CN111579770A - Fluorescent microsphere labeled antibody and preparation method and application thereof - Google Patents
Fluorescent microsphere labeled antibody and preparation method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Abstract
The invention relates to a fluorescent microsphere labeled antibody and a preparation method and application thereof, wherein the preparation method comprises the following steps: adding 100-1000mg/mL chemical cross-linking agent solution into 1-50mg/mL fluorescent microsphere solution for activation; mixing the labeled antibody and the quality control protein to obtain total protein, and adding the total protein into the obtained activated product for reaction; and adding a sealing agent into the obtained solution for sealing, and centrifuging to obtain the fluorescent microsphere labeled antibody. The invention uses the marked antibody/antigen and other protein to be marked on the surface of the marker at the same time, thereby not only reducing the dosage of the marked antibody/antigen, but also reducing the cost; and the original performance of the labeled antibody/antigen can be kept well and stably.
Description
Technical Field
The invention relates to a fluorescent microsphere labeled antibody and a preparation method and application thereof, belonging to the technical field of immunodetection.
Background
The detection of certain antigens or specific proteins is generally applied to immunolabeling techniques. The immunolabeling technologies refer to antigen-antibody reactions carried out by labeling antibodies or antigens with fluorescein, radioisotope, enzyme, ferritin, colloidal gold and chemical (or biological) luminescent agents as tracers, directly microscopic observation or automatic determination of experimental results by means of instruments such as a fluorescence microscope, an enzyme-labeled detector and the like, and can carry out qualitative and positioning research on the antigen-antibody reactions at the cellular, subcellular and molecular levels; or various liquid phase and solid phase immunoassay methods are applied to carry out qualitative and quantitative determination on hapten, antigen or antibody in body fluid. The immune labeled tracer and the antibody or antigen are generally combined by chemical bonds, and the antigen or antibody in the reaction system is qualitatively or quantitatively detected by the enhanced amplification effect of the marker.
Because the labeling substance and the detection method adopted by the labeling are different, the immune labeling technology is mainly divided into: colloidal gold, latex, fluorescein, enzyme, radionuclide labels, and the like. In labeling applications, these immunolabeling techniques all have different drawbacks due to different application principles. For example, in the colloidal gold labeling technique, although the detection operation is simple, the substance itself has the disadvantages of low sensitivity and narrow linear range; in the fluorescein labeling technique, fluorescein used for labeling has problems of fluorescence intensity and fluorescence stability, and the method for labeling the antigen or antibody is complex; in the enzyme labeling technology, the storage environment and the reaction condition of enzyme are strict, and a label is easy to inactivate, so that the detection limit of a substance to be detected is influenced; in the radionuclide labeling technology, although the nuclide detection sensitivity is extremely high, the nuclide has radioactivity, strong potential safety hazards exist, and environmental pollution and health hazards are easily caused in the production operation process. These methods also have different problems in clinical application. For example, colloidal gold and latex are judged by naked eyes and are mostly used for simple qualitative judgment, so certain artificial subjectivity exists, and meanwhile, the content of related detection substances in a sample is required to reach a certain amount or the sensitivity of a marker is required to be good, so false negative and false positive results are easy to occur; the operation processes of the fluorescein labeling technology, the enzyme labeling technology and the radionuclide technology are complex and need to be supported by equipment, the maintenance cost of the equipment is expensive, and the equipment is not convenient to carry and transport.
In addition to the problems of selection of the label and the labeling technique, the specific antibody or antigen used for the immunolabeling is also a key point. The specific antibody or antigen applied to the immune marker at present has different manufacturing processes and levels and different cost performance of the antibody or antigen, which is shown in that although the high-quality antibody or antigen has high specificity and good sensitivity, the price is relatively expensive, and the antigen produced by manufacturers at home and abroad has the problem. The import of antibodies or antigens from foreign manufacturers also has the problems of fixed production or inventory and long transportation period, which causes many immune reagent manufacturers to invest much expense and time for research and development and production. The specific antibody or antigen is more strict in the manufacturing process and specificity requirements, and the price per se is generally higher than that of the general antibody or protein. How to reduce the research and development cost and improve the research and development production efficiency under the condition of ensuring good product performance is a concern of many reagent manufacturers.
Therefore, the market at present has further needs for the development of an immunolabeling method and a detection technology with low cost, simple operation, objective result interpretation and high sensitivity.
Disclosure of Invention
In view of this, the main objective of the present invention is to provide a fluorescent microsphere labeled antibody, and a preparation method and an application thereof.
In order to achieve the above object, the present invention provides a method for preparing a fluorescent microsphere-labeled antibody, comprising the following steps:
1) adding 100-1000mg/mL chemical cross-linking agent solution into 1-50mg/mL fluorescent microsphere solution for activation;
2) mixing the labeled antibody and the quality control protein to obtain total protein, and adding the total protein into the activated product obtained in the step 1) for reaction;
3) adding a sealing agent into the solution obtained in the step 2) for sealing, and centrifuging to obtain the fluorescent microsphere labeled antibody.
As a preferable scheme of the preparation method of the fluorescent microsphere labeled antibody, in the step 1), the weight ratio of the fluorescent microsphere solution to the chemical crosslinking agent solution is 1 (0.1-5), preferably 1:2.5, so as to ensure that the chemical crosslinking agent is supersaturated and the activation is more sufficient; the activation time is 5-30min, preferably 30min, to ensure complete activation.
As a preferable scheme of the preparation method of the fluorescent microsphere labeled antibody, in step 1), the concentration of the chemical cross-linking agent solution is 500mg/mL, and the concentration of the fluorescent microsphere solution is 100mg/mL, which is higher, so that the proportion of the chemical cross-linking agent solution can be reduced.
As a preferable scheme of the preparation method of the fluorescent microsphere labeled antibody, in the step 1), the particle size of the fluorescent microsphere is 100nm-500 nm.
As a preferable embodiment of the method for preparing the fluorescent microsphere labeled antibody, in step 1), the chemical crosslinking agent is at least one selected from 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, glutaraldehyde and periodate, and chloramine-T; preferably, the chemical crosslinker is a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a weight ratio of 1:1, so that activation is preferably assisted synergistically with other parameters.
As a preferable embodiment of the above method for preparing a fluorescent microsphere labeled antibody, in step 2), the weight ratio of the total protein to the fluorescent microsphere is 1: (200-1); the weight ratio of the labeled antibody to the quality control protein is 1 (0.1-20), preferably 1 (1-9), and the ratio can ensure that the total protein and the activated product can be well coupled and labeled, and the cost is greatly reduced.
As a preferable embodiment of the preparation method of the fluorescent microsphere labeled antibody, in the step 2), the rotation speed of the reaction is 50-150rpm, and the time is 2-5h, preferably 100rpm, and 4h, so that the progress of the coupling labeling reaction is preferably promoted by cooperating with other parameters.
As a preferable embodiment of the above method for preparing the fluorescent microsphere labeled antibody, in step 2), the labeled antibody is selected from a human Myoglobin (MYO) labeled antibody, a human neutrophil gelatinase-associated lipocalin (NGAL) labeled antibody, a troponin I (cTnI) labeled antibody, an N-terminal-B type brain-urine peptide precursor (NT-proBNP) labeled antibody, a Procalcitonin (PCT) labeled antibody, a C-reactive protein (CRP) labeled antibody, a D-Dimer (D-Dimer) labeled antibody, a human chorionic gonadotropin beta subunit (beta-HCG) labeled antibody, a lipoprotein-associated phospholipase A2(Lp-PLA2) labeled antibody, an anti-mullerian hormone (AMH) labeled antibody, an interleukin 6(IL-6) labeled antibody, a Serum Amyloid A (SAA) labeled antibody or a cystatin C (Cys-C) labeled antibody; the quality control protein is selected from bovine serum albumin BSA, chicken IgY antibody, rabbit-derived antibody, sheep-derived antibody or donkey-derived antibody, and is preferably bovine serum albumin BSA, so that the coupling labeling reaction is preferably promoted by cooperating with other parameters.
As a preferable embodiment of the preparation method of the fluorescent microsphere labeled antibody, in step 3), the blocking agent is at least one selected from the group consisting of 1-5 wt% bovine serum albumin solution, 0.1-5 wt% casein solution and 10-50mM glycine, preferably 50mM glycine; the volume ratio of the sealant to the solution is 1 (0.01-2); the sealing time is 5min-1h, preferably 30 min; this preferably then promotes the progress of the occlusion in conjunction with other parameters.
As a preferable embodiment of the preparation method of the fluorescent microsphere labeled antibody, in step 3), the rotation speed of the centrifugation is 10000-20000rpm for 8-20min, preferably 14000rpm for 10min, so that the centrifugation is complete and the time is not too long.
In order to achieve the purpose, the invention also provides a fluorescent microsphere labeled antibody, which is prepared by the method.
In order to achieve the purpose, the invention also provides application of the fluorescent microsphere labeled antibody in lateral immunochromatography.
The invention focuses primarily on the process of immunoantibody labeling, where the label is coupled to both the labeled antibody/antigen and another protein simultaneously by chemical cross-linking agents. The fluorescent microspheres with fixed quantity are coupled with the two proteins in the labeling process, so that the proportion of the labeled antibody/antigen in the labeling process can be reduced, the cost is saved, meanwhile, the added proteins can ensure that the protein content in the labeling process is enough, the condition of low binding efficiency of the fluorescent microspheres due to the reduction of the protein quantity can be avoided, and the performance is ensured to be unchanged. When the detection is carried out, the labeled antibody/antigen labeled by the fluorescent microsphere and the protein labeled by the fluorescent microsphere are added at the same time, and the nonspecific protein labeled by the fluorescent microsphere has the effect of maintaining the signal stability of the quality control line, so that the accuracy in the detection is ensured.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses the marked antibody/antigen and other protein to be marked on the surface of the marker at the same time, thereby not only reducing the dosage of the marked antibody/antigen, but also reducing the cost; and the protein which can replace part of labeled antibody/antigen for labeling can keep better stability of the quality control line, so that the accuracy, sensitivity and specificity of the labeled antibody/antigen are kept well and stable; the type and proportion of the protein used in the labeling can be adjusted according to the performance of different items of antibodies, and the protein with unobvious cross reaction is selected.
Drawings
Fig. 1 is a schematic structural diagram of an immunochromatographic test strip in an embodiment of the present invention;
FIG. 2A is a schematic view of an inner structure of a bottom cap of an embodiment of the immunochromatographic detection device of the present invention;
FIG. 2B is a schematic diagram of an internal structure of an upper cover in an embodiment of the immunochromatographic detection device of the present invention.
The test strip detection device comprises a PVC plate 1, a sample pad 2, a coating pad 3, a water absorption pad 4, a detection line 31, a quality control line 32, an upper cover 11, a bottom groove 12, a sample adding hole 13, an observation window 14, a test strip placing area 15, a positioning column 16, a positioning hole 17, a first limiting part 18, a second limiting part 19 and a third limiting part 20.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
The following materials or reagents, unless otherwise specified, were all purchased.
Example 1
1.1 preparation of fluorescent microsphere-labeled antibody
1.1.1 microsphere cleaning:
the original concentration of the fluorescent microspheres is 2.0 wt%, 10mM phosphate buffer (pH6.0) is used for centrifugal cleaning, the rotating speed is 14000rpm, the time is 10min, the supernatant is discarded, and the steps are repeated twice to obtain microsphere precipitates.
1.1.2 activation:
1) preparing a chemical cross-linking agent solution:
the chemical cross-linking agent selects N-hydroxysuccinimide and 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide with the weight ratio of 1:1, and respectively uses 10mM (pH6.0) phosphate buffer solution to prepare 500mg/ml solution for standby;
2) activation of fluorescent microspheres:
adding 10mM phosphate buffer (pH6.0) into the washed microsphere precipitate, ultrasonically mixing (40kHz, 6min) to obtain 100mg/mL fluorescent microsphere solution, mixing the N-hydroxysuccinimide prepared in the previous step 1) with the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide mixed solution (fluorescent microspheres, adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in a weight ratio of 1:2.5:2.5) and ultrasonically mixing uniformly (40kHz, 6min), wherein the concentration of the fluorescent microspheres in the obtained solution is 10mg/ml, the concentration of the N-hydroxysuccinimide is 25mg/ml, and the concentration of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 25 mg/ml; the solution was then placed on a shaker and reacted at 37 ℃ for 24min with shaking at 100 rpm.
3) Activated microsphere centrifugation:
the activated microsphere solution was washed by centrifugation with 10mM phosphate buffer (pH6.0) at 14000rpm for 9min, and the supernatant was discarded and repeated twice.
1.1.3 coupling labeling:
adding 10mM phosphate buffer (pH6.0) into the microsphere precipitate obtained by activation centrifugation, and ultrasonically mixing (40kHz, 6min) to make the final concentration of the fluorescent microsphere be 10 mg/ml; and then adding a human Myoglobin (MYO) labeled antibody and bovine serum albumin BSA (50mM, PB, 2 wt% BSA, pH7.4), wherein the weight ratio of the human Myoglobin (MYO) labeled antibody to the bovine serum albumin BSA is 1:1, 1:3 or 1:9, the weight ratio of the fluorescent microspheres to the mixture of the human Myoglobin (MYO) labeled antibody and the bovine serum albumin BSA is 10:1, ultrasonically mixing (40kHz, 6min), placing on a shaking table, and carrying out oscillation reaction at 37 ℃ and 100rpm for 4 hours. In the reaction process, the mixture is ultrasonically mixed once every half hour (40kHz, 6min), so that the generation of precipitates is avoided.
1.1.4 sealing
After the labeling is finished, 50mM glycine solution (pH7.4) with the same volume as the microsphere solution is added into the microsphere solution, the mixture is ultrasonically mixed (40kHz and 6min), and the mixture is placed on a shaker for reaction for 14min at 37 ℃ and 100rpm in a shaking way. After the reaction was completed, the reaction mixture was centrifuged and washed with 50mM glycine solution (pH7.4) at 14000rpm for 9min, and the supernatant was discarded and repeated twice.
1.1.5 preservation:
the storage solution (50mM Tris, 0.5 wt% BSA, pH7.8) was added to the pellet obtained after the completion of the blocking centrifugation so that the final fluorescent microsphere concentration was 5mg/ml, and the mixture was ultrasonically mixed (40kHz, 6min) and stored at 4 ℃.
1.2 preparation of coated antibodies
Diluting human myoglobin (Myo) labeled antibody to fixed concentration (2.0mg/ml) with coating buffer solution, diluting control reagent (goat anti-mouse IgG) to fixed concentration (2.0mg/ml), coating the 2 liquids (coating amount is 1 μ l/cm) onto Sidolisi CN95 (nitrocellulose membrane) by using special coating equipment (XYZ 3060 of BIODOT company), and drying in a drying oven at 37 ℃ for 4 hours for later use; wherein the coating buffer consists of: 0.01M Phosphate Buffered Saline (PBS) +3 wt% sucrose (as a protectant).
1.3 marker preparation
And (3) spraying the 5 groups of fluorescent microsphere markers on a test tube according to the required concentration and the volume of 5 microlitres, and drying for later use (vacuum drying).
1.4 sample pad preparation
The sample treatment solution is dipped or spotted in or on the sample pad at the desired concentration (which can be passed through the sample processor), and dried for use. The specific formula of the sample treatment solution is as follows: 50mmol/L Tris-HCl, 0.5 wt% Casein, 0.5 wt% BSA, 0.1 wt% Tween-20, 0.05 wt% PEG, 0.1 wt% Tween-80, 0.05% PVP, 0.3 wt% sodium citrate dihydrate, 2 wt% sucrose.
Abbreviations: PB: a phosphate buffer; BSA: bovine serum albumin, Casein: casein, Tris-HCl: tris hydrochloride buffer, PEG: polyethylene glycol, Tween-20: tween 20, Tween-80: tween 80, PVP: polyvinylpyrrolidone.
1.5 Assembly of test strips
Firstly, a coating pad 3 is bonded on a PVC plate 1, then, one end of a quality control line 32 close to the coating pad 3 is lapped with a water absorption pad 4, one end of a detection line 31 close to the coating pad 3 is lapped with a sample pad 2, a strip cutting machine is used for cutting the test strip (shown in figure 1) into test strips with the thickness of 4mm +/-0.1 mm, and the test strips are put into a card shell to prepare the immunochromatography detection card.
The card housing is selected from the prior art, for example, the card housing (as shown in fig. 2A and 2B) may include: a bottom tank 12 connected to the PVC plate 1; an upper cover 11 connected to the bottom tank 12, the upper cover 11 having a sample application hole 13 for applying a sample to the sample pad 2; and the observation window 14 is arranged on the upper cover 11 and is used for data acquisition of the detection line 31 and the quality control line 32.
As shown in fig. 2B, the bottom tank 12 includes: a plurality of positioning holes 17 which are symmetrically distributed and positioned on the inner surface of the test strip, wherein a plurality of first limiting parts 18 used for limiting the test strip to move transversely and second limiting parts 19 used for limiting the test strip to move longitudinally are arranged among the plurality of positioning holes 17; the first limiting part 18 and the second limiting part 19 which are symmetrically arranged enclose a paper strip placing area 15 (a dotted line area) for placing the test paper strip;
as shown in fig. 2A, the upper cover 11 includes: a plurality of positioning posts 16 which are matched with a plurality of positioning holes 17, and are matched with each other to fix the upper cover 11 and the bottom groove 12 together; the upper cover 11 further includes a third limiting portion 20 for limiting the up and down movement of the test strip.
An observation window 14 for data acquisition is arranged above the coated pad 3 to expose all the detection lines 31 and quality control lines 32 for collecting detection results; and the observation window 14 is arranged on the upper cover 11 at a position corresponding to the middle part of the test strip placement area 15. The upper cover 11 is provided with a sample adding hole at a position corresponding to the sample pad 2, so as to be used for dropwise adding a sample on the sample pad 2. The distance between the detection line and the sample adding hole is 15-25 mm.
1.6 detection
Adding 75 mu L of sample to be tested into a test tube (the content of the fluorescent microspheres is 5 mu g/per person) with the fluorescent microsphere labeled antibody, adding 300 mu L of sample diluent, repeatedly blowing and beating for 20 times, dissolving the fluorescent substance, and carrying out immunoreaction with the object to be tested in the sample to be tested to obtain a well-mixed sample solution. And (3) dropwise adding 75 mu L of the mixed sample solution into the sample adding hole 13 of the card shell, carrying out forward lateral chromatography under the capillary action, and reading data at 15 minutes to obtain a detection result. The specific formula of the sample diluent is as follows: 50mmol/L Tris-HCl, 1 wt% Tween-80, 0.72 wt% trisodium citrate dihydrate. Abbreviations: Tris-HCl: tris-hcl buffer, Tween-80: tween 80.
1.7 evaluation of test strips
Comparative example 1 differs from example 1 in the steps of labeling the antibody: and (3) without adding BSA, carrying out fluorescent labeling on the human myoglobin (Myo) labeled antibody by using fluorescent microspheres (the mass ratio of the fluorescent microspheres to the human myoglobin (Myo) labeled antibody is 10: 1).
Comparative example 2 differs from example 1 in the steps of labeling the antibody: and (3) labeling the human myoglobin (Myo) labeled antibody with a fluorescent microsphere (the mass ratio of the fluorescent microsphere to the human myoglobin (Myo) labeled antibody is 100:1) without adding BSA.
The fluorescence signal values of the 3 groups of fluorescent microsphere labeled antibodies of example 1 and the calibrator (Myo solution) of the labeled antibodies of comparative examples 1 and 2 were measured, and the measurement data are shown in table 1 below.
TABLE 1
As is clear from the data in Table 1, in the inventive example 1, the human myoglobin (Myo) -labeled antibody and the BSA solution (50mM, PB, 2 wt% BSA, pH7.4) are mixed uniformly at the mass ratio of 1:1, 1:3, 1:9, respectively, and then are labeled with the fluorescent microspheres (the mass ratio of the fluorescent microspheres to the Myo-labeled antibody, BSA solution mixture is 10:1), compared with the comparative example 1 (the mass ratio of the fluorescent microspheres to the Myo antibody is 10:1), the amounts of Myo antibodies in the 3 groups of the inventive example 1 are 1/2, 1/4, 1/10 of the comparative example 1, respectively, and the reduced antibodies are labeled with the BSA supplement, the fluorescent value and sensitivity of the detection card are not reduced but increased when the antibodies in the 3 groups of the inventive example 1 are reduced to 1/2 of the comparative example 1, and the antibodies in the 1/4 of the comparative example 1, and when the antibodies in the 3 groups of the inventive example 1 are reduced to 1/10 of the comparative example 1, the fluorescence value of the highest concentration calibrator (1000ng/mL) used is almost not reduced, and the linearity is better; in contrast, in comparative example 2, the total amount of Myo-labeled antibody used for labeling was the same as in group 3 of the present invention, except that the antibody reduced in example 1 of the present invention was labeled with BSA supplement, and as a result, the fluorescence signal value in comparative example 2 was greatly decreased and the sensitivity was insufficient. Therefore, the antibody can be reduced by 90% when the antibody is labeled in the embodiment 1 of the invention, the sensitivity is basically not influenced, and the linearity is better.
Example 2
2.1 preparation of fluorescent microsphere-labeled antibody
2.1.1 microsphere cleaning:
the original concentration of the fluorescent microspheres is 2.0 wt%, 10mM phosphate buffer (pH6.0) is used for centrifugal cleaning, the rotating speed is 14000rpm, the time is 10min, the supernatant is discarded, and the steps are repeated twice to obtain microsphere precipitates.
2.1.2 activation:
1) preparing a chemical cross-linking agent solution:
the chemical cross-linking agent selects N-hydroxysuccinimide and 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide with the weight ratio of 1:1, and respectively uses 10mM (pH6.0) phosphate buffer solution to prepare 500mg/ml solution for standby;
2) activation of fluorescent microspheres:
adding 10mM phosphate buffer (pH6.0) into the washed microsphere precipitate, ultrasonically mixing (40kHz, 6min) to obtain 10mg/mL fluorescent microsphere solution, mixing the N-hydroxysuccinimide prepared in the previous step 1) with the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide mixed solution (fluorescent microspheres, adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in a weight ratio of 1:2.5:2.5) and ultrasonically mixing uniformly (40kHz, 6min), wherein the concentration of the fluorescent microspheres in the obtained solution is 10mg/ml, the concentration of the N-hydroxysuccinimide is 25mg/ml, and the concentration of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 25 mg/ml; the solution was then placed on a shaker and reacted at 37 ℃ for 24min with shaking at 100 rpm.
3) Activated microsphere centrifugation:
the activated microsphere solution was washed by centrifugation with 10mM phosphate buffer (pH6.0) at 14000rpm for 10min, and the supernatant was discarded and repeated twice.
2.1.3 coupling labeling:
adding 10mM phosphate buffer (pH6.0) into the microsphere precipitate obtained by activation centrifugation, and ultrasonically mixing (40kHz, 6min) to make the final concentration of the fluorescent microsphere be 10 mg/ml; then adding a human neutrophil gelatinase-associated lipocalin (NGAL) labeled antibody (NGAL monoclonal antibody) and mouse IgG (50mM, PB, 2 wt% BSA, pH7.4), wherein the weight ratio of the human neutrophil gelatinase-associated lipocalin (NGAL) labeled antibody to the mouse IgG is 1:3, the weight ratio of the fluorescent microspheres to the human neutrophil gelatinase-associated lipocalin (NGAL) labeled antibody to the mouse IgG is 10:1, ultrasonically mixing (40kHz, 6min), placing on a shaking bed, and carrying out shake reaction at 37 ℃ and 100rpm for 4 hours. The mixture is ultrasonically mixed once every half hour during the reaction (40kHz, 6min) to avoid the generation of precipitate.
2.1.4 sealing
After the labeling is finished, 50mM glycine solution (pH7.4) with the same volume as the microsphere solution is added into the microsphere solution, the mixture is ultrasonically mixed (40kHz and 6min), and the mixture is placed on a shaker for shaking reaction at 37 ℃ and 100rpm for 24 min. After the reaction was completed, the reaction mixture was centrifuged and washed with 50mM glycine solution (pH7.4) at 14000rpm for 10min, and the supernatant was discarded and repeated twice.
2.1.5 preservation:
the storage solution (50mM Tris, 0.5 wt% BSA, pH7.8) was added to the pellet obtained after the completion of the blocking centrifugation so that the final fluorescent microsphere concentration was 5mg/ml, and the mixture was ultrasonically mixed (40kHz, 6min) and stored at 4 ℃.
2.2 preparation of coated antibodies
Human neutrophil gelatinase-associated lipocalin (NGAL) -labeled antibody was diluted to a fixed concentration (2.0mg/ml) with coating buffer, a control reagent (goat anti-mouse IgG) was diluted to a fixed concentration (2.0mg/ml), and the above 2 liquids were coated (coating amount 1. mu.l/cm) onto Celadox CN95 (nitrocellulose membrane) using a special coating apparatus (XYZ 3060 of BIODOT), and dried in a drying oven at 37 ℃ for 4 hours for use. The coating buffer was 0.01M Phosphate Buffered Saline (PBS) plus 3 wt% sucrose as a protectant.
2.3 marker preparation
And (3) spraying the 3 groups of fluorescent microsphere markers into a test tube according to the required concentration, and drying for later use (vacuum drying).
2.4 sample pad preparation:
the sample treatment solution is dipped or spotted in or on the sample pad at the desired concentration (which can be passed through the sample processor), and dried for use. The specific formula of the sample treatment solution is as follows: 50mmol/L/L Tris-HCl, 0.5 wt% Casein, 0.5 wt% BSA, 0.1 wt% Tween-20, 0.05 wt% PEG, 0.1 wt% Tween-80, 0.05% PVP, 0.3 wt% sodium citrate dihydrate, 2 wt% sucrose.
Abbreviations: BSA: bovine serum albumin, Casein: casein, Tris-HCl: tris hydrochloride buffer, PEG: polyethylene glycol, Tween-20: tween 20, Tween-80: tween 80, PVP: polyvinylpyrrolidone.
2.5 Assembly of test strips
Same as example 1 in 1.5.
2.6 detection
Adding 75 mu L of sample to be tested into a test tube (the content of the fluorescent microspheres is 5 mu g/per person) with the fluorescent microsphere labeled antibody, adding 300 mu L of sample diluent, repeatedly blowing and beating for 20 times, dissolving the fluorescent substance, and carrying out immunoreaction with the object to be tested in the sample to be tested to obtain a well-mixed sample solution. And (3) dropwise adding 75 mu L of the mixed sample solution into the sample adding hole 13 of the card shell, carrying out forward lateral chromatography under the capillary action, and reading data at 15 minutes to obtain a detection result. The specific formula of the sample diluent is as follows: 50mmol/L Tris-HCl, 1 wt% Tween-80, 0.72 wt% trisodium citrate dihydrate. Abbreviations: Tris-HCl: tris-hcl buffer, Tween-80: tween 80.
2.7 evaluation of the test strips
Comparative example 3 differs from example 2 in the steps of labeling the antibody: without adding BSA, the human neutrophil gelatinase-associated lipocalin (NGAL) labeled antibody is labeled with a fluorescent microsphere (the mass ratio of the fluorescent microsphere to the antibody is 10:1, and the fluorescent microsphere is connected to the surface of the fluorescent microsphere).
Comparative example 4 differs from example 2 in the steps of labeling the antibody: without adding BSA, the human neutrophil gelatinase-associated lipocalin (NGAL) labeled antibody 2 was labeled with fluorescent microspheres (the mass ratio of fluorescent microspheres to antibody was 40:1, attached to the surface of fluorescent microspheres).
The fluorescence signal values of the fluorescent microsphere labeled antibody of example 2 and the calibrator (NGAL solution) of the labeled antibodies of comparative examples 3 and 4 were measured at different concentration gradients, and the measurement data are shown in tables 2, 3, and 4 below.
Table 2 fluorescence detection data for comparative example 3
As can be seen from the data in Table 2, the detection line signal value of the fluorescent microsphere labeled in comparative example 3 reaches a higher level when detecting a low-concentration calibrator (0-100ng/mL), which results in a smaller increase in the detection line signal value and a poorer gradient when detecting a high-concentration calibrator (500-1000 ng/mL).
Table 3 fluorescence detection data for example 2
As can be seen from the data in Table 3, the fluorescent microspheres labeled in example 2 of the present invention have good overall gradient, high signal value, and good detection line fluorescence value, quality control line fluorescence value, and detection line/quality control line ratio gradient.
Table 4 fluorescence detection data for comparative example 4
As can be seen from the data in Table 4, the fluorescent microspheres labeled in comparative example 4 have a certain gradient, but the detection line signal value and the quality control line fluorescence value are lower, and the sensitivity is insufficient.
As can be seen from the data in tables 2 to 4, in example 2 of the present invention, a human neutrophil gelatinase-associated lipocalin (NGAL) labeled antibody and mouse IgG are mixed uniformly according to a mass ratio of 1:3, and then labeled with a fluorescent microsphere (the mass ratio of the fluorescent microsphere to the NGAL labeled antibody and the mouse IgG mixture is 10:1), compared with the normal label of comparative example 3 (the mass ratio of the fluorescent microsphere to the NGAL labeled antibody is 10: 1); the amount of NGAL-labeled antibody in comparative example 4 was the same as that in example 2 of the present invention, but no additional mouse IgG was added, the amount of NGAL antibody in example 2 of the present invention was 1/4, which was the amount of comparative example 3 (normal labeling), the reduced antibody was labeled with mouse IgG (which is inexpensive compared to NGAL-labeled antibody), the amount of antibody in example 2 of the present invention was reduced to 1/4 in comparative example 3, whereas the fluorescence value and sensitivity of example 2 of the present invention were not decreased, but slightly increased, the value of 0ng/mL calibrator was lower, non-specificity was better, and linearity was better; meanwhile, because the NGAL labeled antibody and the mouse IgG are added and then labeled, the NGAL antibody and the mouse IgG are labeled on the fluorescent microsphere, and the goat anti-mouse IgG is coated by the quality control line, the fluorescence value of the quality control line is greatly improved, and the fluorescence value of the quality control line is obviously reduced along with the concentration of the sample from low to high, so that the ratio gradient of the fluorescence value of the detection line to the fluorescence value of the quality control line is more obvious, and the performances of the neutrophil gelatinase-associated lipocalin (NGAL) immunochromatographic detection card such as the linear range and the like can be improved; in contrast, in comparative example 4, the total amount of NGAL antibody used for labeling was the same as that of group 3 in example 2 of the present invention, except that the antibody decreased in example 2 of the present invention was labeled with mouse IgG, and as a result, the fluorescence value was greatly decreased and the sensitivity was insufficient in comparative example 4.
Therefore, when the antibody is labeled in the example 2 of the invention, the dosage of the antibody can be reduced by 75%, the sensitivity is basically not influenced, and the linearity is better.
Example 3
3.1 preparation of fluorescent microsphere-labeled antibody
3.1.1 microsphere cleaning:
the original concentration of the fluorescent microspheres is 2.0 wt%, 10mM phosphate buffer (pH6.0) is used for centrifugal cleaning, the rotating speed is 14000rpm, the time is 9min, supernatant fluid is discarded, and the process is repeated twice, and microsphere precipitation is obtained.
3.1.2 activation:
1) preparing a chemical cross-linking agent solution:
the chemical cross-linking agent selects N-hydroxysuccinimide and 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide with the weight ratio of 1:1, and respectively uses 10mM (pH6.0) phosphate buffer solution to prepare 500mg/ml solution for standby;
2) activation of fluorescent microspheres:
adding 10mM phosphate buffer (pH6.0) into the washed microsphere precipitate, ultrasonically mixing (40kHz, 6min) to obtain 100mg/mL fluorescent microsphere solution, mixing the N-hydroxysuccinimide prepared in the previous step 1) with the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide mixed solution (fluorescent microspheres, adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in a weight ratio of 1:2.5:2.5) and ultrasonically mixing uniformly (40kHz, 6min), wherein the concentration of the fluorescent microspheres in the obtained solution is 10mg/ml, the concentration of the N-hydroxysuccinimide is 25mg/ml, and the concentration of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 25 mg/ml; the solution was then placed on a shaker and reacted at 37 ℃ for 24min with shaking at 100 rpm.
3) Activated microsphere centrifugation:
the activated microsphere solution was washed by centrifugation with 10mM phosphate buffer (pH6.0) at 14000rpm for 10min, and the supernatant was discarded and repeated twice.
3.1.3 coupling labeling:
adding 10mM phosphate buffer (pH6.0) into the microsphere precipitate obtained by activation centrifugation, and ultrasonically mixing (40kHz, 6min) to make the final concentration of the fluorescent microsphere be 10 mg/ml; then adding humanized troponin I (cTnI) labeled antibody and DNP-BSA (dinitrophenol bovine serum albumin conjugate); the weight ratio of the humanized troponin I (cTnI) labeled antibody to the DNP-BSA is 1:1, wherein the weight ratio of the fluorescent microspheres to the humanized troponin I (cTnI) labeled antibody and the DNP-BSA mixture is 10:1, the mixture is ultrasonically mixed uniformly (40kHz and 6min), and the mixture is placed on a shaking table and is subjected to oscillation reaction for 4 hours at 37 ℃ and 100 rpm. The mixture is ultrasonically mixed once every half hour during the reaction (40kHz, 6min) to avoid the generation of precipitate.
3.1.4 sealing
After the labeling is finished, 50mM glycine solution (pH7.4) with the same volume as the microsphere solution is added into the microsphere solution, the mixture is ultrasonically mixed (40kHz and 6min), and the mixture is placed on a shaker for shaking reaction at 37 ℃ and 100rpm for 24 min. After the reaction was completed, the reaction mixture was centrifuged and washed with 50mM glycine solution (pH7.4) at 14000rpm for 10min, and the supernatant was discarded and repeated twice.
3.1.5 preservation:
the storage solution (50mM Tris, 0.5 wt% BSA, pH7.8) was added to the pellet obtained after the completion of the blocking centrifugation so that the final fluorescent microsphere concentration was 5mg/ml, and the mixture was ultrasonically mixed (40kHz, 6min) and stored at 4 ℃.
3.2 preparation of coated antibodies
Diluting a human troponin I (cTnI) labeled antibody to a fixed concentration (2.0mg/ml) by using a coating buffer solution, diluting a contrast reagent (rabbit anti-DNP polyclonal antibody) to a fixed concentration (2.0mg/ml), coating the 2 liquids (the coating amount is 1 mu l/cm) on Sidoides CN95 (nitrocellulose membrane) by using a special coating device (XYZ 3060 of BIODOT company), and drying in a drying oven at 37 ℃ for 4 hours for later use; wherein the coating buffer consists of: 0.01M Phosphate Buffered Saline (PBS) +3 wt% sucrose (as a protectant).
3.3 marker preparation
Comparative example 5: labeling 2 fluorescent microspheres according to the ratio of 1:1 mixing, spraying the mixture into a test tube according to the required concentration, and drying for later use (vacuum drying).
Example 3: and spraying 1 fluorescent microsphere label at the required concentration in a test tube with the volume of 5 microliter, and drying for later use (vacuum drying).
3.4 sample pad preparation
The sample treatment solution is dipped or spotted in or on the sample pad at the desired concentration (which can be passed through the sample processor), and dried for use. The specific formula of the sample treatment solution is as follows: 50mmol/L Tris-HCl, 0.5 wt% Casein, 0.5 wt% BSA, 0.1 wt% Tween-20, 0.05 wt% PEG, 0.1 wt% Tween-80, 0.05% PVP, 0.3 wt% sodium citrate dihydrate, 2 wt% sucrose.
Abbreviations: PB: a phosphate buffer; BSA: bovine serum albumin, Casein: casein, Tris-HCl: tris hydrochloride buffer, PEG: polyethylene glycol, Tween-20: tween 20, Tween-80: tween 80, PVP: polyvinylpyrrolidone.
3.5 Assembly of test strips
Same as example 1 in 1.5.
3.6 detection
Adding 75 mu L of sample to be tested into a test tube (the content of the fluorescent microspheres is 5 mu g/per person) with the fluorescent microsphere labeled antibody/antigen being spotted, adding 300 mu L of sample diluent, repeatedly blowing and beating for 20 times, dissolving the fluorescent substance, and carrying out immunoreaction with the object to be tested in the sample to be tested to obtain a well-mixed sample solution. And (3) dropwise adding 75 mu L of the mixed sample solution into the sample adding hole 13 of the card shell, carrying out forward lateral chromatography under the capillary action, and reading data at 15 minutes to obtain a detection result. The specific formula of the sample diluent is as follows: 50mmol/L Tris-HCl, 1 wt% Tween-80, 0.72 wt% trisodium citrate dihydrate. Abbreviations: Tris-HCl: tris-hcl buffer, Tween-80: tween 80.
3.7 evaluation of test strips
Comparative example 5 differs from example 3 in that the labeled antibody and procedure are different: marking the humanized troponin I (cTnI) marked antibody with a fluorescent microsphere (the mass ratio of the fluorescent microsphere to the cTnI marked antibody is 10: 1); the dinitrophenol bovine serum albumin conjugate (DNP-BSA) is labeled with fluorescent microspheres (the mass ratio of the fluorescent microspheres to the DNP-BSA is 10: 1).
The fluorescence signal values of the fluorescent microsphere labeled antibody of example 3 and the calibrator (NGAL solution) of the labeled antibody of comparative example 5 under different concentration gradients were measured, and the measurement data are shown in tables 5 and 6 below.
TABLE 5 fluorescence detection data for comparative example 5
Table 6 fluorescence detection data for example 3
As can be seen from the data in tables 5 and 6, since the labeled antibody in example 3 of the present invention is humanized troponin i (ctni), different secondary antibodies cannot be used as quality control lines, a set of independent quality control system needs to be added separately, the quality control system added in the system is DNP-BSA for labeling, and the quality control lines coat rabbit anti-DNP polyclonal antibody; comparative example 5, respectively marking a cTnI antibody and DNP-BSA (the mass ratio of the fluorescent microspheres to the antibodies is 10:1), and then mixing the two marked markers according to the mass ratio of 1:1 to prepare marker gun heads; in example 3 of the present invention, a humanized troponin i (cTnI) labeled antibody and DNP-BSA were mixed in a mass ratio of 1:1, and then labeled with a fluorescent microsphere (the mass ratio of the fluorescent microsphere to the cTnI labeled antibody to the DNP-BSA mixture was 10:1), followed by preparation of a marker tip.
As can be seen from the data in tables 5 and 6, in example 3 of the present invention, compared with comparative example 5, the amounts of cTnI-labeled antibody, DNP-BSA, and fluorescent microsphere were halved, but the sensitivities of the highest signal values of the T-line fluorescence values were substantially the same; the quality control line of the comparative example 5 is an independent system, the fluorescence value of the quality control line is basically unchanged, the fluorescent microsphere of the embodiment 3 of the invention is marked with both cTnI antibody and DNP-BSA, the quality control line is coated with rabbit anti-DNP polyclonal antibody, and the fluorescence value of the quality control line is obviously reduced along with the sample concentration from low to high, so that the gradient of the ratio of the fluorescence value of the detection line to the fluorescence value of the quality control line is more obvious, and the performances of the humanized troponin I (cTnI) chromatographic detection card, such as linear range, can be improved.
Therefore, when the antibody, DNP-BSA and fluorescent microspheres are labeled in the embodiment 3 of the invention, the dosage can be reduced by 50%, the sensitivity is basically not affected, and the linearity is better.
The preparation method of the invention uses the marker to mark a protein and a labeled antibody/antigen simultaneously, reduces the ratio of the labeled antibody/antigen in the total amount of the protein combined with the marker, and achieves the purposes of saving the antibody and reducing the cost; meanwhile, the protein which replaces part of labeled antibody/antigen for labeling can keep better stability of the quality control line, and is beneficial to improving the detection accuracy and stability. The type and proportion of the protein used in the labeling can be adjusted according to the performance of different items of antibodies, and the protein with unobvious cross reaction is selected.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.
Claims (10)
1. A preparation method of a fluorescent microsphere labeled antibody is characterized by comprising the following steps:
1) adding 100-1000mg/mL chemical cross-linking agent solution into 1-50mg/mL fluorescent microsphere solution for activation;
2) mixing the labeled antibody and the quality control protein to obtain total protein, and adding the total protein into the activated product obtained in the step 1) for reaction;
3) adding a sealing agent into the solution obtained in the step 2) for sealing, and centrifuging to obtain the fluorescent microsphere labeled antibody.
2. The preparation method according to claim 1, wherein in the step 1), the weight ratio of the fluorescent microsphere solution to the chemical cross-linking agent solution is 1: 0.1-5; the granularity of the fluorescent microsphere is 100nm-500 nm; the activation time is 5-30 min.
3. The method of claim 1, wherein in step 1), the chemical crosslinker is selected from at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, glutaraldehyde and periodate, chloramine-T.
4. The method of claim 1, wherein in step 2), the weight ratio of total protein to fluorescent microspheres is 1: (200-1); the weight ratio of the labeled antibody to the quality control protein is 1 (0.1-20); the rotating speed of the reaction is 50-150rpm, and the time is 2-5 h.
5. The method according to claim 1, wherein in step 2), the labeled antibody is selected from the group consisting of a human myoglobin labeled antibody, a human neutrophil gelatinase-associated lipocalin labeled antibody, a troponin I labeled antibody, an N-terminal-B type brain-urine peptide precursor labeled antibody, a procalcitonin labeled antibody, a C-reactive protein labeled antibody, a D-dimer labeled antibody, a human chorionic gonadotropin β subunit labeled antibody, a lipoprotein-associated phospholipase a2 labeled antibody, an anti-mullerian hormone labeled antibody, an interleukin 6 labeled antibody, a serum amyloid a labeled antibody, and a chalone C labeled antibody; the quality control protein is selected from bovine serum albumin BSA, chicken IgY antibody, rabbit-derived antibody, sheep-derived antibody or donkey-derived antibody.
6. The method according to claim 1, wherein in the step 3), the blocking agent is at least one selected from the group consisting of a 1-5 wt% bovine serum albumin solution, a 0.1-5 wt% casein solution and 10-50mM glycine.
7. The preparation method according to claim 6, wherein in the step 3), the volume ratio of the blocking agent to the solution is 1 (0.01-2).
8. The method according to claim 1, wherein in step 3), the blocking time is 5min to 1 h; the rotation speed of the centrifugation is 10000-20000rpm, and the time is 8-20 min.
9. A fluorescent microsphere-labeled antibody prepared by the method of any one of claims 1 to 8.
10. Use of the fluorescent microsphere-labeled antibody of claim 9 in lateral immunochromatography.
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