CN108205064B - 25OHD3 detection reagent, kit and detection method thereof - Google Patents
25OHD3 detection reagent, kit and detection method thereof Download PDFInfo
- Publication number
- CN108205064B CN108205064B CN201810261201.5A CN201810261201A CN108205064B CN 108205064 B CN108205064 B CN 108205064B CN 201810261201 A CN201810261201 A CN 201810261201A CN 108205064 B CN108205064 B CN 108205064B
- Authority
- CN
- China
- Prior art keywords
- 25ohd
- enzyme
- reagent
- ohd
- hydroxyvitamin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to an immunological detection method and a reagent thereof of homogeneous enzyme assay technology, which are used for measuring 25-hydroxyvitamin D3(25-hydroxyvitamin D3,25 OHD) in a sample3) The content of (a). The method adopts 25OHD resistance3Specific antibody, enzyme-25 OHD3Complex, using enzyme-25 OHD3Complexes with anti-25 OHD3Immunological reaction of specific antibody, forming antigen-antibody complex, enzyme-25 OHD3The activity of the complex is inhibited, and the amount of the combined enzyme marker is obtained by measuring the change of the enzyme activity in a reaction system, so that 25OHD in a sample to be detected is obtained3The content of (a). The method has the advantages that the method can realize immunological determination like a common biochemical enzyme method, thereby simultaneously determining a plurality of samples on a full-automatic biochemical analyzer, meeting the characteristics of rapidness, large batch and economy of clinical detection, and being beneficial to popularization and application.
Description
Technical Field
The invention relates to directional connection of small molecules and proteins, small molecule components and a detection method, belongs to the field of in-vitro diagnosis of medical immunity, and particularly relates to a 25OHD (extracorporeal immune response)3A detection reagent, a kit and a detection method thereof.
Background
25OHD3(25-hydroxyvitamin D3,25OHD3) is also called 25-hydroxyvitamin D3, and the structural formula is shown in figure 1.
Vitamin D is an essential fat-soluble vitamin, and under the natural state, ultraviolet irradiation and food source supplement are main sources of vitamin D of human bodies. Vitamin D2 and vitamin D3, both self-synthesized and food-derived, enter the liver through blood circulation and are converted to 25OHD2 and 25OHD3, collectively referred to as 25OHD, by the action of vitamin D-25-hydroxylase. The 25OHD is mainly converted into 1, 25-dihydroxy vitamin D with physiological activity by the kidney under the catalysis of 25OHD-1 alpha hydroxylase. Vitamin D, 25OHD and 1, 25-dihydroxyvitamin D have half-lives in humans of 24h, 3 weeks and 4h, respectively. Among them, 25OHD is relatively stable and has a high concentration in a human body, and reflects the total amount of food intake and self-synthesized vitamin D, and the vitamin D conversion ability, so 25OHD is considered as an optimal index for measuring the nutritional status of vitamin D. Maintaining normal vitamin D levels is critical for regulating calcium and phosphorus metabolism. Deficiency of vitamin D can lead to rickets in children, osteoporosis in adults, osteomalacia and myasthenia. In addition, recent studies have shown that vitamin D also has many non-skeletal effects associated with autoimmune diseases, diabetes, cardiovascular diseases, tumors, and the like. Molecular mechanism researches show that vitamin D deficiency directly affects the expression of human genes, and the genes are related to a plurality of diseases such as rheumatoid arthritis and diabetes. Therefore, there is an increasing clinical demand for 25OHD testing, and 25OHD3, a representative component of 25OHD, is of great significance in clinical diagnosis.
At present, methods for quantitative determination of 25OHD3 in vitro mainly include High Performance Liquid Chromatography (HPLC), liquid mass spectrometry (HPLC-MS), Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and latex-enhanced immunoturbidimetry. The high performance liquid chromatography and the liquid chromatography-mass spectrometry are not suitable for clinical diagnosis due to complex operation, high instrument requirement, low flux, high cost, time consumption and labor consumption. The enzyme-linked immunosorbent assay for determining 1,25(OH)2D3 has the disadvantages of complicated operation steps, sensitive matrix effect, long time and poor repeatability, and is not favorable for automation and standardization of instruments. Radioimmunoassay methods present the risk of radioactive contamination to operators, and the reported radioimmunoassay methods have low recovery rates. Several patents are pending in 25OHD3 by latex-enhanced immunoturbidimetry (201510578139.9, 201410328010.8, 201510851361.1), but this methodology has the disadvantage of low detection sensitivity.
The anti-25 OHD3 antibody on the market at present has large cross reaction with vitamin D2, vitamin D3, 1,25(OH)2D3 and the like, and has poor specificity and poor specificity of the prepared reagent. The literature reports that the method for preparing the hapten of the small molecule like 25OHD3 determines the specificity and the affinity of the prepared antibody. The binding site of the small molecule to the carrier protein determines the specificity of the antibody, and ensures that the specific antigenic determinant of the small molecule is not destroyed in the process of hapten coupling. The length of the connecting arm of the carrier protein and the hapten can ensure the full exposure of the small molecules, if the length is too long, the immunogenicity of the small molecules can be greatly reduced, and if the length is too short, the carrier protein can influence the structure of the small molecules. The invention adopts specific connecting arms, and fully exposes the specific epitope of 25OHD3 through the directional selection of connecting sites, thereby obtaining the antibody with high affinity and high specificity.
At present, 25OHD3 detection reagents with high sensitivity and strong specificity, in particular high-quality automatic detection reagents, are lacking in the market.
Therefore, in order to solve the defects and drawbacks of the prior art, it is necessary to research a 25OHD3 detection reagent, a kit and a detection method thereof.
Disclosure of Invention
The present invention has been made in view of at least one of the above problems, and it is an object of the present invention to provide a 25OHD3And (3) detecting the reagent.
According to an aspect of the present invention, there is provided a 25OHD3Detection reagent, characterized by comprising anti-25 OHD3Specific antibody against 25OHD3Specific antibody by 25OHD3Immunogen immunized animal, 25OHD3The immunogen has a structural formula shown in formula 1, wherein the carrier is protein with immunogenicity, the carrier is albumin, hemocyanin or thyroglobulin, and the carrier passes through 25OHD3To 25OHD3R is a linking group-CO- (CH)2)n-COO-, n is any integer between 1 and 10; animals to be immunized include, but are not limited to, rabbits, mice, rats, goats, horses, and alpacas.
The invention adopts specific connection carriers such as rabbit albumin, human albumin and mouse albumin, but does not adopt universal connection carriers such as bovine serum albumin, hemocyanin, thyroglobulin and the like, mainly because of 25OHD coupled with bovine serum albumin, thyroglobulin and the like3The antiserum produced by stimulation also contains antibodies against bovine serum albumin, thyroglobulin and the like, possibly resulting in false positive results; the molecular weight of the hemocyanin is larger, and the hapten obtained by coupling is easy to influence and even shield the antigen epitope of the micromolecule, so that the prepared antibody has low affinity. While rabbits immunized with rabbit serum albumin as a carrier are less likely to develop a target for rabbitsAntibodies to proteins of the daughter itself.
According to another aspect of the present invention, the reagent further comprises 25OHD3Enzyme-labeled conjugates in which the enzyme is glucose-6-phosphate dehydrogenase (EC 1.1.1.49), malate dehydrogenase (EC 1.1.1.37), lysozyme (EC3.2.1.17), hexokinase (EC 2.7.1.1), β -D-galactosidase (EC 3.2.1.23) or amylase (EC3.2.1.2), preferably glucose-6-phosphate dehydrogenase (G6PDH), the G6PDH having a high catalytic efficiency for a specific substrate, and the reagents prepared have an extremely high sensitivity.
According to another aspect of the present invention, the 25OHD325OHD for G6PDH enzyme-labeled conjugate3The derivative is coupled with glucose-6-phosphate dehydrogenase to form G6PDH and 25OHD3And (3) cross-linking the derivative.
According to another aspect of the invention, G6PDH and 25OHD3The coupling mode of the derivative comprises a polybasic acid anhydride method, a carbodiimide method, a mixed acid anhydride method or an N-hydroxysuccinimide method.
According to another aspect of the invention, G6PDH and 25OHD3G6PDH enzyme activity protective agent in the derivative coupling process is glucose-6-phosphate, NADH, NADPH; the concentration of the protective agent is 0.01mM-500 mM.
According to another aspect of the present invention, there is also provided a method for measuring 25OHD using the above reagent3A method of concentration characterized by comprising 25OHD3The biological sample of (2) is the same as the 25OHD3Competitive binding of enzyme-labelled conjugates to anti-25 OHD3A specific antibody; 25OHD3Enzyme-labeled conjugates with anti-25 OHD3The activity of the catalytic substrate glucose-6-phosphate is reduced or lost after the specific antibody forms a complex; by measuring 25OHD3Determination of the amount of 25OHD in a biological sample by measuring the amount of NADH, the product of the enzymatic conjugate catalyzing the production of glucose-6-phosphate3And (4) concentration.
According to another aspect of the present invention, the amount of NADH produced by the reaction is finally determined by a spectrophotometer or an automated biochemical analyzer, and the less the amount of NADH produced at the time of data analysis, the 25OHD in the sample is measured3The more the amount of the compound is, the more the biological sample is serum,Plasma, saliva or urine.
According to another aspect of the present invention, there is also provided a method for detecting 25OHD, comprising the above reagent3The kit is characterized by comprising a reagent R1, a reagent R2, a buffer, a stabilizer and 25OHD3A standard liquid or dry powder kit wherein reagent R1 comprises an anti-25 OHD3Specific antibodies and homogeneous enzyme substrates, reagent R2 comprising 25OHD3An enzyme-labeled conjugate.
According to another aspect of the present invention, the buffer solution of the reagent R1 is TRIS buffer solution, glycine buffer solution, citric acid buffer solution or phosphoric acid buffer solution, and the pH value of the buffer solution is 4.0-7.0; the homogeneous enzyme substrate contained in R1 is glucose-6-phosphate, and the cofactor is NAD+(ii) a The concentration of the substrate in the R1 is 0.01-500mM, and the concentration of the cofactor is 0.01-500 mM.
According to another aspect of the invention, the buffer of reagent R2 is TRIS buffer, glycine buffer, citrate buffer or phosphate buffer, the pH of the buffer being between 5.0 and 10.0.
According to another aspect of the invention, the stabilizer is BSA, glycerol, mannitol, sodium azide, EDTA, PEG, sucrose or trehalose; the 25OHD3The standard substance is added with 25OHD3A liquid of pure human serum.
Compared with the prior art, the invention has the beneficial effects that:
25OHD of the invention3The immunogen has strong specificity and high immunogenicity, and the prepared anti-25 OHD3The specific antibody has strong specificity and high titer, and is similar to vitamin D2, vitamin D3 and 1,25(OH)2D3No cross reaction exists; containing the above 25 OHD-resistant3The homogeneous enzyme immunoassay reagent of the specific antibody can conveniently, quickly and accurately determine 25OHD in a sample3Content, and can simultaneously determine a plurality of samples on a full-automatic biochemical analyzer to realize 25OHD3The high-flux rapid determination has high accuracy, strong specificity, and greatly improved precision and detection efficiency compared with the prior art, and simultaneously realizes the full detection processThe automation is realized, the requirement on detection personnel is not high, and the realization and the popularization and the use are easy;
the invention is a homogeneous immunoassay method, has the advantages of high detection speed, simple operation, high sensitivity and strong specificity, can realize high-flux rapid detection of small molecular substances on a full-automatic biochemical analyzer, and has higher application value.
Drawings
FIG. 1 is a 25OHD3Structural formula of (1).
FIG. 2 is a 25OHD used in the present invention3The biochemical structural formula of the derivative is shown in the figure.
FIG. 3 is a 25OHD according to a preferred embodiment of the present invention3Schematic diagram of derivative synthesis steps.
FIG. 4 is a graph of OD340 absorbance at different time points versus reaction rate at different standard concentrations in accordance with a preferred embodiment of the present invention.
FIG. 5 is a schematic diagram of a correlation analysis according to a preferred embodiment of the present invention.
Detailed Description
The best mode for carrying out the present invention will be described in detail with reference to the accompanying drawings, wherein the detailed description is for the purpose of illustrating the invention in detail, and is not to be construed as limiting the invention, as various changes and modifications can be made therein without departing from the spirit and scope thereof, which are intended to be encompassed within the appended claims.
Preferably, the present invention provides a 25OHD3Detection reagent, characterized by comprising anti-25 OHD3Specific antibody against 25OHD3Specific antibody by 25OHD3Immunogen immunized animal, 25OHD3The immunogen has a structural formula shown in formula 1, wherein the carrier is protein with immunogenicity, the carrier is albumin, hemocyanin or thyroglobulin, and the carrier passes through 25OHD3To 25OHD3R is a linking group-CO- (CH)2)n-COO-, n is any integer between 1 and 10; the animals to be immunized include, but are not limited to, rabbit, mouse, and ratRats, goats, horses, and alpacas.
Advantageously, the present invention employs specific ligation vectors rabbit albumin, human albumin and mouse albumin, rather than universal ligation vectors such as bovine serum albumin, hemocyanin and thyroglobulin, among others, primarily because of the 25OHD coupled to bovine serum albumin and thyroglobulin, among others3The antiserum produced by stimulation also contains antibodies against bovine serum albumin, thyroglobulin and the like, possibly resulting in false positive results; the molecular weight of the hemocyanin is larger, and the hapten obtained by coupling is easy to influence and even shield the antigen epitope of the micromolecule, so that the prepared antibody has low affinity. Rabbits immunized with rabbit serum albumin as a carrier are less likely to produce antibodies to the rabbit's own proteins.
Preferably, the reagent is characterized by further comprising 25OHD3An enzyme-labeled conjugate in which the enzyme is glucose-6-phosphate dehydrogenase (EC 1.1.1.49), malate dehydrogenase (EC 1.1.1.37), lysozyme (EC3.2.1.17), hexokinase (EC 2.7.1.1), β -D-galactosidase (EC 3.2.1.23) or amylase (EC3.2.1.2), preferably glucose-6-phosphate dehydrogenase (G6PDH), the G6PDH having a high catalytic efficiency for a specific substrate, and the reagent prepared has an extremely high sensitivity.
Preferably, the 25OHD325OHD for G6PDH enzyme-labeled conjugate3The derivative is coupled with glucose-6-phosphate dehydrogenase to form G6PDH and 25OHD3And (4) crosslinking the derivative.
Preferably, G6PDH and 25OHD3The coupling mode of the derivative comprises a polybasic acid anhydride method, a carbodiimide method, a mixed acid anhydride method or an N-hydroxysuccinimide method.
Preferably, G6PDH and 25OHD3G6PDH enzyme activity protective agent in the derivative coupling process is glucose-6-phosphate, NADH, NADPH; the concentration of the protective agent is 0.01mM-500 mM.
Preferably, the invention also provides a method for determining 25OHD by using the reagent3A method of concentration characterized by comprising 25OHD3The biological sample of (2) is the same as the 25OHD3Competitive binding of enzyme-labelled conjugatesAnti-25 OHD3A specific antibody; 25OHD3Enzyme-labeled conjugates with anti-25 OHD3The activity of the catalytic substrate glucose-6-phosphate is reduced or lost after the specific antibody forms a complex; by measuring 25OHD3Determination of the amount of 25OHD in a biological sample by measuring the amount of NADH, the product of the enzymatic conjugate catalyzing the production of glucose-6-phosphate3And (4) concentration.
Advantageously, the method of the invention employs 25OHD resistance3Specific antibody, enzyme-25 OHD3Complex, using enzyme-25 OHD3Complexes with anti-25 OHD3Immunological reaction of specific antibody, forming antigen-antibody complex, enzyme-25 OHD3The activity of the complex is inhibited, and the amount of the combined enzyme marker is obtained by measuring the change of the enzyme activity in a reaction system, so that 25OHD in a sample to be detected is obtained3The content of (a). The method has the advantages that the method can realize immunological determination like a common biochemical enzyme method, thereby simultaneously determining a plurality of samples on a full-automatic biochemical analyzer, meeting the characteristics of rapidness, large batch and economy of clinical detection, and being beneficial to popularization and application.
Preferably, the amount of NADH produced by the reaction is finally determined by a spectrophotometer or an automated biochemical analyzer, and the less the amount of NADH produced at the time of data analysis, the 25OHD in the sample is determined3The greater the amount of (a), the more the biological sample is serum, plasma, saliva or urine.
Preferably, the present invention also provides a reagent for detecting 25OHD, comprising the above reagent3The kit is characterized by comprising a reagent R1, a reagent R2, a buffer, a stabilizer and 25OHD3A standard liquid or dry powder kit wherein reagent R1 comprises an anti-25 OHD3Specific antibodies and homogeneous enzyme substrates, reagent R2 comprising 25OHD3An enzyme-labeled conjugate.
Preferably, the buffer solution of the reagent R1 is TRIS buffer solution, glycine buffer solution, citric acid buffer solution or phosphoric acid buffer solution, and the pH value of the buffer solution is 4.0-7.0; the homogeneous enzyme substrate contained in R1 is glucose-6-phosphate, and the cofactor is NAD+(ii) a The concentration of the substrate in the above R1 is 0.01-500mM, and adjuvantThe concentration of the cofactor is 0.01-500 mM.
Preferably, the buffer solution of the reagent R2 is TRIS buffer solution, glycine buffer solution, citric acid buffer solution or phosphate buffer solution, and the pH value of the buffer solution is 5.0-10.0.
Preferably, the stabilizer is BSA, glycerol, mannitol, sodium azide, EDTA, PEG, sucrose or trehalose; the 25OHD3The standard substance is added with 25OHD3A liquid of pure human serum.
Preferably, the present invention provides a 25OHD3The homogeneous immunoassay kit has the following detection principle: containing 25OHD3The biological sample of (2) is the same as the 25OHD3Competitive binding of G6 PDH-enzyme-labeled conjugates to anti-25 OHD3A specific antibody; 25OHD3-G6PDH enzyme-labeled conjugate and anti-25 OHD3After the specific antibody forms a complex, the activity of the specific antibody for catalyzing a substrate glucose-6-phosphate is reduced or lost, and the amount of a reaction product NADH is also reduced; determination of 25OHD in samples3Higher concentration of (A) competitive anti-25 OHD3The more specific antibody, the anti-25 OHD3Specific antibody pair 25OHD3The lower the enzyme activity inhibition of the G6PDH enzyme-labeled conjugate, the more NADH (nicotinamide adenine dinucleotide) is generated in the reaction; calculation of amount of NADH measurement 25OHD in measurement sample3And (4) content.
Preferably, wherein 25OHD is prepared3Immunogen for preparing anti-25 OHD3Specific antibodies, preferably using 25OHD3The hydroxyl group of (A) is crosslinked with a carrier protein, preferably the linking group-CO- (CH)2)n-COO-, preferably n is 2 to 6; the carrier protein is preferably rabbit albumin (RSA); the immunized animal is preferably New Zealand white rabbit; the chemical coupling mode is preferably a succinic anhydride method and a carbodiimide method; 25 OHD-resistant prepared by the preferred method3Specific antibody pair 25OHD3High affinity and good specificity, and has the same functions as vitamin D2, vitamin D3 and 1,25(OH)2D3No cross reaction occurred.
Preferably, wherein 25OHD is prepared3Enzyme-labeled conjugates for determination of 25OHD3An assay kit, preferably the enzyme is glucose-6-phosphate dehydrogenase (EC 1.1.1.49); preferred use of25OHD3Crosslinking the hydroxy derivative with glucose-6-phosphate dehydrogenase; 25OHD3The crosslinking method of the derivative and glucose-6-phosphate dehydrogenase is preferably a mixed anhydride method; 25OHD3The enzyme activity protectant crosslinked with glucose-6-phosphate dehydrogenase is preferably glucose-6-phosphate and NADH; the concentration of glucose-6-phosphate as the protective agent is preferably 10mM-100mM, and the concentration of NADH is preferably 10-100 mM.
Preferably, 25OHD thereof3The R1 reagent buffer solution in the determination kit is preferably TRIS buffer solution; the pH is preferably 5.0-7.0; the concentration of glucose-6-phosphate is preferably 10-100 mM; the NAD + concentration is preferably 10-100 mM.
Preferably, 25OHD thereof3The R2 reagent buffer solution in the determination kit is preferably TRIS buffer solution; the pH is preferably 7.0 to 9.0.
Preferably, the stabilizer is 0.2-0.5% bovine serum albumin, 5-20% glycerol, 1-5% mannitol, 0.001-0.01% sodium azide, 0.1-2mM EDTA, 0.1-1% PEG20000, 1-10% sucrose, 1-10% trehalose.
Preferably, the 25OHD described therein3The standard substance is added with 25OHD3Pure human serum or other serum matrix-like liquids. Preferably, the 25OHD is selectively added3A liquid of pure human serum.
Preferably, the invention also provides a method for utilizing 25OHD3The detection method of the immunodetection reagent is characterized by comprising the following steps of:
1) mixing the measurement sample with anti-25 OHD3Specific antibody contact;
2) according to 25OHD in a sample to be tested3And 25OHD resistance3Binding of specific antibodies;
preferably, the rate of NADH production is measured using an autoanalyzer to determine 25OHD in the sample3The content of (A);
the sample to be detected is serum, plasma, saliva or urine.
It can be understood that the invention provides a method for detecting 25OHD in a sample to be detected safely, quickly, efficiently, sensitively and accurately3Content of 25OHD3Homogeneous enzyme immunoassay reagent andthe preparation method can be used together with various automatic biochemical analyzers, and has low requirements on detection personnel.
Example one 25OHD3Synthesis of derivatives and confirmation of their structures
25OHD used in the following examples3The biochemical structure of the derivative is shown in figure 2.
Referring to FIG. 3, the 25OHD3The specific synthetic steps of the derivative are as follows:
10.8mg of 25OHD were weighed3And 18mg succinic anhydride, adding 3.0mL anhydrous pyridine, refluxing at 80 deg.C for 2h, rotary evaporating to remove pyridine, adding 5mL ultrapure water, extracting with 5mL × 4 ethyl acetate, combining organic phases, and rotary drying under reduced pressure to obtain white solid, i.e. 25OHD3And (3) derivatives.
Carrying out structural identification on the white solid purified product
1) The obtained derivatives were analyzed and identified by chromatography/mass spectrometry (LCMS) using an Agilent tandem quadrupole mass spectrometer LC/MSD1200 series, the ion source being in positive or negative ionization mode. The chromatographic column specification is Agilent ZORBAX Eclipse XDB-C18(50mm × 2.1mm, 1.8 μm), the column temperature is 25 deg.C, the flow rate is 0.8mL/min, the detection wavelength is 224nm, the mobile phase is acetonitrile-water, and gradient elution is carried out. LCMS results showed: purity > 95%; retention time 3.431 min.
From the above results, it was confirmed that the white solid compound was 25OHD as shown in FIG. 23And (3) derivatives.
Example two: RSA-25OHD3Immunogen synthesis
RSA-25OHD3The immunogen is composed of rabbit protein RSA and 25OHD shown in formula 13-CO- (CH) of a derivative2) n-COO-group, in this example, the synthesis of the immunogen is described in detail by taking n ═ 2 as an example, and the specific steps are as follows:
1) dissolving 60mg of RSA in 6ml of 0.1M PBS (pH8.3), and placing the solution in a beaker A;
2) 25mg of 25OHD3Derivative, 0.6ml of dimethylformamide DMF, 0.5ml of ethanol, 1ml of 10mM MES buffer (pH 5.0), 68mg of 1-ethyl-3- (N-dimethylformamide)-3-dimethylaminopropyl) carbodiimide, 22mg of N-hydroxythiosuccinimide were dissolved in a beaker B and reacted with stirring at room temperature for 1 hour.
3) Slowly dripping the solution in the beaker B into the beaker A to obtain a mixed solution, and stirring the mixed solution at the temperature of between 2 and 8 ℃ overnight; dialyzing and purifying the stirred mixed solution by neutral PBS buffer solution to obtain RSA-25OHD3Immunogen, stored at-70 ℃.
Similarly, when n is other integers within the range of 1-10, 25OHD shown in the formula 1 can be prepared by the same method3An immunogen. Of course, the carrier is still an immunogenic protein, and may be bovine serum albumin, hemocyanin (KLH) and thyroglobulin.
The invention only discloses that the connecting group R is-CO- (CH)2) 25OHD of n-COO-, and n ═ 23Synthesis examples of derivatives and related subsequent experiments were carried out, the linking group mainly functions to link the small molecule derivatives to the carrier, but the different length connecting arm pairs 25OHD3The structure exposure degree is different; immunogenicity and synthesized 25OHD3Derivatives molecular Structure and selected vector species Using 25OHD with different values of n325OHD prepared from derivatives3The immunogens all have different immunogenicity, and the specific antibodies prepared correspondingly all have different properties.
Example three: anti-25 OHD3Preparation of specific antibodies
RSA-25OHD prepared as above3The immunogen is used for immunizing a New Zealand white rabbit by a conventional method, and antiserum is taken after the immunization is strengthened, and the specific steps are as follows:
the synthesized RSA-25OHD was treated with PBS3Immunogen is diluted to 2.0mg/ml to obtain antigen solution, and then 1.0ml antigen solution is mixed with Freund's complete adjuvant to carry out subcutaneous multipoint immunization on the back of New Zealand white rabbits.
After 2-3 weeks, the New Zealand white rabbits were immunized once with 1.0ml of the same antigen solution and Freund's incomplete adjuvant, and then immunized once every three weeks for a total of 4 times.
Collecting the above New Zealand white rabbitsSeparating and purifying blood to obtain 25 OHD-resistant3A specific antibody.
Example four: 25OHD3Preparation of-glucose-6-phosphate dehydrogenase-labeled conjugate
1) Preparation of glucose-6-phosphate dehydrogenase (G6PDH) solution:
a. 38mg of 200KU G6PDH was weighed and dissolved in 10ml of a solution containing 0.1M Tris and 2mM MgCl at room temperature2And 0.9% NaCl, pH 8.3;
b. adding 390mg of nicotinamide adenine dinucleotide NADH in a reduced state, 250mg of glucose-6-phosphate (G-6-P) and 1mL of carbitol;
c. 2.5mL of dimethyl sulfoxide was added dropwise;
2)25OHD3activation of derivatives
a) Weighing 50mg of 25OHD under nitrogen blowing condition3Derivative, dissolved in 800 μ L DMF;
b) reducing the temperature of the solution to-10 ℃;
c) adding 7.2 mu L of tributylamine;
d) 4.3 μ L of isobutyl chloroformate was added;
e) stirring at-10 ℃ for 1 hour;
3) g6PDH and 25OHD3Coupling of derivatives
a) 25OHD to be activated as described above3The derivative solution is slowly added into the dissolved G6PDH solution;
b) stirring at 2-8 deg.C overnight;
4) purification of the product
The ligation product was purified by dialysis to obtain the final product glucose-6-phosphate dehydrogenase-25 OHD3The enzyme-labeled conjugate is stored at 2-8 ℃.
Example five: 25OHD3Preparation of immunoassay reagent
25OHD3A homogeneous enzyme immunoassay reagent comprising: the above-mentioned 25OHD resistance3Specific antibody for detecting anti-25 OHD3Specific antibody-25 OHD3An enzymatic reagent of the complex comprising: 25OHD3-G6PDH enzyme-labeled conjugate and substrate for the enzyme.
25OHD3Before the homogeneous enzyme immunoassay reagent is used, in order to avoid the enzyme-labeled conjugate in the enzyme reagent from reacting with the enzyme substrate, the enzyme-labeled conjugate and the enzyme substrate are separately placed, so that the enzyme substrate is combined with the above-mentioned anti-25 OHD3The specific antibodies are mixed together. That is, 25OHD3The homogeneous enzyme immunoassay reagent comprises two separately arranged reagents, specifically as follows:
preparation of reagent R1: dissolving 20mM NAD, 30mM glucose-6-phosphate G6P, and 55mM Tris buffer (pH 8.0) to obtain a homogeneous enzyme substrate; adding 1% -0.1% of anti-25 OHD3Specific antibody to the above-mentioned homogeneous enzyme substrate, the specific ratio in this example was 0.5%.
Preparation of reagent R2: tris buffer 0.1-0.1M, pH ═ 8.5, to which 25OHD prepared at 0.01% -1% was added3G6PDH enzyme-labeled conjugate, in this example the specific ratio was 0.05%.
The above 25OHD3A method of using an immunoassay reagent comprising the steps of:
1) mixing the measurement sample with anti-25 OHD3Specific antibody contact;
2) according to 25OHD in a sample to be tested3And 25OHD resistance3Determination of binding of specific antibodies 25OHD in samples Using enzymatic reagents3The content of (A);
specifically, in the detection, a sample to be detected is added to the reagent R1, and 25OHD in the sample to be detected3With anti-25 OHD in reagent A3Specific binding of specific antibody to generate anti-25 OHD3Specific antibody-25 OHD3A complex; further addition of reagent R2 at 25OHD in reagent R23-G6PDH enzyme-labeled conjugate and anti-25 OHD in reagent R13Specific binding of specific antibody, not with anti-25 OHD3Specific antibody-bound 25OHD3the-G6 PDH enzyme-labeled conjugate is mixed and contacted with a substrate to carry out enzymatic reaction to form the detection of the anti-25 OHD3Specific antibody-25 OHD3Enzyme reagent of complex, enzyme reagent according to 25OHD in sample to be tested3The above-mentioned 25OHD resistance3Determination of the binding of specific antibodies in a sample to be tested25OHD3The content of (a).
Due to 25OHD3-G6PDH enzyme-labeled conjugate and 25OHD in a sample to be tested3Competitive binding against 25OHD3Specific antibody, therefore, 25OHD in the sample to be tested3The larger the amount of (A), the free 25OHD in the homogeneous enzyme solution3The greater the amount of-G6 PDH-enzyme-labeled conjugate, the faster the enzymatic reaction, resulting in OD340The faster the rise.
The sample to be tested is a physiological sample, such as serum, plasma, urine, saliva, etc.
Preferably, the sample to be tested is serum or plasma.
Example six: 25OHD3Homogeneous enzyme immunoassay
1. And (4) obtaining a standard curve, and setting reaction parameters of a Hitachi 7060 full-automatic biochemical analyzer (see table 1). The operation process is as follows: adding the reagent R1, adding the standard substance, incubating at 37 ℃ for 5min, and finally adding the reagent R2. After the reagent R2 was added, the OD340 absorbance at different time points was measured, and the reaction rate at different concentrations of the standard was calculated. As shown in fig. 4.
TABLE 1 Hitachi 7060 reaction parameters of full-automatic biochemical analyzer
25OHD obtained by the invention3And (3) clinically collecting 10 samples according to a standard curve of the homogeneous enzyme immunoassay reagent, repeatedly detecting for 10 times, calculating a mean value, and calculating a variation coefficient. When the coefficient of variation is less than 10%, the kit has better repeatability.
TABLE 2 precision test
| Serum | Mean value (ng/mL) | Coefficient of variation |
| Sample 1 | 1.5 | 5.9％ |
| Sample 2 | 2.8 | 3.1％ |
| Sample 3 | 4.3 | 4.5％ |
| Sample No. 4 | 6.1 | 6.0％ |
| Sample No. 5 | 8.8 | 2.0％ |
| Sample No. 6 | 16.1 | 2.7％ |
| Sample 7 | 23.8 | 3.3％ |
| Sample 8 | 59.9 | 5.2％ |
| Sample 9 | 73.2 | 3.2％ |
| Sample 10 | 90.5 | 3.8％ |
As can be seen from the data of the graph, the detected variation coefficients of the ten samples are all less than 10%, which indicates that 25OHD3The homogeneous enzyme immunoassay kit has good repeatability and high precision.
Example seven: interferent testing
Several common drugs, or endogenous substances in the human body, were selected for the interference test. The results are shown in Table 3
TABLE 3 common interferent test
25OHD3The cross reaction rate of the homogeneous enzyme immunoreagent and the interferent is less than 0.1 percent, which indicates that the 25OHD prepared by the invention3The antibody specifically recognizes 25OHD3And the homogeneous reagent enables accurate determination of 25OHD3The content of (a).
Example eight: correlation analysis
Will be the 25OHD of the invention3Homogeneous enzyme immunoassay reagent and high performance liquid chromatography for measuring 25OHD in human serum3The results of the process comparisons are shown in Table 4.
The correlation curve is shown in fig. 5, and it can be seen that the two fit well.
In conclusion, the beneficial effects of the invention are as follows:
25OHD of the invention3The immunogen has strong specificity and high immunogenicity, and the prepared anti-25 OHD3The specific antibody has strong specificity and high titer, and is similar to vitamin D2, vitamin D3 and 1,25(OH)2D3No cross reaction exists; containing the above 25 OHD-resistant3The homogeneous enzyme immunoassay reagent of the specific antibody can conveniently, quickly and accurately determine 25OHD in a sample3Content, and can simultaneously determine a plurality of samples on a full-automatic biochemical analyzer to realize 25OHD3The high-flux rapid detection has high accuracy, strong specificity, greatly improved precision and detection efficiency compared with the prior art, realizes the full automation of the detection process, has low requirement on detection personnel, and is easy to realize, popularize and use;
the invention is a homogeneous immunoassay method, has the advantages of high detection speed, simple operation, high sensitivity and strong specificity, can realize high-flux rapid detection of small molecular substances on a full-automatic biochemical analyzer, and has higher application value.
It should be noted that the above-mentioned embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent flow transformations made by using the contents of the specification and the drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (3)
1. The detection reagent for 25 hydroxyvitamin D3 is characterized by comprising an anti-25 hydroxyvitamin D3 specific antibody, wherein the anti-25 hydroxyvitamin D3 specific antibody is obtained by immunizing a rabbit with a 25 hydroxyvitamin D3 immunogen, and the structural formula of the 25 hydroxyvitamin D3 immunogen is shown in formula 1:
in the formula 1, the carrier is immunogenic protein, the carrier is rabbit albumin, the carrier is connected to 25 hydroxy vitamin D3 through a hydroxy group of 25 hydroxy vitamin D3, and R is a connecting group-CO- (CH)2)2-COO-；
The reagent also comprises a 25-hydroxy vitamin D3-enzyme-labeled conjugate, and the enzyme in the enzyme-labeled conjugate is glucose-6 phosphate dehydrogenase.
2. The reagent according to claim 1, wherein the glucose-6-phosphate dehydrogenase activity-protecting agent in the coupling of glucose-6-phosphate dehydrogenase with 25-hydroxyvitamin D3 derivative is glucose-6-phosphate and NADH; the concentration of the protective agent is 10mM-100 mM.
3. A kit for the detection of 25 hydroxyvitamin D3 comprising the reagent of any one of claims 1-2, wherein the kit is a liquid or dry powder kit comprising reagent R1, reagent R2, a buffer, a stabilizer, a 25 hydroxyvitamin D3 standard, wherein reagent R1 comprises an anti-25 hydroxyvitamin D3 specific antibody and a homogeneous enzyme substrate, and reagent R2 comprises a 25 hydroxyvitamin D3-enzyme labeled conjugate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810261201.5A CN108205064B (en) | 2018-03-28 | 2018-03-28 | 25OHD3 detection reagent, kit and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810261201.5A CN108205064B (en) | 2018-03-28 | 2018-03-28 | 25OHD3 detection reagent, kit and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108205064A CN108205064A (en) | 2018-06-26 |
| CN108205064B true CN108205064B (en) | 2020-08-21 |
Family
ID=62606692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810261201.5A Active CN108205064B (en) | 2018-03-28 | 2018-03-28 | 25OHD3 detection reagent, kit and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108205064B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114924086A (en) * | 2022-03-29 | 2022-08-19 | 北京世纪沃德生物科技有限公司 | 25-hydroxyvitamin D3 determination kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1592170A (en) * | 1977-01-07 | 1981-07-01 | Hoffmann La Roche | 1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay |
| JPS5892656A (en) * | 1981-11-30 | 1983-06-02 | Teijin Ltd | Novel vitamin d3 derivative and its preparation |
| JPH0641058A (en) * | 1992-07-21 | 1994-02-15 | Asahi Chem Ind Co Ltd | New vitamin d derivatives containing residue on hydroxyl group at 25-position, their production and their use |
| CN101273062A (en) * | 2005-09-29 | 2008-09-24 | 霍夫曼-拉罗奇有限公司 | Antibodies against 25-hydroxyvitamin D |
| CN103857698A (en) * | 2011-05-20 | 2014-06-11 | 西门子医疗保健诊断公司 | Antibodies to 25-hydroxyvitamin D2 and D3 and uses thereof |
| CN105753969A (en) * | 2016-04-06 | 2016-07-13 | 苏州博源医疗科技有限公司 | Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method |
| CN106290903A (en) * | 2016-08-03 | 2017-01-04 | 北京德奥平生物技术有限公司 | A kind of ADMA immunization measures reagent and detection method |
-
2018
- 2018-03-28 CN CN201810261201.5A patent/CN108205064B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1592170A (en) * | 1977-01-07 | 1981-07-01 | Hoffmann La Roche | 1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay |
| JPS5892656A (en) * | 1981-11-30 | 1983-06-02 | Teijin Ltd | Novel vitamin d3 derivative and its preparation |
| JPH0641058A (en) * | 1992-07-21 | 1994-02-15 | Asahi Chem Ind Co Ltd | New vitamin d derivatives containing residue on hydroxyl group at 25-position, their production and their use |
| CN101273062A (en) * | 2005-09-29 | 2008-09-24 | 霍夫曼-拉罗奇有限公司 | Antibodies against 25-hydroxyvitamin D |
| CN103857698A (en) * | 2011-05-20 | 2014-06-11 | 西门子医疗保健诊断公司 | Antibodies to 25-hydroxyvitamin D2 and D3 and uses thereof |
| CN105753969A (en) * | 2016-04-06 | 2016-07-13 | 苏州博源医疗科技有限公司 | Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method |
| CN106290903A (en) * | 2016-08-03 | 2017-01-04 | 北京德奥平生物技术有限公司 | A kind of ADMA immunization measures reagent and detection method |
Non-Patent Citations (2)
| Title |
|---|
| L.J.FRAHER, et al..Radioimmunoassay of 1,25-dihydroxy vitamin D2: studies on the metabolism of vitamin D2 in man..《Clinical Endocrinology》.1983,第151-165页. * |
| Radioimmunoassay of 1,25-dihydroxy vitamin D2: studies on the metabolism of vitamin D2 in man.;L.J.FRAHER, et al.;《Clinical Endocrinology》;19830930;第151-165页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108205064A (en) | 2018-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2374942T3 (en) | RELEASE REAGENT FOR VITAMIN D COMPOUNDS. | |
| CN104447984B (en) | Docetaxel immunogene, anti-Docetaxel specific antibody and Docetaxel detection reagent | |
| EP2962105B1 (en) | Methods and reagents for determining isomeric analytes | |
| CN104788560B (en) | Ciclosporin A immunogene, anti-Ciclosporin A specific antibody and Ciclosporin A detection reagent | |
| CN110950820A (en) | Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent | |
| CN107436351B (en) | Detection kit for reconstituted milk adulteration in fresh milk and detection method thereof | |
| CN109824599B (en) | Albendazole hapten as well as preparation method and application thereof | |
| EP0070033B1 (en) | Quantitative determination of adenosine | |
| JP3392868B2 (en) | Competitive immunoassay using binding proteins in a multiclonal antibody format | |
| CN109704954B (en) | 3- (3-hydroxyphenyl) -3-hydroxypropionic acid derivative, homogeneous enzyme immunoassay reagent and preparation method thereof | |
| CN108205064B (en) | 25OHD3 detection reagent, kit and detection method thereof | |
| CN109239368A (en) | Measure the method and kit of progesterone | |
| CN110078711B (en) | Carbamazepine derivative and application thereof in immunodetection | |
| CN108490195A (en) | A kind of vitamin B12 immunization assay method and its reagent | |
| CN110357886B (en) | Methotrexate hapten and complete antigen as well as preparation method and application thereof | |
| CN110713986A (en) | Vitamin B1Monoclonal antibody hybridoma cell strain CBDD and application thereof | |
| EP2660603B1 (en) | Immunological assay method | |
| JP3154724B2 (en) | Methods for detecting monoclonal antibodies, hybridomas and diarrheal shellfish poisons specific to diarrheal shellfish toxins | |
| CN110981791A (en) | Paraquat derivative, preparation method thereof and paraquat detection reagent | |
| CN111620931A (en) | Vancomycin derivative and preparation method and application thereof | |
| CN110954707B (en) | Valproic acid derivatives and their use in immunoassays | |
| Newton et al. | Radioimmunoassay of cytidine 3′, 5′-cyclic monophosphate: Unambiguous assay by means of an optimized protocol incorporating a trilayer column separation to obviate cross-reactivity problems | |
| JPS6247554A (en) | Immunochemical measuring method of human epithelia cell growing factor and kit thereof | |
| JPS59142466A (en) | Measuring method of material provided with antigen- determining group | |
| KR100309141B1 (en) | Immunoassay using 5'-deoxy-5'-methylthioadenosine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: 25ohd3 detection reagent, kit and detection method thereof Effective date of registration: 20211015 Granted publication date: 20200821 Pledgee: Zhongguancun Technology Leasing Co., Ltd Pledgor: BEIJING DIAGREAT BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021980010818 |