hb separately with buffer, mix them, and incubate with shaking;
B、孵育样品离心取上清，层析柱凝胶过滤，分离Hb-Aβ复合体；B. Incubate the sample, centrifuge the supernatant, filter the column for gel filtration, and separate the Hb-Aβ complex;
C、用质谱分析方法，确定Aβ占Hb-Aβ复合体的占比。C. Using mass spectrometry to determine the proportion of Aβ in the Hb-Aβ complex.
进一步的，在本发明的一个具体实施方式中，上述步骤2)是通过使用封闭液稀释抗Aβ抗体来进行的。Further, in a specific embodiment of the present invention, the above step 2) is carried out by diluting the anti-Aβ antibody with a blocking solution.
还一方面，本发明还提供了Aβ的检测方法，其包括以下步骤：In still another aspect, the present invention also provides a method for detecting Aβ, which comprises the following steps:
1)抗Hb抗体捕获Hb；和1) anti-Hb antibody captures Hb; and
2)抗Aβ抗体检测与Hb结合的Aβ。2) Anti-Aβ antibody detects Aβ bound to Hb.
进一步的，上述Aβ的检测方法，具体包括：Further, the method for detecting Aβ mentioned above specifically includes:
S1：准备待检样品；S1: preparing a sample to be tested;
S2：向固相载体中加入抗Hb抗体，孵育，冲洗；S2: adding an anti-Hb antibody to the solid phase carrier, incubating and rinsing;
S3：向固相载体中加入封闭液，孵育，冲洗；S3: adding a blocking solution to the solid phase carrier, incubating and rinsing;
S4：向固相载体中加入酶标记的抗Aβ抗体，孵育，冲洗；或 S4: adding an enzyme-labeled anti-Aβ antibody to the solid phase carrier, incubating and rinsing; or
向固相载体中加入抗Aβ抗体，孵育，冲洗；加入酶标二抗，孵育，冲洗；加入OPD显色液显色；加入终止液终止显色；或，Adding anti-Aβ antibody to the solid phase carrier, incubating and rinsing; adding enzyme-labeled secondary antibody, incubating and rinsing; adding OPD coloring solution to develop color; adding stop solution to stop color development; or
向固相载体中加入生物素化的抗Aβ抗体，孵育，冲洗；加入亲和素标记的碱性磷酸酶，孵育，冲洗；加入对硝基苯磷酸显色液显色。The biotinylated anti-Aβ antibody was added to the solid phase carrier, incubated, washed; avidin-labeled alkaline phosphatase was added, incubated, washed; and the p-nitrophenyl phosphate coloring solution was added for color development.
优选地，本发明中Aβ的检测包括对Aβ的定性和定量检测。Preferably, the detection of A[beta] in the present invention comprises qualitative and quantitative detection of A[beta].
本发明还提供了Aβ试剂盒在检测Aβ中的应用。The invention also provides the use of an Aβ kit for detecting Aβ.
优选地，在本发明所述的抗体为单克隆抗体，例如能够特异性结合Hb的抗体和能够特异性结合Aβ的抗体。示例性地，所述抗体可为完整抗体、双特异性抗体、抗体片段、基因工程抗体等，例如可以为(Fab’)2、Fab’、Fab、scFv、diabody、minibody、鼠源抗体和人源化抗体等等。Preferably, the antibody of the present invention is a monoclonal antibody, for example, an antibody capable of specifically binding to Hb and an antibody capable of specifically binding to Aβ. Illustratively, the antibody may be an intact antibody, a bispecific antibody, an antibody fragment, a genetically engineered antibody, or the like, and may be, for example, (Fab') 2 , Fab', Fab, scFv, diabody, minibody, murine antibody, and human Sourced antibodies and the like.
优选地，在本发明中，与二抗相偶联的酶可选自辣根过氧化物酶(Horseradish Peroxidase，HRP)、碱性磷酸酶(Alkaline Phosphatase，AP)或葡萄糖氧化酶(Glucose Oxidase，GO)。Preferably, in the present invention, the enzyme coupled to the secondary antibody may be selected from Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP) or Glucose Oxidase (Glucose Oxidase). GO).
优选地，本发明的试剂盒还含有使用说明书。Preferably, the kit of the invention further contains instructions for use.
本发明提供的Aβ试剂盒及其检测方法和相关的应用是以抗Hb抗体为捕获抗体，用于捕获Hb，再以抗Aβ抗体为检测抗体，检测与捕获的Hb相结合的Aβ。示例性地，本发明至少具有以下优势之一：The Aβ kit provided by the present invention and the detection method thereof and related applications are an anti-Hb antibody as a capture antibody, which is used for capturing Hb, and an anti-Aβ antibody as a detection antibody to detect Aβ combined with the captured Hb. Illustratively, the present invention has at least one of the following advantages:
1)本发明提供的Aβ检测试剂盒适用于所有与Hb结合的Aβ含量的定量检测，包括与外周血红细胞中的与Hb结合的Aβ含量的定量检测；1) The Aβ detection kit provided by the present invention is suitable for quantitative detection of all Aβ content bound to Hb, including quantitative detection of Aβ content bound to Hb in peripheral blood red blood cells;
2)血液红细胞样本采集与保存便捷，适合大规模人群筛选；2) Blood red blood cell samples are easy to collect and store, suitable for large-scale population screening;
3)测试结果稳定； 3) The test results are stable;
4)可反映机体组织尤其是神经组织中Aβ含量的变化；4) can reflect changes in Aβ content in body tissues, especially nerve tissues;
5)能够解决现有的从脑脊液、血浆和血清样本中检测Aβ的技术所存在的溶血对样本的污染问题。5) It can solve the problem of contamination of samples by hemolysis existing in the existing technology for detecting Aβ from cerebrospinal fluid, plasma and serum samples.
附图说明DRAWINGS
图1为本发明实施例中红细胞胞浆蛋白分离过程示意图；1 is a schematic view showing a process of separating erythrocyte cytoplasmic proteins in an embodiment of the present invention;
图2为本发明实施例5中Aβ试剂盒检测结果；2 is a detection result of an Aβ kit in Example 5 of the present invention;
图3为本发明实施例5中Aβ试剂盒检测结果的ROC曲线图。Fig. 3 is a graph showing the ROC curve of the detection result of the Aβ kit in Example 5 of the present invention.
具体实施方式Detailed ways
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention are clearly and completely described below. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
需要说明的是，本发明中β-淀粉样肽或蛋白、amyloid、amyloid β-peptide、β-protein、Amyloid beta、Aβ以及Abeta等均为同一物质，且英文名称、简写等不区分大小写。It should be noted that in the present invention, the β-amyloid peptide or protein, amyloid, amyloid β-peptide, β-protein, Amyloid beta, Aβ, and Abeta are all the same substance, and the English name, short form, and the like are not case sensitive.
本发明中部分试剂的配制方法如下，其他试剂均为常规配制方法。The preparation method of some reagents in the present invention is as follows, and other reagents are conventional preparation methods.
PBS的浓度为0.01mol/L，配制方法为：The concentration of PBS is 0.01 mol/L, and the preparation method is as follows:
Figure PCTCN2017112086-appb-000001
Figure PCTCN2017112086-appb-000001
Figure PCTCN2017112086-appb-000002
Figure PCTCN2017112086-appb-000002
NaHCO3缓冲液的浓度为200mmol/L，pH 9.6，配制方法为：The concentration of NaHCO 3 buffer was 200 mmol/L, pH 9.6, and the preparation method was as follows:
NaHCO3           0.84gNaHCO 3 0.84g
20％叠氮钠(NaN3) 50μl20% sodium azide (NaN 3 ) 50μl
DDW              50mlDDW 50ml
封闭液的浓度为2.5％，配制方法为：The concentration of the blocking solution is 2.5%, and the preparation method is:
明胶             2.5gGelatin 2.5g
PBST             100mlPBST 100ml
PBST为含0.05％Tween-20的0.01mol/L PBS，配制方法为：PBST is 0.01 mol/L PBS containing 0.05% Tween-20, and the preparation method is as follows:
Figure PCTCN2017112086-appb-000003
Figure PCTCN2017112086-appb-000003
实施例1 Aβ试剂盒的制备方法Example 1 Preparation method of Aβ kit
1.配制抗Hb抗体包被液1. Preparation of anti-Hb antibody coating solution
采用缓冲溶液将抗Hb抗体稀释至0.2μg/mL-10μg/mL；所述缓冲溶液选自：Tris-HCl缓冲液、PBS缓冲液、CBS缓冲液或缓冲生理盐水。本实施例采用NaHCO3缓冲液。本实施例中对抗Hb抗体不做限定，可为任意能够特异性识别血红蛋白的抗体。The anti-Hb antibody is diluted to 0.2 μg/mL to 10 μg/mL with a buffer solution; the buffer solution is selected from the group consisting of Tris-HCl buffer, PBS buffer, CBS buffer or buffered saline. This example employed a NaHCO 3 buffer. The anti-Hb antibody in the present embodiment is not limited, and may be any antibody capable of specifically recognizing hemoglobin.
2.配制封闭液2. Preparation of blocking solution
封闭液选自2％-8％的新生牛血清或明胶含量为2.5％的PBST溶液(含0.05％Tween-20的0.01mol/L PBS)。本实施例采用明胶含量为2.5％的PBST溶液。 The blocking solution is selected from 2% to 8% of newborn calf serum or PBST solution having a gelatin content of 2.5% (0.01 mol/L PBS containing 0.05% Tween-20). This example uses a PBST solution having a gelatin content of 2.5%.
3.包被用固相载体3. Solid phase carrier for coating
固相载体可为酶标板、微孔板、试管或微孔滤膜。本实施例中采用酶标板，且对酶标板进行修饰，其中所述的修饰通过如下进行：将酶标板置于装有紫外灯的医用净化操作台上，固定紫外灯与微孔板基底的垂直距离，对微孔板进行紫外处理。The solid phase carrier can be an ELISA plate, a microplate, a test tube or a microporous membrane. In this embodiment, an ELISA plate is used, and the ELISA plate is modified, wherein the modification is performed by placing the ELISA plate on a medical purification operation table equipped with an ultraviolet lamp, and fixing the ultraviolet lamp and the microplate. The microplate is UV treated by the vertical distance of the substrate.
4.配制标准品(定量标准曲线的建立)4. Preparation of standard products (establishment of quantitative standard curve)
1)用500μL的0.01mol/L PBS溶解人Aβ和Hb，使终浓度分别为2mol/L与1mol/L，混合于37℃、230rpm、振荡孵育24h；1) Dissolve human Aβ and Hb with 500 μL of 0.01 mol/L PBS to a final concentration of 2 mol/L and 1 mol/L, respectively, and mix at 37 ° C, 230 rpm, and incubate for 24 h with shaking;
2)孵育样品在10000×g离心5min，吸取上清，以PBS为流动相，HiPrep 26/60Sephacryl S-200High Resolution层析柱凝胶过滤，分离Hb-Aβ复合体；2) The incubation sample was centrifuged at 10000×g for 5 min, the supernatant was aspirated, the mobile phase was PBS, and the HiPrep 26/60 Sephacryl S-200 High Resolution column was gel-filtered to separate the Hb-Aβ complex;
3)用SWATH(Sequential Window Acquisition of all THeoretical Mass Spectra)定量蛋白组方法，确定平均每个Hb结合的Aβ蛋白分子数，从而计算出Aβ占复合体的重量百分比或摩尔百分比。据此，配制不同浓度的Hb-Aβ蛋白复合体溶液，将其作为标准蛋白制作标准曲线。3) Using the SWATH (Sequential Window Acquisition of all THeoretical Mass Spectra) quantitative proteome method to determine the average number of Aβ protein molecules bound per Hb, thereby calculating the weight percentage or mole percentage of Aβ in the complex. Accordingly, different concentrations of the Hb-Aβ protein complex solution were prepared and used as a standard protein to prepare a standard curve.
5.配制抗Aβ抗体溶液5. Preparation of anti-Aβ antibody solution
将抗Aβ抗体用酶偶联物稳定剂稀释而成。本实施例中对抗Aβ抗体不做限定，可为任意能够特异性识别Aβ的抗体。The anti-Aβ antibody is diluted with an enzyme conjugate stabilizer. The anti-Aβ antibody in the present embodiment is not limited, and may be any antibody capable of specifically recognizing Aβ.
酶偶联物稳定剂能够保持抗体与酶偶联物之间稳定性的试剂，能够保持抗体及酶的活性。优选的，其可为HRP酶偶联物稳定剂，本发明中酶偶联物稳定剂可为市售产品。The enzyme conjugate stabilizer is an agent capable of maintaining the stability between the antibody and the enzyme conjugate, and is capable of maintaining the activity of the antibody and the enzyme. Preferably, it may be an HRP enzyme conjugate stabilizer, and the enzyme conjugate stabilizer in the present invention may be a commercially available product.
6.配制酶标二抗溶液6. Preparation of enzyme-labeled secondary antibody solution
用封闭液稀释辣根过氧化物酶(HRP)标记的抗Aβ抗体。Horseradish peroxidase (HRP)-labeled anti-Aβ antibody was diluted with blocking solution.
7.洗液 7. lotion
洗液为含Tween-20的PBS溶液(简称PBST溶液)，其中，PBST溶液中可包含生物液体防腐剂如ProClin300。The washing solution is a Tween-20-containing PBS solution (abbreviated as PBST solution), wherein the PBST solution may contain a biological liquid preservative such as ProClin300.
8.底物溶液8. Substrate solution
底物溶液可为OPD(邻苯二胺)、OT(邻联甲苯胺)、ABTS(2,2'-连氮-双(3-乙基苯并噻唑-6-磺酸))、p-NPP(对硝基苯磷酸酯)或四甲基联苯胺(3,3′,5,5′-Tetramethylbenzidine，TMB)。优选的为OPD。The substrate solution may be OPD (o-phenylenediamine), OT (o-toluidine), ABTS (2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid)), p- NPP (p-nitrophenyl phosphate) or tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB). Preferred is OPD.
9.终止液9. Stop solution
终止液可为10％(V/V)硫酸溶液。The stop solution can be a 10% (v/v) sulfuric acid solution.
10.样品稀释液10. Sample diluent
样品稀释液为PBS溶液。The sample diluent was a PBS solution.
本发明提供的试剂盒中，各种试剂分别包装，优选使用包装管，每个包装管装入试剂的量以够一个样本使用量为基本量，可以扩大到10个，100个，1000个样本的使用量。In the kit provided by the invention, the various reagents are respectively packaged, preferably using a packaging tube, and the amount of the reagents in each packaging tube is sufficient for one sample to be used, and can be expanded to 10, 100, 1000 samples. The amount of use.
实施例2 Aβ试剂盒的检测方法(OPD法)Example 2 Method for detecting Aβ kit (OPD method)
S1：如图1所示，取5-10ml抗凝血，混匀，贴壁加入至离心管中，记录全血的体积，按照一定的稀释比例加入PBS，充分混匀。S1: As shown in Figure 1, take 5-10ml anticoagulation, mix, add to the centrifuge tube, record the volume of whole blood, add PBS according to a certain dilution ratio, and mix well.
S2：将稀释后的全血缓慢加至淋巴细胞分离液上，离心，分离红细胞层。将红细胞转移到新的离心管中，加PBS，离心，-80℃保存。S2: The diluted whole blood is slowly added to the lymphocyte separation solution, centrifuged, and the red blood cell layer is separated. Transfer the red blood cells to a new centrifuge tube, add PBS, centrifuge, and store at -80 °C.
S3：将冻存红细胞在室温融化后放入离心机，离心，上层即为红细胞胞浆蛋白。S3: The frozen red blood cells are thawed at room temperature, placed in a centrifuge, and centrifuged, and the upper layer is a erythrocyte cytoplasmic protein.
S4：包被：用NaHCO3缓冲液稀释抗Hb抗体至终浓度为0.5-4μg/mL。向酶标板各孔加入该抗体稀释液，孵育过夜。冲洗。 S4: Coat: The anti-Hb antibody was diluted with NaHCO3 buffer to a final concentration of 0.5-4 μg/mL. The antibody dilution was added to each well of the plate and incubated overnight. rinse.
S5：封闭：向酶标板各孔中加入封闭液，孵育2h。冲洗。S5: Blocking: Add blocking solution to each well of the plate and incubate for 2 h. rinse.
S6：反应1：向酶标板各孔中加入红细胞胞浆样品，或血红蛋白-Aβ复合物标准蛋白，孵育2h。冲洗。S6: Reaction 1: Add erythrocyte cytoplasmic sample or hemoglobin-Aβ complex standard protein to each well of the plate to incubate for 2 h. rinse.
S7：反应2：用封闭液稀释鼠抗Aβ抗体至终浓度为0.1-2μg/mL。向酶标板的各孔加入该抗体稀释液，孵育2h。冲洗。S7: Reaction 2: The mouse anti-Aβ antibody was diluted with blocking solution to a final concentration of 0.1-2 μg/mL. The antibody dilution was added to each well of the plate and incubated for 2 h. rinse.
S8：用封闭液稀释辣根过氧化物酶标记的羊抗鼠二抗(1:5000稀释)，向酶标板的各孔加入该酶稀释液，每孔100μL。孵育0.5-2h。冲洗。S8: The horseradish peroxidase-labeled goat anti-mouse secondary antibody (1:5000 dilution) was diluted with a blocking solution, and the enzyme dilution was added to each well of the plate to 100 μL per well. Incubate for 0.5-2 h. rinse.
S9：显色：向酶标板的各孔加入OPD显色液，室温避光显色15min。S9: Color development: OPD coloring solution was added to each well of the microplate, and the color was developed at room temperature for 15 min.
S10：终止：用10％H2SO4终止显色。S10: Termination: The color development was terminated with 10% H 2 SO 4 .
S11：测定含量：在紫外分光光度计490nm处测定酶标板各孔的吸光度值。S11: Determination of content: The absorbance value of each well of the microplate was measured at 490 nm of an ultraviolet spectrophotometer.
S12：计算：根据体外制备的Hb-Aβ复合体标准品的浓度与490nm处吸光值之间的关系曲线，计算样品中与血红蛋白结合的Aβ复合体的含量。S12: Calculation: The content of the Aβ complex bound to hemoglobin in the sample was calculated based on the relationship between the concentration of the Hb-Aβ complex standard prepared in vitro and the absorbance at 490 nm.
本实施例试验了不同的抗Hb抗体浓度及样品浓度对试剂盒检测结果的影响。其中，包被抗体(抗Hb抗体)分别选择0.5μg/mL、1μg/mL、2μg/mL和4μg/mL四个条件；样品采用PBS缓冲液稀释，样品浓度分别选择原样、1:1、1:2、1:4四个条件。其对试剂盒检测结果的影响如下表1。This example tested the effects of different anti-Hb antibody concentrations and sample concentrations on the kit test results. Among them, the coating antibody (anti-Hb antibody) was selected under the conditions of 0.5 μg/mL, 1 μg/mL, 2 μg/mL and 4 μg/mL; the sample was diluted with PBS buffer, and the sample concentration was selected as follows, 1:1, 1 respectively. : 2, 1:4 four conditions. Its effect on the test results of the kit is shown in Table 1 below.
表1 不同的抗Hb抗体浓度及样品浓度对试剂盒的影响Table 1 Effect of different anti-Hb antibody concentrations and sample concentrations on the kit
Figure PCTCN2017112086-appb-000004
Figure PCTCN2017112086-appb-000004
Figure PCTCN2017112086-appb-000005
Figure PCTCN2017112086-appb-000005
从上述表1中可以看出，血液采用原样和稀释度为1:1比例的OD值相差不大。考虑到成本问题，选择1:1比例稀释血液进行上样。包被抗体为1-2μg/mL时，测定的OD值在线性范围内，并且包被抗体达不到饱和状态，使得测量数值更加准确。综合考虑，上样样品浓度选择1:1的稀释比例，包被抗体选择2μg/mL。As can be seen from Table 1 above, the OD values of the blood as it is and the dilution ratio of 1:1 are not much different. Considering the cost issue, choose a 1:1 dilution of blood for loading. When the coated antibody was 1-2 μg/mL, the measured OD value was in the linear range, and the coated antibody did not reach saturation, making the measured value more accurate. Considering the concentration of the sample, select a dilution ratio of 1:1, and coat the antibody to choose 2 μg/mL.
本实施例还试验了不同的检测抗体(抗Aβ抗体)浓度对试剂盒检测结果的影响。其浓度选择及实验结果见下表2。This example also tested the effect of different detection antibody (anti-Aβ antibody) concentrations on the kit detection results. The concentration selection and experimental results are shown in Table 2 below.
表2 不同的捕获抗体浓度对试剂盒的影响Table 2 Effect of different capture antibody concentrations on the kit
检测抗体(μg/ml)Detection antibody (μg/ml) 0.50.5 11 22
OD值OD value 0.3710.371 1.4581.458 2.2272.227
从上述表2中可以看出，检测抗体为0.5-2μg/mL时，测定的OD值在线性范围内。综合考虑，检测抗体选择1μg/mL。As can be seen from the above Table 2, when the detection antibody was 0.5-2 μg/mL, the measured OD value was in the linear range. Taken together, the detection antibody was selected at 1 μg/mL.
实施例3 Aβ试剂盒的检测方法(生物素法)Example 3 Method for detecting Aβ kit (biotin method)
S1：如图1所示，取5-10ml抗凝血，混匀，贴壁加入至离心管中，记录全血的体积，按照一定的稀释比例加入PBS，充分混匀。S1: As shown in Figure 1, take 5-10ml anticoagulation, mix, add to the centrifuge tube, record the volume of whole blood, add PBS according to a certain dilution ratio, and mix well.
S2：将稀释后的全血缓慢加至淋巴细胞分离液上，离心，分离红细胞层。将红细胞转移到新的离心管中，加PBS，离心，-80℃保存。S2: The diluted whole blood is slowly added to the lymphocyte separation solution, centrifuged, and the red blood cell layer is separated. Transfer the red blood cells to a new centrifuge tube, add PBS, centrifuge, and store at -80 °C.
S3：将冻存红细胞在室温融化后放入离心机，离心，上层即为红细胞胞浆蛋白。S3: The frozen red blood cells are thawed at room temperature, placed in a centrifuge, and centrifuged, and the upper layer is a erythrocyte cytoplasmic protein.
S4：包被：用NaHCO3缓冲液稀释抗Hb抗体至终浓度为0.5-4μg/mL。向酶标板各孔加入该抗体稀释液，孵育过夜。冲洗。 S4: Coat: The anti-Hb antibody was diluted with NaHCO 3 buffer to a final concentration of 0.5-4 μg/mL. The antibody dilution was added to each well of the plate and incubated overnight. rinse.
S5：封闭：向酶标板各孔中加入封闭液，孵育2h。冲洗。S5: Blocking: Add blocking solution to each well of the plate and incubate for 2 h. rinse.
S6：反应1：向酶标板各孔中加入红细胞胞浆样品，或Hb-Aβ复合物标准蛋白，孵育2h。冲洗。S6: Reaction 1: Add erythrocyte cytoplasmic sample or Hb-Aβ complex standard protein to each well of the plate to incubate for 2 h. rinse.
S7：反应2：用封闭液稀释生物素化鼠抗Aβ抗体至终浓度为0.5-4μg/mL。向酶标板的各孔加入该抗体稀释液，孵育2h。冲洗。S7: Reaction 2: The biotinylated murine anti-Aβ antibody was diluted with blocking solution to a final concentration of 0.5-4 μg/mL. The antibody dilution was added to each well of the plate and incubated for 2 h. rinse.
S8：用封闭液稀释亲和素标记的碱性磷酸酶(1:5000稀释)，向酶标板的各孔加入该酶稀释液，每孔100μL。孵育0.5-2h。冲洗。S8: Avidin-labeled alkaline phosphatase (1:5000 dilution) was diluted with blocking solution, and the enzyme dilution was added to each well of the plate to 100 μL per well. Incubate for 0.5-2 h. rinse.
S9：显色：向酶标板的各孔加入对硝基苯磷酸显色液，37℃水浴避光显色30min。S9: Color development: p-nitrophenyl phosphate coloring solution was added to each well of the microplate, and the color was developed in a water bath at 37 ° C for 30 min.
S10：终止：用10％H2SO4终止显色。S10: Termination: The color development was terminated with 10% H 2 SO 4 .
S11：测定含量：在紫外分光光度计405nm处测定酶标板各孔的吸光度值。S11: Determination of content: The absorbance value of each well of the microplate was measured at 405 nm of an ultraviolet spectrophotometer.
S12：计算：根据体外制备的Hb-Aβ复合体标准品的浓度与405nm处吸光值之间的关系曲线，计算样品中与Hb结合的Aβ含量。S12: Calculation: The Aβ content bound to Hb in the sample was calculated based on the relationship between the concentration of the Hb-Aβ complex standard prepared in vitro and the absorbance at 405 nm.
同样，本实施例试验了不同的包被抗体(抗Hb抗体)浓度、样品浓度及检测抗体(抗Aβ抗体)浓度对试剂盒检测结果的影响。其中，包被抗体(抗Hb抗体)分别选择0.5μg/mL、1μg/mL、2μg/mL和4μg/mL四个条件；样品采用PBS缓冲液稀释，样品浓度分别选择原样、1:1、1:2、1:4四个条件；检测抗体(抗Aβ抗体)浓度选择0.5μg/mL、1μg/mL、2μg/mL三个条件。Similarly, this example tested the effects of different coating antibody (anti-Hb antibody) concentrations, sample concentrations, and detection antibody (anti-Aβ antibody) concentrations on the kit detection results. Among them, the coating antibody (anti-Hb antibody) was selected under the conditions of 0.5 μg/mL, 1 μg/mL, 2 μg/mL and 4 μg/mL; the sample was diluted with PBS buffer, and the sample concentration was selected as follows, 1:1, 1 respectively. : 2, 1:4 conditions; detection antibody (anti-Aβ antibody) concentration was selected under conditions of 0.5 μg/mL, 1 μg/mL, and 2 μg/mL.
通过实验，最终选择包被抗体的浓度为2μg/mL；样品浓度为1:1的稀释比例；检测抗体浓度为1μg/mL。Through experiments, the concentration of the coated antibody was finally selected to be 2 μg/mL; the sample concentration was a dilution ratio of 1:1; and the detection antibody concentration was 1 μg/mL.
实施例4 Aβ试剂盒重复性、精密度试验Example 4 Aβ kit repeatability, precision test
以实施例2中选出的最佳条件制备Aβ试剂盒，分别取三批， 每批中随机抽取20个试剂盒进行如下试验。The Aβ kit was prepared under the optimal conditions selected in Example 2, and three batches were taken. Twenty kits were randomly selected from each batch for the following tests.
精密度试验：采用上述抽取出的试剂盒分别测定三份Aβ蛋白值分别为0.5mmol/L(高)、0.125mmol/L(中)和0.03125mmol/L(低)的样本，各8次。计算测定浓度的变异系数。Precision test: Three samples of Aβ protein values of 0.5 mmol/L (height), 0.125 mmol/L (middle), and 0.03125 mmol/L (low) were measured, respectively, using the above extracted kits, 8 times each. Calculate the coefficient of variation of the measured concentration.
实验结果表明，上述三批试剂盒的检测结果的变异系数小于8.0％。重复性试验：采用上述抽取出的试剂盒分别对相同样本重复试验3次，其结果显示相对标准差RSD为0.60％，The experimental results show that the coefficient of variation of the detection results of the above three batches of kits is less than 8.0%. Repeatability test: The same sample was repeated three times using the above-mentioned extracted kits, and the results showed that the relative standard deviation RSD was 0.60%.
以上实验结果表明各试剂盒对样品的检测结果的离散程度较小，重复性较好，均可用于Aβ的检测。The above experimental results show that the detection results of the samples of each kit are less discrete and have good repeatability, and can be used for the detection of Aβ.
实施例5 Aβ试剂盒的应用Example 5 Application of Aβ Kit
利用本发明实施例2选出的最佳条件制备Aβ试剂盒，检测50例正常人群，50例AD病人血液红细胞中与Hb结合的Aβ的OD值，然后绘制成散点图，如图2所示。The Aβ kit was prepared according to the optimal conditions selected in Example 2 of the present invention, and the OD value of Aβ bound to Hb in the blood red blood cells of 50 normal patients and 50 AD patients was detected, and then plotted as a scatter plot, as shown in FIG. 2 Show.
图2中实心圆圈代表每个正常的人Hb-Aβ的OD值。空心圆圈代表每个AD病人Hb-Aβ的OD值。图中的横线代表正常人与AD的均值水平。The solid circle in Fig. 2 represents the OD value of each normal human Hb-Aβ. Open circles represent the OD values of Hb-Aβ in each AD patient. The horizontal line in the figure represents the mean level of normal people and AD.
从图2中可知，AD病人血液红细胞中Hb-Aβ的量较正常人群增高显著。As can be seen from Figure 2, the amount of Hb-Aβ in the blood red blood cells of AD patients is significantly higher than that of the normal population.
图3为根据试剂盒检测的OD值绘制的ROC曲线，初步检测了Aβ试剂盒的敏感性为87.5％；特异性为81.25％；95％可信区间为0.7615-0.9612。Figure 3 is a plot of ROC plotted against the OD value detected by the kit. The sensitivity of the Aβ kit was initially determined to be 87.5%; the specificity was 81.25%; and the 95% confidence interval was 0.7615-0.9612.
据此可知，本发明实施例提供的Aβ试剂盒敏感性、特异性均较高，能够适用于血液红细胞中Hb-Aβ的检测。且利用本发明提供的Aβ试剂盒，能够避免溶血对样本的污染，使得检测结果更加准确可靠，依据Aβ蛋白循环于血液、脑脊液和脑间质液的特征， 可间接判定Aβ蛋白在其他部位的含量。It can be seen that the Aβ kit provided by the embodiments of the present invention has high sensitivity and specificity, and can be applied to the detection of Hb-Aβ in blood red blood cells. Moreover, by using the Aβ kit provided by the invention, the contamination of the sample by hemolysis can be avoided, and the detection result is more accurate and reliable, and the characteristics of the Aβ protein circulating in the blood, the cerebrospinal fluid and the interstitial fluid are determined according to the characteristics. The content of Aβ protein in other parts can be determined indirectly.
再者，含有血红蛋白的样本易于采集，容易被患者所接受，使得本发明提供的Aβ试剂盒能够适用于大规模的筛查，以便尽早的检测出或确定是否为AD病人，也便于对神经保护药物疗效的评估，其应用价值非常广泛。Furthermore, the sample containing hemoglobin is easy to collect and is easily accepted by the patient, so that the Aβ kit provided by the present invention can be applied to large-scale screening, so as to detect or determine whether it is an AD patient as early as possible, and also to facilitate neuroprotection. The evaluation of the efficacy of drugs has a wide range of applications.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., which are within the spirit and principles of the present invention, should be included in the scope of the present invention. within.
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Claims (10)

  1. 试剂盒，包括抗血红蛋白(Hb)抗体和抗Aβ抗体。Kits, including anti-hemoglobin (Hb) antibodies and anti-Aβ antibodies.
  2. 如权利要求1所述的试剂盒，还包括Hb-Aβ复合体标准蛋白。The kit of claim 1 further comprising a Hb-Aβ complex standard protein.
  3. 如权利要求1或2所述的试剂盒，所述抗Aβ抗体可为酶标记的抗Aβ抗体，或荧光或化学发光材料或胶体金标记的抗Aβ抗体，或生物素化的抗Aβ抗体。The kit according to claim 1 or 2, wherein the anti-Aβ antibody is an enzyme-labeled anti-Aβ antibody, or a fluorescent or chemiluminescent material or a colloidal gold-labeled anti-Aβ antibody, or a biotinylated anti-Aβ antibody.
  4. 如权利要求1或2所述的试剂盒，所述抗Aβ抗体为未标记的抗Aβ抗体，所述试剂盒还包括酶，或荧光素，或化学发光材料，或胶体金标记的二抗，或生物素化的二抗，所述二抗可特异性地与抗Aβ抗体相结合。The kit according to claim 1 or 2, wherein the anti-Aβ antibody is an unlabeled anti-Aβ antibody, and the kit further comprises an enzyme, or a fluorescein, or a chemiluminescent material, or a colloidal gold-labeled secondary antibody, Or a biotinylated secondary antibody that specifically binds to an anti-A[beta] antibody.
  5. 抗Hb抗体在制备用于检测待测样品中Aβ含量的试剂盒或试剂中的用途。Use of an anti-Hb antibody in the preparation of a kit or reagent for detecting Aβ content in a sample to be tested.
  6. 抗Hb抗体和抗Aβ抗体在制备用于检测待测样品中Aβ含量的试剂盒或试剂中的用途。Use of an anti-Hb antibody and an anti-Aβ antibody in the preparation of a kit or reagent for detecting Aβ content in a sample to be tested.
  7. 抗Hb抗体在制备用于检测待测样品是否患有阿尔茨海默(AD)或患有AD风险的试剂盒或试剂中的用途。Use of an anti-Hb antibody in the preparation of a kit or reagent for detecting whether a sample to be tested has Alzheimer's (AD) or is at risk of AD.
  8. 如权利要求5-7中任一项所述的用途，所述待测样品来源于人、动物和/或培养细胞，优选来源于AD患者或者具有患AD风险的人群。The use according to any one of claims 5 to 7, wherein the sample to be tested is derived from a human, an animal and/or a cultured cell, preferably from an AD patient or a population at risk of developing AD.
  9. 如权利要求5-7中任一项所述的用途，所述待测样品为血液样品，或来自于血液的红细胞胞浆蛋白，或其它任何含有血红蛋白的组织。The use according to any one of claims 5 to 7, wherein the sample to be tested is a blood sample, or a erythrocyte cytoplasmic protein derived from blood, or any other hemoglobin-containing tissue.
  10. 检测Aβ的方法，其包括在检测过程中使用抗Hb抗体和 抗Aβ抗体。 A method for detecting Aβ, which comprises using an anti-Hb antibody and a detection process Anti-Aβ antibody.
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