CN117092330A - Casein signal amplification luminescence detection method - Google Patents
Casein signal amplification luminescence detection method Download PDFInfo
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- CN117092330A CN117092330A CN202311006013.5A CN202311006013A CN117092330A CN 117092330 A CN117092330 A CN 117092330A CN 202311006013 A CN202311006013 A CN 202311006013A CN 117092330 A CN117092330 A CN 117092330A
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- 238000000034 method Methods 0.000 title claims abstract description 49
- 238000000504 luminescence detection Methods 0.000 title claims abstract description 43
- 230000003321 amplification Effects 0.000 title claims abstract description 42
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 42
- 239000005018 casein Substances 0.000 title description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical group NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 title description 2
- 235000021240 caseins Nutrition 0.000 title description 2
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical group [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 claims abstract description 80
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 claims abstract description 79
- 229960003732 tyramine Drugs 0.000 claims abstract description 79
- 239000011324 bead Substances 0.000 claims abstract description 46
- 238000001514 detection method Methods 0.000 claims abstract description 35
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 238000007789 sealing Methods 0.000 claims abstract description 19
- 239000000203 mixture Substances 0.000 claims abstract description 14
- 239000003550 marker Substances 0.000 claims description 42
- 239000000243 solution Substances 0.000 claims description 37
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 238000005406 washing Methods 0.000 claims 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 description 24
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 19
- 230000005284 excitation Effects 0.000 claims description 18
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 108010090804 Streptavidin Proteins 0.000 claims description 12
- 229960002685 biotin Drugs 0.000 claims description 12
- 235000020958 biotin Nutrition 0.000 claims description 12
- 239000011616 biotin Substances 0.000 claims description 12
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000004020 luminiscence type Methods 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- 239000011248 coating agent Substances 0.000 claims description 8
- 238000000576 coating method Methods 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 239000006180 TBST buffer Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 150000002148 esters Chemical class 0.000 claims description 3
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000002038 chemiluminescence detection Methods 0.000 abstract description 5
- 239000006228 supernatant Substances 0.000 description 10
- 239000011259 mixed solution Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- 239000003761 preservation solution Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 3
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000006287 biotinylation Effects 0.000 description 2
- 238000007413 biotinylation Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- -1 acridine esters Chemical class 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 230000010460 detection of virus Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/5375—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning
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- 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
- G01N21/763—Bioluminescence
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- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
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Abstract
The invention discloses a tyramine signal amplification luminescence detection method, which has the advantages of remarkably reducing the background value during tyramine signal amplification luminescence detection, remarkably improving the signal-to-noise ratio, greatly improving the chemiluminescence detection sensitivity, saving the antibody consumption and improving the detection efficiency by optimizing the composition of magnetic bead sealing liquid, adjusting at least one of the use amount of capture antibody, detection antibody, HRP, tyramine solution concentration and the like.
Description
Technical Field
The invention relates to the detection field, in particular to a low background value tyramine signal amplification luminescence detection method.
Background
Tyramine signal amplification systems (Tyramine signal amplification, TSA) are commonly used for detection of bacteria, viruses and cells, the principle of which is: peroxidase reaction with tyramine Tyramide (tyramine salt in HRP catalyzed H 2 O 2 Covalent bond binding sites are formed), a large amount of enzymatic products are generated, the products can be combined with surrounding protein residues (comprising tryptophan, histidine and tyrosine residues), the phenol groups of Tyramine (TYR) and tyrosine residues of protein can be converted into free radical intermediates, the free radical intermediates are mutually and covalently combined, the Tyramine is mutually crosslinked or connected with protein, TYR is coupled with signal substances such as fluorescein, and signal amplification can be caused after triggering phenol polymerization reaction.
Although the signal amplification effect can be achieved by applying the TSA technology principle, the problem that the background value is high and the detection sensitivity is affected because tyramine can be combined with surrounding protein residues (comprising tryptophan, histidine and tyrosine residues) exists.
Therefore, in order to solve the above problems, there is a need for an optimized luminescence detection method based on tyramine signal amplification technology.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a tyramine signal amplification luminescence detection method with a low background value.
The technical scheme adopted by the invention is as follows:
a tyramine signal amplification luminescence detection method comprises the following steps:
s1) coating magnetic beads by using a capture antibody, then sealing the magnetic beads by using a magnetic bead sealing liquid, and separating to obtain capture magnetic beads;
s2) using sample diluent to resuspend and capture magnetic beads, adding a sample to be detected, a detection antibody marked by a first marker, horseradish peroxidase marked by a second marker, and washing after incubation is completed, wherein the first marker and the second marker are mutually affinitive;
s3) adding a tyramine solution marked by a third marker, and washing after incubation is completed;
s4) adding a luminescent substance marked by a fourth marker, washing after incubation, adding a pre-excitation liquid and an excitation liquid, and reading a luminescent value, wherein the third marker and the fourth marker are mutually affinitive;
s5) determining the quantity of the to-be-detected object in the sample according to the luminous value.
In some examples of tyramine signal amplification luminescence detection methods, the composition of the magnetic bead blocking solution is: 10-200 mM Tris-HCl, 100-500 mM sodium chloride, 0.1-5% CE210, 0.1-5% CE510, and pH 6.5-8.5.
In some examples of the tyramine signal amplification luminescence detection method, 2 to 3wt.% CE210 and 2 to 3wt.% CE510 are contained in the magnetic bead sealing liquid.
In some examples of tyramine signal amplification luminescence detection methods, capture antibodies are used at a concentration of 5mg/ml to 15mg/ml when the capture antibodies are coated with magnetic beads.
In some examples of tyramine signal amplification luminescence detection methods, the magnetic beads are blocked for a period of time ranging from 1.5 to 3 h.
In some examples of the tyramine signal amplification luminescence detection method, the total concentration of the detection antibody marked by the first marker and the horseradish peroxidase marked by the second marker in the reaction system is 2-6 mug/mL.
In some examples of the tyramine signal amplification luminescence detection method, the mixing ratio by mass of the detection antibody labeled with the first label to the horseradish peroxidase labeled with the second label is (3.5 to 4.5): 1.
in some examples of the tyramine signal amplification luminescence detection method, the total concentration of the detection antibody marked by the first marker and the horseradish peroxidase marked by the second marker in a reaction system is 2-6 mug/mL, and the mass mixing ratio of the detection antibody marked by the first marker and the horseradish peroxidase marked by the second marker is (3.5-4.5): 1.
in some examples of the tyramine signal amplification luminescence detection method, the stock solution of the first label-labeled detection antibody and the second label-labeled horseradish peroxidase is TBST buffer+1 wt.% bsa+1wt.% trehalose.
In some examples of the method for detecting amplification luminescence of a tyramine signal, the concentration of tyramine in the tyramine solution labeled with the third label is 0.75. Mu.g/ml to 1.25. Mu.g/ml.
In some examples of the tyramine signal amplification luminescence detection method, the composition of the tyramine solution marked by the third marker is Tris-HCl buffer solution, 0.08-0.12 mg/mL tyramine, a proper amount of hydrogen peroxide and pH 7.2-7.6.
In some examples of the tyramine signal amplification luminescence detection method, the first label is selected from one of biotin, streptavidin, and FITC.
In some examples of the tyramine signal amplification luminescence detection method, the third label is selected from one of biotin, streptavidin, and FITC.
In some examples of the tyramine signal amplification luminescence detection method, the first label is selected from one of biotin, streptavidin, and FITC, and the third label is selected from one of biotin, streptavidin, and FITC.
In some examples of tyramine signal amplification luminescence detection methods, the luminophore is selected from one of acridinium ester, oxalic ester, and ferric-luminol.
The beneficial effects of the invention are as follows:
according to the method provided by the embodiment of the invention, the working solution (at least one of the magnetic bead sealing solution, the coating solution, the detection antibody, the HRP mixed solution and the tyramine solution) is improved, so that the sealing effect is effectively submitted, the background value of the detection is reduced, the detection sensitivity is greatly improved, and the detection sensitivity is improved by a plurality of times. Compared with the traditional chemiluminescence detection, the method can obviously improve the chemiluminescence detection sensitivity, and has the advantages of saving the antibody consumption and improving the detection efficiency.
Drawings
Fig. 1 is the effect of different coating concentrations on signal to noise ratio.
FIG. 2 is a graph showing the effect of different detection antibodies and HRP concentrations on signal to noise ratio.
Figure 3 is the effect of different biotinylated tyramine solution concentrations on signal to noise ratio.
Detailed Description
A tyramine signal amplification luminescence detection method comprises the following steps:
s1) coating magnetic beads by using a capture antibody, then sealing the magnetic beads by using a magnetic bead sealing liquid, and separating to obtain capture magnetic beads;
s2) using sample diluent to resuspend and capture magnetic beads, adding a sample to be detected, a detection antibody marked by a first marker, horseradish peroxidase marked by a second marker, incubating for 9-11 min, and washing, wherein the first marker and the second marker are mutually affinitive;
s3) adding tyramine solution marked by a third marker, incubating for 9-11 min, washing, further adding luminescent material marked by a fourth marker, incubating for 9-11 min, washing, adding pre-excitation liquid and excitation liquid, and reading a luminescent value, wherein the third marker and the fourth marker are mutually affinitive;
s4) determining the quantity of the to-be-detected object in the sample according to the luminous value.
In some examples of tyramine signal amplification luminescence detection methods, the composition of the magnetic bead blocking solution is: 10-200 mM Tris-HCl, 100-500 mM sodium chloride, 0.1-5% CE210, 0.1-5% CE510, and pH 6.5-8.5.
In some examples of the tyramine signal amplification luminescence detection method, 2 to 3wt.% CE210 and 2 to 3wt.% CE510 are contained in the magnetic bead sealing liquid. Experimental data show that the background value of tyramine signal amplified luminescence detection can be obviously reduced under the dosage.
In some examples of tyramine signal amplification luminescence detection methods, capture antibodies are used at a concentration of 5mg/ml to 15mg/ml when the capture antibodies are coated with magnetic beads.
In some examples of tyramine signal amplification luminescence detection methods, the magnetic beads are blocked for a period of time ranging from 1.5 to 3 h.
In some examples of the tyramine signal amplification luminescence detection method, the total concentration of the detection antibody marked by the first marker and the horseradish peroxidase marked by the second marker in the reaction system is 2-6 mug/mL.
In some examples of the tyramine signal amplification luminescence detection method, the mixing ratio by mass of the detection antibody labeled with the first label to the horseradish peroxidase labeled with the second label is (3.5 to 4.5): 1.
in some examples of the tyramine signal amplification luminescence detection method, the total concentration of the detection antibody marked by the first marker and the horseradish peroxidase marked by the second marker in a reaction system is 2-6 mug/mL, and the mass mixing ratio of the detection antibody marked by the first marker and the horseradish peroxidase marked by the second marker is (3.5-4.5): 1.
the preservation solution has no special requirement, and can meet the requirement of chemiluminescence detection. In some examples of the tyramine signal amplification luminescence detection method, the stock solution of the first label-labeled detection antibody and the second label-labeled horseradish peroxidase is TBST buffer+1 wt.% bsa+1wt.% trehalose. The preservation solution has good adaptability and can meet the requirements of most detection.
In some examples of the method for detecting amplification luminescence of a tyramine signal, the concentration of tyramine in the tyramine solution labeled with the third label is 0.75. Mu.g/ml to 1.25. Mu.g/ml.
In some examples of the tyramine signal amplification luminescence detection method, the composition of the tyramine solution marked by the third marker is Tris-HCl buffer solution, 0.08-0.12 mg/mL tyramine, a proper amount of hydrogen peroxide and pH 7.2-7.6.
In some examples of the tyramine signal amplification luminescence detection method, the first label is selected from one of biotin, streptavidin, and FITC. The corresponding second label may be streptavidin, biotin and anti-FITC antibody.
In some examples of the tyramine signal amplification luminescence detection method, the third label is selected from one of biotin, streptavidin, and FITC.
In some examples of the tyramine signal amplification luminescence detection method, the first label is selected from one of biotin, streptavidin, and FITC, and the third label is selected from one of biotin, streptavidin, and FITC.
These markers are common markers, and the marking method is mature, which is more beneficial to reducing the cost.
The luminescent material is not particularly required, but can be used for various chemiluminescent detection. In some examples of tyramine signal amplification luminescence detection methods, the luminophore is selected from one of acridinium ester, oxalic ester, and ferric-luminol.
CE210, CE510 were purchased from JSR corporation. The pre-excitation liquid and the excitation liquid are commercial pre-excitation liquid and excitation liquid for chemiluminescence detection.
Abbreviations used herein have the meaning common in the art, and some of the abbreviations have the following specific meaning:
AE: acridine esters
Bio: biotin
BSA: bovine serum albumin
EDC: 1-ethyl- (3-dimethylaminopropyl) carbodiimide
FITC: fluorescein isothiocyanate
LAM: lipoarabinomannan
MES: morpholinoethanesulfonic acid buffer
NHS: n-hydroxysuccinimide
SA: streptavidin.
The technical scheme of the invention is further described below by combining examples.
In the following examples, LAM detection is taken as an example for corresponding explanation.
Antibody 2109 is a commercial LAM capture antibody produced by rad biotechnology limited, guangzhou. Antibody 2107 is a commercial LAM detection antibody produced by rad biotechnology limited, guangzhou.
In the following examples, the preparation method of the antibody-coated magnetic bead solution using LAM capture antibody and LAM detection antibody is as follows:
adding LAM capture antibody into the coating liquid, adding magnetic beads, incubating, washing, and re-suspending with buffer solution.
1) 2mg of carboxyl magnetic microspheres are taken and diluted to 1ml by MES, and vortexed for 30s;
2) Washing: placing on a magnetic rack, removing the supernatant, then adding 1ml MES, and swirling for 30s; repeating for 3 times;
3) 10ul EDC working solution (0.1 mg EDC) was added and vortexed for 30s;
4) 2.5 μl of NHS working solution (0.025 mg NHS) was added and vortexed for 30s;
5) Placing the mixture on a rotary table mixing instrument, and uniformly mixing the mixture at room temperature and in a dark place (30 rpm) for 30 minutes;
6) Washing: placing on a magnetic rack, removing the supernatant, then adding 1ml MES, and swirling for 30s; repeating for 3 times; removing the supernatant after washing;
7) 10ug of coated antibody was added and vortexed for 30s;
8) Placing the mixture on a rotary table mixing instrument, and uniformly mixing the mixture at room temperature and in a dark place (30 rpm) for 2 hours;
9) Washing: placing on a magnetic rack, removing the supernatant, then adding 1ml TBST, and swirling for 30s; repeating for 3 times; removing the supernatant after washing;
10 Closing: 1ml of the blocking solution was added and vortexed for 30s;
11 Placing on a rotary table mixer, and uniformly mixing at room temperature (30 rpm) overnight;
12 Placing on a magnetic rack, removing the supernatant, adding 0.4ml of preservation solution, and swirling for 30s, wherein the microsphere concentration is 5mg/ml;
13 Marking and storing at 2-8 ℃.
Embodiment one: influence of different magnetic bead sealing liquids on background values
The operation steps are as follows:
s1) respectively taking LAM standard solution (0 pg/ml, 1000 pg/ml) and adding 1.5. 1.5mL into 50 mu L of antibody coated magnetic bead solution, wherein the antibody coated magnetic beads are sealed by using sealing solutions with different compositions, and each group is repeated for two times and incubated for two hours;
s2) separating magnetic beads, discarding supernatant, and adding 200 mu L of sample diluent for resuspension;
s3) taking 100 mu L of the sample, adding 50 mu L of a mixed solution of biotinylation antibody bio-2107 and SA-HRP, incubating for 10 minutes, and washing for 4 times;
s4) adding 50 mu L of biotinylated tyramine solution, incubating for 10min and washing for 4 times, adding 50 mu L of SA-AE, incubating for 10min, washing for 4 times, and adding pre-excitation liquid and excitation liquid;
s5) reading the luminescence value.
Wherein the composition of the biotinylated tyramine solution is: 50mM Tris-HCl, 0.1. 0.1 mg/mL B-T,0.01% H 2 O 2 The pH7.4, the content of the magnetic beads in the antibody coating magnetic bead solution is 0.5 mg/mL.
The mass compositions of the magnetic bead sealing solutions of different groups are respectively as follows:
experiment group 1:50mM Tris-HCl,150mM sodium chloride, 2.5% CE210,2.5% CE510, pH7.4
Experiment group 2:10mM Tris-HCl,500mM sodium chloride, 2.0% CE210,3.0% CE510, pH7.4
Experiment group 3:200mM Tris-HCl,100mM sodium chloride, 3.0% CE210,2.0% CE510, pH7.4
Experiment group 4:200mM Tris-HCl,100mM sodium chloride, 4.0% CE210,1.0% CE510, pH7.4
Experimental group 5:200mM Tris-HCl,100mM sodium chloride, 1.0% CE210,4.0% CE510, pH7.4
Experiment group 6:200mM Tris-HCl,100mM sodium chloride, 1.5% CE210,3.5% CE510, pH7.4
Control group 1:50mM Tris-HCl,150mM sodium chloride, 1% BSA, pH7.4
Control group 2:50 mM Tris-HCl,150mM sodium chloride, 1% BSA,100mM glycine, pH7.4
Control group 3:50mM Tris-HCl,150mM sodium chloride, 5% CE210, pH7.4
Control group 4:50mM Tris-HCl,150mM sodium chloride, 5% CE510, pH7.4
Control group 5:50mM Tris-HCl,150mM sodium chloride, 5% CE210,1% CE510, pH7.4
Control group 6:50mM Tris-HCl,150mM sodium chloride, 1% CE210,5% CE510, pH7.4.
The test results are shown in Table 1.
TABLE 1 influence of different bead sealing solutions on luminescence values
Signal to noise ratio = average luminescence at 1000 pg/ml/average luminescence at 0pg/ml
As can be seen from Table 1, the background (0 pg/ml) luminescence values of the experimental groups were significantly lower and the signal to noise ratio was higher. The higher the signal-to-noise ratio is, the higher the sensitivity is, and the magnetic bead sealing liquid of the experimental group can effectively reduce the background value and greatly improve the detection sensitivity.
Embodiment two: influence of different coating concentrations on background values
S1) adding 1.5mL of LAM standard solution (0 pg/mL, 1000 pg/mL) into 50 mu L of magnetic bead solution coated with capture antibodies with different concentrations, and incubating for two hours;
s2) separating magnetic beads, discarding supernatant, and adding 200 mu L of sample diluent for resuspension;
s3) then taking 100 mu L of the sample, adding 50 mu L of a mixed solution of biotinylated antibody bio-2107 and SA-HRP, incubating for 10 minutes, washing for 4 times,
s4) adding 50 mu L of biotinylated tyramine solution, incubating for 10min and washing for 4 times, adding 50 mu L of SA-AE, incubating for 10min, washing for 4 times, and adding pre-excitation liquid and excitation liquid;
s5) reading the luminescence value.
The experimental results are shown in FIG. 1. As can be seen from FIG. 1, the signal to noise ratio is increased and then decreased when the coated antibody concentration is measured at different intervals ranging from 0mg/ml to 20mg/ml, wherein the peak value occurs at 10mg/ml, and the signal to noise ratio is higher at 5mg/ml to 15mg/ml, so that the sensitivity is higher and the optimal 10mg/ml is seen when the coated antibody concentration is ranging from 5mg/ml to 15mg/ml.
Example three Effect of mixture concentration of different biotinylated antibodies bio-2107 and SA-HRP
Preparation of the mixed solution: bio-2107 and SA-HRP are mixed according to the mass ratio of 4:1 to be mixed into a mixed solution with the concentration of 10 mug/ml, wherein the preservation solution is TBST+1% BSA+1% trehalose;
1) Respectively adding 1.5. 1.5mL of LAM standard solutions (0 pg/ml and 1000 pg/ml) into 50 mu L of antibody-coated magnetic bead solution, and incubating for two hours;
2) Separating magnetic beads, discarding supernatant, adding 200 μl of sample diluent for resuspension, taking 100 μl of the sample, adding 50 μl of mixed solution of biotinylated antibodies bio-2107 and SA-HRP with different concentrations, incubating for 10min, and washing for 4 times;
3) Then 50. Mu.L of biotinylated tyramine solution was added, incubated for 10min and washed 4 times;
4) Adding 50 mu L of SA-AE, incubating for 10min, washing for 4 times, and adding pre-excitation solution and excitation solution;
5) The luminescence value is read.
The experimental results are shown in FIG. 2. As can be seen from FIG. 2, when the signal to noise ratio is measured at different mixed solution concentrations ranging from 0 mug/ml to 10 mug/ml, the peak value appears at 5 mug/ml, and the signal to noise ratio is higher at 2.5 mug/ml to 7.5 mug/ml, the sensitivity is higher and the optimum is 5 mug/ml when the mixed solution concentration is 2.5 mug/ml to 7.5 mug/ml.
Example four Effect of different biotinylated tyramine solution concentrations
1) Respectively adding 1.5. 1.5mL of LAM standard solutions (0 pg/ml and 1000 pg/ml) into 50 mu L of antibody-coated magnetic bead solution, and incubating for two hours;
2) Separating magnetic beads, discarding supernatant, adding 200 μl of sample diluent for resuspension, taking 100 μl of the sample, adding 50 μl of mixed solution of biotinylation antibody bio-2107 and SA-HRP, incubating for 10min, and washing for 4 times;
3) Then 50 mu L of biotinylated tyramine solution with different concentrations is added, incubated for 10min and washed 4 times;
4) Adding 50 mu L of SA-AE, incubating for 10min, washing for 4 times, and adding pre-excitation solution and excitation solution;
5) The luminescence value is read.
The experimental results are shown in FIG. 3. As can be seen from FIG. 3, when the signal to noise ratio is measured at different levels of biotinylated tyramine solutions ranging from 0. Mu.g/ml to 2. Mu.g/ml, the signal to noise ratio is increased and then decreased, and the signal to noise ratio is higher at a level ranging from 0.75. Mu.g/ml to 1.25. Mu.g/ml, it can be seen that the sensitivity is higher and the optimum 1. Mu.g/ml is obtained at a level ranging from 0.75. Mu.g/ml to 1.25. Mu.g/ml.
The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.
Claims (10)
1. A tyramine signal amplification luminescence detection method comprises the following steps:
coating magnetic beads by using a capture antibody, then sealing the magnetic beads by using a magnetic bead sealing liquid, and separating to obtain capture magnetic beads;
re-suspending and capturing magnetic beads by using a sample diluent, adding a sample to be tested, a detection antibody marked by a first marker and horseradish peroxidase marked by a second marker, and washing after incubation is completed, wherein the first marker and the second marker are mutually affinitive;
adding a tyramine solution marked by a third marker, washing after incubation is completed,
adding a luminescent substance marked by a fourth marker, washing after incubation, adding a pre-excitation liquid and an excitation liquid, and reading a luminescent value, wherein the third marker and the fourth marker are mutually affinitive;
and determining the amount of the to-be-detected object in the sample according to the luminescence value.
2. The method for amplified luminescence detection of tyramine signal according to claim 1, wherein the magnetic bead sealing liquid comprises the following components: 10-200 mM Tris-HCl, 100-500 mM sodium chloride, 0.1-5% CE210, 0.1-5% CE510, pH 6.5-8.5; and/or
When the magnetic beads are sealed, the sealing time is 1.5-3 h.
3. The method for amplified luminescence detection of tyramine signal according to claim 2, wherein the bead sealing solution contains 2 to 3wt.% CE210 and 2 to 3wt.% CE510.
4. The method for amplified luminescence detection of tyramine signal according to claim 1, wherein the capture antibody is used at a concentration of 5mg/ml to 15mg/ml when the capture antibody is coated on the magnetic beads.
5. The method for amplified luminescence detection of tyramine signal according to claim 1, wherein the total concentration of the detection antibody labeled with the first marker and the horseradish peroxidase labeled with the second marker in the reaction system is 2-6 μg/mL.
6. The tyramine signal amplified luminescence detection method according to claim 1 or 5, wherein the mass mixing ratio of the detection antibody labeled with the first label to the horseradish peroxidase labeled with the second label is (3.5-4.5): 1, a step of; and/or
The stock solution of the first label-labeled detection antibody and the second label-labeled horseradish peroxidase is TBST buffer+1 wt.% BSA+1wt.% trehalose.
7. The method for amplified luminescent detection of tyramine signal according to claim 1, wherein the concentration of tyramine in the tyramine solution labeled with the third label is 0.75 μg/ml to 1.25 μg/ml.
8. The method for amplified luminescence detection of tyramine signal according to claim 1, wherein the composition of tyramine solution marked by the third marker is Tris-HCl buffer solution, 0.08-0.12 mg/mL tyramine, a proper amount of hydrogen peroxide, and pH is 7.2-7.6.
9. The method for amplified luminescent detection of tyramine signals according to claim 1, wherein the first label is one selected from the group consisting of biotin, streptavidin and FITC; and/or
The third marker is selected from one of biotin, streptavidin and FITC.
10. The method for amplified luminescent detection of tyramine signals according to claim 1, wherein the luminescent substance is one selected from the group consisting of acridinium ester, oxalic ester and ferric-luminol.
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| CN117405874B (en) * | 2023-12-15 | 2024-04-05 | 迪亚莱博（张家港）生物科技有限公司 | Preparation method of PIC antibody magnetic bead reagent and kit |
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