CN112098652B - Paper-based enzyme-linked immunosorbent assay for fixing and capturing antibody based on covalent bonding method - Google Patents
Paper-based enzyme-linked immunosorbent assay for fixing and capturing antibody based on covalent bonding method Download PDFInfo
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Abstract
The invention discloses a paper-based double-antibody sandwich method for fixing and capturing an antibody based on a covalent bonding method, belongs to the technical field of immunoassay, and is used for detecting the content of an allergy-like related MRGPRX2 receptor in human blood. MRGPRX2 mouse monoclonal antibody is used as a capture antibody, surface chemical modification is not needed to be carried out on filter paper, Bovine Serum Albumin (BSA) molecules with carboxyl and amino functional groups are introduced, and an antibody-BSA network structure is formed by utilizing the amide condensation reaction between amino and carboxyl in antibody molecules and BSA molecules, so that the capture antibody is fixed; a paper-based double-antibody sandwich method of MRGPRX2 is established by taking horseradish peroxidase (HRP) -labeled MRGPRX2 rabbit polyclonal antibody as a detection antibody and taking MRGPRX2 solution as a standard substance. The fixing method of the capture antibody is simple and efficient, has low cost, has high detection flux, high speed, detection limit, precision and other technical indexes meeting the requirements, has good accuracy and reproducibility of a quantitative result, and is suitable for clinical examination and blood epidemiological investigation.
Description
Technical Field
The invention belongs to the technical field of immunoassay, and relates to a paper-based enzyme-linked immunosorbent assay, in particular to a paper-based ELISA double-antibody sandwich method for detecting the content of a hypersensitivity-like related MRGPRX2 receptor in human blood by fixing and capturing an antibody by a covalent bonding method.
Background
Enzyme-linked immunosorbent assay (ELISA) is a technology that known antigen or antibody is adsorbed on the surface of a solid phase carrier by utilizing the specific reaction of antigen and antibody, an object to be detected is captured by the specific reaction, then the object to be detected reacts with the antigen or antibody labeled by enzyme, and then the object to be detected is quantitatively detected by the color reaction generated by the enzyme and a substrate. The technology can be used for detecting macromolecular antigens, specific antibodies and the like, has the advantages of rapidness, sensitivity, simplicity and convenience and the like, and can be used for clinical examination and investigation of serum epidemiology. Compared with the traditional 96-pore plate desk type enzyme-linked immunosorbent assay, the paper-based enzyme-linked immunosorbent assay has obvious advantages. The paper has good biocompatibility, and the microporous structure with high specific surface area improves the quantity of immobilized antibodies. Another notable feature of the paper is that small amounts of antibody, sample and substrate solutions are required for each test zone, sample and reagent consumption is low, analysis is fast, and operation is simple, so this method is both cheap and fast (less than 1 hour). Most importantly, the method is compatible with a variety of detection methods, and the results can be obtained by a simple desktop scanner or mobile phone and analyzed by ImageJ software, making the method applicable to resource-poor countries. Therefore, paper-based enzyme-linked immunosorbent assay has become an interesting cheap detection platform, and can replace glass and high polymer chips to carry out on-site analysis and detection in some cases.
A typical ELISA double antibody sandwich requires many steps and several washes after each step, which presents a challenge to the strength of the immobilization of the capture antibody. In general, the greater the number of antibodies immobilized on the substrate surface, the greater the number of molecules of analyte captured during the assay, and the greater the sensitivity of the assay detection. Methods for immobilizing antibodies include physical adsorption and chemical binding. Physical adsorption is currently the most widely used immobilization method due to its simplicity and versatility. However, about 40% of the antibody molecules adsorbed on cellulose paper can be desorbed, thereby making the paper-based ELISA method based on physisorption method for immobilizing antibodies insensitive to the detection of very low concentration of target. Therefore, developing an effective immobilization strategy to improve sensitivity is crucial to paper-based enzyme-linked immunosorbent assays. Compared with the physical adsorption method for fixing the antibody, the chemical covalent method for fixing the antibody can improve the fixing strength of the antibody and ensure the fixing quantity of the antibody, thereby improving the sensitivity of analysis and detection. Patent CN201810960085.6 reports that a paper-based sensor that complexes His-tag allergen detects allergic reactions associated with immunoglobulin IgE in serum. The chelating coordination fixing method is firm and reliable in fixing the antigen, but the fixing process needs to firstly carry out chemical modification on the surface of a paper base to generate aldehyde groups, then the aldehyde groups are combined with complex nickel-nitrilotriacetic acid, and then the allergen containing a His label is chelated. Therefore, the method for covalently immobilizing the capture antibody has the advantages of simple development steps, low cost and high efficiency and is of great significance.
G Protein Coupled Receptors (GPCRs) are the largest seven-transmembrane receptors, can transmit extracellular signals into cells to trigger biological response, regulate and control the proliferation, survival and metabolism of the cells, and play an important role in the transmission process of nerve signals. MRGPRX2 is a novel class of GPCRs, with high expression mainly in human skin mast cells. Researches show that MRGPRX2 is an action target of many small-molecule drugs for causing anaphylactoid and pseudo-anaphylaxis. In recent years, along with monitoring of various adverse drug reactions, the frequency of anaphylactoid reactions tends to increase, and due to the high hazard, the anaphylactoid reactions are more and more concerned. Early laboratory studies found that the content of the MRGPRX2 receptor in human blood basophilic granulocytes is positively correlated with the sensitivity of the study subject to anaphylactoid reaction. Patent cn201580053612.x and international patent WO2018232316(a1) report methods for screening pro-and anti-anaphylactoid drugs based on MRGPRX2/MRGPRB2 expressing cells, but clinical patients prone to anaphylactoid reaction are not predicted from a population perspective. Patent CN110240643A reports that the conventional ELISA method based on the physical adsorption method for immobilizing capture antibodies measures the content of MRGPRX2 in blood, and the method has limited sensitivity.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a paper-based enzyme-linked immunoassay method for fixing a capture antibody based on a covalent bonding method for detecting the content of an allergy-like related MRGPRX2 receptor in human blood, and the method can solve the problems of various steps, complex operation, high cost and low sensitivity of the conventional covalent bonding technology.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a paper-based enzyme-linked immunosorbent assay for fixing and capturing an antibody based on a covalent bond method, which comprises the following steps:
1) introducing bovine serum albumin with carboxyl and amino functional groups into an MRGPRX2 mouse monoclonal antibody serving as a capture antibody, and forming an antibody-BSA network structure through an amide condensation reaction of the MRGPRX2 mouse monoclonal antibody and the bovine serum albumin;
2) fixing the capture antibody on filter paper, establishing a standard curve by taking horseradish peroxidase-labeled MRGPRX2 rabbit polyclonal antibody as a detection antibody and MRGPRX2 protein solution as a standard substance;
3) the content of MRGPRX2 in the human blood sample to be detected is obtained by comparing the color development intensity of the human blood sample to be detected with a standard curve, and the rapid quantitative detection of the anaphylactoid-related receptor MRGPRX2 in the human blood is realized.
Preferably, a circular hydrophobic boundary is constructed on the filter paper by a wax sealing technology, and the fixation of the capture antibody and the detection of the target object are limited in a series of circular hydrophilic areas with the diameter of 0.5-1.0 cm.
Preferably, an activating agent is added when bovine serum albumin is introduced for the amide condensation reaction, and the activating agent is one or a mixture of 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide and N-hydroxysuccinimide.
Preferably, the reaction solvent for the amide condensation reaction of the MRGPRX2 murine monoclonal antibody and the bovine serum albumin is triple distilled water, 2- (N-morpholine) ethanesulfonic acid buffer solution with the pH value of 5-8 or phosphate buffer solution with the pH value of 5-8.
Preferably, the blocking solution for blocking and capturing the antibody in the quantitative detection process of the enzyme-linked immunosorbent assay is bovine serum albumin or milk powder solution with the concentration of 2-10%.
Preferably, the concentration of the capture antibody is 5-100 mug/mL.
Preferably, the concentration of the detection antibody is 1-10 mug/mL.
Compared with the prior art, the invention has the following beneficial effects:
the method disclosed by the invention takes MRGPRX2 mouse monoclonal antibody as a capture antibody, introduces Bovine Serum Albumin (BSA) with amino and carboxyl functional groups, and realizes the fixation of the capture antibody by utilizing the amide condensation reaction between the capture antibody and the amino and carboxyl in the BSA. The surface of the filter paper does not need to be subjected to functionalization pretreatment, the operation is simple and easy, and the cost is low; the addition of BSA also plays a role in isolation, minimizes antibody-antibody cross-linking, avoids the formation of a highly cross-linked network between antibodies, is beneficial to the maintenance of antibody conformation, and promotes the stability of the antibodies. Stability experiments showed that the paper-capture antibody immobilized by this covalent bonding method can be stably stored in a 4 degree refrigerator for at least 80 days (fig. 5). Meanwhile, the method has accurate and reliable quantitative result, less reagent dosage and short protein antibody reaction time, and can realize high-flux and rapid determination of the MRGPRX2 content in blood within 45 minutes. The conventional ELISA method has long binding time of the protein with the capture antibody and the detection antibody, and usually requires 2.5-3.5 hours to complete detection. The limit of the method for detecting the MRGPRX2 protein in human blood is 1.5ng/mL, and compared with the traditional ELISA method (the limit of the method is 7.75ng/mL, patent CN110240643A), the method has higher sensitivity. Therefore, the invention uses the MRGPRX2 receptor as a substance to be tested, aims to develop a low-cost, simple and efficient covalent bonding fixed capture antibody method and a high-flux and high-sensitivity ELISA double-antibody sandwich method for determining the content of the MRGPRX2 receptor in human blood, and has important significance for predicting the sensitivity of clinical patients to anaphylactoid reaction and guiding clinical medication.
Drawings
FIG. 1 is a schematic diagram of the preparation process of a paper-based ELISA microplate;
FIG. 2 is a schematic illustration of the immobilization of a capture antibody;
FIG. 3 is a schematic diagram of the detection process of the paper-based ELISA double antibody sandwich method;
FIG. 4 is a standard curve of ELISA double antibody sandwich method;
FIG. 5 shows the stability test results of paper-based ELISA microplates stored at 25 and 4 degrees.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
referring to fig. 3, the paper-based enzyme-linked immunosorbent assay for detecting the content of the receptor MRGPRX2 related to the anaphylactoid in human blood disclosed by the invention comprises the following steps:
(1) the hydrophobic region was prepared by wax-sealing filter paper as follows (fig. 1):
1) knocking out a series of small holes with the diameter of 0.5-1.0 cm by using a puncher and a small hammer, and covering the punched waxed paper on filter paper for later use;
2) preheating the steel plate at 300 ℃ for 5min, and then pressing the filter paper covered with the perforated wax paper to prepare the wax-sealed paper-based ELISA microporous plate.
(2) Immobilization of the capture antibody on filter paper:
respectively sucking a certain amount of MRGPRX2 mouse monoclonal antibody, reaction solvent, activator and Bovine Serum Albumin (BSA) solution, mixing, vortexing, taking out a certain amount of MRGPRX2 mouse monoclonal antibody, dripping the MRGPRX2 mouse monoclonal antibody, the reaction solvent, the activator and Bovine Serum Albumin (BSA) solution on a small hole of wax-sealed filter paper, and airing at room temperature (see figure 2).
(3) Washing:
and adding PBST buffer solution into the small holes of the dried filter paper for washing, and drying at room temperature.
(4) And (3) sealing:
BSA solution was added to the wells for blocking and air-dried at room temperature.
(5) Adding MRGPRX2 standard protein:
adding MRGPRX2 protein solution into the small hole, wherein the concentration of MRGPRX2 protein is 15 ng/mL-1000 ng/mL, and drying at room temperature.
(6) Add detection antibody and wash:
horseradish peroxidase (HRP) -labeled MRGPRX2 rabbit polyclonal antibody (HRP-rabbit polyclonal antibody) was added to the wells as a detection antibody, and washed four times with PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition:
substrate TMB was added and developed in the dark, then photographed with a cell phone or camera and analyzed for color intensity by ImageJ software.
(8) Drawing a standard curve:
the standard curve was established with the intensity of color development as ordinate and the concentration of MRGPRX2 protein as abscissa, and as a result, see fig. 4, it can be seen from fig. 4 that the intensity of color development increases linearly with the increase in concentration when the concentration of MRGPRX2 protein increases from 0 to around 250 ng/mL.
(9) Pretreatment of human blood samples:
collecting fresh blood sample, using human mononuclear cell separating medium, separating and purifying to obtain 1mL basophilic granulocyte in the anticoagulated blood of the patient; and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of human blood samples:
and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding the membrane protein extracted in the step (9) into the small hole, repeating the steps (6) and (7) to obtain the color development intensity of the human blood sample, comparing the color development intensity with the standard curve obtained in the step (8) to obtain the content of MRGPRX2 in the human sample to be detected, and predicting the sensitivity degree of the patient to anaphylactoid reaction through the content of MRGPRX 2. Experiments show that the content of the MRGPRX2 receptor of a normal person is 2-30 ng/mL, and the content of the MRGPRX2 receptor of an asthma patient is higher than 40 ng/mL. The method can simply, quickly and sensitively realize the determination of the content of the MRGPRX2 receptor in human blood, thereby predicting the sensitivity degree of clinical patients to anaphylactoid reaction and guiding clinical medication.
Example 1
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 6mm from the wax paper by using a puncher with the inner diameter of 6mm and a small hammer, and covering the wax paper with the punched holes on filter paper; and (3) putting the steel plate into a box type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare wax-sealed filter paper.
(2) Immobilization of the capture antibody: mu.L of 600. mu.M BSA, 50. mu.L of 100. mu.M MES pH 6.0, 60. mu.L of 100. mu.M EDC and 25. mu.L of 40. mu.g/mL murine mAb were pipetted into a centrifuge tube and mixed, 5. mu.L was dropped onto wax-sealed filter paper wells and air-dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 8% BSA to each well for blocking and air dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody at a concentration of 2. mu.g/mL was added to each well and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 2
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 8mm from the wax paper by using a puncher with the inner diameter of 8mm and a small hammer, and covering the wax paper with the punched holes on the filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: 50 μ L of 600 μ M BSA, 80 μ L of triple distilled water, 40 μ L of 100 μ M EDC, 40 μ L of 100 μ M NHS and 50 μ L of 30 μ g/mL murine mAb were pipetted into a centrifuge tube and mixed well, and 8 μ L of each was dropped onto a small hole of wax-sealed filter paper and dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 10% BSA to each well for blocking and air dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody at a concentration of 5. mu.g/mL was added to each well and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 3
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 10mm from the wax paper by using a puncher with the inner diameter of 10mm and a small hammer, and covering the wax paper with the punched holes on the filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: 25 μ L of 300 μ M BSA, 50 μ L of 100 μ M PBS with pH 6.0, 50 μ L of 100 μ M NHS and 25 μ L of 10 μ g/mL murine mAb were pipetted into a centrifuge tube and mixed, 10 μ L was dropped into a wax-sealed filter paper well, and the filter paper well was dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 20. mu.L of 5% milk powder solution to each well, seal, and dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody was added to each well at a concentration of 6. mu.g/mL, and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 4
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 6mm from the wax paper by using a puncher with the inner diameter of 6mm and a small hammer, and covering the wax paper with the punched holes on filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: 50 μ L of 400 μ M BSA, 50 μ L of 100 μ M MES pH 8.0, 80 μ L of 100 μ M EDC, 20 μ L of 100 μ M NHS and 25 μ L of 100 μ g/mL murine mAb were pipetted into a centrifuge tube and mixed, 5 μ L was dropped onto wax-sealed filter paper wells, and then air-dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 10% BSA to each well for blocking and air dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: 5 μ L of HRP-rabbit polyclonal antibody at a concentration of 8 μ g/mL was added to each well and washed four times with 10 μ L PBST 1min after reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) The pretreatment of human blood samples to collect fresh blood samples, and separating and purifying the fresh blood samples by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004) to obtain 1mL of basophilic granulocytes in the anticoagulated blood of patients; and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 5
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 8mm from the wax paper by using a puncher with the inner diameter of 8mm and a small hammer, and covering the wax paper with the punched holes on the filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: 50 μ L of 300 μ M BSA, 100 μ L of triple distilled water, 80 μ L of 100 μ M EDC, 10 μ L of 100 μ M NHS and 25 μ L of 10 μ g/mL murine mAb were pipetted into a centrifuge tube and mixed well, and 8 μ L of each was dropped onto a small hole of wax-sealed filter paper and dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 5% milk powder solution to each well, seal, and dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody at a concentration of 10. mu.g/mL was added to each well and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 6
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 10mm from the wax paper by using a puncher with the inner diameter of 10mm and a small hammer, and covering the wax paper with the punched holes on the filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: 50 μ L of 200 μ M BSA, 70 μ L of 100 μ M PBS with pH 8.0, 80 μ L of 100 μ M EDC, 40 μ L of 100 μ M NHS and 25 μ L of 80 μ g/mL murine mAb were pipetted into a centrifuge tube and mixed, 10 μ L was dropped onto wax-sealed filter paper wells, and then dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 4% BSA to each well and block and dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody at a concentration of 3. mu.g/mL was added to each well and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 7
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 8mm from the wax paper by using a puncher with the inner diameter of 8mm and a small hammer, and covering the wax paper with the punched holes on the filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: mu.L of 100. mu.M BSA, 100. mu.M MES at pH 7.0, 60. mu.L of 100. mu.M EDC and 25. mu.L of 30. mu.g/mL murine mAb were pipetted into a centrifuge tube and mixed, 5. mu.L was dropped onto wax-sealed filter paper wells, and the filter paper wells were dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 5% BSA to each well for blocking and air dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody at a concentration of 10. mu.g/mL was added to each well and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 8
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 8mm from the wax paper by using a puncher with the inner diameter of 8mm and a small hammer, and covering the wax paper with the punched holes on the filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: 25 μ L of 800 μ M BSA, 50 μ L of 100 μ M PBS at pH 7.0, 90 μ L of 100 μ M EDC, 20 μ L of 100 μ M NHS and 25 μ L of 40 μ g/mL murine mAb were pipetted into a centrifuge tube and mixed, 10 μ L was dropped onto wax-sealed filter paper wells, and the tube was dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 10% BSA to each well for blocking and air dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody at a concentration of 10. mu.g/mL was added to each well and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 9
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 6mm from the wax paper by using a puncher with the inner diameter of 6mm and a small hammer, and covering the wax paper with the punched holes on filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: mu.L of 200. mu.M BSA, 50. mu.L of 100. mu.M MES pH 5.0, 50. mu.L of 100. mu.M EDC, 30. mu.L of 100. mu.M NHS and 25. mu.L of 50. mu.g/mL murine mAb were pipetted into a centrifuge tube and mixed, 5. mu.L was dropped onto wax-sealed filter paper wells, and the mixture was dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 4% milk powder solution to each well, seal, and dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody was added to each well at a concentration of 6. mu.g/mL, and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) The pretreatment of human blood samples to collect fresh blood samples, and separating and purifying the fresh blood samples by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004) to obtain 1mL of basophilic granulocytes in the anticoagulated blood of patients; and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
Example 10
The steps for detecting the content of MRGPRX2 in human blood by a paper-based ELISA double-antibody sandwich method based on antibody fixed by a covalent bonding method are as follows:
(1) wax sealing with filter paper: knocking out a series of small holes with the inner diameter of 10mm from the wax paper by using a puncher with the inner diameter of 10mm and a small hammer, and covering the wax paper with the punched holes on the filter paper; and (3) putting the steel plate into a box-type resistance furnace at 300 ℃ to heat for 5min, taking out the steel plate, and pressing the steel plate on wax paper for 5min to prepare the wax-sealed filter paper.
(2) Immobilization of the capture antibody: mu.L of BSA (300. mu.M), 50. mu.L of PBS (100. mu.M, pH 5.0), 50. mu.L of EDC (100. mu.M), 25. mu.L of NHS (100. mu.M) and 25. mu.L of murine mAb (80. mu.g/mL) were pipetted into a centrifuge tube, mixed, and 10. mu.L was dropped into a small hole of wax-sealed filter paper, and then dried at room temperature.
(3) Washing: after drying in the air, 10 μ L of PBST was added to the pores of the filter paper, and the filter paper was washed three times with PBST and then dried in the air at room temperature.
(4) And (3) sealing: add 10. mu.L of 2% BSA to each well and block, and dry at room temperature.
(5) Addition of standard protein solution: mu.L of 2% BSA diluted MRGPRX2 protein solution was added to each well at concentrations ranging from 15ng/mL to 1000ng/mL of MRGPRX2 and allowed to air dry at room temperature.
(6) Addition and washing of detection antibody: mu.L of HRP-rabbit polyclonal antibody at a concentration of 7. mu.g/mL was added to each well and washed four times with 10. mu.L of PBST immediately after 1min of reaction.
(7) And (3) color development and image acquisition: 5 μ L of TMB was added to each well and developed in the dark, and after 10min, the image was taken and the intensity of the development was analyzed by ImageJ software.
(8) Drawing a standard curve: a standard curve was established with the intensity of color development as the ordinate and the concentration of MRGPRX2 as the abscissa.
(9) Pretreatment of human blood samples: collecting fresh blood sample, and separating and purifying to obtain 1mL basophilic granulocyte in anticoagulated blood of patients by using human mononuclear cell separating medium (Oriental Huahui, product batch number: 25171004); and (3) cracking the cells by using a Thermo membrane protein extraction kit to extract membrane protein.
(10) ELISA detection of blood samples: and (3) repeating the steps (3) and (4) on a small hole of the filter paper fixed with the capture antibody, adding 5 mu L of membrane protein into each hole, repeating the steps (6) and (7) to obtain the chromogenic intensity, comparing with a standard curve to obtain the content of MRGPRX2 in the blood sample to be detected, and experimentally measuring that the content of MRGPRX2 in human blood is within the range of 2-160 ng/mL.
In conclusion, the invention aims to detect the anaphylactoid-related receptor MRGPRX2 in human blood, uses MRGPRX2 mouse monoclonal antibody as a capture antibody, adopts a chemical bonding method to fix the capture antibody, introduces Bovine Serum Albumin (BSA) molecules with carboxyl and amino functional groups, and utilizes the amide condensation reaction between the amino and carboxyl in the antibody molecules and the BSA molecules to form an antibody-BSA network structure, thereby realizing the economical, simple and efficient fixation of the capture antibody without chemically modifying the surface of filter paper; the prepared paper-capture antibody can be stably stored in a 4-degree refrigerator for 80 days; a paper-based ELISA double-antibody sandwich method of MRGPRX2 is established by taking horseradish peroxidase (HRP) -labeled MRGPRX2 rabbit polyclonal antibody as a detection antibody and taking MRGPRX2 solution as a standard substance. ELISA detection is completed within 45 minutes, the result can be obtained by a simple desktop scanner or a mobile phone and analyzed by ImageJ software, the instrument dependence is low, and field analysis and detection can be carried out. The fixing method of the capture antibody is simple, efficient and low in cost, the paper-based ELISA double-antibody sandwich method has the advantages that the detection flux is high, the speed is high, the detection limit, the precision and other technical indexes meet the requirements, the accuracy and the repeatability of the quantitative result are good, and the method is suitable for clinical examination and blood epidemiological investigation.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (3)
1. A paper-based enzyme-linked immunosorbent assay for immobilizing and capturing antibodies based on a covalent bonding method is characterized by comprising the following steps:
1) introducing bovine serum albumin with carboxyl and amino functional groups into an MRGPRX2 mouse monoclonal antibody serving as a capture antibody, and forming an antibody-BSA network structure through an amide condensation reaction of the MRGPRX2 mouse monoclonal antibody and the bovine serum albumin; the concentration of the capture antibody is 5-100 mug/mL; wherein, an activating agent is added when bovine serum albumin is introduced for carrying out amide condensation reaction, and the used activating agent is one or a mixture of 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide and N-hydroxysuccinimide;
2) fixing the capture antibody on filter paper, establishing a standard curve by taking horseradish peroxidase-labeled MRGPRX2 rabbit polyclonal antibody as a detection antibody and MRGPRX2 protein solution as a standard substance; the concentration of the detection antibody is 1-10 mug/mL; constructing a circular hydrophobic boundary on filter paper by a wax sealing technology, and limiting the fixation of a capture antibody and the detection of a target object in a series of circular hydrophilic areas with the diameter of 0.5-1.0 cm;
3) the content of MRGPRX2 in the human blood sample to be detected is obtained by comparing the color development intensity of the human blood sample to be detected with a standard curve, and the rapid quantitative detection of the anaphylactoid-related receptor MRGPRX2 in the human blood is realized.
2. The paper-based enzyme-linked immunosorbent assay for immobilizing the capture antibody based on the covalent bonding method, according to claim 1, wherein the reaction solvent for the amide condensation reaction of the MRGPRX2 murine monoclonal antibody and bovine serum albumin is triple distilled water, 2- (N-morpholine) ethanesulfonic acid buffer with a pH value of 5-8, or phosphate buffer with a pH value of 5-8.
3. The paper-based enzyme-linked immunosorbent assay for immobilizing the capture antibody based on the covalent bonding method as claimed in claim 1, wherein the blocking solution for blocking the capture antibody in the quantitative detection process of the enzyme-linked immunosorbent assay is bovine serum albumin or milk powder solution with the concentration of 2% -10%.
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| CN111381038A (en) * | 2020-03-06 | 2020-07-07 | 南京农业大学 | Method for simultaneously and rapidly quantitatively detecting tetracycline and chloramphenicol based on paper chip color comparison |
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