CN108279306A - A kind of PGI detection kits, method of preparation and use based on protein fragments complementary technology - Google Patents
A kind of PGI detection kits, method of preparation and use based on protein fragments complementary technology Download PDFInfo
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- CN108279306A CN108279306A CN201711271968.8A CN201711271968A CN108279306A CN 108279306 A CN108279306 A CN 108279306A CN 201711271968 A CN201711271968 A CN 201711271968A CN 108279306 A CN108279306 A CN 108279306A
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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Abstract
The present invention provides a kind of PGI diagnostic kits based on protein fragments complementary technology.The kit includes：The reaction substrate of the zymoprotein N-terminal segment of anti-PGI antibody couplings, the zymoprotein C-terminal segment and enzyme of anti-PGI antibody couplings；The invention also discloses a kind of preparation method of the PGI diagnostic kits based on protein fragments complementary technology, this method includes：The preparation of the zymoprotein N-terminal segment of anti-PGI antibody couplings, the preparation of the zymoprotein C-terminal segment of anti-PGI antibody couplings；Finally also disclose the application method of the kit；Kit of the present invention has many advantages, such as that easy to operate, specificity is good, free of cleaning, accuracy is high, convenient for clinical detection use, can early warning gastric cancer occur, to prevent gastric cancer have considerable meaning, have great market value.
Description
Technical field
A kind of content the present invention relates to protein fragments complementary technology for PGI in ion vitro immunization diagnosis detection human body, belongs to
In medical diagnosis on disease detection field.
Background technology
Gastric cancer is the fourth-largest common cancer in the whole world, is the second largest cancer cause of the death, is only second to lung cancer.The incidence gastric cancer of China
Rate has been more than Japan, and in rising trend.If gastric cancer can early diagnose, survival rate is generally more than 90% within 5 years, if when diagnosis
It it is late period, survival rate can be solely 10%-20% within 5 years.One retrospective study proves that 90% or more patients with gastric cancer is associated with
Atrophic gastritis.It generally believes the precancerous lesion that atrophic gastritis is critically important, is played in the pathogenesis of cancer heavy to closing
The effect wanted.
Propepsin is that pepsin does not digest active precursor substance in gastric juice, is divided into pepsinogen I (PGI)
With two classes of pepsinogen I I (PGII), most propepsin enters gastral cavity, and only minute quantity (about 1%) penetrates capillary
Blood vessel enters blood, and the concentration in serum reflects its secretion level.Wherein PGI is mainly thin by the chief cell of gastric gland and mucus neck
Intracrine, PGI are the pointers for detecting oxyntic gland cell function, and gastric acid secretion increases PGI raisings, and secretion reduces or gastric mucosa gland
Body atrophy PGI is reduced.Atrophic gastritis is the main precancerous lesion of gastric cancer, when atrophic gastritis occurs, body of gland and chief cell
Quantity is reduced, and pepsinogen I secretion is caused to decline.Due in superficial gastritis-gastric mucosal erosion ulcer-atrophic gastritis-
In the evolution of gastric cancer, along with the variation of propepsin, propepsin not only becomes the good of these three lesions
Diagnosis analysis index, and be to treat and prevent the monitoring index most wanted in intervention.
PGI detection kits are used to detect the content of the pepsinogen I in serum or blood plasma, have easy, quick
Advantage, avoid X-ray to the infringement of human body and the inconvenience of gastroscope.The measurement of conventional PGI is mainly using immune suppression
Preparation method and enzyme linked immunosorbent assay, immunosuppression assay for detecting is rapid, time saving, there is higher sensibility, but influence factor and
Defect is numerous.Enzyme linked immunosorbent assay has easy to operate, the feature of stable reagent phase length, but detection sensitivity is relatively low.For solution
The certainly above problem, using protein fragments complementary technology, with the antibody construction technology platform of PGI, develop it is a kind of it is easy, intuitive,
Sensitive PGI quick detection reagents.It is applied to the detection of detection oxyntic gland cell function, atrophic gastritis can be improved and examine
Disconnected accuracy rate has great market value.
Invention content
For the technical problems in the prior art, the present invention provides a kind of detection examination can be used for quantitative detection PGI
Agent box, its method of preparation and use.
The invention is realized by the following technical scheme：
A method of it prepares based on protein fragments complementary technology PGI detection kits, includes the following steps：
1) anti-PGI antibody couplings zymoprotein N-terminal segment；
2) anti-PGI antibody couplings zymoprotein C-terminal segment；
In the above-mentioned technical solutions, the anti-PGI antibody is the monoclonal antibody or Anti-TNF-α for PGI different epitopes
Body.
In the above scheme, in the step of anti-PGI antibody couplings zymoprotein N-terminal segment, zymoprotein N-terminal segment with it is anti-
The mass ratio of PGI antibody is 1: 1-10.
In the above scheme, in the step of anti-PGI antibody couplings zymoprotein C-terminal segment, zymoprotein C-terminal segment with it is anti-
The mass ratio of PGI antibody is 1: 1-10.
According to the PGI detection kits based on protein fragments complementary technology prepared described in any of the above technical solution.
It is mainly formed：
1) the zymoprotein N-terminal segment of anti-PGI antibody couplings；
2) the zymoprotein C-terminal segment of anti-PGI antibody couplings.
The application method of kit described in any of the above technical solution, it is characterised in that：Include the following steps：
1) sample, the zymoprotein N-terminal segment of anti-PGI antibody couplings and anti-PGI antibody are added in the reacting hole of kit
The zymoprotein C-terminal segment of coupling, hybrid reaction 5-10min are added zymolyte and react 1-5min；
2) light absorption value of each reacting hole is measured.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Description of the drawings
Fig. 1 is the PGI detection kit principle schematics provided in an embodiment of the present invention based on protein fragments complementary technology,
Wherein, the anti-PGI antibody of 1-, 2- bridging agents, 3- zymoprotein N-terminal segments, 4- determinands (PGI), the anti-PGI antibody of 5-, 6- zymoproteins
C-terminal segment, 7- bridging agents.
Fig. 2 is that the PGI detection kits provided in an embodiment of the present invention based on protein fragments complementary technology detect linear model
Enclose figure.
Fig. 3 is the PGI detection kit results relevances provided in an embodiment of the present invention based on protein fragments complementary technology
Compare.
Specific implementation mode
Below with reference to attached drawing to the PGI detection kits based on protein fragments complementary technology of the present invention, prepare and its make
It is described in detail with method.
Embodiment 1
Anti- PGI antibody couplings zymoprotein N-terminal segment, with the 26-196 amino acid of beta-lactamase (β-lactamase)
For segment β-lactamaseN, specific implementation process is：
1) 0.1mg β-lactamaseN albumen is added in centrifuge tube, with 0.05mol/L pH9.5 carbonate buffer solutions
(CB) it prepares and β-lactamaseN albumen is diluted to 1mg/mL.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) desalting column Sephadex G-25 or 0.05mol/L pH9.5 CB dialysis is used to remove excessive glutaraldehyde.
5) the anti-PGI antibody of 0.1mg is taken, 1mg/mL antibody is prepared with 0.05mol/L pH9.5 CB, by the β-of activation
LactamaseN albumen and antibody mixing.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ L 0.2mol/L lysine solutions are added, room temperature closes 2h, to close remaining aldehyde radical, terminates anti-
It answers.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by Sephadex G-200 gel columns or with 0.45 μm of filter membrane, used
0.01 mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 2
Anti- PGI antibody couplings zymoprotein C-terminal segment, with the 198-290 amino acid of beta-lactamase (β-lactamase)
For segment β-lactamaseC, specific implementation process is：
1) 0.1mg β-lactamaseC albumen is added in centrifuge tube, with 0.05mol/L pH9.5 carbonate buffers
Liquid (CB) is prepared is diluted to 1mg/mL by β-lactamaseC albumen.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) desalting column Sephadex G-25 or 0.05mol/L pH9.5 CB dialysis is used to remove excessive glutaraldehyde.
5) the anti-PGI antibody of 0.1mg is taken, 1mg/mL antibody is prepared with 0.05mol/L pH9.5CB, by the β-of activation
LactamaseC albumen and antibody mixing.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ L 0.2mol/L lysine solutions are added, room temperature closes 2h, to close remaining aldehyde radical, terminates anti-
It answers.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by Sephadex G-200 gel columns or with 0.45 μm of filter membrane, used
0.01 mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 3
Kit mainly forms：
1) the zymoprotein N-terminal segment of anti-PGI antibody couplings；
2) the zymoprotein C-terminal segment of anti-PGI antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) it is even that sample, the zymoprotein N-terminal segment of anti-PGI antibody couplings, anti-PGI antibody are added in the reacting hole of kit
The zymoprotein C-terminal segment of connection, hybrid reaction 5-10min are added zymolyte and react 1-5min；
2) each reacting hole light absorption value is measured.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Embodiment 5
Kit method evaluation of the present invention：
1. linear
Compound concentration is 0ng/mL, 1ng/mL, 5ng/mL, 25ng/mL, 100ng/mL, 200ng/mL, 400ng/mL's
PGI standard solutions.The zymoprotein N-terminal piece for being separately added into 10 μ L standard items in reacting hole, the anti-PGI antibody couplings of 50 μ L being added
The zymoprotein C-terminal segment of the anti-PGI antibody couplings of 50 μ L, 37 DEG C of incubation 5min are added in section.Enzyme chromogenic substrate Nitrocefin is added
After reacting 1-5min, each reacting hole light absorption value is measured under 492nm.
Using light absorption value as ordinate, standard concentration is abscissa, draws standard working curve (see attached drawing 2).
2. accuracy
Recovery test：It is added in the serum specimen of normal person with known quantity PGI standard items, measures concentration value after being added
It is compared with the theoretical value of addition, calculates the rate of recovery of PGI.Testing result is as follows：
| Sample number | PGI concentration (ng/mL) is added | Measure the concentration (ng/mL) of PGI | The rate of recovery (%) |
| 1 | 5 | 4.9 | 98.0 |
| 2 | 20 | 19.3 | 96.5 |
| 3 | 70 | 68.5 | 97.8 |
| 4 | 150 | 154.5 | 103.0 |
3. precision
Choose the sample of 3 parts of various concentrations, respectively duplicate measurements 20 times according to the method described in the present invention.According to 20 times
Measurement result calculates average deviation CV values.
4. sensitivity for analysis
The definition of sensitivity for analysis is：It refer to the amount that can be distinguished statistically with zero-dose.It is repeated 20 times measurement
Zero-dose point, calculates its average value (X) and standard deviation (SD), and the concentration value with the calculating of X+2SD is the analysis of the kit
Sensitivity.The sensitivity for analysis of kit of the present invention is 0.5ng/mL.
5. anti-interference
The immunological assay reagents based on protein fragments complementary technology of the present invention are detected in interference substance (haemolysis, high blood
Fat, high bilirubin) in the presence of detect sample accuracy.Hemoglobin solutions are taken respectively and are added to PGI sun in right amount
In property serum specimen, it is respectively 0.5mg/mL, 1.0mg/mL to make the content of hemoglobin in serum.Triglycerides solution is distinguished
It takes and is added in PGI positive serum samples in right amount, it is respectively 0.5mg/mL, 1.0mg/mL to make the content of Triglycerides in Serum.
Bilirubin solution is taken respectively and is added in PGI positive serum samples in right amount, it is respectively 25 μ g/ to make the content of serum mesobilirubin
mL、 50μg/mL.PGI positive samples to adding hemoglobin, triglycerides and bilirubin are measured.By theoretical concentration
Ratio with measured concentration is as the rate of recovery, and the rate of recovery is between 97.3%-102.2%.Show to be based on protein fragments complementation skill
The PGI reagents of art are not interfered when detecting serum sample by hemoglobin, triglycerides, bilirubin.
6. correlation
As shown in figure 3, being with the correlation of Wuxi river original industry PGI kits：Y=1.034x-0.397, R2=
0.998。
The present invention is compared with existing method and product, and with detection sensitivity height, specificity is good, cost is relatively low, to detection
The low advantage of instrument requirements.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art within the technical scope disclosed by the invention, the change or replacement that can be readily occurred in, all
It is covered by the protection scope of the present invention.
Claims (7)
1. a kind of PGI detection kits, method of preparation and use based on protein fragments complementary technology, it is characterised in that：
1) kit mainly forms：The zymoprotein N-terminal segment of anti-PGI antibody couplings, the zymoprotein C-terminal piece of anti-PGI antibody couplings
The reaction substrate of section and enzyme；
2) application method：The zymoprotein N-terminal segment and PGI bodies of sample, anti-PGI antibody couplings are added in the reacting hole of kit
The zymoprotein C-terminal segment of antibody coupling, hybrid reaction 5-10min are added zymolyte and react 1-5min；
3) detection method：Measure the light absorption value of each reacting hole.
2. a kind of preparation method of the PGI detection kits based on protein fragments complementary technology, which is characterized in that including walking as follows
Suddenly：
1) preparation of anti-PGI antibody couplings zymoprotein N-terminal segment；
2) preparation of anti-PGI antibody couplings zymoprotein C-terminal segment.
3. anti-PGI antibody according to claim 1 is the monoclonal antibody or polyclonal antibody for PGI different epitopes.
4. zymoprotein according to claim 1, including but not limited to beta galactosidase, dihyrofolate reductase, β-are interior
Amidase, firefly luciferase, Renilla luciferase, beetle luciferase, long ascites flea luciferase.
5. method prepared by a kind of PGI detection kits based on protein fragments complementary technology according to claim 2,
It is characterized in that, in the anti-PGI antibody couplings zymoprotein N-terminal segment step, the quality of zymoprotein N-terminal segment and PGI antibody
Than being 1: 1-10.
6. method prepared by the PGI detection kits according to claim 2 based on protein fragments complementary technology, feature
It is, in the anti-PGI antibody couplings zymoprotein C-terminal segment step, the mass ratio of zymoprotein C-terminal segment and PGI antibody is 1
∶1-10。
7. reacting hole according to claim 1, including but not limited to microwell plate, micro-fluidic reagent disc, reaction cup, reaction
Pipe.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711271968.8A CN108279306A (en) | 2017-11-27 | 2017-11-27 | A kind of PGI detection kits, method of preparation and use based on protein fragments complementary technology |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711271968.8A CN108279306A (en) | 2017-11-27 | 2017-11-27 | A kind of PGI detection kits, method of preparation and use based on protein fragments complementary technology |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202854148U (en) * | 2012-05-16 | 2013-04-03 | 江苏省原子医学研究所 | Pepsinogen I time-resolved fluoresence immunoassay kit |
-
2017
- 2017-11-27 CN CN201711271968.8A patent/CN108279306A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202854148U (en) * | 2012-05-16 | 2013-04-03 | 江苏省原子医学研究所 | Pepsinogen I time-resolved fluoresence immunoassay kit |
Non-Patent Citations (2)
| Title |
|---|
| CLIFF I. STAINS ET AL.: "A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins utilizing Split-Luciferase Reassembly", 《ACS CHEMICAL BIOLOGY》 * |
| 黄欣媛等: "蛋白片段互补分析技术研究进展", 《中国生物工程杂志》 * |
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Application publication date: 20180713 |