CN109541226A - A kind of pepsinogen Cgene/II bigeminy check reagent box and preparation method thereof - Google Patents
A kind of pepsinogen Cgene/II bigeminy check reagent box and preparation method thereof Download PDFInfo
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- CN109541226A CN109541226A CN201811329541.3A CN201811329541A CN109541226A CN 109541226 A CN109541226 A CN 109541226A CN 201811329541 A CN201811329541 A CN 201811329541A CN 109541226 A CN109541226 A CN 109541226A
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses a kind of pepsinogen Cgene/II bigeminy check reagent boxes, are grouped as by following group: double antibody magnetic microballoon, I/II calibration object of PG, double antibody marker, analysis buffer, cleaning solution, enhancement solution and RFID card.This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, overcome the physical sorption reaction time of ELISA Plate longer, the slower drawback of testing result, the reaction time is greatly shortened, while also having that time resolution detection technique accuracy height, high sensitivity, high specificity, the range of linearity is wide, detection is stable and convenient advantage.The present invention advanced optimizes traditional two-step method detection, and detection only needs one-step method to detect, and result can be detected in 20 minutes.And it further, realizes two joint inspections of PG I/II using time resolution double labelling advantage, further shortens project result detection time.
Description
Technical field
The present invention relates to a kind of pepsinogen Cgene/II bigeminy check reagent boxes and preparation method thereof, and in particular to one kind is based on
Magnetic microsphere combines pepsinogen Cgene/II bigeminy check reagent box preparation method with Timed-resolved fluoroimmunoassay.
Background technique
Propepsin is the nonactive precursor of pepsin in gastric juice, can be divided into amphitypy in immunology: pepsinogen Cgene
(PG I) and Pepsinogen II (PG II), PG I are mainly secreted by the chief cell of fundus gland and mucus neck cell, and PG II remove by
Chief cell and mucus the neck cell secretion of fundus gland are outer, the mucus neck cell and duodenum of the pyloric gland of cardiac gland and antrum
Upper section secretion.During fundus gland mucosal atrophy, the chief cell of secretion PG I is reduced, pyloric gland cytosis, to cause PG
I/PG, II ratio reduces, horizontal by the detection of propepsin, especially I/PG of PG, II ratio and PG I, for diagnosing chronic
Atrophic gastritis, erosive gastritis, gastric ulcer, duodenal ulcer, atrophic gastritis and intestinal metaplasia have very high value.
1 part of serum sample detects two pepsinogen Cgene, Pepsinogen II projects simultaneously；Time-resolved fluoroimmunoassay
The multiple labeling advantage for analyzing (TRFIA), marks pepsinogen Cgene antibody, Pepsinogen II using two kinds of lanthanide series respectively
The mixed mark object of antibody, two kinds be coated with respectively pepsinogen Cgene antibody, Pepsinogen II antibody magnetic microsphere mixing
Liquid, then the double antibody sandwich method of double labelling can be formed in conjunction with two kinds of corresponding antibody in serum sample.Both the time is inherited
The advantages of Gao Min of resolved immuno fluorometric analytic approach, the wide line, high-accuracy property, while also the liquid phase with magnetic microsphere solution is special
Property, in conjunction with double labelling, further shorten the reaction time, detects result faster.It is sent out compared to chemiluminescence (CLIA), electrochemistry
Light (ECL) detection method, while reaching same performance detection, it may have inexpensive advantage.
Summary of the invention
Based on this, it is an object of the invention to overcome the associated disadvantages of the prior art, provide it is a kind of faster, more convenient and fast stomach
I/II bigeminy check reagent box of proproteinase and preparation method thereof.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of pepsinogen Cgene/II bigeminy check reagent box, is grouped as: double antibody magnetic microballoon, I/II school PG by following group
Quasi- product, double antibody marker, analysis buffer, concentration washing lotion (cleaning solution) and enhancement solution, RFID card.
Preferably, the magnetic microsphere is by micron-sized Fe2O3Or Fe3O4Magnetic particle and high-molecular organic material
Carry out compound, being formed has superparamagnetism, the micron-sized microballoon that can combine with immunising antigen or antibody, that is, is generally called
For " magnetic bead ".It is 0.1~5 μm that magnetic microsphere, which should be able to meet diameter, and magnetic microsphere can have various active function by surface modification
It can group, including but not limited to hydroxyl (- OH), amino (- NH2), carboxyl (- COOH).
Preferably, the double antibody magnetic microballoon is prepared using following steps:
By PG I, II antibody of PG respectively after 2~8 DEG C of superspeed refrigerated centrifuges are by buffer system replacement Treatment, with cleaning,
Magnetic microsphere mixing and constant-temperature incubation after activation, clean magnetic bead and abandon supernatant, then carried out with magnetic bead confining liquid after incubation
Closing, cleaning magnetic bead abandon supernatant again, and two kinds of high concentration antibody magnetic microspheres are saved 2~8 DEG C of refrigerators in liquid in magnetic bead
It is upright to save, then two kinds of magnetic beads are diluted to mix after working concentration, double antibody magnetic microballoon is made.
It is 10 μ g that every milligram of magnetic microsphere of EDC and Sulfo-NHS, which is preferably loaded quality, in the activation of the magnetic microsphere
~1000 μ g.Activation method includes but is not limited to EDC, Sulfo-NHS one such and two kinds.
Preferably, the antibody in the double antibody marker and lanthanide series are completed by intermediate chelating agent, group of the lanthanides
Element 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 benzene
Methyl D TTA, P- isothiocyanatobenzyl-DTTA, diethylene triamine pentaacetic acid aminophenyl-EDTA.
The present invention also provides a kind of methods using mentioned reagent box detection PG I/II, and described method includes following steps:
(1) double antibody marker is diluted to working solution with analysis buffer；
(2) working solution is diluted to by washing lotion is concentrated with purified water；
(3) double antibody magnetic microballoon is added in reaction cup；
(4) sample to be examined or calibration object are added in above-mentioned reaction cup；
(5) the double antibody marker working solution diluted in (1) is added in reaction cup；
(6) reaction cup is incubated at room temperature；
(7) magnetic microsphere after using the cleaning liquid in (2) to add magnetic in washing reaction cup；
(8) after washing, degaussing is added enhancement solution and is incubated for；
(9) after being incubated for, the fluorescent collecting for carrying out corresponding wavelength is detected and is analyzed.
Preferably, for the present invention in order to further reduce manual steps, self-produced SmartTRF grinds certainly in cooperation company
The relevant parameter of detection method, operating procedure are all copied to RFID card by complete series Immunofluorescence test equipment
In.In actual mechanical process, it is only necessary to RFID card is adapted to above-mentioned Immunofluorescence test equipment can be automatically finished it is above-mentioned
The operating procedure of experiment.RFID (Radio Frequency Identification) technology, also known as radio frequency identification, 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 pepsinogen Cgene/II bigeminy check reagent box preparation methods, and the method includes as follows
Step:
(1) double antibody magnetic microballoon is prepared；
(2) double antibody marker is prepared；
(3) I/II calibration object of PG is prepared；
(4) analysis buffer, concentration washing lotion and enhancement solution are prepared；
(5) each component is dispensed respectively into corresponding storage container；
(6) RFID card duplicate copy；
(7) coding, labelling；
(8) it is assembled into finished product kit.
Compared with prior art, the beneficial effects of the invention are that:
This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, that is, overcomes the physical absorption of ELISA Plate anti-
Longer between seasonable, the slower drawback of testing result greatly shortens the reaction time, while it is accurate also to have time resolution detection technique
Property height, high sensitivity, high specificity, the range of linearity is wide, detection is stable and convenient advantage.The present invention is for traditional two-step method
Detection advanced optimizes, and detection only needs one-step method to detect, and result can be detected in 20 minutes.And it further, uses
Time resolution double labelling advantage realizes two joint inspections of PG I/II, further shortens project result detection time.In addition, due to
The fluid behaviour of magnetic microsphere buffer system is no longer limited by the frame limitation of traditional ELISA Plate, in arbitrary reaction cup and small
Detection can be completed in test tube, while volume size and the equipment cost of detecting instrument equipment can be reduced to meet two, three line cities
The full-automatic detection demand in city also can carry out big flux detection as ELISA Plate, the reflective detection of chemistry, meet a line city
Full-automatic detection demand.
Detailed description of the invention
Fig. 1 is to be used to store double antibody magnetic microballoon, double antibody marker, analysis using detection kit of the present invention
The schematic top plan view of the reagent strip of buffer, cleaning solution and enhancement solution.
Fig. 2 is to be used to store double antibody magnetic microballoon, double antibody marker, analysis using detection kit of the present invention
The schematic perspective view of the reagent strip of buffer, cleaning solution and enhancement solution.
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；Contrast agents box is that (chemiluminescence is micro- for Abbott Laboratories' pepsinogen Cgene assay kit
Particle immunodetection).
Embodiment 1
A kind of pepsinogen Cgene/II bigeminy check reagent box, the kit include: double antibody magnetic microballoon, I/II school PG
Quasi- product, I/II double antibody marker of PG, analysis buffer, concentration washing lotion and enhancement solution, RFID card.
The present invention also provides above-mentioned pepsinogen Cgene/II bigeminy check reagent box preparation methods, and the method includes as follows
Step:
(1) double antibody magnetic microballoon is prepared: by I/II antibody of PG respectively through 2~8 DEG C of superspeed refrigerated centrifuges by buffer body
It is to be mixed simultaneously constant-temperature incubation 1~3 hour, after incubation after replacement Treatment with 1 μm of carboxyl magnetic microsphere of diameter after cleaning, activation
Magnetic bead is cleaned using magnetic bead cleaning solution and abandons supernatant, is then closed with magnetic bead confining liquid, and cleaning magnetic bead abandons again
Supernatant, by I/II antibody magnetic microsphere of PG being coated in magnetic bead saves in liquid, 2~8 DEG C of refrigerators uprightly save.It again will coating
Good I/II antibody magnetic microsphere of PG is diluted to after working solution concentration with magnetic bead preservation liquid and is mixed and made into double antibody magnetic microballoon, is dispensed
At 10mL/ bottles.Preferably, magnetic microsphere and the coated mass ratio of I/II antibody of PG are divided into 30:1,20:1；Preferably, described
Displacement buffer and magnetic bead cleaning solution are the MES buffers of 0.05~0.5M PH, 5.8~PH 7.0；Preferably, magnetic microsphere
Activation in every milligram of magnetic microsphere of EDC and Sulfo-NHS be preferably loaded quality be 10 μ of μ g~1000 g；Preferably, magnetic
The Tris-HCl buffer that the confining liquid and preservation liquid of microballoon are 0.1~0.5M PH 7.0~8.5 containing 0.1%~8%BSA；
(2) prepare I/II double antibody marker of PG: it is 10000 ultra-filtration centrifuge tubes that I/II antibody of PG, 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 it respectively and in advance with the solvent DTTA-EU of carbonate buffer solution3、DTTA-SM3++Mixing, I/II antibody of PG and europium, samarium matter
Amount mixes 24 ± 2 hours than respectively 5:1,3:1,2~8 DEG C of oscillations.Label solution is buffered through 0.05M PH 7.8Tris-Hcl
The Sephadex of liquid balanceTMG-75 gel columnChromatographic purifying monitors in A280 and collects first peak.It will collect
I/II double antibody marker 0.2%BSA, 0.05M the PH 7.8Tris-Hcl buffer of PG arrived is diluted to the 1/ of optium concentration
It mixes, and is dispensed to 1.0mL/ bottles after 20 times.
(3) prepare I/II calibration object of PG: by from stomach lining II antigen of PGI, PG be diluted to respectively low, high two it is dense
Point is spent, and low, high concentration spot is mixed I/II calibration object of PG is made respectively.
(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, TritonX-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 above-mentioned pepsinogen Cgene/II bigeminy check reagent box detection method, the method concrete operations
It is as follows:
(1) reagent prepares
1. kit restores in being placed at room temperature for room temperature；
2. I/II double antibody marker of PG is diluted to working solution concentration 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 double antibody magnetic microballoon, mixes stand-by before experiment；
(2) experimental implementation
1. the double antibody magnetic microballoon for drawing 50 μ L mixing is added in reaction cup；
2. sample to be tested or I/II calibration object of PG, 100 μ L are added into each reaction cup；
3. the 100 μ L of double antibody marker working solution diluted is added into each reaction cup again；
4. room temperature blending incubation 20 minutes；
5. after being incubated for, being cleaned 4 times using cleaning solution；
6. 100 μ L of enhancement solution is added into each reaction cup, room temperature blending incubation 3 minutes；Fluorescence is completed in 30 minutes
It acquires 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 pepsinogen Cgene/II bigeminy check reagent box, it is essentially identical with detection kit described in embodiment 1, it is different
It is:
(1) pepsinogen Cgene/II bigeminy check reagent box component only includes: reagent strip, I/II calibration object of PG, RFID
Card.
(2) in the present embodiment, reagent strip is by double antibody magnetic microballoon, I/II double antibody mark of PG in the detection kit
It is formed after sealer in note object, analysis buffer, cleaning solution and enhancement solution, packing to the corresponding hole of reagent strip.Wherein cleaning solution is
Washing lotion is concentrated in embodiment 1 adds 25 times of purified water dilution to form；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
I/II double antibody magnetic microballoon of PG, 50 μ L PG, I/II double antibody marker, 200 μ L analysis buffers, 3000 μ L cleaning solutions, 200
μ L enhancement solution is dispensed respectively to the 1st of reagent strip the, 3,5,6,12 holes, and the reagent strip is made after sealer.
The present invention also provides above-mentioned pepsinogen Cgene/II bigeminy check reagent box preparation methods, and examination is obtained in the above describe manner
After agent item, kit is constituted with I/II calibration object of PG, RFID card.In addition to each component packing mode and storage container are different
Outside, remaining is in the same manner as in Example 1.
The present invention also provides above-mentioned pepsinogen Cgene/II bigeminy check reagent box detection methods, it is only necessary to by mentioned reagent
Item is inserted into the reagent clamp bar slot of SmartTRF serial equipment after gently shaking mixing, and equipment reads RFID card relevant information can entirely certainly
It is dynamic to complete detection process.The related information parameters and detecting step of RFID card are in the same manner as in Example 1.
Embodiment 3
Pepsinogen Cgene of the present invention/II bigeminy check reagent box performance evaluation:
By the kit and detection method prepared in embodiment, detected with from Abbott Laboratories, hospital Architect i2000
The clinical sample of pepsinogen Cgene 240.The 2.5th percentiles of PG I is 63.8ng/mL in embodiment 1,2；PG II
95 percentiles are 22.5ng/mL.
Embodiment 1, embodiment 2 detect 240 Abbott Laboratories' pepsinogen Cgene, Pepsinogen II clinical samples, as a result such as
Under:
1 embodiment 1 of table, the comparison of 2 clinical samples
I/PG of PG, II ratio negative match-rate, positive coincidence rate are 100% in the embodiment of the present invention 1, embodiment 2.
What the concentration washing lotion (cleaning solution) referred in embodiment 1 is that the high concentration to be diluted to working solution is cleaned
Liquid；What cleaning solution referred in example 2 is the working solution for not needing any processing.
It should be understood that pepsinogen Cgene/II bigeminy check reagent box detection method and preparation method in embodiment 1
It is to be invented to meet big flux testing goal, instrument and equipment bulky and cost is relatively high, in order to further full
The detection demand in two, three line cities of foot, correspondingly, we have further made some detection methods on the basis of embodiment 1
And the 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 double antibody magnetic microballoon, double antibody marker, analysis buffer, cleaning solution and the enhancing in embodiment 1
Liquid is dispensed into special reagent strip (as shown in Figure 1 and Figure 2) simultaneously, thus in embodiment 2 component of kit only have reagent strip,
RFID card, I/II calibration object of PG.Wherein it is consistent in RFID card, I/II calibration object of PG and embodiment 1.
It should be understood that the detection method of the present invention in example 2 is after reagent strip is inserted into detection device, to use simultaneously
Corresponding RFID card is adapted to equipment, and equipment full automatic working step is consistent with embodiment 1；
It should be understood that the preparation method of the detection kit of the present invention in example 2 it is different from embodiment 1
Become in, step (5) " calibration object is dispensed into calibration object bottle, remaining each component is dispensed respectively to the corresponding aperture of reagent strip,
Sealer coding immediately after having dispensed ".
Claims (6)
1. a kind of pepsinogen Cgene/II bigeminy check reagent box, it is characterised in that be grouped as by following group: double antibody magnetic microballoon,
I/II calibration object of PG, double antibody marker, analysis buffer, cleaning solution, enhancement solution and RFID card.
2. pepsinogen Cgene described in claim 1/II bigeminy check reagent box, which is characterized in that the magnetic microsphere be by
Micron-sized Fe2O3Or Fe3O4Magnetic particle and high-molecular organic material progress are compound, and being formed has superparamagnetism, Neng Gouyu
The micron-sized microballoon that immunising antigen or antibody combine.
3. pepsinogen Cgene described in claim 1/II bigeminy check reagent box, which is characterized in that the diameter of magnetic microsphere is
0.1~5 μm, magnetic microsphere has various active functional group, including but not limited to hydroxyl, amino or carboxylic by surface modification
Base.
4. pepsinogen Cgene described in claim 1/II bigeminy check reagent box, which is characterized in that the double antibody magnetic is micro-
Ball is prepared using following steps:
By PG I, II antibody of PG respectively after 2~8 DEG C of superspeed refrigerated centrifuges are by buffer system replacement Treatment, with cleaning, activation
Magnetic microsphere mixing and constant-temperature incubation afterwards, clean magnetic bead and abandon supernatant, then closed with magnetic bead confining liquid after incubation,
Cleaning magnetic bead abandons supernatant again, and two kinds of high concentration antibody magnetic microspheres are saved 2~8 DEG C of refrigerators in liquid in magnetic bead and are uprightly protected
It deposits, then two kinds of magnetic beads is diluted to mix after working concentration, double antibody magnetic microballoon is made.
5. pepsinogen Cgene described in claim 1/II bigeminy check reagent box, which is characterized in that the double antibody marker
In antibody and lanthanide series be to be completed by intermediate chelating agent, lanthanide series be europium or samarium, chelating agent be isothiocyanic acid benzene
Base-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-DTTA or diethylene triamine pentaacetic acid aminophenyl-
EDTA。
6. pepsinogen Cgene described in claim 1/II bigeminy check reagent box preparation method, which is characterized in that including as follows
Step:
(1) double antibody magnetic microballoon is prepared；
(2) double antibody marker is prepared；
(3) I/II calibration object of PG is prepared；
(4) analysis buffer, concentration washing lotion and enhancement solution are prepared；
(5) each component is dispensed respectively into corresponding storage container；
(6) RFID card duplicate copy；
(7) coding, labelling；
(8) it is assembled into finished product kit.
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| CN111398591A (en) * | 2020-04-03 | 2020-07-10 | 北京清分稳同科技有限公司 | Pepsinogen I and pepsinogen II combined detection kit and application thereof |
| CN112147333A (en) * | 2020-08-31 | 2020-12-29 | 浙江博实生物科技有限公司 | Immunoassay kit based on pepsinogen PG II, and preparation method and detection method thereof |
| CN112147328A (en) * | 2020-08-31 | 2020-12-29 | 浙江博实生物科技有限公司 | Immunoassay kit based on pepsinogen PG I, and preparation method and detection method thereof |
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Application publication date: 20190329 |