CN113721012B - Composition and application of kit thereof in detection of glycocholic acid - Google Patents
Composition and application of kit thereof in detection of glycocholic acid Download PDFInfo
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Abstract
A composition and a use of a kit thereof in detection of glycocholic acid, wherein the composition comprises glycocholic acid-biotin derivatives formed by combining glycocholic acid and biotin. The invention designs a glycocholic acid detection kit based on an immunoturbidimetry platform, wherein the glycocholic acid-biotin micromolecule synthesis method is quick, simple and convenient, and a brand new reaction principle is designed by utilizing the binding characteristic of biotin-avidin binding, so that the defects of the prior art are overcome, the glycocholic acid-biotin micromolecule is not required to be combined with macromolecular protein or microspheres, and is directly applied to a latex immunoturbidimetry platform as a competitor, and the complicated process of preparing the macromolecular competitor is avoided.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a composition and application of a kit thereof in detection of glycocholic acid.
Background
Glycocholic acid (CG) is a bound form of cholic acid that is formed by binding cholic acid and glycine. Glycocholic acid is mainly synthesized in the liver and excreted into the intestinal tract along with bile, and under normal conditions, the concentration of glycocholic acid in blood is extremely low.
The structural formula of glycocholic acid is as follows:
when the liver cells are damaged, the capacity of the liver cells to take glycocholic acid is reduced, so that the content of glycocholic acid in blood is increased; when bile stagnates, the liver fails to excrete bile acid, and the content of glycocholic acid in the blood circulation is increased, which also increases the content of glycocholic acid in the blood. Therefore, glycocholic acid in blood is one of the sensitive indicators for evaluating the function of liver cells and the circulation function of the hepatobiliary substances thereof. In addition, glycocholic acid is one of the ideal indicators for diagnosing and evaluating the early sensitivity of patients with Intrahepatic Cholestasis of Pregnancy (ICP). ICP is a specific complication of the middle and late gestation, is a reversible cholestatic liver disease, and is clinically manifested as cutaneous pruritus and jaundice in the middle and late gestation. Bile excretion disorder in ICP patients leads to accumulation of glycocholic acid in peripheral blood circulation, resulting in increased levels of glycocholic acid in the blood.
The detection methods of glycocholic acid clinically applied at present mainly comprise a homogeneous enzyme immunoassay method, a chemiluminescence method and an immunoturbidimetry method. The homogeneous enzyme immunoassay method has the defects of poor thermal stability and easy interference of endogenous enzymes in a sample. The chemiluminescence method has high sensitivity and wide linearity, but has high cost and high instrument requirement, and the processes of preparing competitors and labeling luminescence are complicated. The immunoturbidimetry has fast detection speed and low cost, but the antibody and the small molecule can not form turbidity due to direct combination, and the sensitivity is low, so a competitive method is needed. Wherein, the preparation of the competitor is complicated, and the small molecule can be combined with the antibody to form turbidity only by forming the competitor after being combined with the macromolecular protein or the microsphere after being modified. FIG. 1 is a schematic diagram showing the reaction of the disclosed glycocholic acid detection reagent (latex immunoturbidimetry). The detection reagent for glycocholic acid (latex immunoturbidimetry) disclosed in chinese patents with publication numbers CN105301255B, CN105988000A, CN106841596A, CN108982860A, CN109444399A, CN109557301A, and CN112285345A, wherein the competitor is glycocholic acid-protein or glycocholic acid-protein-microsphere, and the process of preparing the competitor requires activation, protein coupling, desalting and purification; or the processes of activation, microsphere coupling, sealing, washing and the like are very complicated.
Disclosure of Invention
According to a first aspect, in one embodiment, there is provided a composition comprising a glycocholic acid-biotin derivative formed by binding glycocholic acid and biotin.
According to a second aspect, in one embodiment, a composition is provided that comprises an antibody-microsphere complex comprising an antibody that specifically binds to glycocholic acid bound to latex microspheres, and a binding protein-microsphere complex comprising a binding protein bound to latex microspheres.
According to a third aspect, in an embodiment, there is provided a reagent combination comprising at least one of a first reagent, a second reagent; the first reagent comprises glycocholic acid-biotin derivatives formed by binding glycocholic acid and biotin, and the second reagent comprises antibody-microsphere complexes formed by binding antibodies capable of being specifically bound with glycocholic acid and latex microspheres and binding protein-microsphere complexes formed by binding proteins and latex microspheres.
According to a fourth aspect, in an embodiment, a kit comprises a combination of reagents according to the third aspect.
According to a fifth aspect, in an embodiment, there is provided a composition according to the first aspect, or a composition according to the second aspect, or a combination of reagents according to the third aspect, or a kit according to the fourth aspect, for use in the detection of glycocholic acid.
According to the composition and the application of the kit in detection of glycocholic acid, the invention designs a glycocholic acid detection kit based on an immunoturbidimetry platform, wherein the glycocholic acid-biotin micromolecule synthesis method is quick, simple and convenient, and a brand new reaction principle is designed by utilizing the combination characteristic of biotin-avidin combination, so that the defects of the prior art are overcome, the glycocholic acid-biotin micromolecule is not required to be combined with macromolecular protein or microspheres, and the glycocholic acid-biotin micromolecule can be directly used as a competitor to be applied to a latex immunoturbidimetry platform, and the complicated process of preparing a macromolecular competitor is avoided. The preparation process of the kit is simple, so that the preparation cost of the kit is greatly reduced, and the two microspheres form a cross-linked network structure through the combination principle of antibody-antigen and biotin-avidin, so that the turbidity change is obvious and the sensitivity is high. In the aspect of reagent performance, the detection shows that the sensitivity, the linear range, the precision and the recovery rate all meet the requirements. Compared with the chemiluminescence method for carrying out methodology, the method has excellent correlation.
Drawings
FIG. 1 is a schematic diagram of the reaction of the disclosed glycocholic acid detection reagent (latex immunoturbidimetry);
FIG. 2 is a schematic diagram of an exemplary reaction of a glycocholic acid detection reagent (latex immunoturbidimetry);
FIG. 3 is a graph of an example glycocholic acid detection reagent calibration;
FIG. 4 is a graph of the linear range of the glycocholic acid detection reagent of an example;
FIG. 5 is a comparison of different methodologies of an exemplary glycocholic acid detection reagent.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments have been given like element numbers associated therewith. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.
The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning. The terms "connected" and "coupled" when used herein, unless otherwise indicated, include both direct and indirect connections (couplings).
Herein, unless otherwise specified, the concentrations of the components in the composition all refer to the final concentrations of the corresponding components in the corresponding composition.
According to a first aspect, in one embodiment, there is provided a composition comprising a glycocholic acid-biotin derivative formed by binding glycocholic acid and biotin. The method for synthesizing the glycocholic acid-biotin derivative micromolecules is quick, simple and convenient, does not need to be combined with macromolecular proteins or microspheres, is directly used as a competitor to be applied to a latex immunoturbidimetry platform, and avoids the complicated process of preparing the macromolecular competitor. Glycocholic acid-biotin derivatives are used as a component of the first reagent for competitively binding with glycocholic acid in the sample to be tested to the antibody in the antibody-microsphere complex from the second reagent. After a sample to be detected, a first reagent and a second reagent are mixed to form a reaction liquid, the lower the concentration of glycocholic acid from the sample to be detected, the more glycocholic acid-biotin derivatives which are competitively bound to the antibody-microsphere complex, the higher the crosslinking degree of a crosslinking structure formed by the glycocholic acid-biotin derivatives, the antibody-microsphere complex and the binding protein-microsphere complex, the higher the turbidity of the reaction liquid, and conversely, the higher the concentration of glycocholic acid from the sample to be detected, the less glycocholic acid-biotin derivatives which are competitively bound to the antibody-microsphere complex, the lower the crosslinking degree of a crosslinking structure formed by the glycocholic acid-biotin derivatives, the antibody-microsphere complex and the binding protein-microsphere complex, the lower the turbidity of the reaction liquid, and the inversely proportional ratio of the concentration of glycocholic acid in the sample to be detected to the turbidity of the reaction liquid, by measuring the turbidity of the reaction solution, the concentration of glycocholic acid in the sample to be measured can be calculated.
In one embodiment, the composition comprises 1-200 μ g/mL glycocholic acid-biotin derivative.
In one embodiment, the composition comprises 10-50 μ g/mL glycocholic acid-biotin derivative.
In one embodiment, the composition further comprises at least one of the following components: buffer, electrolyte, stabilizer, promoter, surfactant and preservative.
In one embodiment, the composition further comprises at least one of the following components in concentrations: 10 to 200mM buffer, 0.1 to 1% w/w electrolyte, 0.1 to 15% w/w stabilizer, 0.5 to 1.5% w/w accelerator, 0.05 to 1% w/w surfactant, 0.01 to 0.1% w/w preservative.
In one embodiment, the composition further comprises at least one of the following components in concentrations: 20 to 100mM buffer, 0.1 to 0.5% w/w electrolyte, 0.1 to 0.5% w/w stabilizer, 0.5 to 1.5% w/w accelerator, 0.05 to 0.25% w/w surfactant, 0.01 to 0.05% w/w preservative.
In one embodiment, the buffer comprises at least one of the following buffers: phosphate Buffer (PBS), Tris buffer, 2-morpholinoethanesulfonic acid buffer (MES), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), boric acid buffer, glycine buffer.
In one embodiment, the electrolyte contains at least one of the following ions: sodium ion (Na) + ) Magnesium ion (Mg) 2+ ) Potassium ion (K) + ) Calcium ion (Ca) 2+ )。
In one embodiment, the electrolyte includes, but is not limited to, at least one of the following compounds: sodium chloride (NaCl), magnesium chloride (MgCl) 2 ) Potassium chloride (KCl) and calcium chloride (CaCl) 2 )。
In one embodiment, the stabilizing agent includes, but is not limited to, at least one of bovine serum albumin, gelatin, trehalose, lactose, sucrose.
In one embodiment, the accelerant includes, but is not limited to, polyethylene glycol (PEG).
In one embodiment, the molecular weight of the polyethylene glycol is 200 to 20000, preferably 1000 to 10000, more preferably 3000 to 9000, and more preferably 5000 to 7000.
In one embodiment, the surfactant includes, but is not limited to, at least one of Triton X-100, Triton X-405, Tween 20, Tween 80.
In one embodiment, the preservative includes, but is not limited to, at least one of sodium azide, ProClin 300.
In one embodiment, the pH of the composition is 7.0 to 9.0.
In one embodiment, the glycocholic acid-biotin derivative is obtained by reacting glycocholic acid with an amino-polyethylene glycol-biotin derivative.
In one embodiment, the glycocholic acid-biotin derivative is obtained by reacting glycocholic acid with an amino-polyethylene glycol-biotin derivative in the presence of a solvent and under the action of a condensing agent and a catalyst.
In one embodiment, the molecular weight of the polyethylene glycol in the amino-polyethylene glycol-biotin derivative is 300-3500, preferably 700-3500, more preferably 700-3000, and more preferably 1000-3000.
According to a second aspect, in one embodiment, there is provided a composition comprising an antibody-microsphere complex comprising an antibody capable of specifically binding to glycocholic acid bound to latex microspheres, and a binding protein-microsphere complex comprising a binding protein bound to latex microspheres. The binding protein in the binding protein-microsphere complex is used for binding with biotin in a glycocholic acid-biotin derivative in a first reagent, the antibody in the antibody-microsphere complex is used for binding with glycocholic acid in a sample to be tested or glycocholic acid in a glycocholic acid-biotin derivative, and glycocholic acid from the sample to be tested and glycocholic acid-biotin derivative from the first reagent are competitively bound with the antibody in the antibody-microsphere complex of a second reagent.
After the sample to be tested, the first reagent and the second reagent are mixed to form a reaction solution, glycocholic acid from the sample to be tested and glycocholic acid-biotin derivative from the first reagent are competitively combined with the antibody in the antibody-microsphere complex. If the content of glycocholic acid from a sample to be detected in the reaction solution is relatively high, the content of glycocholic acid-biotin derivatives from the first reagent is relatively low, and each glycocholic acid molecule only has one binding site capable of being bound to an antibody, the glycocholic acid from the sample to be detected cannot form a cross-linked structure after being bound with the antibody-microspheres from the second reagent, and the turbidity of the reaction solution is low; if the content of glycocholic acid in the reaction solution from the sample to be detected is relatively low, the content of glycocholic acid-biotin derivative from the first reagent is relatively high, glycocholic acid in the glycocholic acid-biotin derivative is bound to the antibody in the antibody-microsphere complex, biotin in the glycocholic acid-biotin derivative is bound to the binding protein in the binding protein-microsphere complex to form a cross-linked structure, and the turbidity of the reaction solution is high. The concentration of the glycocholic acid in the sample to be detected is inversely proportional to the turbidity of the reaction solution, and the concentration of the glycocholic acid in the sample to be detected can be calculated by measuring the turbidity of the reaction solution.
In one embodiment, the latex microspheres include, but are not limited to, at least one of polystyrene microspheres, polyacrylic microspheres, polyacrylate microspheres. Latex microspheres are commercially available.
In one embodiment, the latex microspheres may have a diameter of 50 to 450 nm.
In one embodiment, the latex microspheres include, but are not limited to, carboxylated latex microspheres. Carboxylated latex microspheres are generally commercially available.
In one embodiment, the antibody includes, but is not limited to, at least one of a monoclonal antibody, a polyclonal antibody. Antibodies are commercially available, may be obtained by techniques such as recombinant DNA and recombinant RNA, or may be purified from natural antibodies.
In one embodiment, the antibody includes, but is not limited to, at least one of an antibody of murine or rabbit origin. The biological source of the antibody is not limited, and antibodies capable of specifically binding to glycocholic acid are all suitable for use in the present invention.
In one embodiment, the composition contains the following components in the following concentrations: 0.05-0.2% w/w antibody-microsphere complex, 0.05-0.2% w/w binding protein-microsphere complex. The concentrations referred to above are all the final concentrations of the respective components in the composition.
In one embodiment, the composition further comprises at least one of the following components: buffer solution, electrolyte, stabilizer, surfactant and preservative.
In one embodiment, the composition further comprises at least one of the following components in concentrations: 10 to 200mM buffer, 0.1 to 1% w/w electrolyte, 0.1 to 15% w/w stabilizer, 0.05 to 1% w/w surfactant, 0.01 to 0.1% w/w preservative.
In one embodiment, the composition further comprises at least one of the following components in concentrations: 20 to 100mM buffer, 0.1 to 0.5% w/w electrolyte, 0.1 to 0.5% w/w stabilizer, 0.05 to 0.25% w/w surfactant, 0.01 to 0.05% w/w preservative.
In one embodiment, the buffer comprises at least one of the following buffers: phosphate Buffer (PBS), Tris buffer, 2-morpholinoethanesulfonic acid buffer (MES), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), boric acid buffer, glycine buffer.
In one embodiment, the electrolyte contains at least one of the following ions: sodium ion (Na) + ) Magnesium ion (Mg) 2+ ) Potassium ion (K) + ) Calcium ion (Ca) 2+ )。
In one embodiment, the electrolyte includes, but is not limited to, at least one of the following compounds: sodium chloride (NaCl), magnesium chloride (MgCl) 2 ) Potassium chloride (KCl) and calcium chloride (CaCl) 2 )。
In one embodiment, the stabilizing agent includes, but is not limited to, at least one of bovine serum albumin, gelatin, trehalose, lactose, sucrose.
In one embodiment, the surfactant includes, but is not limited to, at least one of Triton X-100, Triton X-405, Tween 20, Tween 80.
In one embodiment, the preservative includes, but is not limited to, at least one of sodium azide, ProClin 300.
In one embodiment, the pH of the composition is 7.0 to 9.0.
According to a third aspect, in an embodiment, there is provided a reagent combination comprising at least one of a first reagent, a second reagent; the first reagent comprises glycocholic acid-biotin derivatives formed by binding glycocholic acid and biotin, and the second reagent comprises antibody-microsphere complexes formed by binding antibodies capable of being specifically bound with glycocholic acid and latex microspheres and binding protein-microsphere complexes formed by binding proteins and latex microspheres. The first reagent and the second reagent are not mixed before use, are independently stored in different containers or different chambers of the same container, and are added into a sample to be detected according to a detection program when used for detecting the sample, and the biochemical analyzer automatically calculates the concentration of a target substance glycocholic acid in the sample to be detected by detecting the turbidity change of a reaction liquid.
The main principle of glycocholic acid detection by using the first reagent and the second reagent is as follows: after a sample to be detected, a first reagent (a composition containing glycocholic acid-biotin derivatives) and a second reagent (a composition containing antibody-microsphere complexes and binding protein-microsphere complexes) are mixed to form a reaction solution, glycocholic acid from the sample to be detected and glycocholic acid-biotin derivatives from the first reagent are competitively bound with a glycocholic acid antibody from the second reagent, if the content of glycocholic acid from the sample to be detected in the reaction solution is high and the content of glycocholic acid-biotin derivatives from the first reagent is relatively low, a target glycocholic acid has only one binding site, and cannot form a cross-linked structure after being bound with the antibody-microsphere complexes, and the turbidity of the reaction solution is low; if the content of the target substance glycocholic acid from the sample to be detected in the reaction solution is low, the content of glycocholic acid-biotin derivatives from the first reagent is relatively high, and after the glycocholic acid-biotin derivatives are combined with the antibody from the second reagent, the biotin part of the glycocholic acid-biotin derivatives is combined with the binding protein in the binding protein-microsphere complex to form a cross-linked structure, so that the turbidity is high. The concentration of the target glycocholic acid in the sample to be detected is inversely proportional to the turbidity formed by the reaction, and the concentration of the glycocholic acid in the sample to be detected can be calculated by measuring the turbidity of the reaction liquid.
In one embodiment, the combination of reagents comprises all of the first reagent and the second reagent.
In one embodiment, the first agent comprises a composition of the first aspect and the second agent comprises a composition of the second aspect.
In one embodiment, the volume ratio of the first reagent to the second reagent is (1-5): 1, preferably 4: 1.
in one embodiment, the reagent combination further comprises a calibrator. The calibration material is typically used to create a calibration curve that is used as a basis for subsequent calculations of the glycocholic acid concentration in the sample to be tested.
In one embodiment, the calibrator comprises glycocholic acid.
In one embodiment, the calibrator further comprises at least one of the following components: buffering agent, surfactant and preservative.
According to a fourth aspect, in an embodiment, a kit comprises the reagent combination of the third aspect. In one embodiment, the kit can be used to detect the concentration of glycocholic acid in a sample of a human or animal body fluid. Body fluid samples include, but are not limited to, serum.
In one embodiment, the kit further comprises containers for separately storing the first reagent and the second reagent. The first reagent and the second reagent can be independently stored in two different containers, or can be independently stored in two independent chambers of one container, and are packaged into a kit product which can be sold.
In one embodiment, where the reagent combination comprises a calibrator, the kit further comprises a container for separately storing the calibrator.
In one embodiment, the kit further comprises instructions for use to instruct a user to use the kit.
According to a fifth aspect, in an embodiment, there is provided the use of a composition of the first aspect, or a composition of the second aspect, or a combination of reagents of the third aspect, or a kit of reagents of the fourth aspect, in the detection of glycocholic acid.
In one embodiment, the use is the use for detecting the concentration of glycocholic acid in a sample to be tested. Glycocholic acid concentrations include, but are not limited to, mass concentration, volume concentration, mass-volume concentration, and the like.
In one embodiment, the method for detecting glycocholic acid by using the kit is as follows: and mixing the sample to be detected, the first reagent and the second reagent, and calculating to obtain the concentration of glycocholic acid in the sample to be detected according to the turbidity change of the reaction liquid. The method can be generally automated by a biochemical analyzer.
In one embodiment, the sample to be tested may be serum of a human or animal body.
Since the detection result of glycocholic acid is only an intermediate result of disease diagnosis and usually requires combination with other factors such as clinical symptoms of a subject to obtain a final diagnosis result, the above-mentioned use is not a method for diagnosing and treating a disease.
In some embodiments, the glycocholic acid detection kit is designed based on an immunoturbidimetry platform, wherein the glycocholic acid-biotin small molecule synthesis method is rapid, simple and convenient, and a brand new reaction principle is designed by utilizing the binding characteristic of biotin-avidin binding, so that the defects of the prior art are overcome, the glycocholic acid-biotin small molecule is not required to be bound with macromolecular protein or microspheres, and is directly used as a competitor to be applied to a latex immunoturbidimetry platform, and the complicated process of preparing the macromolecular competitor is avoided. The method has the advantages that the process is simple, the preparation cost of the kit is greatly reduced, and the reagent forms a cross-linked network structure by the combination principle of the two microspheres through the antibody-antigen and biotin-avidin, so that the turbidity change is obvious and the sensitivity is high. In the aspect of reagent performance, the detection shows that the sensitivity, the linear range, the precision and the recovery rate all meet the requirements, and the reagent has excellent correlation with a method for carrying out a chemiluminescence method.
In some embodiments, the present invention designs a glycocholic acid detection kit using glycocholic acid-biotin small molecules as competitors. The reaction principle is shown in fig. 2, a sample containing glycocholic acid (i.e. a sample to be detected, referred to as a sample for short) is mixed and incubated with a reagent 1 containing glycocholic acid-biotin, and a reagent 2 containing glycocholic acid antibody-microspheres and streptavidin microspheres is added to start the reaction. Glycocholic acid in the sample and glycocholic acid biotin in the reagent 1 are competitively combined with a glycocholic acid antibody, if the glycocholic acid content in the sample is high, the glycocholic acid-biotin content is relatively low, because each glycocholic acid molecule only has one binding site capable of being bound to the glycocholic acid antibody (for an IgG antibody, each antibody molecule has two Fab antigen binding fragments, so that 2 glycocholic acid molecules can be bound), the glycocholic acid in the sample cannot form a cross-linked structure after being combined with the glycocholic acid antibody-microsphere in the reagent 2, and the turbidity of a reaction solution is low; if the content of glycocholic acid in the sample is low, the glycocholic acid-biotin content is relatively high, after the glycocholic acid-biotin is combined with a glycocholic acid antibody, the biotin part of the glycocholic acid-biotin is combined with the streptavidin-microsphere to form a cross-linked structure, and the turbidity of the reaction solution is high. The concentration of glycocholic acid in the sample is inversely proportional to the turbidity formed by the reaction.
In some embodiments, the glycocholic acid detection reagent comprises the following components:
the glycocholic acid detection reagent consists of a reagent 1, a reagent 2 and a calibrator, and the following components of each part.
TABLE 1 ingredient Table of reagent 1
| Serial number | Name (R) | Concentration of |
| 1 | Glycocholic acid-biotin | 1-200μg/mL |
| 2 | Buffering agent | 10-200mM |
| 3 | Electrolyte | 0.1-1％w/w |
| 4 | Stabilizer | 0.1-15％w/w |
| 5 | Accelerator | 0.5-1.5％w/w |
| 6 | Surface active agent | 0.05-1％w/w |
| 7 | Preservative | 0.01-0.1％w/w |
The pH of the reagent 1 is 7.0-9.0.
TABLE 2 reagent 2 ingredients Table
| Serial number | Name (R) | Concentration of |
| 1 | Glycocholic acid antibody-microspheres | 0.05-0.2％w/w |
| 2 | Streptavidin microspheres | 0.05-0.2％w/w |
| 3 | Buffering agent | 10-200mM |
| 4 | Electrolyte | 0.1-1％w/w |
| 5 | Stabilizer | 0.1-15％w/w |
| 6 | Surface active agent | 0.05-1％w/w |
| 7 | Preservative | 0.01-0.1％w/w |
The pH of the reagent 2 is 7.0-9.0.
TABLE 3 ingredient Table of calibrator
| Serial number | Name (R) | Concentration of |
| 1 | Glycocholic acid | 0-80mg/L |
| 2 | Buffering agent | 10-200mM |
| 3 | Stabilizer | 0.1-15％w/w |
| 4 | Preservative | 0.01-0.1％w/w |
The pH value of the calibrator is 7.0-9.0.
In some embodiments, the glycocholic acid-biotin derivative is a derivative obtained by connecting glycocholic acid to biotin through polyethylene glycol (PEG), and the molecular weight of the PEG can be 700-3500. In some embodiments, the buffer includes, but is not limited to, at least one of Phosphate Buffer (PBS), Tris buffer, 2-morpholinoethanesulfonic acid buffer (MES), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer, boric acid buffer, glycine buffer.
In some embodiments, the electrolyte comprises at least one of sodium ions, magnesium ions, potassium ions, calcium ions.
In some embodiments, the stabilizing agent includes, but is not limited to, at least one of bovine serum albumin, gelatin, trehalose, lactose, sucrose.
In some embodiments, the enhancer includes, but is not limited to, polyethylene glycol, which can have a molecular weight of 200 to 20000.
In some embodiments, the surfactant includes, but is not limited to, at least one of Triton X-100, Triton X-405, Tween 20, Tween 80.
In some embodiments, the preservative includes, but is not limited to, at least one of sodium azide, ProClin 300.
In some embodiments, the microspheres include, but are not limited to, polystyrene carboxyl microspheres having a diameter of 50-450 nm.
In some embodiments, the glycocholic acid antibody comprises at least one of a monoclonal antibody, a polyclonal antibody.
In some embodiments, there is provided a method of making a glycocholic acid detection kit, comprising:
1) preparation of glycocholic acid-biotin
The principle of glycocholic acid-biotin is as follows:
the raw material comprises glycocholic acid; NH (NH) 2 The PEG-Biotin is an amino PEG Biotin derivative, and the molecular weight of the PEG can be 300-3000; the condensing agent includes but is not limited to at least one of N, N '-dicyclohexylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N, N' -diisopropylcarbodiimide, O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate; the catalyst includes but is not limited to at least one of 4-dimethylamino pyridine, 1-hydroxybenzotriazole and 4-pyrrolidinyl pyridine; the solvent includes but is not limited to at least one of dichloromethane, tetrahydrofuran, diethyl ether, ethyl acetate, dimethyl sulfoxide, N-dimethylformamide, toluene, pyridine and piperidine; glycocholic acid in the reaction: NH (NH) 2 -PEG-Biotin: condensing agent: the molar ratio of the catalyst is 1 (1-10) to 1-10; (0.1-1); the reaction temperature is 0-100 ℃; the reaction time is 1-72 h; the reaction concentration of the glycocholic acid is 0.01-1 mmol/mL; and (3) purification conditions: the eluent is dichloromethane/methanol mixed solution.
The preparation method comprises the following specific steps: glycocholic acid, NH 2 Adding PEG-Biotin, a condensing agent, a catalyst and a solvent into a reaction vessel, and reacting for 1-72 hours at 0-100 ℃ under the condition of nitrogen. After the reaction is finished, the glycocholic acid-biotin is obtained by silica gel column chromatography purification.
2) Preparation of reagent 1
Adding glycocholic acid-biotin into a container filled with purified water, stirring at room temperature until the solid is completely dissolved, and preparing into a glycocholic acid-biotin mother liquor.
Adding the buffer solution, the electrolyte, the stabilizer, the accelerator, the surfactant and the preservative into a container filled with purified water, stirring at room temperature until the solid is completely dissolved, adjusting the pH, adding the glycocholic acid-biotin mother liquor, and uniformly mixing to obtain the reagent 1.
3) Preparation of reagent 2
Adding the buffer solution, the electrolyte, the stabilizer, the surfactant and the preservative into a container filled with purified water, stirring at room temperature until the solid is dissolved, and adjusting the pH value to obtain a reagent 2 mother solution.
Adding polystyrene carboxyl microspheres, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into a container filled with MES buffer solution, activating the microspheres, and reacting at room temperature. Centrifuging, washing for 2-4 times, adding MES buffer solution, and ultrasonic re-suspending to obtain activated microsphere. Adding glycocholic acid antibody, and reacting at room temperature. Glycine solution was added to stop the reaction. Centrifuging, washing for 2-4 times, and finally adding the reagent 2 mother liquor for ultrasonic resuspension to obtain the glycocholic acid antibody-microsphere solution.
Adding polystyrene carboxyl microspheres, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into a solvent filled with MES buffer solution to activate the microspheres, and reacting at room temperature. Centrifuging, washing for 2-4 times, adding MES buffer solution, and ultrasonic re-suspending to obtain activated microsphere. Streptavidin was added and the reaction was carried out at room temperature. Glycine solution was added to stop the reaction. Centrifuging, washing for 2-4 times, and finally adding the reagent 2 mother solution for ultrasonic resuspension to obtain the streptavidin-microsphere solution.
And uniformly mixing the glycocholic acid antibody-microsphere solution and the streptavidin-microsphere solution to obtain the reagent 2.
4) Preparation of calibrator
And adding the buffer solution, the stabilizer and the preservative into a container filled with purified water, stirring at room temperature until the solid is dissolved, and adjusting the pH (which can be 7.0-9.0) to obtain the mother liquor of the calibrator. And adding glycocholic acid into the mother liquor of the calibrator according to the required concentration, and uniformly mixing to obtain the calibrator.
Example 1
In this example, the reagents used to adjust the pH were 2.0M hydrochloric acid and 2.0M aqueous sodium hydroxide.
In this example, the source and product information of each raw material are as follows:
the manufacturer of polystyrene carboxyl microspheres: JSR (beijing bolmeyer agency); the product name is as follows: latex microspheres; product goods number: p0113.
The manufacturer of glycocholic acid: (ii) alatin; the product name is as follows: glycocholic acid; product goods number: G131002.
antibody: the manufacturer: exploring a life science and technology; the product name is as follows: CG rabbit polyclonal antibody; product goods number: FAB-F005.
NH 2 -PEG-Biotin: the manufacturer: shanghai Tuo Yang; the product name is as follows: biotin polyethylene glycol amino; product goods number: p002009.
1. Glycocholic acid reagent preparation
1.1 preparation of Glycocholic acid-Biotin derivatives
To a dry round bottom flask was added glycocholic acid (46.5mg, 0.10mmol), NH 2 PEG-Biotin (PEG molecular weight of about 2000, 240.0mg, about 0.12mmol), N' -dicyclohexylcarbodiimide (24.8mg,0.12mmol), 4-dimethylaminopyridine (2.4mg,0.02mmol) and tetrahydrofuran (5.0mL) were added three times with replacement of nitrogen and stirred at room temperature for 12 hours. After completion of the reaction, insoluble solids were removed by filtration. Concentrating the reaction solution, performing silica gel column chromatography, and performing gradient elution by using an eluent (the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 10:1 to 3:1), wherein the volume ratio of the dichloromethane to the methanol in the eluent is 10: 1. 5:1, 3:1, to give glycocholic acid-biotin derivative as a white solid (155.3mg, 63% yield). MS (+): the highest peak of the molecular weight signal was 2139.5. The molar ratio of biotin to glycocholic acid is slightly greater than 1: 1, the glycocholic acid is reacted more completely, and the subsequent purification is facilitated.
2. Preparation of reagent 1
The materials and amounts of reagent 1 are shown in Table 4.
TABLE 4 reagent 1 preparation materials and amounts thereof
| Serial number | Name (R) | Concentration of | 1L dosage |
| 1 | Glycocholic acid-biotin | 35μg/mL | 35.0mg |
| 2 | Tris | 50mM | 6.06g |
| 3 | Sodium chloride | 0.5％w/w | 5.00g |
| 4 | Bovine serum albumin | 0.2％w/w | 2.00g |
| 5 | PEG6000 | 0.75％w/w | 7.50g |
| 6 | Triton X-100 | 0.05％w/w | 0.50g |
| 7 | Sodium azide | 0.05％w/w | 0.50g |
The glycocholic acid-biotin derivative (35.0mg) was added to a beaker containing 35.0mL of purified water, and stirred at room temperature until completely dissolved, to prepare a 1.0mg/mL glycocholic acid-biotin mother liquor.
Tris (6.06g), sodium chloride (5.00g), bovine serum albumin (2.00g), PEG6000(7.50g), Triton X-100(0.50g) and sodium azide (0.50g) were put in a pure water vessel containing 800mL, and the mixture was stirred at room temperature until the solid was completely dissolved, and the pH was adjusted to 7.5. And adding glycocholic acid-biotin mother liquor (1.0mg/mL,35mL), diluting to 1L, and mixing uniformly to obtain a reagent 1.
3. Preparation of reagent 2
The materials and amounts of reagent 2 are shown in Table 5.
TABLE 5 amount of reagent 2 preparation materials
Tris (1.51g), sodium chloride (1.25g), bovine serum albumin (0.50g), Triton X-100(125.0mg) and sodium azide (125.0mg) were added to a vessel containing 200mL of purified water, and the mixture was stirred at room temperature until the solid was dissolved, adjusted to pH 8.0, and then the volume was adjusted to 250mL to obtain a reagent 2 mother solution.
1-Ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (20.6mg) and N-hydroxysuccinimide (154.7 mg) were added to a vessel containing MES buffer (11.25mL), and polystyrene carboxyl microspheres (10% w/w solution, 1250. mu.L, 200nm) were added to activate the microspheres, followed by reaction at room temperature for 1 hour. After the reaction is finished, centrifuging, removing clear liquid, adding MES buffer solution, carrying out ultrasonic washing, and repeating the centrifugal washing process for 2 times. After centrifugation, MES buffer (12.50mL) was added and resuspended by sonication to give activated microspheres (1% w/w, solution 12.50mL), rabbit-derived glycocholic acid antibody (8.9mg, this is a polyclonal antibody) was added and reacted at room temperature for 4 h. The reaction was stopped by the addition of 1M aqueous glycine (1.50mL), centrifuged, the supernatant discarded, washed ultrasonically with MES buffer and the centrifugation and washing process repeated 2 times. After centrifugation, reagent 2 mother liquor (125mL) was added and resuspended by sonication to give a glycocholic acid antibody-microsphere solution (0.1% w/w,125 mL).
1-Ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (20.6mg) and N-hydroxysuccinimide (154.7 mg) were added to a vessel containing MES buffer (11.25mL), and polystyrene carboxyl microspheres (10% w/w solution, 1250. mu.L, 200nm) were added to activate the microspheres, followed by reaction at room temperature for 1 hour. After the reaction is finished, centrifuging, removing supernatant, adding MES buffer solution, ultrasonically washing, and repeating the centrifuging and washing process for 2 times. After centrifugation, MES buffer (12.50mL) was added and resuspended by sonication to give activated microspheres (1% w/w, solution 12.50mL), streptavidin (8.9mg) was added and the reaction was carried out at room temperature for 4 h. The reaction was stopped by the addition of 1M aqueous glycine (1.50mL), centrifuged, the supernatant discarded, washed ultrasonically with MES buffer and the centrifugation and washing process repeated 2 times. After centrifugation, reagent 2 mother liquor (125mL) was added and resuspended by sonication to give a streptavidin-microsphere solution (0.1% w/w,125 mL).
And (3) uniformly mixing the glycocholic acid antibody-microsphere solution (125mL) and the streptavidin-microsphere solution (125mL) to obtain a reagent 2(250mL), wherein the concentration of the glycocholic acid antibody-microsphere and the concentration of the streptavidin-microsphere in the reagent 2 are both 0.05% w/w.
Table 6 calibrant preparation material usage
Phosphate buffer (800.0 mg of sodium chloride; 20.0mg of potassium chloride; 144.0mg of disodium hydrogen phosphate; 24.0mg of potassium dihydrogen phosphate), bovine serum albumin (200.0mg) and ProClin 300(100.0mg) were added to a vessel containing 80mL of purified water, stirred at room temperature until the solid dissolved, the pH was adjusted to 7.4, and the volume was fixed to 100mL to obtain a stock solution of the calibrator. And (3) adding glycocholic acid into the mother liquor of the calibrator according to the required concentration to prepare the calibrator with the concentrations of 0, 2.50mg/L, 5.00mg/L, 10.00mg/L, 30.00mg/L and 80.00 mg/L.
4. Evaluation of reagent Properties
4.1 test parameter and calibration Curve plotting
In the embodiment, a Hitachi 7180 full-automatic biochemical analyzer is adopted for detection, and the instrument parameters are set as follows:
reagent 1: 200 mu L; reagent 2: 50 mu L of the solution; sample size (both serum samples): 10 mu L of the solution;
the detection method comprises the following steps: a two-point end-point method;
the reaction direction is as follows: rising;
measuring wavelength: 600 nm;
reaction time: 10min (the time for adding the sample to be detected is taken as the starting time, and the time for automatically calculating the detection result by the automatic biochemical analyzer is taken as the ending time);
reading points: 18 to 34;
the calibration method comprises the following steps: a Spline;
measuring temperature: 37 ℃ is carried out.
The liquid adding process of the Hitachi 7180 full-automatic biochemical analyzer comprises the following steps: adding a sample to be detected, adding the reagent 1, incubating for 5 minutes, adding the reagent 2, and reacting for 5 minutes. The process is automatically carried out by a biochemical analyzer.
A6-point calibration method is adopted, a Hitachi 7180 full-automatic biochemical analyzer is used for calibration, the concentrations of the calibrants are respectively 0, 2.50mg/L, 5.00mg/L, 10.00mg/L, 30.00mg/L and 80.00mg/L, calibration curves are prepared, and fig. 3 is a calibration curve chart of the glycocholic acid detection reagent of the embodiment.
TABLE 7 Glycocholic acid detection reagent 7180 calibration results
4.2 assay sensitivity
A serum sample was collected at a concentration of (2.5. + -. 0.5) mg/L, and the collected sample had a value of about 2.5 mg/L, which was measured by the indicated chemiluminescence method. The test was carried out using the reagents 1 and 2 of the present example, and the change in absorbance (. DELTA.OD) produced under the specified parameters of the reagents was recorded Sample(s) ) Deducting the change in absorbance (. DELTA.OD) produced under the specified parameters for the zero concentration calibrator 0 ) Converted into the absolute value (| DeltaOD |) of the change in absorbance of the sample (2.5. + -. 0.5). After 3 times of detection, the mean value of the concentration of glycocholic acid in the serum sample is about 2.70mg/L, and DeltaOD I is 1614, which indicates that the analysis sensitivity is high.
TABLE 8 Glycocholic acid detection reagent assay sensitivity test results
4.3 Linear Range
A high concentration sample (82.87 mg/L) and a low concentration sample (0.49mg/L) of glycocholic acid were collected, and the collected samples were mixed at the ratio shown in Table 9 with the chemiluminescence detection concentration value as a reference, and each linear sample was tested. The average value of the measured concentration is compared with a theoretical value to obtain an absolute deviation or a relative deviation, and linear regression analysis is performed.
TABLE 9 Linear sample preparation
TABLE 10 Glycocholic acid detection reagent Linear Range test results
Table 10 shows the results of the linear range test of the reagent for detecting glycocholic acid, and FIG. 4 is a graph of the linear range of the reagent for detecting glycocholic acidObtaining the regression equation y as 0.9988x-0.3078 and the correlation coefficient R 2 The result shows that the linear range of the glycocholic acid detection kit can reach 0.5-80 mg/L.
4.4 precision
Selecting low-value serum samples (2.50 +/-0.50 mg/L) and high-value serum samples (20.00 +/-5.00 mg/L), and taking the collected samples as reference for detecting concentration values by a chemiluminescence method. The samples were measured 10 times in succession using the reagent, and the coefficient of variation CV was calculated. The test results showed that the coefficient of variation of the low-value sample was 2.87% and the coefficient of variation of the high-value sample was 0.88%, which resulted in good surface precision.
TABLE 11 Glycocholic acid detection reagent precision test results
4.5 recovery
Adding a certain volume of standard solution (specifically a calibrator with glycocholic acid concentration of 80.00 mg/L) into a human serum sample (2.70mg/L), wherein the volume ratio of the standard solution to the human serum sample is 1:9, repeating the detection for 3 times, and calculating the recovery rate. The test results are shown in table 12. The calculated recovery rate is 106.7%, and the accuracy requirement of the measured value is met within 90-110%.
TABLE 12 recovery test
The recovery rate calculation formula is as follows:
in the formula:
R recovering -recovery,%;
v is the volume of the standard solution added;
V 0 -a sample volume of human origin;
c, adding the human source sample into the standard solution to obtain a detection concentration mean value;
C 0 -mean value of detected concentration of human samples;
C S -concentration of standard solution.
4.6 methodological alignment
The glycocholic acid detection kit (latex immunoturbidimetry) of the present example was compared with the glycocholic acid detection kit (chemiluminescence method) on the market, and the collected samples had values measured by the chemiluminescence method. The test was performed on Hitachi 7180 full-automatic biochemical analyzer, and the test was performed on 40 cases of fresh serum samples according to the respective parameters of the reagents, and the results are shown in Table 13. Linear regression analysis is carried out on the measured values, a graph of the comparison result of glycocholic acid detection reagent methodology is shown in FIG. 5, the regression equation y is calculated to be 1.0467x-0.6625, and the correlation coefficient R is calculated 2 0.9926. The result shows that the glycocholic acid detection kit (latex immunoturbidimetry) of the embodiment has good correlation with the chemiluminescence method reagent in the market for testing serum glycocholic acid.
TABLE 13 Glycocholic acid detection reagent latex immunoturbidimetry and chemiluminescence method methodological comparison test result
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (34)
1. A kit comprising a combination of reagents comprising a first reagent, a second reagent; the first reagent comprises glycocholic acid-biotin derivatives formed by binding glycocholic acid and biotin, the second reagent comprises antibody-microsphere complexes formed by binding antibodies capable of being bound with glycocholic acid and latex microspheres and binding protein-microsphere complexes formed by binding proteins and latex microspheres, and the glycocholic acid-biotin derivatives are obtained by reacting glycocholic acid and amino-polyethylene glycol-biotin derivatives; the binding protein comprises at least one of streptavidin and avidin;
the glycocholic acid-biotin derivative is obtained by reacting glycocholic acid with amino-polyethylene glycol-biotin derivative in the presence of a solvent under the action of a condensing agent and a catalyst;
the molecular weight of polyethylene glycol in the amino-polyethylene glycol-biotin derivative is 300-3500.
2. The kit of claim 1, wherein the first reagent comprises 1-200 μ g/mL glycocholic acid-biotin derivative.
3. The kit of claim 1, wherein the first reagent comprises 10-50 μ g/mL glycocholic acid-biotin derivative.
4. The kit of claim 1, wherein the first reagent further comprises at least one of the following components: buffer, electrolyte, stabilizer, promoter, surfactant and preservative.
5. The kit of claim 1, wherein the first reagent further comprises at least one of the following components in concentrations: 10 to 200mM buffer, 0.1 to 1% w/w electrolyte, 0.1 to 15% w/w stabilizer, 0.5 to 1.5% w/w accelerator, 0.05 to 1% w/w surfactant, 0.01 to 0.1% w/w preservative.
6. The kit of claim 1, wherein the first reagent further comprises at least one of the following components in concentrations: 20 to 100mM buffer, 0.1 to 0.5% w/w electrolyte, 0.1 to 0.5% w/w stabilizer, 0.5 to 1.5% w/w accelerator, 0.05 to 0.25% w/w surfactant, and 0.01 to 0.05% w/w preservative.
7. The kit of any one of claims 4 to 6, wherein the buffer comprises at least one of the following buffers: phosphoric acid buffer, tris buffer, 2-morpholinoethanesulfonic acid buffer, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid buffer, boric acid buffer, glycine buffer.
8. The kit according to any one of claims 4 to 6, wherein the electrolyte comprises at least one of the following ions: sodium ion, magnesium ion, potassium ion, calcium ion.
9. The kit according to any one of claims 4 to 6, wherein the electrolyte comprises at least one of the following compounds: sodium chloride, magnesium chloride, potassium chloride and calcium chloride.
10. The kit according to any one of claims 4 to 6, wherein the stabilizer comprises at least one of bovine serum albumin, gelatin, trehalose, lactose, sucrose.
11. The kit of any one of claims 4 to 6, wherein the enhancer comprises polyethylene glycol.
12. The kit of any one of claims 4 to 6, wherein the surfactant comprises at least one of Triton X-100, Triton X-405, Tween 20, Tween 80.
13. The kit of any one of claims 4 to 6, wherein the preservative comprises at least one of sodium azide and ProClin 300.
14. The kit according to claim 11, wherein the polyethylene glycol contained in the accelerator has a molecular weight of 200 to 20000.
15. The kit of claim 1, wherein the first reagent has a pH of 7.0 to 9.0.
16. The kit of claim 1, wherein in the second reagent, the latex microspheres comprise at least one of polystyrene microspheres, polyacrylic microspheres, and polyacrylate microspheres.
17. The kit of claim 1, wherein the latex microspheres have a diameter of 50 to 450 nm.
18. The kit of claim 1, wherein the antibody comprises at least one of a monoclonal antibody, a polyclonal antibody.
19. The kit of claim 1, wherein the antibody comprises at least one of an antibody of murine or rabbit origin.
20. The kit of claim 1, wherein the latex microspheres are carboxylated latex microspheres.
21. The kit of claim 1, wherein the second reagent comprises the following concentrations of components: 0.05-0.2% w/w antibody-microsphere complex, 0.05-0.2% w/w binding protein-microsphere complex.
22. The kit of claim 1, wherein the second reagent further comprises at least one of the following components: buffer, electrolyte, stabilizer, surfactant, preservative.
23. The kit of claim 1, wherein the second reagent further comprises at least one of the following components in concentrations: 10 to 200mM buffer, 0.1 to 1% w/w electrolyte, 0.1 to 15% w/w stabilizer, 0.05 to 1% w/w surfactant, 0.01 to 0.1% w/w preservative.
24. The kit of claim 1, wherein the second reagent further comprises at least one of the following components in concentrations: 20 to 100mM buffer, 0.1 to 0.5% w/w electrolyte, 0.1 to 0.5% w/w stabilizer, 0.05 to 0.25% w/w surfactant, 0.01 to 0.05% w/w preservative.
25. The kit of any one of claims 22 to 24, wherein the buffer comprises at least one of the following buffers: phosphoric acid buffer, tris buffer, 2-morpholinoethanesulfonic acid buffer, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid buffer, boric acid buffer, glycine buffer.
26. The kit of any one of claims 22 to 24, wherein the electrolyte comprises at least one of the following ions: sodium ion, magnesium ion, potassium ion, calcium ion.
27. The kit of any one of claims 22 to 24, wherein the electrolyte comprises at least one of the following compounds: sodium chloride, magnesium chloride, potassium chloride and calcium chloride.
28. The kit of any one of claims 22 to 24, wherein the stabilizer comprises at least one of bovine serum albumin, gelatin, trehalose, lactose, sucrose.
29. The kit of any one of claims 22 to 24, wherein the surfactant comprises at least one of Triton X-100, Triton X-405, Tween 20, Tween 80.
30. The kit of any one of claims 22 to 24, wherein the preservative comprises at least one of sodium azide and ProClin 300.
31. The kit of claim 1, wherein the pH of the second reagent is 7.0 to 9.0.
32. The kit of claim 1, wherein the volume ratio of the first reagent to the second reagent is (1-5): 1.
33. the kit of claim 32, wherein the volume ratio of the first reagent to the second reagent is 4: 1.
34. the kit of claim 1, further comprising a container for separately storing the first reagent and the second reagent.
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| NL7902396A (en) * | 1978-04-26 | 1979-10-30 | Abbott Lab | ANALYSIS OF BALIC ACIDS. |
| US4200625A (en) * | 1978-07-12 | 1980-04-29 | Becton, Dickinson And Company | Immobilized binder for automated assay |
| DE3822750A1 (en) * | 1988-07-05 | 1990-01-11 | Boehringer Mannheim Gmbh | Method for determining a specific binding substance |
| DE4011601A1 (en) * | 1990-04-10 | 1991-10-17 | Boehringer Mannheim Gmbh | HAPTEN-BIOTIN CONJUGATES AND THEIR USE |
| US5747352A (en) * | 1994-05-23 | 1998-05-05 | Beckman Instruments, Inc. | Reagents and methods for the rapid and quantitative assay of pharmacological agents |
| US5627080A (en) * | 1994-07-29 | 1997-05-06 | Beckman Instruments, Inc. | Detergent-facilitated immunoassay for the rapid and quantitative assay of pharmacological agents |
| US5705353A (en) * | 1995-06-07 | 1998-01-06 | Beckman Instruments, Inc. | Method of reducing interferences in assays |
| JP3544962B2 (en) * | 2000-08-21 | 2004-07-21 | エフ．ホフマン−ラ ロシュ アーゲー | Improved binding assay |
| CN105988000B (en) * | 2015-03-02 | 2019-03-19 | 上海铭浦企业管理有限公司 | A kind of kit and method measuring glycocholic acid concentration |
| WO2018011691A1 (en) * | 2016-07-12 | 2018-01-18 | Nestec S.A. | Competitive immunoassay methods |
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