CN112014382A - Chemiluminescence kit for detecting myoglobin content and application thereof - Google Patents
Chemiluminescence kit for detecting myoglobin content and application thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The invention provides a chemiluminescence immunoassay kit for detecting myoglobin content, which comprises quality control streptavidin magnetic microspheres, a sealant, a biotinylation MYO antibody, MYO standard antigen, an alkaline phosphatase labeled antibody with MYO recognition sites, a washing buffer solution, an enzyme labeled antibody diluent, a standard diluent and a substrate solution. The kit disclosed by the invention has the advantages that through double-antibody sandwich detection, enzymatic luminescence is realized, the detection result is stable, the kit can be suitable for various chemiluminescence apparatuses, and the chemiluminescence performance of other streptavidin magnetic microspheres to be tried can be evaluated.
Description
Technical Field
The invention relates to the field of chemical detection, in particular to a chemiluminescence kit for detecting myoglobin content.
Background
Myoglobin (Myoglobin, MYO) is a protein that stores and distributes oxygen to mammalian cells, primarily muscle cells, and measurement of serum Myoglobin is the most sensitive indicator of the early stage of Acute Myocardial Infarction (AMI) diagnosis. In scientific research activities and clinical detection, myoglobin detection methods and means are mature, and enzyme-linked immunosorbent assay, immunoturbidimetry, chemiluminescence and the like are mainly used at present.
Myoglobin detection is a mature detection item, and a plurality of commercial enzyme linked immunosorbent assay kits are available on the market, but a few commercial chemiluminescence assay kits which can be sold individually are available. The chemiluminescence immunoassay is a detection item with high automation degree, and is generally sold together with instrument reagents, so that the quality control is easier, and the maximization of industrial benefits can be realized. However, for some non-clinical testing units that do not have the need for large-scale and automated testing, a chemiluminescent kit that can be provided separately and adapted to different brands of chemiluminescent instruments is necessary.
As an important reagent in chemiluminescence detection, magnetic particles are required to be tried on a chemiluminescence platform continuously in the research and development process and improved. However, the purchased commercial chemiluminescence kits are not only expensive, but also require additional equipment, thus increasing much research and development cost. The MYO chemiluminescence detection kit provided by the invention has the advantages of good stability and high reproducibility, and can be used as a detection tool in the research and development process of chemiluminescence magnetic particles.
Disclosure of Invention
In order to achieve the purpose, the invention provides a chemiluminescence immunoassay kit for detecting the myoglobin content, the kit detects through a double antibody sandwich method, performs enzymatic luminescence, has stable detection results, can be matched with various chemiluminescence instruments, and can be used for evaluating the chemiluminescence properties of other streptavidin magnetic microspheres to be tried.
In order to achieve the purpose, the invention adopts the following technical scheme:
the chemiluminescence kit for detecting the myoglobin content comprises quality control streptavidin magnetic microspheres, a sealant, a biotinylated MYO antibody, a MYO standard antigen, an alkaline phosphatase-labeled antibody with a MYO recognition site, a washing buffer solution, an enzyme-labeled antibody diluent, a standard diluent and a substrate solution.
The quality control streptavidin magnetic microsphere is a streptavidin magnetic microsphere which is subjected to pre-coating coupling by using a biotinylated MYO antibody and then is sealed by using a sealing agent; the blocking agent is free biotin with the concentration of 2-10 mu g/ml.
The quality control streptavidin magnetic microsphere is subjected to pre-coating coupling by using a biotinylated MYO antibody and is sealed by using a sealing agent, and the method specifically comprises the following steps: dynal (T1) SA1 mu m beads of Thermo fisher are used as streptavidin magnetic microspheres to be coupled with biotinylated MYO antibody, the coupling is sealed by a sealing agent after coupling, the magnetic microspheres are incubated with MYO standard antigen and ALP-MYO antibody and substrate liquid after coupling and sealing are finished, and then the magnetic microspheres are placed into a chemiluminescence instrument for reading. ALP-MYO antibody: biotinylated MYO antibodies.
The quality control streptavidin magnetic microsphere is subjected to pre-coating coupling by using a biotinylated MYO antibody and is sealed by using a sealing agent, and the method specifically comprises the following steps: coupling is carried out by taking Dynal (T1) SA1 mu m beads of Thermo fisher as streptavidin magnetic microspheres, the coupling concentration of biotinylated MYO antibody is 10-20 mu g/ml, 1mg of the streptavidin magnetic microspheres are added with 2-3ml of biotinylated antibody diluent, the antibody is mansion concentric rat Anti-human Anti-MYO monoclonal antibody with the product number XJ21, and the antibody diluent is 1 XPBS containing 0.1% Tween20 (PH 7.4); the blocking agent is free biotin with the concentration of 2-10 mu g/ml, the blocking agent is used for blocking after the coupling is finished, 500-1000 mu L of magnetic microspheres are added into 1mg of magnetic microspheres, the magnetic microspheres are diluted to 0.5mg/ml after the coupling and blocking are finished, 50 mu L of magnetic microspheres, 50 mu L of MYO standard substance and 100 mu L of LALP-MYO antibody are added into each hole, after the uniform mixing reaction is carried out for 15 minutes, 150 mu L of substrate liquid is added into each hole for incubation for 5 minutes after the washing, and the holes are placed into a plate-type chemiluminescence instrument for reading. Different reading systems of the chemiluminescence apparatus are different, and the displayed readings are different, so that for the ampere-plate chemiluminescence apparatus used in the invention, the background readings of the quality control microspheres are required to be lower than twenty thousand, the standard curve readings are higher than one million, and the weak positive readings are higher than 2.6 times of the background readings.
Wherein the labeling method of the biotinylated MYO antibody is as follows: dissolving a MYO antibody in PBS (phosphate buffer solution) to prepare an antibody solution, and dissolving Biotin sulfo-NHS-Biotin in anhydrous DMF (dimethyl formamide) to obtain a Biotin/DMF solution; adding Biotin/DMF solution into the antibody solution, reacting, and purifying to remove free Biotin micromolecules.
Specifically, the labeling method of the biotinylated MYO antibody is as follows: adding 125 μ L MYO antibody (4mg/mL, from Xiamen concentric) into 375 μ L PBS to dilute to 1mg/mL, centrifuging at 4 deg.C and 12000 rpm in an ultrafiltration tube for 10 min/time, repeating for three times, removing original buffer, adding 500 μ L PBS, and placing in ice bath to obtain antibody solution; dissolving 3.1mg of sulfo-NHS-Biotin in 700. mu.L of anhydrous DMF to obtain 10mM Biotin/DMF solution; adding 16.7 mu L of 10mM Biotin/DMF solution into the antibody solution, quickly and uniformly mixing, and reacting for 2h on ice; after the reaction is finished, centrifuging the reaction solution at 4 ℃, 12000 r for 10 min/time, repeating for three times, and removing unreacted and hydrolyzed small molecules such as biotin and the like; adding 500 μ L PBS, repeatedly blowing for 10min, taking out, subpackaging with 50 μ L each tube, and storing at 4 deg.C.
Wherein, the alkaline phosphatase labeled antibody with MYO recognition site is labeled with Abnova (KA0001) alkaline phosphatase labeling kit. The marking steps are as follows: firstly, adding 100 mu L of Washing buffer into a filter tube, adding 37 mu L of antibody into the filter tube, wherein the antibody is a rat Anti-human-MYO monoclonal antibody of Xiamen with the product number XJ22, uniformly blowing, and centrifuging at 8000g for 10 min; adding 100 μ L Washing buffer, blowing, mixing, centrifuging at 8000g for 10 min; add 10. mu.L of Reaction buffer to ALP-NH2Blowing and beating the powder to dissolve; dissolving ALP-NH2Adding the solution into a filter tube, blowing, beating and uniformly mixing; incubating the filter tube at 37 ℃ for 2 h; add 190 u L Storage buffer, blow for 10min, make the antibody solution dissociation, antibody solution transfer to another EP tube, 4 degrees preservation. In use, 5% BSA in PBS 1; and (5) diluting by 2000.
Wherein, the MYO standard antigen is purchased from the same core of mansion, and when in use, the MYO standard antigen is diluted from 250ng/ml to 3.906 ng/ml.
Wherein the substrate liquid is purchased from Shenzhen MeyKate, the platform period is short, and the reaction can be completed in 1 min; high sensitivity, measurable < 10-19mol ALP; the addition amount of ALP and the final luminous value are proved to have good linearity before the experiment; the temperature requirement is not strict, and the reaction can be carried out at 25-35 ℃.
Wherein the washing buffer solution is TBST solution containing 20mM Tris, 30mM NaCl and 0.1% Tween 20. Prepare 2L of 5 × mother solution TBS solution: weighing 24.228g of Tris powder and 17.532g of NaCl powder, and placing the powder in a 2L beaker with a magnetic stirrer; pouring 1600mL of purified water, placing the beaker on a magnetic stirrer, adjusting the rotating speed, and stirring at room temperature until the powder reagent is completely dissolved; inserting into a pH meter electrode, dropwise adding 1M hydrochloric acid, adjusting pH to 8.0 +/-0.1, and rotationally stirring at room temperature (25 ℃) for 3 minutes; finally, the volume is determined to be 2L by a volumetric flask; taking a sterilized and cleaned 2L blue-cap reagent bottle, installing a suction filter and a 0.22 mu m filter membrane, and adding the solution into the suction filter for filtering; and (6) bottling. When used, the solution is diluted by 5 times, and 0.1 percent of Tween20 is added.
The invention also provides application of the chemiluminescence kit for detecting the myoglobin content, and the chemiluminescence kit for detecting the myoglobin content is used for detecting the myoglobin content and evaluating the chemiluminescence performance of the streptavidin magnetic microspheres.
The invention can detect the MYO content in the serum sample and can detect the serum sample as low as 4 ng/ml.
The invention relates to a Biotin-avidin system, namely SA magnetic beads and Biotin-MYO antibodies are adopted, and the chemiluminescence performance of SA magnetic microspheres to be detected is judged by comparing the test luminescence value, the calibration curve property, the background height and the separation degree of the luminescence values of standards with different concentrations with control (quality control) SA magnetic microspheres.
The principle of the invention is that a double-antibody sandwich method is adopted to determine the MYO content. The streptavidin magnetic microsphere is a solid phase carrier, biotinylated MYO antibody is combined on the streptavidin magnetic microsphere through a streptavidin-biotin system, then the antibody is combined with antigen in reaction solution, then the antibody which is marked with alkaline phosphatase ALP and has different MYO antigen sites is combined with MYO, and finally substrate is added. The substrate contains adamantyl, wherein a luminescent group in a molecule is an aromatic group and a group acted by enzyme, chemiluminescence is caused under the action of enzyme and a starting luminescent reagent, a phosphate group is hydrolyzed under the action of alkaline phosphatase ALP to remove a phosphate group, a medium stable intermediate AMPPD (half life of 2-30 min) is obtained, the intermediate is cracked into a molecule of adamantanone and a molecule of methyl meta-oxybenzoate anion in an excited state through intramolecular electron transfer, when the intermediate returns to a ground state, 470nm light is generated, and the number of emitted photons is positively correlated with the amount of enzyme in the reaction. The MYO concentration in the sample is quantified according to a Log (X) -Log (Y) mathematical model established by the MYO concentration of the calibrator and the corresponding RLU, so that the MYO content in human serum and plasma is detected.
The key technical points of the invention are as follows:
1) selection of anti-MYO antibody pairs
The ability of an antigen to specifically bind to an antibody is based on the structural complementarity and affinity between the antigenic determinant (epitope) and the hypervariable region of the antibody. This property is determined by the spatial configuration of the antigen and antibody molecules. Besides the high complementarity of the two molecular configurations, the antigen epitope and the antibody hypervariable region must be in close contact to have sufficient binding force. The affinity of an antibody refers to the attractive force between an antigen binding site and a corresponding epitope on an antibody molecule, which is the inherent binding force between antigen and antibody. It represents the strength of binding between the antibody binding site and the epitope, and is related to the antibody's binding valency. The term "multivalent" means that the antibody has high affinity, binds to the antigen firmly, and is not easily dissociated. The selection of antibody pairs is particularly critical in immunoassays based on the double antibody sandwich method. First, the antibody selected should have a high affinity for the antibody. In the whole immune reaction, all the participated molecules are chain-connected, if the affinity of the antibody is insufficient and the binding with the antigen is not firm, the antigen can be easily dissociated, and the whole reaction chain is broken. Second, the two antibodies in the selected pair not only have high affinity for each other, but also the antigen-binding epitopes cannot overlap. Once the two antibodies have overlapping antigen-binding epitopes, they will compete for binding to the antigen, making the antibody unable to bind to the antigen in a post-reaction step. After investigation and testing of various antibody pairs, the invention selects the Xiamen rat Anti-human Anti-MYO monoclonal antibody pair, selects the antibody with the code XJ21 to couple with the magnetic microspheres, and selects the antibody with the code XJ22 to carry out enzyme labeling. The choice of this antibody pair has also proved to be very suitable in the subsequent formulation and use of the kit.
2) Purification after labeling of biotin with antibody
When the antibody is used to label biotin, an excess of biotin is generally added to allow the antibody to be sufficiently labeled, and after the reaction is completed, unbound free biotin is removed because free biotin competes with the biotinylated antibody for the binding site of streptavidin. Biotin is a kind of small molecular substance with molecular weight of 244D, and the molecular weight of general IgG antibody is 150KD, and the molecular weights of the two are greatly different. Most of the general experimental schemes are to remove free biotin by dialysis, but the dialysis method is not only time-consuming and very costly, but also damages the dialysis bag under the condition of unknown condition, and the dialysis bag is hard to mark and is in the east. The invention selects Millpore with 50KD molecular weight cut-off
An Ultra-4 filter tube was used to remove free biotin. Centrifuging the reaction solution at 4 ℃ under 12000 rpm for 10 min/time, and repeating the centrifuging and centrifuging for three times to remove unreacted and hydrolyzed small molecules such as biotin. The method is simple and convenient to operate, saves time and has little loss.
3) Encapsulation of magnetic microspheres
After coupling the streptavidin magnetic microspheres with biotinylated antibody, there may be some SA protein (streptavidin) present in the phantom to await detection. Compared with free biotin, the biotinylated antibody has greatly increased molecular weight and a more complex spatial structure. The biotinylated antibody may be sterically hindered when it reacts with the SA protein on the magnetic microspheres, resulting in masking of some sites where SA can bind to biotin. If these sites are not blocked, they will react with biotin analogues in the serum or plasma sample, resulting in non-specific adsorption on the magnetic microspheres. According to the invention, after the streptavidin magnetic microsphere is coupled with the biotinylated antibody, free biotin with a certain concentration is used for sealing, so that the nonspecific adsorption of the magnetic microsphere is effectively reduced.
The invention is a MYO chemiluminescence kit prepared by selecting a proper antibody pair based on a double-antibody sandwich immunoassay method. The standard curve detected by the quality-control streptavidin magnetic microspheres has good linearity, wherein R2 is more than or equal to 0.99, S1/S0 is more than 2.6, S7/S0 is more than 500, and CV values detected by the same sample for 10 times are less than or equal to 10%. In a sample recovery experiment, the sample recovery rate is more than 85 percent and less than 115 percent, and the sample recovery rate meets the national requirements of the national medical and pharmaceutical industry standards of the people's republic of China.
Drawings
FIG. 1 is the result of the biotinylated antibody test of example 2.
FIG. 2 shows the results of the test of example 4.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents also fall within the scope of the invention.
The chemiluminescence kit for detecting the myoglobin content comprises quality control streptavidin magnetic microspheres, a sealing agent, a biotinylated MYO antibody, a MYO standard antigen, an alkaline phosphatase-labeled antibody with a MYO recognition site, a washing buffer solution, an enzyme-labeled antibody diluent, a standard diluent and a substrate solution.
The quality control streptavidin magnetic microsphere is Dynal (T1) SA1 mu m beads of Thermo fisher, which are pre-coated and coupled by a biotinylated MYO antibody and then blocked by a blocking agent. The coupling concentration of the biotinylated MYO antibody is 10-20 mug/ml, and the coupling concentration of the biotinylated MYO antibody is 1mg of streptavidin magnetic microspheres and 2-3ml of biotinylated antibody diluent, wherein the antibody diluent is 1 × PBS containing 0.1% Tween20 (PH 7.4). The blocking agent is free biotin with the concentration of 2-10 mu g/ml, the blocking agent is blocked by the free biotin with the concentration of 2-10 mu g/ml after the coupling is finished, and 1000 mu L of the magnetic microsphere with the concentration of 1mg is added. After the coupling and sealing of the magnetic microspheres are finished, the magnetic microspheres are diluted to 0.5mg/ml, 50 mu L of the magnetic microspheres, 50 mu L of MYO standard and 100 mu L of ALP-MYO antibody are added into each hole of a chemiluminescence plate, the mixture is uniformly mixed and reacted for 15 minutes, 150 mu L of substrate liquid is added into each hole after washing, incubation is carried out for 5 minutes, and the plate-type chemiluminescence apparatus is placed for reading.
The labeling method for biotinylated MYO antibody was as follows: adding 125 μ L of antibody (4mg/mL, from Xiamen concentric) into 375 μ L of PBS to dilute to 1mg/mL, centrifuging at 4 deg.C and 12000 r in an ultrafiltration tube for 10 min/time for three times, removing the original buffer, adding 500 μ L of PBS, and placing in an ice bath; dissolving 3.1mg of sulfo-NHS-Biotin in 700. mu.L of anhydrous DMF to obtain 10mM Biotin/DMF solution; adding 16.7 mu L of 10mM Biotin/DMF solution into the antibody solution, quickly and uniformly mixing, and reacting for 2h on ice; after the reaction is finished, centrifuging the reaction solution at 4 ℃, 12000 r for 10 min/time, repeating for three times, and removing unreacted and hydrolyzed small molecules such as biotin and the like; adding 500 μ L PBS, repeatedly blowing for 10min, taking out, subpackaging with 50 μ L each tube, and storing at 4 deg.C.
The ALP labeling of MYO antibody adopts Abnova (KA0001) alkaline phosphatase labeling kit, and the labeling steps are as follows: firstly, adding 100 mu L Washing buffer into a filter tube, adding 37 mu L antibody into the filter tube, blowing and uniformly mixing, and centrifuging at 8000g for 10 min; adding 100 μ L Washing buffer, blowing, mixing, centrifuging at 8000g for 10 min; add 10. mu.L of Reaction buffer to ALP-NH2Blowing and beating the powder to dissolve; dissolving ALP-NH2Adding the solution into a filter tube, blowing, beating and uniformly mixing; the filter tube was incubated at 37 ℃ for 2 h. Add 190 u L Storage buffer, blow for 10min, make the antibody solution dissociation, antibody solution transfer to another EP tube, 4 degrees preservation. In use, 5% BSA in PBS 1; and (5) diluting by 2000.
The MYO standard antigen or standard is purchased from concentric centers of Xiamen, and is packaged in 5 μ L tubes after arrival, and stored at-20 deg.C without dilution. After thawing, the mixture is not returned to-20 ℃ so as to avoid repeated freeze thawing and inactivation. In use, MYO standard is diluted from 250ng/ml times to 3.906 ng/ml.
The substrate liquid is purchased from Shenzhen MeyKate, the platform period is short, and the reaction can be completed in 1 min; the sensitivity is high, and the ALP can be measured to be less than 10-19 mol; the addition amount of ALP and the final luminous value are proved to have good linearity before the experiment; the temperature requirement is not strict, and the reaction can be carried out at 25-35 ℃.
The washing buffer was TBST solution containing 20mM Tris, 30mM NaCl, 0.1% Tween 20. Prepare 2L of 5 × mother solution TBS solution: weighing 24.228g of Tris powder and 17.532g of NaCl powder, and placing the powder in a 2L beaker with a magnetic stirrer; pouring 1600mL of purified water, placing the beaker on a magnetic stirrer, adjusting the rotating speed, and stirring at room temperature until the powder reagent is completely dissolved; inserting into a pH meter electrode, dropwise adding 1M hydrochloric acid, adjusting pH to 8.0 +/-0.1, and rotationally stirring at room temperature (25 ℃) for 3 minutes; finally, the volume is adjusted to 2L by a volumetric flask. A2L blue-capped reagent bottle which had been sterilized and cleaned was filled with a suction filter and a 0.22 μm filter membrane, and the solution was filtered by adding it to the suction filter. Finally, a label is pasted on the blue cap bottle body. When in use, the solution is diluted by 5 times, and 0.1 percent of Tween20 is added.
Example 1: MYO antibody biotin labeling
Reagent consumable
1.PBS：10mM PBS，pH7.4
2. Anhydrous DMF (removing water before use)
Sulfo-NHS-Biotin (BBI Life sciences) C10021350 mg powder
Anti-MYO antibody (Cat XJ-21, 4mg/mL in 10mM PBS, pH7.4, Xiamen concentric)
5. Ultrafiltration tube Millpore
The Ultra-4MWCO is 50 KD. The ultrafilter tube was soaked in sterile PBS for half a day before use, and placed in a refrigerator at 4 deg.C.
6. Molecular sieve, 5A type, 3-5mm
The method comprises the following steps:
1. adding 125 μ L of antibody (XJ21, 4mg/mL) into 375 μ L PBS, diluting to 1mg/mL, centrifuging at 4 deg.C under 12000 rpm in an ultrafiltration tube for 10 min/time for three times, removing the buffer, adding 500 μ L PBS, and placing in ice bath;
2. dissolving 3.1mg of sulfo-NHS-Biotin in 700. mu.L of anhydrous DMF to obtain 10mM Biotin/DMF solution;
3. adding 16.7 mu L of 10mM Biotin/DMF solution into the antibody solution according to the calculation that the amount of Biotin is 50 times of that of the antibody, quickly mixing the mixture evenly, and reacting the mixture for 2 hours on ice;
4. after the reaction is finished, centrifuging the reaction solution at 4 ℃, 12000 r for 10 min/time, repeating for three times, and removing unreacted and hydrolyzed small molecules such as biotin and the like;
5. adding 500 μ L PBS, repeatedly blowing for 10min, taking out, subpackaging with 50 μ L each tube, and storing at 4 deg.C.
Example 2 biotinylated antibody assay
Reagent:
wash Buffer: PBST buffer (1 × PBS with 0.1% Tween 20);
2.5% BSA solution: weighing 0.25g of BSA, dissolving with 5mL of 1 xPBST, mixing uniformly, and storing at 4 ℃ for 24 h;
biotin-mouse IgG standard master solutions: 1mg/mL diluted 1000 times to 1 mug/mL;
4. goat anti-mouse-HRP secondary antibody: diluted 10000 times with 5% BSA;
TMB reaction stop solution: 2M concentrated H2SO454.5mL of 98% sulfuric acid solution is weighed, added dropwise into 350mL of ultrapure water, stirred while dropwise adding, and after cooling to room temperature, ultrapure water is continuously added to a constant volume of 500 mL.
The method comprises the following steps:
1. biotin-mouse IgG label and biotinylated MYO antibody (diluted with PBST) were diluted to the concentrations in Table 1;
TABLE 1
| Reference numerals | Concentration of | Preparation method | Volume of dilution liquid | |
| Stock solution | 1mg/ml | |||
| 1 | 1μg/ml | Suck 1. mu.L of stock solution | Adding 1ml | |
| 2 | 500ng/ | Suck | 500 μ L of the solution from above | Adding 500 mu L |
| 3 | 250ng/ | Suck | 500 μ L of the solution from above | Adding 500 mu L |
| 4 | 125ng/ | Suck | 500 μ L of the solution from above | Adding 500 mu L |
| 5 | 62.5ng/ | Suck | 500 μ L of the solution from above | Adding 500 mu L |
| 6 | 31.25ng/ | Suck | 500 μ L of the solution from above | Adding 500 mu L |
| 7 | 15.625ng/ | Suck | 500 μ L of the solution from above | Adding 500 muL |
| 8 | 0 | 0 | Adding 500 mu L |
2. Taking three coated SA plates, adding 100 mu L of the monoclonal antibody solution into each hole, and incubating for 2 hours at 37 ℃;
3. after the incubation time is up, the liquid in the wells is spun off and washed three times with 200 μ L of washing buffer, three minutes each time;
4. diluting goat anti-mouse secondary antibody, placing on a shaker at a temperature of 1: 10000, and mixing and incubating for 1 h;
5. after the incubation time had elapsed, the well was drained and washed five times with 200 μ L of wash buffer, three minutes each time;
6. adding 100 mu L of TMB color development solution, incubating for 15min in a dark place, and then adding 50 mu L of stop solution;
7. moving to the OD450 position of a microplate reader, and operating the microplate reader: newly building, operating, measuring photometry, and operating on a filter of 450 nm.
The results are shown in FIG. 1, indicating that: when reacting with SA plate, the concentration of biotinylation antibody has good linear relation with OD value, and the plate can be developed at nanogram level, which shows that the biotin labeling efficiency is good.
Example 3 enzyme labeling of antibodies
Antibody: 5.4mg/ml of concentric MYO (Cat. XJ22) of mansion door for 1 mg;
labeling kit: the molecular mass of the Abnova (KA0001) protein is more than 50KD (the antibody is 150KD, which meets the requirement);
each time, the label is 50-200. mu.g, and this time, the label is 200. mu.g.
TABLE 2 kit Components
1, adding 100 mu L Washing buffer into a filter tube, adding 37 mu L antibody into the filter tube, and blowing, beating and uniformly mixing;
2,8000 g, centrifuging for 10 min;
3, adding 100 mu L Washing buffer, and blowing and uniformly mixing;
4,8000 g, centrifuging for 10 min;
5, add 10. mu.L Reaction buffer to ALP-NH2Blowing and beating the powder to dissolve;
6, dissolving ALP-NH2Adding the solution into a filter tube, blowing and uniformly mixing the solution and the antibody;
7, incubating the filter tube at 37 ℃ for 2 h;
8, 190. mu.L of Storage buffer was added, and the antibody solution was dissociated by pipetting for 10min, transferred to another EP tube, and stored at 4 ℃.
Example 4 different streptavidin magnetic microspheres this chemiluminescent kit reagent was used:
1, Wash Buffer: : TBST buffer (20mM Tris 30mM NaCl 0.1% Tween 20);
enzyme-labeled antibody diluent: PBST containing 5% BSA, ALP-MYO antibody 1: 2000 diluted, 1 μ L diluted to 2 ml;
biotinylated antibody dilution: 1 PBS containing 0.1% Tween20, 10ug/ml, 10. mu.L biotinylated antibody was diluted to 1 ml;
4, antigen dilution: 1 PBS containing 0.1% Tween20, diluted from 1000ng, a total of 10 wells blanked, and 8 wells used;
5, a sealant: 2 μ g/ml free biotin diluted with 1 × PBS containing 0.1% Tween 20;
6, substrate solution: shenzhen MeiKate;
7, SA magnetic microspheres: after Dynal 1um biotinylated antibody coupling, blocking with free organisms;
and 8, testing the magnetic beads.
TABLE 3 antigen preparation method
| Reference numerals | Concentration of | Preparation method | Volume of dilution liquid | |
| Stock solution | 0.34mg/ml | |||
| 1 | 1μg/ml | Suck 3. mu.L of the stock solution | Adding 1ml | |
| 2 | 500ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 3 | 250ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 4 | 125ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 5 | 62.5ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 6 | 31.25ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 7 | 15.625ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 8 | 7.8125ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 9 | 3.90625ng/ | Suck | 200 μ L of the solution from above | Adding 200 mu L of |
| 10 | 0ng/ml | Adding 200 mu L of |
The method comprises the following steps:
1, preparing 1ml of 10ug/ml biotinylated MYO antibody, and diluting 10 mu L of MYO antibody to 1 ml;
2, taking 25 mu L (10mg/ml) of each of two SA magnetic microspheres (quality control streptavidin magnetic microspheres and to-be-detected streptavidin magnetic microspheres) to magnetically adsorb supernatant, and adding 500 mu L of 10ug/ml biotinylated MYO antibody to incubate for 1h at room temperature;
3, after washing for 3 times, adding 100 mu L of blocking agent respectively, namely 2 mu g/ml of free biotin for blocking, and incubating for 1h at room temperature; after washing for 3 times, each was diluted to 0.5mg/ml with 500. mu.L of PBST;
4, taking two pieces of chemiluminescent plates, adding 50 mu L of magnetic microspheres into a 96-well plate, adding a test standard, namely 50 mu L/well (3-10) of MYO standard antigen, and then adding 100 mu L of enzyme-labeled antibody (alkaline phosphatase labeled antibody with MYO recognition sites) into each well; fully shaking and resuspending the magnetic microspheres, incubating for 15min in a thermostat at 37 ℃, performing magnetic separation, sucking supernatant by using a vacuum liquid sucking pump, and taking down a 96-well plate from a magnetic separator;
adding 200 mu L of Washing buffer into each hole, fully shaking and resuspending the magnetic microspheres, carrying out magnetic separation, sucking off supernatant by using a vacuum imbibition pump, taking down a 96-hole plate from a magnetic separator, repeating the step for 2 times, and Washing for 3 times in total;
6, adding 150 mu L of substrate solution into each hole, fully shaking and resuspending the magnetic microspheres, and incubating for 5min at 37 ℃ in a dark place;
7, putting the 96-well plate into a chemiluminescence instrument for reading, and pointing and installing a figure 2.0-password and user name login-quick measurement-allowing oscillation (low intensity 5S, all holes) -starting measurement-Ctrl-C-Ctrl-V to a U disk.
The results are shown in Table 4 for the machine readings and in FIG. 2, a standard curve fitted to the luminescence values versus the concentration of the standard. As can be seen from FIG. 2, the obtained standard curve is good in linearity, R2≥0.99，S1/S0＞2.6，S7/S0＞500。
TABLE 4 chemiluminescence measurement values of magnetic microsphere to be detected and reference magnetic microsphere standards
| Concentration of MYO ng/ml | Luminous value (Dynal T1) | Luminous value (magnetic microsphere to be detected) |
| 125 | 12406350 | 11061994 |
| 62.5 | 6098591 | 5524763 |
| 31.25 | 2645590 | 2370637 |
| 15.625 | 1017961 | 959306 |
| 7.8125 | 422230 | 426806 |
| 3.90625 | 182384 | 208620 |
| 0 | 17164 | 20178 |
TABLE 5 separation degree of luminescence values of magnetic microsphere to be detected and quality control magnetic microsphere Dynal T1
| Degree of separation | Dynal T1 | Magnetic microsphere to be detected |
| S1/S0 | 10.626 | 10.339 |
| S2/S0 | 24.600 | 21.152 |
| S3/S0 | 59.308 | 47.542 |
| S4/S0 | 154.136 | 117.486 |
| S5/S0 | 355.313 | 273.801 |
| S6/S0 | 722.812 | 548.221 |
| S7/S0 | 1175.337 | 908.312 |
Note: the separation degree is the ratio of each measured value except the background in the standard product to the background, and the higher the ratio is, the higher the separation degree is, the more sensitive the whole test system is.
The above results show that: the standard curve of the MYO chemiluminescence detection kit has good linearity, R2The detection kit is more than or equal to 0.99, S1/S0 is more than 2.6, and S7/S0 is more than 500, which indicates that the detection kit is a mature and stable chemiluminescence detection kit, has stable detection results, and can be applied to various chemiluminescence apparatuses. In addition, the invention can judge the chemiluminescence property of the SA magnetic microsphere to be tested by comparing the test luminescence value, the calibration curve property, the background height and the separation degree of the luminescence values of the standard products with different concentrations with the control (quality control) SA magnetic microsphere, and can be used for evaluating the chemiluminescence properties of other streptavidin magnetic microspheres to be tested. This is also another practical use of the invention in addition to measuring myoglobin content.
The invention has stable detection result through double-antibody sandwich detection and enzymatic luminescence, can be suitable for various chemiluminescence apparatuses, and can evaluate the chemiluminescence performance of other streptavidin magnetic microspheres to be tried.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications that can be made by the present invention as an equivalent structure or an equivalent process, or directly or indirectly applied to other related technical fields are also included in the scope of the present invention.
Claims (10)
1. The chemiluminescence kit for detecting the myoglobin content is characterized by comprising quality control streptavidin magnetic microspheres, a sealant, a biotinylation MYO antibody, a MYO standard antigen, an alkaline phosphatase labeled antibody with a MYO recognition site, a washing buffer solution, an enzyme labeled antibody diluent, a standard diluent and a substrate solution.
2. The chemiluminescence kit for detecting the myoglobin content according to claim 1, wherein the quality-controlled streptavidin magnetic microspheres are streptavidin magnetic microspheres which are pre-coated and coupled by biotinylated MYO antibody and then sealed by a sealant; the blocking agent is free biotin with the concentration of 2-10 mu g/ml.
3. The chemiluminescence kit for detecting myoglobin content according to claim 2, wherein the quality control streptavidin magnetic microspheres are pre-coated with biotinylated MYO antibody for coupling and sealed with a sealant, specifically: dynal (T1) SA1 mu m beads of Thermo fisher are used as streptavidin magnetic microspheres to be coupled with biotinylated MYO antibody, the coupling is sealed by a sealing agent after coupling, the magnetic microspheres are incubated with MYO standard antigen and ALP-MYO antibody and substrate liquid after coupling and sealing are finished, and then the magnetic microspheres are placed into a chemiluminescence instrument for reading.
4. The chemiluminescent kit for detecting myoglobin content according to claim 1, wherein the labeling method of the biotinylated MYO antibody is as follows: dissolving a MYO antibody in PBS (phosphate buffer solution) to prepare an antibody solution, and dissolving Biotin sulfo-NHS-Biotin in anhydrous DMF (dimethyl formamide) to obtain a Biotin/DMF solution; adding Biotin/DMF solution into the antibody solution, reacting, and purifying to remove free Biotin micromolecules.
5. The chemiluminescence kit for detecting the myoglobin content according to claim 1, wherein the alkaline phosphatase labeled antibody with MYO recognition site is labeled with Abnova KA0001 alkaline phosphatase labeling kit.
6. The chemiluminescent kit for detecting myoglobin according to claim 1, wherein the MYO standard antigen is purchased from concentric centers of mansion and is diluted from 250ng/ml to 3.906ng/ml in use.
7. The chemiluminescence kit for detecting the myoglobin content according to claim 1, wherein the washing buffer is TBST solution containing 20mM Tris, 30mM NaCl and 0.1% Tween 20.
8. The chemiluminescence kit for detecting the myoglobin content according to claim 1, wherein the enzyme-labeled antibody diluent is PBST containing 5% BSA.
9. The chemiluminescence kit for detecting the myoglobin content according to claim 1, wherein the standard diluent comprises a biotinylated antibody diluent and an antigen diluent, and each is 1 × PBS containing 0.1% Tween 20.
10. The application of the chemiluminescence kit for detecting myoglobin according to any one of claims 1 to 9, wherein the chemiluminescence kit for detecting myoglobin is used for quantitatively detecting myoglobin and evaluating chemiluminescence performance of streptavidin magnetic microspheres.
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