CN117031046A - TAT enzyme-labeled antibody stock solution - Google Patents

TAT enzyme-labeled antibody stock solution Download PDF

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Publication number
CN117031046A
CN117031046A CN202310756384.9A CN202310756384A CN117031046A CN 117031046 A CN117031046 A CN 117031046A CN 202310756384 A CN202310756384 A CN 202310756384A CN 117031046 A CN117031046 A CN 117031046A
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enzyme
labeled antibody
storage buffer
antibody
kit
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谢永华
王肖楠
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Shanghai Sunbio Technology Co ltd
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Shanghai Sunbio Technology Co ltd
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    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • 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
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8121—Serpins
    • G01N2333/8128—Antithrombin III
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

Abstract

The invention relates to the technical field of biology, in particular to TAT enzyme-labeled antibody stock solution. The invention provides application of proteins, saccharides and aromatic amino acids in preparing a thrombin-antithrombin III complex (TAT) chemiluminescent assay kit. The kit reduces the non-specific binding of the antibody, reduces the interference of other substances in the sample on the detection result, improves the calibration curve of chemiluminescent detection, and improves the repeatability of antigen detection.

Description

TAT enzyme-labeled antibody stock solution
Technical Field
The invention relates to the field of biotechnology, in particular to a composition and application thereof.
Background
Thrombin (Thrombin) is a multifunctional serine proteolytic enzyme comprising A, B two polypeptide chains linked by an interchain disulfide linkage. Thrombin acts directly on the last step of the blood coagulation process to promote the conversion of soluble fibrinogen in the blood plasma into insoluble fibrin, thereby achieving the purpose of quick-acting hemostasis. Prothrombin is activated and rapidly covalently binds to antithrombin III to form thrombin-antithrombin III complex (TAT). TAT is used as a molecular marker for activating a coagulation system, which suggests that thrombin activation and thrombosis are closely related to diseases such as Disseminated Intravascular Coagulation (DIC), deep Vein Thrombosis (DVT), pulmonary Embolism (PE), atrial fibrillation and the like, so that the determination of the thrombin-antithrombin III complex content has important significance for diagnosis and treatment of some diseases. Because thrombin half-life is very short, only a few seconds, it is difficult to directly detect, and thrombin-antithrombin III complex (TAT) half-life is long, which can reach tens of minutes, the existing method is used for detecting the content of thrombin-antithrombin III complex (TAT). Currently available methods for detecting TAT are mainly enzyme-linked immunosorbent assay (ELISA) and tubular chemiluminescence. The ELISA method has the advantages of complicated operation, long time consumption, unstable test result, poor repeatability and inconvenience in measurement and emergency treatment in hospitals; the tubular chemiluminescence method also has the problems of poor stability of the enzyme-labeled antibody, low result reproducibility and the like. The magnetic particle chemiluminescence method is combined with a magnetic separation technology, an immunoassay technology and a chemiluminescence technology to finish quantitative determination of specific antigens, and the existing TAT magnetic particle chemiluminescence determination kit adopts streptavidin-biotin or radioactive element for marking, so that the detection result is easily interfered by excessive markers and has radioactive harm.
Disclosure of Invention
In view of the above, the composition and the application thereof, the kit provided by the invention combine with an immunoassay technology and a chemiluminescent technology, and the alkaline phosphatase hydrolyzes a chemiluminescent substrate for detection, so that the composition in the enzyme-labeled antibody can stabilize the three-dimensional structure of the complex, reduce the non-specific binding of the antibody and improve the repeatability of chemiluminescent assay.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides the use of a saccharide in any of the following:
(I) Improving the reproducibility of the chemiluminescent assay kit;
(II) a storage buffer solution for preparing the enzyme-labeled antibody;
the saccharide comprises one or more of trehalose, glucose, mannose, hyaluronic acid or galactose;
the chemiluminescent assay kit comprises a chemiluminescent assay kit for thrombin-antithrombin III complex.
The invention also provides the application of the aromatic amino acid in any of the following:
(I) Improving the calibration curve of the chemiluminescent assay kit;
(II) a storage buffer solution for preparing the enzyme-labeled antibody;
the aromatic amino acid comprises one or more of tryptophan, tyrosine or phenylalanine;
the chemiluminescent assay kit comprises a chemiluminescent assay kit for thrombin-antithrombin III complex.
The invention also provides compositions comprising proteins, carbohydrates and aromatic amino acids;
the protein comprises one or more of bovine serum albumin, human serum albumin or rabbit serum albumin; the saccharide comprises one or more of trehalose, glucose, mannose, hyaluronic acid or galactose;
the aromatic amino acids include one or more of tryptophan, tyrosine, or phenylalanine.
In some embodiments of the invention, in the above composition:
the protein is bovine serum albumin;
the saccharide is trehalose;
the aromatic amino acid is tyrosine.
The invention also provides the application of the composition in any of the following:
(I) Improving the calibration curve of the chemiluminescent assay kit;
(II) improving the reproducibility of the chemiluminescent assay kit;
(III) preparing an enzyme-labeled antibody storage buffer;
the chemiluminescent assay comprises a chemiluminescent assay for thrombin-antithrombin III complex.
The invention also provides an enzyme-labeled antibody storage buffer, which comprises the composition and:
(A) Buffer solution; and/or
(B) A preservative; and/or
(C) Acceptable auxiliary materials or auxiliary agents.
In some embodiments of the invention, the concentration of the saccharide in the above-described enzyme-labeled antibody storage buffer is 10mg/mL.
In some embodiments of the invention, the saccharide in the enzyme-labeled antibody storage buffer is trehalose; the aromatic amino acid is tyrosine.
In some embodiments of the invention, the enzyme-labeled antibody storage buffer comprises 50mM Tris,5mg/mL BSA, 10mg/mL trehalose, 2.5mg/mL tyrosine, and 1mg/mL NaN 3 。
In some embodiments of the invention, the pH of the above-described enzyme-labeled antibody storage buffer is 8.0.
The invention also provides a kit comprising acceptable auxiliary materials or auxiliary agents, and:
(a) The above composition; or (b)
(b) And the enzyme-labeled antibody storage buffer.
In some embodiments of the invention, the kit further comprises an enzyme-labeled antibody;
the preparation method of the enzyme-labeled antibody comprises the following steps: mixing the activated enzyme with the thiolated antibody according to a mass ratio of 2:1 to obtain the enzyme-labeled antibody;
the enzyme includes alkaline phosphatase;
the antibodies include antithrombin III antibodies.
In some embodiments of the invention, the kit further comprises immunomagnetic beads;
the preparation method of the immunomagnetic beads comprises the following steps: activating carboxyl magnetic beads by using an activating agent, and adding an antibody for coupling after activation to obtain the immune magnetic beads;
the antibodies include thrombin antibodies;
the feeding ratio of the carboxyl magnetic beads to the antibody is 50:1 (w/w);
the feeding ratio of the carboxyl magnetic beads to the activator is 20:1 (w/w);
the activator is EDC and Sulfo-NHS.
The composition and application thereof and the kit have the following effects:
according to the invention, a certain amount of thrombin antibodies are combined to the surface of the activated magnetic beads through a chemical coupling method, and the used magnetic beads have superparamagnetism, so that quick magnetic response can be generated without hysteresis effect; the enzyme-labeled antibody adopts a heterobifunctional reagent crosslinking method, and alkaline phosphatase after being activated by Sulfo-SMCC is coupled with an antithrombin III antibody after being thiolated by Traut's. The storage buffer solution contains protein, sugar and aromatic amino acid, so that the nonspecific binding of the antibody can be effectively reduced, the interference of other substances in a sample on a detection result is reduced, and the repeatability of the detection result is improved. The thrombin antibody and the antithrombin III antibody used in the invention are combined with thrombin and antithrombin III antigen with high specificity and high affinity, thrombin-antithrombin III complex in a sample can be rapidly and accurately captured, and the thrombin-antithrombin III complex is matched with substrate liquid containing chromogenic substrates such as AMPPD or APS-5 and the like for use in a full-automatic immunoassay system to complete quantitative detection. The kit has good calibration curve and test repeatability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIGS. 1 and 2 show different storage buffer set calibration curves;
FIG. 3 shows a test consistency analysis of the kit of the present invention and a sample of the Hizimeric kit.
Detailed Description
The invention discloses a composition and application thereof, and a person skilled in the art can use the content of the composition to appropriately improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention relates to a preparation and detection method of a thrombin-antithrombin III complex (TAT) chemiluminescent assay kit, in particular to an enzyme-labeled antibody storage buffer solution and a preparation method of the kit. The enzyme-labeled antibody storage buffer solution comprises an enzyme-labeled antibody storage buffer solution containing protein, aromatic amino acid, preservative and the like. The invention also relates to a preparation method of the thrombin antibody coated magnetic particle suspension (immunomagnetic beads) and the alkaline phosphatase marked antithrombin III antibody (enzyme-labeled antibody).
The kit of the invention develops a preparation method which is simple to operate and can rapidly and accurately detect thrombin-antithrombin III complex based on a magnetic particle chemiluminescence immunoassay method. The magnetic nanoparticle has the advantages of large specific surface area, quick separation, good reproducibility, coupling labeling and the like, and the magnetic particle chemiluminescence immunoassay (CLIA) based on the magnetic nanoparticle combines a chemiluminescence measuring technology with high sensitivity with high-specificity immunoreaction, and has the characteristics of high sensitivity, wide linear range, high specificity, stable measurement value, high automation and the like.
The invention uses alkaline phosphatase to mark antithrombin III antibody, and uses the alkaline phosphatase to match with substrate liquid containing AMPPD or APS-5 chromogenic substrate for full-automatic immune inspection system to complete quantitative detection. The antigen is combined with thrombin antibody coated on the surface of the magnetic bead to form stable antigen-thrombin antibody complex, the enzyme-labeled antibody is combined with other sites of the antigen to form antigen, thrombin antibody and antithrombin III antibody complex, and alkaline phosphatase marked on the antibody catalyzes and hydrolyzes luminous substrate to emit light signals. The optical signal value is positively correlated with the concentration of alkaline phosphatase, and quantitative test of the double-antibody sandwich complex (antigen) is realized through detection and analysis by a chemiluminescent instrument. The enzyme-labeled antibody storage buffer solution contains protein and aromatic amino acid, so that the nonspecific binding of the antibody can be effectively reduced, the interference of other substances in a sample on the activity of alkaline phosphatase and the detection result of an on-machine is reduced, and the repeatability of detection is improved. The concrete steps are as follows:
the kit enzyme-labeled antibody storage buffer solution is added with the protein, the sugar and the aromatic amino acid, so that the nonspecific binding of the antibody can be reduced, the interference of other substances in a sample on a detection result can be reduced, and the repeatability of antigen detection is improved.
The invention provides the following technical scheme:
the invention provides a TAT chemiluminescence detection kit, the detection principle is a double-antibody sandwich method and an enzymatic chemiluminescence method, and the kit comprises a magnetic particle suspension (immunomagnetic beads) coated with thrombin antibody and an alkaline phosphatase-labeled antithrombin III antibody (enzyme-labeled antibody).
Wherein, the magnetic particles have superparamagnetism, the core material can be one or more of ferric oxide, zinc ferrite or ferrous oxide, and the surface of the magnetic particles is modified with uniform and sufficient carboxyl groups.
The magnetic particles may have a particle diameter in the range of 0.5 μm to 5. Mu.m, preferably 1.5. Mu.m.
The antibody may be a monoclonal antibody, a modified antibody fragment having Fab activity, an antibody or an antibody fragment multimer, or the like, may specifically bind to an epitope, and may be derived from animals such as mice, rabbits, sheep, dogs, and the like, preferably a murine monoclonal antibody.
The immunomagnetic beads can be coupled with antibodies by activators such as EDC, EDC and NHS, EDC and Sulfo-NHS, and the like, preferably EDC and Sulfo-NHS, and the coupling steps are as follows:
a) Uniformly dispersing magnetic particles in an activation buffer;
b) Dissolving an activating agent by using an activating buffer solution, adding the activating agent into the magnetic particle suspension, uniformly mixing, and incubating for 30 min-2 h at room temperature (10-30 ℃) to obtain activated magnetic beads;
c) Removing residual activator by magnetic separation, adding crosslinking buffer solution to redisperse magnetic particles;
d) Diluting thrombin antibody by using a crosslinking buffer solution, adding the thrombin antibody into the activated magnetic particle suspension, uniformly mixing, and incubating for 0.5-12 h at 4-30 ℃ to obtain an immunomagnetic bead suspension;
e) Dissolving a blocking agent by using a crosslinking buffer solution, adding the blocking agent into the immune magnetic bead suspension after the reaction is finished, and incubating for 0.5-3 h at the temperature of 4-30 ℃;
f) After magnetic separation, the immunomagnetic beads are dispersed by using a storage buffer solution and stored for later use.
The feeding ratio of the magnetic beads to the antibodies is 10:1-200:1 (w/w), preferably 50:1.
The feeding ratio of the magnetic beads to the activator is 0.1:1-20:1 (w/w), preferably 20:1.
The blocking agent can be one or more of substances containing free amino groups such as ethanolamine, bovine serum albumin, casein, amino acid and the like.
The activating and crosslinking buffer solution can be one of buffer solutions without amino and carboxyl, such as MES buffer solution, boric acid buffer solution, PBS buffer solution, MOPS buffer solution, HEPES buffer solution and the like, and is preferably MES buffer solution;
the pH of the activating and crosslinking buffer is in the range of 5.5 to 7.0, preferably 6.5.
The storage buffer system can be one of MOPS buffer, tris-HCl buffer, PBS buffer, HEPES buffer, glycine buffer and the like, and is preferably Tris-HCl buffer.
The storage buffer solution contains Bovine Serum Albumin (BSA) and preservative NaN 3 ；
The pH of the storage buffer is in the range of 6.0 to 9.0, preferably 7.5.
The enzyme-labeled antibody can be prepared by glutaraldehyde method, sodium periodate method or heterobifunctional reagent crosslinking method (Sulfo-SMCC) and the like, and the heterobifunctional reagent crosslinking method is preferred. The preparation process can utilize Sulfo-SMCC to activate alkaline phosphatase, 2-iminothiophene (Traut's)/Dithiothreitol (DTT)/2-mercaptoethylamine (2-MEA) sulfhydryl antithrombin III antibody, or use Sulfo-SMCC to activate antithrombin III antibody, traut's/DTT/2-MEA reagent sulfhydryl alkaline phosphatase.
The preparation steps are as follows:
a) Dissolving/diluting alkaline phosphatase with enzyme-labeled buffer, adding a certain amount of Sulfo-SMCC, mildly incubating for 0.5-3 h at 4-30 ℃, and desalting the product for later use;
b) Dissolving/diluting an antithrombin III antibody by using an enzyme-labeled buffer solution, adding a certain amount of Traut's reagent, incubating for 0.5-3 h at 4-30 ℃, adding 5-25 mg/mL glycine solution, quenching for 5min, and desalting the product for later use;
c) Mixing the activated alkaline phosphatase with the thiolated antithrombin III antibody, then carrying out mild stirring incubation for 2-30 h at the temperature of 4-30 ℃, and adding 5-25 mg/mL of cysteine solution to seal for 0.5-2 h;
d) The product is desalted and then stored in a storage buffer solution for standby.
The alkaline phosphatase/antibody to sulfoSMCC feed ratio is 2-30:1 (w/w), preferably 15:1.
The alkaline phosphatase/antibody to Traut's feed ratio is 20-250:1 (w/w), preferably 20:1.
The feeding ratio of alkaline phosphatase to antibody is 1-5:1 (w/w), and the feeding ratio of alkaline phosphatase to antibody can influence the three-dimensional structure of enzyme-labeled antibody complex and the number of antibodies bound on alkaline phosphatase, so that the formation of immunomagnetic beads, antigens and enzyme-labeled antibody complex is influenced, the difference of chemiluminescent signals is caused, and the repeatability of the on-machine test result is influenced, preferably 2:1.
The enzyme-labeled buffer can be one of MES buffer, boric acid buffer, PBS buffer and other buffers without amino and carboxyl, preferably PBS buffer.
The pH of the enzyme-labeled buffer is 7.0-8.0, preferably 7.0.
The storage buffer solution mainly comprises a buffer system, a stabilizer (protein, saccharide, aromatic amino acid and the like), a preservative and the like.
The concentration of the amino acid in the storage buffer solution is 1-3 mg/mL, preferably 2.5mg/mL;
the saccharide in the storage buffer solution can be trehalose, glucose, mannose, hyaluronic acid, galactose and the like, preferably trehalose; the concentration of the saccharide may be 5 to 15mg/mL, preferably 10mg/mL;
the aromatic amino acid can be one or more of tryptophan, tyrosine, phenylalanine and the like, preferably tyrosine;
the storage buffer system can be one of MOPS buffer, tris-HCl buffer, PBS buffer, HEPES buffer, glycine buffer and the like, and is preferably Tris-HCl buffer.
The pH of the enzyme-labeled buffer is 7.0-8.0, preferably 8.0.
The storage buffer stabilizer can be one or more of polyethylene glycol, bovine Serum Albumin (BSA), human serum albumin, rabbit serum albumin, aromatic amino acids, celluloses (sodium carboxymethyl cellulose, ethylcellulose and the like), aminoglycans (chondroitin sulfate, chitosan and the like) and the like, the aromatic amino acids can be phenylalanine, tyrosine and tryptophan, and the stabilizer can reduce the nonspecific binding of antibodies, can also bind with interfering substances in a sample, and improves the repeatability of detection.
The storage buffer preservative may be NaN 3 One or more of Proclin300, proclin 600;
the TAT chemiluminescent assay kit can be applied to a chemiluminescent instrument with an enzymatic glow type chemiluminescent detection system.
The TAT chemiluminescence method determination kit has the immunomagnetic bead on-machine test concentration of 0.1-1 mg/mL (calculated by the magnetic bead concentration), and preferably 0.5mg/mL.
The TAT chemiluminescence method determination kit has the enzyme-labeled antibody on-machine test concentration of 0.1-1 mug/mL (calculated by antibody concentration), preferably 0.5 mug/mL.
Further, the linear range of the kit is 0.5-120 ng/mL.
Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all commercially available and commercially available.
The invention is further illustrated by the following examples:
preparation example
1. Preparation of immunomagnetic beads
The carboxyl magnetic beads are fully and uniformly mixed by using a vortex mixer, 5mg of magnetic bead solution is taken in a centrifuge tube, an activation buffer (0.1M MES, pH 6.5) is added to fix the volume to 1mL, and after uniform mixing, magnetic separation is carried out for 1min, and the supernatant is removed. Adding 1mL of activating buffer again, uniformly mixing, magnetically separating for 1min, removing supernatant, adding 1mL of activating buffer, and uniformly mixing for later use.
After adding 100. Mu.L of activator (2.5 mg/mL EDC &2.5mg/mL Sulfo-NHS, as prepared) the mixture was homogenized, and incubated for 30min with spin mixing at room temperature.
Removing the supernatant after magnetic separation, taking 100 mug thrombin antibody, adding a crosslinking buffer solution (0.1M MES, pH 7.0) to a volume of 1mL, adding the mixture into the activated magnetic beads, mixing uniformly, and incubating for 6 hours at room temperature by rotating and mixing.
The supernatant was removed after magnetic separation, 1mL of a crosslinking buffer containing 10mg/mL BSA was added, and the mixture was blocked by spin mixing at room temperature for 1h, and the supernatant was removed after magnetic separation.
Dispersing the coupled immunomagnetic beads by using 1mL of storage buffer solution, storing at 2-8 ℃, and diluting to 0.5mg/mL by using the storage buffer solution before on-machine testing.
2. Enzyme-labeled antibody preparation
2mg of alkaline phosphatase is dissolved/diluted into 10mg/mL of alkaline phosphatase solution by using 0.2mL of enzyme-labeled buffer, 33.4 mu L of Sulfo-SMCC (4 mg/mL) is added, the mixture is mildly incubated for 1h at room temperature, and the product is dispersed for later use by using the enzyme-labeled buffer after desalting;
diluting 2mg of antithrombin antibody into 10mg/mL of antibody solution by using 0.2mL of enzyme-labeled buffer solution, adding 10 mu L of Traut's (10 mg/mL), carrying out mild incubation for 1h at room temperature, adding 40 mu L (5 mg/mL) of glycine solution, uniformly mixing, continuing to incubate for 5min, and dispersing the product for later use by using enzyme-labeled buffer solution after desalting;
after mixing the activated alkaline phosphatase and the thiolated antithrombin antibody uniformly, after mild incubation for 24 hours at 4 ℃, 100 mu L (10 mg/mL) of cysteine is added to mix uniformly, incubation is continued for 30min, the product is diluted with an enzyme-labeled storage buffer after desalting, and the enzyme-labeled storage buffer is used to dilute to 0.5 mu g/mL before on-machine testing.
Effect example 1: storage buffer composition
1. Concentration of trehalose
The compositions each contained 0mg/mL, 5mg/mL, 7.5mg/mL, 10mg/mL, 12.5mg +.Enzyme-labeled antibody storage buffer (50 mM Tris,5mg/mL BSA,1mg/mL NaN) with 15mg/mL trehalose 3 pH 8.0), diluting the enzyme-labeled antibody (the preparation method is shown in the preparation example), matching with immunomagnetic beads (the preparation method is shown in the preparation example), performing on-machine testing of a plasma sample, performing testing by using a one-step method, adding 50 mu L of immunomagnetic beads and 50 mu L of enzyme-labeled antibody, incubating 10 mu L of sample for 30min, washing three times by using a washing liquid, adding 200 mu L of substrate liquid, incubating for 5min, and detecting the photon quantity. The test was repeated five times, the coefficient of variation CV (%) of the test results was calculated, the repeatability of the test results of the upper machine was examined, the results are shown in Table 1, the coefficient of variation of the test results was reduced after adding trehalose to the enzyme-labeled antibody storage buffer compared with the 0mg/mL trehalose group, the repeatability was improved, wherein the coefficient of variation of the enzyme-labeled antibody storage buffer group containing 10mg/mL trehalose was the least, and the repeatability was the best, so that the enzyme-labeled antibody was stored in the enzyme-labeled antibody storage buffer containing 10mg/mL trehalose.
Table 1: analysis of results of reproducibility tests for different storage buffer groups
2. Alkaline phosphatase to antibody ratio
The enzyme-labeled antibody is prepared according to the method shown in the preparation example, wherein the mass ratio of alkaline phosphatase to antibody is 1:1, 2:1, 3:1, 4:1 and 5:1 respectively, the plasma sample is tested by matching with immunomagnetic beads (the preparation method is shown in the preparation example) in an on-machine manner, the test is repeated five times, the variation coefficient CV (%) of the test result is calculated, the repeatability of the on-machine test result is examined, the result is shown in the table 2, when the ratio of Alkaline Phosphatase (AP) to antibody (lgG) is 2:1, the variation coefficient of test sample 1 and sample 2 is the smallest, the repeatability is the best, the three-dimensional structure of the enzyme-labeled antibody prepared at this time and the combination ratio of alkaline phosphatase and antibody in the complex are more favorable for detection of antigen, the stability of the complex is favorable for improving the repeatability of the measurement value, and therefore the enzyme-labeled antibody is prepared by selecting the ratio of alkaline phosphatase to antibody to 2:1.
Table 2: analysis of results of reproducibility tests of different enzyme-labeled antibody groups
3. Calibration curve test
Preparing enzyme-labeled antibody storage buffers according to table 3, respectively diluting enzyme-labeled antibody (preparation method is shown in preparation example), matching with immunomagnetic beads (preparation method is shown in preparation example), performing on-machine test on TAT antigens with different concentrations, performing linear regression according to antigen concentration and measured photon quantity results, and comparing the results with the results shown in fig. 1 and fig. 2 (corresponding data are shown in table 4), wherein the correlation coefficients of the rest four groups of standard curves are increased compared with the comparison group storage buffer 1 (without amino acid), and the calibration curve is improved after amino acid is added into the enzyme-labeled antibody storage buffer; compared with the storage buffer solution 2 groups (containing non-aromatic amino acid-glycine), the correlation coefficient of the standard curve of the storage buffer solution 3-5 groups (containing aromatic amino acid) is further improved, wherein the correlation coefficient gamma= 0.9976 of the standard curve of the storage buffer solution 5 has good linear relation, and the measured photon magnitude is higher, so that the addition of the aromatic amino acid tyrosine into the storage buffer solution is more beneficial to the stability of the enzyme-labeled antibody, the three-dimensional structure of the enzyme-labeled antibody is maintained, the calibration curve of a TM (TM) assay kit is further improved, and the repeatability of the test is improved. Therefore, a stock buffer 5-group formulation was selected to formulate a stock buffer for the enzyme-labeled antibodies.
Table 3: storage buffer composition
Table 4: calibration curve test results
Antigen concentration (ng/mL) Storage buffer 1 Storage buffer 2 Storage buffer 3 Storage buffer 4 Storage buffer 5
0 27132 24158 21143 27131 18148
5 114157 129365 123435 153733 259075
10 1531726 1651357 842121 1132731 1251516
40 4521154 4649776 3205465 4630903 4849576
80 8012570 8656245 7423578 8548846 11453243
100 8424028 10629964 9463820 9071871 15629639
120 11876420 14829144 12973709 11511218 17829144
Effect example 2: sample test comparison
A total of 100 blood samples were randomly drawn from hospitalization and outpatient clinics of the attached Dragon hospital at Shanghai university of traditional Chinese medicine. The blood is evenly mixed with 0.109mol/L sodium citrate anticoagulation solution according to the proportion of 9:1, and is centrifuged at 2500rpm for 15 minutes, and the plasma is obtained after separation. The same sample was tested with the kit of the present invention (immunomagnetic beads 0.5mg/mL, 50. Mu.L, enzyme-labeled antibody 0.5. Mu.g/mL, 50. Mu.L, preparation method as shown in preparation example, wherein the storage buffer was prepared according to the formulation of storage buffer 5 described in effect example 1) and the obtained values were subjected to linear regression and correlation coefficients of the two were calculated. As a result, as shown in fig. 3, the correlation coefficient γ=0.996 of the two kits, and the linear regression equation is y=0.932x+0.6155, and the detection data of the kit of the present invention and the hson-meric kit have good consistency.
Effect example 3: repeatability test
The kit (immunomagnetic beads 0.5mg/mL, enzyme-labeled antibody 0.5. Mu.g/mL, and enzyme-labeled antibody 50. Mu.L) of the invention, the preparation method is shown in the preparation example, wherein the storage buffer solution is prepared according to the formula of the storage buffer solution 5 in the effect example 1), the Hiziram kit and the plasma sample are placed at the designated position of a chemiluminescent apparatus, the test is carried out according to the following procedure, the test is repeated for 10 times, and the average value and the variation coefficient CV of the test results are calculated:
a) Uniformly mixing 50 mu L of immunomagnetic beads, 50 mu L of enzyme-labeled antibody and 10 mu L of sample, and incubating at 37 ℃ for 30min;
b) Magnetically separating, and cleaning the compound by using a chemiluminescent instrument cleaning liquid;
c) Adding 200 mu L of substrate solution for a full-automatic immunoassay system into the cleaned magnetic bead compound, and incubating for 5min at 37 ℃ in a dark place;
d) Measuring the photon quantity of the substrate liquid after the reaction by using a photoelectric reactor;
e) Sample concentrations were calculated from the fitted calibration curve.
Table 5: repeatability test results
The test results are shown in Table 5, and the test results of the kit have smaller variation coefficients, which indicate that the test repeatability is better.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Use of a saccharide in any of the following:
(I) Improving the reproducibility of the chemiluminescent assay kit;
(II) a storage buffer solution for preparing the enzyme-labeled antibody;
the saccharide comprises one or more of trehalose, glucose, mannose, hyaluronic acid or galactose;
the chemiluminescent assay kit comprises a chemiluminescent assay kit for thrombin-antithrombin III complex.
2. Use of an aromatic amino acid in any of the following:
(I) Improving the calibration curve of the chemiluminescent assay kit;
(II) a storage buffer solution for preparing the enzyme-labeled antibody;
the aromatic amino acid comprises one or more of tryptophan, tyrosine or phenylalanine;
the chemiluminescent assay kit comprises a chemiluminescent assay kit for thrombin-antithrombin III complex.
3. A composition comprising a protein, a carbohydrate, and an aromatic amino acid;
the protein comprises one or more of bovine serum albumin, human serum albumin or rabbit serum albumin;
the saccharide comprises one or more of trehalose, glucose, mannose, hyaluronic acid or galactose;
the aromatic amino acids include one or more of tryptophan, tyrosine, or phenylalanine.
4. Use of a composition according to claim 3 in any of the following:
(I) Improving the calibration curve of the chemiluminescent assay kit;
(II) improving the reproducibility of the chemiluminescent assay kit;
(III) preparing an enzyme-labeled antibody storage buffer;
the chemiluminescent assay comprises a chemiluminescent assay for thrombin-antithrombin III complex.
5. An enzyme-labeled antibody storage buffer comprising the composition of claim 3, and:
(A) Buffer solution; and/or
(B) A preservative; and/or
(C) Acceptable auxiliary materials or auxiliary agents.
6. The enzyme-labeled antibody storage buffer of claim 5, wherein the saccharide is trehalose; the aromatic amino acid is tyrosine.
7. The enzyme-labeled antibody storage buffer of claim 5 or 6 comprising 50mM Tris,5mg/mL BSA, 10mg/mL trehalose, 2.5mg/mL tyrosine and 1mg/mL NaN 3 The method comprises the steps of carrying out a first treatment on the surface of the The pH value of the enzyme-labeled antibody storage buffer solution is 8.0.
8. The kit is characterized by comprising acceptable auxiliary materials or auxiliary agents and:
(a) A composition according to claim 3; or (b)
(b) The enzyme-labeled antibody storage buffer of any one of claims 5 to 7.
9. The kit of claim 8, further comprising an enzyme-labeled antibody;
the preparation method of the enzyme-labeled antibody comprises the following steps: mixing the activated enzyme with the thiolated antibody according to a mass ratio of 2:1 to obtain the enzyme-labeled antibody;
the enzyme includes alkaline phosphatase;
the antibodies include antithrombin III antibodies.
10. The method for preparing the kit according to claim 9, comprising the step of mixing the activated enzyme with the thiolated antibody in a mass ratio of 2:1 to obtain the enzyme-labeled antibody;
the enzyme includes alkaline phosphatase;
the antibodies include antithrombin III antibodies.
CN202310756384.9A 2023-06-26 2023-06-26 TAT enzyme-labeled antibody stock solution Pending CN117031046A (en)

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