CN112285353A - Method for improving anti-biotin interference capability and sensitivity of chemiluminescence kit of streptavidin-biotin reaction system - Google Patents
Method for improving anti-biotin interference capability and sensitivity of chemiluminescence kit of streptavidin-biotin reaction system Download PDFInfo
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- CN112285353A CN112285353A CN202011140213.6A CN202011140213A CN112285353A CN 112285353 A CN112285353 A CN 112285353A CN 202011140213 A CN202011140213 A CN 202011140213A CN 112285353 A CN112285353 A CN 112285353A
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- 239000011616 biotin Substances 0.000 title claims abstract description 97
- 229960002685 biotin Drugs 0.000 title claims abstract description 97
- 238000000034 method Methods 0.000 title claims abstract description 34
- 230000035945 sensitivity Effects 0.000 title claims abstract description 30
- 230000001745 anti-biotin effect Effects 0.000 title claims abstract description 25
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 142
- 235000020958 biotin Nutrition 0.000 claims abstract description 71
- 239000011324 bead Substances 0.000 claims abstract description 59
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims abstract description 43
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims abstract description 43
- 239000003550 marker Substances 0.000 claims abstract description 35
- 239000012224 working solution Substances 0.000 claims abstract description 28
- 239000011248 coating agent Substances 0.000 claims abstract description 24
- 238000000576 coating method Methods 0.000 claims abstract description 24
- 239000003085 diluting agent Substances 0.000 claims abstract description 20
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 238000004140 cleaning Methods 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 32
- 238000002156 mixing Methods 0.000 claims description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 15
- -1 cyclohexane-1-carboxylic acid succinimide ester Chemical class 0.000 claims description 13
- 239000012190 activator Substances 0.000 claims description 12
- 239000012452 mother liquor Substances 0.000 claims description 11
- 238000002372 labelling Methods 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 239000002808 molecular sieve Substances 0.000 claims description 9
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 239000010413 mother solution Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 12
- 238000007789 sealing Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 19
- 108010090804 Streptavidin Proteins 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 9
- 229940098773 bovine serum albumin Drugs 0.000 description 9
- 238000004020 luminiscence type Methods 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 230000006287 biotinylation Effects 0.000 description 4
- 238000007413 biotinylation Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000003359 percent control normalization Methods 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- WZELXJBMMZFDDU-UHFFFAOYSA-N Imidazol-2-one Chemical group O=C1N=CC=N1 WZELXJBMMZFDDU-UHFFFAOYSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical group C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L Zinc chloride Inorganic materials [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
- 230000001064 anti-interferon Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000002619 bicyclic group Chemical group 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000003670 easy-to-clean Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N pentanoic acid group Chemical group C(CCCC)(=O)O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
-
- 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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/543—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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- 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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Abstract
The invention discloses a method for improving the anti-biotin interference capability and sensitivity of a chemiluminescence kit of a streptavidin-biotin reaction system, and belongs to the technical field of biology. A method for improving the anti-biotin interference capability and sensitivity of a chemiluminescence kit of a streptavidin-biotin reaction system comprises the steps of adding SA magnetic beads into a magnetic bead diluent to form a magnetic bead coating working solution; adding an alkaline phosphatase labeled antibody into an enzyme label diluent to form an alkaline phosphatase label working solution; adding a biotin labeled antibody into the biotin labeled substance diluent to form a biotin labeled substance working solution; adding the sample, the magnetic bead coating working solution and the biotin marker working solution into a reaction cup in sequence, incubating for a certain time, performing reaction cleaning, adding the alkaline phosphatase marker working solution, reacting and cleaning; according to the invention, by sealing the free SA magnetic beads which are not combined with the biotin antibody, non-specific signals are reduced, and the detection sensitivity is improved.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for improving the anti-biotin interference capability and sensitivity of a chemiluminescence kit of a streptavidin-biotin reaction system.
Background
Streptavidin is a protein secreted by streptomyces that specifically binds to biotin, with the strongest non-covalent binding known to date. Because of this property, the streptavidin-biotin reaction system has been widely used in the field of purification and detection. Streptavidin exists in a homotetramer form, and each mole of tetramer molecules can be combined with four moles of biotin molecules, so that the streptavidin has a signal amplification effect in the field of immunoassay.
The basic structure of biotin is a bicyclic structure: the I ring is an imidazolone ring and is a part combined with avidin; II is thiophene ring, contains a valeric acid side chain, and the terminal carboxyl group can be connected with biomacromolecule to form biotin-labeled antigen, antibody or enzyme. After being combined with the biological macromolecules, the biotin does not influence the original activities of the biological macromolecules and the biotin, namely the biotin-labeled antibody has the characteristic of being specifically combined with avidin and corresponding antigens.
The characteristic of strong binding force of streptavidin and biotin is utilized, and the date of announcement is as follows: 2011-10-05, patent No.: CN102207501A introduces a biotinylation bovine serum albumin and streptavidin enzyme-labeled reaction plate and a preparation method thereof, and discloses that biotinylation bovine serum albumin and streptavidin are wrapped in micropores of an enzyme-labeled plate, and a biotin-labeled antibody and an antigen to be detected are simultaneously added on the plate, so that the antigen-antibody reaction is in a liquid phase, the reaction efficiency is improved, the reaction time is shortened, and the detection range is widened. The defects of the traditional enzyme labeling reaction plate coating technology are overcome, and because the protein concentration in the coating liquid is very low and antibody molecules cannot completely cover all sites, in order to avoid non-specific adsorption during reaction, high-concentration bovine serum albumin needs to be added to achieve the purpose of closing blank sites.
In a chemiluminescence system, the luminescence modes comprise direct luminescence, indirect luminescence and electrochemical luminescence, and the current researches are more on enzymatic luminescence, mainly comprising an HRP-luminol system and an ALP-AMPPD system. In an enzymatic chemiluminescence system, in order to improve the sensitivity and specificity of a kit, a streptavidin-biotin reaction system is widely used, but biotin interference is always a problem to be solved by an immunoassay kit, particularly the streptavidin-biotin immunoassay kit, SA magnetic beads have the characteristic of adsorbing biotin, the surface areas of magnetic beads with different particle sizes are different from non-specific adsorption capacity, small-particle-size magnetic beads are larger than the surface areas of large-particle-size magnetic beads, the streptavidin protein loading capacity is higher, so more biotin can be combined, the interference caused by the occupation of biotin in a sample can be reduced, but small-particle-size magnetic beads are not easy to clean, the surface non-specific adsorption is higher, and the sensitivity of a reagent is influenced.
Disclosure of Invention
The invention mainly aims to solve the defect that the non-specific adsorption of small-particle-size magnetic beads is difficult to control in the prior art, and provides a method for improving the anti-biotin interference capability and sensitivity of a chemiluminescence kit of a streptavidin-biotin reaction system.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for improving the anti-biotin interference capability and sensitivity of a chemiluminescence kit of a streptavidin-biotin reaction system comprises the following steps:
the method comprises the following steps: adding SA magnetic beads into the magnetic bead diluent to form magnetic bead coating material working solution;
step two: adding an alkaline phosphatase labeled antibody into an enzyme label diluent to form an alkaline phosphatase label working solution;
step three: adding a biotin labeled antibody into the biotin labeled substance diluent to form a biotin labeled substance working solution;
step four: and adding the sample, the magnetic bead coating working solution and the biotin marker working solution into the reaction cup in sequence, incubating for a certain time, performing reaction cleaning, adding the alkaline phosphatase marker working solution, reacting, and cleaning.
Preferably, the specific preparation method of the magnetic bead coating in the first step comprises the following steps:
s1: the supernatant was replaced before use and washed three times with PBS buffer, pH 7.2;
s2: then, the magnetic beads are resuspended in an appropriate amount of basic buffer solution to prepare a magnetic bead coating mother solution.
Preferably, the concentration of the mother solution of the coating material of the magnetic beads in the step S2 is 10 mg/mL.
Preferably, the specific preparation method of the alkaline phosphatase marker in the second step comprises the following steps:
s1: weighing a certain mass of an antibody activator 2 IT;
s2: dissolving activator 2IT to 10mg/mL with an aqueous solution containing 50mM Tris, 0.1M NaCl, and 0.005M EDTA at pH 8.5 ± 0.05;
s3: accurately measuring a certain mass of HBsAg monoclonal antibody, and putting the antibody in a solution with the concentration of 3.5mg/mL in a concentration or constant volume mode, wherein the solvent of the 2IT solution is used as the solvent and is placed at the bottom of a reaction test tube;
s4: adding an activating agent 2IT solution into a test tube containing 3.5mg/mL of HBsAg monoclonal antibody solution for activation and uniform mixing, and reacting for 20 minutes at room temperature;
s5: removing excessive activator 2IT in the solution by molecular sieve chromatography to obtain activated antibody
S6: accurately measuring a certain mass of alkaline phosphatase, and placing the alkaline phosphatase at the bottom of a reaction test tube;
s7: weighing a proper amount of cyclohexane-1-carboxylic acid succinimide ester, and dissolving the cyclohexane-1-carboxylic acid succinimide ester with dimethylformamide to 5 mg/ml;
s8: adding 1/20 volume of cyclohexane-1-carboxylic acid succinimide ester solution into a test tube containing alkaline phosphatase, mixing uniformly, and reacting at room temperature for 15 minutes;
s9: removing excessive cyclohexane-1-carboxylic acid succinimide ester in the solution by molecular sieve chromatography to obtain activated alkaline phosphatase;
s10: mixing the activated antibody in S5 and the activated alkaline phosphatase in S9 at a certain ratio, adding 6. mu.L of 1M MgCl2Uniformly mixing the solution, and reacting for 24 hours at the temperature of 2-8 ℃;
s11: after the reaction is finished, purifying by a molecular sieve chromatography method to obtain a final alkaline phosphatase labeled antibody;
s12: protein concentration was determined by BCA assay and diluted with buffer plus protectant.
Preferably, the mixing ratio in the step S10 is 1: 0.8 by mass ratio.
Preferably, the dilution concentration in step S12 is 0.5 mg/mL.
Preferably, the mixing volume ratio of the HBsAg monoclonal antibody solution to the activator 2IT solution in step S4 is 1/20.
Preferably, the specific preparation method of the biotin marker in step three comprises the following steps:
s1: biotin takes an antibody to be marked, and a phosphate buffer solution with pH of 7.425mM is used for replacing a buffer solution carried by the antibody before marking;
s2: dissolving biotin in anhydrous N, N-dimethylformamide into 0.1-100 mg/mL biotin mother liquor before labeling biotin;
s3: under the condition of keeping out of the sun, taking the antibody and the biotin mother liquor which have been replaced with the buffer solution to label according to the molar concentration of 1: 1-200;
s4: suspending and mixing under the condition of keeping out of the sun, wherein the marking buffer solution is pH7.425mM PB;
s5: after the reaction is finished, an AKTA purification instrument is used for purification to obtain a biotin-labeled antibody;
s6: protein concentration was determined by BCA assay and diluted with buffer plus protectant.
Preferably, the suspension labeling time in step S4 is 1 to 12 hours under light-shielding conditions.
Preferably, the dilution concentration in step S6 is 0.5 mg/mL.
Compared with the prior art, the invention provides a method for improving the anti-biotin interference capability and sensitivity of the chemiluminescence kit of the streptavidin-biotin reaction system, and the method has the following beneficial effects:
1. this improve streptavidin-biotin reaction system's chemiluminescence kit anti biotin interference ability and sensitivity's method, use the problem that little particle size magnetic bead peridium streptavidin reduces because of biotin in the sample and the surperficial streptavidin of magnetic bead combine the occupy-place and lead to the appearance deviation of reagent testing result through the screening, and add biotinylation bovine serum albumin in enzyme marker component simultaneously and seal the free SA magnetic bead that does not combine with the biotin antibody, reduce SA magnetic bead nonspecific binding enzyme-labeled antibody and produce nonspecific signal, improve detectivity.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to data in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Preparing magnetic bead coating working solution, wherein SA magnetic beads can be purchased commodities, and adding the SA magnetic beads into the magnetic bead diluent to form the magnetic bead coating working solution;
before use, supernatant is replaced, washed three times by PBS buffer solution with pH7.2, and then the magnetic beads are resuspended in a proper amount of basic buffer solution to prepare magnetic bead coating mother solution, wherein the concentration of the magnetic bead mother solution is kept at 10 mg/mL;
preparing an alkaline phosphatase marker working solution, namely adding an alkaline phosphatase marker antibody into an enzyme marker diluent to form the alkaline phosphatase marker working solution;
first, the antibody was activated: weighing a certain mass of an antibody activator 2IT, dissolving the antibody activator into 10mg/mL by using an aqueous solution containing 50mM Tris, 0.1M NaCl and 0.005M EDTA and having the pH of 8.5 +/-0.05, accurately weighing a certain mass of HBsAg monoclonal antibody, and putting the antibody activator into the bottom of a reaction test tube in a concentration or constant volume mode to ensure that the antibody concentration in the solution is 3.5mg/mL, wherein the solvent is the solvent of the 2IT solution; adding 2IT solution with the volume of 1/20 proportion into a test tube containing 3.5mg/mL HBsAg monoclonal antibody solution for activation and uniform mixing, reacting at room temperature for 20 minutes, and removing excessive 2IT in the solution by using a molecular sieve chromatography mode after the reaction is finished to obtain an activated antibody;
secondly, activating alkaline phosphatase: accurately measuring a certain mass of alkaline phosphatase, and placing the alkaline phosphatase at the bottom of a reaction test tube; weighing a proper amount of cyclohexane-1-carboxylic acid succinimide ester, and dissolving the cyclohexane-1-carboxylic acid succinimide ester with dimethylformamide to 5 mg/mL; adding 1/20 volume of cyclohexane-1-carboxylic acid succinimide ester solution into a test tube containing alkaline phosphatase, mixing uniformly, reacting at room temperature for 15 minutes, and removing excessive cyclohexane-1-carboxylic acid succinimide ester in the solution by molecular sieve chromatography to obtain activated alkaline phosphatase;
mixing the above activated antibody and activated alkaline phosphatase at a mass ratio of 1: 0.8, adding 6. mu.L of 1M MgCl2Uniformly mixing the solution, reacting for 24 hours at the temperature of 2-8 ℃, after the reaction is finished, purifying by using a molecular sieve chromatography method to obtain the final alkaline phosphatase labeled antibody, determining the protein concentration by using a BCA method, and diluting to 0.5mg/mL by using a buffer solution added with a protective agent;
preparing a biotin marker working solution, namely adding a biotin marker antibody into a biotin marker diluent to form the biotin marker working solution;
the method comprises the following steps that biotin takes an antibody to be marked, a phosphate buffer solution with the pH value of 7.425mM is used for replacing a buffer solution carried by the antibody before marking, and the biotin is dissolved into 0.1-100 mg/mL of biotin mother liquor in anhydrous N, N-dimethylformamide in advance before marking;
under the dark condition, taking the antibody and the biotin mother liquor which are completely replaced with the buffer solution to label according to the molar concentration of 1:1-200, wherein the labeling buffer solution is pH7.425mM PB, suspending and labeling for 1-12 hours under the dark condition, purifying by using an AKTA (alkyl ketene dimer) purifier after the reaction is finished to obtain the biotin-labeled antibody, determining the protein concentration by using a BCA (burst amplification) method, and diluting to 0.5mg/mL by using the buffer solution added with a protective agent;
the present scheme is described in detail below with reference to examples 1-4 in conjunction with the actual composition of the components and selection of the starting materials.
Example 1:
magnetic bead coating material component:
SA magnetic beads: the grain diameter is 1 mu m;
magnetic bead coating dilution:
Tris：50-100mmol/L；
BSA：0.5％-2％；
tween 20: 0.05% -0.2%;
PC300：0.05％；
BND：0.02％；
filtering with 0.2 μm filter membrane at pH7.0, and storing at 2-8 deg.C;
calculating the volume of the needed magnetic bead mother liquor according to the preparation volume of the working solution of the magnetic bead coating object and the concentration of the magnetic bead of 0.5mg/mL, measuring a proper amount of the magnetic bead mother liquor by using a liquid transfer device, putting the magnetic bead mother liquor into a clean centrifugal tube, carrying out magnetic separation, replacing a supernatant, adding the supernatant into a magnetic bead coating object diluent, and uniformly mixing the supernatant and the diluent for later use.
Alkaline phosphatase marker component:
alkaline phosphatase-labeled antibody: alkaline phosphatase marker fraction-1 and alkaline phosphatase marker fraction-2, wherein the concentration of the mother liquor: 0.5 mg/mL;
enzyme label diluent:
MES：50-100mmol/L；
Nacl：2％-5％；
Mgcl2：2mM-5mM；
Zncl2：0.1mM-0.5mM；
BSA：0.5％-2％；
tween 20: 0.05% -0.2%;
Mouse IgG：500μg/mL；
Goat IgG：500μg/mL；
MAK33 POLY：250μg/mL；
PC300：0.05％；
BND：0.02％；
filtering with 0.5 μm filter membrane at pH of 6.5, and storing at 2-8 deg.C;
and (3) determining that the alkaline phosphatase marker component-1 and the alkaline phosphatase marker component-2 are added into an enzyme marker diluent according to the ratio of 1:1 through signal preparation, wherein the dilution is as follows: 1: 500-1: 1500.
Biotin marker component:
biotin-labeled antibody: biotin marker component-1 and biotin marker component-2, wherein the mother liquor concentration: 0.5 mg/mL;
biotin marker dilution:
Tris：50-100mmol/L；
Nacl：2％-5％；
tween 20: 0.05% -0.2%;
TritonX-100：0.1％；
Mouse IgG：500μg/mL；
Bovine IgG：500μg/mL；
MAK33 POLY：250μg/mL；
Mutein AP：150μg/mL；
Polybrene：0.05％；
biotinylation of bovine serum albumin: 0.5 percent;
PC300：0.05％；
BND：0.02％；
filtering with 0.2 μm filter membrane at pH7.4, and storing at 2-8 deg.C;
determining that the biotin marker-1 and the biotin marker component-2 are added into the biotin marker diluent according to the ratio of 10:1 through signal preparation, wherein the dilution of the biotin marker component-2 is as follows: 1: 500-1: 1500;
example 2: on the basis of the embodiment 1, the SA magnetic beads are replaced by 2 μm in particle size, and other components and processes are the same as those in the embodiment 1;
example 3: on the basis of the embodiment 1, 0.2 percent of biotinylated bovine serum albumin is added into the enzyme labeling diluent, and other components and processes are the same as those of the embodiment 1;
example 4: on the basis of the embodiment 1, 1.0 percent of biotinylated bovine serum albumin is added into the enzyme labeling diluent, and other components and processes are the same as those of the embodiment 1;
control group 1: on the basis of the example 1, the particle size of the SA magnetic bead is changed to be 5 μm, and other components and processes are the same as those of the example 1;
control group 2: on the basis of the embodiment 1, 0.5 percent of biotinylated bovine serum albumin is not added into the enzyme marker diluent, and other components and processes are the same as the embodiment 1;
the following test is performed by taking an HBsAg determination kit as an example, wherein the HBsAg kit is a detection kit based on an enzymatic chemiluminescence and streptavidin-biotin amplification reaction system. The kit is divided into 3 components, namely streptavidin-coated magnetic beads, biotin-labeled antibodies and alkaline phosphatase-labeled antibodies, and the reaction mode is two-step cleaning: the first step is that antigen, biotin labeled antibody and SA magnetic bead coating matter in a sample are combined into a compound; the second step is that the antigen-biotin labeled antibody-SA magnetic bead coating compound is combined with the alkaline phosphatase labeled antibody, and the test method is as follows:
firstly, adding 50 mu L of sample, 50 mu L of magnetic bead coating working solution and 50 mu L of biotin labeling working solution into a reaction cup in sequence, incubating for 10 minutes, adding cleaning solution, and cleaning for 5 times;
secondly, continuously adding 50 mu L of alkaline phosphatase marker working solution into the reaction cup, incubating for 10 minutes, adding a cleaning solution, and cleaning for 5 times;
and thirdly, injecting a luminescent substrate into the reaction mixture, catalyzing the substrate to emit light by alkaline phosphatase, and detecting the quantity of generated photons by a PMT (scanning electron microscope) instrument, wherein the quantity of the generated photons is in direct proportion to the concentration of the HBsAg.
Selecting experimental instruments: CL1200i, detection sensitivity and anti-interferon ability using the reagents prepared in control 1-2 and examples 1-4, and accuracy, linearity and repeatability of the retests of examples 1-4 were determined as follows:
(1) and (3) sensitivity detection: measuring blank samples and low-concentration samples with HBsAg concentration of 0.05IU/mL, 0.10IU/mL and 0.15IU/mL, measuring each sample for 10 times, calculating a mean value, a standard deviation and a variation coefficient, wherein CV is less than 10% and the deviation is less than 10% so as to meet the use requirement; in addition, calculating the signal-to-noise ratio RLU1/RLU0 of the luminescence value RLU1 of the concentration point of 0.05IU/mL and the luminescence value RLU0 of the blank sample;
(2) detection of anti-biotin interference ability: preparing biotin interference negative serum and biotin interference positive serum, wherein the biotin interference negative serum can be prepared by adding biotin into the biotin interference negative serum, the content of the biotin is 100ng/mL, the two samples are repeatedly measured for 3 times respectively, and the concentration deviation is calculated; in addition, 50 clinical serum samples were assayed and compared for clinical relevance to roche reagents;
(3) linear range detection: diluting a serum sample approaching to the upper limit of a linear range of 0.05-250 IU/mL to the lower limit of the linear range step by step, setting 7-11 concentration gradients at the lower limit, testing each concentration point for 3 times, calculating a mean value and a theoretical value, repeatedly measuring each concentration for 3 times, and calculating a correlation coefficient of the mean value and the theoretical value; the relative deviation of each point is within 10 percent, so that the use requirement can be met;
(4) and (3) accuracy detection: measuring a serum sample assigned by an enterprise reference product, and calculating the Bias (the measurement mean value-target value)/target value multiplied by 100 percent according to a high value H and a low value L, wherein the Bias is within the range of 85-115 percent, and the requirement is met;
(5) and (3) repeatability detection: measuring a low value L and a high value H of the repetitive sample, repeating the measurement for 10 times, and calculating a mean value M, a standard deviation SD and a coefficient of variation CV;
the detection data are as follows:
(1) sensitivity:
table 1 examples 1-4 sensitivity data
TABLE 2 control 1-2 sensitivity data
TABLE 3 SNR data
From the data, the measured value of the low-concentration sample measured by the control group 2 is low, even the value can not be measured, the concentration of the sample is the same as that of the blank liquid measured value, the CV is high and can not meet the requirement, the signal-to-noise ratio of the low-concentration sample signal and the local signal is small, and the low end of the signal can not be separated; the control group 1 and the examples 1-4 can detect the concentration of 0.05IU/mL, the CV of the low-concentration sample is better, the signal-to-noise ratio of the signal of the low-concentration sample to the local signal can reach more than 4, and the verified functional sensitivity can reach 0.05 IU/mL.
(2) Anti-biotin interference capacity:
TABLE 4 Biotin interference bias data
| Examples | Sample c measurement (IU/Ml) | Sample d measurement (IU/mL) | Deviation of concentration |
| Example 1 | 190.36 | 189.63 | -0.38％ |
| Example 2 | 187.98 | 179.42 | -4.55％ |
| Example 3 | 189.20 | 190.37 | 0.62％ |
| Example 4 | 198.01 | 190.42 | -3.83％ |
| Control group 1 | 187.68 | 116.83 | -37.75％ |
| Control group 2 | 189.01 | 196.48 | 3.95％ |
TABLE 5 data relating to Roche reagent clinical samples
The data show that when the control group 1 uses SA magnetic beads with large particle sizes to measure the biotin adding samples, obvious measurement value deviation occurs, and the correlation is poor when the SA magnetic beads are compared with Roche clinical data, which indicates that the anti-biotin interference capability is poor; the control group 2 and the examples 1 to 4 use the small-particle-size SA magnetic beads to detect the biotin-added samples, have no obvious measurement value deviation, and have better correlation with Roche clinical samples, which shows that the small-particle-size SA magnetic beads can effectively improve the anti-biotin interference capability of the reagent.
(3) Linear range:
TABLE 6 Linear data
From the above data, it can be seen that the linear gradient samples measured in examples 1-4 have a concentration correlation coefficient r greater than 0.990, which satisfies the performance requirements, and example 1 is the most optimal, so the conditions described in example 1 (SA magnetic bead particle size 1 μm, 0.5% biotinylated bovine serum albumin is added to the enzyme marker dilution) are preferred.
(4) Accuracy:
TABLE 7 accuracy data
| Sample(s) | Target value | Example 1 | Example 2 | Example 3 | Example 4 |
| Accuracy sample L | 0.56 | 0.51 | 0.53 | 0.59 | 0.52 |
| Accuracy sample H | 88.64 | 86.34 | 80.21 | 92.19 | 79.9 |
| Deviation of L | / | -1.79％ | -5.36％ | 5.36％ | -7.14％ |
| Deviation from H | / | -2.59％ | -9.51％ | 4.00％ | -9.86％ |
From the above data, the samples of examples 1-4 were determined with accuracy and concentration deviations of less than 10% that meet the performance requirements, and example 1 performed optimally, so the conditions described in example 1 were preferred.
(5) Repeatability:
table 8 repeatability data
From the above data, it can be seen that the coefficient of variation cv of the repetitive samples measured in examples 1-4 is less than 10%, and the performance requirements are met.
In conclusion, the HBsAg kit based on the alkaline phosphatase-streptavidin-biotin system prepared by the invention has strong anti-biotin interference capability and high sensitivity, and other properties can meet the clinical use requirements. The method of the present invention for improving the anti-biotin interference ability and detection sensitivity can be used in other kits using an enzymatic chemiluminescence-streptavidin-biotin reaction system.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A method for improving the anti-biotin interference capability and sensitivity of a chemiluminescence kit of a streptavidin-biotin reaction system is characterized by comprising the following steps:
the method comprises the following steps: adding SA magnetic beads into the magnetic bead diluent to form magnetic bead coating material working solution;
step two: adding an alkaline phosphatase labeled antibody into an enzyme label diluent to form an alkaline phosphatase label working solution;
step three: adding a biotin labeled antibody into the biotin labeled substance diluent to form a biotin labeled substance working solution;
step four: and adding the sample, the magnetic bead coating working solution and the biotin marker working solution into the reaction cup in sequence, incubating for a certain time, performing reaction cleaning, adding the alkaline phosphatase marker working solution, reacting, and cleaning.
2. The method for improving the anti-biotin interference capability and sensitivity of the chemiluminescent kit of the streptavidin-biotin reaction system as claimed in claim 1, wherein the specific preparation method of the magnetic bead coating in the first step comprises:
s1: the supernatant was replaced before use and washed three times with PBS buffer, pH 7.2;
s2: then, the magnetic beads are resuspended in an appropriate amount of basic buffer solution to prepare a magnetic bead coating mother solution.
3. The method for improving the anti-biotin interference capability and sensitivity of the chemiluminescent kit of the streptavidin-biotin reaction system of claim 2 wherein the concentration of the mother solution of the magnetic bead coating substance in step S2 is 10 mg/mL.
4. The method for improving the anti-biotin interference capability and sensitivity of the chemiluminescence kit of the streptavidin-biotin reaction system according to claim 1, wherein the specific preparation method of the alkaline phosphatase marker in step two comprises:
s1: weighing a certain mass of an antibody activator 2 IT;
s2: dissolving activator 2IT to 10mg/mL with an aqueous solution containing 50mM Tris, 0.1M NaCl, and 0.005M EDTA at pH 8.5 ± 0.05;
s3: accurately measuring a certain mass of HBsAg monoclonal antibody, and putting the antibody in a solution with the concentration of 3.5mg/mL in a concentration or constant volume mode, wherein the solvent of the 2IT solution is used as the solvent and is placed at the bottom of a reaction test tube;
s4: adding an activator 2IT solution into a test tube containing 3.5mg/mLHBsAg monoclonal antibody solution for activation and uniform mixing, and reacting for 20 minutes at room temperature;
s5: removing excessive activator 2IT in the solution by molecular sieve chromatography to obtain activated antibody
S6: accurately measuring a certain mass of alkaline phosphatase, and placing the alkaline phosphatase at the bottom of a reaction test tube;
s7: weighing a proper amount of cyclohexane-1-carboxylic acid succinimide ester, and dissolving the cyclohexane-1-carboxylic acid succinimide ester with dimethylformamide to 5 mg/mL;
s8: adding 1/20 volume of cyclohexane-1-carboxylic acid succinimide ester solution into a test tube containing alkaline phosphatase, mixing uniformly, and reacting at room temperature for 15 minutes;
s9: removing excessive cyclohexane-1-carboxylic acid succinimide ester in the solution by molecular sieve chromatography to obtain activated alkaline phosphatase;
s10: mixing the activated antibody in S5 and the activated alkaline phosphatase in S9 at a certain ratio, adding 6. mu.L of 1M MgCl2Uniformly mixing the solution, and reacting for 24 hours at the temperature of 2-8 ℃;
s11: after the reaction is finished, purifying by a molecular sieve chromatography method to obtain a final alkaline phosphatase labeled antibody;
s12: protein concentration was determined by BCA assay and diluted with buffer plus protectant.
5. The method for improving the anti-biotin interference capability and sensitivity of the chemiluminescent kit of the streptavidin-biotin reaction system of claim 4 wherein the mixing ratio in step S10 is 1: 0.8 by mass.
6. The method for improving the anti-biotin interference capability and sensitivity of the chemiluminescent kit of the streptavidin-biotin reaction system of claim 5 wherein the dilution concentration in step S12 is 0.5 mg/mL.
7. The method for improving the anti-biotin interference capability and sensitivity of the chemiluminescence kit of the streptavidin-biotin reaction system according to claim 6, wherein the mixing volume ratio of the HBsAg monoclonal antibody solution to the activator 2IT solution in step S4 is 1/20.
8. The method for improving the anti-biotin interference capability and sensitivity of the chemiluminescence kit of the streptavidin-biotin reaction system according to claim 1, wherein the specific preparation method of the biotin marker in step three comprises:
s1: biotin takes an antibody to be marked, and a phosphate buffer solution with pH of 7.425mM is used for replacing a buffer solution carried by the antibody before marking;
s2: dissolving biotin in anhydrous N, N-dimethylformamide into 0.1-100 mg/mL biotin mother liquor before labeling biotin;
s3: under the condition of keeping out of the sun, taking the antibody and the biotin mother liquor which have been replaced with the buffer solution to label according to the molar concentration of 1: 1-200;
s4: the suspension mixing of the marking buffer solution is pH7.425mM PB under the condition of keeping out of the light;
s5: after the reaction is finished, an AKTA purification instrument is used for purification to obtain a biotin-labeled antibody;
s6: protein concentration was determined by BCA assay and diluted with buffer plus protectant.
9. The method for improving the anti-biotin interference ability and sensitivity of the chemiluminescent kit of the streptavidin-biotin reaction system of claim 8 wherein the labeling time of step S4 is 1-12 hours under dark conditions.
10. The method for improving the anti-biotin interference ability and sensitivity of the chemiluminescent kit of the streptavidin-biotin reaction system of claim 9 wherein the dilution concentration in step S6 is 0.5 mg/mL.
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