CN109696550B - Aqueous solution for stabilizing luminescent ELISA body fluid sample for cerebral apoplexy - Google Patents
Aqueous solution for stabilizing luminescent ELISA body fluid sample for cerebral apoplexy Download PDFInfo
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Abstract
The invention provides an aqueous solution for stabilizing a body fluid sample for luminescence ELISA, wherein the body fluid sample contains a nerve injury marking protein component, solutes in the aqueous solution for stabilizing the body fluid sample comprise human serum, animal serum albumin, inorganic alkali metal salt, Tris alkali, a protein denaturant and a nonionic surfactant, and the pH value of the aqueous solution is 6.7-7.6. When the nerve cell damage marker protein component NF-H is detected from a body fluid sample (for example, peripheral blood) by using an ELISA method, the body fluid sample stabilizing aqueous solution can effectively reduce the interference of other components in the body fluid sample, improve the accuracy and repeatability of a detection result, and stably and accurately detect pg-grade NF-H in the body fluid, so that the disease degree, the treatment effect, the prognosis and the like of a cerebral apoplexy patient can be diagnosed.
Description
Technical Field
The invention relates to the field of biomedical detection, in particular to an aqueous solution for stabilizing a body fluid sample of a patient suffering from stroke (cerebral stroke), an in-vitro detection kit and a detection system.
Background
Stroke is commonly called stroke, and is caused by brain injury caused by cerebrovascular occlusion or rupture, so that the damage is serious. Epidemiological research finds that the disability rate of stroke ranks first and the fatality rate ranks second in China, and is only second to cancer. No disease can cause people to have facial distortion, numbness of limbs and paralysis in bed instantly like stroke, so that people lose dignity in life. At present, more than 370 million stroke cases are newly added in China every year, the number of surviving stroke patients is nearly 2000 million, and the number of the patients is in a rapid rise period. Hypertension, diabetes and heart disease are all risk factors for cerebral apoplexy, and middle-aged and elderly people are high risk groups for apoplexy. Therefore, there is a need to establish a fast and accurate method for diagnosing stroke.
The present diagnosis method of cerebral apoplexy is mainly based on imaging, has low accuracy and high cost, and brings great burden to the social health industry. In addition, the patients with apoplexy have rapid changes of illness state and obvious individual differences, which brings great difficulties to diagnosis and treatment.
In addition to the diagnostic imaging, there is a method for diagnosing cerebral stroke by measuring the content of neurofilament protein (NFP) in cerebrospinal fluid by ELISA. Neurofilament protein (NFP) is one of the most abundant proteins in the central nervous system, and is a major component of the channels that constitute neurofilament-nerve cell intercommunication. The neurofilament protein is assembled by three specific protein subunits (NF-L, NF-M and NF-H). Acute ischemic stroke directly causes death of a large number of nerve cells, which are then broken down and release a large number of structural proteins, including NF-H. Due to the blood-brain barrier, NF-H is mostly retained in cerebrospinal fluid. To detect NF-H in the cerebrospinal fluid, a spinal puncture method is generally used. However, the method causes serious physical and psychological pains to the patients again, and brings huge medical burden to the society and families.
As a method for detecting NF-H in cerebrospinal fluid, an enzyme-linked immunosorbent assay (ELISA) method is more popular, and the basic principle is to use the specific reaction of an antigen and an antibody and detect an unknown antigen or antibody by using a known antigen or antibody. The ELISA test method has the characteristics of sensitivity, specificity, economy, simplicity, safety and the like, and is particularly widely applied to the aspects of disease diagnosis, curative effect observation, preventive medicine and the like at present.
As a classical method for measuring trace antigens and antibodies, the ELISA method is widely applied to clinical examination and diagnosis, and has the advantages of mature technology, wide application, high analysis sensitivity, good analysis specificity and simple operation. The basic operation process is as follows: in the first step, antigen (or antibody) is coated on a solid phase carrier (such as a polystyrene plastic enzyme label plate) and placed in a refrigerator at 4 ℃ overnight. And step two, washing the coated plate with buffer solution for three times, adding the sample to be detected, and then placing the sample in a 37 ℃ incubator (or room temperature) for reaction for 1-2.5 hours. And thirdly, washing the plate three times by using a buffer solution, adding an enzyme-labeled antibody, and then placing the plate in a 37 ℃ incubator (or room temperature) for reaction for 1-2.5 hours. And step four, washing the plate three times by using buffer solution, adding substrate solution for color development, stopping reaction by using sulfuric acid, and processing the plate on a machine to judge a result.
As a latest technology for ELISA development, luminescence ELISA couples a luminescent group to a detection antibody, and directly measures the luminescence amount thereof to quantify the antigen, thereby reducing additional interference caused by enzyme/substrate reaction and greatly improving detection sensitivity.
No matter through imaging diagnosis or ELISA cerebrospinal fluid detection, at present, the main difficulties of cerebral apoplexy diagnosis are as follows: early asymptomatic or mild symptoms, difficult to detect; real-time diagnosis makes it difficult to distinguish between hemorrhagic or ischemic stroke; the type of damaged cells and/or treatment effect cannot be judged for long-term treatment, so that the diagnosis level of stroke is generally low. Therefore, early diagnosis, rapid diagnosis and accurate diagnosis, particularly the diagnosis can be completed without spinal puncture to obtain cerebrospinal fluid, and the method has particularly important effect on reducing the disability rate and the fatality rate of the cerebral apoplexy.
Disclosure of Invention
The inventor of the present invention has conducted long-term and intensive studies on body fluid samples, particularly peripheral blood, of patients with cerebral stroke, and found that a very small amount (< 100pg/mL) of NF-H also enters the peripheral blood system. Based on this finding, the present invention provides a technical means for diagnosing stroke by performing luminescence ELISA detection on body fluids typified by peripheral blood serum and measuring the concentration of a marker protein component (for example, NF-H) in the peripheral blood serum, and also avoiding high-risk spinal puncture.
The luminescence ELISA is characterized by the ability of recognizing weak signals, and the sensitivity of the luminescence ELISA is 10-1,000 times that of the traditional enzyme-catalyzed ELISA. The detection sensitivity is greatly improved, and extremely high requirements are provided for the capability of distinguishing true and false positive signals of a detection system, especially the effect of a detection reagent.
In order to reduce noise and significantly reduce false positives while enhancing a signal in luminescence ELISA, a first aspect of the present invention provides an aqueous solution for stabilizing a body fluid sample for luminescence ELISA, wherein the body fluid sample contains a neural injury marker protein component, solutes in the aqueous solution include human serum, animal serum albumin, an inorganic alkali metal salt, Tris base, a protein denaturing agent, and a nonionic surfactant, and the pH of the aqueous solution is 6.7 to 7.6.
Preferably, the pH value of the aqueous solution for stabilizing a body fluid sample is 7.0 to 7.4.
The animal serum albumin is derived from cattle, sheep, horses, rabbits, chickens, and is preferably bovine serum albumin.
Preferably, the concentration of human serum is 10 to 50% by volume per 100ml of the aqueous solution for body fluid sample stabilization; the concentration of the bovine serum albumin is 0.1-10 mass/volume percent; the concentration of the inorganic alkali metal salt is 110 mM-200 mM; the concentration of Tris alkali is 10 mM-25 mM; the concentration of the protein denaturant is 0.01 to 0.5mM, and the concentration of the nonionic surfactant is 0.05 to 5.0 vol%.
The inorganic alkali metal salt is preferably selected from alkali metal hydrochlorides; the protein denaturant is selected from urea and sodium dodecyl sulfate; the non-ionic surfactant is selected from Tween-20, Tween-40, Tween-60, Tween-80 and Triton X-100.
Preferably, the inorganic alkali metal salt is sodium chloride and potassium chloride, the protein denaturant is urea, and the nonionic surfactant is tween-20.
More preferably, the human serum has a concentration of 10 to 35% by volume per 100ml of the aqueous solution for stabilizing a body fluid sample; the concentration of the bovine serum albumin is 0.5 mass/volume percent to 5 mass/volume percent; the concentration of the Tween-20 is 0.1 to 3.0 volume percent; the concentration of the urea is between 0.05 and 0.25mM, and the concentration of the sodium chloride is between 110 and 150 mM; the concentration of the potassium chloride is 2.0 mM-3 mM; and the concentration of Tris base is 15 mM-25 mM.
At least a portion of the sodium chloride, potassium chloride, and the Tris base are provided in the form of Tris buffered saline.
The nerve injury marker protein component is selected from Glial Fibrillary Acidic Protein (GFAP), neurofilament protein (NFP) heavy chain (NF-H), neurofilament protein (NFP) medium chain (NF-M), neurofilament protein (NFP) light chain (NF-L), and neuroenolase.
The body fluid is blood, serum, cerebrospinal fluid, interstitial fluid, urine, saliva or sweat, preferably human peripheral blood serum. More preferably, the human peripheral blood serum contains NF-H as the nerve injury marker protein component; even more preferably, the human peripheral serum contains NF-H at pg level per ml.
The human peripheral blood serum is from asymptomatic volunteers, slightly symptomatic patients with cerebral apoplexy, patients with acute stage of cerebral apoplexy, patients with convalescent stage of cerebral apoplexy, or patients with sequela stage of cerebral apoplexy.
The luminescence ELISA is double-antibody sandwich ELISA.
In a second aspect, the present invention provides an in vitro diagnostic kit for luminescence ELISA, comprising the aqueous solution for stabilization of a body fluid sample according to the first aspect, and optionally an antibody against the nerve injury marker protein component.
The antibody against the nerve injury marking protein component is preferably an anti-NF-H protein antibody.
The anti-NF-H protein antibody comprises: a capture antibody and a detector antibody; wherein the capture antibody and the detection antibody are anti-NF-H antibodies respectively aiming at different epitopes of NF-H protein.
Preferably, a luminophore is coupled to the detector antibody.
The luminescent means of the luminescent group includes a chemical means, preferably including enzymatic or chemical luminescence, or a physical means, preferably including any one of electroluminescence, fluorescence, and fluorescence resonance energy transfer.
Preferably, the capture antibody is a monoclonal antibody or a polyclonal antibody, and the detector antibody is a monoclonal antibody or a polyclonal antibody.
The polyclonal antibody is from any one of mice, rabbits, chickens, sheep, guinea pigs, donkeys and horses.
The in vitro diagnostic kit of the invention can be used for in vitro diagnosis of cerebral apoplexy, hemorrhagic brain disorder, traumatic brain disorder, neurodegenerative disease (Alzheimer disease, cerebrovascular dementia, Parkinson disease, Huntington's chorea, amyotrophic lateral sclerosis, etc.). Preferably, the in vitro diagnostic kit is used for the diagnosis of cerebral stroke.
In a third aspect, the invention provides an in vitro test device for cerebral stroke diagnosis or prognosis evaluation, which comprises a luminescence ELISA determinator and an in vitro diagnostic kit for luminescence ELISA according to the second aspect.
Preferably, the ELISA meter is a high sensitivity meter with a detection sensitivity of less than 10 pg.
The invention has the beneficial technical effects that:
1. the aqueous solution for stabilizing the body fluid sample can effectively reduce the interference of matrix effect, improve the signal-to-noise ratio and improve the accuracy and the repeatability of a detection result, and is particularly suitable for luminescence ELISA.
2. The in-vitro diagnostic kit can accurately detect the concentration of NF-H protein in peripheral blood of a patient with cerebral apoplexy, can be used for clinical diagnosis and early screening of cerebral apoplexy, and can also be used for monitoring treatment effect and evaluating prognosis and recurrence risk.
3. The invention can effectively avoid spinal puncture with high risk. Through technical innovation, the cerebral apoplexy is diagnosed based on detecting trace NF-H in peripheral blood, and the detection cost can be greatly reduced.
Drawings
FIG. 1 shows the differences in NF-H content in serum samples from patients with stroke and healthy control populations in test example 2.
FIG. 2 shows the increase of NF-H levels in serum samples from patients with stroke over time.
FIG. 3 is a MSD NF-H ELISA standard curve.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting of the invention. Various modifications and improvements may be made by those skilled in the art based on the basic idea of the invention, but it is within the scope of the invention as long as it does not depart from the basic idea of the invention.
Aqueous solution for stabilizing body fluid sample
Although the inventor finds that a trace amount of NF-H protein exists in body fluid of a patient with cerebral apoplexy, the content of the marker protein in the body fluid is extremely small, generally in the pg level, and a large amount of non-antigen components exist in the body fluid, so that the detection cannot be completed by adopting a conventional ELISA method.
In view of the above problems, the present inventors have found that, if a body fluid sample is appropriately diluted with a specific body fluid sample-stabilizing aqueous solution, it is possible to significantly shield an interfering component in the body fluid sample to be tested, so that the signal/noise ratio is greatly improved, and thus, it is possible to stably and reliably perform luminescence ELISA detection of cerebral apoplexy using the body fluid sample.
In a first aspect, the present invention provides an aqueous solution for stabilizing a body fluid sample for diluting a body fluid of a patient suffering from cerebral stroke to thereby detect the NF-H protein content therein. In the present invention, the body fluid sample may include blood, urine, saliva, sweat, interstitial fluid, cerebrospinal fluid, and the like. The body fluid sample is preferably blood in view of the NF-H content, the ease of collection and handling of the sample, and the like. The blood may be fresh blood or dried blood sample. Particularly preferably, the blood sample is peripheral blood serum obtained by standing fresh whole blood of a patient with cerebral apoplexy or a suspected patient and centrifuging.
In the aqueous solution for stabilizing the body fluid sample for ELISA, solutes include human serum, animal serum albumin, an inorganic alkali metal salt, Tris base, a protein denaturant and a nonionic surfactant, and the pH of the aqueous solution is 6.7 to 7.6.
The animal serum albumin may be derived from mammals other than humans, more preferably from cattle, sheep, horses, rabbits, chickens, etc., and still more preferably bovine serum albumin.
The inorganic alkali metal salt may be an alkali metal salt of hydrochloric acid, sulfuric acid, or phosphoric acid. The concentration of the inorganic alkali metal salt may be in the range of 110 to 200mM, more preferably 120 to 150mM, per 100ml of the aqueous solution for stabilizing a body fluid sample.
The protein denaturant may be urea or sodium lauryl sulfate. The concentration of the protein denaturing agent may be in the range of 0.01 to 0.5mM, more preferably in the range of 0.05 to 0.25mM, per 100ml of the aqueous solution for stabilizing a body fluid sample.
The nonionic surfactant can be Tween-20, Tween-40, Tween-60, Tween-80 and Triton X-100. The concentration of the nonionic surfactant may be in the range of 0.05 to 5.0 vol%, more preferably 0.1 to 3.0 vol%, per 100ml of the aqueous solution for stabilizing a body fluid sample.
Preferably, the aqueous solution for body fluid sample stabilization is an aqueous solution for serum sample stabilization.
In a particularly preferred embodiment, 100ml of the aqueous solution for stabilizing a body fluid sample comprises 10 to 50 vol% of human serum, 0.1 to 5.0 mass/vol% of bovine serum albumin, 0.05 to 5.0 vol% of Tween-20, 0.05 to 0.25mM of urea, 110 to 150mM of sodium chloride, 2.0 to 3.0mM of potassium chloride, 15 to 25mM of Tris base, and sterile distilled water. The aqueous stabilizing solution is preferably used for diluting a blood sample to be tested. The aqueous solution for stabilizing the blood sample can complete the treatment and dilution of the blood sample at one time, is simple and convenient to operate, and effectively reduces the interference of other components in serum.
For example, the aqueous solution for stabilizing a body fluid sample can be prepared as follows:
a small amount of sterile distilled water is prepared, normal human serum (BioRecamationIVT, USA) is added according to the proportion of 10-50% of the volume, bovine serum albumin (Sigma, USA) is added according to the proportion of 0.1-5.0 mass/volume, Tween 20(Sigma-Aldrich, USA) is added according to the proportion of 0.05-5.0% of the volume, urea (Sigma-Aldrich, USA) is added according to the proportion of 0.05-0.25 mM of the weight, sodium chloride (Sigma-Aldrich, USA) is added according to the proportion of 110-150 mM of the weight, potassium chloride (Sigma-Aldrich, USA) is added according to the proportion of 2.0-3.0 mM of the weight, alkali (Sigma-Aldrich, USA) is added according to the proportion of 15-25 mM of the weight, and sterile distilled water is added to be mixed uniformly to make the total volume be 100 ml.
Second, in vitro diagnostic kit
In a second aspect, the invention provides an in vitro diagnostic kit for luminescence ELISA, preferably for the detection of NF-H proteins using an ELISA double antibody sandwich method.
The ELISA method is a common antibody or antigen determination method. The ELISA method mainly comprises three links of antigen separation and purification, antiserum preparation and antiserum reaction. ELISA is based primarily on the determination of the enzyme-catalyzed reaction of antigen-antibody immune complexes by the associated enzyme. For detection of an antigen, methods such as direct antigen measurement, competitive antigen measurement, and antigen measurement by a double antibody sandwich method are commonly used in ELISA.
The double antibody sandwich method is currently the most common solution for determining antigens. In the double antibody sandwich method, known antibodies are adsorbed in small holes on a microtiter plate (plastic plate) and washed once; specific antigen-antibody binding occurs by adding a test antigen (e.g., test serum containing NF-H protein); then washing away the excess unbound antibody; adding a detection antibody which is in specific reaction with the antigen to be detected and is coupled with enzyme or a luminescent group in advance to form a sandwich; adding a substrate for the enzyme, or a necessary luminescence-inducing agent. The substrate can generate colored substances after being degraded by enzyme, or the luminescent group can generate visible light or fluorescence, and the color or the light can be quantitatively detected by a corresponding ELISA tester, so that the existence and the quantity of the antigen can be determined.
The inventor finds that the content of the nerve injury marker protein (such as NF-H) in the body fluid of a cerebral apoplexy patient is extremely small, generally in the pg level, and in order to accurately detect the content, the invention provides a luminous ELISA diagnostic kit.
Specifically, the luminescent ELISA diagnostic kit of the present invention comprises the aqueous solution for sample stabilization according to the above first aspect, further comprises a capture antibody, a probe antibody, a corresponding enzyme substrate as needed, and instructions for use and the like.
In the luminescent ELISA diagnostic kit of the present invention, a luminescent group is coupled to the detection antibody. Different from the traditional ELISA, the reaction carrier of the luminescence ELISA is an opaque plate (hereinafter, sometimes referred to as an enzyme label plate), the luminescence system is photon absorption, the detection instrument utilizes the photon absorption of a photomultiplier, and the sensitivity of the luminescence ELISA is higher.
The light emitting means of the light emitting group may include chemical means or physical means. The chemical means includes enzymatic or chemiluminescent and the physical means includes electroluminescence, fluorescence, or fluorescence resonance energy transfer.
The chemiluminescence immunoassay method has the advantages of strong specificity, stability, rapidness, wide detection range, simple operation and the like. The principle of the chemiluminescent system is to act an enzyme on a light-emitting substrate. Under the action of enzyme, the substrate reacts chemically and releases a great deal of energy to generate excited intermediate. Such excited state intermediates, when they return to a stable ground state, can simultaneously emit photons. The light quantum yield, which is proportional to the amount of the substance to be measured in the sample, can be measured by using the luminescence signal measuring instrument. From this, a standard curve can be established and the content of the substance to be measured in the sample can be calculated. Specifically, horseradish peroxidase (HRP) catalyzed Luminol (Luminol) substrate luminescence system may be used.
As an example of the chemiluminescent solution, for example, a solution of tris (hydroxymethyl) aminomethane containing luminol or p-cresol is prepared as solution A, and citric acid and anhydrous Na are separately prepared 2 HPO 4 And mixing the solution A and the solution B in a ratio of 1: 1 before use.
Electrochemiluminescence (ECL) is a process in which a light-emitting substance forms a high-energy excited state after electrochemical and chemical reactions on the surface of an electrode, and then relaxes to generate light. A common ECL reagent is 9 which,10-Diphenylanthracene (DPA), ruthenium bipyridine (Ru (byp) 3 2+ ) Oxalate peroxide, luminol, quantum dots, and the like.
Fluorescence and Fluorescence Resonance Energy Transfer (FRET) are used as a tool capable of detecting nano-scale distance change in living organisms and in vitro, and have wide application in biomacromolecule interaction, immunoassay, nucleic acid detection and the like. FRET has the advantages of high sensitivity, wide application, high analysis speed and the like. Among them, Quantum Dots (QDs) have unique optical properties (wide absorption, narrow emission, photobleaching resistance, and fluorescence tunability), making them very suitable for FRET studies.
The in vitro diagnostic kit for luminescence ELISA according to the present invention preferably employs mouse NF-H monoclonal antibody as a capture antibody. Herein, the term "capture antibody" refers to an antibody coated on a solid phase microplate (e.g., microtiter plate). In addition, the in vitro diagnostic kit also preferably comprises chicken-derived NF-H polyclonal antibody as a detection antibody. The term "detection antibody" as used herein refers to a specific antibody in the kit that binds to the antigen to be detected and is not coated on a solid-phase microplate. The "capture antibody" and "detection antibody" can be prepared from commercially available products, or by hybridoma cell technology or immunized animals, using techniques that are conventional in the art.
The in vitro diagnostic kit for luminescence ELISA of the present invention can be used for rapid diagnosis of traumatic brain injury or primary brain injury. For example, the in vitro diagnostic kit of the present invention can be used for rapid diagnosis of diseases such as cerebral stroke, hemorrhagic brain disorder, traumatic brain disorder, neurodegenerative disease (Alzheimer's disease, cerebrovascular dementia, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, etc.).
Preferably, the in vitro diagnostic kit for luminescence ELISA of the present invention can be used for detecting NF-H protein in serum of patients with cerebral stroke.
Any reagents or means required for detection, such as pre-coated plates, washing solutions, color developers, stop solutions, and the like, may also be included in the kit of the present invention. If a solid phase microplate (e.g., a microtiter plate) is included in the kit, the wells of the microplate are preferably pre-coated plates coated with the capture antibody.
Third, external detection system
The invention also provides an in vitro detection device for cerebral apoplexy diagnosis or prognosis evaluation, which comprises a luminescence ELISA determinator and an in vitro diagnosis kit for luminescence ELISA according to the second aspect.
Examples of the luminescence ELISA assay used in the present invention include, but are not limited to, MSD ELISA Imagers of Meso Scale Discovery, AlphaLISA of Perkinelmer, and Simoa HD1 of Quanterix.
Specifically, if the blood of a patient with cerebral apoplexy is taken as a body fluid sample to be detected, the basic operation steps of the ELISA tester for detection comprise:
preparation of serum to be tested
Blood of a patient suffering from cerebral apoplexy is extracted intravenously; standing for 1-2 h at room temperature to separate blood cells and serum naturally; and uniformly mixing the serum with the aqueous solution for stabilizing the serum sample according to the ratio of 2: 1-1: 10 for later use.
Enzyme linked immunosorbent assay
A48-or 96-well microtiter plate (microplate) was prepared. Adsorbing the NF-H monoclonal antibody in a small hole on a microtiter plate, and washing once by PBS; adding the diluted serum of a patient suffering from cerebral apoplexy prepared as above into the pores of the microtiter plate; then washing away the excess unbound antibody; a detector antibody, preferably an antiserum of animal origin (i.e., a polyclonal antibody), which has been previously coupled to a luminophore is then added to the wells. The wells of the microplate are then washed 3-4 times.
Antigen content determination
After washing, if desired, an inducer of the luminophore (e.g., a chemiluminescent enzyme substrate, etc.) is added to the wells of the microplate, which inducer causes the luminophore to produce color or luminescence. And (3) immediately putting the ELISA plate into a corresponding high-sensitivity ELISA detector after adding the substrate, and reading signal data.
By diluting a serum sample and using a high-sensitivity ELISA detector, the method adopts an ELISA method to detect the anti-neurofilament protein NF-H antibody, and can detect pg-level NF-H protein content in the serum of a patient with cerebral apoplexy.
The experimental research of the inventor discovers that the content of the NF-H protein in the serum caused by brain injury after the cerebral apoplexy is abnormally increased. The serum NF-H protein level directly reflects the brain damage degree and the damaged cell type (whether the ischemic or hemorrhagic brain cell is damaged) caused by the stroke, and can be used as an important reference index for evaluating the curative effect of the stroke patient and making a rehabilitation scheme. According to the protein content, the invention can be used for diagnosing the type and relative quantity of the brain cell damage of a patient with cerebral apoplexy, and can diagnose asymptomatic or slightly symptomatic cerebral apoplexy, the acute stage of cerebral apoplexy, the convalescent stage of cerebral apoplexy, the sequela stage of cerebral apoplexy and the like. The invention can also be used for early warning and monitoring the curative effect and the course of disease of the cerebral apoplexy, and guiding the prevention of the cerebral apoplexy and the physical examination and detection.
In addition to blood samples, the in vitro test device for cerebral stroke diagnosis or prognosis evaluation of the present invention is also suitable for testing other body fluid samples, such as cerebrospinal fluid, interstitial fluid, urine, saliva, sweat, etc.
The present invention will be further explained or illustrated below by way of examples, which should not be construed as limiting the scope of the invention.
Examples
Preparation example 1
Preparation of aqueous solution 1 for stabilization of body fluid sample
Prepare 50mL of sterile distilled water, add 10mL of normal human serum (BioReclamatoniivt, USA), 1g of bovine serum albumin (Sigma, USA), 2mL of Tween 20(Sigma-Aldrich, USA), urea (Sigma-Aldrich, USA) at a final concentration of 0.05mM, sodium chloride (Sigma-Aldrich, USA) at a final concentration of 110mM, potassium chloride (Sigma-Aldrich, USA) at a final concentration of 2.0mM, and Tris base (Sigma-Aldrich, USA) at a final concentration of 10mM, and finally add the balance of sterile distilled water to mix well and make up to 100 mL.
Preparation example 2
Preparation of aqueous solution 2 for stabilization of body fluid sample
Prepare 50mL sterile distilled water, add 20mL normal human serum (BioReclamatoIVT, USA), 2g bovine serum albumin (Sigma, USA), 2mL Tween 20(Sigma-Aldrich, USA), urea (Sigma-Aldrich, USA) at a final concentration of 0.08mM, sodium chloride (Sigma-Aldrich, USA) at a final concentration of 125mM, potassium chloride (Sigma-Aldrich, USA) at a final concentration of 2.3mM, Tris base (Sigma-Aldrich, USA) at a final concentration of 15mM, add the balance sterile distilled water, mix well and make up to 100 mL.
Preparation example 3
Preparation of aqueous solution for stabilization of body fluid sample 3
Prepare 50mL sterile distilled water, add 30mL normal human serum (BioReclamatoIVT, USA), 2g bovine serum albumin (Sigma, USA), 2mL Tween 20(Sigma-Aldrich, USA), urea (Sigma-Aldrich, USA) at a final concentration of 0.1mM, sodium chloride (Sigma-Aldrich, USA) at a final concentration of 137mM, potassium chloride (Sigma-Aldrich, USA) at a final concentration of 2.7mM, Tris base (Sigma-Aldrich, USA) at a final concentration of 19mM, finally add sterile distilled water and mix well and make up to 100 mL.
Preparation example 4
Preparation of aqueous solution 4 for stabilization of body fluid sample
20mL of sterile distilled water was prepared, 50mL of normal human serum (BioReclamatoIVT, USA), 1g of sheep serum albumin (Sigma, USA), 5mL of Tween 20(Sigma-Aldrich, USA), urea (Sigma-Aldrich, USA) at a final concentration of 0.25mM, sodium chloride (Sigma-Aldrich, USA) at a final concentration of 150mM, potassium chloride (Sigma-Aldrich, USA) at a final concentration of 3mM, Tris base (Sigma-Aldrich, USA) at a final concentration of 25mM were added, and sterile distilled water was added to mix well and made to volume of 100 mL.
Preparation example 5
Preparation of NF-H Capture antibody (mouse NF-H monoclonal antibody) solution
A mouse NF-H monoclonal antibody (Novus, anti-NF-H mouse monoclonal antibody NAP4) was diluted to 1. mu.g/mL with Phosphate Buffered Saline (PBS), and stored at 4 ℃ until use.
Preparation example 6
Preparation of Probe antibody (polyclonal antibody)
Chicken polyclonal antibody (Abcam, anti-NF-H, ab4680) was coupled to the SULFO-tagged NHS Ester of MSD (SULFO-Tag-NHS-Ester) according to the manufacturer's instructions (MSD, R91AO-1) and stored at 4 ℃ until use.
Test example 1 efficacy test for detecting serum NF-H protein of the present invention
1. Experimental method
(1) MSD ELISA NF-H kit detection step:
a96-well MSD ELISA plate was prepared for coating NF-H capture antibody. The specific procedures are as follows: mu.L of an antibody solution (1. mu.g/mL, mouse NF-H monoclonal antibody, prepared according to the preparation method of preparation example 5) was added to the wells of the microplate, and the antibody was uniformly spread on the bottom of the wells by gentle shaking. The wells were left overnight at 4 ℃ to immobilize the antibody to the bottom of the wells. The wells of the plate wells were washed 2 times with 120. mu.L of PBS to remove components such as the unfixed antibody. Add 120. mu.L blocking solution (3% BSA/PBS) to each microplate well, place the microplate in the microplate shaker at 200-500 RPM speed and mix for 1 hour at room temperature.
A standard Bovine NF-H protein sample (USbiological Life Sciences, Bovine NF-H) was prepared. The standard bovine NF-H protein sample was diluted with the body fluid sample-stabilizing aqueous solution 1 obtained in preparation example 1 to 8 concentration gradients set in step (2). Two 25 microliter diluted samples are added into two adjacent enzyme-labeled plate holes (used as repeated samples), and the enzyme-labeled plates are placed on a microplate oscillator and mixed for 1 hour at room temperature at the speed of 200-500 RPM.
The detection antibody (chicken-derived NF-H polyclonal antibody) was coupled in advance (Sulfo-tag according to the product manual of MSD company; 4C storage).
Before the experiment, the coupling detection antibody is diluted to 2 μ g/mL by a solution (obtained by dissolving 2% bovine serum albumin and 0.2% Tween 20 in a tris buffer solution), 25 μ L of the antibody diluent is added to an ELISA plate hole, and the ELISA plate is placed in a microplate shaker and mixed for 1 hour at room temperature at a speed of 200-500 RPM.
The microplate was washed 4 times with a TBST wash (prepared by diluting Tween 20 to 0.2% by volume with Tris Buffer (TBS)). mu.L of MSD luminescence solution (1X), which was obtained by diluting 4XMSD luminescence solution (MSD Read Buffer (4X) Cat # R92TC) with sterile distilled water, was added to the wells of the microplate, and immediately the ELISA plate was placed in an MSD Imager 2400 to Read the signal data and the concentration was automatically calculated with reference to MSD Benchwork preset program.
(2) MSD ELISA NF-preparation of the Standard Curve and measurement of the sensitivity (Lower Limit of Detection; LLOD): standard bovine NF-H protein was diluted with aqueous body fluid sample stabilization solution 1 according to the following gradient concentrations: 0, 2.5, 9.8, 39.1, 156.3, 625, 2500, and 10000 (pg/mL). And (3) calculating the related concentration and the lowest measured concentration according to the step (1) to prepare a standard curve.
(3) Measuring the stability and precision of the MSD NF-H ELISA kit:
3 experiments were performed according to experimental steps (1) and (2) to obtain 3 independent standard curve measurements. Each experiment was 8 months apart. The detection stability and the measurement precision of the kit for at least two years are obtained by comparing the results of the 3 experiments.
2. Results of the experiment
(1) MSD NF-H ELISA Standard Curve and sensitivity:
the experiments were performed 3 times according to experimental procedures (1) and (2).
The results of 3 times of experiments show that the method has wide linear range and high sensitivity. The detection sensitivity (lowest detection concentration; LLOD) of 3 experiments on blood NF-H protein is respectively 5.62, 7.99 and 8.71pg/mL, and the concentrations are all lower than 10pg/mL, so that the kit has great clinical diagnosis value. The results are shown in Table 1 and FIG. 3.
Table 1: MSD NF-H ELISA standard curve and sensitivity
Experiment 1
Experiment 3
(2) Stability and precision of MSD NF-H ELISA kit:
according to the independent 6 times of experiment results separated by 4 months, the NF-H kit disclosed by the invention has high stable detection results and detection accuracy. The Relative Error (RE) and Coefficient of Variation (CV) calculated from the results of 6 independent experiments were less than 10% in the concentration range of 39pg/mL or more. The results of the experiment are shown in Table 2.
Table 2: stability and precision of MSD NF-H ELISA kit
Statistical results of 6 experiments:
test example 2 MSD ELISA method for detecting NF-H content in serum of patient with cerebral apoplexy
1. Experimental methods
(1) The conditions of the apoplexy patients are as follows:
bringing into standard: the patient is 40-85 years old, and the hospitalization time is within 48 h; the diagnosis of ischemic or hemorrhagic acute cerebral apoplexy is carried out by adopting the classical methods such as CT and the like.
Exclusion criteria: with mental illness or severe disturbance of consciousness or language communication, the examination cannot be completed; the history of serious head trauma or stroke occurs within 3 months; performing arterial puncture at a non-compression part in 14d or performing major surgery; intracranial arteriovenous malformations or aneurysms.
Thirdly, a sample collection method: 3-5 serum samples were collected for each case, including specifically: in the acute stage: within 7 days after stroke occurred: collecting serum samples for 1 time every other day, and collecting 2-3 times in total; a recovery period: 8-30 days after the occurrence of stroke: collecting a serum sample for 1 time; in the sequela stage: serum samples were collected 1 time.
34 stroke patients, and 25 normal persons of age and sex matched were included in the experiment.
(2) Collecting and processing blood samples:
1mL of blood samples were collected from the patients at 0, 8 hours, 24 hours, 72 hours (day 3), 120 hours (day 5), 192 hours (day 8) and 240 hours (day 10) with the time of admission as 0h, respectively. Blood samples were collected from normal persons at 8 am. Standing for 1-2 h at room temperature, separating serum, preparing into dry powder by using a low-temperature freeze dryer, and storing for later use.
(3) Detecting the NF-H protein content of the blood sample:
the dried blood sample was dissolved in 1mL of purified water. The sample was mixed with the aqueous solution for sample stabilization 1 prepared in preparation example 1 at a ratio of 1: 1, 25. mu.L of the diluted sample (as a duplicate sample) was added to each of 2 wells of the microplate, and the NF-H content in the sample was measured in accordance with the experimental procedure (1) in test example 1.
2. Results of the experiment
According to the detection result of total 67 blood samples (42 samples belong to stroke patients and 25 samples belong to healthy people), the average value of the NF-H content of the blood of the stroke patient group is 116.92pg/mL, which is obviously higher than that of the healthy people and the control group by 3.61 pg/mL. The results are shown in Table 3 and FIG. 1. And, as shown in fig. 2, it was found that the NF-H content in blood was in an increasing trend as the time after the occurrence of the cerebral stroke was increased.
Table 3: NF-H content in blood of cerebral apoplexy patient and healthy person control group
The invention adopts ELISA method to detect the NF-H protein content in the serum of the patient with apoplexy, thus obtaining extremely high sensitivity. The invention can accurately measure the content change level of NF-H protein caused by stroke in the peripheral blood of a stroke patient, makes up for the defects of the traditional imaging method or cerebrospinal fluid analysis method, and can carry out asymptomatic or slightly symptomatic stroke diagnosis. The popularization and the application of the invention are expected to reduce the incidence rate of the cerebral apoplexy, guide the formulation of a apoplexy treatment scheme, evaluate the curative effect of the apoplexy and reduce the sequelae of the apoplexy. Therefore, the method has important and wide clinical diagnostic value.
Claims (2)
1. An aqueous solution for stabilizing a body fluid sample of a luminescent ELISA for cerebral apoplexy, the body fluid sample being human peripheral blood serum containing a neurofilament heavy chain NF-H as a nerve injury marker protein component, the human peripheral blood serum being from an asymptomatic volunteer, a patient with mild symptoms of cerebral apoplexy, a patient in an acute stage of cerebral apoplexy, a patient in a convalescent stage of cerebral apoplexy, or a patient in a sequela stage of cerebral apoplexy, and the luminescent ELISA being a double antibody sandwich ELISA, and
100ml of the aqueous solution for stabilizing a body fluid sample comprises 10 to 50 vol% of human serum, 0.1 to 5.0 mass/vol% of bovine serum albumin, 0.05 to 5.0 vol% of tween-20, 0.05 to 0.25mM of urea, 110 to 150mM of sodium chloride, 2.0 to 3.0mM of potassium chloride, 15 to 25mM of Tris alkali, and sterile distilled water, and the pH value of the aqueous solution is 6.7 to 7.6.
2. The aqueous solution for body fluid sample stabilization according to claim 1, wherein,
NF-H contained in the peripheral serum of the human per milliliter is at a level of pg grade.
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