CN117491650A - Quantitative detection kit, detection method and application of peripheral blood Hb-Abeta complex - Google Patents
Quantitative detection kit, detection method and application of peripheral blood Hb-Abeta complex Download PDFInfo
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- CN117491650A CN117491650A CN202311439856.4A CN202311439856A CN117491650A CN 117491650 A CN117491650 A CN 117491650A CN 202311439856 A CN202311439856 A CN 202311439856A CN 117491650 A CN117491650 A CN 117491650A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
The invention relates to a quantitative detection kit of peripheral blood Hb-Abeta complex, a detection method and application, wherein the method comprises the steps of fixing Protein G or Streptavidin (SA) on a solid phase carrier, then adding an anti-Hb antibody (AHb) or a biotinylated anti-Hb antibody (B-AHb) to form a combined body, reacting with Hb-Abeta complex in a sample, and then respectively reacting with an enzyme-labeled anti-Abeta antibody (E-Abeta) and a chemiluminescent solution, or respectively reacting with a biotin-labeled anti-Abeta antibody (B-Abeta), an enzyme-labeled avidin (E-SA) and a chemiluminescent solution; the invention also relates to a step of forming Hb-Abeta complex in the liquid phase reaction solution, and further combining the complex with anti-Hb antibody to perform chemiluminescence reaction; the invention further comprises the step of adding polyethylene glycol (PEG) into the reaction liquid in the reaction step containing the enzyme-labeled antibody or enzyme-labeled avidin to enhance the binding efficiency of antigen and antibody and the efficiency of enzyme-catalyzed chemiluminescent reaction, thereby greatly improving the sensitivity of the detection method.
Description
Technical Field
The invention belongs to the field of immunoassay, relates to a quantitative detection kit, a detection method and application of a hemoglobin-beta-amyloid peptide (Hb-Abeta) complex, and in particular relates to a kit, a method and application of the quantitative detection kit for the Hb-Abeta complex in peripheral blood, wherein the Hb-Abeta complex is mainly from peripheral red blood cells.
Technical Field
beta-Amyloid peptide (aβ) refers to a polypeptide molecule consisting of 36 to 43 amino acids, and is a polypeptide degradation product of a beta Amyloid precursor protein (β -Amyloid precursor protein, APP) produced by the action of a beta secretase. Aβ can be produced by neurons, astrocytes, activated microglia, and into extracellular fluids, including interstitial fluid, cerebrospinal fluid, plasma, lymph, saliva and urine. Aβ has strong aggregation properties, especially Aβ1-42 or Aβ42 consisting of 42 amino acids; the toxic effect of the aβ abnormal aggregates on neurons by directly acting on the neurons or activating the mechanism such as the glial inflammatory reaction is an important cause for inducing the development of Alzheimer's Disease (AD). The extracellular deposition of abnormal aggregates of aβ forms the hallmark pathological structure of AD, senile Plague (SP), which is the main basis for AD pathology and imaging diagnosis.
Given that aβ plays an important role in the pathogenesis of AD and forms senile plaques of the marked pathological structure in the brain of AD patients, aβ is considered as a Core marker (Core biomarker) capable of reflecting the neuropathological changes of AD. For many years, efforts have been directed to research into effective aβ detection methods and open detection kits. For example, INNOTEST Beta-AMYLOID (1-42) ELISA detection kit developed by Fujirbio, ADx-EUROIMMUN Beta-Amyloid (1-42) ELISA kit developed by EUROIMMUN, roche Diagnosticsbeta-Amyloid (1-42) CSF II electrochemistryLuminescence Immunoassay (ECLIA) kits, all of which are certified and widely used for detection of aβ42 in cerebrospinal fluid of AD patients. However, these techniques have unsatisfactory effects when applied to the detection of aβ42 in plasma, and the accuracy rate (AUC) of diagnosis is between 0.672 and 0.727 (AUC is the area under the curve of the working characteristic curve of a subject, and is preferably represented by auc=0.85 to 0.95, and is preferably represented by auc=0.70 to 0.84, and is generally represented by auc=0.50 to 0.69, and is relatively low). In view of the easy availability of blood samples, small damage to patients, relatively low cost, ease of repeated testing and dynamic tracking of changes in target molecules, detecting aβ0 in blood samples has unique advantages and is favored by most clinicians. To date, a number of ultrasensitive techniques have been developed internationally for detecting aβ1 in plasma or serum, including aβ242, aβ40 and aβ42/aβ40 ratios. Currently, among various techniques for directly detecting aβ in plasma, only Immunoprecipitation (IP) combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), i.e., IP-MS, and immunomagnetic decay (Immunomagnetic reduction, IMR) gave good results, with AUC that differentiated between AD and Healthy Control (HC) of greater than 0.85; in addition, sysmex Corporation (the Hissen health group) developed a full-automatic immunity test platform of HISCL series, whose accuracy of detecting plasma Abeta 42 and Abeta 42/Abeta 40 ratios predicted Abeta deposition in the brain was 0.738 and 0.868, respectively. The other detection techniques include super-sensitive single molecule immunity detection technique (SIMOA-Quantix, SIMOA-N4 PE) and- >When the technologies such as beta-Amyloid (1-42), INNOTEST beta-AMYLoid (1-42), IP-MS-UDOT and the like detect Abeta in blood plasma, the AUC for distinguishing AD from HC does not exceed 0.85. In addition to direct detection of aβ in plasma, there are also methods for detecting the amount of aβ aggregates with seeding activity in plasma using PMCA (Protein misfolding cyclic amplification, PMCA) technology. InBlood was developed by Korean human organism company (Peoplebio Inc., gyeonggido, korea) TM Aβ O Test commercial kit was obtained by PMCA amplification of Aβ aggregates in plasma and then ELISAThe AUC of this method to distinguish AD from HC reached 0.85, to determine the amplified aβ42 oligomer content. The university of capital medical science Xuan Wu hospital Gu Jianping teaches a team to use PMCA technology to detect seeding activity of aβ42 aggregates in plasma with an accuracy of 0.85-0.86 for distinguishing AD from HC. In addition, detection of aβ (Neuron-derived exosome A β, NDE-aβ) in neuronal-derived exosomes in plasma may lead to better diagnostic results, with AUC distinguishing between AD and HC of more than 0.90.
However, although the IP-MS, PMCA, IMR, NDE-aβ detection techniques achieve better diagnostic results, these techniques require more cumbersome sample handling procedures (IP-MS, NDE-aβ), more expensive equipment (IP-MS, IMR), more sample volumes (NDE-aβ) or longer detection times (PMCA); furthermore, since erythrocytes in blood contain high concentrations of aβ, the latter can cause a decrease in the stability of the erythrocyte membrane and hemolysis, leading to release of high concentrations of aβ into the plasma, affecting the accuracy of the aβ test in the plasma. These factors limit the accuracy of the above techniques for testing plasma aβ and its clinical applications, especially for community high risk group screening and dynamic tracking of disease progression.
Circulating peripheral red blood cells are known to adsorb and ingest aβ in plasma, including aβ40 and aβ42; the red blood cells contain abundant Abeta, and the content of Abeta is far higher than that of blood plasma; the content of Aβ in erythrocyte membranes and cytoplasm of AD patients is significantly higher than that of healthy controls; interactions of aβ with erythrocytes can induce intravascular hemolysis, leading to massive amounts of aβ entering the plasma, affecting accurate testing of aβ content in the plasma or serum. We demonstrate that aβ40 and aβ42 in erythrocytes can bind to hemoglobin (Hb) to form Hb-aβ complexes, which are present in AD patients in significantly higher amounts than in healthy controls. Therefore, the detection of Hb-Abeta complex content in peripheral red blood cells can be used as another marker for reflecting Abeta pathology of AD patients, and can avoid the influence of factors such as hemolysis and the like on the Abeta content test in blood plasma or blood serum.
Chinese patent 201711132802.8 discloses an ELISA method for quantitatively detecting Hb-Abeta complex, which comprises coating an ELISA plate with an anti-Hb antibody as a capture antibody, using a biotin-labeled or enzyme-labeled anti-Abeta antibody as a detection antibody, and displaying the content of the Hb-Abeta complex to be detected by using an enzymatic pigment or an enzymatic chemiluminescent reaction. Chinese patent CN103852579A discloses a quantitative detection method of human serum Abeta, which comprises the steps of carrying out a capture immune reaction on Abeta in a plasma sample treated by adopting a kit and a specific mouse monoclonal antibody which is paved with an Abeta peptide structure N-terminal selective amino acid sequence in advance and a temperature-controlled and constant-speed centrifugation, obtaining a reaction complex, carrying out a secondary antigen-antibody reaction on the reaction complex and an Abeta antibody of rabbit anti-human with Abeta specific amino acid sequence, carrying out a color development reaction on the antibody by a rabbit anti-mouse polyclonal antibody with an enzyme substrate, and quantitatively measuring by a spectrophotometer. Chinese patent CN103547593a discloses antibodies, kits and methods for determining amyloid peptides, and specifically discloses that the kit comprises a first antibody capable of specifically binding to aβ (1-17) peptide and a second antibody capable of recognizing a region of said aβ (1-17) peptide different from the region recognized by said first antibody. In the detection of the sample, the peptide present in the sample is captured by a first antibody which specifically binds to the Abeta (1-17) peptide, and the peptide captured by the first antibody is contacted with a second antibody to form an immune complex for measurement.
The aβ detection method according to the above patent faces the following problems: 1) An ELISA method for quantitatively detecting Hb-Abeta complex disclosed in China patent 201711132802.8 is low in sensitivity, needs a red blood cell lysate with higher concentration, is easy to cause non-specific binding, and cannot directly utilize peripheral blood whole blood to test the Hb-Abeta complex; 2) The methods for detecting Abeta in serum or plasma disclosed in Chinese patent CN103852579A and CN103547593A have the interference of potential interference factors such as heterophagic antibodies, lipoproteins and the like due to lower Abeta content in serum or plasma, and are easily influenced by the accuracy and stability of a test result caused by the fact that Abeta in high-concentration erythrocytes enter the plasma due to hemolysis.
Disclosure of Invention
The invention discloses a quantitative detection method of a Hb-Abeta compound of peripheral blood, wherein the Hb-Abeta compound is mainly from peripheral red blood cells. The method comprises the steps of immobilizing streptococcal Protein G (Protein G) or Streptavidin (SA) on a solid phase carrier, then adding anti-Hb antibody (AHb) or biotinylated anti-Hb antibody (B-AHb) to form a combined body, and reacting with Hb-Abeta complex, enzyme-labeled anti-Abeta antibody (E-AA beta) or biotin-labeled anti-Abeta antibody (B-AA beta) and enzyme-labeled avidin, chemiluminescent solution in a sample respectively. Wherein polyethylene glycol (Polyethylene glycol, PEG) is added to the reaction solution of the enzyme-labeled antibody or the enzyme-labeled avidin to enhance the efficiency of the enzymatic chemiluminescent reaction.
Wherein the solid support is selected from the group consisting of: the solid phase carrier is preferably an ELISA plate, a microplate, a test tube, magnetic beads or a microporous filter membrane; the solid phase carrier is made of the following materials: polystyrene, polyvinyl chloride, nitrocellulose, nylon, and the like.
The detection method of the present invention further comprises collecting a blood sample, wherein the AA beta forms Hb-aβ -AA beta complexes with Hb-aβ complexes in the sample in a liquid phase reaction solution, wherein AA beta is an enzyme-labeled antibody against aβ, including aβ42, aβ40 or any other aβ peptide, preferably aβ42; wherein the liquid phase reaction solution contains a certain amount of PEG, the complex is combined with the above-mentioned combination (i.e. the combination formed by fixing Protein G or SA and AHb or B-AHb), and the antigen-antibody combination is detected by adopting a chemiluminescence method.
According to the detection method, the binding efficiency of antigen-antibody and the reaction efficiency of enzymatic chemiluminescence reaction are enhanced by forming a Proten G or SA and AHb or B-AHb conjugate in a solid phase in advance, forming an Hb-Abeta complex in a liquid phase in advance and adding PEG into an enzyme-labeled antibody or enzyme-labeled avidin reaction solution, so that the sensitivity of the detection method is greatly improved.
The detection method, the preferable method is as follows:
and (3) coating an ELISA plate: adding the prepared Protein G or SA coating liquid into the hole of the ELISA plate, incubating overnight, and flushing; adding the prepared sealing liquid into the hole of the ELISA plate, incubating for 2-4h at constant temperature, and flushing.
Adding AHb or B-AHb solution into the coated ELISA plate, incubating for 0.5-2h at constant temperature, and flushing.
The Protein G coating liquid is prepared by the following steps: the solvent is buffer solution or physiological saline, and the concentration of Protein G is 0.001-0.05 μg/mL, preferably 0.002-0.02 μg/mL, more preferably 0.005 μg/mL.
The SA coating liquid is prepared by the following steps: the solvent used is a buffer solution or physiological saline, and the concentration of SA is 0.1-10. Mu.g/mL, preferably 1-5. Mu.g/mL, more preferably 4. Mu.g/mL.
Wherein, the preparation method of the AHb solution or the B-AHb solution comprises the following steps: the concentration of the anti-AHb antibody or the B-AHb antibody is 0.1 to 5.0. Mu.g/mL, preferably 0.5 to 2.0. Mu.g/mL, more preferably 1.0. Mu.g/mL, using a buffer solution or physiological saline as the solvent.
Wherein the blocking solution is selected from 1-10% bovine serum albumin solution or 1-2% gelatin solution or any other liquid capable of preventing non-specific binding of antigen and antibody; the preparation method of the 1% -10% bovine serum albumin solution comprises the following steps: the bovine serum albumin is added into buffer solution or physiological saline to be uniformly mixed, and the preparation method of the 1-2% gelatin solution comprises the following steps: dissolving gelatin in buffer solution or physiological saline, and mixing.
Wherein, the PEG can be PEG with any molecular weight, including PEG-200-PEG-20000, preferably PEG-4000-PEG-8000, more preferably PEG-6000. Wherein the concentration of PEG is 0.1-12%, preferably 2-8%, more preferably 5%.
Wherein the buffer solution is selected from the group consisting of: tris-HCl buffer, phosphate Buffer (PBS), carbonate Buffer (CBS).
Based on the above method of the present invention, the method can avoid problems that may exist with directly coating antibodies, including uncertainty in orientation, denaturation, poor fixation efficiency, or binding of contaminants to target molecules, affecting the binding of antibodies to antigen molecules. Thus, the method is advantageous for improving the binding efficiency of AHb to antigens, including Hb, hb-Abeta complex, hb-Abeta complex, by forming a conjugate of an AHb antibody or B-AHb with Protein G or SA previously immobilized on a solid support.
Based on the method, PEG is added into the reaction liquid of the enzyme-labeled anti-Abeta antibody (E-Abeta) or the enzyme-labeled avidin (E-SA), and the PEG can increase the effective concentration of the reactants, thereby being beneficial to the collision between the reactants, on one hand, the combination efficiency of the enzyme-labeled Abeta and the antigen can be improved, and on the other hand, the collision and reaction efficiency of the enzyme and the chemiluminescent substrate can be obviously improved, so that the sensitivity of the detection method is greatly improved.
The method comprises the steps of adding prepared Protein G coating liquid into a solid phase carrier, incubating overnight, and flushing; adding the prepared sealing liquid into the hole of the ELISA plate, and incubating at constant temperature; adding AHb solution into the coated solid phase carrier, and incubating at constant temperature.
Based on the above method of the present invention, the present invention further provides a method for detecting Hb-aβ complex and content in a sample in its entirety, the method comprising the steps of:
s1: preparing a sample to be detected;
s2: adding Protein G solution into the solid phase carrier, incubating and flushing;
s3: adding a sealing liquid, incubating and flushing;
s4: adding AHb solution, incubating and flushing;
s5: adding a sample to be detected (containing Hb-Abeta complex), incubating and flushing;
s6: adding enzyme-labeled anti-Abeta antibody (E-Abeta), incubating, washing, and adding chemiluminescent liquid to detect the value; or adding biotin-labeled anti-Abeta antibody (B-Abeta), incubating, washing, adding enzyme-labeled avidin, incubating, washing, and adding chemiluminescent liquid to detect the value;
wherein, a certain amount of PEG can be added into the reaction liquid of enzyme-labeled AA beta (E-AA beta) or enzyme-labeled avidin (E-SA), thereby greatly enhancing the reaction efficiency of enzymatic chemiluminescence; the Hb-Abeta complex content in the sample can be obtained through numerical calculation.
The method of the present invention may further comprise a method of coating SA on a solid support to detect Hb-Abeta complex and content in a sample, the method comprising the steps of:
s1: preparing a sample to be detected;
s2: adding SA solution into the solid phase carrier, incubating and flushing;
s3: adding a sealing liquid, incubating and flushing;
s4: adding a B-AHb solution, incubating and flushing;
s5: adding a sample to be detected (containing Hb-Abeta complex), incubating and flushing;
s6: adding E-AA beta reaction solution, incubating, washing, and adding chemiluminescent solution to detect the value;
the PEG with a certain concentration is added into the E-AA beta reaction liquid, so that the reaction efficiency of enzymatic chemiluminescence is greatly enhanced; the Hb-Abeta complex content in the sample can be obtained through numerical calculation.
The present invention can also employ the following Hb-Abeta complexes preformed in the liquid phase to achieve the object of further improving the sensitivity of detecting Hb-Abeta complexes, the method comprising the steps of:
s1: preparing a sample to be detected;
s2: adding Protein G into the solid phase carrier, incubating and flushing;
s3: adding a sealing liquid, incubating and flushing;
s4: adding AHb solution, incubating and flushing;
S5: adding a preformed Hb-Abeta complex (wherein Abeta refers to enzyme-labeled Abeta), incubating and flushing; adding chemiluminescent liquid to detect the value;
the PEG with a certain concentration is added into the liquid phase reaction liquid to enhance the combination efficiency of antigen-antibody and the efficiency of enzymatic chemiluminescence reaction, so as to further improve the sensitivity of the detection method; the Hb-Abeta complex content can be obtained through calculation.
Wherein, the preparation method of the Hb-Abeta complex comprises the following steps:
an enzyme (e.g., alkaline phosphatase, AP) labeled anti-Abeta antibody is prepared, and the labeled antibody binds to Abeta in Hb-Abeta complexes in the sample in a liquid phase, thereby forming Hb-Abeta complexes.
In the above method, in order to calculate the Hb-Abeta complex content in the sample to be measured, a Hb-Abeta complex standard is prepared by the following preparation method;
A. respectively dissolving the purified Abeta peptide and Hb protein by using a buffer solution, mixing the Abeta peptide and the Hb protein, and carrying out shaking incubation;
B. incubating a sample, centrifuging, taking supernatant, and filtering by a gel filtration column to separate Hb-Abeta complex;
C. mass spectrometry was used to determine the mass fraction of aβ peptide to Hb-aβ complex.
The invention also includes the preparation of Hb-Abeta complex standards for this purpose. The Hb-Abeta complex standard can be combined with antibodies AHb and Abeta and used for preparing a standard curve of the relation between a chemiluminescent signal and concentration, so as to obtain a mathematical formula, and quantitatively calculate the content of the Hb-Abeta complex in a sample through the mathematical formula.
Based on the method of the invention, the invention further provides a detection kit, which comprises a specific anti-Hb antibody (AHb), a Hb-Abeta standard, a solid phase carrier, protein G and PEG. On the basis, the detection kit can also contain biotin or enzyme-labeled anti-Abeta antibody (B-Abeta or E-Abeta), enzyme-labeled avidin (E-SA) which can be specifically combined with the B-Abeta, a luminescent agent, a necessary solvent, a use instruction and the like.
In a specific embodiment provided by the invention, the detection kit can further comprise Hb-Abeta standard, a solid phase carrier, streptavidin (SA), biotin-labeled anti-Hb antibody (B-AHb), enzyme-labeled anti-Abeta antibody (E-AA beta), a luminescent agent, a necessary solvent, a use instruction and the like.
In one embodiment provided by the invention, the detection kit of the invention can be used to detect Hb-aβ complex content in a test sample to further determine whether a subject has aβ accumulation, has Alzheimer's Disease (AD), or is at risk for AD. Wherein the sample to be tested is derived from a human, preferably from a blood sample of an AD patient or a person at risk for AD or from erythrocytes in a blood sample.
In a specific embodiment provided by the invention, the detection kit of the invention, wherein the antibody AA beta is an enzyme-labeled anti-aβ42 antibody. The enzyme is an enzyme that can catalyze chemiluminescent reactions, such as alkaline phosphatase (Alkaline Phosphatase, AP), horseradish peroxidase (Horseradish Peroxidase, HRP), or Glucose Oxidase (GO).
In a specific embodiment provided by the invention, the detection kit further comprises antibodies and avidin labeled by fluorescein, colloidal gold and other substances, wherein the antibodies and the avidin can be anti-Abeta antibodies or avidin which can be specifically combined with the Abeta antibodies.
In a specific embodiment provided by the invention, the detection kit further comprises a buffer solution, a blocking solution and the like.
The invention also provides application of Protein G, SA, AHb, B-AHb, PEG, E-AA beta or B-AA beta, E-SA in preparation of a kit or a reagent for quantitatively detecting the Hb-A beta complex content in a sample to be detected.
The invention also provides application of Protein G, SA, AHb, B-AHb, PEG, E-AA beta or B-AA beta, E-SA in preparation of a kit or a reagent for detecting whether a sample to be detected has AD or is at risk of AD. Antibodies of the invention are monoclonal antibodies, e.g., capable of specifically binding Hb and monoclonal antibodies capable of specifically binding aβ (including aβ36-43, preferably aβ40 and aβ42, and aβ42 is used in this study). The antibody may be illustratively an intact antibody, an antibody fragment, e.g., may be (Fab) 2, fab', scFv, or the like. The antibody may be of any animal species origin, recognizing Hb, aβ of any species, preferably human Hb, aβ.
The Hb-Abeta complex detection kit, the detection method and the related application thereof provided by the invention coat a solid-phase carrier such as a 96-well ELISA plate with Protein G or SA, so that the solid-phase carrier is combined with AHb or B-AHb serving as a capture antibody, and the Hb-Abeta complex in a sample is captured. This method of immobilizing antibodies can increase the capture capacity of the AHb antibodies and increase their binding efficiency to the antigen in the sample, i.e., hb-aβ complex. And then AA beta is used as a detection antibody, so that the sensitivity of the detection method can be greatly improved.
Wherein, in the case of Protein G coated on a solid support, AHb is immobilized to the solid support by saturation binding to Protein G, because Protein G binds mainly to the Fc-segment of an antibody, the Fab-segment of an antibody binding to an antigen can be made to face upwards, i.e. head (Fab-segment) up and tail (Fc-segment) down. The AHb head is facing up, relative to facing down, lying sideways and lying flat. The treatment can avoid problems possibly caused by directly passively adsorbing and coating the antibody on an ELISA plate or other solid-phase carriers, such as uncertainty of direction, antibody denaturation, poor fixing efficiency and incapability of normally combining the antibody with a target molecule due to space structure problems, so that the combining efficiency of the antibody and the antigen is greatly improved.
Under the condition that SA is coated on a solid-phase carrier, the biotinylated anti-Hb antibody (B-AHb) is fixed on the solid-phase carrier through combination with SA, because the antibody is combined with SA through biotin, the combination efficiency is high, and meanwhile, the problems that the antibody is denatured and poor in fixation efficiency and the antibody and a target molecule cannot be normally combined due to space structure problems and the like possibly generated on an ELISA plate or other solid-phase carriers are avoided, so that the combination efficiency of the antibody and the antigen is greatly improved.
The invention further provides a using method of the kit based on the kit, and the method comprises the following steps:
a step of immobilizing Protein G or SA on a solid phase carrier, followed by adding AHb to form a conjugate. The method specifically comprises the following steps:
and (3) coating an ELISA plate: adding the prepared Protein G or SA coating liquid into the hole of the ELISA plate, incubating overnight, and flushing; adding the prepared sealing liquid into the hole of the ELISA plate, incubating for 2-4h at constant temperature, and flushing.
Adding AHb solution or B-AHb solution into the coated ELISA plate, incubating for 0.3-2h at constant temperature, and washing.
The Protein G coating liquid is prepared by the following steps: the solvent used is a buffer solution or physiological saline, and the concentration of the Protein G solution is 0.001-0.05. Mu.g/mL, preferably 0.002-0.02. Mu.g/mL, more preferably 0.005. Mu.g/mL.
The SA coating liquid is prepared by the following steps: the solvent used is buffer solution or physiological saline, and SA is prepared at a concentration of 0.1-10 μg/mL, preferably 1-5 μg/mL, more preferably 4 μg/mL.
Wherein, the AHb solution is prepared by the following steps: the solvent used is a buffer solution or physiological saline, and the concentration of the AHb or B-AHb solution is formulated to be 0.1-5.0. Mu.g/mL, preferably 0.5-2.0. Mu.g/mL, more preferably 1.0. Mu.g/mL.
Wherein the blocking solution is selected from a 1% -10% bovine serum albumin solution or a 1-2% gelatin-containing solution or any solution that can be used for non-specific binding of an antigen or antibody; the preparation method of the 1% -10% bovine serum albumin solution comprises the following steps: adding bovine serum albumin into buffer solution or normal saline, and uniformly mixing, wherein the preparation method of the 1-2% gelatin solution comprises the following steps: dissolving gelatin in buffer solution or physiological saline, and mixing.
Wherein the buffer solution is selected from the group consisting of: tris-HCl buffer, PBS buffer, CBS buffer.
The method of using the kit of the present invention may further comprise any step selected from the group consisting of the prior art, for example, collection of a blood sample and preparation of a sample to be tested, preparation of E-AA beta, preparation of B-AA beta, preparation of E-SA, preparation of Hb-A beta complex, preparation of Hb-A beta-AA beta complex, preparation of a buffer solution and physiological saline, and detection of an antigen-antibody conjugate using a known chemiluminescent assay using a chemiluminescent assay device, as required.
PEG is added into the E-AA beta and E-SA incubation liquid, and the improvement can greatly enhance the antigen-antibody combination efficiency and the enzymatic chemiluminescence reaction efficiency, so that the sensitivity of the detection method is greatly improved, and the detection time is shortened;
liquid phase reactions with Protein G or SA, PEG and Hb-Abeta complexes, these improvements make three advances in detection indicators: firstly the sensitivity of the detection, secondly the accuracy of the diagnosis, and again the detection time.
The invention further comprises application of the reagent in the kit in preparation of the kit for detecting Alzheimer's disease.
The following is an explanation and explanation of the name terms of the present invention:
hb-aβ complex: a complex formed by binding hemoglobin to aβ.
Streptococcal protein G: i.e., protein G, a cell wall Protein on the surface of Streptococcus, has a molecular weight of about 65kDa and binds to the Fc segment of immunoglobulins, i.e., antibodies.
Avidin and streptavidin: avidin is a glycoprotein which can be extracted from egg white and has a molecular weight of 60kDa, and each molecule consists of 4 subunits and can be tightly combined with 4 biotin molecules. Streptavidin (SA) is a protein with similar biological properties to Avidin (Avidin). Is a protein product secreted by Streptomyces Avidinii bacteria in the culture process, and the streptavidin SA can also be produced by a genetic engineering means. Streptavidin SA has a molecular weight of 65kDa and consists of 4 peptide chains with the same sequence, and each streptavidin SA peptide chain can be combined with 1 biotin molecule. Thus, as with A, each streptavidin SA molecule also has 4 binding sites for biotin molecules with a binding constant of 1015mol/L as with A.
Solid phase carrier: solid phase media (including ELISA plates, microwell plates, microporous filters, magnetic beads, colloidal gold, etc.) for Enzyme-linked immunosorbent assay (ELISA) or ChemiLuminescent immunoassay (ChemiLuminescent ImmunoAssay, CLIA) reactions.
ELISA plates or microplates: a special plastic plate is used for enzyme-linked immunosorbent assay or chemiluminescent immunoassay. May be of different numbers and sizes, such as 48 wells, 96 wells, 384 wells, different colors, such as transparent, white, black, etc., and different bottoms, such as flat bottom, round bottom, etc. Can be used for fixing molecules such as antigen, antibody, protein G, SA and the like.
Microporous filter membrane: special microporous membranes for immobilization of Protein G, SA or antibodies, such as nylon membranes, cellulose acetate membranes, nitrocellulose membranes, polyvinylidene fluoride membranes (PVDF membranes).
Magnetic beads: the magnetic beads can be used as carriers of antigen-antibody reaction by binding active proteins through functional groups with surface external modification.
Colloidal gold: gold particles capable of binding to antibody molecules, antigen molecules or other protein molecules.
Polyethylene glycol: i.e., polyethylene glycol, PEG, is a generic term for ethylene glycol polymers containing α, ω -double terminal hydroxyl groups. Polyethylene glycol is a high molecular polymer, and has a chemical formula of HO (CH 2CH 2O) nH.
Biotin: biotin, a synthetic vitamin, is used to label antibody molecules that bind to specific antigens in immunoassays.
Alkaline phosphatase: alkaline Phosphatase (AP), a specific phosphatase, is used in immunoassays to label detection antibodies or avidin and to quantify the immune response by catalyzing the luminescence or color forming substrate. Horseradish peroxidase: horseradish peroxidase (HRP), an enzyme from plant horseradish, is a glycoprotein formed by combining colorless enzyme protein and brown iron porphyrin. Is a common enzyme in clinical test reagents. The enzyme is used for labeling antibodies or avidin in immunodetection, and the immune response is quantified by catalyzing luminescence or forming a substrate.
Glucose oxidase: glucose Oxidase (GO), an enzyme extracted from Penicillium aureum, is used to label antibodies or avidin in immunoassays, and quantifies immune responses by catalyzing luminescent or chromogenic substrates.
anti-Hb antibody: an antibody that specifically binds to hemoglobin is abbreviated as AHb in this specification. Anti-aβ antibodies: antibodies that specifically bind to aβ molecules are abbreviated herein as AA β. Biotin-labeled antibody: antibodies that bind biotin, such as biotin-labeled anti-Hb antibody (B-AHb) and biotin-labeled anti-aβ antibody (B-AA β) in the present specification.
Enzyme-labeled antibodies: antibodies that bind to specific enzymes, such as anti-aβ antibodies that specifically bind to alkaline phosphatase (AP-AA β) or antibodies that bind to the enzyme-labeled antibody aβ (E-AA β).
Enzyme-labeled avidin: avidin (E-SA) which binds to a specific enzyme, such as alkaline phosphatase, horseradish peroxidase, etc.
Fab fragment of anti-Hb antibody (AHb): specific antigen-binding fragments of anti-hemoglobin antibodies. Is the product of the antibody after the hydrolysis of papain.
Hb-aβ -AA beta complex: a complex of hemoglobin and aβ and an antibody specifically recognizing aβ. Fluorescein: fluorescein is also known as fluorescein, fluorogenic, and fluorogenic red, and can automatically emit fluorescence or be excited to emit fluorescence by excitation light with a certain wavelength. In immunoassays, the immune response is often quantitatively detected or quantified as a chemiluminescent substrate.
Monoclonal antibodies: is a highly homogeneous, antibody directed against only one specific epitope produced by a single B cell clone. Typically prepared using hybridoma technology.
Sealing liquid: a liquid for blocking non-specific binding of an antigen or antibody to a reaction interface or other non-target protein.
Incubation: the reaction process of biological molecules under certain conditions.
Chemiluminescent liquid: a solution capable of emitting light of a specific wavelength under the catalysis of an enzyme.
Aβ40: is a small molecule peptide consisting of 40 amino acids produced by Amyloid Precursor Protein (APP) under the action of beta secretase.
Aβ42: is a small molecule peptide consisting of 42 amino acids produced by Amyloid Precursor Protein (APP) under the action of beta secretase. Aβ42 plays an important role in the pathogenesis of alzheimer's disease.
Erythrocyte lysate: erythrocytes are formed to contain a liquid by repeated freeze thawing or rupture of the cell membrane in hypotonic solutions. The erythrocyte lysate contains the target protein to be detected, such as Abeta.
Chemiluminescent detection instrument: a special instrument for detecting chemiluminescent intensity.
Comparison of the inventive method with the methods of the prior patent
Methods 1 and 2: ELISA (ELISA) in which the capture antibody is directly coated on an ELISA plate and bound with biotin or an enzyme-labeled detection antibody: the capture antibody (AHb in this patent) is first coated directly on the ELISA plate, then reacts with antigen molecules (such as Hb-Abeta complex) in the sample, then reacts with biotin-labeled Abeta antibody (B-Abeta) and enzyme-labeled avidin (such as AP-SA) (method 1) or directly reacts with enzyme-labeled anti-Abeta antibody (AP-Abeta in this patent) (method 2), finally reacts with chromogenic solution containing chromogenic substrate (such as nitrophenylphosphoric acid, pNPP), and the ELISA plate records absorbance value of specific wavelength.
Methods 3 and 4: chemiluminescent immunoassay (CLIA) in which the capture antibody is directly coated on an elisa plate to bind biotin or an enzyme-labeled detection antibody: the capture antibody (AHb in this patent) is first coated directly onto the ELISA plate, then reacted with antigen molecules (e.g. Hb-Abeta complex) in the sample, then reacted with biotin-labeled Abeta antibody (B-Abeta) and enzyme-labeled SA (e.g. AP-SA) (method 3), or reacted directly with enzyme-labeled anti-Abeta antibody (AP-Abeta in this patent) (method 4), finally reacted with chemiluminescent liquid, and the light intensity of specific wavelength is recorded.
Method 5: chemiluminescent immunoassay (CLIA) in which a capture antibody is immobilized to an elisa plate via SA in combination with an enzyme-labeled detection antibody: SA is coated on an ELISA plate, then combined with a biotinylation capture antibody (biotinylation AHb, namely B-AHb in the patent), then reacted with an antigen molecule (such as Hb-Abeta complex) in a sample, then reacted with an enzyme-labeled anti-Abeta antibody (such as AP-Abeta), finally reacted with chemiluminescent liquid, and the chemiluminescent intensity of specific wavelength is recorded.
Methods 6 and 7: chemiluminescent immunoassay (CLIA) of capture antibodies conjugated with PEG-enhanced enzyme-labeled antibodies or avidin immobilized to an elisa plate via Protein G: protein G is coated on an ELISA plate, then is combined with a capture antibody (AHb in the patent), then reacts with antigen molecules (such as Hb-Abeta complex) in a sample, then reacts with biotin-labeled anti-Abeta antibody (B-Abeta) and enzyme-labeled avidin (such as AP-SA) (method 6) or directly reacts with enzyme-labeled anti-Abeta antibody (such as AP-Abeta) (method 7), finally reacts with chemiluminescent liquid, and the chemiluminescent intensity of specific wavelength is recorded. Wherein, a certain amount of PEG is added into the reaction liquid of the enzyme-labeled anti-Abeta antibody or the enzyme-labeled avidin to enhance the efficiency of the enzymatic chemiluminescent reaction.
Method 8: chemiluminescent immunoassay (CLIA) in which a capture antibody is immobilized to an elisa plate via Protein G and a PEG-conjugated enhanced liquid phase elisa detects the antibody-antigen complex reaction: protein G was first coated on an elisa plate and AHb was bound. Simultaneously, antigen (namely Hb-Abeta complex) in the sample reacts with enzyme (such as alkaline phosphatase) marked anti-Abeta antibody in a liquid phase containing PEG to form Hb-Abeta complex, then reacts with AHb combined on Protein G on an ELISA plate, finally reacts with chemiluminescent liquid, and the chemiluminescent intensity of specific wavelength is recorded.
The technical improvements of the invention compared with the prior method include: 1) Coating an ELISA plate by Protein G or SA, and then combining with a capture antibody AHb or B-AHb, so that the combination efficiency of AHb and an antigen Hb-Abeta complex is improved; 2) The formation of Hb-aβ -AA beta complexes in the liquid phase further increases antigen-antibody binding efficiency; 3) The introduction of PEG greatly enhances the efficiency of enzyme-catalyzed chemiluminescent reaction. The technical improvements increase the sensitivity of the detection method by times, obviously shorten the detection time and obviously improve the diagnosis accuracy. These technical solutions all belong to the protection scope of the present invention.
The invention has at least one of the following advantages:
1) Binding of AHb to the antigen Hb-aβ complex can be enhanced by binding of Protein G or SA to the anti-AHb or B-AHb coated solid support.
2) PEG is added into the enzyme-labeled antibody or enzyme-labeled avidin incubation liquid, so that on one hand, the binding efficiency of the enzyme-labeled antibody and antigen is improved, and on the other hand, the efficiency of enzymatic chemiluminescence reaction is remarkably improved, and the sensitivity of the detection method is greatly improved.
The enzyme-labeled anti-Abeta antibody reacts with the antigen in the sample in the liquid phase in advance to form an antigen-antibody-enzyme complex, and meanwhile, PEG is added into the liquid phase reaction liquid, so that the sensitivity of the detection method is further improved, and the detection time is obviously shortened.
The Hb-Abeta complex detection kit and the detection method provided by the invention are suitable for quantitative detection of all kinds of Abeta (Abeta 36-43) content combined with Hb, including quantitative detection of various Abeta content combined with Hb in peripheral red blood cells.
Since the improved method greatly improves the sensitivity of the kit, not only is the amount of sample required for detecting Hb-Abeta complexes in peripheral red blood cells greatly reduced, but also a whole blood sample can be used for testing Hb-Abeta complexes. The improved method ensures that the blood sample is collected and processed more conveniently and is suitable for large-scale crowd screening.
The test result is more stable, and the test accuracy is greatly improved.
Whereas aβ in body tissue, especially brain tissue, may enter plasma, which may further enter erythrocytes and bind to Hb in erythrocytes, the amount of Hb-aβ complex tested using the detection method according to the invention may reflect changes in aβ content in body tissue, especially brain tissue.
The method can solve the problems of the existing technology for detecting the Abeta in the blood plasma and serum samples, including the problems of the Abeta in the red blood cells released to the blood plasma or the blood serum due to hemolysis, the accuracy of the Abeta test results of the blood plasma and the blood serum and the interference of the plasma lipoprotein or the autophagic antibody on the test accuracy.
The main innovation points of the method provided by the invention are as follows:
protein G or SA is coated on the ELISA plate in advance, and then the ELISA plate is reacted with an anti-Hb antibody (AHb) or a biotinylated anti-Hb antibody (B-AHb). The method has the advantages that: the problems of antibody denaturation, poor fixing efficiency or combination of pollutants and target molecules and the like which possibly occur when the anti-Hb antibody is directly coated on the ELISA plate are avoided. In addition, in the case of coating by using Protein G, the problem of uncertainty of the head-tail orientation of the coated antibody can be solved. Therefore, the method provided by the patent greatly improves the capturing capability of the anti-Hb antibody and increases the binding efficiency of the anti-Hb antibody with the antigen in the sample, namely Hb-Abeta complex. PEG is added into enzyme-labeled anti-Abeta antibody (E-Abeta) or enzyme-labeled avidin (E-SA) reaction liquid, so that the combination efficiency of the enzyme-labeled anti-Abeta antibody and antigen can be improved, and the efficiency of enzymatic chemiluminescence reaction can be obviously enhanced, thereby greatly improving the sensitivity of a detection method and a kit. The method comprises the steps of firstly reacting an antigen, namely Hb-Abeta complex, with an enzyme-labeled anti-Abeta antibody (E-Abeta) in a liquid phase to form the Hb-Abeta complex, then combining the Hb-Abeta complex with a capture antibody, namely anti-Hb antibody (AHb), immobilized on an ELISA plate, and adding PEG into a liquid phase reaction solution of the Hb-Abeta complex. The improvement further improves the binding efficiency of the antigen and the antibody and the efficiency of the enzymatic chemiluminescence reaction, so that the sensitivity of the detection method and the kit is further improved.
The beneficial effects of the invention include:
the Hb-Abeta complex detection kit provided by the invention greatly improves the sensitivity of a detection method, and only a small amount of red blood cell sample (1-2 mu L) is needed to carry out accurate quantitative detection of Hb-Abeta complex in the sample.
Because the Hb-Abeta complex content in the peripheral blood is high, and the detection sensitivity is greatly improved by the method provided by the invention, the method can directly utilize a small amount of whole blood sample (3-4 mu L) to test the Hb-Abeta complex.
Compared with the method for directly testing the Abeta in the blood plasma and serum samples, the method has the advantages that the Hb-Abeta complex content in the peripheral blood is high, abeta pollution in the blood plasma and serum samples caused by potential hemolysis can be avoided, the interference of plasma lipoprotein and autophagic antibodies on the test is avoided, and the test result is stable, the accuracy is high and the repeatability is good. The blood sample, especially the whole blood sample, is easy to obtain, has small invasiveness and low cost, has little sample requirement, does not need complex pretreatment, and is suitable for large-scale screening of the high-risk group of Alzheimer's disease or tracking of the change condition of the Abeta content in the bodies of Alzheimer's disease patients and the high-risk group through dynamic repeated test.
Compared with the prior patent application, the sensitivity, the specificity and the accuracy of detection are greatly improved. Compared with the original method, the detection sensitivity can be improved by more than 20 times by the current method. The Hb-Abeta complex concentration in the red blood cells can be detected only when the dilution ratio of the sample is lower than 10 times by the prior method, the Hb-Abeta complex concentration ratio (AD/HC) of the AD patient and the healthy control is 1.50, the Hb-Abeta complex signal value is obviously reduced or even can not be detected when the sample is further diluted to 50 times, and the AD/HC ratio is obviously reduced (note: the greater the AD/HC ratio is, the better the distinguishing degree is). The new method can obviously increase the AD/HC ratio to be close to or even more than 2.0 on the premise of greatly improving the sensitivity of the detection method, and can obtain the ideal AD/HC ratio in a 50-400-time dilution range. A more desirable AD/HC ratio can also be obtained when testing with whole blood samples. Compared with the prior application, the sensitivity, the specificity and the accuracy of diagnosis are improved. Subject operating characteristic curve (ROC) analysis shows that the novel method of the present application provides a significant improvement in several indicators of sensitivity, specificity and Accuracy (AUC) for distinguishing AD from HC. Compared with the current technology for detecting the Aβ in blood: a) Compared with various technologies for detecting the plasma Abeta, the AD/HC ratio, the sensitivity, the specificity and the AUC for distinguishing the AD from the HC have obvious advantages; b) Compared with various technologies for detecting the apo Abeta of the plasma neuron source, various technical indexes are equivalent, but the required sample size is obviously reduced, the complex process of apo separation is not required, the cost is obviously reduced, the operation is simple, and the community high risk group screening and dynamic tracking are convenient.
Drawings
FIG. 1 is a schematic diagram of an example detection method. Methods 1 and 2 are enzyme-linked immunosorbent assay methods, and methods 3 to 8 are chemiluminescent immunoassay methods. The method 1-7 is solid phase immune detection, namely, capturing antibody, antigen, detecting antibody and avidin react in the solid phase in sequence. Method 8 is a liquid phase immunoassay in which the antigen and the enzyme-labeled detection antibody first form a complex in the liquid phase and then react with a capture antibody immobilized to a solid phase by Protein G. PEG is added to a dilution containing enzyme-labeled antibodies or enzyme-labeled avidin.
FIG. 2 is a bar graph of different gain effects of two detection steps of biotin-labeled antibody and enzyme-labeled avidin in the PEG-6000 pair detection method. The capture antibody (AHb) directly coats the ELISA plate and reacts sequentially with Hb-A beta complex (red blood cell lysate from AD and HC), B-AA beta, AP-SA and chemiluminescent fluid to test chemiluminescent signal values. Wherein, adding PEG-6000 with a certain concentration into the B-AA beta or/and AP-SA diluent, and comparing the influence of the PEG-6000 on the chemiluminescence intensity of different reaction steps. AHb: an anti-hemoglobin antibody; B-AA beta: biotin-labeled anti-aβ antibodies; AP-SA: alkaline phosphatase labeled streptavidin; AD: alzheimer's disease; HC: healthy controls.
FIG. 3 shows bar graphs of different gains of Protein G and PEG-6000 on luminescent signals in enzyme-labeled detection antibody chemiluminescent immunoassay. Protein G coats the ELISA plate, binds to the capture antibody AHb, and reacts sequentially with Hb-Abeta complex (red blood cell lysate from AD and HC), AP-Abeta and chemiluminescent fluid to test chemiluminescent signal values. And adding PEG-6000 with a certain concentration into the Protein G coated ELISA plate or the AP-AA beta diluent, and comparing the influence of two gain conditions on the chemiluminescence intensity. AHb: an anti-hemoglobin antibody; AP-AA beta: alkaline phosphatase-labeled anti-aβ antibody; AD: alzheimer's disease; HC: healthy controls.
FIG. 4A graph of Hb-Abeta standard concentration versus chemiluminescent signal for various detection methods is tested in accordance with an example of the present invention. FIG. 5A chart of the various detection methods for the Hb-Abeta complex chemiluminescence signals and concentration values in peripheral erythrocytes and whole blood and samples and a chart of ROC curve analysis were tested.
Detailed Description
The following description of the technical solution in the embodiments of the present invention is clear and complete. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the present invention, the β -Amyloid peptide or protein, beta-Amyloid, amyloid β -peptide, amylase beta, aβ, abeta, etc. are the same substance, and the english name, shorthand, etc. are not in any case.
The preparation method of part of the reagents in the invention is as follows, and other reagents are all conventional preparation methods.
The preparation method of the 0.01mol/L PBS comprises the following steps: na (Na) 2 HPO 4 2.72g，NaH 2 PO 4 0.28 g, naCl9.00g, DDW (double distilled water) 1000mL.
200mmol/L NaHCO 3 The preparation method of the buffer solution (pH 9.6) comprises the following steps: naHCO (NaHCO) 3 0.84 g,20% sodium azide (NaN) 3 )50μL，DDW50 mL。
The preparation method of the 1% sealing liquid comprises the following steps: BSA1 g, PBST100 mL.
The preparation method of the PBS (PBST) containing 0.05% Tween-20 and 0.01mol/L PBS comprises the following steps: na (Na) 2 HPO 4 2.72 g，NaH 2 PO 4 0.28 g，NaCl9.00 g，DDW1000 mL，Tween-20 500μL。
Example 1A method for preparing A beta kit
1. Preparing a coating liquid: diluting Protein G to 0.001 mu G/mL-0.1 mu G/mL by adopting a buffer solution; or adopting buffer solution to dilute SA to 0.1-10 mug/mL; the buffer solution is selected from: tris-HCl buffer, PBS buffer, CBS buffer or buffered physiological saline. The embodiment adopts NaHCO 3 And (3) a buffer solution.
2. Preparing a sealing liquid: the blocking solution is selected from 1% -10% of new born calf serum or PBST solution with gelatin content of 2.5% (0.01 mol/L PBS containing 0.05% Tween-20). This example uses a PBST solution containing 1% bsa.
3. After the blocking of the binding capture antibody is completed, the AHb is diluted to 0.1 mu g/mL-4 mu g/mL by adopting buffer solution; or diluting the B-AHb antibody to 0.1-4. Mu.g/mL, preferably 0.5-2. Mu.g/mL, more preferably 1. Mu.g/mL, with a buffer; the buffer solution is selected from: PBS buffer, PBST solution, CBS buffer or buffered saline. Incubating for 1-2h at constant temperature, and washing. The present example uses PBST solution. Coating with a solid phase carrier:
the solid phase carrier can be an ELISA plate, a microplate, a magnetic bead, a test tube or a microporous filter membrane. In this embodiment, an elisa plate is used, and the elisa plate is modified, where the modification is performed as follows: and placing the ELISA plate on a medical purification operation table provided with an ultraviolet lamp, fixing the vertical distance between the ultraviolet lamp and the substrate of the micro-pore plate, and carrying out ultraviolet treatment on the micro-pore plate.
4. Standard formulation (establishment of quantitative standard curve):
1) Dissolving Abeta peptide and Hb protein with 500. Mu.L of 0.01mol/L PBS to obtain final concentration of 2mol/L and 1mol/L respectively, mixing, and incubating at 37deg.C and 230rpm under shaking for 24 hr; the aβ selected in this example is aβ42.
2) Incubating the sample, centrifuging at 10000 Xg for 5min, absorbing supernatant, taking PBS as a mobile phase, performing gel filtration on HiPrep 26/60Sephacryl S-200High Resolution chromatographic column, and separating Hb-Abeta complex;
3) The average number of aβ molecules bound per Hb molecule was determined using the SWATH (Sequential Window Acquisition of all Theoretical Mass Spectra) quantitative proteome method, whereby the weight or mole percent of aβ in the complex was calculated. Accordingly, hb-Abeta complex solutions having different concentrations were prepared, and a standard curve was prepared using the Hb-Abeta complex solutions as a standard protein.
5. Preparing an anti-Abeta antibody dilution solution: and diluting the AA beta with an enzyme conjugate stabilizer. In this example, the AA β is not limited, and any antibody capable of specifically recognizing the AA β may be used.
The enzyme conjugate stabilizer is a reagent capable of maintaining 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 Alkaline Phosphatase (AP) conjugate stabilizer, which in the present invention may be a commercially available product.
6. Preparing an enzyme-labeled antibody solution: alkaline Phosphatase (AP) -labeled anti-Abeta antibodies, AP-AA beta, were diluted with blocking solution.
7. Washing liquid: the washing solution is PBS solution (PBST solution for short) containing Tween-20 (Tween-20), wherein the PBST solution can contain biological liquid preservative such as Proclin300.
8. Substrate solution: the substrate solution may be AMPPD (1, 2-dioxan derivative), APS-5, preferably AMPPD in this case.
9. Sample dilution: the sample diluent was a PB solution.
In the kit provided by the invention, various reagents are packaged respectively, preferably using packaging tubes, and the amount of the reagents packaged in each packaging tube is basically one sample, and can be expanded to 10, 100 and 1000 samples.
Example 2 different detection methods of kit principle and procedure (FIG. 1)
Method 1: ELISA (enzyme-linked immunosorbent assay) method for directly coating capture antibody on ELISA plate and combining biotin-labeled detection antibody
S1: taking 5-10mL of peripheral blood, uniformly mixing, adding into a centrifuge tube by adherence, and taking part of peripheral blood whole blood for preservation at-80 ℃. The rest is added with PBS according to a certain dilution ratio and is fully and uniformly mixed.
S2: the diluted whole blood was slowly added to the lymphocyte separation medium, and centrifuged to separate the erythrocyte layer. The red blood cells were transferred to a new centrifuge tube, added with PBS, centrifuged, and stored at-80 ℃.
S3: and (3) thawing the frozen red blood cells at room temperature, and adding a sample diluent according to a certain dilution ratio to obtain the cytosol sample.
S4: coating: with NaHCO 3 The anti-Hb antibody (AHb) was diluted in buffer to a final concentration of 1. Mu.g/mL. 100 μl of the diluent was added to each well of the elisa plate and incubated overnight. And (5) flushing.
S5: closing: to each well of the ELISA plate, 300. Mu.L of blocking solution was added and incubated for 2 hours. And (5) flushing.
S6: reaction 1: adding diluted peripheral blood whole blood or erythrocyte lysate or Hb-Abeta complex standard protein into each hole of the ELISA plate, and incubating for 2h. And (5) flushing.
S7: reaction 2: the biotin-labeled anti-Abeta antibody (B-AA beta) was diluted with blocking solution to a final concentration of 0.1-2. Mu.g/mL. The antibody dilutions were added to each well of the elisa plate and incubated for 2h. And (5) flushing.
S8: reaction 3: alkaline phosphatase-labeled avidin (AP-SA) was diluted with blocking solution (1:5000 dilution), and 100. Mu.L of the enzyme-diluted solution was added to each well of the ELISA plate. Incubate for 1h. And (5) flushing.
S9: color development: and adding a color development solution into each hole of the ELISA plate, and developing for 0.5h at room temperature in a dark place.
S10: and (3) terminating: with 10% H 2 SO 4 The color development was terminated.
S11: and (3) measuring the content: the absorbance values of each well of the microplate were measured at a specific wavelength of the ultraviolet spectrophotometer using the microplate reader.
S12: and (3) calculating: the Hb-Abeta complex content in the sample is calculated according to a relation curve between the concentration of the Hb-Abeta complex standard prepared in vitro and the absorbance at a specific wavelength.
Method 2: ELISA method for directly coating capture antibody on ELISA plate and combining enzyme-labeled detection antibody
S1-S3: method 1.
S4: coating: with NaHCO 3 The anti-Hb antibody (AHb) was diluted in buffer to a final concentration of 1. Mu.g/mL. 100 μl of the diluent was added to each well of the elisa plate and incubated overnight. And (5) flushing.
S5: closing: to each well of the ELISA plate, 300. Mu.L of blocking solution was added and incubated for 2 hours. And (5) flushing.
S6: reaction 1: adding diluted peripheral blood whole blood or erythrocyte lysate or Hb-Abeta complex standard protein into each hole of the ELISA plate, and incubating for 2h. And (5) flushing.
S7: reaction 2: alkaline phosphatase-labeled anti-Abeta antibody (AP-AA beta) was diluted with blocking solution to a final concentration of 0.1-2. Mu.g/mL. The antibody dilutions were added to each well of the elisa plate and incubated for 2h. And (5) flushing.
S8: color development: and adding a color development solution into each hole of the ELISA plate, and developing for 0.5h at room temperature in a dark place.
S9: and (3) terminating: with 10% H 2 SO 4 The color development was terminated.
S10: and (3) measuring the content: the absorbance values of each well of the microplate were measured at a specific wavelength of the ultraviolet spectrophotometer using the microplate reader.
S11: and (3) calculating: and calculating the content of Hb-Abeta complex in the sample according to a relation curve between the concentration and the absorbance of the Hb-Abeta complex standard prepared in vitro.
Method 3: chemiluminescent immunoassay (CLIA) wherein a capture antibody is directly coated onto an elisa plate and a biotin-labeled detection antibody is bound thereto
S1-S3: as shown in method 1.
S4: coating: with NaHCO 3 The anti-Hb antibody (AHb) was diluted in buffer to a final concentration of 1. Mu.g/mL. 100 μl of the diluent was added to each well of the elisa plate and incubated overnight. And (5) flushing.
S5: closing: to each well of the ELISA plate, 300. Mu.L of blocking solution was added and incubated for 2 hours. And (5) flushing.
S6: reaction 1: adding diluted peripheral blood whole blood or erythrocyte lysate or Hb-Abeta complex standard protein into each hole of the ELISA plate, and incubating for 1h. And (5) flushing.
S7: reaction 2: the biotinylated anti-Abeta antibody (B-AA beta) was diluted with blocking solution to a final concentration of 0.5-4. Mu.g/mL. The antibody dilutions were added to each well of the elisa plate and incubated for 1h. And (5) flushing.
S8: reaction 3: alkaline phosphatase-labeled avidin (AP-SA) was diluted with blocking solution (1:10000 dilution), and the enzyme-diluted solution was added to each well of the ELISA plate at 100. Mu.L per well. Incubate for 1h. And (5) flushing.
S9: chemiluminescence: chemiluminescent solution is added to each well of the microplate.
S10: and (3) measuring the content: the chemiluminescent value detector measures the chemiluminescent value of each well of the ELISA plate.
S11: and (3) calculating: the complex content in the sample was calculated from the relationship between the concentration of Hb-aβ complex standard prepared in vitro and the chemiluminescent value.
Method 4: chemiluminescent immunoassay (CLIA) wherein a capture antibody is directly coated on an ELISA plate and an enzyme-labeled detection antibody is bound thereto
S1-S3: as shown in method 1.
S4: coating: with NaHCO 3 The anti-Hb antibody (AHb) was diluted in buffer to a final concentration of 1. Mu.g/mL. To the direction ofThe wells of the ELISA plate were incubated overnight with 100. Mu.L of diluent. And (5) flushing.
S5: closing: to each well of the ELISA plate, 300. Mu.L of blocking solution was added and incubated for 2 hours. And (5) flushing.
S6: reaction 1: adding diluted peripheral blood whole blood or erythrocyte cytoplasmic samples or Hb-Abeta complex standard proteins into each hole of the ELISA plate, and incubating for 1h. And (5) flushing.
S7: reaction 2: alkaline phosphatase-labeled anti-Abeta antibody (AP-AA beta) was diluted with blocking solution to a final concentration of 0.1-2. Mu.g/mL. The antibody dilutions were added to each well of the elisa plate and incubated for 1h. And (5) flushing.
S8: chemiluminescence: chemiluminescent solution is added to each well of the microplate.
S9: and (3) measuring the content: the chemiluminescent signal detection instrument measures the chemiluminescent value of each well of the ELISA plate.
S10: and (3) calculating: the content of Hb-Abeta complex in the sample is calculated according to a relation curve between the concentration of Hb-Abeta complex standard prepared in vitro and the chemiluminescence value.
Method 5 chemiluminescent immunoassay method in which a capture antibody is immobilized to an ELISA plate via SA and an ELISA detection antibody is bound
S1-S3: as shown in method 1.
S4: coating: with NaHCO 3 The buffer dilutes Streptavidin (SA) to a final concentration of 0.1-10. Mu.g/mL. 100. Mu.L of this streptavidin SA dilution was added to each well of the ELISA plate and incubated overnight. And (5) flushing.
S5: closing: to each well of the ELISA plate, 300. Mu.L of blocking solution was added and incubated for 2 hours. And (5) flushing.
S6: combining: biotinylated anti-Hb antibody (B-AHb) was added to each well of the ELISA plate and incubated for 0.5h. And (5) flushing.
S7: reaction 1: adding diluted peripheral blood whole blood or erythrocyte lysis or Hb-Abeta complex standard protein into each hole of the ELISA plate, and incubating for 1h. And (5) flushing.
S8: reaction 2: alkaline phosphatase-labeled anti-Abeta antibody (AP-AA beta) was diluted with blocking solution to a final concentration of 0.1-2. Mu.g/mL. The antibody dilutions were added to each well of the elisa plate and incubated for 1h. And (5) flushing.
S9: chemiluminescence: chemiluminescent solution is added to each well of the microplate.
S10: and (3) measuring the content: the chemiluminescent signal detection instrument measures the chemiluminescent value of each well of the ELISA plate.
S11: and (3) calculating: the content of Hb-Abeta complex in the sample is calculated according to a relation curve between the concentration of Hb-Abeta complex standard prepared in vitro and the chemiluminescence value.
Method 6: chemiluminescent immunoassay (CLIA) of capture antibodies conjugated to PEG-enhanced enzyme-labeled avidin immobilized to an elisa plate via Protein G
S1-S3: as shown in method 1.
S4: coating: with NaHCO 3 The buffer dilutes Protein G to a final concentration of 0.001-0.1. Mu.g/mL. The Protein G dilution was added to each well of the ELISA plate and incubated overnight. And (5) flushing.
S5: closing: blocking solution was added to each well of the ELISA plate and incubated for 2h. And (5) flushing.
S6: combining: anti-Hb antibodies (AHb) were added to each well of the elisa plate and incubated for 0.5h. And (5) flushing.
S7: reaction 1: adding diluted peripheral blood whole blood or erythrocyte lysate or Hb-Abeta complex standard protein into each hole of the ELISA plate, and incubating for 1h. And (5) flushing.
S8: reaction 2: the blocking solution is used for diluting the biotinylation anti-Abeta antibody (B-AA beta) to a final concentration of 0.5-4 mug/mL. The antibody dilutions were added to each well of the elisa plate and incubated for 1h. And (5) flushing.
S9: reaction 3: alkaline phosphatase-labeled streptavidin (AP-SA) (1:10000 dilution) was diluted with a PEG-6000-containing diluent, and 100. Mu.L of the enzyme diluent was added to each well of the ELISA plate. Incubate for 0.5h. And (5) flushing.
S10: chemiluminescence: chemiluminescent solution is added to each well of the microplate.
S11: and (3) measuring the content: the chemiluminescent value detector measures the chemiluminescent value of each well of the ELISA plate.
S12: and (3) calculating: the complex content in the sample was calculated from the relationship between the concentration of Hb-aβ complex standard prepared in vitro and the chemiluminescent value.
Method 7 chemiluminescent immunoassay (CLIA) wherein a capture antibody is immobilized to an elisa plate via Protein G and a PEG-enhanced elisa detection antibody is conjugated
S1-S3: as shown in method 1.
S4: coating: with NaHCO 3 The buffer dilutes Protein G to a final concentration of 0.001-0.1. Mu.g/mL. 100 μl of this Protein G dilution was added to each well of the elisa plate and incubated overnight. And (5) flushing.
S5: closing: to each well of the ELISA plate, 300. Mu.L of blocking solution was added and incubated for 2 hours. And (5) flushing.
S6: combining: anti-Hb antibodies were added to each well of the ELISA plate and incubated for 0.5h. And (5) flushing.
S7: reaction 1: adding diluted peripheral blood whole blood or erythrocyte lysate or Hb-Abeta complex standard protein into each hole of the ELISA plate, and incubating for 1h. And (5) flushing.
S8: reaction 2: the alkaline phosphatase-labeled anti-Abeta antibody (AP-AA beta) was diluted with a PEG-6000 solution to a final concentration of 0.1-2. Mu.g/mL. The antibody dilutions were added to each well of the elisa plate and incubated for 0.5h. And (5) flushing.
S9: chemiluminescence: chemiluminescent solution is added to each well of the microplate.
S10: and (3) measuring the content: the chemiluminescent signal detection instrument measures the chemiluminescent value of each well of the ELISA plate.
S11: and (3) calculating: the content of Hb-Abeta complex in the sample is calculated according to a relation curve between the concentration of Hb-Abeta complex standard prepared in vitro and the chemiluminescence value.
Method 8: chemiluminescent immunoassay (CLIA) in which a capture antibody is immobilized to an elisa plate via Protein G and a PEG-conjugated liquid phase elisa is used to detect antibody-antigen complex reaction
S1-S3: as shown in method 1.
S4: coating: with NaHCO 3 The buffer dilutes Protein G to a final concentration of 0.001-0.1. Mu.g/mL. 100 μl of this Protein G dilution was added to each well of the elisa plate and incubated overnight. And (5) flushing.
S5: closing: to each well of the ELISA plate, 300. Mu.L of blocking solution was added and incubated for 2 hours. And (5) flushing.
S6: combining: anti-Hb antibodies (AHb) were added to each well of the elisa plate and incubated for 0.5h. And (5) flushing.
S7: reaction 1: adding peripheral blood whole blood or erythrocyte lysate diluted by PEG-6000 diluent and alkaline phosphatase labeled anti-Abeta antibody (AP-Abeta) into an EP tube, and incubating for 0.5h.
S8: 100. Mu.L of the preformed Hb-Abeta complex was added to each well of the ELISA plate, incubated for 0.5h, and rinsed.
S9: chemiluminescence: chemiluminescent solution is added to each well of the microplate.
S10: and (3) measuring the content: the chemiluminescent signal detection instrument measures the chemiluminescent value of each well of the ELISA plate.
S12: and (3) calculating: the content of Hb-Abeta complex in the sample is calculated according to a relation curve between the concentration of Hb-Abeta complex standard prepared in vitro and the chemiluminescence value.
Example 3 sensitivity, repeatability and precision test of Hb-Abeta detection kit
The Hb-Abeta kit was prepared under the optimal conditions selected in method 7, and three batches of 20 kits were randomly extracted from each batch, and the following test was performed.
Precision test: three samples were measured 8 times each using the above extracted kit. The coefficient of variation of the measured concentration was calculated. Experimental results show that the variation coefficient of the detection results of the three batches of kits is less than 8.0%.
Repeatability test: the test was repeated 3 times for the same sample using the above extracted kit, and the result showed that the relative standard deviation RSD was 0.60%.
The experimental results show that the detection results of the kits on the samples have smaller discrete degree and better repeatability, and can be used for detecting Abeta.
Example 4 gain Effect of different methods on sensitivity of kits
Gain effect of PEG-6000 on Hb-Abeta detection kit
AHb directly coats an ELISA plate, then sequentially reacts with antigen to be detected (namely Hb-Abeta complex in AD and HC red blood cell samples), biotin-marked Abeta (B-Abeta) and AP-marked avidin (AP-SA), and chemiluminescent liquid is added to detect a chemiluminescent signal value. A certain amount of PEG-6000 was added to the B-AA beta or/and AP-SA dilutions, and the effect on the chemiluminescent signal values was observed. The results showed that adding PEG to the B-AA β or AP-SA dilutions increased the signal values by a factor of 1.42 and 6.07, respectively, calculated as the signal value without PEG, whereas the signal value increased by a factor of 8.88 with both the B-AA β and AP-SA dilutions added PEG. The results indicate that PEG has some promoting effect on antigen-antibody binding, but more remarkable enhancing effect on enzymatic chemiluminescent reaction (fig. 2).
Combined gain action of Protein G and PEG-6000 on Hb-Abeta detection kit
Coating an ELISA plate with Protein G, combining with AHb, then sequentially reacting with antigen to be detected (namely Hb-Abeta complex in AD and HC red blood cell samples) and alkaline phosphatase marked Abeta (AP-Abeta), and adding chemiluminescent liquid to detect chemiluminescent signal intensity. And (3) adding a certain amount of PEG-6000 into the Protein G coated ELISA plate or the AP-AA beta diluent, or observing the influence of the Protein G coated ELISA plate or the AP-AA beta diluent on the chemiluminescent signal intensity under the condition that the Protein G coated ELISA plate and the AP-AA beta diluent are added with a certain amount of PEG-6000 at the same time. The results show that the chemiluminescent signal values are improved by a factor of 2 or more in the case of Protein G coating compared to the case of no Protein G coating. Furthermore, the addition of PEG in the presence of Protein G coating can still greatly increase the value of the chemiluminescent signal. The signal value is increased by 2.46 times and 5.48 times respectively by adding PEG to the Protein G coated ELISA plate and the AP-AA beta diluent, and the signal value is increased by 14.48 times under the condition that the Protein G coated ELISA plate and the AP-AA beta diluent are added with PEG simultaneously, wherein the signal value is calculated as 1 under the condition that the signal value without PEG. The results indicate that both Protein G coating and PEG have an enhancing effect on antigen-antibody binding and enzymatic chemiluminescent reaction efficiency, but that PEG has a stronger enhancing effect on enzymatic chemiluminescent reaction efficiency, and that the gain effect of adding PEG in the case of Protein G coated elisa plate is not a mere additive effect, but a multiplicative effect (fig. 3).
3. Testing Hb-Abeta standard concentration and chemiluminescent signal relation curve by different detection methods
And detecting Hb-Abeta standard substances by using different methods, and drawing a relationship curve of Hb-Abeta concentration and chemiluminescence signals to obtain indexes such as the minimum detection limit, curve slope and the like of the different methods. The ELISA method was used for both methods 1 and 2, and the sensitivity was not significantly changed (FIG. 4A). Methods 3 and 6 both employ CLIA methods, wherein the capture antibodies, after binding to the antigen, react with B-AA β and AP-SA, respectively. As the method 6 increases the Protein G coating and adds PEG-6000 into the AP-SA diluent, the detection sensitivity is greatly improved (FIG. 4B). In the CLIA method by directly combining the antigen with the AP-AA beta, the sensitivity of the capture antibody is higher than that of the direct coating ELISA plate (method 5) through the SA coating ELISA plate (method 4), and the sensitivity of the detection method is obviously enhanced by adding PEG-6000 into the AP-AA beta diluent through the combination of the Protein G coating ELISA plate (method 7). Protein G coats the ELISA plate, forms Hb-Abeta complex in the liquid phase, and PEG-6000 is added into the liquid phase complex reaction solution, so that the detection sensitivity is further improved (method 8) (FIG. 4C). Various methods test the slope, R of Hb-Abeta concentration and chemiluminescent Signal relationship curve obtained from Hb-Abeta standards 2 The values and the lowest limit of detection (LOD) are shown in table 1.
Example 5 comparison of the effects of different Hb-Abeta detection kits to differentiate AD and HC
The different methods selected in example 2 of the present invention were used to prepare Hb-Abeta detection kits, 51 Healthy Control (HC) subjects were examined, and the chemiluminescent values of Hb-Abeta in peripheral blood whole blood and erythrocytes of 51 AD patients were then calculated for each subject based on a standard curve, i.e., hb-Abeta ng/mL (FIGS. 5A and A ') or Hb-Abeta ng/mg Hb (FIGS. 5B and B'). Scatter plots and subject operating characteristics (Receiver operating characteristic curve, ROC) were plotted according to the test results (fig. 5C and C'). The results are shown in Table 1. The improved methods (methods 5-8) used in this patent have different degrees of improvement in sensitivity, specificity and AUC for distinguishing AD from HC when detecting Hb-Abeta complexes in erythrocytes, wherein the improvement effect of methods 7 and 8 is most obvious. These methods have significantly improved sensitivity, specificity and AUC effects in distinguishing AD from HC when detecting Hb-aβ complexes in peripheral blood whole blood samples. FIG. 5 shows a scatter plot and ROC curve of the test values of Hb-Abeta complexes in AD and HC red blood cells and whole blood samples using a partial kit method.
Table 1: instance verification of various technical indexes of different detection methods
Description: LOD: a minimum detection limit; AUC: the area under the operating characteristic curve of the subject represents the accuracy of diagnosis, and the higher the numerical value, the higher the accuracy.
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Claims (10)
1. A method for quantitatively detecting Hb-aβ complexes derived from peripheral red blood cells, comprising the steps of immobilizing Protein G or Streptavidin (SA) on a solid support, subsequently adding an anti-Hb antibody (AHb) or a biotinylated anti-Hb antibody (B-AHb) to form a conjugate, and reacting with Hb-aβ complexes in a sample, followed by a step of reacting with an enzyme-labeled anti-aβ antibody (E-AA β), a chemiluminescent solution, respectively, or a step of reacting with a biotin-labeled anti-aβ antibody (B-AA β), an enzyme-labeled avidin (E-SA), a chemiluminescent solution, respectively; wherein, AA beta may be an antibody that specifically recognizes aβ42, aβ40 or any other aβ peptide; in the reaction step of enzyme labeling AA beta or enzyme labeling avidin, polyethylene glycol (PEG) is added into the reaction solution; wherein the concentration of PEG is 0.1-12%, preferably 2-8%, more preferably 5%.
2. The method according to claim 1, further comprising the step of collecting a blood sample, forming Hb-Abeta complex in the liquid phase reaction solution containing PEG, and detecting the antigen-antibody conjugate by a luminescence method.
3. The detection method according to claim 1, comprising the steps of:
and (3) coating an ELISA plate: adding the prepared Protein G or SA coating liquid into the hole of the ELISA plate, incubating overnight, and flushing; adding the prepared sealing liquid into the holes of the ELISA plate, incubating for 2-4h at constant temperature, and flushing;
adding AHb or B-AHb solution into the coated ELISA plate, incubating for 0.5-2h at constant temperature, and flushing.
4. The method for detecting according to claim 1,
the Protein G coating liquid is prepared by the following steps: the solvent is buffer solution or physiological saline, and the concentration of Protein G is 0.001-0.05 μg/mL, preferably 0.002-0.02 μg/mL, more preferably 0.005 μg/mL;
the SA coating liquid is prepared by the following steps: the solvent is buffer solution or physiological saline, and SA concentration is 0.1-10 μg/mL, preferably 1-5 μg/mL, more preferably 4 μg/mL;
wherein, the preparation method of the AHb or B-AHb solution comprises the following steps: the solvent is buffer solution or physiological saline, and the concentration of AHb or B-AHb is 0.1-5.0 mug/mL, preferably 0.5-2.0 mug/mL, more preferably 1.0 mug/mL;
wherein the blocking solution is selected from 1% -10% bovine serum albumin solution or 1-2% gelatin solution or any other liquid which can be used for preventing the non-specific binding of antibodies; the preparation method of the 1% -10% bovine serum albumin solution comprises the following steps: adding bovine serum albumin into buffer solution or normal saline, and uniformly mixing, wherein the preparation method of the 1-2% gelatin solution comprises the following steps: dissolving gelatin in buffer solution or physiological saline solution, and mixing uniformly;
Wherein the buffer solution is selected from the group consisting of: tris-HCl buffer, phosphate Buffer (PBS), carbonate Buffer (CBS).
5. The method for detecting according to claim 1,
the method comprises the steps of adding prepared Protein G or SA coating liquid into a solid phase carrier, incubating overnight, and flushing; adding the prepared sealing liquid into the hole of the ELISA plate, and incubating at constant temperature; adding AHb or B-AHb solution into the coated solid phase carrier, and incubating at constant temperature.
6. The detection method according to claim 1, the method comprising the steps of:
s1: preparing a sample to be detected;
s2: adding Protein G solution into the solid phase carrier, incubating and flushing;
s3: adding a sealing liquid, incubating and flushing;
s4: adding AHb solution, incubating and flushing;
s5: adding a sample to be detected (containing Hb-Abeta complex), incubating and flushing;
s6: adding enzyme-labeled Abeta, incubating, washing, and adding chemiluminescent liquid to detect the value;
or adding biotin-marked AA beta into the solid phase carrier, incubating, washing, adding enzyme-labeled avidin secondary antibody, incubating, washing, and adding chemiluminescent liquid to detect the value;
wherein, adding PEG into enzyme marked AA beta or enzyme marked avidin reaction liquid; the Hb-Abeta complex content in the sample can be obtained through numerical calculation.
7. The detection method according to claim 1, the method comprising the steps of:
s1: preparing a sample to be detected;
s2: adding a streptavidin SA solution into the solid phase carrier, incubating and flushing;
s3: adding a sealing liquid, incubating and flushing;
s4: adding a biotinylated anti-Hb antibody solution, incubating, and rinsing;
s5: adding a sample to be detected (containing Hb-Abeta complex), incubating and flushing;
s6: adding enzyme-labeled anti-Abeta antibody, incubating, washing, and adding chemiluminescent liquid to detect the value;
wherein, PEG is added into the enzyme-labeled anti-Abeta antibody solution; the Hb-Abeta complex content in the sample can be obtained through numerical calculation.
8. The detection method according to claim 1, the method comprising the steps of:
s1: preparing a sample to be detected;
s2: adding Protein G into the solid phase carrier, incubating and flushing;
s3: adding a sealing liquid, incubating and flushing;
s4: adding anti-Hb antibody, incubating and washing;
s5: adding Hb-Abeta complex preformed in a liquid phase, incubating and flushing; adding chemiluminescent liquid to detect the value;
wherein, PEG is added into the liquid phase reaction liquid; the Hb-Abeta compound content can be obtained through calculation;
Wherein, the preparation method of the Hb-Abeta complex comprises the following steps:
and (3) preparing an enzyme-labeled anti-Abeta antibody (E-Abeta), and combining the Abeta in the Hb-Abeta complex in the sample in a liquid phase to form the Hb-Abeta complex.
9. The detection method according to claim 1, wherein a Hb-aβ complex standard is prepared for calculating the Hb-aβ complex content in the sample to be measured, the preparation method being as follows;
A. respectively dissolving the purified Abeta polypeptide and Hb protein by using a buffer solution, mixing, shaking and incubating;
B. incubating a sample, centrifuging, taking supernatant, and filtering by a gel filtration column to separate Hb-Abeta complex;
C. mass spectrometry was used to determine the mass fraction of aβ in the Hb-aβ complex.
10. A detection kit comprising a specific anti-Hb antibody (AHb), a solid support, protein G, PEG; the kit may also contain an anti-aβ antibody (AA β), a luminescent agent, biotin or an enzyme, necessary solvents, instructions for use; the kit may further comprise Hb-aβ standard, streptavidin (SA), biotin-labeled anti-aβ antibody (B-AA β), or enzyme-labeled anti-aβ antibody (E-AA β); enzyme-labeled avidin (E-SA) which specifically binds to B-AA beta may also be included; the enzyme is an enzyme that can catalyze chemiluminescent reactions, such as alkaline phosphatase (Alkaline Phosphatase, AP), horseradish peroxidase (Horseradish Peroxidase, HRP), or Glucose Oxidase (GO); the kit can also comprise antibodies or avidin marked by other substances such as fluorescein, colloidal gold and the like, the antibodies can be specifically combined with the anti-Abeta antibodies, and the kit can also comprise buffer solution and blocking solution; the PEG can be PEG with any molecular weight, including PEG-200-PEG-20000, preferably PEG-4000-PEG-8000, more preferably PEG-6000; the invention further comprises application of the reagent in the kit in preparation of the kit for detecting Alzheimer's disease.
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