CN111381028A - Kit, system, application, detection antibody composition and preparation method thereof - Google Patents
Kit, system, application, detection antibody composition and preparation method thereof Download PDFInfo
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- CN111381028A CN111381028A CN201811647322.XA CN201811647322A CN111381028A CN 111381028 A CN111381028 A CN 111381028A CN 201811647322 A CN201811647322 A CN 201811647322A CN 111381028 A CN111381028 A CN 111381028A
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- detection antibody
- detection
- antibody composition
- fluorescence
- related protein
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Abstract
The invention discloses a kit, a system, an application, a detection antibody composition and a preparation method thereof, wherein the detection antibody composition comprises: the kit comprises a detection antibody, a fluorescent marker and fluorescence enhancement particles which are respectively connected with the detection antibody, wherein the fluorescence enhancement particles comprise metal nanoparticles. By the mode, the method can simplify the operation process, effectively amplify the fluorescence detection signal and improve the detection sensitivity.
Description
Technical Field
The invention relates to the field of in vitro diagnosis and detection, in particular to a kit, a system, application, a detection antibody composition and a preparation method thereof.
Background
Immunofluorescence detection technology is a commonly used immunoassay means, and mainly determines the result of a corresponding detection index by detecting the fluorescence intensity of a cell complex containing a fluorescent marker. With the continuous development of immunofluorescence detection technology, the requirement for detection sensitivity is continuously improved, and how to improve the detection sensitivity becomes a hotspot of research of people.
At present, the detection sensitivity is usually improved by increasing the type of the fluorescent substance or increasing the dosage of the fluorescent substance, however, the fluorescent intensity of the fluorescent marker is limited, and the requirement on the fluorescent intensity in the high-sensitivity detection process cannot be met.
In the long-term research and development process, the inventor of the application finds that the detection sensitivity is low in the conventional immunofluorescence detection process.
Disclosure of Invention
The invention mainly solves the technical problem of providing a kit, a system, an application, a detection antibody composition and a preparation method thereof, which can simplify the operation process, effectively amplify fluorescence detection signals and improve the detection sensitivity.
In order to solve the technical problems, the invention adopts a technical scheme that: a detection antibody composition is provided.
Wherein the detection antibody composition comprises: the kit comprises a detection antibody, a fluorescent marker and fluorescence enhancement particles which are respectively connected with the detection antibody, wherein the fluorescence enhancement particles comprise metal nanoparticles.
In order to solve the technical problem, the invention adopts another technical scheme that: provides a preparation method of a detection antibody composition.
The method comprises the following steps:
adding the fluorescence-enhancing particles to a buffer solution to form a fluorescence-enhancing particle suspension;
adding a cross-linking agent into the fluorescence-enhanced particle suspension and activating to obtain a first product;
adding a detection antibody suspension to the first product to obtain a second product.
In order to solve the technical problem, the invention adopts another technical scheme that: a kit is provided.
Wherein, the kit includes:
a kit body;
the first reagent holding position is arranged on the kit body and is used for holding the detection antibody composition;
wherein the first reagent holding site comprises at least two of the detection antibody compositions, and the detection antibodies in different detection antibody compositions are of different types.
In order to solve the technical problems, the invention adopts a technical scheme that: the application of a detection antibody composition in immunofluorescence analysis is provided, wherein the detection antibody composition is used for detecting at least one of thyroid function related protein, cardiovascular function related protein, cardiac troponin, hepatic fibrosis related protein, tumor related protein, gonadal function related protein, renal function related protein, bone metabolism function related protein, carbohydrate metabolism function related protein, infectious disease related protein, autoimmune function related protein, prenatal screening project related protein, drug detection related protein, type 4 human herpesvirus related protein and inflammation related protein.
In order to solve the technical problems, the invention adopts a technical scheme that: an immunofluorescence analysis system is provided.
The immunofluorescence analysis system includes:
the kit described;
a sample analyzer for performing detection of different analytes by using the immunodetection composition and the detection antibody composition in the kit, and outputting a detection result.
The invention has the beneficial effects that: different from the situation of the prior art, the metal nano particles and the fluorescent marker are connected on the detection antibody, and the plasma resonance generated by the metal nano particles enables the fluorescent signal emitted by the fluorescent marker to be obviously enhanced, so that the detection sensitivity can be effectively improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:
FIG. 1 is a schematic structural diagram of a first embodiment of a detection antibody composition of the present invention;
FIG. 2 is a schematic structural diagram of a second embodiment of a detection antibody composition of the present invention;
FIG. 3 is a schematic structural view of a first embodiment of a kit according to the present invention;
FIG. 4 is a schematic structural view of a second embodiment of a kit according to the present invention;
FIG. 5 is a schematic flow chart diagram illustrating one embodiment of a method for preparing a detection antibody composition according to the present invention;
FIG. 6 is a schematic diagram of an embodiment of an immunofluorescence analysis system according to the present invention.
Detailed Description
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.
Referring to fig. 1, fig. 1 is a block diagram of a first embodiment of a detection antibody composition 100 according to the present invention, comprising: a detection antibody 110, a fluorescent label 130 and a fluorescence-enhancing particle 120 respectively linked to the detection antibody 110, wherein the fluorescence-enhancing particle 120 comprises a metal nanoparticle.
In the embodiment, the metal nanoparticles and the fluorescent marker 130 are connected to the detection antibody, and the plasma resonance generated by the metal nanoparticles enhances the fluorescent signal emitted by the fluorescent marker, so that the detection sensitivity can be effectively improved.
Specifically, in the present embodiment, since the fluorescent substance in the fluorescent marker 130 is labeled on the antibody linked to the metal nanoparticle at a distance, plasmon resonance generated by the metal nanoparticle increases the quantum yield of the fluorescent group in the fluorescent marker 130, so that the fluorescent signal emitted from the fluorescent marker 130 is enhanced.
Optionally, the fluorescent marker 130 includes an organic fluorescent dye and quantum dots; the organic fluorescent dye comprises one or more of fluorescein and derivatives thereof, rhodamine and derivatives thereof, cyanine fluorescent dye, coumarin fluorescent dye, boron fluoride fluorescent dye and phthalocyanine fluorescent dye. The specific type of the fluorescent marker can be specifically determined according to the type of the fluorescence-enhancing particle, the detection conditions, and other indicators, and is not limited herein.
Further, the fluorescence-enhancing particle 120 has a functional group (not shown) on the surface, and the fluorescence-enhancing particle 120 is connected to the detection antibody 110 through the functional group. Further, the functional group comprises one or more of carboxyl, hydroxyl, amino, tosyl, chloromethyl, sulfydryl, aldehyde group, hydrazide, silicon hydroxyl, succinimide ester, and epoxy group. The linking group is linked to a group on the surface of the antibody 110 by physical adsorption or covalent coupling.
Optionally, the metal constituting the metal nanoparticles includes one or more of silver, gold, platinum, aluminum, or an alloy thereof. Gold nanoparticles and silver nanoparticles for better fluorescence signal enhancement.
Alternatively, the shape of the metal nanoparticles includes spherical, ellipsoidal, rod-like, triangular, or other irregular shapes. Further, the metal nanoparticles are spherical, and the size of the metal nanoparticles is 5 to 200 nm.
Optionally, referring to fig. 2, fig. 2 is a schematic structural diagram of a second embodiment of a detection antibody composition of the present invention, wherein the detection antibody composition 200 includes a detection antibody 210, a fluorescent marker 230 and a fluorescence-enhanced particle 220 respectively connected to the detection antibody 210, the fluorescence-enhanced particle 220 further includes a spacer layer 222, and the spacer layer 222 is coated on the periphery of the metal nanoparticle 221. The spacer layer 222 provides a dielectric layer with a certain thickness to separate the fluorescent marker 230 and the metal nanoparticle 221 by a certain distance, and also facilitates modification of various functional groups on the spacer layer 222 to achieve binding with antibodies and/or antigens.
Optionally, the spacer layer 222 coated on the periphery of the metal nanoparticle 221 includes one or more of a silicon dioxide spacer layer, a titanium dioxide spacer layer, an aluminum oxide spacer layer, an organic polymer spacer layer, and a biomacromolecule spacer layer; wherein the organic polymer spacing layer comprises a polymer and a spacing layer, the polymer comprises polyesters, polyolefins and the like, and particularly, the polymer comprises but is not limited to polyesters, polyamides, polyethers, polythioethers, polyureas, polycarbonates, polycarbonamides, proteins, polysaccharides and polyarylates. The biomacromolecule spacer layer comprises a protein spacer layer and/or a nucleic acid spacer layer. In one embodiment, the metal nanoparticles 221 are gold spheres and the spacer layer is silicon dioxide. The price of the silicon dioxide is low, which is beneficial to reducing the cost; the silicon dioxide is transparent, the absorption of the metal nanoparticles to light cannot be influenced, meanwhile, other various functional groups can be easily modified on the surface of the silicon dioxide, the silicon dioxide can be conveniently connected with antibodies and/or antigens, the operation process can be simplified, and the detection efficiency can be improved.
The thickness of the spacer layer 222 separates the fluorescent labeling substance and the metal nanoparticles by a certain distance. It is apparent that the increase in fluorescence intensity is related to the kind of fluorescent substance of the fluorescent marker 230, the size of the metal nanoparticles, and the distance between the metal nanoparticles 221. For better fluorescence enhancement, the thickness of the spacer layer 222 is preferably 5nm to 100 nm, such as 5nm, 50nm, or 100 nm.
In order to solve the technical problem, the invention adopts another technical scheme that: a kit is provided.
Referring to fig. 3, fig. 3 is a schematic structural diagram of a first embodiment of a kit according to the present invention, wherein the kit 300 includes: a cartridge body 310; a first reagent holding portion 320 disposed on the cartridge body 310 for holding the detection antibody composition; wherein the first reagent holding site comprises at least two of the detection antibody compositions, and the detection antibodies in different detection antibody compositions are of different types.
In this embodiment, the antibody composition comprises: the kit comprises a detection antibody, a fluorescent marker and fluorescence enhancement particles which are respectively connected with the detection antibody, wherein the fluorescence enhancement particles comprise metal nanoparticles. According to the embodiment, the metal nanoparticles and the fluorescent marker are connected to the detection antibody, and the plasma resonance generated by the metal nanoparticles enables the fluorescent signal emitted by the fluorescent marker to be enhanced, so that the detection sensitivity can be effectively improved. In addition, the detection antibody in different detection antibody compositions is different in type, and different detection antibodies can detect different objects to be detected, so that multi-project joint detection is facilitated, and the detection efficiency is improved.
The technical details and technical advantages of detecting antibody compositions have been set forth in detail above and will not be described herein.
Further, referring to fig. 4, fig. 4 is a schematic structural diagram of a second embodiment of the kit according to the present invention, and the kit 300 further includes: a second reagent holding portion 320, disposed on the kit body 310, for holding at least two immunoassay compositions, wherein the immunoassay compositions are specifically combined with the corresponding detection antibody compositions and the analyte to obtain different detection complexes, so as to detect different analytes; wherein different of the detection complexes have different fluorescence intensities. Further, the immunoassay composition comprises a capture antibody composition and/or a labeled antigen.
Specifically, in the detection process using the sandwich method, the capture antibody composition comprises a capture antibody, a magnetic bead linked to the capture antibody, and a luminescent substance. In order to realize joint detection, when detecting capture antibody compositions of different analytes, classification of different antigens to be detected can be realized by five methods: 1) the magnetic beads have different particle sizes and the luminescent substances connected to the magnetic beads are the same; 2) when the luminescent substances attached to the magnetic beads are the same but have different fluorescence intensities; 3) the fluorescent substances connected to the magnetic beads are different in types; 4) the magnetic balls are the same, but the types of fluorescent substances connected with the capture antibodies are different; 5) the magnetic balls are the same and the fluorescent substances are the same in type, but the fluorescent intensities of the fluorescent substances on the capture antibodies are different; 6) the magnetic beads do not have a fluorescent substance but have different particle sizes, and the fluorescent substance on the capture antibody may be the same kind. And when different capture antibodies in the capture antibody composition specifically bind to corresponding different antigens to be detected and different antigens to be detected specifically bind to corresponding different types of detection antibodies, the detection antibody composition with different fluorescence intensities can be obtained by any one of the 6 methods. Thus, in the fluorescence detection system, different types of detection antibody compositions can pass through the detector in a row, and different fluorescence signals are acquired, processed and analyzed, wherein the first path of fluorescence signal can realize classification of the antigen or antibody to be detected in the sample to be detected through data processing according to any one of the 6 modes, and the antigen or antibody to be detected in the sample to be detected is quantitatively detected through data processing according to the fluorescence marker of the detection immune composition. Of course, the capture antibody in the capture antibody composition may also have the fluorescence enhancement particles attached thereto to increase the fluorescence signal intensity.
Similarly, in the process of detection by using the competition method, the labeled antigen comprises an antigen and a luminescent marker connected to the antigen, and as with the 6 modes, detection antibody compositions with different fluorescence intensities can be obtained, in a fluorescence detection system, different detection antibody compositions can be sequentially subjected to the detector in a single row, and different fluorescence signals are acquired, processed and analyzed, wherein the first fluorescence signal can be used for classifying the antigen to be detected in the sample to be detected through data processing according to any condition of the 6 modes, and the antigen to be detected or the antibody to be detected in the sample to be detected is quantitatively detected through data processing according to the fluorescent marker for detecting the immune composition.
Optionally, the kit 300 further comprises a diluent comprising Bovine Serum Albumin (BSA), newborn bovine serum, sheep serum, horse serum, dithiothreitol, tris, 2-morpholinoethanesulfonic acid hydrate (e.g., 2-morpholinoethanesulfonic acid monohydrate), ethylene glycol, glycerol, tween-80, casein, and disodium edetate; the solvent of the diluent is preferably water. And the concentration of each component of the diluent is as follows: 1-10 g/L BSA, 1-50% by volume newborn bovine serum, 0.1-10% by volume goat serum, 0.1-10% by volume horse serum, 1-100 mmol/L dithiothreitol, 1-100 mmol/L tris (hydroxymethyl) aminomethane, 1-100 mmol/L hydrated 2-morpholinoethanesulfonic acid, 0.1-10% by volume ethylene glycol, 0.1-10% by volume glycerol, 0.01-2% by volume Tween-80, 0.1-10 g/L casein and 0.1-10 g/L disodium ethylenediamine tetraacetate. The diluent also preferably further comprises 0.01-1 g/L of preservative.
The addition of the diluent in the detection process can eliminate various factors interfering with immune reaction, such as Rheumatoid Factors (RF), human anti-mouse antibodies (HAMA), heterophile antibodies, antinuclear antibodies (ANA), and the like, thereby providing a condition more favorable for the reaction of the antibodies and the antigens. Therefore, the addition of the diluent can significantly reduce the nonspecific binding of the sample detection, thereby further improving the reaction sensitivity and the detection accuracy.
Optionally, the diluent further comprises an antiseptic selected from one or more of potassium sorbate, sodium benzoate, sodium azide, sodium nitrite, Proclin 300 (one of common antiseptic for immunodiagnostics, and 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one as main active ingredients) and antibiotics. The preservative is added, so that the components in the immunoreagent can be prevented from deteriorating, the detection quality of the kit can be improved, and the reliability of a detection result can be improved.
Further, the magnetic beads are prepared by mixing nanoscale Fe2O3Or Fe3O4The magnetic particles and the organic polymer material are compounded to form the micron-sized solid phase microsphere with superparamagnetism and extremely large protein adsorption capacity, and the micron-sized solid phase microsphere has the properties that the micron-sized solid phase microsphere can be quickly magnetized under the action of an external magnetic field and the remanence is zero after the magnetic field is removed. In this embodiment, the magnetic beads used should be such that the diameter is from 0.1 to 5 micronsThe magnetic beads can also be surface modified to have various active functional groups, including but not limited to carboxyl, hydroxyl, amino, tosyl, chloromethyl, thiol, aldehyde, hydrazide, silicon hydroxyl, succinimidyl ester, epoxy, and covalently coupled to the capture antibody through the active functional groups. In this embodiment, the magnetic bead size is 5 μm.
In order to solve the technical problem, the invention adopts another technical scheme that: provides a preparation method of a detection antibody composition.
Referring to fig. 5, fig. 5 is a schematic flow chart of an embodiment of a method for preparing a composition for detecting antibodies according to the present invention, wherein the method comprises the steps of:
and S510, adding the fluorescence enhancement particles into a buffer solution to form a fluorescence enhancement particle suspension.
In step S510, the fluorescence-enhancing particles are silica-coated metal nanoparticles or metal nanoparticles; the buffer solution includes 2-morpholinoethanesulfonic acid biological buffer (MES), Phosphate Buffer Solution (PBS), etc.
And S520, adding a cross-linking agent into the fluorescence enhancement particle suspension and activating to obtain a first product.
In step S520, the crosslinking agent includes 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or hydroxysuccinimide; in order to ensure better crosslinking effect, the crosslinking agent is prepared at present. The activation time may be 20 to 60 minutes, for example, 20 minutes, 30 minutes, or 60 minutes, and the specific time is determined according to the kind of the activator and the property of the fluorescence enhancing particle. And the first product is the activated fluorescence enhancement particles, and the pre-activation of the fluorescence enhancement particles is beneficial to improving the combination efficiency of the fluorescence enhancement particles and the antibody.
S530, dropwise adding a detection antibody solution into the first product to obtain a second product.
In step 530, a detection antibody solution is added dropwise to the activated fluorescence-enhanced particles, and then the activated fluorescence-enhanced particles are crosslinked with the detection antibody. The reaction time may be 1 to 3 hours, e.g., 1 hour, 2 hours, or 3 hours, etc. The second product is a detection antibody to which a fluorescence-enhancing particle is attached.
In this embodiment, the antibody composition comprises: the kit comprises a detection antibody, a fluorescent marker and fluorescence enhancement particles which are respectively connected with the detection antibody, wherein the fluorescence enhancement particles comprise metal nanoparticles. According to the embodiment, the metal nanoparticles and the fluorescent marker are connected to the detection antibody, and the plasma resonance generated by the metal nanoparticles enables the fluorescent signal emitted by the fluorescent marker to be enhanced, so that the detection sensitivity can be effectively improved.
The technical details and technical advantages of detecting antibody compositions have been set forth in detail above and will not be described herein.
Further, the method further comprises fluorescently labeling the detection antibody. Specifically, the method for carrying out fluorescence labeling on the detection antibody comprises the steps of adding a fluorescence label into the second product, carrying out a light-shielding reaction for a period of time, diluting the obtained fluorescence-enhanced particle-antibody-fluorescence label to a preset concentration by using a diluent, and storing at 2-8 ℃.
In one embodiment, a blocking agent is added to the second product and reacted for a period of time before the detection antibody is fluorescently labeled, so that sites on the detection antibody where the fluorescent marker conjugate is not needed are occupied, and the fluorescent marker is bound to a specific site on the detection antibody, thereby obtaining a better fluorescent labeling effect. Wherein the blocking agent comprises polyhydroxy saccharide compound, protein compound or primary amino (-NH) group2) At least one of the small molecule compounds of (1). The polyhydroxy carbohydrate is at least one of glucose, sucrose, lactose, trehalose, dextran, mannitol or polysucrose; the protein compound is at least one of bovine serum albumin, human serum albumin, casein, gelatin, casein hydrolysate, immunoglobulin, milk powder and human or animal serum; the small molecular compound is at least one of trihydroxymethyl aminomethane, ethanolamine, hydroxylamine, hexylamine or glycine. In one embodiment, the blocking agent comprises at least two blocking agents, and the addition of at least two blocking agents can improve the blocking effect and is beneficial to reducing background informationAnd the detection sensitivity is further improved. Optionally, in this embodiment, the blocking time is 2-4 hours, e.g., 2 hours, 3 hours, or 4 hours, etc. In another embodiment, after fluorescently labeling the detection antibody, the method further comprises: and (4) centrifuging the product after the light-shielding reaction, and removing the fluorescent marker which is not combined with the detection antibody.
In order to solve the technical problems, the invention adopts a technical scheme that: the use of a detection antibody composition in immunofluorescence assays is provided.
In this embodiment, the detection antibody composition is used for chemofluorescence immunoassay of various specific total antigens in human serum; for example, the detection antibody composition is used for detecting at least one of thyroid function related protein, cardiovascular function related protein, cardiac troponin, liver fibrosis related protein, tumor related protein, gonad function related protein, renal function related protein, bone metabolism function related protein, carbohydrate metabolism function related protein, infectious disease related protein, autoimmune function related protein, prenatal screening program related protein, drug detection related protein, type 4 human herpesvirus related protein and inflammation related protein.
In order to solve the technical problems, the invention adopts a technical scheme that: an immunofluorescence analysis system is provided.
Referring to fig. 6, fig. 6 is a schematic structural diagram of an embodiment of an immunofluorescence analysis system according to the present invention, the immunofluorescence analysis system 600 includes: the kit 610; a sample analyzer 620, wherein the sample analyzer 620 performs detection of different analytes by using the immunoassay composition and the detection antibody composition in the kit 610, and outputs a detection result.
In this embodiment, the detection antibody used by the sample analyzer 620 is connected to the metal nanoparticles and the fluorescent marker, and the plasma resonance generated by the metal nanoparticles enhances the fluorescent signal emitted by the fluorescent marker, thereby effectively improving the detection sensitivity.
The immunofluorescence analyzer 620 includes a flow cytometry analyzer, a microplate reader, or a fluorescence microscope, and the like, which performs qualitative and/or quantitative detection by fluorescence signals. The specific technical benefits and technical details of the kit 610 have been explained in detail above and are therefore not described in detail herein.
The technical solution of the present invention is explained in detail by the following examples
Comparative example 1:
mu.L of Alexa Fluor 488-N-hydroxysuccinimide (NHS) solution in dimethyl sulfoxide was added to 20. mu.g of renin antibody, and the mixture was reacted at room temperature in the dark for 2 hours, and the fluorescent label not bound to the renin antibody was removed by passing through a desalting column and diluted to a final concentration of 2. mu.g/mL with PBS-TBN buffer solution having pH of 7.4 to obtain detection antibody composition 01.
Comparative example 2:
mu.L of Fluorescein Isothiocyanate (FITC) -N-hydroxysuccinimide (NHS) dimethyl sulfoxide solution is added into 100 mu g of the renin antibody, the mixture is subjected to a light-shielding reaction at room temperature for 2 hours, a fluorescent marker which is not combined with the renin antibody is removed through a desalting column, and the mixture is diluted to a final concentration of 2 mu g/mL by PBS-TBN buffer solution with the pH value of 7.4, so that the detection antibody composition 02 is obtained.
Comparative example 3:
50 μ L of Cy 5-N-hydroxysuccinimide (NHS) in dimethylsulfoxide was added to 100 μ g of interleukin-6 (IL-6) antibody, and the mixture was reacted at room temperature in the dark for 2 hours, and the fluorescent label not bound to the IL-6 antibody was removed by passing through a desalting column and diluted with PBS-TBN buffer solution having a pH of 7.4 to a final concentration of 2 μ g/mL, to obtain a detecting antibody composition 03.
Example 1:
0.5mg of silica-coated gold nanoparticles (maximum absorption wavelength 520nm) with carboxylated surfaces were taken, the thickness of the silica layer was 20nm, and the particle size of the gold nanoparticles was 15 nm. After two washes with 50mM MES buffer, pH 5, centrifugation, resuspension with MES buffer, 50. mu.g each of succinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) were dissolved in 50 mmol/L2- (N-morpholino) ethanesulfonic acid (MES) buffer, pH 5, and the solution was added to the suspension. After centrifugation, the cells were washed twice with PBS buffer containing Tween 20 at a concentration of 10mmol/L and containing 0.1% by mass of Tween 20. Adding a renin antibody dissolved in PBS into the gold nanoparticles coated with the silica, wherein the addition amount of the renin antibody is 20 mu g, and reacting at room temperature for 2 hours to obtain the gold nanoparticle-renin antibody conjugate coated with the silica.
Adding 20 mu L of Alexa Fluor 488-NHS dimethyl sulfoxide solution into 200 mu L of the silica-coated gold nanoparticle-renin antibody solution, reacting at room temperature in a dark place for 2 hours, centrifuging to remove the fluorescent marker which is not combined with the renin antibody, and diluting with PBS-TBN buffer solution with pH of 7.4 to the final concentration of 2 mu g/mL to obtain the detection antibody composition 1.
Example 2:
1mg of gold nanoparticles (maximum absorption wavelength 520nm) coated with surface-carboxylated polystyrene were taken, the thickness of the polystyrene layer was 20nm, and the particle size of the gold nanoparticles was 15 nm. After two washes with 50mM MES buffer, pH 5, centrifugation, resuspension with MES buffer, 100. mu.g each of succinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) were dissolved in 50 mmol/L2- (N-morpholino) ethanesulfonic acid (MES) buffer, pH 5, and the solution was added to the suspension. After centrifugation, the cells were washed twice with PBS buffer containing Tween 20 at a concentration of 10mmol/L and containing 0.1% by mass of Tween 20. Adding a renin antibody dissolved in PBS into the solution, wherein the addition amount of the renin antibody is 20 mu g, and reacting for 2 hours at room temperature to obtain the polystyrene-coated gold nanoparticle-renin antibody conjugate.
Adding 20 mu L of Alexa Fluor 488-NHS dimethyl sulfoxide solution into 200 mu L of the polystyrene coated gold nanoparticle-renin antibody solution, reacting at room temperature in a dark place for 2 hours, centrifuging to remove the fluorescent marker which is not combined with the renin antibody, and diluting with PBS-TBN buffer solution with pH of 7.4 to the final concentration of 2 mu g/mL to obtain the detection antibody composition 2.
Example 3:
1mg of silica-coated silver nanoparticles (maximum absorption wavelength 480nm) with carboxylated surfaces were taken, the thickness of the silica layer was 5nm, and the particle size of the gold nanoparticles was 70 nm. After two washes with 50mM MES buffer, pH 5, centrifugation, resuspension with MES buffer, 50. mu.g each of succinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) were dissolved in 50 mmol/L2- (N-morpholino) ethanesulfonic acid (MES) buffer, pH 5, and the solution was added to the suspension. After centrifugation, the cells were washed twice with PBS buffer containing Tween 20 at a concentration of 10mmol/L and containing 0.1% by mass of Tween 20. Adding the renin antibody dissolved in PBS to the solution, wherein the addition amount of the renin antibody is 20 mug, and reacting at room temperature for 2 hours to obtain the silica-coated silver nanoparticle-renin antibody conjugate.
And adding 20 mu L of the dimethyl sulfoxide solution of FITC-NHS into 200 mu L of the silver nanoparticle-renin antibody solution coated with the silicon dioxide, carrying out a light-shielding reaction at room temperature for 2 hours, centrifuging to remove a fluorescent marker which is not combined with the renin antibody, and diluting with PBS-TBN buffer solution with the pH of 7.4 to the final concentration of 2 mu g/mL to obtain a detection antibody composition 3.
Example 4
0.5mg of surface-aminated gold nanorods (maximum absorption wavelength 650nm) were washed twice with Phosphate Buffer (PBS) at a concentration of 1mmol/L at pH 8.4, centrifuged, resuspended in PBS buffer, 200. mu.g of carboxy-polyethylene glycol-succinimidyl ester (COOH-PEG-NHS) was added, reacted at room temperature for 4 hours, centrifuged, and washed twice with PBS buffer containing Tween 20 at a concentration of 10mmol/L and containing 0.1% by mass of Tween 20.
50 μ g each of succinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) was dissolved in 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution at a concentration of 50mmol/L and pH 5, and the solution was added to gold nanorods and activated at room temperature for 30 min. After centrifugation, the cells were washed twice with PBS buffer containing Tween 20 at a concentration of 10mmol/L and containing 0.1% by mass of Tween 20. IL-6 antibody (40. mu.g) dissolved in PBS was added to the washed gold nanorods, and the reaction was carried out at room temperature for 2 hours to obtain a conjugate of the gold nanorods and the IL-6 antibody.
And adding 20 mu L of Cy5-NHS dimethyl sulfoxide solution into 20 mu g of the composition of the gold nanorods and the renin antibody, carrying out a light-shielding reaction at room temperature for 2 hours, centrifuging to remove the fluorescent marker which is not combined with the renin antibody, and diluting with PBS-TBN buffer solution with the pH of 7.4 to the final concentration of 2 mu g/mL to obtain a detection antibody composition 4.
2. Preparation of capture antibody composition
1) Preparation of renin Capture antibody composition
1mg of the magnetic beads were washed twice with 50mM MES buffer at pH 6 and the magnetic separation was followed by resuspension of the beads in MES buffer. 50. mu.g each of succinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) was dissolved in 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution at a concentration of 50mmol/L and pH 5, and the solution was added to the suspension of magnetic beads and activated at room temperature for 30 min. After magnetic separation, the column was washed twice with PBS buffer containing Tween 20 at a concentration of 10mmol/L and containing 0.1% by mass of Tween 20. A PBS solution containing 20. mu.g of renin antibody was added to the magnetic beads and reacted at room temperature for 2 hours. After magnetic separation, the sample was washed twice with 10mmol/L PBS buffer containing 0.1% Tween 20 in parts by mass. Adding 0.5mL of a first blocking agent (the first blocking agent comprises 5% of ethanolamine by mass, pH 7.4 and PBS buffer solution with the concentration of 10 mM), and blocking for 0.5 hour at room temperature; then, 0.5mL of a second blocking agent (the second blocking agent includes 1% by mass of casein, pH 7.4, and 10mM PBS buffer) was added for blocking at room temperature for 3 hours. After the sealing is completed, the magnetic separation is washed twice. Adding PBS buffer solution with the concentration of 10mmol/L and containing 0.1% of Tween 20 in parts by mass, and diluting until the concentration of magnetic beads is 0.2mg/mL to obtain the renin capture antibody composition.
2) Preparation of IL-6 Capture antibody composition
1mg of the magnetic beads were washed twice with 50mM MES buffer at pH 6 and the magnetic separation was followed by resuspension of the beads in MES buffer. Succinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) each 80 μ g were dissolved in 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution at a concentration of 50mmol/L, pH 5, and the solution was added to the magnetic bead suspension and activated at room temperature for 30 min. After magnetic separation, the column was washed twice with PBS buffer containing Tween 20 at a concentration of 10mmol/L and containing 0.1% by mass of Tween 20. The PBS solution containing 20. mu. gIL-6 antibody was added to the magnetic beads and reacted at room temperature for 2 hours. After magnetic separation, the sample was washed twice with 10mmol/L PBS buffer containing 0.1% Tween 20 in parts by mass. Adding 0.5mL of a first blocking agent (the first blocking agent comprises 5% of ethanolamine by mass, pH 7.4 and PBS buffer solution with the concentration of 10 mM), and blocking for 0.5 hour at room temperature; then, 0.5mL of a second blocking agent (the second blocking agent includes 1% by mass of casein, pH 7.4, and 10mM PBS buffer) was added for blocking at room temperature for 3 hours. After the sealing is completed, the magnetic separation is washed twice. Adding 10mmol/L Tween 20 containing 0.05% by mass and 0.1% by mass of bovine serum albumin PBS-TBN buffer solution, and diluting until the concentration of the magnetic beads is 0.1mg/mL to obtain the IL-6 capture antibody composition.
3. Flow-through fluorescence detection
1) Preparation of renin calibrator
The renin recombinant antigen is dissolved in PBS buffer solution with pH 7.4, concentration 10mM and including 2% bovine serum albumin by mass part to prepare a series of calibrators with different concentrations. It was assigned with an international calibrator (NIBSC 68/356). The concentrations of calibrant were 0, 12.5, 25, 50, 125, 250, 500, and 1000 pg/mL.
2) Renin flow-type fluorescence assay
Performing flow type fluorescence immunoassay by adopting a double antibody sandwich method mode:
adding 10 mu L of sample (calibrator) and 50 mu L of renin-capturing antibody composition solution into a reaction cup, reacting for 20min, then adding 100 mu L of detection antibody composition 01 solution, reacting for 20min, carrying out magnetic separation, adding 100 mu L of PBS-TBN cleaning solution to obtain a detection sample 01, and placing the detection sample 01 into a flow cytometer to detect fluorescence intensity (MFI). And repeating the above process using different concentrations of calibrator to obtain the linear range and sensitivity of the detection antibody composition 01.
Adding 10 mu L of sample (calibrator) and 50 mu L of renin-capturing antibody composition solution into a reaction cup, reacting for 20min, then adding 100 mu L of detection antibody composition 02 solution, reacting for 20min, carrying out magnetic separation, adding 100 mu L of PBS-TBN cleaning solution to obtain a detection sample 02, and placing the detection sample 02 into a flow cytometer to detect fluorescence intensity (MFI). And repeating the above process using different concentrations of calibrator to obtain the linear range and sensitivity of the detected antibody composition 02.
Adding 10 mu L of sample (calibrator) and 50 mu L of renin-capturing antibody composition solution into a reaction cup, reacting for 20min, then adding 100 mu L of detection antibody composition 1 solution, reacting for 20min, carrying out magnetic separation, adding 100 mu L of PBS-TBN cleaning solution to obtain a detection sample 1, and placing the detection sample 1 into a flow cytometer to detect fluorescence intensity (MFI). And repeating the above process using different concentrations of calibrator to obtain the linear range and sensitivity of the detection antibody composition 1.
Adding 10 mu L of sample (calibrator) and 50 mu L of renin-capturing antibody composition solution into a reaction cup, reacting for 20min, then adding 100 mu L of detection antibody composition 2 solution, reacting for 20min, carrying out magnetic separation, adding 100 mu L of PBS-TBN cleaning solution to obtain a detection sample 2, and placing the detection sample 2 into a flow cytometer to detect fluorescence intensity (MFI). And repeating the above process using different concentrations of calibrator to obtain the linear range and sensitivity of the detection antibody composition 2.
Adding 10 mu L of sample (calibrator) and 50 mu L of renin-capturing antibody composition solution into a reaction cup, reacting for 20min, then adding 100 mu L of detection antibody composition 3 solution, reacting for 20min, carrying out magnetic separation, adding 100 mu L of PBS-TBN cleaning solution to obtain a detection sample 3, and placing the detection sample 3 into a flow cytometer to detect fluorescence intensity (MFI). And the above process was repeated using different concentrations of calibrator to obtain the linear range and sensitivity of the detection antibody composition 3.
3) Preparation of IL-6 calibrator
The IL-6 recombinant antigen was dissolved in PBS buffer at pH 7.4, 10mM and containing 2% by mass bovine serum albumin to make a series of calibrators of different concentrations. The concentration of the calibrator was 0,0.5,50,500,1000,5000,10000,20000 pg/mL.
4) IL-6 flow fluorescence detection
Adding 10 mu L of sample (calibrator) and 50 mu L of IL-6 capture antibody composition solution into a reaction cup, reacting for 20min, then adding 100 mu L of detection antibody composition 03 solution, reacting for 20min, performing magnetic separation, adding 100 mu L of PBS-TBN cleaning solution to obtain a detection sample 03, and placing the detection sample 03 into a flow cytometer for detecting fluorescence intensity (MFI). And the above process is repeated using calibrators of different concentrations to obtain the linear range and sensitivity of the detection antibody composition 03.
Adding 10 mu L of sample (calibrator) and 50 mu L of IL-6 capture antibody composition solution into a reaction cup, reacting for 20min, then adding 100 mu L of detection antibody composition 4 solution, reacting for 20min, performing magnetic separation, adding 100 mu L of PBS-TBN cleaning solution to obtain a detection sample 4, and placing the detection sample 4 into a flow cytometer to detect fluorescence intensity (MFI). And repeating the above process using different concentrations of calibrator to obtain the linear range and sensitivity of the detection antibody composition 4.
4. Analysis of results
1) Determination of the Linear Range
Table 1 table of detection ranges of detection antibody compositions comprising renin antibodies
TABLE 2 detection scope Table for detection antibody composition comprising interleukin-6 antibody
The regression equations in tables 3 and 4 were obtained and the regression constants in tables 4 and 3 were obtained with the linear range as abscissa (unit: pg/mL) and the measured concentration as ordinate (unit: pg/mL).
Table 3 table of theoretical concentrations of detection antibody composition/reagent concentrations comprising renin antibody
As can be seen from table 3 above, the data of the test antibody composition 01 standard is determined to be in a linear range, where the linear range is reliable when the regression equation is y is 0.976x +8.422 and the regression constant R2 is 0.999; the data for the test antibody composition 02 standard was determined to be in the linear range, with the regression equation being y 0.978x +7.848, and the regression constant R2 being 0.999, the linear range 50-1000pg/mL is more reliable; the data for the test antibody composition 1 standard was determined to be in the linear range, which was determined from the data in table 6, with a regression equation of y 1x +0.042 and a regression constant of R2 1, which is reliable in the linear range of 5-1000 pg/mL. The data of the standard product of the detection antibody composition 2 is determined to be in a linear range, the regression equation is that y is 1.017x-3.206, and the regression constant R2 is 0.998, so that the linear range of 5-1000pg/mL is reliable; the data for the test antibody composition 3 standard was determined to be in the linear range with a regression equation of y 1x +0.085 and a regression constant of R2 1, the linear range of 5-1000pg/mL was more reliable.
TABLE 4 theoretical concentration/reagent concentration Table for test antibody compositions comprising interleukin-6 antibody
As can be seen from table 4 above, the data for the test antibody composition 03 standard was determined to be in the linear range, with the regression equation being 1.043x-172.0 with the regression constant R2The linear range of 100-; the data for the 4 standard test antibody composition was determined to be in the linear range with the regression equation being 1.035x 114.3 and the regression constant R2A linear range of 20-20000pg/mL is more reliable at 0.992.
Referring to the data in tables 1 to 4, and comparing composition 01, composition 1 and composition 2, and composition 02 and composition 3, composition 03 and composition 4, it can be seen that, after the fluorescence-enhancing particles are added, the detection range of the detection antibody composition containing the renin antibody or the detection antibody composition containing interleukin-6 is significantly widened, indicating that the detection range can be widened by adding the fluorescence-enhancing particles.
Whereas at the same calibrator concentration, the fluorescence signal intensity was stronger in compositions 1 and 2 compared to composition 01; the fluorescence signal intensity was stronger for composition 3 compared to composition 02; the fluorescence signal intensity was stronger in composition 4 compared to composition 03; specifically, the signal intensity changes by orders of magnitude, and the difference can reach 100 times. It can be seen that the addition of the fluorescence-enhancing particles can effectively enhance the intensity of the fluorescence signal.
2) Determination of sensitivity
The reagent is used to test a blank sample (5% BSA), the test is repeated 20 times, and the mean X and standard deviation SD are calculated for the 20 test results, where X +2SD should not be greater than the blank limit.
The sensitivity of the detection antibody composition comprising the renin antibody is shown in table 5 below.
TABLE 5 sensitivity Table of detection antibody compositions comprising renin antibodies
As can be seen from table 5, the sensitivity of composition 01 was 6.89, the sensitivity of composition 02 was 4.1, the sensitivity of composition 1 was 1.05, the sensitivity of composition 2 was 1.9, and the sensitivity of composition 3 was 1.06.
TABLE 6 sensitivity Table for detecting antibody compositions comprising interleukin-6 antibodies
As can be seen from table 5, the sensitivity of composition 03 was 2.03 and the sensitivity of composition 4 was 0.06.
Referring to the data in tables 5 to 6, and comparing composition 01, composition 1 and composition 2, and composition 02 and composition 3, composition 03 and composition 4, it can be seen that, when the fluorescence-enhancing particles are added, the detection sensitivity of the detection antibody composition containing the renin antibody or the detection antibody composition containing interleukin-6 is significantly enhanced, that is, the fluorescence intensity of the detection sample corresponding to the calibrator at a lower concentration can be distinguished from the fluorescence intensity at the time when the calibrator is at a concentration of 0. After the fluorescence enhancement particles are added, the detection sensitivity of the renin antibody composition is improved, and the sensitivity of the interleukin-6 detection antibody composition is improved, so that the fluorescence intensity of the metal nanoparticles is enhanced by nearly 100 times, and the detection sensitivity is greatly improved.
In summary, the addition of fluorescence-enhancing particles to the detection antibody can expand the detection range, increase the intensity of the fluorescence signal, and improve the detection sensitivity.
In summary, the invention discloses a kit, a detection antibody composition and a preparation method thereof, wherein the detection antibody composition comprises: the kit comprises a detection antibody, a fluorescent marker and fluorescence enhancement particles which are respectively connected with the detection antibody, wherein the fluorescence enhancement particles comprise metal nanoparticles. By the mode, the method can simplify the operation process, effectively amplify the fluorescence detection signal and improve the detection sensitivity.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (18)
1. A detection antibody composition, wherein the detection antibody composition comprises:
the kit comprises a detection antibody, a fluorescent marker and fluorescence enhancement particles which are respectively connected with the detection antibody, wherein the fluorescence enhancement particles comprise metal nanoparticles.
2. The detection antibody composition of claim 1, wherein the fluorescence-enhancing particle has a functional group on the surface thereof, and the fluorescence-enhancing particle is linked to the detection antibody via the functional group.
3. The detection antibody composition of claim 2, wherein the functional group comprises one or a combination of carboxyl, hydroxyl, amino, tosyl, chloromethyl, thiol, aldehyde, hydrazide, siloxyl, succinimidyl ester, epoxy.
4. The detection antibody composition of claim 1, wherein the metal comprising the metal nanoparticles comprises one or a combination of silver, gold, aluminum, platinum, or alloys thereof.
5. The detection antibody composition of claim 1, wherein the fluorescent label comprises an organic fluorescent dye and/or a quantum dot.
6. The detection antibody composition of claim 1, wherein the metal nanoparticles are spherical and have a particle size of 3-200 nm.
7. The detection antibody composition of claim 1, wherein the fluorescence-enhancing particle further comprises a spacer layer coated on the outer periphery of the metal nanoparticle.
8. The detection antibody composition of claim 7, wherein the spacer layer coated on the periphery of the metal nanoparticle comprises one or more of a silica spacer layer, a titanium dioxide spacer layer, an alumina spacer layer, an organic polymer spacer layer, and a biomacromolecule spacer layer.
9. The detection antibody composition of claim 7, wherein the spacer layer has a thickness of 5 to 50 nanometers.
10. A kit, comprising:
a kit body;
a first reagent holding site provided on the cartridge body for holding the detection antibody composition of any one of claims 1 to 9;
wherein the first reagent holding site comprises at least two of the detection antibody compositions, and the detection antibodies in different detection antibody compositions are of different types.
11. The kit of claim 10, further comprising:
the second reagent holding position is arranged on the kit and used for holding at least two immunodetection compositions, and the immunodetection compositions are respectively matched with the corresponding detection antibody compositions to obtain different detection compounds so as to detect different objects to be detected;
wherein different of the detection complexes have different fluorescence intensities.
12. The kit of claim 11, wherein the immunoassay composition comprises a capture antibody composition and/or a labeled antigen.
13. A method of preparing a detection antibody composition, the method comprising:
adding the fluorescence-enhancing particles to a buffer solution to form a fluorescence-enhancing particle suspension;
adding a cross-linking agent into the fluorescence-enhanced particle suspension and activating to obtain a first product;
and adding a detection antibody solution into the first product to obtain a second product.
14. The method of claim 12, wherein after adding the detection antibody suspension to the first product to obtain a second product, the method further comprises;
adding a fluorescent marker into the second product, and reacting in a dark place.
15. The method of claim 13, wherein prior to adding the fluorescent label to the second product, the method further comprises:
adding a blocking agent to the second product and reacting for a period of time, wherein the blocking agent comprises a polyhydroxy carbohydrate compound, a proteinaceous compound, or a compound containing primary amino groups (-NH)2) At least one of the small molecule compounds of (1).
16. The method of claim 15,
the polyhydroxy carbohydrate is at least one of glucose, sucrose, lactose, trehalose, dextran, mannitol or polysucrose;
the protein compound is at least one of bovine serum albumin, human serum albumin, casein, gelatin, casein hydrolysate, immunoglobulin, milk powder and human or animal serum;
the small molecular compound is at least one of trihydroxymethyl aminomethane, ethanolamine, hydroxylamine, hexylamine or glycine.
17. An immunofluorescence assay system, comprising:
a kit comprising the kit of any one of claims 10-12;
a sample analyzer for performing detection of different analytes by using the immunodetection composition and the detection antibody composition in the kit, and outputting a detection result.
18. Use of a detection antibody composition of any one of claims 1-9 in an immunofluorescence assay;
the detection antibody composition is used for detecting at least one of thyroid function related protein, cardiovascular function related protein, cardiac troponin, liver fibrosis related protein, tumor related protein, gonadal function related protein, renal function related protein, bone metabolism function related protein, carbohydrate metabolism function related protein, infectious disease related protein, autoimmune function related protein, prenatal screening project related protein, drug detection related protein, type 4 human herpesvirus related protein and inflammation related protein.
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