CN109541230A - A kind of detection anti-HBc kit and preparation method thereof - Google Patents
A kind of detection anti-HBc kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of detection anti-HBc kits and preparation method thereof, are grouped as by following group: being coated with the magnetic microsphere, anti-HBc calibration object, anti-HBc marker, analysis buffer, cleaning solution, enhancement solution and RFID card in conjunction with lanthanide series of HBcAg.This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, overcome the physical sorption reaction time of ELISA Plate longer, the slower drawback of testing result, the reaction time is greatly shortened, while also having that time resolution detection technique accuracy height, high sensitivity, high specificity, the range of linearity is wide, detection is stable and convenient advantage.Magnetic microsphere is coated with corresponding antigen or antibody and greatly increases the contact surface area of immune response because of the characteristic of magnetic microsphere solid, to greatly shorten detection time, detecting anti-HBc only needs 30 minutes can detect result.
Description
Technical field
The present invention relates to a kind of detection anti-HBc kits and preparation method thereof, and in particular to a kind of
The preparation side of detection anti-HBc kit is combined with Timed-resolved fluoroimmunoassay based on magnetic microsphere
Method.
Background technique
Hepatitis B (abbreviation hepatitis B) is to lead to liver property inflammation and liver function energy loss caused by hepatitis type B virus (HBV)
A kind of harmful communicable disease.The anti-HBc of hepatitis type B virus is most sensitive HBV infection index, anti-HBc content fluctuation and disease
Feelings are in parallel relation, related with virus multiplication and hepatocellular damage, and content commonly uses high level or low-level clinically to sentence
Disconnected hepatitis B lapse to and prognosis, this index are to reflect the presence infected recently, the B-mode liver in content and liver cell
Scorching virus core antigen content is related, is the index of hbv replication, and high-caliber anti-HBc indicates that HBV is persistently replicated in vivo, low
It is horizontal then indicate previous infection mistake.Early stage acute hepatitis B, anti-HBc is significantly raised, equal in the entire acute stage of hepatitis B
It can detect, then be low-level in convalescence and recovery period.And anti-HBc is that the mark that occurs earliest after HBV infection in serum is anti-
Body is the good index of epidemiological survey.
The project of hepatitis B Serological testing at present generally comprises HBsAg, Anti-HBs, HBeAg, Anti-HBe, Anti-
HBc, that is, " Hepatitis B virus " being commonly called as.The detection method of the existing anti-HBe of mainstream has enzyme linked immunological (ELISA), colloidal gold method, change
Learn luminous (CLIA), electrochemical luminescence (ECL), time-resolved fluoroimmunoassay (TRFIA) etc..ELISA is examined as sxemiquantitative
Survey method is now most widely used detection method, but its sensitivity, linear extent range are not up to higher level, it is difficult to be adapted to
The demand of market development.Colloidal gold method and ELISA have the shortcomings that identical.Though and CLIA and ECL high sensitivity but presence inspection
Measurement equipment cost is high, and corresponding marker research and development threshold is high, and the country can be under one's control in short-term, equally also limits
Popularization at home.And time-resolved fluoroimmunoassay (TRFIA) sensitivity can reach the consistent level of CLIA, not only detect
Equipment manufacturing cost is cheap, and labelling technique is quite mature at home, testing cost is low simultaneous.And traditional time resolution detection,
Still based on the physical absorption of blank ELISA Plate, time-consuming for detection process.Shorten the reaction time, reduce testing cost, improves inspection
Sensitivity and the range of linearity are surveyed, it will be with good market prospects.
Summary of the invention
Based on this, it is an object of the invention to overcome defect in the prior art and the relevant technologies testing cost is high lacks
Point provides the preparation method of a kind of detection anti-HBc kit and the kit.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of detection anti-HBc kit, is grouped as by following group: be coated with HBcAg magnetic microsphere,
Anti- HBc calibration object, the anti-HBc marker in conjunction with lanthanide series, analysis buffer, concentration washing lotion (cleaning solution) and enhancing
Liquid, RFID card.
Preferably, the magnetic microsphere being coated in the magnetic microsphere of HBcAg is by micron-sized Fe2O3Or Fe3O4
Magnetic particle and high-molecular organic material carry out it is compound, formed have superparamagnetism, can be with immunising antigen or antibody phase
In conjunction with micron-sized microballoon, that is, be commonly referred to as " magnetic bead ".It is 0.1~5 μm that magnetic microsphere, which should be able to meet diameter, and magnetic microsphere passes through
Surface modification can have various active functional group, including but not limited to hydroxyl (- OH), amino (- NH2), carboxyl (- COOH).
Preferably, the HBcAg being coated in the magnetic microsphere of HBcAg can be recombinant protein, be also possible to
Native protein.
Preferably, the magnetic microsphere for being coated with HBcAg is prepared using following steps:
By HBcAg after 2~8 DEG C of superspeed refrigerated centrifuges are by buffer system replacement Treatment, with the magnetic microsphere after cleaning, activation
Simultaneously constant-temperature incubation is mixed, magnetic bead is cleaned after incubation and abandons supernatant, is then closed with magnetic bead confining liquid, cleaning magnetic bead is again
Supernatant is abandoned, the HBcAg being coated with is saved in liquid in magnetic bead and is uprightly saved with 2~8 DEG C of refrigerators.
It is 10 μ g that every milligram of magnetic microsphere of EDC and Sulfo-NHS, which is preferably loaded quality, in the activation of the magnetic microsphere
~1000 μ g.Activation method includes but is not limited to EDC, Sulfo-NHS one such and two kinds.
Preferably, the antibody in the anti-HBc marker in conjunction with lanthanide series is either monoclonal antibody also can be more
It is anti-.Lanthanide series therein is to be completed in conjunction with anti-HBc by intermediate chelating agent, lanthanide series include but is not limited to europium (EU),
Samarium (Sm), chelating agent include but is not limited to isothiocyanic acid phenyl-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyano benzyl
Base-DTTA, diethylene triamine pentaacetic acid aminophenyl-EDTA.
The present invention also provides a kind of methods for detecting anti-HBc using mentioned reagent box, and described method includes following steps:
(1) working solution is diluted to by washing lotion is concentrated with purified water；
(2) marker is diluted to working solution with analysis buffer；
(3) magnetic microsphere of the working concentration diluted is added in reaction cup；
(4) sample to be examined or calibration object are added in above-mentioned reaction cup；
(5) the marker working solution diluted in (2) is added in reaction cup；
(6) reaction cup is incubated at room temperature；
(7) magnetic microsphere after using the cleaning liquid in (1) to add magnetic in washing reaction cup；
(8) after washing, degaussing is added enhancement solution and is incubated for；
(9) after being incubated for, the fluorescent collecting for carrying out corresponding wavelength is detected and is analyzed.
Preferably, for the present invention in order to further reduce manual steps, self-produced SmartTRF grinds certainly in cooperation company
The relevant parameter of detection method, operating procedure are all copied to RFID card by complete series Immunofluorescence test equipment
In.In actual mechanical process, it is only necessary to RFID card is adapted to above-mentioned Immunofluorescence test equipment can be automatically finished it is above-mentioned
The operating procedure of experiment.RFID(Radio Frequency Identification) technology, also known as radio frequency identification are one
The kind communication technology can be identified specific objective by radio signals and read and write related data, without identifying system and specific mesh
Mechanical or optical contact is established between mark.
The present invention also provides a kind of preparation methods for detecting anti-HBc kit, and described method includes following steps:
(1) preparation is coated with the magnetic microsphere of HBcAg；
(2) anti-HBc marker of the preparation in conjunction with lanthanide series；
(3) calibration object is prepared；
(4) analysis buffer, concentration washing lotion and enhancement solution are prepared；
(5) each component is dispensed respectively into corresponding storage container；
(6) RFID card duplicate copy；
(7) coding, labelling；
(8) it is assembled into finished product kit.
Compared with prior art, the beneficial effects of the invention are that:
This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, that is, when overcoming the physical adsorption reaction of ELISA Plate
Between it is longer, the slower drawback of testing result greatly shortens the reaction time, while also having time resolution detection technique accuracy
Height, high sensitivity, high specificity, the range of linearity are wide, detection is stable and convenient advantage.Magnetic microsphere be coated with corresponding antigen or
Antibody greatly increases the contact surface area of immune response because of the characteristic of magnetic microsphere solid, so that detection time is greatly shortened,
Detecting anti-HBc only needs 30 minutes can detect result.In addition, being no longer limited by traditional enzyme due to the fluid behaviour of magnetic microsphere
The frame of target limits, and detection can be completed in arbitrary reaction cup and small test tube, while can reduce detecting instrument equipment
Volume size and equipment cost, also can be such as ELISA Plates, the reflective inspection of chemistry to meet the full-automatic detection demand in two, three line cities
Survey equally carries out big flux detection, meets the full-automatic detection demand in a line city.
Detailed description of the invention
Fig. 1 is to be used to store the magnetic microsphere and lanthanide series for being coated with HBcAg using detection kit of the present invention
In conjunction with anti-HBc marker, analysis buffer, cleaning solution and enhancement solution reagent strip schematic top plan view.
Fig. 2 is to be used to store the magnetic microsphere and lanthanide series for being coated with HBcAg using detection kit of the present invention
In conjunction with anti-HBc marker, analysis buffer, cleaning solution and enhancement solution the stereochemical structure mark of reagent strip be intended to schematic diagram.
Specific embodiment
The present invention is further illustrated with attached drawing with reference to embodiments, and it is special that technology of the invention is better described
Point, technical solution.Following embodiment does not cause any restrictions to the present invention.The antigen of anti-HBc, antibody in following embodiment
Derive from Wuhan AudioCodes Bo Tai Biotechnology Co., Ltd；Magnetic microsphere derives from GE company；Europium label is purchased from Finland
Wallac company；Anti-HBc National reference is provided by National Institute for Food and Drugs Control；Control examination
Agent box is Abbott Laboratories' anti-HBc assay kit (chemiluminescence particulate immunodetection).
Embodiment 1
A kind of detection anti-HBc kit, the kit include: the magnetic microsphere for being coated with HBcAg,
Anti- HBc calibration object, the anti-HBc marker of europium label, analysis buffer, concentration washing lotion and enhancement solution, RFID card.
The present invention also provides the preparation method of above-mentioned detection anti-HBc kit, the method includes
Following steps:
(1) preparation is coated with the magnetic microsphere of HBcAg: HBcAg is replaced buffer system through 2~8 DEG C of superspeed refrigerated centrifuges
After processing, is mixed simultaneously constant-temperature incubation 1~3 hour with 1 μm of carboxyl magnetic microsphere of diameter after cleaning, activation, magnetic is used after incubation
Pearl cleaning solution cleaning magnetic bead simultaneously abandons supernatant, is then closed with magnetic bead confining liquid, and cleaning magnetic bead abandons supernatant again,
The anti-HBc magnetic microsphere being coated with is saved in liquid in magnetic bead and is uprightly saved with 2~8 DEG C of refrigerators.The anti-HBc that will be coated with again
Magnetic microsphere saves liquid with magnetic bead and is diluted to working solution concentration, is distributed into 10mL/ bottles.Preferably, magnetic microsphere and HBcAg are coated with
Mass ratio be one of 10:1,20:1,30:1,40:1；Preferably, the displacement buffer and magnetic bead cleaning solution are
The MES buffer of 0.05~0.5M PH, 5.8~PH 7.0；Preferably, EDC and Sulfo-NHS is every in the activation of magnetic microsphere
It is 10 μ of μ g~1000 g that milligram magnetic microsphere, which is preferably loaded quality,；Preferably, the confining liquid of magnetic microsphere and save liquid be containing
The Tris-HCl buffer of 0.1~0.5M PH 7.0~8.5 of 0.1%~8% BSA；
(2) prepare europium label anti-HBc marker: by anti-HBc be placed in molecular cut off be 10000 ultra-filtration centrifuge tubes in,
10000rpm is centrifuged 7~10min, discards filtrate.Add 9.6 carbonate buffer solution 10000rpm of 0.05M PH centrifugation 7~
10min 2~3 times repeatedly, centrifuge tube filter membrane reversion 3000rpm is centrifuged 6min, collects 200 μ L solution being finally concentrated.And it will
Itself and in advance with the solvent DTTA-EU of carbonate buffer solution3+Mixing, anti-HBc and europium mass ratio are 2:1, and 2~8 DEG C of oscillations mix
48 ± 2 hours.Sephadex TM G-75 gel column that label solution is balanced through 7.8 Tris-Hcl buffer of 0.05M PH (
1.0*50cm) chromatographic purifying monitors in A280 and collects first peak.By the anti-HBc marker of the europium being collected into label with 0.2%
7.8 Tris-Hcl buffer of BSA, 0.05M PH is diluted to 1/20 times of optium concentration, dispenses to 1.0mL/ bottles.
(3) anti-HBc calibration object is prepared: using 2% BSA, 0.5% Tween-20,0.02% Proclin300, pH 7.5
Anti- HBc is diluted to lower concentration values point 1IU/mL, high concentration value point 8IU/mL by 0.02M Tris-HCl buffer.
(4) it prepares analysis buffer: containing Tween-20, Proclin300, EDTA, BSA, Tris-HCl buffer, dividing
It is filled to 30~40mL/ bottles.
(5) preparation concentration washing lotion: containing Tween-20, Proclin300, Tris-Hcl buffer, dispense to 30~
40mL/ bottles.
(6) it prepares enhancement solution: containing sodium acetate, β-NTA, TOPO, glacial acetic acid, dehydrated alcohol, Triton X-100, dividing
It is filled to 30~40mL/ bottles.
(7) it prepares RFID card: blank RFID card is subjected to relative parameters setting by detection method in the present embodiment of the present invention；
(8) coding, labelling assemble kit.
The present invention also provides the detection method of above-mentioned detection anti-HBc kit, the method is specific
It operates as follows:
(1) reagent prepares
Kit restores in being placed at room temperature for room temperature；
The anti-HBc marker of europium label is diluted to working solution concentration using analysis buffer, is mixed stand-by；
Cleaning solution: purified water is added by 1:25 in concentration washing lotion and is diluted to work cleaning solution；
Upright light rolling is coated with the magnetic microsphere of HBcAg before experiment, mixes stand-by；
(2) experimental implementation
The magnetic microsphere for drawing 50 μ L mixing is added in reaction cup；
Sample to be tested or 50 μ L of calibration object are added into each reaction cup；
The anti-100 μ L of HBc marker working solution diluted is added into each reaction cup again；
Room temperature blending incubation 30 minutes；
After incubation, cleaned 4 times using cleaning solution；
The addition 100 μ L of enhancement solution into each reaction cup, room temperature blending incubation 3 minutes；Fluorescent collecting is completed in 30 minutes
And carry out data analysis.
In the present embodiment, actual laboratory operating procedures and relevant parameter are copied in matched RFID card in advance, real
It tests after operating process only needs to be ready to by reagent preparation process, RFID card is adapted to fully-automatic equipment can be completed from information
Read, be loaded onto the overall process of detection.
Embodiment 2
A kind of detection anti-HBc kit, it is essentially identical with detection kit described in embodiment 1, it is different
It is:
(1) the detection anti-HBc kit component only includes: reagent strip, anti-HBc calibration object, RFID
Card.
(2) in the present embodiment, reagent strip is by being coated with the magnetic microsphere of HBcAg, europium label in the detection kit
Anti- HBc marker, analysis buffer, cleaning solution and enhancement solution, formed after sealer in packing to the corresponding hole of reagent strip.Its
Middle cleaning solution is that concentration washing lotion adds 25 times of purified water dilution to form in embodiment 1；Remaining each component is in the same manner as in Example 1.
In the present embodiment, just as shown in Figure 1 and Figure 2, each hole bit function of reagent strip is described as follows in the detection kit:
Reagent strip is from left to right arranged successively, and title is followed successively by the 1st~13 hole.1st, 2 holes are instrument connection, the 3rd, 4 holes be
Fluorescent marker hole, the 5th hole be analysis buffer hole, the 6th, 7 holes be cleaning fluid apertures, the 8th, 9 be Sample Dilution fluid apertures, the 12nd for increase
Strong fluid apertures, the 10th, 11,13 be preparation hole.1st and 2 holes are the reacting hole storing magnetic microsphere and being immunoreacted, and maximum can
Storage liquid volume is 800 μ L；3rd, 4 holes can be disassembled into from entire reagent strip and be independent component, convenient for fluorescence mark
Note object carries out packing storage.3rd, 4,5 holes can store maximum liquid volume be 400 μ L；6th, 7 holes can store maximum liquid
Volume is 3000 μ L；It is 400 μ L that 8th ~ 12 hole, which can store maximum liquid volume,；13rd hole can store maximum liquid volume
600μL。
In the present embodiment, the reagent strip in the detection kit is made after carrying out sealer as follows:
300 μ L are coated with HBcAg magnetic microsphere, 50 μ L europiums mark anti-HBc marker, 200 μ L analysis buffers, 3000 μ L
Cleaning solution, 200 μ L enhancement solutions are dispensed respectively to the 1st of reagent strip the, 3,5,6,12 holes, and the reagent strip is made after sealer.
The present invention also provides the preparation methods of above-mentioned detection anti-HBc kit, make in a manner described
After obtaining reagent strip, kit is constituted with anti-HBe calibration object, RFID card.In addition to each component dispenses mode and storage container not
Equally outer, remaining is in the same manner as in Example 1.
The present invention also provides the detection methods of above-mentioned detection anti-HBc kit, it is only necessary to will be above-mentioned
Reagent strip is inserted into the reagent clamp bar slot of SmartTRF serial equipment after gently shaking mixing, and equipment reads RFID card relevant information
It is automatically finished detection process.The related information parameters and detecting step of RFID card are in the same manner as in Example 1.
Embodiment 3
The performance evaluation of detection anti-HBc kit of the present invention:
By the kit and detection method prepared in embodiment, B-mode liver of the detection buying from National Institute for Food and Drugs Control
Scorching virus core antibody National reference, and collect and detect facing for anti-HBc from Abbott Laboratories, hospital Architect i2000
Bed 300, sample.
Embodiment 1, embodiment 2 detect anti-HBc National reference, as a result as follows:
(1) negative match-rate: 15 parts of negative National reference N1~N15, embodiment 1, embodiment 2 detect coincidence rate (-/-)
It is 15/15；
(2) positive coincidence rate: 15 parts of positive National reference P1~P10, embodiment 1, embodiment 2 detect coincidence rate (+/+)
It is 15/15；
(3) accuracy: accuracy reference material repeats detection 10 times, and embodiment 1, embodiment 2 detect accuracy C.V≤20%；
(4) limit of identification: detection sensitivity reference material, embodiment 1, the detection of embodiment 2 meet≤1IU/mL.
Embodiment 1, embodiment 2 detect the anti-HBe300 clinical sample of Abbott Laboratories, as a result as follows:
The comparison of 1 embodiment of table, 1 clinical sample
Detection kit described in the embodiment of the present invention 1 detects hepatitis B e antigen and contrast agents negative match-rate 100%, sun
Property coincidence rate 100%.
What the concentration washing lotion (cleaning solution) referred in embodiment 1 is that the high concentration to be diluted to working solution is cleaned
Liquid；What cleaning solution referred in example 2 is the working solution for not needing any processing.
It should be understood that detecting the detection method of anti-HBc kit in embodiment 1 and preparation method is to meet
Big flux testing goal and invent, instrument and equipment bulky and cost is relatively high, for further satisfaction two, three line cities
The detection demand in city, correspondingly, we have further made some detection methods and kit preparation on the basis of embodiment 1
Modification in method, as in embodiment 2.
It should be understood that difference of the present invention in example 2 with detection method and preparation method in embodiment 1 exists
In, embodiment 2 be by the magnetic microsphere for being coated with HBcAg in embodiment 1, the anti-HBc marker in conjunction with lanthanide series,
Analysis buffer, cleaning solution and enhancement solution are dispensed into special reagent strip (as shown in Figure 1 and Figure 2) simultaneously, therefore in embodiment 2
The component of kit only has reagent strip, RFID card, anti-HBc calibration object.Wherein in RFID card, anti-HBc calibration object and embodiment 1
It is consistent.
It should be understood that the detection method of the present invention in example 2 is after reagent strip is inserted into detection device, to use simultaneously
Corresponding RFID card is adapted to equipment, and equipment full automatic working step is consistent with embodiment 1；
It should be understood that the preparation method of the detection kit of the present invention in example 2 is with the difference in embodiment 1,
Concentration washing lotion becomes cleaning solution in step (4), and step (5) becomes " dispensing to the corresponding aperture of reagent strip each component respectively, dividing
Sealer coding immediately after installing ".
Claims (6)
1. a kind of detection anti-HBc kit, it is characterised in that be grouped as by following group: being coated with HBcAg
Magnetic microsphere, anti-HBc calibration object, anti-HBc marker, analysis buffer, cleaning solution, enhancement solution in conjunction with lanthanide series
And RFID card.
2. detection anti-HBc kit as described in claim 1, it is characterised in that described is coated with
Magnetic microsphere in the magnetic microsphere of HBcAg is by micron-sized Fe2O3Or Fe3O4Magnetic particle and high-molecular organic material into
Row is compound, forms micron-sized microballoon having superparamagnetism, can combining with immunising antigen or antibody.
3. as claimed in claim 2 detection anti-HBc kit, which is characterized in that magnetic microsphere it is straight
Diameter be 0.1~5 μm, magnetic microsphere by surface modification have various active functional group, including but not limited to hydroxyl, amino or
Carboxyl.
4. detection anti-HBc kit as described in claim 1, which is characterized in that described is coated with
The magnetic microsphere of HBcAg is prepared using following steps:
By HBcAg after 2~8 DEG C of superspeed refrigerated centrifuges are by buffer system replacement Treatment, with the magnetic microsphere after cleaning, activation
Simultaneously constant-temperature incubation is mixed, magnetic bead is cleaned after incubation and abandons supernatant, is then closed with magnetic bead confining liquid, cleaning magnetic bead is again
Supernatant is abandoned, the HBcAg being coated with is saved in liquid in magnetic bead and is uprightly saved with 2~8 DEG C of refrigerators.
5. detection anti-HBc kit as described in claim 1, which is characterized in that described and group of the lanthanides
The antibody in anti-HBc marker that element combines is monoclonal antibody or resists more that lanthanide series is to chelate in conjunction with anti-HBc by centre
Agent is completed, and lanthanide series is europium or samarium, and chelating agent is isothiocyanic acid phenyl-EDTA, isothiocyanic acid benzyl-DTTA, the different sulphur of P-
Cyanobenzyls-DTTA or diethylene triamine pentaacetic acid aminophenyl-EDTA.
6. the preparation method of detection anti-HBc kit described in claim 1, which is characterized in that including
Following steps:
(1) preparation is coated with the magnetic microsphere of HBcAg；
(2) anti-HBc marker of the preparation in conjunction with lanthanide series；
(3) calibration object is prepared；
(4) analysis buffer, concentration washing lotion and enhancement solution are prepared；
(5) each component is dispensed respectively into corresponding storage container；
(6) RFID card duplicate copy；
(7) coding, labelling；
(8) it is assembled into finished product kit.
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| CN110954692A (en) * | 2019-12-20 | 2020-04-03 | 蓝怡科技集团股份有限公司 | Reducing agent buffer solution and preparation method and application thereof |
| CN113030469B (en) * | 2021-03-18 | 2024-04-09 | 贵州省分析测试研究院 | Novel coronavirus detection method |
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Application publication date: 20190329 |