CN109060783A - A kind of single part chemical luminous immune detection method and system based on bar magnet method - Google Patents
A kind of single part chemical luminous immune detection method and system based on bar magnet method Download PDFInfo
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- 238000002156 mixing Methods 0.000 claims abstract description 27
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- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 8
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- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 4
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 4
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 2
- XYIPYISRNJUPBA-UHFFFAOYSA-N [3-(3'-methoxyspiro[adamantane-2,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC(C3)CC2C4)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 XYIPYISRNJUPBA-UHFFFAOYSA-N 0.000 claims description 2
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- UKWLRLAKGMZXJC-QIECWBMSSA-L disodium;[4-chloro-3-[(3r,5s)-1-chloro-3'-methoxyspiro[adamantane-4,4'-dioxetane]-3'-yl]phenyl] phosphate Chemical compound [Na+].[Na+].O1OC2([C@@H]3CC4C[C@H]2CC(Cl)(C4)C3)C1(OC)C1=CC(OP([O-])([O-])=O)=CC=C1Cl UKWLRLAKGMZXJC-QIECWBMSSA-L 0.000 claims description 2
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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
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Abstract
The invention discloses a kind of single part chemical luminous immune detection method and system based on bar magnet method, this method is drawn magnetic bead and be added in reagent using bar magnet is reacted or is cleaned, traditional chemical luminous immune detection method then utilizes magnetic Reagent Tube side wall, again with taking liquid needle to siphon away liquid from bottom, compared with the latter, method of the invention carries pollution less, and thoroughly, wash number is few for cleaning.Method of the invention is also reacted by the way of the mixing of certain frequency sustained oscillation, be conducive to magnetic particle and be constantly in even suspension state, keeps the reaction between antigen, antibody molecule free, unimpeded, in conjunction with abundant, shorten the pattern detection time, improves detection sensitivity.Method and detection system of the invention is detected suitable for single part chemiluminescence immunoassay, avoids the problem of current 100 person-portion detects rotten existing reagent, sensitivity decrease, the wasting of resources and is not suitable for basic medical unit.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of single part chemiluminescence immunoassay based on bar magnet method
Detection method and system.
Background technique
Chemiluminescence immunoassay technology is with spies such as its detection sensitivity height, wide, the Testing index broad covered areas of analyst coverage
Put and become the mainstream technology of clinical examination analysis.But according to the feedback of our observational study and infective use, tradition
Chemiluminescence immunoassay technology still has multiple defects.
Firstly, traditional chemical electrochemiluminescent immunoassay kit package amount is excessive, it is not able to satisfy the use demand of basic hospital.It is big by three
First hospital only accounts for very little part in all hospitals, and most groups of hospital are sample size community less than normal, small towns, at county level etc.
Basic hospital.Conventional luminescent kit is generallyd use to be packed with 100 tests/box, i.e. a box reagent contains the amount of 100 person-portions,
This will lead to 100 tests/box reagent can not be finished in corkage validity period (1 month).And reagent was stored for a long time
Cheng Zhong due to that need to open use repeatedly, and tosses about that reagent each component liquid can be caused in 2 DEG C~8 DEG C refrigerators and instrument reagent storehouse
Serious volatilization.In addition, by the even same one man operation of different people, may all go out since kit reuse number is excessive
Now sealing or lid lid reversed order, chaotic situation, can make to generate cross contamination between reagent each component in this way, lead to component
Deterioration failure is not available.Above various situations will lead to the waste of hospital and producer's resource, and due to ingredient volatilization and
Interference of the reagent contamination increase detection ambient noise to detection sensitivity.
Secondly, traditional chemical luminescence immunoassay system is huge, structure is complicated, cumbersome.Traditional chemical electrochemiluminescent immunoassay
Analyzer as described in patent CN107831322A (Fig. 1) and patent CN108226549A (Fig. 2), have reagent disc, sampling probe,
The structures such as syringe needle, wiper mechanism and its corollary apparatus, multiple moving arms and its motor, drawing liquid pump and its motor, lead to system body
Product is huge, takes up a large area, and the scale and place to hospital laboratory all propose high requirement, and it is difficult to increase producer's production
Degree.Extremely complex sequential logic such as patent must be taken into consideration in the operation module of traditional chemical luminescence immunoassay instrument
Described in CN108226549A, operating system is caused to require enterprise technology support staff couple when installation is checked and accepted every time using complexity
Hospital professional carries out detailed written give lessons and operation training.
In addition, traditional chemical chemiluminescence immunoassay method mostly mixes insufficient ask there is also pollution is carried with reaction solution
Topic.
The reaction of each test of traditional chemical chemiluminescence immunoassay method, cleaning, detection and etc. in a by-reaction pipe
Middle completion adsorbs magnetic bead in outer wall with magnet when cleaning, washing lotion is injected into pipe and siphons away (or toppling over) waste liquid alternately, but
Since in a pipe, the suction-operated of tube wall will lead to tube wall and remain various liquid, such as sample, magnetic bead working solution, enzyme always
Working solution etc., cleaning step must carry out 3~6 times, cumbersome, complicated, and can not accomplish thoroughly to clean, and inevitably begin
Pollution is carried eventually: and carry and pollute this examination chemiluminescence system most critical, most crucial index, it is always traditional chemical hair
The problem that light immune Research personnel can not capture.Separately not thorough enough due to cleaning, high level sample, enzyme conjugates, washing lotion etc. are in pipe
Wall can generate absorption and residual, and then generate detection background value height, and signal-to-noise ratio is undesirable, the bad result of sensitivity.
Traditional chemical chemiluminescence immunoassay method, the either antigen-reactive in the antibody and sample on magnetic particle, still
Antibody response in antigen antibody complex and enzyme conjugates on magnetic particle forms exempting from for double-antibody sandwich " sandwich " structure
Epidemic disease compound, reaction process are in 37 DEG C of (or other set temperatures) static conditions, and magnetic particle can be because gravity influences at this time
Gradually sink, even suspension state can not be constantly in, this results in the reaction compartment between antigen, antibody molecule to be obstructed, and resists
It combines insufficient between former, antibody molecule, the reaction platform phase cannot be reached in a short time, when this both will affect pattern detection
Between, it also will affect detection sensitivity.
Although disclosing some spirits in the patent applications such as CN106546732A, CN104090101A, CN106290864A
The chemiluminescence immunoassay kit and its detection method that sensitivity is high, accuracy is good, but all fail to solve the problem above-mentioned.
Summary of the invention
In view of the deficienciess of the prior art, one of the objects of the present invention is to provide a kind of single parts based on bar magnet method
Chemical luminous immune detection method, this method magnetic bead clean thorough, carrying pollution less, and mixing is abundant, suitable for single part chemistry
Electrochemiluminescent immunoassay kit, using flexible, ambient noise are low.
The second object of the present invention is to provide a kind of chemiluminescence immune detection system for being able to achieve the above method.This is
Equipment instrument needed for uniting is small and exquisite, can be carried around into diagnosis and treatment scene, to laboratory sample size without particular/special requirement, price is low
It is honest and clean, substantially without maintenance cost, basic medical unit is suitble to promote the use of chemiluminescence immunoassay technology.
Above-mentioned purpose of the invention adopts the following technical scheme that realization:
A kind of single part chemical luminous immune detection method based on bar magnet method, agents useful for same includes: analysis buffer, magnetic
Pearl working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, substrate solution, this method comprising the following specific steps
1) analysis buffer is added in sample to be tested, obtains mixed liquor A；
2) it takes out magnetic bead from magnetic bead working solution by way of magnetic, is added in mixed liquor A, then to mix up and down
Mode is reacted certain time, is obtained mixed liquid B with certain frequency oscillation；
3) magnetic bead is removed from mixed liquid B by way of magnetic, is added in cleaning solution 1, obtains mixed liquor C, then again
It is shaken up and down with certain frequency and mixes cleaning；
4) magnetic bead is taken out from mixed liquor C by way of magnetic, and is added in enzyme working solution, then again to mix up and down
Mode with certain frequency oscillation, react certain time, obtain mixed liquor D；
5) magnetic bead is removed from mixed liquor D by way of magnetic, is added in cleaning solution 2, obtains mixed liquor E, then again
It is shaken up and down with certain frequency and mixes cleaning；
6) magnetic bead is taken out from mixed liquor E by way of magnetic, is added in substrate solution, is mixed up and down with certain frequency,
Obtain mixed liquor F；
7) mixed liquor is subjected to luminous signal detection.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, certain frequency choosing
From 1 beat/min~360 beats/min.
The present invention uses 1 beat/min~360 beats/min of mode of averaging up and down, and it is mixed to overcome traditional instrument eccentric force vortex
Following defect existing for even method: mixing frequency is unstable, and reaction solution easily splashes, and causes cleaning solution that can not wash and occur seriously jumping
, there are false positive test results in point；The salt component of another cleaning solution easily oozes out corrosion control motor, and motor is caused easily to damage, this
It will lead to motor frequently to replace, increase use cost.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, analysis buffer, magnetic bead
Working solution, enzyme working solution, cleaning solution 1, cleaning solution 2 and substrate solution addition volume be 1~1000 μ l.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, step 2) and 4) anti-
It is 30 seconds~60 minutes between seasonable.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, the partial size of the magnetic bead
It is 0.01 μm~50 μm.Magnetic bead in primary test can be the magnetic bead of same partial size, be also possible to the mixing of different-grain diameter
Magnetic bead.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, step 7) is described to shine
The principle of signal detection includes but is not limited to that alkaline phosphatase inspires light, horseradish peroxidase inspires light, acridinium ester is directly sent out
Light.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, the substrate solution include
But it is not limited to AMPPD, APS-5, CDP-Star, luminol and its derivative, hydrogen peroxide dilute alkaline soln.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, the enzyme working solution are
Pass through the product or other marked products of alkali phosphatase enzyme mark, horseradish peroxidase-labeled or acridinium ester label.
Further, single part chemical luminous immune detection method above-mentioned based on bar magnet method, the alkaline phosphatase
Including but not limited to calf intestine alkaline phosphatase, bacterial alkaline phosphatase or P-ALP.
Detection method of the invention, the analyzer finally by PMT or comprising PMT carry out the result detection that shines.
The above-mentioned detection method of the present invention can also can be completed with manual operation by instrument system.When manual operation, only
It needs to carry out the single part kit for fixing packaging by above step, not need accurate from the conventional reagents box of 100 person-portions
It draws 1 person-portion dosage to be tested, therefore eliminates the reliance on traditional large scale equipment, can flexibly be realized in basic medical unit.
When completing above-mentioned detection method using instrument detection system, have benefited from the present invention cleverly magnetic bead and liquid separate mode, inspection
Examining system no longer needs various cumbersome mixing mechanisms and liquid assimilating mechanism, it is only necessary to which multiple steps can be completed in a bar magnet
After magnetic bead and liquid separation.And this separate mode makes cleaning more thorough, efficiently reduces wash number, saves
About reagent.Its blending manner makes mixed liquor be constantly in even suspension state, and magnetic bead will not sink, between antigen, antibody molecule
Reaction it is free, unimpeded, in conjunction with abundant, the reaction platform phase can be reached in the short time, can both shorten the pattern detection time, also
Detection sensitivity can be improved.
According to above-mentioned, it is using what the above method carried out single part chemiluminescence immunoassay detection the present invention also provides a kind of
System, the system include reagent modules, magnetic suction module, isolation and mix module and control module；The reagent modules, magnetic
Module and mixing module are connected with control module, and the reagent modules include reagent tube assembly 1 and fixating reagent tube assembly
Workbench 2, the magnetic suction module include moving in the horizontal direction with the bar magnet 3 of vertical direction movement and driving bar magnet 3
Dynamic motor, the isolation and mixing module include the separation sleeve 7 that can be moved in the horizontal direction with vertical direction and driving
The mobile motor of separation sleeve 7.
Further, the system of the single part chemiluminescence immunoassay detection of above-mentioned carry out, the magnetic suction module further includes bar magnet
The vertical arm 6 of fixed block 4, bar magnet horizontal arm 5, bar magnet and horizontal arm fixed block 10, the bar magnet fixed block 4 grip bar magnet 3,
And can be horizontally moved under the drive of the motor along bar magnet horizontal arm 5, the bar magnet horizontal arm 5 is by horizontal arm fixed block 10
It is fixed on the vertical arm 6 of bar magnet, horizontal arm fixed block 10 can be along the vertical arm 6 of bar magnet in vertical side under the driving of another motor
It moves up.Realize that bar magnet 3 is without being limited thereto with the mode of vertical direction movement in the horizontal direction, this is that the embodiment of the present invention mentions
A kind of mode supplied.
Further, the system of the single part chemiluminescence immunoassay detection of above-mentioned carry out, the isolation and mixing module are also wrapped
The vertical arm 9 of horizontal extension bar 8, separation sleeve and telescopic rod fixed block 11 are included, one end of the horizontal extension bar 8 is flexibly connected isolation
Set 7, the other end are fixed on the vertical arm 9 of separation sleeve by telescopic rod fixed block 11, and horizontal extension bar 8 carries out under motor driven
Horizontal extension moves separation sleeve 7 in the horizontal direction, and telescopic rod fixed block 11 is vertical along separation sleeve under another motor driven
Arm 9 moves in the vertical direction.Realize that separation sleeve 7 is without being limited thereto with the mode of vertical direction movement in the horizontal direction, this is
A kind of mode provided in an embodiment of the present invention.
Further, the system of the single part chemiluminescence immunoassay detection of above-mentioned carry out, further includes being connected to the control module
Detection module, the detection module include detection handgrip, detection handgrip motor, the detection plate with hole location and PMT analyzer.
Technical solution of the present invention has the advantage that
1. single part chemical luminous immune detection method based on bar magnet method thoroughly solves traditional detection method and carries dirt
The problem of dye.Since detection background value is low, it is only necessary to which lower signal value is able to achieve compared with high s/n ratio, improves detection sensitivity:
The reaction of each test, cleaning, detection and etc. carried out in differential responses pipe, cleaning step only needs 1~2 time, cleaning
Mode is magnetic bead by bar magnet toward moving in the differential responses pipe for being respectively provided with the solution such as sample, washing lotion, enzyme working solution, be put into
And suction, it is thus completely absent the problem of tube wall absorption is remained with liquid, magnetic bead cleaning effect is more thorough, and is all made of one
Secondary property consumptive material, this just completely avoids the presence for carrying pollution.Thoroughly, completely just because of cleaning, detection background value is low, it is only necessary to
Lower signal value is able to achieve compared with high s/n ratio, improves detection sensitivity.
2. single part chemical luminous immune detection method based on bar magnet method realizes the lasting mixing to reaction solution.No matter
It is in the antigen antibody complex and enzyme conjugates on the antigen-reactive or magnetic particle in the antibody and sample on magnetic particle
Antibody response forms the immune complex of double-antibody sandwich " sandwich " structure, entire reaction process be in 37 DEG C it is (or other
Set temperature) state is persistently mixed, magnetic particle is constantly in even suspension state at this time, will not sink, and makes antigen, antibody molecule
Between reaction it is free, unimpeded, between antigen, antibody molecule combine sufficiently, the reaction platform phase can be reached in a short time, both
The pattern detection time can be shortened, detection sensitivity can also be improved.
3. single part chemical luminous immune detection method based on bar magnet method using single part, independent packaging, with tear open with
Mode, make to measure for the basic hospitals such as community, small towns: kit is using single part, independent packaging by the way of, to hospital's day
Equal sample size is without any requirement, and patient is completely absent box examination with just breaking a seal 1 person-portion reagent to survey, detecting a patient
Between the volatilization of reagent components caused by agent Reusability, reagent each component the problem of cross contamination, hospital and factory less will cause
The wasting of resources of family.
4. single part chemiluminescence immune detection system based on bar magnet method be omitted legacy system multiple transfer devices,
Cleaning device, rotating device motor corresponding with them, compact is easy to operate, and the requirement to timing reduces, ordinary people
Member can be operated and use.And instrument is desktop computer, does not have any requirement at all to storage place and place.
Detailed description of the invention
Fig. 1 is Full-automatic chemiluminescence immunoassay analysis meter device structural schematic diagram disclosed in prior art CN107831322A；
Fig. 2 is the disclosed sequential control system knot for being used for chemical illumination immunity analysis instrument of prior art CN108226549A
Structure schematic diagram；
Fig. 3 is that a kind of single part chemiluminescence immune detection system based on bar magnet method of the embodiment of the present invention is mainly tied
Structure schematic diagram；
1- reagent tube assembly, 2- workbench, 3- bar magnet, 4- bar magnet fixed block, 5- bar magnet horizontal arm, 6- bar magnet are vertical
Arm, 7- separation sleeve, 8- horizontal extension bar, the vertical arm of 9- separation sleeve, 10- horizontal arm fixed block, 11- telescopic rod fixed block.
Specific embodiment
It is illustrated below by way of specific embodiment is further to summary of the invention of the invention, those skilled in the art should
Know, the present invention is based on single part chemical luminous immune detection methods of bar magnet method can be used in a variety of chemiluminescence detections, and
It is not limited to HIV, HCV and TP of following test examples；Realize that the system of chemical luminous immune detection method of the present invention can be according to reality
Border needs for existing some functional modules to be combined in following embodiments, these all basic inventions are easy to get, and still belongs to
In the scope that the present invention protects.
Embodiment 1
A kind of single part chemiluminescence immune detection system based on bar magnet method, including reagent modules, magnetic suction module, isolation
With mixing module and control module；The reagent modules, magnetic suction module, isolation and mixing module are connected with control module.
The reagent modules include the workbench 2 of reagent tube assembly 1 and fixating reagent tube assembly, and the magnetic suction module includes being capable of edge
The mobile motor of the bar magnet 3 and driving bar magnet 3 of horizontal direction and vertical direction movement, the isolation and mixing module include
The mobile motor of the separation sleeve 7 and driving separation sleeve 7 that can be moved in the horizontal direction with vertical direction.
With reference to Fig. 3, under control of the control system, bar magnet 3 and separation sleeve 7 are shifted under the driving of corresponding motor,
The surface for arriving magnetic bead working solution Reagent Tube first, by vertical shift, bar magnet 3 is protruded into separation sleeve 7, further protrudes into magnetic
In pearl working solution Reagent Tube, magnetic bead is drawn.Then bar magnet 3 and separation sleeve 7, which are lifted straight up and are moved to simultaneously, has added sample
Analysis buffer Reagent Tube in, bar magnet 3 lifts, and magnetic bead stays in pipe, and separation sleeve 7 does certain amplitude and frequency under motor driven
The concussion up and down of rate mixes.Then bar magnet 3 protrudes into pipe, adsorbs magnetic bead, then upward together with separation sleeve 7, and magnetic bead is transferred to
In next Reagent Tube, until reaction and cleaning terminate.
Realize that a kind of mode mobile with vertical direction in the horizontal direction, the magnetic suction module are also wrapped as realization bar magnet 3
The vertical arm 6 of bar magnet fixed block 4, bar magnet horizontal arm 5, bar magnet and horizontal arm fixed block 10 are included, the bar magnet fixed block 4 grips
Bar magnet 3, and can be horizontally moved under the drive of the motor along bar magnet horizontal arm 5, the bar magnet horizontal arm 5 is consolidated by horizontal arm
Determine block 10 to be fixed on the vertical arm 6 of bar magnet, horizontal arm fixed block 10 can exist under the driving of another motor along the vertical arm 6 of bar magnet
It is moved on vertical direction.
A kind of mode mobile with vertical direction in the horizontal direction, the isolation and mixing are realized as realization separation sleeve 7
Module further includes horizontal extension bar 8, the vertical arm 9 of separation sleeve and telescopic rod fixed block 11, one end activity of the horizontal extension bar 8
Separation sleeve 7 is connected, the other end is fixed on the vertical arm 9 of separation sleeve by telescopic rod fixed block 11, and horizontal extension bar 8 drives in motor
The dynamic lower horizontal extension that carries out moves separation sleeve 7 in the horizontal direction, telescopic rod fixed block 11 another motor driven lower edge every
It is moved in the vertical direction from vertical arm 9 is covered.The separation sleeve 7 of flexible connection removes replacement after each test, for disposable consumption
Material.
As preferred embodiment mode, the system of the above-mentioned single part chemiluminescence immunoassay detection of carry out further includes and controls
The connected detection module of module, the detection module include detection handgrip, detection handgrip motor, the detection plate with hole location and
PMT analyzer.
Test example 1
The single part chemical luminescence detection method (alkaline phosphoric acid enzyme method) of HIV 1+2 type antibody based on bar magnet method
The single part reagent of HIV 1+2 type antibody is provided by Chengdu Bo Aoxin scape medical science and technology Co., Ltd.Reagent components packet
Contain: analysis buffer, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, substrate solution, detection method can pass through implementation
The detection system of example 1 is realized, can also be realized with manual operation.When with detection system, analysis buffer, enzyme working solution, cleaning solution
1, cleaning solution 2, substrate solution are placed in advance in reagent tube assembly.This method specifically includes the following steps:
Step 1: sample to be tested is added in analysis buffer, mixed liquor A is obtained.It (draws sample and is put into reagent tube assembly
Analysis buffer pipe in, then reagent tube assembly is fixed on workbench 2.)
Step 2: taking out magnetic bead from the magnetic bead working solution of HIV 1+2 type antibody reagent by way of magnetic, and it is added
In mixed liquor A, then in a manner of mixing up and down, with 120 beats/min of frequency oscillation, reacts 5 minutes, obtain mixed liquid B.
(bar magnet 3 and separation sleeve 7 are moved to above magnetic bead work liquid pipe in motor driven, are extended downwardly into pipe, then bar magnet 3 is drawn
Magnetic bead is then transferred in analysis buffer pipe, and bar magnet 3 lifts straight up, and magnetic bead is fallen, separation sleeve 7 under motor driven with
Small concussion reaction 5 minutes in 120 beats/min of frequency.)
Step 3: removing magnetic bead by way of magnetic from reaction mixture B again, it is added in cleaning solution 1, is mixed
Then liquid C carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.(bar magnet extends downwardly into separation sleeve 7, suction pipe
Interior magnetic bead is shifted with separation sleeve 7 together in magnetic bead manages to cleaning solution 1, and bar magnet 3 lifts straight up, and magnetic bead is fallen, separation sleeve 7
Concussion cleaning up and down is carried out with 180 beats/min of frequency under motor driven.)
Step 4: taking out magnetic bead from mixed liquor C by way of magnetic, and HIV 1+2 type antibody reagent enzyme work is added
Make in liquid, then again in a manner of mixing up and down, is mixed with 120 beats/min of frequency oscillation, react 15 minutes, mixed
Liquid D.(bar magnet extends downwardly into separation sleeve 7, and magnetic bead in suction pipe shifts in magnetic bead to enzyme work liquid pipe, magnetic together with separation sleeve 7
Stick 3 lifts straight up, and magnetic bead is fallen, and separation sleeve 7 is mixed instead under motor driven with shaking above and below 120 beats/min of frequency
It answers 15 minutes.)
Step 5: removing magnetic bead by way of magnetic from reaction mixture D again, it is added in cleaning solution 2, is mixed
Then liquid E carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.(bar magnet extends downwardly into separation sleeve 7, suction pipe
Interior magnetic bead is shifted with separation sleeve 7 together in magnetic bead manages to cleaning solution 2, and bar magnet 3 lifts straight up, and magnetic bead is fallen, separation sleeve 7
Cleaning is mixed under motor driven with about 180 beats/min concussions.)
Step 6: taking out magnetic bead from mixed liquor E by way of magnetic, move on in substrate solution, with the side mixed up and down
Formula is mixed with 120 beats/min of frequency oscillation, obtains mixed liquor F, then carries out luminous signal detection.(bar magnet extends downwardly into
Separation sleeve 7, magnetic bead in suction pipe, is shifted in magnetic bead to substrate solution pipe, bar magnet 3 lifts straight up, magnetic bead together with separation sleeve 7
It falls, separation sleeve 7 is mixed under motor driven with 120 beats/min of frequency oscillation.)
Step 7: mixed liquor is carried out luminous signal detection.(detection handgrip crawl substrate liquid pipe is put into the hole of detection plate
It is interior, signal detection is carried out with PMT analyzer.)
With the diaghostic reagent of human immunodeficiency virus antibody box (chemiluminescence of Beijing Kemei Biological Technology Co., Ltd.
Method) and 1500 Full-automatic chemiluminescence immunoassay analysis meter of CHEMCLIN detection result be used as reference, with investigate the present invention detect
The accuracy of method.
Testing result is as follows:
Conclusion: detecting through the single part chemoluminescence method of bar magnet method HIV 1+2 type antibody, sample yin and yang attribute testing result and ginseng
It is completely the same according to producer's testing result.
Test example 2
The single part chemical luminescence detection method (alkaline phosphoric acid enzyme method) of HIV-1p24 antigen based on bar magnet method
HIV-1p24 antigenic agents are provided by Chengdu Bo Aoxin scape medical science and technology Co., Ltd.Reagent components include: analysis
Buffer, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, substrate solution, specific step is as follows, completes when using system
When, it is similar with test example 1:
Step 1: sample to be tested is added in analysis buffer, mixed liquor A is obtained.
Step 2: taking out magnetic bead from the magnetic bead working solution of HIV-1p24 antigenic agents by way of magnetic, and it is added
In mixed liquor A, then in a manner of mixing up and down, with 120 beats/min of frequency oscillation, reacts 5 minutes, obtain mixed liquid B.
Step 3: removing magnetic bead by way of magnetic from reaction mixture B again, it is added in cleaning solution 1, is mixed
Then liquid C carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.
Step 4: taking out magnetic bead from mixed liquor C by way of magnetic, and the work of HIV-1p24 antigenic agents enzyme is added
In liquid, then again in a manner of mixing up and down, is mixed with 120 beats/min of frequency oscillation, react 15 minutes, obtain mixed liquor
D。
Step 5: removing magnetic bead by way of magnetic from reaction mixture D again, it is added in cleaning solution 2, is mixed
Then liquid E carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.
Step 6: taking out magnetic bead from mixed liquor E by way of magnetic, move on in substrate solution, with the side mixed up and down
Formula is mixed with 120 beats/min of frequency oscillation, obtains mixed liquor F, then carries out luminous signal detection.
Step 7: mixed liquor is carried out luminous signal detection.
(National Institute for Food and Drugs Control, article No.: 22015- are purchased from HIV-1p24 antigen National reference
20150505) testing result is as reference, to investigate the accuracy of detection method.
Testing result is as follows:
Conclusion: it is detected through the single part chemoluminescence method of bar magnet method HIV-1p24 antigen, sample yin and yang attribute result and HIV-
The requirement of 1p24 antigen National reference is completely the same.
Test example 3
The single part chemical luminescence detection method (alkaline phosphoric acid enzyme method) of anti-HCV based on bar magnet method
Anti-HCV reagent is provided by Chengdu Bo Aoxin scape medical science and technology Co., Ltd.Reagent components include: analysis buffering
Liquid, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, substrate solution, operation is similar with test example 1, and specific step is as follows,
It is similar with test example 1 when being completed using system:
Step 1: sample to be tested is added in analysis buffer, mixed liquor A is obtained.
Step 2: taking out magnetic bead from the magnetic bead working solution of anti-HCV reagent by way of magnetic, and mixing is added
In liquid A, then in a manner of mixing up and down, with 120 beats/min of frequency oscillation, reacts 5 minutes, obtain mixed liquid B.
Step 3: removing magnetic bead by way of magnetic from reaction mixture B again, it is added in cleaning solution 1, is mixed
Then liquid C carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.
Step 4: taking out magnetic bead from mixed liquor C by way of magnetic, and anti-HCV reagent enzyme working solution is added
In, it then again in a manner of mixing up and down, is mixed with 120 beats/min of frequency oscillation, reacts 15 minutes, obtain mixed liquor D.
Step 5: removing magnetic bead by way of magnetic from reaction mixture D again, it is added in cleaning solution 2, is mixed
Then liquid E carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.
Step 6: taking out magnetic bead from mixed liquor E by way of magnetic, move on in substrate solution, with the side mixed up and down
Formula is mixed with 120 beats/min of frequency oscillation, obtains mixed liquor F, then carries out luminous signal detection.
Step 7: result detects.Result detection is carried out by semi-automatic chemical illumination immunity analysis instrument.
With the antibody of HCV assay kit (chemiluminescence particulate immunodetection) of Abbott Laboratories, Abbott Laboratories
ARCHITECT i2000SRThe result of detection is as reference, to investigate the accuracy of detection method.
Testing result is as follows:
Conclusion: detecting through the single part chemoluminescence method of bar magnet method anti-HCV, and sample yin and yang attribute result is examined with producer is compared
It is completely the same to survey result.
Test example 4
The single part chemical luminescence detection method (alkaline phosphoric acid enzyme method) of anti-TP based on bar magnet method
Anti-TP reagent comes from Chengdu Bo Aoxin scape medical science and technology Co., Ltd.Reagent components include: enzyme working solution 1, magnetic
Pearl working solution, enzyme working solution 2, cleaning solution 1, cleaning solution 2, substrate solution, operate similar with test example 1, and specific step is as follows, when adopting
It is similar with test example 1 when being completed with system:
Step 1: sample to be tested is added in enzyme working solution 1, mixed liquor A is obtained.
Step 2: taking out magnetic bead from the magnetic bead working solution of anti-TP reagent by way of magnetic, and mixed liquor is added
In A, then in a manner of mixing up and down, with 120 beats/min of frequency oscillation, reacts 5 minutes, obtain mixed liquid B.
Step 3: removing magnetic bead by way of magnetic from reaction mixture B again, it is added in cleaning solution 1, is mixed
Then liquid C carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.
Step 4: taking out magnetic bead from mixed liquor C by way of magnetic, and it is added in anti-TP reagent enzyme working solution,
Then it again in a manner of mixing up and down, is mixed with 120 beats/min of frequency oscillation, reacts 15 minutes, obtain mixed liquor D.
Step 5: removing magnetic bead by way of magnetic from reaction mixture D again, it is added in cleaning solution 2, is mixed
Then liquid E carries out concussion up and down again with 180 beats/min of frequency and mixes cleaning.
Step 6: taking out magnetic bead from mixed liquor E by way of magnetic, move on in substrate solution, with the side mixed up and down
Formula is mixed with 120 beats/min of frequency oscillation, obtains mixed liquor F, then carries out luminous signal detection.
Step 7: result detects.Result detection is carried out by semi-automatic chemical illumination immunity analysis instrument.
With the treponema pallidum antibody diagnostic kit (chemoluminescence method) of Beijing Kemei Biological Technology Co., Ltd.,
The result of 1500 Full-automatic chemiluminescence immunoassay analysis meter of CHEMCLIN detection is as reference, to investigate detection method
Accuracy.
Testing result is as follows:
Conclusion: detecting through the single part chemoluminescence method of bar magnet method anti-TP, and sample yin and yang attribute result is detected with producer is compared
As a result completely the same.
When detecting sample with other producer's methods, because the reagent of producer is 100 person-portions, it is used for multiple times and is not run out,
It is detected with 20+ days behind Kaifeng reagents, testing result shows noise background more higher than testing result of the present invention.Its
Reason includes that magnetic bead absorption branch mode is different and reagent is changed by time effects.The method of the present invention has higher spirit
Quick property.
The above test example show the testing result that detection method of the invention obtains be accurately, can be with multiple producers
Testing result keeps height consistent.Moreover, detection method of the invention carries, pollution is low, and wash number is few, simplifies program, drops
The difficulty of low software control and staff's operation.The characteristics of being detected due to the cleaning way of the method for the present invention and single part,
The noise signal that pollutant carries and reagent variation generates is effectively reduced, the raising of sensitivity is more conducive to.In addition, using this
The detection system of invention detection method has saved multiple transfer devices, mixing mechanism and corresponding motor, has reduced the body of instrument
Product, makes the traditional detection instrument miniaturization as huge monster, can be applied in more basic medical units of limited area, and
And detection time is shorter.
Claims (10)
1. a kind of single part chemical luminous immune detection method based on bar magnet method, agents useful for same includes: analysis buffer, magnetic bead
Working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, substrate solution, which is characterized in that method includes the following steps:
1) analysis buffer is added in sample to be tested, obtains mixed liquor A；
2) magnetic bead is taken out from magnetic bead working solution by way of magnetic, is added in mixed liquor A, then in a manner of mixing up and down
With certain frequency oscillation, certain time is reacted, mixed liquid B is obtained；
3) magnetic bead is removed from mixed liquid B by way of magnetic, is added in cleaning solution 1, obtains mixed liquor C, then again with certain
Kind frequency is shaken up and down mixes cleaning；
4) magnetic bead is taken out from mixed liquor C by way of magnetic, and is added in enzyme working solution, then side to mix up and down again
Formula is reacted certain time, is obtained mixed liquor D with certain frequency oscillation；
5) magnetic bead is removed from mixed liquor D by way of magnetic, is added in cleaning solution 2, obtains mixed liquor E, then again with certain
Kind frequency is shaken up and down mixes cleaning；
6) magnetic bead is taken out from mixed liquor E by way of magnetic, is added in substrate solution, is mixed, is obtained up and down with certain frequency
Mixed liquor F；
7) mixed liquor F is subjected to luminous signal detection.
2. single part chemical luminous immune detection method according to claim 1 based on bar magnet method, which is characterized in that institute
Stating certain frequency is 1 beat/min~360 beats/min；Analysis buffer, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning
The addition volume of liquid 2 and substrate solution is 1~1000 μ l.
3. single part chemical luminous immune detection method according to claim 1 based on bar magnet method, which is characterized in that step
The rapid reaction time 2) and 4) is 30 seconds~60 minutes；The partial size of the magnetic bead is 0.01 μm~50 μm.
4. single part chemical luminous immune detection method according to claim 1 based on bar magnet method, which is characterized in that step
The principle of rapid 7) the described luminous signal detection is that alkaline phosphatase inspires light, horseradish peroxidase inspires light or acridinium ester is direct
It shines；The enzyme working solution be by the product of alkali phosphatase enzyme mark, horseradish peroxidase-labeled or acridinium ester label or
Other marked products of person.
5. single part chemical luminous immune detection method according to claim 1 based on bar magnet method, which is characterized in that institute
Stating substrate solution is AMPPD, APS-5, CDP-Star, luminol and its derivative or hydrogen peroxide dilute alkaline soln.
6. single part chemical luminous immune detection method according to claim 4 based on bar magnet method, which is characterized in that institute
Stating alkaline phosphatase is calf intestine alkaline phosphatase, bacterial alkaline phosphatase or P-ALP.
7. a kind of system for carrying out chemiluminescence immunoassay detection using method described in claim 1~6 any one, feature
It is, which includes reagent modules, magnetic suction module, isolation and mix module and control module；The reagent modules, magnetic
It inhales module, isolation and mixes module and be connected with control module, the reagent modules include reagent tube assembly (1) and fixating reagent
The workbench (2) of tube assembly (1), the magnetic suction module include can in the horizontal direction the bar magnet (3) mobile with vertical direction,
And the motor that driving bar magnet (3) is mobile, the isolation and mixing module include can be mobile with vertical direction in the horizontal direction
Separation sleeve (7) and the mobile motor of driving separation sleeve (7).
8. the system according to claim 7 for carrying out chemiluminescence immunoassay detection, which is characterized in that the magnetic suction module is also
It is fixed including bar magnet fixed block (4), bar magnet horizontal arm (5), the vertical arm of bar magnet (6) and horizontal arm fixed block (10), the bar magnet
Block (4) grips bar magnet (3), and can be horizontally moved under the drive of the motor along bar magnet horizontal arm (5), the bar magnet
Horizontal arm (5) is fixed on the vertical arm of bar magnet (6) by horizontal arm fixed block (10), and horizontal arm fixed block (10) is in another motor
It can be moved in the vertical direction along the vertical arm of bar magnet (6) under driving.
9. the system according to claim 7 for carrying out chemiluminescence immunoassay detection, which is characterized in that the isolation and mixing
Module further includes horizontal extension bar (8), the vertical arm of separation sleeve (9) and telescopic rod fixed block (11), the horizontal extension bar (8)
One end is flexibly connected separation sleeve (7), and the other end is fixed on the vertical arm of separation sleeve (9) by telescopic rod fixed block (11), horizontal
Telescopic rod (8) carries out horizontal extension under motor driven moves separation sleeve (7) in the horizontal direction, telescopic rod fixed block (11)
It is moved in the vertical direction under another motor driven along the vertical arm of separation sleeve (9).
10. carrying out the system of chemiluminescence immunoassay detection according to claim 7~9 any one, which is characterized in that should
System further includes being connected to the control module detection module, and the detection module includes detection handgrip, detection handgrip motor, has
The detection plate and PMT analyzer of hole location.
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| CN201811246644.3A CN109060783A (en) | 2018-10-25 | 2018-10-25 | A kind of single part chemical luminous immune detection method and system based on bar magnet method |
| PCT/CN2019/085653 WO2020082720A1 (en) | 2018-10-25 | 2019-05-06 | Method and system for single-part chemiluminescence immunoassay on basis of magnetic rod method |
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| CN114236129B (en) * | 2021-12-20 | 2022-09-23 | 江苏集萃中科纳米科技有限公司 | In-vitro immunodiagnosis reagent background luminescence processing system |
| CN114216897A (en) * | 2021-12-22 | 2022-03-22 | 武汉生之源生物科技股份有限公司 | sST2 chemiluminescence detection kit and detection method thereof |
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