CN116482371A - Hepatitis B E antigen detection kit and preparation method and application thereof - Google Patents
Hepatitis B E antigen detection kit and preparation method and application thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims description 17
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- 239000000126 substance Substances 0.000 claims abstract description 6
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 42
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- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 abstract description 4
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 abstract 1
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- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
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Classifications
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- 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
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5762—Hepatitis B core antigen
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a hepatitis B E antigen detection kit, which comprises a sample diluent, coated magnetic beads, an antibody mark, a washing liquid, a pre-excitation liquid, an excitation liquid and a standard substance, wherein the surface of the coated magnetic beads is combined with biotin-marked hepatitis B E antibody, the antibody mark is acridine ester-marked hepatitis B E antibody, the pre-excitation liquid is nitric acid solution containing hydrogen peroxide, and the excitation liquid is sodium hydroxide solution. Can realize the rapid detection of hepatitis B e antigen, and can obtain the result detection result about 10 minutes while maintaining good performance.
Description
Technical Field
The invention belongs to the technical field of detection kits, and relates to a hepatitis B E antigen detection kit, in particular to a hepatitis B E antigen detection kit based on an acridine ester chemiluminescence method, and a preparation method and application thereof.
Background
Many carriers of Hepatitis B Virus (HBV) in China are paid attention to diagnosis and detection of hepatitis B for a long time. For the detection of hepatitis b, five items are generally involved, including hepatitis b virus e antigen (abbreviated as hepatitis b e antigen, HBeAg). The hepatitis B e antigen is usually detected together with other projects, is one of important indexes for diagnosing hepatitis B major three positive (positive hepatitis B virus surface antigen, positive hepatitis B virus e antigen and positive hepatitis B virus core antibody), can effectively reflect the activity degree of virus replication in hepatitis B patients, and is an important reference index in the treatment stage.
The current common hepatitis B e antigen detection methods mainly comprise an enzyme-linked immunosorbent assay (Enzyme Immunoassay, EIA), a chemiluminescent immunoassay (Chemiluminesent Immunoassay, CLIA) and electrochemiluminescence (Electrogenerated Chemiluminescene, ECL), wherein the three methods are immune reactions based on antigen antibodies, and corresponding results are obtained by using different markers and fluorescent detection methods. Among them, CLIA technology has the characteristics of high sensitivity, wide linear range, more automation realization and the like, and is widely used.
Although chemiluminescent immunoassay (CLIA) is currently in widespread use, a plate-based CLIA kit is still mainly used in the market. The Chinese patent application No. 200610030783.3 discloses an ELISA kit for detecting hepatitis B virus core antigen in serum and a use method thereof. The main components of the kit are as follows: a micro-pore plate pre-coated with a monoclonal hepatitis B core antibody, a sample treating agent and a horseradish peroxidase marked polyclonal hepatitis B core antibody. When in use, the sample to be detected is pretreated by a sample treating agent, then is added into a micro-pore plate pre-coated by a monoclonal hepatitis B core antibody, an enzyme-labeled polyclonal hepatitis B core antibody is added, a substrate is added for color development, then a stop solution is added for stopping the reaction, and finally the colorimetric determination result is obtained. The plate-type CLIA kit still adopts enzymatic reaction, cross-links antibody by horseradish enzyme or alkaline phosphatase, detects dynamic luminescence, causes quicker attenuation of detection signal values and inconsistent attenuation of high-concentration and low-concentration signals, and limits the sensitivity, stability and repeatability of low-end detection in quantitative analysis.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a hepatitis B E antigen detection kit.
In order to achieve the above object, the present invention provides a hepatitis B E antigen detection kit comprising a sample diluent, coated magnetic beads, an antibody label, a wash solution, a pre-excitation solution, an excitation solution and a standard,
the surface of the coated magnetic bead is combined with biotin-marked hepatitis B e antibody,
the antibody is marked as an acridinium ester marked hepatitis B e antibody,
the pre-excitation liquid is nitric acid solution containing hydrogen peroxide,
the excitation liquid is sodium hydroxide solution.
Optimally, the preparation method of the coated magnetic beads comprises the following steps:
(a1) Antibody dialysis: b, adjusting the raw material of the hepatitis B e antibody to a required first concentration by using a first PBS buffer solution, and then dialyzing to obtain a first e antibody solution;
(a2) Coating an antibody: mixing the first e antibody solution with water-soluble biotin according to a proportion, performing oscillation reaction, and then dialyzing with a second PBS buffer solution to obtain biotin-labeled e antibody solution;
(a3) Coating magnetic beads: mixing the biotin-marked e antibody solution with SA magnetic beads, performing oscillation reaction, and cleaning to obtain coated magnetic beads.
Optimally, the preparation method of the antibody label comprises the following steps:
(b1) Antibody dialysis: adjusting the raw material of the hepatitis B e antibody to a second concentration by using a third PBS buffer solution, and then dialyzing to obtain a second e antibody solution;
(b2) Labeling the antibody: mixing the second e antibody solution and acridinium ester in proportion, and carrying out oscillation reaction to obtain a marked e antibody solution;
(b3) Purifying: purifying the marked e antibody solution by using a chromatographic column to obtain the acridinium ester marked hepatitis B e antibody.
Optimally, the sample diluent is Tris-HCl buffer solution and is mixed with tween and preservative, and the washing liquid is Tris-HCl buffer solution and is mixed with tween and preservative; the Tween is a mixture composed of one or more of Tween 20, tween 40, tween 60 and Tween 80, and the concentration of the Tween is 0.1-1 ml/L; the preservative is a mixture of one or more selected from sodium azide, gentamicin and thimerosal, and the concentration of the preservative is 5-15 ml/L.
Optimally, the concentration of hydrogen peroxide in the pre-excitation liquid is 5-10 ml/L, and the concentration of nitric acid is 5-10 ml/L; the concentration of sodium hydroxide in the excitation liquid is 5-10 ml/L.
Optimally, the hepatitis B E antigen detection kit further comprises a box assembly and a magnetic rod assembly, wherein the box assembly comprises a liquid containing unit and a box body formed on any side surface of the liquid containing unit, the liquid containing unit comprises at least one diluent containing groove, a magnetic bead Cheng Cao, a diluted sample containing groove, a labeled antibody containing groove, at least one washing liquid containing groove and a pre-excitation liquid containing groove which are mutually independent, and the magnetic rod assembly comprises a sleeve and a magnetic rod which can be inserted into the sleeve and is used for adsorbing the magnetic bead.
Further, the cartridge assembly further includes a first empty slot disposed between the diluted sample containing slot and the labeled antibody containing slot, and a second empty slot disposed between the wash solution Cheng Cao and the pre-excitation solution containing slot.
The invention aims to provide a preparation method of the hepatitis B E antigen detection kit, which comprises the following steps:
(a) Respectively preparing sample diluent, washing liquid, pre-excitation liquid and excitation liquid;
(b) Using the freeze-dried product of hepatitis B e antigen with specified concentration as a standard substance;
(c) Binding biotin-labeled hepatitis B e antibody to the surface of the magnetic bead to obtain coated magnetic bead;
(d) The antibody mark is obtained by using acridinium ester to mark hepatitis B e antibody.
Still another object of the present invention is to provide an application of the above hepatitis b E antigen detection kit, comprising the steps of:
(S1) adding a sample to be tested containing hepatitis B e antigen into a sample diluent for dilution to obtain a sample diluted solution;
(S2) combining the sample dilution solution with the coated magnetic beads through immune reaction, and diluting to obtain a magnetic bead e antibody solution;
(S3) reacting the magnetic bead e antibody solution with an antibody label, and washing to obtain a magnetic bead e antibody/acridinium ester antibody complex;
(S4) adding the magnetic bead e antibody/acridinium ester antibody complex into a pre-excitation liquid to react, then adding the excitation liquid to react, and detecting the luminescence value of the chemiluminescent reaction.
Optimally, the method further comprises the following steps: (S5) processing the obtained luminescence value by using a calibrator and a calibration card
The hepatitis B E antigen detection kit has excellent sensitivity and specificity by adopting the coated magnetic beads and the hepatitis B E antibody marked by acridinium ester for matching; based on the existing SuperFlex platform, the rapid detection of hepatitis B e antigen can be realized, and the result detection result can be obtained about 10 minutes while the good performance is maintained. Compared with the prior art such as ELISA, the ELISA has more excellent performance (such as sensitivity, specificity, precision, linear range, stability, interference resistance, carrying pollution resistance, detection time and the like).
Drawings
FIG. 1 is a schematic diagram of a hepatitis E antigen detection kit of the present invention.
FIG. 2 is a diagram showing the use of the kit for detecting hepatitis E antigen of the present invention.
FIG. 3 is a schematic diagram of a hepatitis E antigen detection kit of the present invention.
FIG. 4 is a flow chart showing the preparation of coated magnetic beads in the hepatitis E antigen detection kit of the present invention.
FIG. 5 is a flow chart showing the preparation of antibody markers in the hepatitis E antigen detection kit of the present invention.
Detailed Description
The invention relates to a hepatitis B E antigen detection kit, which comprises a sample diluent, coated magnetic beads, an antibody label, a washing liquid, a pre-excitation liquid, an excitation liquid and a standard (the standard contains a freeze-dried product with specified concentration of E antigen and is used for curve calibration), wherein the surface of the coated magnetic beads is combined with biotin-labeled hepatitis B E antibody, the antibody is labeled with acridine ester-labeled hepatitis B E antibody, the pre-excitation liquid is a nitric acid solution containing hydrogen peroxide, and the excitation liquid is a sodium hydroxide solution. The coating magnetic beads and the hepatitis B e antibody marked by the acridinium ester are adopted to be matched so as to combine with the e antigen, so that the kit has excellent sensitivity and specificity; based on the existing SuperFlex platform, the rapid detection of hepatitis B e antigen can be realized, and the result detection result can be obtained about 10 minutes while the good performance is maintained. Compared with the prior art such as ELISA, the ELISA has more excellent performance (such as sensitivity, specificity, precision, linear range, stability, interference resistance, carrying pollution resistance, detection time and the like).
The preparation method of the coated magnetic beads comprises the following steps: (a 1) antibody dialysis: b, adjusting the raw material of the hepatitis B e antibody to a required first concentration by using a first PBS buffer solution, and then dialyzing to obtain a first e antibody solution; (a 2) coating antibody: mixing the first e antibody solution with water-soluble biotin according to a proportion, performing oscillation reaction, and then dialyzing with a second PBS buffer solution to obtain biotin-labeled e antibody solution; (a 3) coating magnetic beads: mixing the biotin-marked e antibody solution with SA magnetic beads, performing oscillation reaction, and cleaning to obtain coated magnetic beads; thus, coated magnetic beads of high purity can be obtained. The preparation method of the antibody label comprises the following steps: (b 1) antibody dialysis: adjusting the raw material of the hepatitis B e antibody to a second concentration by using a third PBS buffer solution, and then dialyzing to obtain a second e antibody solution; (b 2) labeled antibody: mixing the second e antibody solution and acridinium ester in proportion, and carrying out oscillation reaction to obtain a marked e antibody solution; (b 3) purification: purifying the marked e antibody solution by using a chromatographic column to obtain an acridinium ester marked hepatitis B e antibody; this enables to obtain a high-purity antibody label.
The sample diluent is Tris-HCl buffer solution and is mixed with tween and preservative, and the washing liquid is Tris-HCl buffer solution and is mixed with tween and preservative; the Tween is a mixture composed of one or more of Tween 20, tween 40, tween 60 and Tween 80, and the concentration of the Tween is 0.1-1 ml/L; the preservative is a mixture composed of one or more selected from sodium azide, gentamicin and thimerosal, and the concentration of the preservative is 5-15 ml/L; when no tween or preservative is contained in the Tris-HCl buffer, this can lead to contamination of the kit with one or more microorganisms during preparation, storage and use. The concentration of hydrogen peroxide in the pre-excitation liquid is 5-10 ml/L, and the concentration of nitric acid is 5-10 ml/L; the concentration of sodium hydroxide in the excitation liquid is 5-10 ml/L; the existing conventional pre-excitation liquid and excitation liquid can also be selected, so that the detection effect can be reduced.
The hepatitis b E antigen detection kit further comprises a box assembly 1 and a magnetic rod assembly 2, wherein the box assembly 1 comprises a liquid containing unit and a box body 10 formed on any side surface of the liquid containing unit, the liquid containing unit comprises at least one diluent containing groove 11, a magnetic bead Cheng Cao, a diluted sample containing groove 13, a labeled antibody containing groove 15, at least one washing liquid containing groove 16 and a pre-excitation liquid containing groove 18 which are mutually independent, and the magnetic rod assembly 2 comprises a sleeve 21 and a magnetic rod 22 which can be inserted into the sleeve 21 and is used for adsorbing the magnetic beads. The cartridge assembly 1 further comprises a first blank slot 14 arranged between the diluted sample holding slot 13 and the labeled antibody holding slot 15, and a second blank slot 17 arranged between the washing liquid holding slot 16 and the pre-excitation liquid holding slot 18; the diluent storage tank 11 and the washing liquid storage tank 16 are two independent from each other. Specifically, the liquid containing unit in the box assembly 1 consists of 10 liquid containing tanks which are arranged in sequence and continuously (the liquid containing tanks are positioned on the same straight line); two diluent holding tanks 11 (holding sample diluent), one magnetic bead Cheng Cao (holding coated magnetic bead), one diluted sample holding tank 13 (holding diluted sample), one first blank tank 14, one labeled antibody holding tank 15 (holding labeled antibody), two wash liquid holding tanks 16 (holding wash liquid), one second blank tank 17 and a pre-excitation liquid holding tank 18 (holding pre-excitation liquid and adding excitation liquid) are sequentially arranged. Hepatitis b E antigen detection kits also typically have a calibration card containing a primary calibration curve.
The preparation method of the hepatitis B E antigen detection kit comprises the following steps: (a) Respectively preparing sample diluent, washing liquid, pre-excitation liquid and excitation liquid; (b) Using the freeze-dried product of hepatitis B e antigen with specified concentration as a standard substance; (c) Binding biotin-labeled hepatitis B e antibody to the surface of the magnetic bead to obtain coated magnetic bead; (d) The antibody mark is obtained by using acridinium ester to mark hepatitis B e antibody.
The application of the hepatitis B E antigen detection kit comprises the following steps: (S1) adding a sample to be tested containing hepatitis B e antigen into a sample diluent for dilution to obtain a sample diluted solution; (S2) combining the sample dilution solution with the coated magnetic beads through immune reaction, and diluting to obtain a magnetic bead e antibody solution; (S3) reacting the magnetic bead e antibody solution with an antibody label, and washing to obtain a magnetic bead e antibody/acridinium ester antibody complex; (S4) adding the magnetic bead e antibody/acridinium ester antibody complex into a pre-excitation liquid for reaction, then adding the excitation liquid for reaction, and detecting the luminescence value of the chemiluminescent reaction; (S5) processing the obtained luminous value by using a calibrator and a calibration card. The hepatitis B E antigen detection kit has more excellent performance (such as more excellent sensitivity, specificity, precision, linear range, stability, interference resistance, carrying pollution resistance, detection time and the like) compared with the prior art (such as ELISA) through performance test, and is shown in a table 1. The hepatitis B E antigen detection kit is matched with the existing SuperFlex platform, is simple to use, and can obtain a detection result only about 8 minutes and 30 seconds after the sample is started.
In order that the present invention may be better understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which it is to be understood that the invention is illustrated in the appended drawings. All other embodiments obtained under the premise of equivalent changes and modifications made by those skilled in the art based on the embodiments of the present invention shall fall within the scope of the present invention.
Example 1
The embodiment provides a coated magnetic bead and a preparation method thereof, as shown in fig. 4, comprising the following steps:
(a1) Antibody dialysis: the hepatitis b e antibody stock was adjusted to a concentration of 2mg/ml with a PBS buffer at ph=8.0, 0.05M (i.e. 0.5 mol/L), and then dialyzed for 16-24 hours to obtain an e antibody solution.
And (3) condition selection:
preparing by using different buffers as a reaction buffer system, and detecting a sample luminescence value;
the judging method comprises the following steps: the signal to noise ratios (parallel test, 5 samples) of the results of the samples at different concentrations were compared as shown in table 1:
TABLE 1
Results and analysis: under the same pH conditions, the 0.05M PBS is used as a reaction buffer system, and the signal value and the signal to noise ratio are slightly higher than those of the 0.05 MTSA. The reaction pH was further confirmed by selecting 0.05M PBS as the reaction buffer system.
Preparing by using 0.1M PBS with different pH values as a biotin labeling reaction buffer solution, and detecting the luminous value of a sample;
the judging method comprises the following steps: the signal to noise ratios of the results of the different concentrations were compared as shown in table 2:
TABLE 2
Results and analysis: at pH8.0, the signal value and signal to noise ratio of the detected sample were the highest, confirming that 0.05M PBS at pH8.0 was used as the buffer for the biotin-labeled reaction.
(a2) Coating an antibody: mixing the dialyzed antibody (namely the e antibody solution) with water-soluble biotin according to the mass ratio of 40:1, and then carrying out shaking reaction for 2 hours at 25 ℃; the reacted solution was then dialyzed against PBS buffer at ph=7.2, 0.05M for 16-24 hours to obtain a biotin-labeled e antibody solution.
And (3) condition selection:
preparing by using different biotin labeling ratios, and detecting a sample luminescence value;
the judging method comprises the following steps: the signal to noise ratios of the results of the samples at the different concentrations were compared as shown in table 3:
TABLE 3 Table 3
Results and analysis: the mass ratio of the antibody to the biotin is too low or too high, and the signal value and the signal-to-noise ratio are low; when the ratio is 1 to 30, 1 to 40 and 1 to 60, the signal value and the signal to noise ratio of the detection sample are close; the intermediate ratio 1 to 40 was chosen as the biotin-labeling ratio. Further confirm the labeling time of biotin.
Preparing by using different biotin labeling time, and detecting a sample luminescence value;
the judging method comprises the following steps: the signal to noise ratios of the results of the samples at the different concentrations were compared as shown in table 4:
TABLE 4 Table 4
Results and analysis: when the antibody reacts with biotin for 1.5 hours, the signal value reaches the highest value, the reaction is completed, and after the reaction is prolonged, the signal value and the signal-to-noise ratio are basically unchanged; considering in combination, the time after completion of the reaction, i.e., the reaction time 2h, was chosen.
(a3) Coating magnetic beads: mixing SA magnetic beads and biotin-labeled e antibody solution according to the mass ratio of 50:1, and then carrying out shaking reaction at about 37 ℃ for 16-24 hours; the coated magnetic beads were obtained after washing with a magnetic bead washing solution (commercially available, recording number Su Suxie, 20155019).
And (3) condition selection:
preparing by using different magnetic bead coating ratios, and detecting a sample luminescence value;
the judging method comprises the following steps: the signal to noise ratios of the results of the samples at the different concentrations were compared as shown in table 5:
TABLE 5
Results and analysis: when the mass ratio of the antibody to the magnetic beads is changed from 1 to 30 to 1 to 50, the signal value and the signal to noise ratio are relatively close, and the mass ratio is from 1:50 to 1: and 200, the signal value is in a descending trend, and the mass ratio of the antibody to the magnetic beads is 1 to 50 for coating by comprehensively considering.
Example 2
The embodiment provides an antibody label and a preparation method thereof, as shown in fig. 5, comprising the following steps:
(b1) Antibody dialysis: dialyzing the hepatitis B e antibody raw material for 16-24 hours by using PBS buffer solution with pH value of 8.0 and 0.05M to obtain an e antibody solution;
the method comprises the following steps: reacting acridinium ester and antibody in PBS buffer solutions with different pH values, and detecting a sample luminescence value;
the judging method comprises the following steps: comparing the signal to noise ratio; the experimental results are shown in table 6:
TABLE 6
Conclusion: the labeling reaction gradually increases with the increase of the pH value, the signal value is highest when the pH value reaches 9.09, and then gradually decreases, but the background is correspondingly higher as the pH value is higher, so that the reaction condition with the highest signal-to-noise ratio, namely the 0.05M PBS buffer solution with the pH value of 8.0, is selected.
(b2) Labeling the antibody: mixing the dialyzed antibody solution (namely, the e antibody solution) and acridinium ester according to the mass ratio of 35:1, and then carrying out light-shielding and vibration reaction for 2 hours at 25 ℃ to obtain a labeled e antibody solution;
the method comprises the following steps: different ratios of the antibody and the acridinium ester are selected for carrying out a labeling reaction, and the luminous value of a sample is detected;
the judging method comprises the following steps: comparing the signal to noise ratio; the experimental results are shown in table 7:
TABLE 7
Conclusion: when the mass ratio of the antibody to the acridinium ester is 35 to 1, the signal to noise ratio of a detection sample is the highest, and the luminescence value is also in an acceptable range, so that the antibody is selected: the acridinium ester is labeled at a mass ratio of 35:1.
(b3) Purifying: the labeled solution (i.e., labeled e antibody solution) was purified by a G50 column chromatography to obtain acridinium ester-labeled e antibody (i.e., antibody label).
The method comprises the following steps: different reaction times are selected for carrying out the labeling reaction of the antibody and the acridinium ester, and the luminous value of the sample is detected;
the judging method comprises the following steps: comparing the signal to noise ratio; the experimental results are shown in table 8:
TABLE 6
Conclusion: the reaction of the antibody with the acridinium ester was fully completed at 1h, taking into account the overall 2h reaction time.
The method comprises the following steps: in combination with the existing production conditions, the ultrafiltration tube cannot meet the requirement of mass production, so that the purification mode of the G50 purification column is compared with the purification mode of ultrafiltration, the luminous value of a sample is detected, and the purification mode of mass production is determined;
the judging method comprises the following steps: comparing the signal to noise ratio; the experimental results are shown in table 9:
conclusion: the signal value and the signal to noise ratio of the G50 purifying column are relatively close to those of the ultrafiltration purifying column, so that the purifying mode of the G50 purifying column can be used in mass production.
Example 3
The present embodiment provides a kit for detecting hepatitis b E antigen, as shown in fig. 1 and 2, which includes a sample diluent, coated magnetic beads (in embodiment 1), an antibody label (in embodiment 2), a washing liquid, a pre-excitation liquid, an excitation liquid, a standard substance and a calibration card, wherein the pre-excitation liquid is a nitric acid solution containing hydrogen peroxide, and the excitation liquid is a sodium hydroxide solution.
The hepatitis B E antigen detection kit mainly comprises a kit component 1 and a matched magnetic rod component 2 in structure. The magnetic rod assembly 2 includes a sleeve 21 and a magnetic rod 22 inserted into the sleeve 21 for adsorbing magnetic beads (coated magnetic beads). The cartridge assembly 1 includes a cartridge body 10 and a liquid containing unit formed on the cartridge body 10; the liquid containing unit comprises at least one diluent containing groove 11, a magnetic bead Cheng Cao, a diluted sample containing groove 13, a marked antibody containing groove 15, at least one washing liquid containing groove 16 and a pre-excitation liquid containing groove 18 which are mutually independent; the cartridge assembly 1 further comprises a first blank slot 14 arranged between the diluted sample holding slot 13 and the labeled antibody holding slot 15, and a second blank slot 17 arranged between the washing liquid holding slot 16 and the pre-excitation liquid holding slot 18; the diluent storage tank 11 and the washing liquid storage tank 16 are two independent from each other. In the present embodiment, the liquid containing unit in the cartridge assembly 1 is composed of 10 liquid containing tanks which are arranged in sequence and continuously (the liquid containing tanks are on the same straight line); two diluent holding tanks 11 (holding sample diluent), one magnetic bead Cheng Cao (holding coated magnetic bead), one diluted sample holding tank 13 (holding diluted sample), one first blank tank 14, one labeled antibody holding tank 15 (holding labeled antibody), two wash liquid holding tanks 16 (holding wash liquid), one second blank tank 17 and a pre-excitation liquid holding tank 18 (holding pre-excitation liquid and adding excitation liquid) are sequentially arranged.
The sample diluent is Tris-HCl buffer solution containing tween and preservative; tween is Tween 20, and the concentration of the tween is 0.1-1 ml/L; the preservative is sodium azide, and the concentration of the preservative is 5-15 ml/L. In this embodiment, the sample dilutions in both diluent reservoirs 11 are the same and are each 0.4ml. The magnetic beads Cheng Cao contained 50ng of coated magnetic beads, and contained 123ng/ml of a magnetic bead coating liquid, specifically 0.4ml. The diluted sample container 13 contains 0.5ml of the sample diluent and receives the reacted magnetic beads in the magnetic beads Cheng Cao. The labeled antibody containing vessel 15 contains an antibody label (0.4 ml of sample dilution containing the antibody label). The wash solution is identical to the sample diluent and is placed in two wash solution reservoirs 16, respectively. The concentration of hydrogen peroxide in the pre-excitation liquid is 5-10 ml/L, and the concentration of nitric acid is 5-10 ml/L; the concentration of sodium hydroxide in the excitation liquid is 5-10 ml/L.
The preparation method of the hepatitis B E antigen detection kit comprises the following steps: (a) Respectively preparing sample diluent, washing liquid, pre-excitation liquid and excitation liquid; (b) Using the freeze-dried product with the specified concentration of hepatitis B e antigen as a standard (the freeze-dried product containing the specified concentration of hepatitis B e antigen is used for curve calibration; the specified concentration is 10 PEIU/ml); (c) Binding biotin-labeled hepatitis b e antibody to the surface of the magnetic beads to obtain coated magnetic beads (see example 1 for details); (d) Antibody labeling was obtained by labeling hepatitis b e antibody with acridinium esters (see example 2 in particular).
The application of the hepatitis B E antigen detection kit comprises the following steps:
(S1) adding a sample to be tested containing hepatitis B e antigen into a sample diluent for dilution to obtain a sample diluted solution;
the method comprises the following steps: after adding the sample to be measured (blood sample) to the first diluent container 11 (left side in fig. 1) and stirring uniformly, 0.2ml is put into the second diluent container 11 (right side in fig. 1); stirring uniformly to obtain a sample diluted solution.
(S2) combining the sample diluted solution with the coated magnetic beads through immune reaction, and diluting to obtain a magnetic bead e antibody solution;
the method comprises the following steps: taking 0.2ml of the diluted sample solution, and putting the diluted sample solution into a solution containing coated magnetic beads (namely, the magnetic beads Cheng Cao) so that the e antigen and the coated magnetic beads react in an immune way (shown in figure 3) to obtain a magnetic bead e antibody solution; the magnetic beads are sucked by the magnetic rod assembly 2 so as to be transferred into the diluted sample holding groove 13.
(S3) reacting the diluted magnetic bead e antibody solution with an antibody label, and washing to obtain a magnetic bead e antibody/acridinium ester antibody complex;
the method comprises the following steps: sucking the magnetic bead e antibody in the diluted sample containing groove 13 by using the magnetic rod assembly 2, transferring the magnetic bead e antibody into the labeled antibody containing groove 15 to react with the antibody label (shown in fig. 3); then sucking the magnetic bead e antibody after the reaction with the antibody mark by using a magnetic rod assembly 2, and respectively transferring the magnetic bead e antibody to two washing liquid holding tanks 16 (transferring by using the magnetic rod assembly 2) for washing liquid to obtain a magnetic bead e antibody/acridinium ester antibody compound;
(S4) adding the magnetic bead e antibody/acridinium ester antibody complex into a pre-excitation liquid, then adding the excitation liquid, and detecting the luminescence value of the chemiluminescent reaction;
the method comprises the following steps: sucking the magnetic bead e antibody/acridinium ester antibody complex by using a magnetic rod assembly 2, transferring the magnetic bead e antibody/acridinium ester antibody complex into a pre-excitation liquid containing tank 18 (containing pre-excitation liquid), adding excitation liquid, and starting a SuperFlex platform to detect a luminescence value;
(S5) the obtained luminescence value is also processed by using a calibrator and a calibration card (about 8 minutes and 30 seconds in the whole operation flow).
Table 1 comparison of the effects of the detection kit in example 3 with conventional ELISA techniques
The foregoing is merely a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; while the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
Claims (10)
1. A hepatitis B E antigen detection kit is characterized in that: the hepatitis B E antigen detection kit comprises sample diluent, coated magnetic beads, antibody marks, washing liquid, pre-excitation liquid, excitation liquid and standard substances,
the surface of the coated magnetic bead is combined with biotin-marked hepatitis B e antibody,
the antibody is marked as an acridinium ester marked hepatitis B e antibody,
the pre-excitation liquid is nitric acid solution containing hydrogen peroxide,
the excitation liquid is sodium hydroxide solution.
2. The hepatitis b E antigen detection kit of claim 1, wherein the preparation method of the coated magnetic beads comprises the steps of:
(a1) Antibody dialysis: b, adjusting the raw material of the hepatitis B e antibody to a required first concentration by using a first PBS buffer solution, and then dialyzing to obtain a first e antibody solution;
(a2) Coating an antibody: mixing the first e antibody solution with water-soluble biotin according to a proportion, performing oscillation reaction, and then dialyzing with a second PBS buffer solution to obtain biotin-labeled e antibody solution;
(a3) Coating magnetic beads: mixing the biotin-marked e antibody solution with SA magnetic beads, performing oscillation reaction, and cleaning to obtain coated magnetic beads.
3. The hepatitis b E antigen detection kit of claim 1, wherein the preparation method of the antibody label comprises the steps of:
(b1) Antibody dialysis: adjusting the raw material of the hepatitis B e antibody to a second concentration by using a third PBS buffer solution, and then dialyzing to obtain a second e antibody solution;
(b2) Labeling the antibody: mixing the second e antibody solution and acridinium ester in proportion, and carrying out oscillation reaction to obtain a marked e antibody solution;
(b3) Purifying: purifying the marked e antibody solution by using a chromatographic column to obtain the acridinium ester marked hepatitis B e antibody.
4. The hepatitis b E antigen detection kit of claim 1, wherein: the sample diluent is Tris-HCl buffer solution and is mixed with tween and preservative, and the washing liquid is Tris-HCl buffer solution and is mixed with tween and preservative; the Tween is a mixture composed of one or more of Tween 20, tween 40, tween 60 and Tween 80, and the concentration of the Tween is 0.1-1 ml/L; the preservative is a mixture of one or more selected from sodium azide, gentamicin and thimerosal, and the concentration of the preservative is 5-15 ml/L.
5. The hepatitis b E antigen detection kit of claim 1, wherein: the concentration of hydrogen peroxide in the pre-excitation liquid is 5-10 ml/L, and the concentration of nitric acid is 5-10 ml/L; the concentration of sodium hydroxide in the excitation liquid is 5-10 ml/L.
6. The hepatitis b E antigen detection kit of claim 1, wherein: the hepatitis B E antigen detection kit further comprises a box assembly (1) and a magnetic rod assembly (2), wherein the box assembly (1) comprises a liquid containing unit and a box body (10) formed on any side surface of the liquid containing unit, the liquid containing unit comprises at least one diluent containing groove (11), magnetic beads Cheng Cao (12), a diluent sample containing groove (13), a labeled antibody containing groove (15), at least one washing liquid containing groove (16) and a pre-excitation liquid containing groove (18) which are mutually independent, and the magnetic rod assembly (2) comprises a sleeve (21) and a magnetic rod (22) which can be inserted into the sleeve (21) and is used for adsorbing the magnetic beads.
7. The hepatitis b E antigen detection kit of claim 6, wherein: the cartridge assembly (1) further comprises a first blank groove (14) arranged between the diluted sample holding groove (13) and the labeled antibody holding groove (15) and a second blank groove (17) arranged between the washing liquid Cheng Cao (16) and the pre-excitation liquid holding groove (18).
8. The method for preparing a hepatitis b E antigen detection kit as claimed in any one of claims 1 to 7, characterized by comprising the steps of:
(a) Respectively preparing sample diluent, washing liquid, pre-excitation liquid and excitation liquid;
(b) Using the freeze-dried product of hepatitis B e antigen with specified concentration as a standard substance;
(c) Binding biotin-labeled hepatitis B e antibody to the surface of the magnetic bead to obtain coated magnetic bead;
(d) The antibody mark is obtained by using acridinium ester to mark hepatitis B e antibody.
9. Use of the hepatitis b E antigen detection kit as claimed in any one of claims 1 to 7, comprising the steps of:
(S1) adding a sample to be tested containing hepatitis B e antigen into a sample diluent for dilution to obtain a sample diluted solution;
(S2) combining the sample dilution solution with the coated magnetic beads through immune reaction, and diluting to obtain a magnetic bead e antibody solution;
(S3) reacting the magnetic bead e antibody solution with an antibody label, and washing to obtain a magnetic bead e antibody/acridinium ester antibody complex;
and (S4) adding the magnetic bead e antibody/acridinium ester antibody complex into a pre-excitation liquid for reaction, then adding the excitation liquid for reaction, and detecting the luminescence value of the chemiluminescent reaction.
10. The use of a hepatitis b E antigen detection kit as claimed in claim 9, further comprising the steps of: (S5) processing the obtained luminous value by using a calibrator and a calibration card.
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