CN118033114A - 17-Ketosteroid latex enhanced turbidimetric immunoassay reagent and preparation method thereof - Google Patents
17-Ketosteroid latex enhanced turbidimetric immunoassay reagent and preparation method thereof Download PDFInfo
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- CN118033114A CN118033114A CN202211373670.9A CN202211373670A CN118033114A CN 118033114 A CN118033114 A CN 118033114A CN 202211373670 A CN202211373670 A CN 202211373670A CN 118033114 A CN118033114 A CN 118033114A
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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
- G01N33/53—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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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (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)
Abstract
The invention uses polystyrene latex microsphere (amino microsphere) with amino modification on the surface, firstly uses maleimide-polyethylene glycol-acrylic acid succinimidyl ester (Mal-PEG-NHS ester) to carry out coupling reaction with the amino microsphere, modifies the latex microsphere, then uses- (PEG) n-Mal in the modified latex microsphere-polyethylene glycol-maleimide coupling body (Latex microspheres- (PEG) n-Mal) as a connecting molecule to react with sulfhydryl on the surface of an antibody, and obtains the latex microsphere-connecting molecule-antibody protein coupling body (namely the latex microsphere coated by the anti-17-ketosteroid specific antibody. The coupling method can enlarge the space distance between the antibody and the surface of the latex microsphere, increase the space distance between the antibody and the antibody, avoid the mutual extrusion of antibody molecules, ensure that the antibody has enough space to carry out recognition and binding reaction with the antigen, effectively improve the reaction efficiency of the antibody, reduce the use amount of the antibody, save the cost of reagents and obviously improve the sensitivity of 17-ketosteroid detection. The detection reagent can realize high-throughput and rapid detection of the 17-ketosteroid content on a full-automatic biochemical analyzer, has the advantages of good stability, high accuracy, strong specificity, convenient use, obviously improved detection efficiency and easy popularization and application.
Description
Technical Field
The invention belongs to the technical field of biological detection reagents, and particularly relates to a 17-ketosteroid latex enhanced immunonephelometric detection reagent and a preparation method thereof.
Background
17-Ketosteroid (17-ketosteroids, 17-KS) of formula VI:
And the formula VI.
17-KS in urine is a metabolic product of some steroid hormones in adrenal cortex and testes and ovaries, and under normal conditions, 17-KS in urine is an adrenal cortex product in females, 2/3 of 17-KS in males is produced by adrenal cortex, and 1/3 is from testes. It is well known that 17-KS is the index of the activity of testis and adrenal cortex androgens, and any factor affecting the function of testis and adrenal gland may cause the corresponding change in the quantity and quality of 17-KS in urine, thus reflecting the total situation of adrenal cortex hormone, glucocorticoid and gonadal secretion, which is of great value for evaluating the function of adrenal gland to secrete androgens. Such as hypoadrenocortical function, adrenocortical dysplasia, cortical hyperplasia, and increased urinary-17 ketosteroid output. Hypercortisolism: is a syndrome of hypersecretion of adrenoglucocorticoid (mainly cortisol). The main causes are adrenocortical adenoma, adrenocortical carcinoma, pituitary tumor or hypothalamic-pituitary dysfunction, ectopic ACTH-like tumors and iatrogenic glucocorticoid hyperplasia. The patient's 17-ketosteroid levels often exceeded 40.3. Mu. Mol/24h (11.6 mg/24 h) (male) and 37.3. Mu. Mol/24h (8.5 mg/24 h) (female). Anterior pituitary hypofunction: any injury that causes damage to the anterior pituitary or hypothalamus may cause hypophysis function. Postpartum pituitary necrosis and tumors around the pituitary are common etiologies. The patient had urine 17-KS, 17-OHCS and urine free cortisol all below normal values for 24 hours. Chronic adrenocortical insufficiency: when the adrenal glands on both sides are mostly destroyed, various corticoids are insufficient, which is called adrenocortical hypofunction. Primary and secondary can be separated. Primary chronic adrenocortical insufficiency, also known as Addison's disease, is rare; secondary hypothalamic-hypophysis patients may be suffering from atrophy of the adrenal cortex due to insufficient secretion of CRF or ACTH. The patient's urine 17-OHCS and 17-KS output was below normal, with the degree of reduction being in functional parallel relationship with the adrenal cortex. Cushing's syndrome (hypercortisolism), congenital adrenal hyperplasia, hyperthyroidism, simple obesity, ACTH treatment, hypertension, etc. often result in an elevation of 17-KS. Primary adrenocortical insufficiency (Addison's disease), anterior pituitary hypofunction, hypothyroidism, cirrhosis, systemic wasting disease, etc. cause a decrease in 17-KS. In several diseases, the amount of 17-KS in urine is altered, and chronic severe tuberculosis may be accompanied by adrenal cortex dysfunction, thus the amount of 17-KS in urine is reduced.
Classical 17-ketosteroid detection methods include spectrophotometry (colorimetric assay), high Performance Liquid Chromatography (HPLC), and the like. Spectrophotometry: most of 17-ketosterols in urine are water-soluble glucuronic acid esters or sulfuric acid esters, and are hydrolyzed into free fat-soluble sterols by heating and acidolysis during measurement, then extracted by organic solvents such as diethyl ether and the like, alkalized and washed to remove acidic substances such as estrone and the like, and then evaporated to dryness by using absolute ethyl alcohol to dissolve neutral 17-ketosterols in the urine. The ketone-methylene (-CO-CH 2-) structure in 17-ketosteroid molecule can react with m-dinitrobenzene in alkaline solution to generate purple red compound (i.e. Zimmermann reaction), and the purple compound is colorized by the colored substance produced by the same treatment as androsterone standard solution to obtain the 17-ketosteroid content. The method has the advantages of comparatively backward technology, poor stability and unsuitable clinical mass use. High performance liquid chromatography: directly taking methanol-water or isopropanol-water as a mobile phase system to perform gradient elution. Gradient elution is time consuming and in order to reduce analysis time, it is often determined using chromatographic conditions for isocratic elution. However, the HPLC method requires special equipment, the packed column is expensive, the consumption of mobile phase is large, and the detection cost is high.
Therefore, aiming at the defects existing in the prior art, a 17-ketosteroid detection reagent which has high sensitivity, strong specificity, good stability and accurate result and can be applied to high-throughput and rapid detection of a full-automatic biochemical analyzer is developed to reach clinical detection requirements, and becomes a hotspot in the in-vitro diagnostic reagent industry at home and abroad.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention adopts the following technical scheme:
in one aspect, the invention provides a 17-ketosteroid latex enhanced turbidimetric immunoassay reagent comprising: r1 reagent and R2 reagent;
The R1 reagent consists of a 17-ketosteroid-conjugated protein conjugate and an R1 buffer solution, and the R2 reagent consists of latex microspheres coated by an anti-17-ketosteroid specific antibody and an R2 buffer solution;
The 17-ketosteroid-conjugated protein conjugate is formed by coupling 17-ketosteroid derivatives with conjugated proteins, and has a structural formula shown in formula I:
A formula I;
the structural formula of the 17-ketosteroid derivative is shown as a formula II:
a formula II;
The conjugated protein is serum albumin; the R1 buffer solution is a Tirs buffer solution containing polyethylene glycol 20000 and pregnenin 300;
The anti-17-ketosteroid specific antibody is an antibody obtained after the experimental animal is immunized by the 17-ketosteroid immunogen; the 17-ketosteroid immunogen is formed by coupling 17-ketosteroid derivatives shown in a formula II with carrier proteins, and the structural formula of the 17-ketosteroid immunogen is shown in a formula III:
A formula III;
The carrier protein is protein or polypeptide with immunogenicity; the experimental animal is a mammal;
the latex microsphere coated by the anti-17-ketosteroid specific antibody is formed by coupling the anti-17-ketosteroid specific antibody with the latex microsphere through a connecting molecule;
the connecting molecule is maleimide-polyethylene glycol-succinimidyl acrylate, and the structural formula is shown in formula IV:
A formula IV;
N in the formula IV is any integer between 1 and 12;
The latex microsphere is a polystyrene latex microsphere with any one of amino, carboxyl, hydroxyl, mercapto, hydrazide or chloromethyl groups on the surface for modification, and the diameter range is 50-450nm;
the R2 buffer solution is sodium phosphate buffer solution containing the pregnening 300.
Preferably, the serum albumin is one of bovine serum albumin, rabbit serum albumin, sheep serum albumin, horse serum albumin or human serum albumin. More preferably, the serum albumin is rabbit serum albumin.
Preferably, the protein or polypeptide having immunogenicity is one of serum albumin, thyroglobulin, gamma globulin, ovalbumin, hemocyanin or polylysine; the mammal is one of rabbit, goat, mouse, sheep, guinea pig or horse. More preferably, the protein or polypeptide having immunogenicity is bovine thyroglobulin; the mammal is rabbit.
Preferably, the connecting molecule is maleimide-pentapolyethylene glycol-succinimidyl acrylate, and the structural formula is shown in formula V:
Formula V;
preferably, the latex microsphere is a polystyrene latex microsphere with amino modified surface, and the diameter is 200nm.
The invention also provides a preparation method of the 17-ketosteroid latex enhanced turbidimetric immunoassay reagent, which comprises the following steps:
(A1) Preparation of R1 reagent: dissolving 17-ketosteroid-conjugated protein conjugate in purified water, adding polyethylene glycol 20000, pregnening 300 and tris (hydroxymethyl) aminomethane, stirring uniformly, and regulating pH value to 7.0-9.0 to obtain R1 reagent;
(A2) Preparation of R2 reagent: adding the latex microsphere coated by the anti-17-ketosteroid specific antibody into purified water, then adding disodium hydrogen phosphate, monopotassium hydrogen phosphate and pregnening 300, uniformly stirring, and regulating the pH value to 7.0-9.0 to prepare the R2 reagent.
Specifically, the preparation method of the 17-ketosteroid latex enhanced turbidimetric immunoassay reagent comprises the following steps:
(A1) Preparation of R1 reagent: dissolving 17-ketosteroid-conjugated protein conjugate in purified water according to the mass fraction of 0.01%, adding polyethylene glycol 20000 with the mass fraction of 0.1% and pregnenin 300 with the mass fraction of 0.05%, uniformly stirring tris (hydroxymethyl) aminomethane with the molar concentration of 100.0mmol/L, and regulating the pH value to 8.0 to prepare an R1 reagent;
(A2) Preparation of R2 reagent: adding the latex microsphere coated by the anti-17-ketosteroid specific antibody into purified water according to the mass fraction of the final concentration of 3.0%, then adding disodium hydrogen phosphate with the final concentration of 8.0 mmol/L and potassium dihydrogen phosphate with the final concentration of 5.0 mmol/L, and stirring uniformly with the mass fraction of 0.05% of the pregnening 300, and regulating the pH value to 8.0 to prepare the R2 reagent.
Preferably, the preparation method of the 17-ketosteroid-conjugated protein conjugate comprises the following steps:
(B1) Dissolving conjugated protein in potassium phosphate buffer to obtain conjugated protein solution;
(B2) Mixing 17-ketosteroid derivative shown in the formula II with dimethylformamide, ethanol, potassium phosphate buffer, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, and stirring for dissolving to obtain 17-ketosteroid derivative solution;
(B3) Adding the 17-ketosteroid derivative solution obtained in the step (B2) into the conjugated protein solution obtained in the step (B1), stirring for reaction, and dialyzing and purifying to obtain the 17-ketosteroid-conjugated protein conjugate.
Specifically, the preparation method of the 17-ketosteroid-conjugated protein conjugate comprises the following steps:
(B1) 500.0mg of rabbit serum albumin was dissolved in 100.0mL (0.5 mol/L, pH =8.0) of potassium phosphate buffer to obtain a rabbit serum albumin solution;
(B2) 200.0mg of the 17-ketosteroid derivative represented by the above formula II, 10.0mL of dimethylformamide, 7.5mL of ethanol, 15.0mL (10.0 mmol/L, pH =7.0) of potassium phosphate buffer, 250.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and 100.0mg of N-hydroxysulfosuccinimide were mixed and dissolved with stirring to obtain a 17-ketosteroid derivative solution;
(B3) Adding the 17-ketosteroid derivative solution obtained in the step (B2) into the rabbit serum albumin solution obtained in the step (B1), stirring and reacting for 12 hours at the temperature of 4 ℃, and dialyzing and purifying to obtain the 17-ketosteroid-conjugated protein conjugate.
Preferably, the preparation method of the latex microsphere coated with the anti-17-ketosteroid specific antibody comprises the following steps:
(D1) Adding polystyrene latex microspheres into a sodium phosphate buffer solution, suspending the polystyrene latex microspheres in the sodium phosphate buffer solution by gentle shaking, centrifuging, removing supernatant, and then re-suspending the precipitate by using the sodium phosphate buffer solution to prepare a latex microsphere suspension; dissolving maleimide-polyethylene glycol-acrylic acid succinimidyl ester in dimethyl sulfoxide to prepare maleimide-polyethylene glycol-acrylic acid succinimidyl ester solution; adding maleimide-polyethylene glycol-succinimidyl acrylate solution into the latex microsphere suspension, stirring thoroughly to dissolve, stirring at room temperature for reaction. After the reaction, the solution was centrifuged to remove the supernatant, and the precipitate was rapidly washed twice with the same sodium phosphate buffer as described above to obtain a latex microsphere-linker conjugate.
(D2) Re-suspending the latex microsphere-connecting molecule conjugate prepared in the step (D1) in a sodium phosphate buffer solution to prepare a latex microsphere-connecting molecule conjugate suspension; diluting an anti-17-ketosteroid specific antibody with a sodium borate buffer solution to obtain an antibody solution, immediately adding the antibody solution into the latex microsphere-connecting molecule conjugate suspension, and then stirring at room temperature for reaction; after the reaction is completed, cysteine is added and the mixture is kept stand for 2 hours to block excessive maleimide reaction sites; centrifuging to remove supernatant, and washing the precipitate with sodium borate buffer solution for 3 times to obtain latex microspheres coated with anti-17-ketosteroid specific antibodies; finally, the latex microsphere coated by the anti-17-ketosteroid specific antibody is resuspended in glycine buffer, and sodium azide preservative is added for preservation at 0-4 ℃.
Specifically, the preparation method of the latex microsphere coated with the anti-17-ketosteroid specific antibody comprises the following steps:
(D1) 10.0mg of polystyrene latex microspheres with a diameter of 200nm are added into 15.0 mL (10.0 mmol/L, pH =7.2) sodium phosphate buffer, the polystyrene latex microspheres are suspended in the sodium phosphate buffer by gentle shaking, the supernatant is removed after centrifugation for 10 minutes at 10000r/min, and then the precipitate is resuspended with 15.0 mL (10.0 mmol/L, pH =7.2) sodium phosphate buffer to prepare a latex microsphere suspension; dissolving maleimide-pentapolyethylene glycol-succinimidyl acrylate in dimethyl sulfoxide (DMSO) to obtain maleimide-pentapolyethylene glycol-succinimidyl acrylate solution with final concentration of 20.0 mmol/L; adding maleimide-pentapolyethylene glycol-succinimidyl acrylate solution into the latex microsphere suspension according to the volume ratio of 1:20, stirring fully to dissolve, and stirring and reacting for 1 hour at room temperature. After the reaction, the solution was centrifuged at 10000r/min for 5 minutes, the supernatant was removed, and the precipitate was rapidly washed twice with the same sodium phosphate buffer as described above to obtain a latex microsphere-linker conjugate.
(D2) Suspending the latex microsphere-linker conjugate prepared in step (D1) in 10.0mL (10.0 mmol/L, pH =7.2) of sodium phosphate buffer to prepare a latex microsphere-linker conjugate suspension; diluting 1.0mg of anti-17-ketosteroid specific antibody with 5.0mL (50.0 mmol/L, pH =8.2) of sodium borate buffer to obtain an antibody solution, immediately adding the antibody solution into the latex microsphere-linker conjugate suspension, and then stirring at room temperature for reaction for 6 hours; after the reaction was completed, 50.0mmol/L cysteine was added and allowed to stand for 2 hours to block the excess maleimide reaction site; centrifuging at 10000 r/mm for 8 min, removing supernatant, and washing precipitate with 10.0mL (50.0 mmol/L, pH =8.0) sodium borate buffer solution for 3 times to obtain latex microsphere coated with anti-17-ketosteroid specific antibody; finally, the latex microsphere coated by the anti-17-ketosteroid specific antibody is resuspended in 15.0mL (50.0 mmol/L, pH =8.0) glycine buffer, and 0.01% sodium azide preservative by mass fraction is added for preservation at 0-4 ℃.
Preferably, the method for preparing the anti-17-ketosteroid specific antibody comprises the following steps:
(E1) Diluting 17-ketosteroid immunogen shown in the structural formula III with sodium phosphate buffer to obtain an antigen solution, mixing the antigen solution with equivalent Freund complete adjuvant, and injecting experimental animals;
(E2) After 15-45 days, mixing the antigen solution with equivalent Freund incomplete adjuvant, injecting the experimental animal once, and then injecting once every 10-20 days for 4-6 times;
(E3) Taking blood from the experimental animal immunized in the step (E2), and separating and purifying antiserum to obtain an anti-17-ketosteroid specific antibody;
specifically, the preparation method of the anti-17-ketosteroid specific antibody comprises the following steps:
(E1) Diluting 17-ketosteroid immunogen shown in the structural formula III with sodium phosphate buffer (0.2 mol/L, pH =7.5) to a final concentration of 3.0mg/mL to obtain an antigen solution, and then mixing 3.0mL of the antigen solution with equivalent Freund's complete adjuvant to inject the rabbit;
(E2) After 30 days, 3.0mL of the antigen solution is mixed with the equivalent Freund incomplete adjuvant, and the rabbit is injected once every 15 days, and the total injection is 5 times;
(E3) And (3) taking blood from the rabbits immunized in the step (E2), and separating and purifying antiserum to obtain the anti-17-ketosteroid specific antibody.
Preferably, the preparation method of the 17-ketosteroid immunogen shown in the structural formula III comprises the following steps:
(F1) Dissolving carrier protein in potassium phosphate buffer solution to obtain carrier protein solution;
(F2) Mixing 17-ketosteroid derivative shown in the formula II with dimethylformamide, ethanol, potassium phosphate buffer, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, and stirring for dissolving to obtain 17-ketosteroid derivative solution;
(F3) Adding the 17-ketosteroid derivative solution obtained in the step (F2) into the carrier protein solution obtained in the step (F1), stirring for reaction, and dialyzing and purifying to obtain the 17-ketosteroid immunogen.
Specifically, the preparation method of the 17-ketosteroid immunogen comprises the following steps:
(F1) 800.0mg of bovine thyroglobulin is dissolved in 100.0mL (0.3 mol/L, pH =8.3) of potassium phosphate buffer to obtain a bovine thyroglobulin solution;
(F2) 120.0mg of the 17-ketosteroid derivative represented by the above formula II, 6.0mL of dimethylformamide, 6.0mL of ethanol, 15.0mL (10.0 mmol/L, pH=6.5) of potassium phosphate buffer, 150.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and 30.0mg of N-hydroxysulfosuccinimide were mixed and dissolved with stirring to obtain a 17-ketosteroid derivative solution;
(F3) Adding the 17-ketosteroid derivative solution obtained in the step (F2) into the bovine thyroglobulin solution obtained in the step (F1), stirring and reacting for 12 hours at the temperature of 4 ℃, and obtaining the 17-ketosteroid immunogen through dialysis and purification.
The beneficial effects of the invention are as follows: the latex microsphere commonly used in the latex enhanced turbidimetric immunoassay reagent in the prior art is a latex microsphere (carboxyl microsphere) with carboxyl modification on the surface, and the carboxyl microsphere can be directly coupled with amino groups on an antibody (IgG). The coupling method is simpler, but the result after coupling is that the space distance between the antibody and the surface of the latex microsphere in the coupling body (namely the latex microsphere coated by the antibody) is small, meanwhile, the space distance between the antibody and the antibody is also small, and a large number of antibody molecules are mutually extruded together, so that the antibody does not have enough space to carry out recognition and binding reaction with the antigen, further the activity and the sensitivity of the antibody are reduced, the utilization efficiency of the antibody is reduced, and the waste of the antibody is caused.
The latex microsphere used in the invention is polystyrene latex microsphere (amino microsphere) with amino modification on the surface, firstly, maleimide-polyethylene glycol-acrylic acid succinimidyl ester (Mal-PEG-NHS ester) is used for carrying out coupling reaction with the amino microsphere, the latex microsphere is modified, and then- (PEG) n-Mal in a latex microsphere-polyethylene glycol-maleimide coupling body (Latex microspheres- (PEG) n-Mal) obtained after the modification is used as a connecting molecule to react with sulfhydryl on the surface of an antibody, so that a latex microsphere-connecting molecule-antibody protein coupling body (namely, the latex microsphere coated by the anti-17-ketosteroid specific antibody in the invention) is obtained. The coupling method can enlarge the space distance between the antibody and the surface of the latex microsphere, increase the space distance between the antibody and the antibody, avoid the mutual extrusion of antibody molecules, ensure that the antibody has enough space to carry out recognition and binding reaction with the antigen, effectively improve the reaction efficiency of the antibody, reduce the use amount of the antibody, save the cost of reagents and obviously improve the sensitivity of 17-ketosteroid detection. The detection reagent can realize high-throughput and rapid detection of the 17-ketosteroid content on a full-automatic biochemical analyzer, has the advantages of good stability, high accuracy, strong specificity, convenient use, obviously improved detection efficiency and easy popularization and application.
Drawings
FIG. 1 is a calibration curve for a 17-ketosteroid latex enhanced turbidimetric immunoassay reagent.
FIG. 2 is a graph showing a correlation analysis between a latex-enhanced turbidimetric immunoassay and a high performance liquid chromatography for detecting 17-ketosteroid.
Detailed Description
The invention will be further described with reference to the accompanying drawings and detailed description, which are simplified schematic illustrations of the basic structure of the invention, which are presented solely by way of illustration, and thus showing only the structures that are relevant to the invention. Unless otherwise indicated, reagents, instruments, equipment, consumables used in the following examples were purchased from regular vendors.
Example 1: preparation of 17-ketosteroid latex enhanced turbidimetric immunoassay reagent
(1) Preparation of 17-ketosteroid-conjugated protein conjugates
A. 500.0mg of rabbit serum albumin was dissolved in 100.0mL (0.5 mol/L, pH =8.0) of potassium phosphate buffer to obtain a rabbit serum albumin solution;
b. 200.0mg of the 17-ketosteroid derivative represented by the above formula II, 10.0mL of dimethylformamide, 7.5mL of ethanol, 15.0mL (10.0 mmol/L, pH =7.0) of potassium phosphate buffer, 250.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and 100.0mg of N-hydroxysulfosuccinimide were mixed and dissolved with stirring to obtain a 17-ketosteroid derivative solution;
c. And c, adding the 17-ketosteroid derivative solution obtained in the step b into the rabbit serum albumin solution obtained in the step a, stirring at 4 ℃ for reaction for 12 hours, and dialyzing and purifying to obtain the 17-ketosteroid-conjugated protein conjugate.
(2) Preparation of 17-ketosteroid immunogens
A. 800.0mg of bovine thyroglobulin is dissolved in 100.0mL (0.3 mol/L, pH =8.3) of potassium phosphate buffer to obtain a bovine thyroglobulin solution;
b. 120.0mg of the 17-ketosteroid derivative represented by the above formula II, 6.0mL of dimethylformamide, 6.0mL of ethanol, 15.0mL (10.0 mmol/L, pH=6.5) of potassium phosphate buffer, 150.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and 30.0mg of N-hydroxysulfosuccinimide were mixed and dissolved with stirring to obtain a 17-ketosteroid derivative solution;
c. and d, adding the 17-ketosteroid derivative solution obtained in the step b into the bovine thyroglobulin solution obtained in the step a, stirring at 4 ℃ for reaction for 12 hours, and dialyzing and purifying to obtain the 17-ketosteroid immunogen.
(3) Preparation of antibodies specific for anti-17-ketosterols
A. Diluting 17-ketosteroid immunogen shown in a structural formula III with sodium phosphate buffer (0.2 mol/L, pH =7.5) to a final concentration of 3.0mg/mL to obtain an antigen solution, and then mixing 3.0mL of the antigen solution with equivalent Freund's complete adjuvant to inject the rabbit;
b.30 days later, then 3.0mL of the antigen solution is mixed with equivalent Freund incomplete adjuvant, and the rabbit is injected once every 15 days, and then the whole rabbit is injected 5 times;
c. And c, taking blood from the rabbits immunized in the step b, and separating and purifying antiserum to obtain the anti-17-ketosteroid specific antibody.
(4) Preparation of latex microspheres coated with anti-17-ketosteroid-specific antibodies
A. 10.0mg of polystyrene latex microspheres with a diameter of 200nm are added into 15.0 mL (10.0 mmol/L, pH =7.2) sodium phosphate buffer, the polystyrene latex microspheres are suspended in the sodium phosphate buffer by gentle shaking, the supernatant is removed after centrifugation for 10 minutes at 10000r/min, and then the precipitate is resuspended with 15.0 mL (10.0 mmol/L, pH =7.2) sodium phosphate buffer to prepare a latex microsphere suspension; dissolving maleimide-pentapolyethylene glycol-succinimidyl acrylate in dimethyl sulfoxide (DMSO) to obtain maleimide-pentapolyethylene glycol-succinimidyl acrylate solution with final concentration of 20.0 mmol/L; adding maleimide-pentapolyethylene glycol-succinimidyl acrylate solution into the latex microsphere suspension according to the volume ratio of 1:20, stirring fully to dissolve, and stirring and reacting for 1 hour at room temperature. After the reaction, the solution was centrifuged at 10000r/min for 5 minutes, the supernatant was removed, and the precipitate was rapidly washed twice with the same sodium phosphate buffer as described above to obtain a latex microsphere-linker conjugate.
B. Resuspending the latex microsphere-linker conjugate prepared in step a in 10.0mL (10.0 mmol/L, pH =7.2) of sodium phosphate buffer to prepare a latex microsphere-linker conjugate suspension; diluting 1.0mg of anti-17-ketosteroid specific antibody with 5.0mL (50.0 mmol/L, pH =8.2) of sodium borate buffer to obtain an antibody solution, immediately adding the antibody solution into the latex microsphere-linker conjugate suspension, and then stirring at room temperature for reaction for 6 hours; after the reaction was completed, 50.0mmol/L cysteine was added and allowed to stand for 2 hours to block the excess maleimide reaction site; centrifuging at 10000 r/mm for 8 min, removing supernatant, and washing precipitate with 10.0mL (50.0 mmol/L, pH =8.0) sodium borate buffer solution for 3 times to obtain latex microsphere coated with anti-17-ketosteroid specific antibody; finally, the latex microsphere coated by the anti-17-ketosteroid specific antibody is resuspended in 15.0mL (50.0 mmol/L, pH =8.0) glycine buffer, and 0.01% sodium azide preservative by mass fraction is added for preservation at 0-4 ℃.
(5) Preparation of 17-ketosteroid latex enhanced turbidimetric immunoassay reagent
Preparation of R1 reagent: dissolving 17-ketosteroid-conjugated protein conjugate in purified water according to the mass fraction of 0.01%, adding polyethylene glycol 20000 with the mass fraction of 0.1% and pregnenin 300 with the mass fraction of 0.05%, uniformly stirring tris (hydroxymethyl) aminomethane with the molar concentration of 100.0mmol/L, and regulating the pH value to 8.0 to prepare an R1 reagent;
Preparation of R2 reagent: adding the latex microsphere coated by the anti-17-ketosteroid specific antibody into purified water according to the mass fraction of the final concentration of 3.0%, then adding disodium hydrogen phosphate with the final concentration of 8.0 mmol/L and potassium dihydrogen phosphate with the final concentration of 5.0 mmol/L, and stirring uniformly with the mass fraction of 0.05% of the pregnening 300, and regulating the pH value to 8.0 to prepare the R2 reagent.
Example 2: preparation of 17-ketosteroid standard
The 17-ketosteroid pure powder is respectively added into 6 parts of Tris-HCl buffer solution with the concentration of 50mmol/L and the pH value of 7.2, stirred and dissolved, the final concentration is respectively 0.00 mug/mL, 4.00 mug/mL, 8.00 mug/mL, 16.00 mug/mL, 32.00 mug/mL and 80.00 mug/mL, and then sodium chloride with the mass fraction of 0.5 percent, bovine serum albumin with the mass fraction of 1.0 percent, ethylenediamine tetraacetic acid with the mass fraction of 0.75 percent and sodium azide with the concentration of 0.05 percent are respectively added into each part of the solution, and the mixture is uniformly stirred, thus the 17-ketosteroid standard (a group of 6 different concentrations) is obtained.
Example 3: 17-ketosteroid latex enhanced turbidimetric immunoassay reagent calibration curve preparation and performance evaluation experiment
(1) Preparing a 17-ketosteroid latex enhanced turbidimetric immunoassay reagent calibration curve:
The 17-ketosteroid latex enhanced turbidimetric immunoassay reagent prepared in the example 1 and the 17-ketosteroid standard prepared in the example 2 are put into an Olympus AU480 full-automatic biochemical analyzer, and then the biochemical analyzer is subjected to reaction parameter setting, wherein the specific parameters are shown in Table 1. In the actual detection process, the R1 reagent is added first, then the standard substance is added, and finally the R2 reagent is added. After adding the R2 reagent, the OD570nm absorbance at different time points was measured, and the reaction rates at different standard concentrations were calculated (see Table 2 for details) and plotted as a reaction standard curve, as shown in FIG. 1.
TABLE 1 reaction parameters of Olympus AU480 full-automatic Biochemical Analyzer
| Project name | 17-Ketosterols |
| R1 reagent | 160.0µl |
| R2 reagent | 40.0µl |
| Sample size | 3.0µl |
| Scaling method | Endpoint method |
| Dominant wavelength | 570nm |
| Sub-wavelength | 412nm |
| Reaction time | For 10 minutes |
| Incubation time | 8 Minutes |
| Reaction direction | Descent down |
| Results | μg/mL |
| Accuracy of results | 0.01 |
| Fitting method | Spline |
| Concentration of standard | 0.00μg/mL、4.00μg/mL、8.00μg/mL、16.00μg/mL、32.00μg/mL、80.00μg/mL |
Table 2: reactivity values at different standard concentrations
| Standard substance concentration (mug/mL) | △OD570nm |
| 0.00 | 0.8201 |
| 4.00 | 0.6759 |
| 8.00 | 0.5704 |
| 16.00 | 0.4343 |
| 32.00 | 0.3315 |
| 80.00 | 0.1770 |
(2) Quality control experiment:
Dissolving 17-ketosteroid pure powder in methanol to obtain 1mg/mL stock solution, and diluting the stock solution in healthy human plasma without 17-ketosteroid to final concentrations of 0.00, 8.00, 30.00 and 80.00 μg/mL to obtain blank, low, medium and high concentration quality control samples. By using the latex-enhanced turbidimetric immunoassay method, the quality control samples are measured, the content of 17-ketosteroid in each quality control sample is calculated according to the latex-enhanced turbidimetric immunoassay calibration curve manufactured in the step 1, the measurement is repeated for 10 times for each quality control sample, and the detection result and the data analysis are shown in Table 3 in detail.
TABLE 3 detection results and data analysis of 17-ketosteroid latex enhanced turbidimetric immunoassay
| Quality control sample | Blank space | Low and low | In (a) | High height |
| Sample concentration (μg/mL) | 0.00 | 8.00 | 30.00 | 80.00 |
| Test 1 | 0.00 | 8.12 | 30.37 | 81.60 |
| Test 2 | 0.00 | 8.18 | 30.72 | 84.53 |
| Test 3 | 0.00 | 7.93 | 29.59 | 82.79 |
| Test 4 | 0.00 | 8.16 | 29.65 | 78.64 |
| Test 5 | 0.00 | 7.96 | 30.33 | 79.31 |
| Test 6 | 0.00 | 8.22 | 30.02 | 80.75 |
| Test 7 | 0.00 | 7.97 | 30.67 | 78.77 |
| Test 8 | 0.00 | 8.04 | 30.20 | 80.84 |
| Test 9 | 0.00 | 7.97 | 30.64 | 77.05 |
| Test 10 | 0.00 | 8.12 | 29.67 | 83.98 |
| Average value (μg/mL) | 0.00 | 8.07 | 30.19 | 80.83 |
| Standard Deviation (SD) | / | 0.11 | 0.44 | 2.44 |
| Precision (CV) | / | 1.36% | 1.46% | 3.02% |
| Recovery rate | / | 100.88% | 100.63 | 101.04% |
The experimental results show that: CV values of 17-ketosteroid contents in quality control samples with different concentrations are all lower than 5%, recovery rates are all between 95% and 105%, and the accuracy of 17-ketosteroid content in biological samples measured by the 17-ketosteroid latex enhanced immunonephelometry detection reagent is higher and the result is accurate.
(3) Performance evaluation experiment of 17-ketosteroid latex enhanced turbidimetric immunoassay reagent
According to GB/T26124-2011 clinical chemistry in vitro diagnostic reagent (kit), the sanitary industry standard of the people's republic of China: the relevant requirements of the linear evaluation of the quantitative determination method refer to the relevant standards of CLSI, and after a large number of experimental verification, the performance indexes which can be achieved by the reagent of the invention are determined as follows:
a. Reagent blank absorbance: when purified water is used as a detection sample, the absorbance is more than or equal to 0.8 (optical path 1 cm; dominant wavelength 570 nm;37 ℃).
B. Analytical sensitivity: the concentration of the 17-ketosteroid sample is measured to be 8.00 mug/mL, and the absorbance difference (delta A) is in the range of 0.01-0.40.
C. Linear range: [2.00, 80.00] mug/mL, and the linear correlation coefficient r is more than or equal to 0.990; within [2.00, 10.00) μg/mL linear range, absolute deviation is within + -1.50 μg/mL; within the linear range of [10.00, 80.00] mug/mL, the relative deviation is within + -15.0%.
D. precision: repeatability CV is less than or equal to 10.0% (n=10); the same sample is measured by reagents of different batch numbers produced in three batches continuously, each batch number is measured three times, and the difference R between batches is less than or equal to 10.0%.
E. Accuracy: the quality control was measured with 17-ketosteroid reagent, 3 times per concentration, averaged, and the relative deviation B was calculated to be within ±15.0% from the average.
F. Assay specificity: interference test researches of albumin <6g/L, creatinine <4000mg/L, hemoglobin less than or equal to 200mg/dL, bilirubin less than or equal to 20mg/dL and triglyceride less than or equal to 2000mg/dL are carried out. The samples of known concentration were measured 3 times and the relative deviation was calculated to be within + -15.0%, indicating that interferents within this concentration range had no effect on the present reagent.
G. reportable range: the reportable range of the 17-ketosteroid latex enhanced turbidimetric immunoassay reagent is 1.70-240.00 mug/mL.
H. Stability: sealing and storing the reagent at 2-8 ℃ for 12 months; after the reagent is unsealed, the effective period is 30 days in a storage environment at 2-8 ℃.
The performance evaluation experiment results show that the performance indexes such as reagent blank absorbance, analysis sensitivity, linear range, precision (repeatability and batch-to-batch difference), accuracy, analysis specificity, reportable range, stability and the like of the 17-ketosteroid latex enhanced turbidimetric immunoassay reagent all meet the technical requirements of related standards.
Example 4: correlation experiment for detecting 17-ketosteroid by using latex enhanced immunonephelometry detection reagent and high performance liquid chromatography
100 Clinical urine samples were detected by using the 17-ketosteroid latex enhanced turbidimetric immunoassay reagent prepared in example 1 and high performance liquid chromatography, and the data obtained by the detection were subjected to a scatter diagram and correlation analysis (as shown in fig. 2), and the obtained linear equation was: y= 0.9911 x+ 0.3781 and a correlation coefficient R 2 =0.998, which shows that the experimental data of the clinical urine sample detected by adopting the 17-ketosteroid latex enhanced immunonephelometry detection reagent has higher consistency with the high performance liquid chromatography, and the detection result has high accuracy.
Example 5: performance verification of reagents prepared with latex microspheres coated with anti-17-ketosteroid specific antibodies of different linker molecules
To illustrate that latex microspheres coated with anti-17-ketosteroid specific antibodies of the present invention using other different linker molecules can be used to prepare 17-ketosteroid latex-enhanced turbidimetric immunoassay reagents, and all have the same properties, corresponding test reagents were prepared using the anti-17-ketosteroid specific antibody coated latex microspheres of various different linker molecules according to the method of example 1, and various experiments were performed on the prepared test reagents according to the methods of examples 3 to 4, with the linker molecules and the reagent performance verification results used in various experimental protocols being shown in table 4.
TABLE 4 results of reagent Performance verification Using different linker molecules
The experimental results show that: the latex microspheres coated by the anti-17-ketosteroid specific antibodies of other different connecting molecules can be used for preparing the 17-ketosteroid latex enhanced immunonephelometric detection reagent, and have excellent performances.
It should be noted that the foregoing embodiments of the present invention are merely examples, and are not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the descriptions of the present invention and the contents of the drawings thereof, or direct or indirect application in other related technical fields, are included in the scope of the claims of the present invention.
Claims (10)
1. A 17-ketosteroid latex enhanced turbidimetric immunoassay reagent comprising: r1 reagent and R2 reagent;
The R1 reagent consists of a 17-ketosteroid-conjugated protein conjugate and an R1 buffer solution, and the R2 reagent consists of latex microspheres coated by an anti-17-ketosteroid specific antibody and an R2 buffer solution;
The 17-ketosteroid-conjugated protein conjugate is formed by coupling 17-ketosteroid derivatives with conjugated proteins, and has a structural formula shown in formula I:
A formula I;
the structural formula of the 17-ketosteroid derivative is shown as a formula II:
a formula II;
The conjugated protein is serum albumin; the R1 buffer solution is a Tirs buffer solution containing polyethylene glycol 20000 and pregnenin 300;
The anti-17-ketosteroid specific antibody is an antibody obtained after the experimental animal is immunized by the 17-ketosteroid immunogen; the 17-ketosteroid immunogen is formed by coupling 17-ketosteroid derivatives shown in a formula II with carrier proteins, and the structural formula of the 17-ketosteroid immunogen is shown in a formula III:
A formula III;
The carrier protein is protein or polypeptide with immunogenicity; the experimental animal is a mammal;
the latex microsphere coated by the anti-17-ketosteroid specific antibody is formed by coupling the anti-17-ketosteroid specific antibody with the latex microsphere through a connecting molecule;
the connecting molecule is maleimide-polyethylene glycol-succinimidyl acrylate, and the structural formula is shown in formula IV:
A formula IV;
N in the formula IV is any integer between 1 and 12;
The latex microsphere is a polystyrene latex microsphere with any one of amino, carboxyl, hydroxyl, mercapto, hydrazide or chloromethyl groups on the surface for modification, and the diameter range is 50-450nm;
the R2 buffer solution is sodium phosphate buffer solution containing the pregnening 300.
2. The 17-ketosteroid latex enhanced turbidimetric immunoassay reagent of claim 1, wherein said serum albumin is one of bovine serum albumin, rabbit serum albumin, sheep serum albumin, horse serum albumin or human serum albumin.
3. The 17-ketosteroid latex enhanced turbidimetric immunoassay of claim 2, wherein said serum albumin is rabbit serum albumin.
4. The 17-ketosteroid latex enhanced turbidimetric immunoassay of claim 1, wherein said protein or polypeptide having immunogenicity is one of serum albumin, thyroglobulin, gamma globulin, ovalbumin, hemocyanin or polylysine; the mammal is one of rabbit, goat, mouse, sheep, guinea pig or horse.
5. The 17-ketosteroid latex enhanced turbidimetric immunoassay of claim 4, wherein said immunogenic protein or polypeptide is bovine thyroglobulin; the mammal is rabbit.
6. The 17-ketosteroid latex enhanced turbidimetric immunoassay reagent of claim 1, wherein said linker molecule is maleimide-pentapolyethylene glycol-acrylic succinimidyl ester having a structural formula as shown in formula v:
Formula V;
the latex microsphere is a polystyrene latex microsphere with amino modified surface, and the diameter is 200nm.
7. A method for preparing the 17-ketosteroid latex enhanced turbidimetric immunoassay according to claim 1, wherein the method comprises the following steps:
(A1) Preparation of R1 reagent: dissolving 17-ketosteroid-conjugated protein conjugate in purified water, adding polyethylene glycol 20000, pregnening 300 and tris (hydroxymethyl) aminomethane, stirring uniformly, and regulating pH value to 7.0-9.0 to obtain R1 reagent;
(A2) Preparation of R2 reagent: adding the latex microsphere coated by the anti-17-ketosteroid specific antibody into purified water, then adding disodium hydrogen phosphate, monopotassium hydrogen phosphate and pregnening 300, uniformly stirring, and regulating the pH value to 7.0-9.0 to prepare the R2 reagent.
8. The method for preparing the 17-ketosteroid latex enhanced turbidimetric immunoassay reagent according to claim 7, wherein the method for preparing the 17-ketosteroid-conjugated protein conjugate comprises the following steps:
(B1) Dissolving conjugated protein in potassium phosphate buffer to obtain conjugated protein solution;
(B2) Mixing the 17-ketosteroid derivative shown in the formula II in claim 1 with dimethylformamide, ethanol, potassium phosphate buffer, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, and stirring for dissolving to obtain a 17-ketosteroid derivative solution;
(B3) Adding the 17-ketosteroid derivative solution obtained in the step (B2) into the conjugated protein solution obtained in the step (B1), stirring for reaction, and dialyzing and purifying to obtain the 17-ketosteroid-conjugated protein conjugate.
9. The method for preparing 17-ketosteroid latex enhanced turbidimetric immunoassay reagent according to claim 7, wherein the method for preparing the anti-17-ketosteroid specific antibody coated latex microsphere comprises the following steps:
(D1) Adding polystyrene latex microspheres into a sodium phosphate buffer solution, suspending the polystyrene latex microspheres in the sodium phosphate buffer solution by gentle shaking, centrifuging, removing supernatant, and then re-suspending the precipitate by using the sodium phosphate buffer solution to prepare a latex microsphere suspension; dissolving maleimide-polyethylene glycol-acrylic acid succinimidyl ester in dimethyl sulfoxide to prepare maleimide-polyethylene glycol-acrylic acid succinimidyl ester solution; adding maleimide-polyethylene glycol-acrylic acid succinimidyl ester solution into latex microsphere suspension, stirring thoroughly to dissolve, stirring at room temperature for reaction; after the reaction is finished, centrifuging the solution, removing supernatant, and rapidly washing precipitate twice by using the same sodium phosphate buffer solution to obtain a latex microsphere-connecting molecule conjugate;
(D2) Re-suspending the latex microsphere-connecting molecule conjugate prepared in the step (D1) in a sodium phosphate buffer solution to prepare a latex microsphere-connecting molecule conjugate suspension; diluting an anti-17-ketosteroid specific antibody with a sodium borate buffer solution to obtain an antibody solution, immediately adding the antibody solution into the latex microsphere-connecting molecule conjugate suspension, and then stirring at room temperature for reaction; after the reaction is completed, cysteine is added and the mixture is kept stand for 2 hours to block excessive maleimide reaction sites; centrifuging to remove supernatant, and washing the precipitate with sodium borate buffer solution for 3 times to obtain latex microspheres coated with anti-17-ketosteroid specific antibodies; finally, the latex microsphere coated by the anti-17-ketosteroid specific antibody is resuspended in glycine buffer, and sodium azide preservative is added for preservation at 0-4 ℃.
10. The method for preparing the 17-ketosteroid latex enhanced turbidimetric immunoassay reagent according to claim 9, wherein the method for preparing the anti-17-ketosteroid specific antibody comprises the following steps:
(E1) Diluting 17-ketosteroid immunogen shown in structural formula III in claim 1 with sodium phosphate buffer to obtain an antigen solution, mixing the antigen solution with equivalent Freund complete adjuvant, and injecting the experimental animal;
(E2) After 15-45 days, mixing the antigen solution with equivalent Freund incomplete adjuvant, injecting the experimental animal once, and then injecting once every 10-20 days for 4-6 times;
(E3) Taking blood from the experimental animal immunized in the step (E2), and separating and purifying antiserum to obtain an anti-17-ketosteroid specific antibody;
The preparation method of the 17-ketosteroid immunogen shown in the structural formula III comprises the following steps:
(F1) Dissolving carrier protein in potassium phosphate buffer solution to obtain carrier protein solution;
(F2) Mixing the 17-ketosteroid derivative shown in the formula II in claim 1 with dimethylformamide, ethanol, potassium phosphate buffer, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, and stirring for dissolving to obtain a 17-ketosteroid derivative solution;
(F3) Adding the 17-ketosteroid derivative solution obtained in the step (F2) into the carrier protein solution obtained in the step (F1), stirring for reaction, and dialyzing and purifying to obtain the 17-ketosteroid immunogen.
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