CN109541228A - A kind of detection Pepsinogen II kit and preparation method thereof - Google Patents
A kind of detection Pepsinogen II kit and preparation method thereof Download PDFInfo
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- CN109541228A CN109541228A CN201811329567.8A CN201811329567A CN109541228A CN 109541228 A CN109541228 A CN 109541228A CN 201811329567 A CN201811329567 A CN 201811329567A CN 109541228 A CN109541228 A CN 109541228A
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Abstract
The invention discloses a kind of detection Pepsinogen II kits and preparation method thereof, are grouped as by following group: being coated with the magnetic microsphere, II calibration object of PG, II antibody marker of PG, analysis buffer, cleaning solution, enhancement solution and RFID card in conjunction with lanthanide series of II antibody of PG.This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, overcome the physical sorption reaction time of ELISA Plate longer, the slower drawback of testing result, the reaction time is greatly shortened, while also having that time resolution detection technique accuracy height, high sensitivity, high specificity, the range of linearity is wide, detection is stable and convenient advantage.Magnetic microsphere is coated with corresponding antigen or antibody, because of the characteristic of magnetic microsphere solid, greatly increase the contact surface area of immune response, to greatly shorten detection time, and the present invention advanced optimizes traditional two-step method detection, detection Pepsinogen II only needs one-step method to detect, and result can be detected in 20 minutes.
Description
Technical field
The present invention relates to a kind of detection Pepsinogen II kits and preparation method thereof, and in particular to one kind is based on magnetism
Microballoon combines the preparation method of detection Pepsinogen II kit 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, slow for diagnosing
Property atrophic gastritis, erosive gastritis, gastric ulcer, duodenal ulcer, atrophic gastritis and intestinal metaplasia have very high value.
Pepsinogen II Serology test has enzyme linked immunological (ELISA), colloidal gold method, chemiluminescence at present
(CLIA), electrochemical luminescence (ECL), time-resolved fluoroimmunoassay (TRFIA) etc..ELISA is as semi-quantitative detection method
It is now most widely used detection method, but its sensitivity, linear extent range are not up to higher level, it is difficult to adapt to market hair
The demand of exhibition.Colloidal gold method and ELISA have the shortcomings that identical.Though and CLIA and ECL high sensitivity but there is detection device
Cost is high, and corresponding marker research and development threshold is high, and the country can be under one's control in short-term, equally also limits at home
Popularization.And time-resolved fluoroimmunoassay (TRFIA) sensitivity can reach the consistent level of CLIA, not only detection device is made
Valence is lower, and labelling technique is quite mature at home, testing cost is low simultaneous.And traditional time resolution detection, still with sky
Based on the physical absorption of white ELISA Plate, time-consuming for detection process.Shorten the reaction time, reduce testing cost, it is sensitive to improve detection
Degree and the range of linearity, will be with good market prospects.
Summary of the invention
It is mentioned based on this it is an object of the invention to overcome the disadvantage that in the prior art and the relevant technologies testing cost is high
For a kind of detection Pepsinogen II kit and preparation method thereof.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of detection Pepsinogen II kit, is grouped as by following group: being coated with magnetic microsphere, II school PG of II antibody of PG
Quasi- product, the II antibody marker of PG in conjunction with lanthanide series, analysis buffer, concentration washing lotion (cleaning solution) and enhancement solution, RFID
Card.
Preferably, the magnetic microsphere for being coated with II antibody of PG is by micron-sized Fe2O3Or Fe3O4Magnetic particle
With high-molecular organic material carry out it is compound, formed have superparamagnetism, can be combined with immunising antigen or antibody it is micro-
The microballoon of meter level is commonly referred to as " magnetic bead ".It is 0.1~5 μm that magnetic microsphere, which should be able to meet diameter, and magnetic microsphere passes through surface modification
Various active functional group, including but not limited to hydroxyl (- OH), amino (- NH can be had2), carboxyl (- COOH).
Preferably, the magnetic microsphere for being coated with II antibody of PG is prepared using following steps:
By II antibody of PG after 2~8 DEG C of superspeed refrigerated centrifuges are by buffer system replacement Treatment, with the magnetism after cleaning, activation
Microballoon mixing and constant-temperature incubation clean magnetic bead and abandon supernatant, then closed with magnetic bead confining liquid, clean magnetic bead after incubation
Supernatant is abandoned again, and the magnetic microsphere for being coated with II antibody of PG is saved in liquid in magnetic bead and is uprightly saved in 2~8 DEG C of refrigerators.
It is 10 μ g that every milligram of magnetic microsphere of EDC and Sulfo-NHS, which is preferably loaded quality, in the activation of the magnetic microsphere
~1000 μ g.Activation method includes but is not limited to EDC, Sulfo-NHS one such and two kinds.
Preferably, the antibody in the II antibody marker of PG in conjunction with lanthanide series and lanthanide series are in
Between chelating agent complete, lanthanide series includes but is not limited to europium (EU), samarium (Sm), and chelating agent includes but is not limited to isothiocyanic acid benzene
Base-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-DTTA, diethylene triamine pentaacetic acid aminophenyl-
EDTA。
The present invention also provides a kind of methods using mentioned reagent box detection Pepsinogen II, and the method includes as follows
Step:
(1) marker is diluted to working solution with analysis buffer；
(2) working solution is diluted to by washing lotion is concentrated with purified water；
(3) magnetic microsphere of the working concentration diluted is added in reaction cup；
(4) sample to be examined or calibration object are added in above-mentioned reaction cup；
(5) the marker working solution diluted in (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 are one
The kind communication technology can be identified specific objective by radio signals and read and write related data, without identifying system and specific mesh
Mechanical or optical contact is established between mark.
The present invention also provides a kind of preparation methods for detecting Pepsinogen II kit, and the method includes walking as follows
It is rapid:
(1) preparation is coated with the magnetic microsphere of II antibody of PG；
(2) PG II antibody marker of the preparation in conjunction with lanthanide series；
(3) 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, when overcoming the physical adsorption reaction of ELISA Plate
Between it is longer, the slower drawback of testing result greatly shortens the reaction time, while also having time resolution detection technique accuracy
Height, high sensitivity, high specificity, the range of linearity are wide, detection is stable and convenient advantage.Magnetic microsphere be coated with corresponding antigen or
Antibody greatly increases the contact surface area of immune response because of the characteristic of magnetic microsphere solid, so that detection time is greatly shortened,
And the present invention advanced optimizes traditional two-step method detection, and detection Pepsinogen II only needs one-step method to detect,
Result can be detected in 20 minutes.In addition, being no longer limited by traditional ELISA Plate due to the fluid behaviour of magnetic microsphere buffer system
Frame limitation, detection can be completed in arbitrary reaction cup and small test tube, while the volume of detecting instrument equipment can be reduced
Size and equipment cost, also can be such as ELISA Plates, the reflective detection one of chemistry to meet the full-automatic detection demand in two, three line cities
Sample carries out big flux detection, meets the full-automatic detection demand in a line city.
Detailed description of the invention
Fig. 1 is to be used to store the magnetic microsphere and group of the lanthanides for being coated with II antibody of PG using detection kit of the present invention
The schematic top plan view of the reagent strip of II antibody marker of PG, analysis buffer, cleaning solution and enhancement solution that element combines.
Fig. 2 is to be used to store the magnetic microsphere and group of the lanthanides for being coated with II antibody of PG using detection kit of the present invention
The schematic perspective view of the reagent strip of II antibody marker of PG, analysis buffer, cleaning solution and enhancement solution that element combines.
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 the assay kit (chemiluminescence of Abbott Laboratories' Pepsinogen II
Particulate immunodetection).
Embodiment 1
A kind of detection Pepsinogen II kit, the kit include: the magnetic microsphere for being coated with II antibody of PG, II school PG
Quasi- product, the II antibody marker of PG 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 Pepsinogen II kit, and the method includes walking as follows
It is rapid:
(1) preparation is coated with the magnetic microsphere of II antibody of PG: by II antibody of PG 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
The II antibody magnetic microsphere of PG being coated with is saved in liquid in magnetic bead and is uprightly saved with 2~8 DEG C of refrigerators by supernatant.It again will coating
Good II antibody magnetic microsphere of PG saves liquid with magnetic bead and is diluted to working solution concentration, is distributed into 10mL/ bottles.Preferably, magnetic microsphere
It is one of 20:1,30:1 with the coated mass ratio of II antibody of PG；Preferably, the displacement buffer and magnetic bead cleaning solution
It is the MES buffer of 0.05~0.5M PH, 5.8~PH 7.0；Preferably, EDC and Sulfo-NHS in the activation of magnetic microsphere
It is 10 μ of μ g~1000 g that every milligram of magnetic microsphere, which is preferably loaded quality,；Preferably, the confining liquid of magnetic microsphere and preservation liquid are
The Tris-HCl buffer of 0.1~0.5M PH 7.0~8.5 containing 0.1%~8% BSA；
(2) prepare the II antibody marker of PG of europium label: it is 10000 ultra-filtration centrifuge tubes that 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 itself and DTTA-EU solvent with carbonate buffer solution in advance3+Mixing, II antibody of PG and europium mass ratio are 5:1,2~8 DEG C of oscillations
It mixes 24 ± 2 hours.The Sephadex that label solution is balanced through 7.8 Tris-Hcl buffer of 0.05M PHTMG-75 gel column
(1.0*50cm) chromatographic purifying monitors in A280 and collects first peak.The II antibody marker of PG of the europium being collected into label is used
7.8 Tris-Hcl buffer of 0.2%BSA, 0.05M PH is diluted to 1/20 times of optium concentration, dispenses to 1.0mL/ bottles.
(3) II calibration object of PG is prepared: will be from II antigen diluent of PG of stomach lining to two concentration points, low concentration point
0.5~10ng/mL, 80~100ng/mL of high concentration spot.
(4) it prepares analysis buffer: containing Tween-20, Proclin300, EDTA, BSA, Tris-HCl buffer, dividing
It is filled to 30~40mL/ bottles.
(5) preparation concentration washing lotion: containing Tween-20, Proclin300, Tris-Hcl buffer, dispense to 30~
40mL/ bottles.
(6) it prepares enhancement solution: containing sodium acetate, β-NTA, TOPO, glacial acetic acid, dehydrated alcohol, Triton X-100, dividing
It is filled to 30~40mL/ bottles.
(7) it prepares RFID card: blank RFID card is subjected to relative parameters setting by detection method in the present embodiment of the present invention；
(8) coding, labelling assemble kit.
The present invention also provides the detection methods of above-mentioned detection Pepsinogen II kit, and the method concrete operations are such as
Under:
(1) reagent prepares
Kit restores in being placed at room temperature for room temperature；
The II antibody marker of PG of europium label is diluted to working solution concentration using analysis buffer, is mixed stand-by；
Cleaning solution: purified water is added by 1:25 in concentration washing lotion and is diluted to work cleaning solution；
Upright light rolling is coated with the magnetic microsphere of II antibody of PG before experiment, mixes stand-by；
(2) experimental implementation
The magnetic microsphere for drawing 50 μ L mixing is added in reaction cup；
Sample to be tested or 100 μ L of calibration object are added into each reaction cup；
II antibody marker working solution of PG, the 100 μ L diluted is added into each reaction cup again；
Room temperature blending incubation 20 minutes；
After incubation, cleaned 4 times using cleaning solution；
The addition 100 μ L of enhancement solution into each reaction cup, room temperature blending incubation 3 minutes；Fluorescent collecting is completed in 30 minutes
And carry out data analysis.
In the present embodiment, actual laboratory operating procedures and relevant parameter are copied in matched RFID card in advance, real
It tests after operating process only needs to be ready to by reagent preparation process, RFID card is adapted to fully-automatic equipment can be completed from information
Read, be loaded onto the overall process of detection.
Embodiment 2
A kind of detection Pepsinogen II kit, essentially identical with detection kit described in embodiment 1, difference is:
(1) the detection Pepsinogen II kit component only includes: reagent strip, II calibration object of PG, RFID card.
(2) in the present embodiment, reagent strip is by being coated with the magnetic microsphere of II antibody of PG, europium mark in the detection kit
In II antibody marker of PG, analysis buffer, cleaning solution and the enhancement solution of note, packing to the corresponding hole of reagent strip after sealer
At.Wherein cleaning solution is that concentration washing lotion adds 25 times of purified water dilution to form in embodiment 1；Remaining each component and phase in embodiment 1
Together.
In the present embodiment, just as shown in Figure 1 and Figure 2, each hole bit function of reagent strip is described as follows in the detection kit:
Reagent strip is from left to right arranged successively, and title is followed successively by the 1st~13 hole.1st, 2 holes are instrument connection, the 3rd, 4 holes be
Fluorescent marker hole, the 5th hole be analysis buffer hole, the 6th, 7 holes be cleaning fluid apertures, the 8th, 9 be Sample Dilution fluid apertures, the 12nd for increase
Strong fluid apertures, the 10th, 11,13 be preparation hole.1st and 2 holes are the reacting hole storing magnetic microsphere and being immunoreacted, and maximum can
Storage liquid volume is 800 μ L；3rd, 4 holes can be disassembled into from entire reagent strip and be independent component, convenient for fluorescence mark
Note object carries out packing storage.3rd, 4,5 holes can store maximum liquid volume be 400 μ L；6th, 7 holes can store maximum liquid
Volume is 3000 μ L；It is 400 μ L that 8th ~ 12 hole, which can store maximum liquid volume,；13rd hole can store maximum liquid volume
600μL。
In the present embodiment, the reagent strip in the detection kit is made after carrying out sealer as follows:
By 300 μ L be coated with II antibody magnetic microsphere of PG, 50 μ L europiums label II antibody marker of PG, 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 examination is made after sealer
Agent item.
The present invention also provides the preparation methods of above-mentioned detection Pepsinogen II kit, and reagent strip is obtained in the above describe manner
Afterwards, kit is constituted with II calibration object of PG, RFID card.Other than each component dispenses mode and storage container is different,
It is remaining in the same manner as in Example 1.
The present invention also provides the detection methods of above-mentioned detection Pepsinogen II kit, it is only necessary to which mentioned reagent item is light
It shakes and is inserted into after mixing in the reagent clamp bar slot of SmartTRF serial equipment, equipment reads RFID card relevant information can be full-automatic complete
At 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 Pepsinogen II kit of the present invention:
By the kit and detection method prepared in embodiment, stomach egg is detected with from Abbott Laboratories, hospital Architect i2000
The clinical sample of white proenzyme II 240.1,2 the 95th percentiles of embodiment are 22.5ng/mL.
Embodiment 1, embodiment 2 detect 240 clinical samples of Abbott Laboratories' Pepsinogen II, as a result as follows:
1 embodiment 1 of table, the comparison of 2 clinical samples
Detection kit described in the embodiment of the present invention 1, embodiment 2 detects Pepsinogen II and contrast agents negative match-rate
100%, positive coincidence rate 100%.
In conjunction with pepsinogen Cgene detection, (I I/PG of critical value 63.8ng/mL, PG of PG, II ratio critical value is 3.0) to tie
Fruit, I/PG of PG, II ratio and Abbott Laboratories' comparing result are as follows:
2 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 in embodiment 1 detect Pepsinogen II kit detection method and preparation method be for
Satisfaction big flux testing goal and invent, instrument and equipment bulky and cost is relatively high, in order to further satisfaction two,
The detection demand in three line cities, correspondingly, we have further made some detection methods and reagent on the basis of embodiment 1
Modification on 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 magnetic microsphere for being coated with II antibody of PG in embodiment 1, the II antibody mark of PG in conjunction with lanthanide series
Note object, analysis buffer, cleaning solution and enhancement solution are dispensed simultaneously into special reagent strip (as shown in Figure 1 and Figure 2), therefore are implemented
The component of kit only has reagent strip, RFID card, II calibration object of PG in example 2.Wherein RFID card, II calibration object of PG and embodiment 1
In be consistent.
It should be understood that the detection method of the present invention in example 2 is after reagent strip is inserted into detection device, to use simultaneously
Corresponding RFID card is adapted to equipment, and equipment full automatic working step is consistent with embodiment 1；
It should be understood that the preparation method of the detection kit of the present invention in example 2 is with the difference in embodiment 1,
Step (5) becomes " dispensing calibration object into calibration object bottle, remaining each component is dispensed respectively to the corresponding aperture of reagent strip, is divided
Sealer coding immediately after installing ".
Claims (6)
1. a kind of detection Pepsinogen II kit, which is characterized in that be grouped as by following group: being coated with the magnetic of II antibody of PG
Property microballoon, II calibration object of PG, the II antibody marker of PG in conjunction with lanthanide series, analysis buffer, cleaning solution, enhancement solution and
RFID card.
2. detection Pepsinogen II kit as described in claim 1, which is characterized in that described is coated with II antibody of PG
Magnetic microsphere be by micron-sized Fe2O3Or Fe3O4Magnetic particle and high-molecular organic material progress are compound, are formed with super
Micron-sized microballoon that is paramagnetic, can combining with immunising antigen or antibody, that is, be commonly referred to as " magnetic bead ".
3. detection Pepsinogen II kit as claimed in claim 2, which is characterized in that the diameter of the 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. detection Pepsinogen II kit as described in claim 1, which is characterized in that described is coated with II antibody of PG
Magnetic microsphere be prepared using following steps:
By II antibody of PG after 2~8 DEG C of superspeed refrigerated centrifuges are by buffer system replacement Treatment, with the magnetism after cleaning, activation
Microballoon mixing and constant-temperature incubation clean magnetic bead and abandon supernatant, then closed with magnetic bead confining liquid, clean magnetic bead after incubation
Supernatant is abandoned again, and the magnetic microsphere for being coated with II antibody of PG is saved in liquid in magnetic bead and is uprightly saved in 2~8 DEG C of refrigerators.
5. detection Pepsinogen II kit as described in claim 1, which is characterized in that described in conjunction with lanthanide series
II antibody marker of PG in antibody and lanthanide series be to be completed by intermediate chelating agent, lanthanide series be europium or samarium, chelating
Agent is isothiocyanic acid phenyl-EDTA, five vinegar of isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-DTTA or diethylenetriamine
Sour aminophenyl-EDTA.
6. detecting the preparation method of Pepsinogen II kit described in claim 1, it is characterised in that include the following steps:
(1) preparation is coated with the magnetic microsphere of II antibody of PG；
(2) PG II antibody marker of the preparation in conjunction with lanthanide series；
(3) 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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| 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 |
| 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 |
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