CN115575631A - Kit for detecting content of inhibin A in blood plasma - Google Patents
Kit for detecting content of inhibin A in blood plasma Download PDFInfo
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- CN115575631A CN115575631A CN202211210066.4A CN202211210066A CN115575631A CN 115575631 A CN115575631 A CN 115575631A CN 202211210066 A CN202211210066 A CN 202211210066A CN 115575631 A CN115575631 A CN 115575631A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
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- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kit for detecting the content of inhibin A in blood plasma, which comprises the following components: the kit comprises a magnetic particle suspension coated with a first site inhibin A antibody, a calibrator, an enzyme conjugate of an enzyme labeled second site inhibin A antibody, a first buffer solution, a second buffer solution, a luminescent substrate and a concentrated washing solution. The invention utilizes the magnetic particle suspension coated with the inhibin A antibody and the enzyme conjugate of the inhibin A antibody of the enzyme-labeled second site to be respectively combined with two antigenic determinants on the inhibin A antigen in a sample to form an immune complex of solid phase antibody-antigen-enzyme-labeled antibody, then adds a luminescent substrate, and calculates according to a standard curve of a calibrator after color development to determine the content of the inhibin A antigen in blood plasma. The invention fully utilizes the rapid and easy automation of the magnetic separation technology and the high sensitivity of the chemiluminescence technology to shorten the detection time and improve the detection accuracy.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to a kit for detecting the content of inhibin A in blood plasma.
Background
Inhibin A (inhibin-A): inhibin is a heterodimeric glycoprotein. The beta-subunit and one beta A-subunit constitute inhibin A. The free subunits are present in the blood as biologically inactive subunits. The majority of inhibin in maternal serum during pregnancy is thought to be derived from the trophoblasts of the placenta. Inhibin A increases and reaches a peak at 10-12 weeks of gestation, falls to a plateau at mid-gestation, but rises again at late gestation, reaching a maximum level at full term. Inhibin A, like hCG, is elevated in the case of concurrent Down syndrome. The serum concentration of inhibin in the mother varies independently of the concentration of hCG. Noble et al found inhibin A >95 percentile in 12.8% of fetal maternal serum from Down syndrome. Wald et al determined that the inhibin a in maternal serum of 77 cases of down syndrome fetuses at early pregnancy was 1.19MoM, and therefore, the detection of the inhibin a content in plasma was also helpful for diagnosing down syndrome.
At present, for the human inhibin A, a time-resolved fluorescence method is mainly adopted in the detection kit, and the defect of long detection time is mainly overcome. The magnetic particle chemiluminescence method is a novel detection method developed by a chemiluminescence method platform, and is a novel analysis method combining a magnetic separation technology, a chemiluminescence technology and an immunoassay technology, the technology fully utilizes the rapidness and easy automation of the magnetic separation technology, the high sensitivity of the chemiluminescence technology and the specificity of immunoassay, and has a wide application prospect in the field of bioanalysis, but when an enzyme-labeled antibody is prepared in the magnetic separation technology, in order to ensure the activity of enzyme, the whole experiment needs to be carried out at the environment of 4 ℃, the requirement on the environment is high, the activity of the enzyme is influenced by careless operation, the detection accuracy of inhibin A is influenced, a reagent needs to be subjected to a long-time water bath reaction at the temperature of more than 35 ℃ in a part of steps, and the operation method can also reduce the activity of the enzyme, make the enzyme unstable, and influence the luminescence value.
Therefore, a kit for detecting the content of inhibin A in blood plasma, which is convenient for operators to detect and can improve the detection accuracy, is urgently needed.
Disclosure of Invention
The invention aims to provide a kit for detecting the content of inhibin A in blood plasma, which is convenient for operators to detect and improves the detection accuracy.
The invention relates to a kit for detecting the content of inhibin A in blood plasma, which comprises 10-30 mu L of magnetic particle suspension coated with a first site inhibin A antibody, 10-300 mu L of enzyme conjugate of an enzyme labeled second site inhibin A antibody, 10-100 mu L of a calibrator, 10-1000 mu L of a first buffer solution, 10-1000 mu L of a second buffer solution, 10-150 mu L of a luminescent substrate and 10-2000 mu L of concentrated washing solution.
The invention relates to a kit for detecting the content of inhibin A in blood plasma, wherein 25 mu L of magnetic particle suspension coated with inhibin A antibody of a first site, 150 mu L of enzyme conjugate of inhibin A antibody of an enzyme-labeled second site, 50 mu L of calibrator, 500 mu L of first buffer solution, 800 mu L of second buffer solution, 130 mu L of luminescent substrate and 1000 mu L of concentrated washing solution are adopted.
The invention relates to a kit for detecting the content of inhibin A in blood plasma, wherein the preparation method of a first buffer solution comprises the following steps:
s1, collecting 8.0g NaCl, 0.2g KCl and 1.44g Na 2 HPO 4 、0.24g KH 2 PO 4 、0.25g Ba(OH) 2 Dissolving in 800mL of distilled water;
s1.1, adjusting the pH value of the solution to 7.7 by using HCl solution with the pH value of 5.5;
s1.2, and finally adding distilled water to a constant volume of 1L to obtain a first buffer solution.
The invention relates to a kit for detecting the content of inhibin A in blood plasma, wherein the preparation method of an enzyme conjugate of an inhibin A antibody of an enzyme-labeled second site comprises the following steps:
s2, weighing 1.5mg of horseradish peroxidase, dissolving in 1ml of 1mM acetate buffer solution with pH5.6, adding 10 mu L C 102 H 151 O 39 N 31 、10μL MgSO 4 、20μL C 5 HO 2 NS stands for 20min, is filtered and precipitated, and then 0.1ml of DNFB absolute ethanol solution is added with 1 percentStirring for 1h to obtain a first solution;
s2.1, adding 0.2ml of newly prepared 0.1mol/L NaIO into the first solution 4 Stirring the solution for 20 minutes at room temperature in the dark, adding 80 mu L of inhibin A antibody and a second buffer solution to adjust the pH value of the solution to 9.0, standing for 12 hours, adding 1ml of 2.5% glycol, slightly stirring for 1 hour at room temperature, and stopping reaction to obtain a second solution;
s2.2, adding 0.1ml of sodium borohydride solution into the second solution, uniformly mixing, and standing for 3 hours at normal temperature to obtain a third solution;
s2.3, dialyzing the third solution for 12 hours by using a first buffer solution, and replacing the first buffer solution every 4 hours to obtain a fourth solution;
s2.4, centrifuging the fourth solution at the speed of 3000r/min for 30min, and filtering the precipitate to obtain a fifth solution;
s2.5, collecting supernatant in the fifth solution, adding a inhibin A antibody of a second site, and preparing an enzyme conjugate of the inhibin A of the enzyme-labeled second site.
The invention relates to a detection method of a kit for detecting the content of inhibin A in blood plasma, which comprises the following steps:
s3, 50 mu L of plasma is taken in a first test tube, is uniformly shaken and then is centrifuged for 30min at 3000r/min in a centrifuge, then supernatant is taken, 200 mu L of first buffer solution and 20 mu L of magnetic particle suspension coated with inhibin A antibody are added into the supernatant, is shaken for 30S at normal temperature and then is kept stand for 20min, and then concentrated washing liquor is fully washed to obtain first mixed liquor;
s3.1, adding 150 mu L of enzyme conjugate of the enzyme-labeled inhibin A antibody at the second site into the first mixed solution, stirring for 30S, standing for 15min, and fully washing with a concentrated washing solution to obtain a third mixed solution;
s3.2, adding 150 mu L of luminescent substrate into the third mixed solution, measuring a luminescent value within 1 minute in a dark place, and fitting the concentration of the inhibin A in the plasma through the luminescent value.
The invention relates to a detection method of a kit for detecting the content of inhibin A in blood plasma, wherein the step of fitting the concentration of inhibin A in blood plasma by a luminous value comprises the following steps:
s4, taking bovine serum albumin and the first buffer solution as solvents, and preparing a series of gradient calibrators with inhibin A antigen concentrations of 0, 25, 100, 500, 1000 and 2000pg/ml respectively;
s4.1, fitting the concentration value of the inhibin A calibrator and the luminescence value in the third mixed solution by using a LOG-LOG _ B mathematical model.
The invention relates to a kit for detecting inhibin A content in plasma, which is different from the prior art in that magnetic polystyrene microspheres with high mechanical strength and easily functionalized surfaces are used in the kit for detecting the inhibin A content in the plasma, inhibin A antibodies can be well crosslinked, then a magnetic particle suspension coated with the inhibin A antibodies and enzyme conjugates of the inhibin A antibodies at second enzyme-labeled sites are respectively combined with two antigenic determinants on the inhibin A antigens in a sample to form an immune complex of solid phase antibody-antigen-enzyme-labeled antibodies, because the inhibin A antibodies in a reaction system and the enzyme conjugates of the inhibin A antibodies at the second enzyme-labeled sites are excessive compared with the inhibin A antigens to be detected, the formation amount of the complex is in direct proportion to the content of the inhibin A antigens, luminescent substrates are added, and the content of the inhibin A antigens in the plasma can be determined according to a calibration curve after color development. Because the catalytic efficiency of the enzyme is very high, the result of the immune reaction is indirectly amplified, so that the determination method achieves very high sensitivity, and the inaccuracy of the test result of the kit caused by the reduction of the enzyme activity is effectively avoided.
The kit for detecting the content of inhibin A in blood plasma of the present invention is further described with reference to the accompanying drawings.
Drawings
FIG. 1 is a line graph showing clinical comparisons between a kit for measuring the amount of inhibin A in plasma and a comparative kit;
FIG. 2 is a table comparing the activities of horseradish peroxidase;
FIG. 3 is a comparative line graph of the activity of horseradish peroxidase.
Detailed Description
A kit for detecting the content of inhibin A in plasma comprises a magnetic particle suspension coated with an inhibin A antibody of a first site, a calibrator, an enzyme-labeled inhibin A antibody enzyme conjugate of a second site, 10-1000 muL of a first buffer solution, 10-1000 muL of a second buffer solution, 10-150 muL of a luminescent substrate and 10-2000 muL of a concentrated washing solution.
The invention uses magnetic polystyrene microspheres with high mechanical strength and easily functionalized surfaces to well crosslink inhibin A antibodies, and then combines the magnetic particle suspension coated with the inhibin A antibodies and enzyme conjugates of the inhibin A antibodies of enzyme-labeled second sites with two antigenic determinants on the inhibin A antigen molecules in a sample respectively to form an immune complex of solid phase antibodies-antigens-enzyme-labeled antibodies. The enzyme conjugate of the inhibin A antibody in the reaction system and the inhibin A antibody of the enzyme-labeled second site is excessive compared with the inhibin A antigen to be detected, so the formation amount of the complex is in direct proportion to the content of the inhibin A antigen, the added luminescent substrate is added, the content of the inhibin A antigen in the blood plasma can be determined by back calculation according to a standard curve of a calibrator after color development, and the result of immunoreaction is indirectly amplified due to high catalytic efficiency of the enzyme, so that the determination method achieves high sensitivity.
The first site inhibin A antibody, the enzyme-labeled second site inhibin A antibody, the inhibin A antigen and the horseradish peroxidase which are used in the invention are all products of the company, and are the prior art.
The preparation method of the magnetic particle suspension standard substance coated with the inhibin A antibody comprises the following steps: firstly, washing magnetic polystyrene microspheres for 2-5 times by using a first buffer solution with 10 times of the volume of a stock solution, then adding 200 mu L of 5% glutaraldehyde for activation for 30-40min, and adding a inhibin A antibody with the concentration of 15-40 mu g/ml into the activated magnetic polystyrene microspheres to coat the magnetic polystyrene microspheres and the inhibin A antibody.
The connection mode of the magnetic polystyrene microspheres and the inhibin A antibody is as follows: and (3) streptavidin connection, sealing the coated magnetic beads by a sealing solution, fixing the volume, subpackaging and storing at the temperature of 2-8 ℃ for later use.
Wherein the concentrated lotion is prepared by mixing 6% of polysorbate-20, 26% of tris hydrochloride, 15% of sodium chloride solvent and 53% of distilled water.
The invention can provide proper ion concentration by adding the chlorinated solvent, so that the immune complex is kept stable; maintaining the proper pH value of the solution by using the Tris hydrochloride so as to keep the immune complex stable, wherein the Tris hydrochloride is a Tris-HCl buffer system formed by Tris and HCl; the polysorbate-20 is also called Tween 20, and is a non-ionic surfactant, so that non-specific binding of antibody antigens is reduced, and unbound enzyme-labeled antibodies can be sufficiently removed through the above contents, so that the detection result is more accurate, the detection sensitivity is improved, and the occurrence of false positive is avoided.
Wherein the luminescent substrate is 1,2-dioxetane disodium salt.
The 1,2-dioxane disodium salt is also called AMPPD, is a latest ultrasensitive horseradish peroxidase substrate in the field of biochemistry, and the 1,2-dioxane disodium salt has high reaction speed and provides correct and reliable results in a short time.
Wherein the second buffer is also called carbonate buffer, which is composed of 1.6% Na 2 CO 3 3.4% NaHCO 3 Mixing with 95% distilled water, and storing at 4 deg.C.
The second buffer solution prepared by the materials can greatly reduce the solution with pH change when a small amount of acid or alkali and water are added, and can maintain the stability of the solution.
Wherein the column diameter of the magnetic polystyrene microsphere is 0.06-0.7 μm.
The magnetic polystyrene microsphere used in the invention has high mechanical strength, easy surface functionalization and weak surface nonionic interaction, the crosslinked polystyrene can keep a stable structure in strong acid and strong alkali, and the particle size of the microsphere is controllable.
Preferably, 25 μ L of the magnetic particle suspension coated with the inhibin A antibody at the first site, 150 μ L of enzyme conjugate labeled with the inhibin A antibody at the second site, 50 μ L of calibrator, 500 μ L of first buffer solution, 800 μ L of second buffer solution, 130 μ L of luminescent substrate and 1000 μ L of concentrated washing solution.
According to the invention, the solutions can react exactly through the proportion of the parts, so that the waste phenomenon is avoided, and the content of inhibin A in blood plasma can be detected more accurately.
Preferably, the preparation method of the first buffer solution comprises the following steps:
s1, collecting 8.0g NaCl, 0.2g KCl and 1.44g Na 2 HPO 4 、0.24g KH 2 PO 4 、0.25g Ba(OH) 2 Dissolving in 800mL of distilled water;
s1.2, adjusting the pH value of the solution to 7.7 by using an HCl solution with the pH value of 5.5;
s1.3, and finally adding distilled water to fix the volume to 1L to obtain the first buffer solution.
The first buffer solution used in the present invention contains Na as a main component 2 HPO 4 、KH 2 PO 4 NaCl and KCl, which serve as dissolution protection reagents. Due to Na 2 HPO 4 、KH 2 PO 4 Has secondary dissociation, the pH value of the buffer is wide, naCl and KCl mainly play a role in increasing the concentration of salt ions, and Ba (OH) 2 Is weak alkali, barium ions can be combined with hydroxide ions in water to release hydrogen ions, thereby achieving the buffering effect.
The traditional buffering agent is calcium hydroxide, and calcium ions can be combined with hydroxide ions in water to release hydrogen ions, so that the buffering effect is achieved, but calcium hydroxide reacts with magnesium sulfate in an enzyme conjugate for preparing an enzyme labeled inhibin A antibody at a second site to generate calcium sulfate, the calcium sulfate cannot reach the concentration of a precipitate, so that all sulfate radicals cannot be removed, or the activity of horseradish peroxidase can be influenced, therefore, the barium hydroxide used in the invention reacts with the magnesium sulfate to generate a white precipitate of barium sulfate, so that the sulfate ions are removed, the buffering effect can be achieved, the activity of the enzyme can be improved, the weight of the barium hydroxide can also be 0.5g, all sulfate ions generated when the enzyme conjugate for preparing the inhibin A antibody at the second site is prepared can be combined into a precipitate, and the detection accuracy is improved.
Preferably, the preparation method of the enzyme conjugate of the inhibin A antibody labeled with the enzyme at the second site comprises the following steps:
s2, weighing 1.5mg of horseradish peroxidase, dissolving in 1ml of 1mM acetate buffer solution with pH5.6, adding 10 mu L C 102 H 151 O 39 N 31 、10μL MgSO 4 、20μL C 5 HO 2 Standing NS for 20min, filtering, precipitating, adding 0.1ml of DNFB absolute ethanol solution with the concentration of 1%, and slightly stirring at room temperature for 1h to obtain a first solution;
in the invention, horseradish peroxidase is added into acetate buffer solution for oxidizing horseradish peroxidase, but the enzyme is deteriorated at normal temperature and high temperature and can reduce activity, so C is added 102 H 151 O 39 N 31 、MgSO 4 、C 5 HO 2 NS improves enzyme stability, and prevents enzyme deterioration and activity reduction at high temperature, wherein C 102 H 151 O 39 N 31 Is gelatin, mgSO 4 Is magnesium sulfate, C 5 HO 2 NS is methionine, the gelatin is a protein rich in lysine, glycine and proline, and the colloid structure of polycation of the gelatin and the reducing environment provided by a small amount of methionine are favorable for the horseradish peroxidase to maintain the natural conformation and the enzymatic activity thereof. The action of magnesium sulfate is complicated. On one hand, horseradish peroxidase requires metal ions to maintain its native conformation, and the size of magnesium ions and the electrostatic potential energy of magnesium ions may provide an optimal environment for horseradish peroxidase; on the other hand, the metal ions can change the surface free energy of the horseradish peroxidase, the structure of the horseradish peroxidase can be more compact under certain conditions, and simultaneously, the MgSO (magnesium sulfate) is used as a result of the change of the surface free energy of the horseradish peroxidase 4 Can hydrolyze in water to generate Mg (OH) 2 And H 2 SO 4 And Mg (OH) 2 The water-soluble part can be completely ionized and exists in ion form, so that magnesium ions can be generated, the surface free energy of horseradish peroxidase can be changed, and H is generated simultaneously 2 SO 4 Is a highly acidic and corrosive substanceIt destroys the activity of the enzyme, changes the structure of the enzyme, and causes the enzyme to deteriorate, and thus it passes through Ba (OH) in the first buffer 2 Can neutralize H 2 SO 4 And generating Ba 2 SO 4 And Ba 2 SO 4 The white precipitate can be conveniently removed by experimenters, so that the activity of horseradish peroxidase is maximally protected.
Among them, the reason why the Horseradish Peroxidase is used in the present invention is that Horseradish Peroxidase (HRP) is most commonly used because of its high activity, stability, small molecular weight, and easy preparation of pure enzyme. HRP is widely distributed in the dry plant world, and the horseradish has high medium content, is glycoprotein formed by combining colorless zymoprotein and brown ferriporphyrin, and has the sugar content of 18 percent. HRP is composed of a plurality of isoenzymes, the molecular weight is 40,000, the isoelectric point is PH 3-9, the optimum PH of the enzyme catalysis is slightly different due to different hydrogen donors, but is mostly about PH5. Enzyme-soluble dry water and ammonium sulfate solution with saturation degree below 58%. The prosthetic group and the enzyme protein of HRP have maximum absorption spectra of 403nm and 275nm, respectively, and the purity of the enzyme is generally expressed as the ratio RZ (ReinheitZahl, germany) of OD403nm/OD275 nm. The RZ value of the high-purity enzyme is about 3.0 (up to 3.4). The smaller the RZ value, the more non-enzyme proteins.
The DNFB was added to block the residual a-and e-amino groups in the enzyme protein.
S2.1, adding 0.2ml of newly prepared 0.1mol/L NaIO into the first solution 4 Stirring the solution for 20 minutes at room temperature in the dark, adding 80 mu L of inhibin A antibody and a second buffer solution to adjust the pH value of the solution to 9.0, standing for 12 hours, adding 1ml of 2.5% glycol, slightly stirring for 1 hour at room temperature, and stopping reaction to obtain a second solution;
wherein, the solution turns from original brown to dark green, and is placed for 30min, and the solution turns from original brown to dark green;
wherein the purpose of adding ethylene glycol is to terminate the reaction; to facilitate pH adjustment after addition of the inhibin A antibody, ethylene glycol was formulated with 0.05mol/L CBS.
S2.2, adding 0.1ml of sodium borohydride solution into the second solution, uniformly mixing, and standing for 3 hours at normal temperature to obtain a third solution;
the invention can reduce horseradish peroxidase into a stable conjugate after the binding reaction of the horseradish peroxidase and the inhibin A antibody.
S2.3, dialyzing the third solution for 12 hours by using a first buffer solution, and replacing the first buffer solution every 4 hours to obtain a fourth solution;
s2.4, centrifuging the fourth solution at the speed of 3000r/min for 30min, and filtering the precipitate to obtain a fifth solution;
s2.5, collecting supernatant in the fifth solution, adding the inhibin A antibody of the second site, and preparing enzyme conjugate of the inhibin A antibody of the enzyme-labeled second site.
According to the invention, through oxidizing the HRP molecules, namely glycosyl which is irrelevant to the enzyme activity in the horseradish peroxidase into aldehyde groups by sodium periodate, and then forming Schiff base with amino of antibody protein. To prevent self-coupling of the amino groups of the zymoprotein to aldehyde groups, the residual a-and e-amino groups of the zymoprotein were blocked with 2,4-Dinitrofluorobenzene (DNFB) prior to labeling. After the binding reaction of the enzyme and the antibody, sodium borohydride is added to reduce the antibody into a stable conjugate. At the same time adding C in the course of reaction 102 H 151 O 39 N 31 、MgSO 4 、C 5 HO 2 Compared with the traditional reaction steps, the NS can improve the stability of the enzyme, enables the reaction to be carried out without low temperature, can facilitate the detection, improves the activity of the enzyme and enables the detection to be more accurate.
The invention relates to a detection method of a kit for detecting the content of inhibin A in blood plasma, which comprises the following steps:
s3, 50 mu L of plasma is taken in a first test tube, the plasma is uniformly shaken and then centrifuged for 30min at 3000r/min in a centrifuge, supernatant is taken, 200 mu L of first buffer solution and 20 mu L of magnetic particle suspension coated with inhibin A antibody are added into the supernatant, the supernatant is shaken for 30S at normal temperature and then kept stand for 20min, and then concentrated washing liquor is fully washed to obtain first mixed liquor;
s3.1, adding 150 mu L of enzyme conjugate of the enzyme-labeled inhibin A antibody at the second site into the first mixed solution, stirring for 30S, standing for 15min, and fully washing with a concentrated washing solution to obtain a third mixed solution;
s3.2, adding 150 mu L of luminescent substrate into the third mixed solution, measuring a luminescent value within 1 minute in a dark place, and fitting the concentration of the inhibin A in the plasma through the luminescent value.
During measurement, the magnetic particle suspension standard product coated with the inhibin A antibody and the enzyme conjugate of the inhibin A antibody of the enzyme-labeled second site are respectively combined with two antigenic determinants on the inhibin A antigen molecule in the sample to form the immune complex of the solid phase antibody-antigen-enzyme-labeled antibody. The enzyme conjugate of inhibin A antibody and inhibin A antibody of enzyme-labeled second site in the reaction system is excessive compared with inhibin A antigen to be detected, so that the formation amount of the complex is in direct proportion to the content of inhibin A antigen, and the content of inhibin A antigen in blood plasma can be determined by adding a colored substance generated after adding a luminescent substrate.
Preferably, the step of "fitting the concentration of inhibin A in plasma by luminescence values" is:
s4, preparing a series of gradient calibrators with inhibin A antigen concentrations of 0, 25, 100, 500, 1000 and 2000pg/ml by taking bovine serum albumin and a first buffer solution as solvents;
s4.1, fitting the concentration value of the inhibin A calibrator and the luminescence value in the third mixed solution by using a LOG-LOG _ B mathematical model.
Experimental data
Detection of Activity of Horseradish peroxidase
mu.L of horseradish peroxidase solution was taken, and 1000. Mu.L of the first buffer solution was added thereto, and simultaneously, the horseradish peroxidase was stored at 37 ℃ for 2 hours and the decrease in absorbance at 360nm was measured, whereby it was found that the horseradish peroxidase was stored at 37 ℃ for 2 hours and the enzyme activity was 87%.
Control group 2
mu.L of horseradish peroxidase solution was taken, and 1000. Mu.L of the first buffer solution was added thereto, and simultaneously, the horseradish peroxidase was stored at 50 ℃ for 2 hours and the decrease in absorbance at 360nm was measured, whereby it was found that the horseradish peroxidase was stored at 50 ℃ for 2 hours and the enzyme activity was 65%.
Control group 3
mu.L of horseradish peroxidase solution was taken, and 1000. Mu.L of the first buffer solution was added thereto, and simultaneously, the horseradish peroxidase was stored at 50 ℃ for 48 hours and the decrease in absorbance at 360nm was measured, whereby it was found that the horseradish peroxidase was stored at 50 ℃ for 48 hours and the enzyme activity was 32%.
Control group 4
10 u L of horseradish peroxidase solution, and in which 1000 u L of the first buffer solution is added, and simultaneously 2 u L C is added 102 H 151 O 39 N 31 、20μLC 5 HO 2 NS, stored at 50 ℃ for 2 hours and measured for the decrease in absorbance at 360nm, whereby it was found that horseradish peroxidase was mixed with 2. Mu.LC at 50 ℃ 102 H 151 O 39 N 31 、20μLC 5 HO 2 NS is stored for 48 hours simultaneously, and the activity of the enzyme is 73%.
Experimental group
Example 1
10 u L of horseradish peroxidase solution, and in which 1000 u L of the first buffer solution is added, and simultaneously 2 u L C is added 102 H 151 O 39 N 31 、10μLMgSO 4 、20μLC 5 HO 2 NS, stored at 37 ℃ for 2 hours and measured for the decrease in absorbance at 360nm, whereby it was found that horseradish peroxidase was mixed with 2. Mu.LC at 37 ℃ 102 H 151 O 39 N 31 、10μLMgSO 4 、20μLC 5 HO 2 The NS is stored for 2 hours at the same time, and the activity of the enzyme is 98%.
Example 2
10 u L of horseradish peroxidase solution, and in which 1000 u L of the first buffer solution is added, and simultaneously 2 u L C is added 102 H 151 O 39 N 31 、10μLMgSO 4 、20μLC 5 HO 2 NS, stored at 50 ℃ for 2 hours and measured for the decrease in absorbance at 360nm, whereby it was found that horseradish peroxidase was mixed with 2. Mu.LC at 50 ℃ 102 H 151 O 39 N 31 、10μLMgSO 4 、20μLC 5 HO 2 NS is stored for 2 hours simultaneously, and the activity of the enzyme is 95%.
Example 3
Taking 10 μ L of horseradish peroxidase solution, adding 1000 μ L of the first buffer solution, and simultaneously adding 2 μ L C 102 H 151 O 39 N 31 、10μLMgSO 4 、20μLC 5 HO 2 NS, stored at 50 ℃ for 2 hours and measured for the decrease in absorbance at 360nm, whereby it was found that horseradish peroxidase was mixed with 2. Mu.LC at 50 ℃ 102 H 151 O 39 N 31 、10μLMgSO 4 、20μLC 5 HO 2 The NS is stored for 48 hours at the same time, and the activity of the enzyme is 92%.
As can be seen from FIGS. 2 and 3, the activity of horseradish peroxidase added with stabilizer is greatly improved, the activity of horseradish peroxidase stored for 48h at 50 ℃ is 5% higher than that of horseradish peroxidase stored for 2h at 37 ℃ without stabilizer, and MgSO (MgSO) (MgSO) is not added in the stabilizer 4 The activity of the enzyme(s) is also less than the addition of MgSO 4 The activity of the enzyme (2) is thus known to be MgSO in a stabilizer 4 Playing an important role, the magnesium ions can provide surface free energy capable of changing horseradish peroxidase, so that the activity of the horseradish peroxidase is improved.
Specificity detection
The cross reaction rate of detecting 1.0 mu g/ml activin A, inhibin B and 2000ml mu/L beta-HCG is not higher than 1 percent, so that the cross risk does not exist.
Clinical validation
In order to judge the consistency of the inhibin A detection result of the kit of the invention and the detection results of the kits produced by other manufacturers, 200 serum samples of the early pregnancy and the middle pregnancy are collected for parallel detection, and the results are shown in figure 1.
The results of the alignment shown in FIG. 1 show that the present invention is testedThe linear correlation r of the kit to the alignment kit was 0.99968 2 Is 0.982, the correlation equation is: y =0.9246x +1.8652, which proves that the kit of the invention has better correlation and higher accuracy with the comparison kit.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (6)
1. A kit for detecting the content of inhibin A in blood plasma is characterized by comprising the following components: 10-30 mu L of magnetic particle suspension coated with a first site inhibin A antibody, 10-300 mu L of enzyme conjugate of an enzyme labeled second site inhibin A antibody, 10-100 mu L of calibrator, 10-1000 mu L of first buffer solution, 10-1000 mu L of second buffer solution, 10-150 mu L of luminescent substrate and 10-2000 mu L of concentrated washing solution.
2. The kit for detecting the content of inhibin A in blood plasma as claimed in claim 1, which is characterized by comprising the following components: 25 mu L of magnetic particle suspension coated with the inhibin A antibody at the first site, 150 mu L of enzyme conjugate of the inhibin A antibody at the enzyme-labeled second site, 50 mu L of calibrator, 500 mu L of first buffer solution, 800 mu L of second buffer solution, 130 mu L of luminescent substrate and 1000 mu L of concentrated washing solution.
3. The kit for detecting the content of inhibin A in blood plasma as claimed in claim 2, wherein said first buffer is prepared by the following steps:
s1, collecting 8.0g NaCl, 0.2g KCl and 1.44g Na 2 HPO 4 、0.24g KH 2 PO 4 、0.25g Ba(OH) 2 Dissolving in 800mL of distilled water;
s1.1, adjusting the pH value of the solution to 7.7 by using HCl solution with the pH value of 5.5;
s1.2, and finally adding distilled water to a constant volume of 1L to obtain a first buffer solution.
4. The kit for detecting the content of inhibin A in blood plasma as claimed in claim 2, wherein: the preparation method of the enzyme conjugate of the inhibin A antibody of the enzyme-labeled second site comprises the following steps: (ratio and step refer to Link 2)
S2, weighing 1.5mg horseradish peroxidase, dissolving in 1ml of 1mM acetate buffer solution with pH5.6, adding 10 mu L C 102 H 151 O 39 N 31 、10μL MgSO 4 、20μL C 5 HO 2 Standing NS for 20min, filtering, precipitating, adding 0.1ml of DNFB absolute ethanol solution with the concentration of 1%, and slightly stirring at room temperature for 1h to obtain a first solution;
s2.1, adding 0.2ml of newly-prepared 0.1mol/L NaIO into the first solution 4 Stirring the solution for 20 minutes at room temperature in the dark, adding 80 mu L of inhibin A antibody and a second buffer solution to adjust the pH value of the solution to 9.0, standing for 12 hours, adding 1ml of 2.5% glycol, slightly stirring for 1 hour at room temperature, and stopping reaction to obtain a second solution;
s2.2, adding 0.1ml of sodium borohydride solution into the second solution, uniformly mixing, and standing for 3 hours at normal temperature to obtain a third solution;
s2.3, dialyzing the third solution for 12 hours by using a first buffer solution, and replacing the first buffer solution every 4 hours to obtain a fourth solution;
s2.4, centrifuging the fourth solution at the speed of 3000r/min for 30min, and filtering the precipitate to obtain a fifth solution;
s2.5, collecting supernatant in the fifth solution, adding the inhibin A antibody of the second site, and preparing enzyme conjugate of the inhibin A antibody of the enzyme-labeled second site.
5. The method for detecting the content of inhibin A in plasma as a kit according to claims 1-4, comprising the steps of:
s3, 50 mu L of plasma is taken in a first test tube, the plasma is uniformly shaken and then centrifuged for 30min at 3000r/min in a centrifuge, supernatant is taken, 200 mu L of first buffer solution and 20 mu L of magnetic particle suspension coated with inhibin A antibody are added into the supernatant, the supernatant is shaken for 30S at normal temperature and then kept stand for 20min, and then concentrated washing liquor is fully washed to obtain first mixed liquor;
s3.1, adding 150 mu L of enzyme conjugate of the enzyme-labeled inhibin A antibody of the second site into the first mixed solution, stirring for 30S, standing for 15min, and fully washing with concentrated washing solution to obtain a third mixed solution;
s3.2, adding 150 mu L of luminescent substrate into the third mixed solution, measuring a luminescence value within 1 minute in a dark place, and fitting the concentration of the inhibin A in the plasma according to the luminescence value.
6. The method for detecting the inhibin A content in plasma of claim 5, wherein said step of fitting the concentration of inhibin A in plasma by luminescence value comprises:
s4, taking bovine serum albumin and the first buffer solution as solvents, and preparing a series of gradient calibrators with inhibin A antigen concentrations of 0, 25, 100, 500, 1000 and 2000pg/ml respectively;
s4.1, fitting the concentration value of the inhibin A calibrator and the luminescence value in the third mixed solution by using a LOG-LOG _ B mathematical model.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116449029A (en) * | 2023-06-15 | 2023-07-18 | 广州市丰华生物股份有限公司 | Kit for quantitatively detecting inhibin A and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104407143A (en) * | 2014-11-28 | 2015-03-11 | 山东博科生物产业有限公司 | Hepatitis c virus antigen-antibody joint detection kit |
| CN104792996A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Rabies virus antibody (IgG) enzyme-linked immunoassay kit and detection method thereof |
| CN109444432A (en) * | 2018-12-15 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of kit detecting inhibin A |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104792996A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Rabies virus antibody (IgG) enzyme-linked immunoassay kit and detection method thereof |
| CN104407143A (en) * | 2014-11-28 | 2015-03-11 | 山东博科生物产业有限公司 | Hepatitis c virus antigen-antibody joint detection kit |
| CN109444432A (en) * | 2018-12-15 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of kit detecting inhibin A |
Non-Patent Citations (1)
| Title |
|---|
| 毛新焕 等: "辣根过氧化物酶的热稳定剂", 生物工程学报, vol. 25, no. 03, 25 March 2009 (2009-03-25), pages 388 - 391 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116449029A (en) * | 2023-06-15 | 2023-07-18 | 广州市丰华生物股份有限公司 | Kit for quantitatively detecting inhibin A and preparation method thereof |
| CN116449029B (en) * | 2023-06-15 | 2023-09-01 | 广州市丰华生物股份有限公司 | Kit for quantitatively detecting inhibin A and preparation method thereof |
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