CN109541229A - A kind of+2 type antibody kit of detection human immunodeficiency virus I and preparation method thereof - Google Patents
A kind of+2 type antibody kit of detection human immunodeficiency virus I and preparation method thereof Download PDFInfo
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- CN109541229A CN109541229A CN201811329568.2A CN201811329568A CN109541229A CN 109541229 A CN109541229 A CN 109541229A CN 201811329568 A CN201811329568 A CN 201811329568A CN 109541229 A CN109541229 A CN 109541229A
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
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- 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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Abstract
The invention discloses a kind of+2 type antibody kits of detection human immunodeficiency virus I and preparation method thereof, are grouped as by following group: being coated with magnetic microsphere, negative controls, positive reference substance 1, positive reference substance 2, HIV antigenic label, analysis buffer, cleaning solution, enhancement solution and the RFID card in conjunction with lanthanide series of HIV antigen.This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, both it is longer the physical sorption reaction time of ELISA Plate had been overcome, the slower drawback of testing result, the reaction time is greatly shortened, while also having time resolution detection technique accuracy height, high sensitivity, high specificity, detecting 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, result can be detected in 20 minutes.
Description
Technical field
The present invention relates to a kind of+2 type antibody kits of detection human immunodeficiency virus I and preparation method thereof, specifically relate to
And a kind of combined based on magnetic microsphere with Timed-resolved fluoroimmunoassay detects+2 type antibody reagent of human immunodeficiency virus I
The preparation method of box.
Background technique
Human immunodeficiency virus (HIV), the i.e. pathogen of acquired immunodeficiency syndrome (AIDS), belong to reverse transcription disease
Malicious family.The route of transmission of HIV include contaminated blood and blood product, property contact or before pregnant, the pregnancy period and it is pregnant after by infecting
Mother of HIV is broadcast to baby.Up to the present, the means of prevention of AIDS still aims at prevention, and HIV detection is AIDS
The important component of Prevention and control occupies extremely important status in the infection that prevents AIDS.Third generation AIDS
Detection reagent is addition 1 type antigen of HIV (gp41 polypeptide), detection sensitivity on the basis of 2 type antigen of HIV (gp36 polypeptide)
It further increases, window phase further reduces.
At present AIDS antibody serum test mainly have enzyme linked immunological (ELISA), colloidal gold method, chemiluminescence (CLIA),
Electrochemical luminescence (ECL), time-resolved fluoroimmunoassay (TRFIA) etc..ELISA is existing application as semi-quantitative detection method
Most wide detection method, but its sensitivity, linear extent range are not up to higher level, it is difficult to adapt to the need of market development
It asks.Colloidal gold method and ELISA have the shortcomings that identical.Though and CLIA and ECL high sensitivity but to there is detection device cost high
It is high, and corresponding marker research and development threshold is high, domestic disadvantage that can be under one's control equally also limits at home in short-term
Popularization.Time-resolved fluoroimmunoassay (TRFIA) sensitivity can reach the consistent level of CLIA, not only detection device cost
It is lower, and the relevant technologies are quite mature at home.Conventional temporal resolved detection is still with the physical absorption of blank ELISA Plate
Main, detection process takes a long time.Shorten the reaction time, reduce testing cost, improves detection sensitivity and the range of linearity, will have
Good market prospects.
Summary of the invention
It is provided based on this it is an object of the invention to overcome the prior art and the high disadvantage of the relevant technologies testing cost
A kind of preparation method of detection+2 type antibody kit of human immunodeficiency virus I and the kit.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of+2 type antibody kit of detection human immunodeficiency virus I, is grouped as by following group: being coated with the magnetic of HIV antigen
Property microballoon, negative controls, positive reference substance 1, positive reference substance 2, the HIV antigenic label in conjunction with lanthanide series, analysis
Buffer, concentration washing lotion (cleaning solution) and enhancement solution, RFID card.
Preferably, the HIV antigenic label in conjunction with lanthanide series, lanthanide series therein are with HIV antigen
It is combined by intermediate chelating agent, lanthanide series includes but is not limited to europium (EU), samarium (Sm), and chelating agent includes but is not limited to different sulphur
Cyanic acid phenyl-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-DTTA, diethylene triamine pentaacetic acid aminobenzene
Base-EDTA.
Preferably, the HIV antigen containing advantages segments such as gp36, gp41 by forming.
The present invention also provides a kind of methods using mentioned reagent box detection anti-HIV 1+2, and the method includes walking as follows
It is rapid:
(1) the HIV antigenic label that europium marks is diluted to working solution with analysis buffer;
(2) working solution is diluted to by washing lotion is concentrated with purified water;
(3) it is added in reaction cup after mixing the magnetic microsphere for being coated with HIV antigen;
(4) sample to be examined or negative controls, positive reference substance 1, positive reference substance 2 are added in above-mentioned reaction cup;
(5) HIV antigenic label working solution is added in reaction cup, is incubated at room temperature;
(6) use the cleaning liquid reaction cup in (2) that magnetic is added to wash after being incubated for;
(7) after washing, enhancement solution is added and is incubated for;
(8) 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 method for detecting+2 type antibody kit of human immunodeficiency virus I, the sides
Method includes the following steps:
(1) it is coated with the preparation of the magnetic microsphere of HIV antigen;;
(2) negative controls, positive reference substance 1, positive reference substance 2 are prepared;
(3) preparation of the HIV antigenic label of europium label;
(4) preparation of analysis buffer, concentration washing lotion and enhancement solution;
(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, has both overcome the physical adsorption reaction of ELISA Plate
Time is longer, and the slower drawback of testing result greatly shortens the reaction time, while also having time resolution detection technique accuracy
Height, high sensitivity, high specificity, detection is stablized and convenient advantage.Magnetic microsphere is coated with corresponding antigen or antibody, because of magnetism
The characteristic of microballoon solid, greatly increases the contact surface area of immune response, so that detection time is greatly shortened, in 20 minutes
Detect result.In addition, the frame limitation of traditional ELISA Plate is no longer limited by, arbitrary due to the fluid behaviour of magnetic microsphere
Detection can be completed in reaction cup and small test tube, while can also reduce volume and the cost of detecting instrument equipment to meet two, three
The full-automatic detection demand in line city also can carry out big flux detection as ELISA Plate, the reflective detection of chemistry, meet a line
The full-automatic detection demand in city.
Detailed description of the invention
Fig. 1 is that use detection kit of the present invention is used to store the magnetic microsphere for being coated with HIV antigen, europium marks
HIV antigenic label, analysis buffer, cleaning solution and enhancement solution reagent strip schematic top plan view.
Fig. 2 is that use detection kit of the present invention is used to store the magnetic microsphere for being coated with HIV antigen, europium marks
HIV antigenic label, 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.Magnetic microsphere is public from GE in following embodiment
Department;Europium label is purchased from Wallac company, Finland;Human immune defect virus antibody National reference is by Chinese food drug assay
Research institute provides;Contrast agents box is human immunodeficiency virus 1+2 type antibody qualitative detection kit (chemiluminescence particulate
Immunodetection).
Embodiment 1
A kind of+2 type antibody kit of detection human immunodeficiency virus I, the kit includes: the magnetic for being coated with HIV antigen
Property microballoon, negative controls, positive reference substance 1, positive reference substance 2, europium label HIV antigenic label, analysis buffer, dense
Contracting washing lotion and enhancement solution, RFID card.
The present invention also provides the preparation method of above-mentioned+2 type antibody kit of detection human immunodeficiency virus I, the sides
Method includes the following steps:
(1) it is coated with the preparation of the magnetic microsphere of HIV antigen: by the disconnected HIV antigen containing 1 type, 2 matrix through 2~8 DEG C of Superfreezings
Centrifuge after buffer system replacement Treatment, will be mixed with 1 μm of carboxyl magnetic microsphere of diameter after cleaning, activation and constant-temperature incubation 1
~3 hours, magnetic bead was cleaned using magnetic bead cleaning solution after incubation and abandons supernatant, is then closed with magnetic bead confining liquid, clearly
It washes magnetic bead and abandons supernatant again, the HIV antigen magnetic microsphere being coated with is upright with 2~8 DEG C of refrigerators in magnetic bead preservation liquid
It saves.It will be coated with HIV antigen magnetic microsphere again and be diluted to working solution concentration with magnetic bead preservation liquid, be distributed into 10mL/ bottles.It is preferred that
The mass ratio of ground, magnetic microsphere and HIV antigen coat is one of 10:1,20:1,30:1,40:1;Preferably, described to set
It changes buffer and magnetic bead cleaning solution is the MES buffer of 0.05~0.5M PH, 5.8~PH 7.0;Preferably, magnetic microsphere
It is 10 μ of μ g~1000 g that every milligram of magnetic microsphere of EDC and Sulfo-NHS, which is preferably loaded quality, in activation;Preferably, magnetic micro-
The Tris-HCl buffer that the confining liquid and preservation liquid of ball are 0.1~0.5M PH 7.0~8.5 containing 0.1%~8% BSA;
(2) negative controls, positive reference substance 1, positive reference substance 2 are prepared: using 5% BSA, 0.5% Tween-20,0.02%
7.8 0.02M Tris-HCl buffer of Proclin300, pH resists human immunodeficiency virus type 1,2 type sheep more is made feminine gender
Reference substance, positive reference substance 1, positive reference substance 2.
(3) prepare the HIV antigenic label of europium label: marker is pressed after being marked respectively with europium by 1 type antigen, 2 type antigens
Certain proportion mixes.It is to put after tying in 14000 bag filters that 1 type antigen, 2 type antigens, which are respectively placed in molecular cut off,
After entering 2~8 DEG C of refrigerators equipped in 9.6 carbonate buffer solution glass of 0.05M PH magnetic agitation displacement 1 hour, more renew
Carbonate buffer solution is stirred for displacement 1 hour, the buffer hereafter more renewed every 4 hours, last time displacement overnight.It will set
1 type antigen after changing, 2 type antigens collect respectively with DTTA-EU solvent with carbonate buffer solution in advance3+Mixing, 1 type HIV are anti-
Former and europium mass ratio is 3:1, and 2 type HIV antigens and europium mass ratio are 2:1, and 2~8 DEG C of oscillations mix 72 ± 2 hours.Label solution
The Sephadex balanced through 7.8 Tris-Hcl buffer of 0.05M PHTMG-50 gel column (1.0*50cm) chromatographic purifying,
First peak is collected in A280 monitoring.1 type antigen of the europium being collected into label, 2 type antigens are used into 0.2% 0.05M PH 7.8 respectively
Tris-Hcl buffer mixes the HIV antigenic label that europium label is made after being diluted to 1/20 times of optium concentration, packing is extremely
1.0mL/ bottle.
(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+2 type antibody kit of detection human immunodeficiency virus I, the sides
Method concrete operations are as follows:
(1) reagent prepares
1. kit restores in being placed at room temperature for room temperature;
2. the HIV antigenic label of europium label is diluted 20 times to working solution using analysis buffer, mix stand-by;
3. cleaning solution: purified water is added by 1:25 in concentration washing lotion and is diluted to work cleaning solution;
4. upright light rolling is coated with the magnetic microsphere of HIV antigen before experiment, mix stand-by;
(2) experimental implementation
1. the magnetic microsphere for being coated with HIV antigen for drawing 50 μ L mixing is added in reaction cup;
2. sample to be tested or negative controls, positive reference substance 1, each 100 μ L of positive reference substance 2 are added into each reaction cup;
3. the HIV antigenic label of europium label is added into each reaction cup again;
4. being stored at room temperature incubation 20 minutes;
5. using cleaning solution plus magnetic cleaning 4 times after being incubated for;
6. 100 μ L enhancement solutions are added into each reaction cup, are stored at room temperature incubation for degaussing after cleaning
3 minutes;Fluorescent collecting is completed in 30 minutes and carries 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+2 type antibody kit of detection human immunodeficiency virus I, with the basic phase of detection kit described in embodiment 1
Together, difference is:
(1)+2 type antibody kit component of detection human immunodeficiency virus I includes: reagent strip, negative controls, the positive
Reference substance 1, positive reference substance 2, RFID card.
(2) in the present embodiment, reagent strip is by being coated with the magnetic microsphere of HIV antigen, europium mark in the detection kit
HIV antigenic label, analysis buffer, cleaning solution and the enhancement solution of note form after dispensing to sealer in the corresponding hole of reagent strip.
Wherein cleaning solution is that concentration is washed in embodiment 1 plus purified water dilutes 25 times and forms;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:
By 300 μ L be coated with the magnetic microsphere of HIV antigen, 50 μ L europiums label HIV antigenic label, 200 μ L analysis buffers,
3000 μ L cleaning solutions, 200 μ L enhancement solutions are dispensed respectively to the 1st of reagent strip the, 3,5,6,12 holes, and institute is made after sealer coding
State reagent strip.
The present invention also provides the preparation methods of above-mentioned+2 type antibody kit of detection human immunodeficiency virus I, by above-mentioned
After reagent strip is made in mode, kit is constituted with negative controls, positive reference substance 1, positive reference substance 2, RFID card.It removes
Each component packing mode and storage container are different outer, remaining is in the same manner as in Example 1.
The present invention also provides the detection methods of above-mentioned+2 type antibody kit of detection human immunodeficiency virus I, it is only necessary to
Mentioned reagent item is gently shaken and is inserted into the reagent clamp bar slot of SmartTRF serial equipment after mixing, equipment reads RFID card correlation letter
Breath can be 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+2 type antibody kit of human immunodeficiency virus I of the present invention:
By the kit and detection method prepared in embodiment, detection buying is exempted from from the mankind of National Institute for Food and Drugs Control
Epidemic disease defective virus antibody National reference, and collect and lacked from Abbott Laboratories, hospital Architect i2000 detection human immunity
Clinical sample 184 for falling into virus 1+2 type antibody.
Embodiment 1, embodiment 2 detect human immune defect virus antibody National reference, as a result as follows:
(1) negative reference product coincidence rate: 20 parts of negative reference product N1~N20, embodiment 1, embodiment 2 detect coincidence rate (-/-)
It is 20/20;
(2) HIV-1 type positive reference product coincidence rate: 18 parts of positive reference product P1~P18, embodiment 1, the detection of embodiment 2 meet
Rate (+/+) it is 18/18;
(3) HIV-2 type positive reference product coincidence rate: 2 parts of positive reference product P19~P20, embodiment 1, the detection of embodiment 2 meet
Rate (+/+) it is 2/2;
(4) minimum detectability: positive reaction (4 >=6), S1 is detected as feminine gender;
(5) accuracy: accuracy reference material repeats detection 10 times, and embodiment 1, embodiment 2 detect accuracy CV≤8.6%;
Embodiment 1, embodiment 2 detect 184 clinical samples of Abbott Laboratories' anti-HIV 1+2, 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+2 type antibody of human immunodeficiency virus I and contrast agents are negative
Sample coincidence rate 100%, positive sample coincidence rate 100%.
The comparison of 2 embodiment of table, 2 clinical sample
Detection kit described in the embodiment of the present invention 2 detects+2 type antibody of human immunodeficiency virus I and contrast agents are negative
Sample coincidence rate 100%, positive sample coincidence rate 100%.
What concentration washing lotion (cleaning solution) referred in embodiment 1 is the high concentration cleaning solution to be diluted to working solution;Cleaning
What liquid referred in example 2 is the working solution for not needing any processing.
It should be understood that in embodiment 1 detect+2 type antibody kit of human immunodeficiency virus I detection method and
Preparation method is invented to meet big flux testing goal, and instrument and equipment volume is larger and cost is relatively high, in order to
Further satisfaction two, the detection demand in three line cities, correspondingly, we further made on the basis of embodiment 1 it is some
Modification in detection method and reagent box preparation 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 is that the magnetic microsphere for being coated with HIV antigen in embodiment 1, the HIV antigenic label of europium label, analysis is slow
Fliud flushing, cleaning solution and enhancement solution are dispensed into special reagent strip (as shown in Figure 1 and Figure 2), therefore kit in embodiment 2 simultaneously
Component only have reagent strip, RFID card, negative controls, positive reference substance 1, positive reference substance 2.Wherein RFID card, feminine gender are right
According to being consistent in product, positive reference substance 1, positive reference substance 2 and embodiment 1.
It should be understood that the detection method of the present invention in example 2 be by reagent strip be inserted into detection device after, will be corresponding
RFID card be adapted to equipment, equipment whole-course automation operating procedure 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,
Step (5) becomes " dispensing positive and negative reference substance to corresponding reference substance bottle, remaining each component is dispensed respectively to reagent strip
In corresponding aperture, sealer coding after having dispensed ".
Claims (4)
1. a kind of+2 type antibody kit of detection human immunodeficiency virus I, it is characterised in that be grouped as: being coated with by following group
Magnetic microsphere, negative controls, positive reference substance 1, positive reference substance 2, the HIV antigen in conjunction with lanthanide series of HIV antigen
Marker, analysis buffer, cleaning solution, enhancement solution and RFID card.
2. detection+2 type antibody kit of human immunodeficiency virus I as described in claim 1, which is characterized in that described
HIV antigenic label in conjunction with lanthanide series, lanthanide series therein and HIV antigen are the lanthanums by conjunction with intermediate chelating agent
Series elements are europium or samarium, and chelating agent is isothiocyanic acid phenyl-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-
DTTA or diethylene triamine pentaacetic acid aminophenyl-EDTA.
3. detection+2 type antibody kit of human immunodeficiency virus I as described in claim 1, which is characterized in that described
HIV antigen containing gp36, gp41 segment by forming.
4. detecting the preparation method of+2 type antibody kit of human immunodeficiency virus I described in claim 1, it is characterised in that packet
Include following steps:
(1) it is coated with the preparation of the magnetic microsphere of HIV antigen;
(2) negative controls, positive reference substance 1, positive reference substance 2 are prepared;
(3) preparation of the HIV antigenic label of europium label;
(4) preparation of analysis buffer, concentration washing lotion and enhancement solution;
(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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Application publication date: 20190329 |