CN109541224A - A kind of detection syphilis helicoid antibody kit and preparation method thereof - Google Patents
A kind of detection syphilis helicoid antibody kit and preparation method thereof Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention discloses a kind of detection syphilis helicoid antibody kits and preparation method thereof, are grouped as by following group: being coated with magnetic microsphere, negative controls, positive reference substance, TP antigenic label, analysis buffer, cleaning solution, enhancement solution and the RFID card in conjunction with lanthanide series of TP 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 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, result can be detected in 30 minutes.
Description
Technical field
The present invention relates to a kind of detection syphilis helicoid antibody kits and preparation method thereof, and in particular to one kind is based on magnetic
Property microballoon combined with Timed-resolved fluoroimmunoassay detection syphilis helicoid antibody kit preparation method.
Background technique
Syphilis is passed caused by being also referred to as Spirochaeta pallida (Treponema pallidum, TP) infection by microspironema pallidum
Metachromia is strong, endangers biggish mankind's sexually transmitted disease, and syphilis only infects the mankind in natural situation, and people is unique infection of syphilis
Source.Syphilis can cause the Multisystem damages such as nerve, angiocarpy or even life-threatening.Syphilis can be caused by placental infection fetus
Spontaneous abortion, stillbirth or congenital syphilis etc..Syphilization can promote the propagation of AIDS.With the development of economy, it spreads through sex intercourse
Disease is consequently increased.
Syphilis non-specificity lipoids antibody serum test at present mainly has toluidine red that the examination of warm blood clearance response element is not added
(TRUST), Rapid plasma reagintest (RPR) are tested, the test of syphilis specific antibody serum mainly has syphilis
Conveyor screw gelatin agglutination test (TPPA), enzyme linked immunological (ELISA), colloidal gold method, chemiluminescence (CLIA), electrochemical luminescence
(ECL), time-resolved fluoroimmunoassay (TRFIA) etc..Syphilis Specific antigen test is related to the course of disease, to tertiary syphilis and controls
Syphilis recall rate is lower after treatment；TPPA method is tested as the confirmation of syphilis in syphilis specific test, is all had to each phase syphilis
Preferable stability, but the mode of judgement result is visually judges, result reliability declines and can not save initial data,
TPPA method kit cost is high simultaneously.Syphilis ELISA is now most widely used detection method as semi-quantitative detection method, but
Its sensitivity, linear extent range are not up to higher level, it is difficult to adapt to the demand of market development.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, and corresponding marker is ground
Threshold height is sent out, domestic disadvantage that can be under one's control equally also limits popularization at home in short-term.Time-resolved fluorescence
Immunoassay (TRFIA) sensitivity can reach the consistent level of CLIA, and not only detection device cost is lower, and the relevant technologies exist
It is domestic quite mature.Conventional temporal resolved detection, still based on the physical absorption of blank ELISA Plate, detection process time-consuming compared with
It is long.Shorten the reaction time, reduce testing cost, improves detection sensitivity and the range of linearity, it will be with 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 syphilis helicoid antibody kit and the kit.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of detection syphilis helicoid antibody kit, is grouped as by following group: being coated with magnetic microsphere, the yin of TP antigen
Property reference substance, positive reference substance, the TP antigenic label in conjunction with lanthanide series, analysis buffer, concentration washing lotion (cleaning solution)
With enhancement solution, RFID card.
Preferably, the TP antigenic label in conjunction with lanthanide series, lanthanide series therein is logical with TP antigen
It crosses intermediate chelating agent to combine, lanthanide series includes but is not limited to europium (EU), samarium (Sm), and chelating agent includes but is not limited to isothiocyanic acid
Phenyl-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-DTTA, diethylene triamine pentaacetic acid aminophenyl-
EDTA。
Preferably, the TP antigen containing advantages segments such as TP15, TP17, TP47 by forming.
The present invention also provides a kind of methods for detecting anti-TP using mentioned reagent box, and described method includes following steps:
(1) TP antigenic label 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 TP antigen；
(4) sample to be examined or negative controls, positive reference substance are added in above-mentioned reaction cup；
(5) TP 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, is 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 syphilis helicoid antibody kit, and the method includes walking as follows
It is rapid:
(1) it is coated with the preparation of the magnetic microsphere of TP antigen；；
(2) negative controls, positive reference substance are prepared；
(3) preparation of TP antigen 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 absorption of ELISA Plate
Reaction time is longer, and the slower drawback of testing result greatly shortens the reaction time, while it is quasi- also to have time resolution detection technique
True property height, high sensitivity, high specificity, the range of linearity is wide, detection is stable and convenient advantage.Magnetic microsphere coating is corresponding anti-
Former or antibody greatly increases the contact surface area of immune response because of the characteristic of magnetic microsphere solid, thus when greatly shortening detection
Between, result can be detected in 30 minutes.In addition, being no longer limited by the frame of traditional ELISA Plate due to the fluid behaviour of magnetic microsphere
Frame limitation, detection can be completed in arbitrary reaction cup and small test tube, at the same can also reduce detecting instrument equipment volume and
Cost can also be led to greatly with meeting the full-automatic detection demand in two, three line cities such as ELISA Plate, the reflective detection of chemistry
Amount detection, meets the full-automatic detection demand in a line 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 TP antigen, europium marks
TP 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 TP antigen, europium marks
TP 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；Syphilis helicoid antibody National reference is by National Institute for Food and Drugs Control
It provides；Contrast agents box is Abbott Laboratories' syphilis helicoid antibody assay kit (chemiluminescence particulate immunodetection).
Embodiment 1
A kind of detection syphilis helicoid antibody kit, the kit include: the magnetic microsphere for being coated with TP antigen, yin
Property reference substance, positive reference substance, the TP antigenic label of europium label, analysis buffer, concentration washing lotion and enhancement solution, RFID card.
The present invention also provides the preparation methods of above-mentioned detection syphilis helicoid antibody kit, and the method includes walking as follows
It is rapid:
(1) it is coated with the preparation of the magnetic microsphere of TP antigen: TP antigen will be buffered through 2~8 DEG C of superspeed refrigerated centrifuges
After system replacement Treatment, mixes simultaneously constant-temperature incubation 1~3 hour, be incubated for 1 μm of carboxyl magnetic microsphere of diameter after cleaning, activation
Magnetic bead is cleaned using magnetic bead cleaning solution afterwards and abandons supernatant, is then closed with magnetic bead confining liquid, and cleaning magnetic bead is lost again
Supernatant is abandoned, the TP antigen magnetic microsphere being coated with is saved in liquid in magnetic bead and is uprightly saved with 2~8 DEG C of refrigerators.It again will coating
Good TP antigen magnetic microsphere saves liquid with magnetic bead and is diluted to working solution concentration, is distributed into 10mL/ bottles.Preferably, magnetic microsphere with
The mass ratio of TP antigen coat is one of 10:1,20:1,30:1,40:1；Preferably, the displacement buffer and magnetic bead
Cleaning solution is the MES buffer of 0.05~0.5M PH, 5.8~PH 7.0；Preferably, in the activation of magnetic microsphere EDC and
It is 10 μ of μ g~1000 g that every milligram of magnetic microsphere of Sulfo-NHS, which is preferably loaded quality,；Preferably, the confining liquid of magnetic microsphere and
Save the Tris-HCl buffer that liquid is 0.1~0.5M PH 7.0~8.5 containing 0.1%~8%BSA；
(2) negative controls, positive reference substance are prepared: using 5%BSA, 0.5%Tween-20,0.02%
Negative control is made in syphilis helicoid antibody strong positive serum by 7.8 0.02M Tris-HCl buffer of Proclin300, pH
Product, positive reference substance.
(3) prepare the TP antigenic label of europium label: it is 10000 ultra-filtration centrifuge tubes that TP antigen, which is placed in molecular cut off,
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
By itself and DTTA-EU solvent with carbonate buffer solution in advance3+Mixing, TP antigen and europium mass ratio are 1:1, and 2~8 DEG C of oscillations are mixed
Even 48 ± 2 hours.The Sephadex that label solution is balanced through 0.05M PH 7.8Tris-Hcl bufferTMG-50 gel columnChromatographic purifying monitors in A280 and collects first peak.The TP antigen 0.2%0.05M that the europium being collected into is marked
PH 7.8Tris-Hcl buffer is diluted to 1/20 times of optium concentration, dispenses to 1.0mL/ bottles.
(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) enhancement solution is prepared: containing sodium acetate, β-NTA, TOPO, glacial acetic acid, dehydrated alcohol, Triton X-100, packing
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 methods of above-mentioned detection syphilis helicoid antibody kit, and the method concrete operations are such as
Under:
(1) reagent prepares
1. kit restores in being placed at room temperature for room temperature；
2. the TP 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 TP antigen before experiment, mix stand-by；
(2) experimental implementation
1. the magnetic microsphere for being coated with TP antigen for drawing 50 μ L mixing is added in reaction cup；
2. sample to be tested or negative controls, 100 μ L of positive reference substance are added into each reaction cup；
3. the TP antigenic label of europium label is added into each reaction cup again；
4. being stored at room temperature incubation 30 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 detection syphilis helicoid antibody kit, essentially identical with detection kit described in embodiment 1, difference exists
In:
(1) the detection syphilis helicoid antibody kit component include: reagent strip, negative controls, positive reference substance,
RFID card.
(2) in the present embodiment, reagent strip is by being coated with the magnetic microsphere of TP antigen, europium label in the detection kit
TP antigenic label, analysis buffer, cleaning solution and enhancement solution dispense to sealer in the corresponding hole of reagent strip after form.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
Hole 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
To enhance fluid apertures, the 10th, 11,13 be preparation hole.1st and 2 holes are the reacting hole storing magnetic microsphere and being immunoreacted, most
It is 800 μ L that liquid volume can be stored greatly；3rd, 4 holes can be disassembled into from entire reagent strip and be independent component, convenient for glimmering
Signal 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 is 600 μ L.
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 TP antigen magnetic microsphere, 50 μ L europiums label TP 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 the reagent strip is made after sealer coding.
The present invention also provides the preparation methods of above-mentioned detection syphilis helicoid antibody kit, and reagent is obtained in the above describe manner
After item, kit is constituted with negative controls, positive reference substance, RFID card.In addition to each component dispenses mode and storage appearance
Device is different outer, remaining is in the same manner as in Example 1.
The present invention also provides the detection methods of above-mentioned detection syphilis helicoid antibody kit, it is only necessary to by mentioned reagent item
Light rolling is inserted into the reagent clamp bar slot of SmartTRF serial equipment after mixing, and equipment reads RFID card relevant information can be full-automatic
Complete 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 syphilis helicoid antibody kit of the present invention:
By the kit and detection method prepared in embodiment, plum of the detection buying from National Institute for Food and Drugs Control
Malicious helicoid antibody National reference, and collect and detect syphilis from Abbott Laboratories, hospital Architect i2000
The clinical sample of body 241.
Embodiment 1, embodiment 2 detect syphilis helicoid 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) positive reference product coincidence rate: 10 parts of positive reference product P1~P10, embodiment 1, embodiment 2 detect coincidence rate
(+/+) it is 10/10；
(3) accuracy: accuracy reference material repeats to detect 10 times, and embodiment 1, the detection of embodiment 2 accuracy CV≤
9.2%；
(4) minimum detectability: L1, L2, L3 detection are positive, L4 detection is feminine gender.
Embodiment 1, embodiment 2 detect anti-241 clinical samples of TP of Abbott Laboratories, as a result as follows:
The comparison of 1 embodiment of table, 1 clinical sample
The detection syphilis helicoid antibody of detection kit described in the embodiment of the present invention 2 meets with contrast agents negative sample
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 the detection method and preparation method of detection syphilis helicoid antibody kit are in embodiment 1
It is invented to meet big flux testing goal, instrument and equipment volume is larger and cost is relatively high, for further satisfaction
Two, the detection demand in three line cities, correspondingly, we further made on the basis of embodiment 1 some detection methods and
Modification on 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 by the TP antigenic label of the magnetic microsphere for being coated with TP antigen, europium label in embodiment 1, analysis buffering
Liquid, 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 has reagent strip, RFID card, negative controls, positive reference substance.Wherein RFID card, negative controls, positive reference substance
It is consistent with 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 it is different from embodiment 1
In step (5) becomes " dispensing yin and yang attribute reference substance to reference substance bottle, remaining each component is dispensed respectively to the phase of reagent strip
It answers in hole, sealer coding after having dispensed ".
Claims (4)
1. a kind of detection syphilis helicoid antibody kit, it is characterised in that be grouped as by following group: being coated with the magnetic of TP antigen
Property microballoon, negative controls, positive reference substance, the TP antigenic label in conjunction with lanthanide series, analysis buffer, cleaning solution,
Enhancement solution and RFID card.
2. detection syphilis helicoid antibody kit as described in claim 1, which is characterized in that described with lanthanide series knot
The TP antigenic label of conjunction, lanthanide series therein and TP antigen are by conjunction with intermediate chelating agent, and lanthanide series is europium or samarium,
Chelating agent is isothiocyanic acid phenyl-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-DTTA or diethylenetriamine
Five acetic acid aminophenyl-EDTA.
3. detection syphilis helicoid antibody kit as described in claim 1, which is characterized in that the TP antigen is by containing
It is made of TP15, TP17 or TP47 segment.
4. the preparation method of detection syphilis helicoid antibody kit described in claim 1, it is characterised in that including walking as follows
It is rapid:
(1) it is coated with the preparation of the magnetic microsphere of TP antigen；
(2) negative controls, positive reference substance are prepared；
(3) preparation of TP antigen 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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Cited By (1)
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| CN110456052A (en) * | 2019-08-22 | 2019-11-15 | 中国科学院武汉病毒研究所 | The rapid detection method of syphilis helicoid antibody and its application in a kind of serum |
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Application publication date: 20190329 |