CN113777298A - Latex enhanced immunosuppression method kit for rapidly detecting glycocholic acid, preparation method and detection method - Google Patents
Latex enhanced immunosuppression method kit for rapidly detecting glycocholic acid, preparation method and detection method Download PDFInfo
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides a latex enhanced immunosuppression method kit for rapidly detecting glycocholic acid, a preparation method and a detection method, which are characterized in that: the kit comprises a reagent R1, a reagent R2 and a calibrator; the reagent R1 contains a glycocholic acid antibody, the reagent R2 contains latex microspheres coated by a glycocholic acid-carrier protein antigen complex, and the glycocholic acid-carrier protein antigen adopted in the glycocholic acid antibody process obtained in the reagent R1 is different from the glycocholic acid-carrier protein antigen adopted in the reagent R2. The raw materials involved in the invention are simple and easy to obtain, complex operation is not needed, and the involved reagent has good stability, good uniformity, high accuracy of detection results, simple operation and convenient clinical popularization and application.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnostic medical examination, in particular to a latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid (reagent R1 is added into a serum sample, the serum sample is incubated for 5 minutes at 37 ℃, the absorbance A1 of the sample is measured at a certain wavelength, then reagent R2 is added, and the serum sample is continuously incubated for 5 minutes at 37 ℃).
Background
The alias name of glycocholic acid (Cholyglycine, CG), N- (3,7, 12-trihydroxy-24-carbonylcholan-24-yl) -glycine, is one of the combined cholic acids formed by combining cholic acid and glycine, and is the most major bile acid component in serum in late pregnancy, and the specific structure is shown in fig. 1. When the hepatocytes are damaged, the ability of the hepatocytes to take up CG decreases, resulting in an increase in the amount of CG in the blood. When bile stagnates, bile acid excretion by the liver becomes difficult, and the content of CG in the blood circulation is increased, which also increases the content of CG in the blood. The normal metabolic pathway of CG is the intestine-liver circulation, and CG is synthesized by liver cells, is discharged into a gall bladder through a bile capillary and a bile duct, enters duodenum along with bile, and helps to digest food. 95% of the bile acid is reabsorbed at the terminal of ileum, returned to the liver via portal vein, and taken up by hepatocytes for reuse. In serum, it is predominantly present in protein-bound form, with less than 1% of the total amount spilled into the systemic circulation. Normally, peripheral blood cholesterol levels are very low, and normal adults, whether fasting or postprandial, have stable low levels of serum CG.
When the liver cells are damaged, the CG uptake capacity of the liver cells is reduced, so that the content of CG in blood is increased; when bile stagnates, bile acid excretion by the liver becomes difficult, and the content of CG in the blood circulation is increased, which also increases the content of CG in the blood. Therefore, the determination of serum glycocholic acid (SCG) by RIA (radioimmunoassay) method is one of the sensitive indicators for evaluating the function of hepatocytes and the circulating function of hepatobiliary substances thereof.
Normal human serum glycocholic acid content: 1.3 plus or minus 0.8mg/L, the range is 0.4-2.98 mg/L, and the hepatitis diagnosis lower limit value is less than 3.18 mg/L.
At present, methods for detecting glycocholic acid mainly include chemiluminescence method, Fluorescence Polarization Immunoassay (FPIA), enzyme-linked immunosorbent assay (ELISA), latex-enhanced immunoturbidimetry, and the like. The enzyme-linked immunosorbent assay can accurately measure the concentration of glycocholic acid, but is time-consuming, can only measure in batches, has low efficiency and complex operation, and is not suitable for routine clinical detection. The fluorescence polarization immunoassay method is difficult to popularize clinically due to high reagent cost.
The Turbidimetric inhibition assay (Turbidimetric inhibition assay) overcomes the above disadvantages and can be combined with clinical requirements to develop an automatic instrument with accurate quantification. Therefore, for immunological detection, the immunoturbidimetry inhibition method has the specificity of combining immunological antigens and antibodies, has the characteristic of biochemical reaction, can be applied to a full-automatic biochemical analyzer for detecting a trace amount of substances to be detected in body fluid, particularly blood, and is a practical clinical test technology with application prospect.
The existing latex-enhanced turbidimetric immunoassay method adopts a mode that a glycocholic acid-carrier protein compound is added in a reagent 1, and a latex microsphere coated with a glycocholic acid antibody is added in a reagent 2, and the principle is that the glycocholic acid-carrier protein compound in the reagent 1 and a glycocholic acid micromolecule competitive binding reagent 2 in a sample are coated with the glycocholic acid antibody on the surface of the latex microsphere. This has a problem in that the sensitivity is insufficient, and the glycocholic acid is not detected when the concentration of glycocholic acid in the sample is low, or the low-value sample is not accurately detected.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid. The raw materials involved in the invention are simple and easy to obtain, complex operation is not needed, and the involved reagent has good stability, good uniformity, high accuracy of detection results, simple operation and convenient clinical popularization and application.
In order to solve the technical problem, the technical scheme adopted by the application is as follows: a latex enhanced immunosuppression method kit for rapidly detecting glycocholic acid comprises a reagent R1, a reagent R2 and a calibrator; the reagent R1 contains a glycocholic acid antibody, the reagent R2 contains latex microspheres coated by a glycocholic acid-carrier protein antigen complex, and the glycocholic acid-carrier protein antigen adopted in the glycocholic acid antibody process obtained in the reagent R1 is different from the glycocholic acid-carrier protein antigen adopted in the reagent R2.
Specifically, the latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid comprises a reagent R1, a reagent R2 and a calibrator, wherein:
the reagent R1 mainly comprises the following components: 10-50 mmol/L, pH of 7-8 buffer solution of 4-hydroxyethyl piperazine ethanesulfonic acid; 50-300 mmol/L sodium chloride; 5-40 g/L of polyethylene glycol; 0.1-2.0 mg/L glycocholic acid antibody; 0.01-0.2% of tween 20; 0.02-0.1% of sodium azide;
the reagent R2 mainly comprises the following components: 10-50 mmol/L, pH is a phosphate buffer solution of 7-8; 50-300 mmol/L sodium chloride; 0.1-0.5 mg/L latex microsphere coated by glycocholic acid-carrier protein antigen complex; 0.01-0.2% of tween 20; 0.02-0.1% of sodium azide;
the calibration product comprises the following main components: 20-100 mmol/L phosphate buffer solution; 50-300 mmol/L sodium chloride; 0-80 mg/L glycocholic acid; 0.01-0.2% of tween 20; 0.02-0.1% of sodium azide.
Preferably, the glycocholic acid antibody in the reagent R1 described herein is a glycocholic acid polyclonal antibody (glycocholic acid polyclonal antibody).
Preferably, the glycocholic acid-carrier protein antigen used by the glycocholic acid antibody obtained in reagent R1 of the present application is different from the glycocholic acid-carrier protein antigen obtained in reagent R2; this is because the polyclonal antibody obtained by immunization with glycocholic acid-carrier protein complex may contain the same antibody against the carrier protein, and if reagent R1 is immunized with the polyclonal antibody obtained by immunization with glycocholic acid-carrier protein complex which is the same as that used in reagent R2, the signal value in the process of detecting glycocholic acid is partially the result caused by the antibody against the carrier protein and glycocholic acid-carrier protein complex, and is not completely the result caused by the anti-glycocholic acid polyclonal antibody and glycocholic acid-carrier protein complex, thereby leading to inaccurate detection results.
The application also provides a method for preparing the latex enhanced immunosuppression method kit for rapidly detecting glycocholic acid, which specifically comprises the following steps:
A) firstly, synthesizing a glycocholic acid-carrier protein antigen compound;
B) the synthesized glycocholic acid-carrier protein antigen complex is used as an immunogen to immunize animals to obtain glycocholic acid polyclonal antibody.
Preferably, the animal described herein may be one of a rabbit, sheep, horse, mouse, etc.
Preferably, the preparation of glycocholic acid-carrier protein complexes in step a) of the present application specifically comprises: 1. activating carboxyl groups on glycocholic acid molecules with EDCI or EDCI-NHS; 2. coupling the activated glycocholic acid with carrier protein to obtain glycocholic acid-carrier protein compound.
Further, the carrier protein described herein is selected from the group consisting of: serum albumin, serum gamma globulin, thyroglobulin, ovalbumin or keyhole limpet hemocyanin of various animal origins.
Preferably, the step B) of the present application, wherein the step of obtaining glycocholic acid polyclonal antibody from the animal immunized with the synthesized glycocholic acid-carrier protein complex as an immunogen comprises: 1. mixing the synthesized glycocholic acid-carrier protein complex with an adjuvant; 2. immunizing an animal multiple times with a mixture of glycocholic acid-carrier protein complex and adjuvant; 3. the antiserum was collected.
The invention also provides a method for quantitatively detecting the content of glycocholic acid in human serum, which comprises the following steps: adding the reagent R1 into a serum sample, incubating for 5 minutes at 37 ℃, and measuring the absorbance A1 of the sample at a certain wavelength; then adding a reagent R2, continuing incubation at 37 ℃ for 5 minutes, then measuring absorbance A2 at the wavelength, and calculating delta A-A2-A1; measuring the absorbance value delta A of the calibration solution by the same method; serum glycocholic acid content (mg/L) ═ Δ a sample/. DELTA.a standard × standard solution concentration.
The application has the advantages and beneficial effects that:
1. the reagent R1 contains a glycocholic acid antibody, and the reagent R2 contains a latex microsphere coated by a glycocholic acid-carrier protein antigen complex; the technical scheme is used for detecting by using a latex enhanced immunoturbidimetry (inhibition method), and the principle is as follows: in the detection process, the glycocholic acid small molecules in the sample and a glycocholic acid-carrier protein complex in a reagent R2 compete for binding a glycocholic acid antibody in a reagent R1, the higher the concentration of glycocholic acid in the sample is, the greater the binding degree of the glycocholic acid antibody by the glycocholic acid antibody is, the lower the turbidity degree of an immune complex formed by the glycocholic acid-carrier protein complex coated on the latex microspheres and the glycocholic acid antibody is, the turbidity of a reaction system is in negative correlation with the concentration of glycocholic acid in the sample, and a calibration curve is established by measuring the absorbance at 600nm to calculate the concentration of glycocholic acid in a serum sample; the detection formed by the scheme can effectively improve the sensitivity, and can be detected even if the concentration of the glycocholic acid in the sample is low.
2. The application also sets that the glycocholic acid-carrier protein antigen adopted in the glycocholic acid antibody process obtained in the reagent R1 is different from the glycocholic acid-carrier protein antigen in the reagent R2; this is done because the polyclonal antibody raised against glycocholic acid-carrier protein complex may also have antibodies raised against carrier protein, if reagent R1 is raised against glycocholic acid-carrier protein complex identical to that used in reagent R2, such that a signal value is partly the result of the antibody raised against carrier protein and glycocholic acid-carrier protein complex, not completely the result of the anti-glycocholic acid polyclonal antibody and glycocholic acid-carrier protein complex; therefore, the setting can effectively improve the detection accuracy; for example, if the glycocholic acid-carrier protein complex in the reagent R2 is glycocholic acid-BSA complex, and if the polyclonal antibody in the reagent R1 is simultaneously obtained by immunizing with the glycocholic acid-BSA complex in the reagent R2, it may further contain an antibody against BSA, and if the polyclonal antibody obtained by immunizing with the glycocholic acid-BSA complex in the reagent R1, a part of the signal values are the result of the antibody against BSA and the glycocholic acid-BSA complex, and not the result of the antibody against glycocholic acid and the glycocholic acid-BSA complex, the present application sets forth different antigen preparations for polyclonal antibodies to effectively avoid the above-mentioned drawbacks.
3. The principle of this application is: the glycocholic acid small molecules in the sample are preferentially combined with the glycocholic acid antibody in the reagent R1 for about five minutes to block the sites of the glycocholic acid antibody, so that all glycocholic acid small molecules in the sample can be completely combined with the glycocholic acid antibody in the reagent R1, and the sensitivity is effectively improved. In the existing scheme, a mode that a reagent R1 is added with a glycocholic acid antigen complex, and a reagent R2 is added with a glycocholic acid antibody is adopted, so that glycocholic acid small molecules in a sample cannot be preferentially combined with the glycocholic acid antibody in a reagent R2, but compete with the glycocholic acid antigen complex in a reagent R1 to be combined with the glycocholic acid antibody in the reagent R2, all glycocholic acid small molecules in the sample cannot be completely combined with the glycocholic acid antibody in a reagent R2, and especially when the content of the glycocholic acid small molecules in the sample is low, the glycocholic acid antigen complex in the reagent R1 cannot be competed, so that the sensitivity is reduced.
4. The reagent R1 and the reagent R2 do not contain BSA, the prior art scheme has the condition that the reagent R1 and the reagent R2 are both added with BSA at the same time, if macromolecular proteins such as BSA and the like are added into the reagent, a steric hindrance effect can be formed, the reaction of a glycocholic acid antibody in the reagent R1 and a latex microsphere coated with a glycocholic acid-carrier protein antigen complex in the reagent R2 is prevented, so that a reaction signal is reduced, the combination of a glycocholic acid small molecule in a sample and a glycocholic acid antibody in the reagent R1 is also prevented, so that the sensitivity is reduced, and particularly, the influence is greater when the content of glycocholic acid in the sample is lower; in the scheme of the application, the technical problem existing in the prior art scheme is effectively solved by arranging that the glycocholic acid-carrier protein antigen adopted in the glycocholic acid antibody process obtained in the reagent R1 is different from the glycocholic acid-carrier protein antigen in the reagent R2.
5. The glycocholic acid polyclonal antibody adopted by the application has the following advantages compared with most glycocholic acid monoclonal antibodies sold in the market: 1. the preparation time is short, and the high-titer antibody can be obtained only in 1.0-1.5 months generally; 2. the required cost is low; 3. polyclonal antibodies recognize multiple epitopes and are more tolerant of minor changes in the antigen than monoclonal antibodies.
Drawings
FIG. 1: the molecular structural formula of glycocholic acid.
FIG. 2: the kit provided by the invention is used for measuring a glycocholic acid calibration curve.
FIG. 3: the kit of the invention detects the relativity of glycocholic acid and a certain chemiluminescence imported kit for detecting glycocholic acid.
Detailed Description
The following examples are intended to illustrate the invention in detail, but without restricting it thereto.
The present invention is described in further detail below with reference to examples.
The invention relates to the abbreviation:
BSA: bovine serum albumin;
KLH: keyhole limpet hemocyanin;
EDCI: 1-Ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride 1-Ethyl-3- (3-methylenepropyl) carbodiimide hydrochloride;
NHS: n-hydroxysuccinimide 1-hydroxypyrrolidine-2, 5-dione;
PBS: phosphate buffer.
The content of the components in% referred to in the present application is not particularly specified as mass%.
Example 1
Reagent R1, specifically table 1 below:
TABLE 1 reagent R1 ingredient content
| Composition (I) | Content (concentration) |
| 4-hydroxyethyl piperazine ethanesulfonic acid | 30mM(mmol/L) |
| Sodium chloride | 150mM |
| Polyethylene glycol 6000 | 20g/L |
| Glycocholic acid antibody (prepared in example 6) | 0.5mg/ |
| Tween | |
| 20 | 0.05％ |
| Sodium azide | 0.05％ |
| pH | 8.0 |
。
Example 2
Reagent R2 is specifically as follows in table 2:
TABLE 2 ingredient content of reagent R2
Example 3
Calibration products:
the glycocholic acid small molecules are dissolved and diluted to the concentration of 0, 2.5, 5.0, 20 and 80mg/L by using a calibrator diluent (30mM phosphate buffer, 150mM sodium chloride, 0.05% Tween 20 and 0.05% sodium azide). Then filtering and sterilizing by using a filter membrane of 0.22 mu m, and storing at 2-8 ℃.
Example 4
Synthesis of glycocholic acid-carrier protein antigen complex:
20mg of glycocholic acid was weighed out and dissolved in 10mL of 0.1M PBS (pH 8.0), 10mg of EDCI and 50mg of NHS were added, and the reaction was stirred at room temperature for 1 hour to completely activate the carboxyl group of glycocholic acid. Then 100mg BSA is slowly added in batches, the mixture is magnetically stirred at room temperature for reaction for 16 hours, the mixture is filtered by a filter membrane with the diameter of 0.45 μm, and the product after crosslinking is purified by ultrafiltration or dialysis to obtain the glycocholic acid-BSA compound.
The glycocholic acid-KLH complex was prepared by substituting KLH for BSA, according to the method described above.
Example 5
Preparation of small-particle-size latex particles labeled with glycocholic acid-carrier protein complexes:
latex particles (polystyrene latex particles) with a particle size of 150nm were diluted to 0.8% (mass percentage content) with 10mM MES pH6.1, EDAC 0.2mg/ml was added, and the mixture was reacted at room temperature for 15min, and then added with an equal volume of MES diluent of glycocholic acid-carrier protein complex (prepared in example 4) at a concentration of 4mg/ml while stirring, and the mixture was reacted at room temperature for 2.25 hours with stirring to give a final concentration of 0.4%. Adding 2% BSA, dispersing the latex system by ultrasonic, and sealing for 1h at room temperature. The mixture was centrifuged at 18000rpm for 30min, the supernatant was removed, and the pellet was suspended in a dispersion containing 50mM hepespH7.5, 0.02% Tween 20 and 0.09% sodium azide to dissolve the latex, to a final concentration of 0.2%, and dispersed by sonication.
Example 6
Preparation of glycocholic acid polyclonal antibody: the glycocholic acid-BSA complex synthesized in example 4 was taken and immunized with rabbits selected from New Zealand white rabbits, and 0.5mg glycocholic acid-BSA complex per rabbit was mixed with an equal volume of Freund's complete adjuvant thoroughly, and then subcutaneous multiple injections were performed to the New Zealand white rabbits. And (3) carrying out next immunization every two worries, selecting Freund incomplete adjuvant as the adjuvant after the first immunization, carrying out carotid artery bleeding on the rabbit after the fourth immunization, and collecting antiserum to obtain the glycocholic acid rabbit polyclonal antibody.
According to the present invention, if glycocholic acid-BSA complex is added to reagent R2, the glycocholic acid polyclonal antibody in reagent R1 cannot be immunized with glycocholic acid-BSA complex (instead of the glycocholic acid-KLH complex prepared by substituting KLH for BSA), and should be a polyclonal antibody obtained by immunizing with a complex coupled with another carrier protein. This is done because the polyclonal antibody, and possibly also the anti-BSA antibody, obtained by immunization of the glycocholic acid-BSA complex, if reagent R2 is immunized with the glycocholic acid-BSA complex, such that a signal value is partly a result of the anti-BSA antibody and the glycocholic acid-BSA complex, but not completely of the anti-glycocholic acid polyclonal antibody and the glycocholic acid-BSA complex.
Example 7
The reagent parameters are specifically as follows in table 3:
TABLE 3 reagent parameters
| Wavelength of light | 600 nm/none (main/auxiliary) | Specimen (variants) | 5μL | Reaction direction | Two-point end point method |
| Optical path of cuvette | 1cm | R1 | 200μL | Calibration type | Log-4p |
| Reaction temperature | 37℃ | R2 | 50μL | Reaction direction | Downwards facing |
。
Example 8
Reagent assay the following table 4 specifically:
TABLE 4 reagent parameters
1. Obtaining a calibration curve: according to the method for quantitatively detecting glycocholic acid content in human serum, absorbances with glycocholic acid concentrations of 0, 2.5, 5.0, 20 and 80mg/L are respectively obtained, and a calibration curve is obtained by taking the glycocholic acid concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and is specifically shown in figure 2; the absorbance value of glycocholic acid in the sample is determined by the method, and the content (mg/L) of glycocholic acid in serum is equal to delta A sample/. DELTA.A standard multiplied by standard solution concentration.
2. Correlation experiments: the reagents obtained according to examples 1, 2 and 3, and the methods described in examples 7 and 8, were used to perform a comparative assay of glycocholic acid content in 50 clinical samples of glycocholic acid (chemiluminescence assay) in which a glycocholic acid assay value (assigned to an inlet emitting chemiluminescent reagent) was determined according to the method of the present invention, the results are shown in FIG. 3: the detection result of the figure shows that the kit has high detection accuracy and better consistency with a contrast method. Therefore, the glycocholic acid kit can meet the clinical performance requirements, has strong effectiveness and high safety, can meet the requirements of automatic analysis, and is suitable for timely and accurate detection of large-scale samples.
3. And (3) detecting the precision: specific examples are shown in tables 5 and 6 below.
TABLE 5 precision results of reagent R1 and reagent R2 without Tween 20
| Serum sample (mg/L) | Low value of 1 | Low value of 2 | In | Height of |
| 1 | 1.12 | 2.74 | 5.67 | 18.1 |
| 2 | 1.14 | 2.85 | 5.64 | 18.85 |
| 3 | 1.11 | 2.64 | 5.71 | 17.65 |
| 4 | 1.01 | 2.66 | 5.51 | 17.14 |
| 5 | 1.05 | 2.79 | 5.61 | 18.55 |
| 6 | 1.07 | 2.65 | 5.66 | 17.14 |
| 7 | 0.95 | 2.71 | 5.81 | 16.65 |
| 8 | 1.01 | 2.81 | 5.42 | 17.77 |
| 9 | 0.88 | 2.65 | 5.41 | 18.02 |
| 10 | 1.1 | 2.88 | 5.31 | 18.01 |
| Mean value (mg/L) | 1.04 | 2.74 | 5.58 | 17.79 |
| Standard Deviation (SD) | 0.08 | 0.09 | 0.16 | 0.67 |
| Precision (CV%) | 7.9％ | 3.3％ | 2.8％ | 3.8％ |
TABLE 6 results of precision measurement of reagent R1 and reagent R2 containing Tween 20
According to the detection, the Tween 20 is added, so that the precision is improved well and can reach 2.4%; when the concentration of glycocholic acid in a serum sample is about 1.0mg/L, the precision (CV%) of the method can reach 2.4%, which shows that the method has good repeatability and high accuracy when measuring a sample with very low glycocholic acid concentration.
Claims (9)
1. A latex enhanced immunosuppression method kit for rapidly detecting glycocholic acid is characterized in that: the kit comprises a reagent R1, a reagent R2 and a calibrator; the reagent R1 contains a glycocholic acid antibody, the reagent R2 contains latex microspheres coated by a glycocholic acid-carrier protein antigen complex, and the glycocholic acid-carrier protein antigen adopted in the glycocholic acid antibody process obtained in the reagent R1 is different from the glycocholic acid-carrier protein antigen adopted in the reagent R2.
2. The latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to claim 1, which is characterized in that: comprising a reagent R1, a reagent R2 and a calibrator, wherein:
the reagent R1 mainly comprises the following components: 10-50 mmol/L, pH of 7-8 buffer solution of 4-hydroxyethyl piperazine ethanesulfonic acid; 50-300 mmol/L sodium chloride; 5-40 g/L of polyethylene glycol; 0.1-2.0 mg/L glycocholic acid antibody; 0.01-0.2% of tween 20; 0.02-0.1% of sodium azide;
the reagent R2 mainly comprises the following components: 10-50 mmol/L, pH is a phosphate buffer solution of 7-8; 50-300 mmol/L sodium chloride; 0.1-0.5 mg/L latex microsphere coated by glycocholic acid-carrier protein antigen complex; 0.01-0.2% of tween 20; 0.02-0.1% of sodium azide;
the calibration product comprises the following main components: 20-100 mmol/L phosphate buffer solution; 50-300 mmol/L sodium chloride; 0-80 mg/L glycocholic acid; 0.01-0.2% of tween 20; 0.02-0.1% of sodium azide.
3. The latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to claim 1, which is characterized in that: the glycocholic acid antibody is a glycocholic acid polyclonal antibody.
4. The method for preparing the latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to claim 1, which is characterized in that: the method specifically comprises the following steps:
A) firstly, synthesizing a glycocholic acid-carrier protein antigen compound;
B) the synthesized glycocholic acid-carrier protein antigen complex is used as an immunogen to immunize animals to obtain glycocholic acid polyclonal antibody.
5. The method for preparing the latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to claim 4, wherein the kit comprises: the animal may be rabbit, sheep, horse, or mouse.
6. The method for preparing the latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to claim 4, wherein the kit comprises: the synthesis of glycocholic acid-carrier protein complex in step a) specifically comprises: 1. activating carboxyl groups on glycocholic acid molecules with EDCI or EDCI-NHS; 2. coupling the activated glycocholic acid with carrier protein to obtain glycocholic acid-carrier protein compound.
7. The method for preparing the latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to claim 4, wherein the kit comprises: the carrier protein is selected from: serum albumin, serum gamma globulin, thyroglobulin, ovalbumin or keyhole limpet hemocyanin of various animal origins.
8. The method for preparing the latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to claim 4, wherein the kit comprises: the step B) of taking the synthesized glycocholic acid-carrier protein complex as an immunogen to immunize animals to obtain glycocholic acid polyclonal antibody specifically comprises the following steps: 1. mixing the synthesized glycocholic acid-carrier protein complex with an adjuvant; 2. immunizing an animal multiple times with a mixture of glycocholic acid-carrier protein complex and adjuvant; 3. the antiserum was collected.
9. The method for quantitatively detecting the content of glycocholic acid in human serum by using the latex-enhanced immunosuppression method kit for rapidly detecting glycocholic acid according to any one of claims 1 to 3, which is characterized in that: the method comprises the following steps: adding the reagent R1 into a serum sample, incubating for 5 minutes at 37 ℃, and measuring the absorbance A1 of the sample at a certain wavelength; then adding a reagent R2, continuing incubation at 37 ℃ for 5 minutes, then measuring absorbance A2 at the wavelength, and calculating delta A-A2-A1; measuring the absorbance value delta A of the calibration solution by the same method; serum glycocholic acid content (mg/L) ═ Δ a sample/. DELTA.a standard × standard solution concentration.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105301255A (en) * | 2014-04-18 | 2016-02-03 | 安徽大千生物工程有限公司 | Preparation method of kit for measuring glycocholic acid content in human body |
| CN105988000A (en) * | 2015-03-02 | 2016-10-05 | 上海高尼企业管理服务有限公司 | Reagent kit and method for measuring concentration of glycocholic acid |
| CN112285345A (en) * | 2020-08-31 | 2021-01-29 | 北京九强生物技术股份有限公司 | Glycocholic acid detection kit |
| CN112730844A (en) * | 2019-10-14 | 2021-04-30 | 上海云泽生物科技有限公司 | Teicoplanin detection reagent composition, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105301255A (en) * | 2014-04-18 | 2016-02-03 | 安徽大千生物工程有限公司 | Preparation method of kit for measuring glycocholic acid content in human body |
| CN105988000A (en) * | 2015-03-02 | 2016-10-05 | 上海高尼企业管理服务有限公司 | Reagent kit and method for measuring concentration of glycocholic acid |
| CN112730844A (en) * | 2019-10-14 | 2021-04-30 | 上海云泽生物科技有限公司 | Teicoplanin detection reagent composition, and preparation method and application thereof |
| CN112285345A (en) * | 2020-08-31 | 2021-01-29 | 北京九强生物技术股份有限公司 | Glycocholic acid detection kit |
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