CN106501532B - It is a kind of for detecting the kit of gastrin 17 - Google Patents
It is a kind of for detecting the kit of gastrin 17 Download PDFInfo
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- CN106501532B CN106501532B CN201610898634.2A CN201610898634A CN106501532B CN 106501532 B CN106501532 B CN 106501532B CN 201610898634 A CN201610898634 A CN 201610898634A CN 106501532 B CN106501532 B CN 106501532B
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/595—Gastrins; Cholecystokinins [CCK]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/062—Gastritis or peptic ulcer disease
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Abstract
The invention discloses a kind of for detecting the kit of gastrin 17.The present invention provides a kind of for detecting the kit of people's gastrin 17, including component 1 and component 2；The component 1 is the microwell plate for being coated with G-17-BSA；The G-17-BSA is polypeptide, and following two sections: G-17 polypeptide and bovine serum albumin(BSA) mature peptide are successively contained from N-terminal to C-terminal；The G-17 polypeptide are as follows: first amino acid residue end of N-terminal of the polypeptide shown in the sequence 1 of sequence table connects the polypeptide that a pyroglutamic acid obtains；The component 2 is enzyme conjugates solution；The anti-G-17 monoclonal antibody of mouse containing HRP label in the enzyme conjugates solution.Kit provided by the invention can be used for detecting the content of the gastrin 17 in human blood sample, so as to for assessing or predicting gastric mucosal state or situation, diagnose atrophic gastritis, the screening of gastric cancer people at highest risk, so as to for evaluating alimentary system physio-pathological condition.
Description
Technical field
The present invention relates to a kind of for detecting the kit of gastrin 17 (G-17), and in particular to one kind is based on chemiluminescence
The kit of method detection gastrin 17.
Background technique
Gastrin is a kind of important gastrointestinal hormone, is that Edkins JS et al. has found and names earliest.Then, Dockray
GJ et al. research discovery gastrin gene is positioned at the long-armed of No. 17 chromosomes, and overall length 4.1kb can encode 101 amino
Polypeptide-gastrin of sour residue composition is former.Gastrin original has biology living by a series of processing and modification
The mature polypeptide of property.The synthesis of gastrin experienced three former gastrin, glycine extended gastrin and mature gastrin ranks
Section, the intermediate product that first two gastrin is synthesized and processed as gastrin, is not amidated gastrin, and mature stomach is secreted
Element is amidated gastrin, and the amidation of c-terminus is that gastrin family member plays necessary to its bioactivity.Stomach is secreted
A variety of hypotypes, including gastrin 34, gastrin-17 1, gastrin 52, gastrin 14, gastrin 17 etc. are known as, in human body content
At most (about 90%) and effect most importantly gastrin 17.
Gastrin is secreted by the G cell (gastrin containing cell) for being present in stomachus pyloricus mucous membrane, into blood
In act on stomach, promote gastric secretion.G cell is mainly distributed on antrum portion, also has in duodenum and jejunum upper section mucous membrane
A small amount of G cell.Mechanical irritation of the pyloric antrum of stomach by food etc., when pH meta-alkalescence of pyloric part, just carry out point of gastrin
It secretes.The secretion of hydrochloric acid and gastrin when pH as a result, the secretion of gastric juice especially hydrochloric acid becomes vigorous, and inside stomach is reduced
With regard to stopping.In addition, gastrin also has the work for promoting pepsinia, pancreatic secretion, bile secretion, insulin secretion etc.
With.Gastrin in antrum is mainly G-17, has G-17 and G-34 about respectively to account for half in duodenal mucosa.It is imitated from biology
Should from the point of view of, G-17 stimulate stomachial secretion effect ratio G-34 it is 5-6 times strong, the speed that G-34 is removed in vivo very slowly (half-life period
About 50min), the half-life period of G-17 usually only 6min.G-17 is the polypeptide being made of 17 amino acid residues, there are two types of
(gastrin I and II): a kind of tyrosine residue has carried out sulfate substitution, and one kind not carrying out the substitution, and the two is living in physiology
It is not variant in property.To the primary structure of the G-17 of various mammals studies have shown that being 17 amino acid compositions, only
It is different because of animal in the amino acid residue from the 5th of amino terminal and the 10th.
Summary of the invention
The object of the present invention is to provide a kind of for detecting the kit of gastrin 17.
The kit (kit first) that the present invention provides a kind of for detecting people's gastrin 17, including component 1 and component
2；
The component 1 is the microwell plate for being coated with G-17-BSA；The G-17-BSA be polypeptide, from N-terminal to C-terminal according to
It is secondary to contain following two sections: G-17 polypeptide and bovine serum albumin(BSA) mature peptide；The G-17 polypeptide are as follows: in the sequence of sequence table
First amino acid residue end of N-terminal of polypeptide shown in 1 connects the polypeptide that a pyroglutamic acid obtains；
The component 2 is enzyme conjugates solution；The anti-G-17 Dan Ke of mouse containing HRP label in the enzyme conjugates solution
Grand antibody.
The C-terminal of G-17 polypeptide is concretely connect by the G-17-BSA with the N-terminal of bovine serum albumin(BSA) mature peptide
The polypeptide of formation.
Bovine serum albumin(BSA) mature peptide specifically can be as shown in the sequence 2 of sequence table.
Also contain casein, Proclin-300 and BSA in the enzyme conjugates solution.
The enzyme conjugates solution is made of solute and solvent；The solvent is the PBS buffer solution of pH7.4,0.02M；Institute
It states solute and its concentration in enzyme conjugates solution is as follows: the volume basis of the anti-G-17 monoclonal antibody of mouse of HRP label
Content is 0.02%, the volumn concentration of casein 0.1g/100ml, Proclin-300 are 0.02%, BSA2g/100ml.
The microwell plate for being coated with G-17-BSA the preparation method is as follows:
(1) G-17-BSA is diluted with the CB buffer of pH9.6,0.05mol/L, obtains the coating that protein concentration is 2 μ g/ml
Liquid；
(2) microwell plate is taken, every hole is added the coating buffer of 100 μ l steps (1) preparation, 2-8 DEG C of standing 18-24h, then reject
Liquid in hole；
(3) after completing step (2), the microwell plate is taken, every hole is added 200 μ l confining liquids, 2-8 DEG C of standing 18-24h, so
Liquid in reject hole afterwards；Confining liquid is made of solute and solvent；The solvent is the PBS buffer solution of pH7.4,0.02M；It is described molten
Matter and its concentration in confining liquid are as follows: sucrose 8g/100ml, BSA0.5g/100ml, casein 0.5g/100ml；
(4) after completing step (3), the microwell plate is taken, the dry 18-24h in 20-30 DEG C, the environment of humidity < 40%.
Further include in the kit component 3 and or component 4 and or component 5.
The component 3 is 20 × concentration washing lotion；20 × concentration washing lotion is made of solute and solvent；Solvent be pH7.4,
The PBS buffer solution of 0.2mol/L；Concentration in the solute and its 20 × concentration washing lotion is as follows: the volume basis of Tween 20
Content is 1%.
The component 4 is luminescent solution A and luminescent solution B；
Luminescent solution A is made of solute and solvent；The solvent is the Tris-HCl buffer of pH8.0,0.2mol/L；It is described
Solute and its concentration in luminescent solution A are as follows: luminol 3mg/ml, to iodophenol 0.25mg/ml, tetraphenylboron sodium 0.5mg/
ml；
Luminescent solution B is made of solute and solvent；The solvent is the Tri s-HCl buffer of pH8.0,0.2mol/L；Institute
It states solute and its concentration in luminescent solution B is as follows: urea peroxide 0.5mg/ml.
The component 5 is G-17 standard solution；G-17 standard solution is by S0Solution, S1Solution, S2Solution, S3Solution,
S4Solution and S5Solution composition；S0Solution is the Proclin-300 that BSA containing 1g/100ml and volumn concentration are 0.02%
The PBS buffer solution of pH7.4,0.02M；S1Solution, S2Solution, S3Solution, S4Solution and S5Solution is containing G-17,1g/100ml
The PBS buffer solution of pH7.4,0.02M of the Proclin-300 that BSA and volumn concentration are 0.02%；S1In solution, G-17
Concentration be 20pg/ml；S2In solution, the concentration of G-17 is 100pg/ml；S3In solution, the concentration of G-17 is 200pg/ml；S4
In solution, the concentration of G-17 is 500pg/ml；S5In solution, the concentration of G-17 is 1000pg/ml.
The kit further includes the carrier for recording following operating method:
(1) normal equation is made
1. taking the microwell plate for being coated with G-17-BSA, 50 μ lG-17 standard solutions and 100 μ l enzyme conjugates are added in every hole
Solution, oscillation mix, and then stand 60 minutes for 37 DEG C；
2. being washed 5 times after completing step 1. with cleaning solution, button is dry；The cleaning solution is will with distilled water or deionized water
20 × concentration washing lotion is diluted to the solution that 20 times of volumes obtain；
3. after completing step 2., 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, oscillation is mixed, and is then sent out in chemistry
Luminous value is detected on light immunity analysis instrument；
Data processing is carried out using log-logit linear fit mode, makes normal equation: ln [percentage Percentage bound/(1-
Percentage Percentage bound)]=Blog10Concentration+A；A, B is straight line parameter；S1Solution is to S5The corresponding luminous value of any solution in solution
Divided by S0The corresponding luminous value of solution is percentage Percentage bound；
(2) solution to be measured is detected
Solution to be measured is the lysate of testing liquid sample, the dilution of testing liquid sample or solid sample to be measured；
1. taking the microwell plate for being coated with G-17-BSA, 50 μ l solution to be measured and 100 μ l enzyme conjugates solution, vibration is added in every hole
Mixing is swung, then stands 60 minutes for 37 DEG C；
2. being washed 5 times after completing step 1. with cleaning solution, button is dry；
3. after completing step 2., after, 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, and oscillation mixes, then changing
It learns and detects luminous value on luminescence immunoassay instrument；
According to the normal equation that step (1) obtains, people's gastrin 17 concentration in solution to be measured is calculated.
The present invention also protects a kind of kit (kit second) for detecting people's gastrin 17, including the G-17-BSA
With the anti-G-17 monoclonal antibody of the mouse.
The present invention also protects a kind of auxiliary diagnosis person under test whether to suffer from the chronic gastritis that corpus atrophy occurs or stomach occurs
The kit (kit the third) of the chronic gastritis of sinus atrophy, kit first or kit second including any description above.
The kit third further includes the carrier for recording following judgment criteria: if the concentration of gastrin 17 is high in serum
In 120pg/ml, person under test is doubtful with the chronic gastritis that corpus atrophy occurs；If the concentration of gastrin 17 is lower than in serum
40pg/ml, person under test are doubtful with the chronic gastritis that antrum atrophy occurs；If the concentration of gastrin 17 is 50- in serum
100pg/ml, person under test are doubtful not with the chronic gastritis that corpus atrophy occurs and not with the chronic stomach that antrum atrophy occurs
It is scorching.
The present invention also protects a kind of enzyme conjugates solution, is made of solute and solvent；The solvent is pH7.4,0.02M's
PBS buffer solution；The solute and its concentration in enzyme conjugates solution are as follows: the anti-G-17 monoclonal of mouse of HRP label is anti-
The volumn concentration of body is 0.02%, the volumn concentration of casein 0.1g/100ml, Proclin-300 are 0.02%,
BSA2g/100ml。
The preparation method of the anti-G-17 monoclonal antibody of mouse of HRP label: the anti-G-17 monoclonal antibody of mouse is subjected to HRP
Label, is then adjusted to the initial volume of the anti-G-17 monoclonal antibody of mouse (that is, every anti-G-17 of 1ml mouse for volume
Monoclonal antibody obtains the anti-G-17 monoclonal antibody of mouse of 1ml HRP label).
The anti-G-17 monoclonal antibody of mouse is concretely: the article No. of Biorbyt company is the Gastrin- of orb302695
17antibody。
First amino acid residue end of N-terminal of people's gastrin 17 concretely polypeptide shown in the sequence of sequence table 1
Connect the polypeptide that a pyroglutamic acid obtains.
Kit provided by the invention can quantitative determine the concentration of the gastrin 17 in human serum sample, have high sensitive
Property, high specific, easy to operate feature, from the point of view of performance indexes and the result of clinical test, kit be suitable for face
Bed conventional detection.
Kit provided by the invention can be used for detecting the content of the gastrin 17 (gastrin-17) in human blood sample,
So as to the screening of atrophic gastritis people at highest risk be diagnosed, so as to be used for for assessing or predicting gastric mucosal state or situation
Evaluate alimentary system physio-pathological condition.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.BSA, that is, bovine serum albumin(BSA).
The anti-G-17 monoclonal antibody (Gastrin-17antibody) of mouse: Biorbyt company, article No. orb302695.
G-17: artificial synthesized G-17 polypeptide (people's gastrin 17), sequence is as follows:
Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe
(pyroglutamic acid-Gly-Pro-tryptophan-Leu-Glu-the-the third ammonia of Glu-Glu-Glu-glutamic acid
Acid-tyrosine-glycine-tryptophan-methionine-aspartate-phenylalanine).G-17 polypeptide are as follows: in the sequence of sequence table
First amino acid residue end of N-terminal of polypeptide shown in column 1 connects the polypeptide that a pyroglutamic acid obtains.
G-17-BSA: artificial synthesized G-17-BSA polypeptide.G-17-BSA polypeptide is by the N of the C-terminal of G-17 polypeptide and BSA
End connects the polypeptide to be formed.BSA is as shown in the sequence 2 of sequence table.
Propepsin (PG I): Fitzgerald company, article No. 30R-AP038.
Propepsin (PG II): Fitzgerald company, article No. 30R-AP039.
The preparation of embodiment 1, kit
One, the preparation of kit components
1, it is coated with the microwell plate of G-17-BSA.
Be coated with the microwell plate of G-17-BSA the preparation method is as follows:
(1) G-17-BSA is diluted with the CB buffer of pH9.6,0.05mol/L, obtains the coating that protein concentration is 2 μ g/ml
Liquid.
(2) microwell plate is taken, every hole is added the coating buffer of 100 μ l steps (1) preparation, 2-8 DEG C of standing 18-24h, then reject
Liquid in hole.
(3) after completing step (2), the microwell plate is taken, every hole is added 200 μ l confining liquids, 2-8 DEG C of standing 18-24h, so
Liquid in reject hole afterwards.
Confining liquid: the PBS buffer solution of solvent pH7.4,0.02M；Solute and its concentration in confining liquid are as follows: sucrose
8g/100ml, BSA0.5g/100ml, casein 0.5g/100ml.
(4) after completing step (3), the microwell plate is taken, the dry 18-24h in 20-30 DEG C, the environment of humidity < 40%.
(5) microwell plate and one that each aluminium foil bag is put into that one is completed step (4) is responsible for a task until it is completed drying prescription, carries out vacuum sealing packet
Dress.
2, enzyme conjugates solution: the PBS buffer solution of solvent pH7.4,0.02M；Solute and its in enzyme conjugates solution
Concentration it is as follows: HRP label the anti-G-17 monoclonal antibody 0.02% (volumn concentration) of mouse, casein 0.1g/
100ml, Proclin-3000.02% (volumn concentration), BSA2g/100ml.
The preparation method of the anti-G-17 monoclonal antibody of mouse of HRP label: by the anti-G-17 monoclonal antibody of mouse according to normal
Rule method carries out HRP label, and volume is then adjusted to the initial volume of the anti-G-17 monoclonal antibody of mouse (that is, every
The anti-G-17 monoclonal antibody of 1ml mouse obtains the anti-G-17 monoclonal antibody of mouse of 1ml HRP label).
3, G-17 standard solution (S0Solution-S5Solution).
S0Solution is pH7.4,0.02M of BSA containing 1g/100ml and 0.02% (volumn concentration) Proclin-300
PBS buffer solution.S1Solution is to S5Solution is containing G-17,1g/100ml BSA and 0.02% (volumn concentration) Proclin-
The PBS buffer solution of 300 pH7.4,0.02M.S1In solution, the concentration of G-17 is 20pg/ml.S2In solution, the concentration of G-17 is
100pg/ml。S3In solution, the concentration of G-17 is 200pg/ml.S4In solution, the concentration of G-17 is 500pg/ml.S5In solution,
The concentration of G-17 is 1000pg/ml.
4,20 × concentration washing lotion: the PBS buffer solution of solvent pH7.4,0.2mol/L；Solute and its 20 × concentration washing lotion
In concentration it is as follows: Tween 201% (volumn concentration).
5, the Tris-HCl buffer of luminescent solution A: solvent pH8.0,0.2mol/L；Solute and its in luminescent solution A
Concentration is as follows: luminol 3mg/ml, to iodophenol 0.25mg/ml, tetraphenylboron sodium 0.5mg/ml.
6, the Tris-HCl buffer of luminescent solution B: solvent pH8.0,0.2mol/L；Solute and its in luminescent solution B
Concentration is as follows: urea peroxide 0.5mg/ml.
Two, the preparation of kit
The component of kit is shown in Table 1.
Table 1
| It is coated with the microwell plate of G-17-BSA | Hole array: 12 × 8 |
| Enzyme conjugates solution | 12ml × 1 bottle |
| G-17 standard solution | S0Solution-S5Solution: each 1.0ml × 1 bottle |
| 20 × concentration washing lotion | 50ml × 1 bottle |
| Luminescent solution A | 6ml × 1 bottle |
| Luminescent solution B | 6ml × 1 bottle |
| Sealing plate film | It is several |
| Valve bag | 1 |
| Specification | 1 |
The preservation condition of kit: 2-8 DEG C.The validity period of kit: 12 months.
Three, the application method (i.e. the content of specification record) of kit
The preparation method of cleaning solution: 20 × concentration washing lotion is diluted to 20 times of volumes with distilled water or deionized water.
1, normal equation is made
(1) microwell plate for being coated with G-17-BSA is taken, 50 μ lG-17 standard solutions and 100 μ l enzyme conjugates are added in every hole
Solution, gently oscillation mixes, and with sealing plate film sealing plate, then stands 60 minutes for 37 DEG C.
(2) it after completing step (1), is sufficiently washed 5 times with cleaning solution, button is dry.
(3) after completing step (2), 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, and gently oscillation mixes, and are then existed
Luminous value is measured on chemical illumination immunity analysis instrument.
Data processing is carried out using log-logit linear fit mode.Equation are as follows: ln [percentage Percentage bound/(1- percentage
Percentage bound)]=Blog10(concentration)+A.A, B is straight line parameter.S1Solution is to S5The corresponding luminous value of any solution removes in solution
With S0The corresponding luminous value of solution is percentage Percentage bound.
2, solution to be measured is detected
Solution to be measured can be testing liquid sample (such as serum), the dilution of testing liquid sample, solid-like to be measured
This lysate etc..
When testing liquid sample is serum, it is suitable for fresh serum sample, is separated in 24 hours after venous blood collection.
(1) microwell plate for being coated with G-17-BSA is taken, 50 μ l solution to be measured and 100 μ l enzyme conjugates solution are added in every hole,
Gently oscillation mixes, and with sealing plate film sealing plate, then stands 60 minutes for 37 DEG C.
(2) it after completing step (1), is sufficiently washed 5 times with cleaning solution, button is dry.
(3) after completing step (2), 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, and gently oscillation mixes, and are then existed
Luminous value is measured on chemical illumination immunity analysis instrument.
The normal equation obtained according to step 1 calculates the G-17 concentration in solution to be measured.
Four, the working principle of kit
Solid phase is made with G-17-BSA coating microwell plate, solution and enzyme conjugates to be measured, the G-17 in solution to be measured is added
With the anti-G-17 monoclonal antibody competitive binding of mouse for G-17-BSA and the HRP label being coated on microwell plate, it is added and shines
Liquid measures its luminous value, the content of gastrin 17 in solution to be measured can be calculated according to calibration curve, luminous value is with solution to be measured
The increase of middle G-17 concentration and reduce.
Gastrin 17 content in the serum of kit detection different crowd prepared by embodiment 2, Application Example 1
208 normal persons form physical examination group, viscous to exclude gastritis, gastric ulcer, duodenal ulcer, gastrinoma, antrum
The healthy person of the stomach trouble such as film hyperplasia, and be the volunteer of informed consent.
The Patients with Chronic Gastritis of 143 generation corpus atrophies made a definite diagnosis through hospital forms corpus atrophy group, and is informed
The volunteer of agreement.
The Patients with Chronic Gastritis of 168 generation antrum atrophys made a definite diagnosis through hospital forms antrum atrophy group, and is informed
The volunteer of agreement.
Solution to be measured is serum.Using the kit of the preparation of embodiment 1 and by the application method of the kit in embodiment 1
It is operated.
The concentration of gastrin 17 is shown in Table 2 in the serum of each group crowd.Following auxiliary diagnosis standard can be established: if in serum
The concentration of gastrin 17 is higher than 120pg/ml, and person under test is doubtful with the chronic gastritis that corpus atrophy occurs；If stomach in serum
The concentration of secretin 17 is lower than 40pg/ml, and person under test is doubtful with the chronic gastritis that antrum atrophy occurs；If gastrin in serum
17 concentration is 50-100pg/ml, and person under test is doubtful not with the chronic gastritis that corpus atrophy occurs and not with generation antrum
The chronic gastritis of atrophy.
Table 2
| Number of cases | The concentration (pg/ml) of gastrin 17 in serum | |
| Physical examination group | 208 | 71.00±17.26 |
| Corpus atrophy group | 143 | 267.21±103.46 |
| Antrum atrophy group | 168 | 20.41±10.15 |
Embodiment 3, the performance of kit
Test agent box are as follows: the kit (batch one, batch two and batch three) of three batches prepared by embodiment 1.
One, minimum detection limit
(1) microwell plate for being coated with G-17-BSA is taken, 50 μ lS are added in every hole0Solution and 100 μ l enzyme conjugates solution, gently
Light oscillation mixes, and with sealing plate film sealing plate, then stands 60 minutes for 37 DEG C.Each test agent box, S020 multiple holes are arranged in solution.
(2) it after completing step (1), is sufficiently washed 5 times with cleaning solution, button is dry.
(3) after completing step (2), 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, and gently oscillation mixes, and are then existed
Luminous value is measured on chemical illumination immunity analysis instrument.
The luminous value in each hole is shown in Table 3.
Data processing is carried out using log-logit linear fit mode.Calculate 20 hole S0The corresponding luminous value of solution is put down
Mean value (Mean) and standard deviation (SD), it is lowest detection that concentration value when luminous value is Mean-2 × SD is calculated by fit equation
Limit.The minimum detection limit of the kit of three batches is in 15pg/ml or less.
Table 3
Two, analysis specificity
The substance for being easy to interfere the detection of G-17 in human blood mainly has propepsin (PG I) and stomach cardia
Proenzyme (PG II).
(1) microwell plate for being coated with G-17-BSA is taken, 50 μ l particular solutions and 100 μ l enzyme conjugates solution are added in every hole,
Gently oscillation mixes, and with sealing plate film sealing plate, then stands 60 minutes for 37 DEG C.
Particular solution is S0Solution, S1Solution, S2Solution, S3Solution, S4Solution, S5II solution of solution, I solution of PG or PG.
PG I: being dissolved in the PBS buffer solution of pH7.4,0.02M by the preparation method of I solution of PG, and it is molten to obtain the PG I that concentration is 200ng/ml
Liquid.PG II: being dissolved in the PBS buffer solution of pH7.4,0.02M by the preparation method of PG II, and it is molten to obtain the PG II that concentration is 50ng/ml
Liquid.2 multiple holes are arranged in each test agent box, every kind of particular solution.
(2) it after completing step (1), is sufficiently washed 5 times with cleaning solution, button is dry.
(3) after completing step (2), 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, then in chemiluminescence immunoassay
Luminous value is measured on analyzer.
The luminous value in each hole is shown in Table 4.
Table 4
Data processing is carried out using log-logit linear fit mode.I solution of PG is calculated according to standard curve or PG II is molten
The corresponding G-17 concentration of the corresponding luminous value of liquid, the cross reaction value of as PG I or PG II.It the results are shown in Table 5.The result shows that examination
The cross reaction value of agent box and PG I or PG II are below 15pg/ml, will not interfere to the detection of G-17.
5 cross reaction value (pg/ml) of table
| Batch one | Batch two | Batch three | |
| PGⅠ | 2.06 | 11.56 | 7.88 |
| PGⅡ | 5.76 | 11.91 | 7.64 |
Three, repeatability and difference between batch
Repeatability is the coefficient of variation (CV) obtained with same a collection of kit replication portion sample, and each quality-control product needs
It does the measurement of 10 hole accuracies at random in one block of plate, the mean concentration (Mean) and standard deviation (SD) of measurement result is calculated, in batch
The coefficient of variation (CV)=SD/Mean × 100%.
Difference between batch is the repeatability between different batches kit, randomly selects three batches of Reagent Kits, with these kits
Measurement quality-control product 3 times calculates the mean concentration (Mean) and standard deviation (SD) of measurement result, interassay coefficient of variation (CV)=SD/
Mean × 100%.
Quality-control product is G-17 polypeptide.
(1) microwell plate for being coated with G-17-BSA is taken, 50 μ l particular solutions and 100 μ l enzyme conjugates solution are added in every hole,
Gently oscillation mixes, and with sealing plate film sealing plate, then stands 60 minutes for 37 DEG C.
Particular solution is particular solution first or particular solution second.Particular solution first is S0Solution, S1Solution, S2Solution, S3It is molten
Liquid, S4Solution or S5Solution.2 multiple holes are arranged in each test agent box, every kind of particular solution first.Particular solution second is different dense
The quality-control product solution of degree.The preparation method of quality-control product solution: quality-control product is dissolved in the PBS buffer solution of pH7.4,0.02M, is obtained
The low concentration quality-control product solution (Qc1) of 100pg/ml and the high concentration quality-control product solution (Qc2) of 500pg/ml.Each test agent
10 multiple holes are arranged in box, every kind of particular solution second.
(2) it after completing step (1), is sufficiently washed 5 times with cleaning solution, button is dry.
(3) after completing step (2), 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, and gently oscillation mixes, and are then existed
Luminous value is measured on chemical illumination immunity analysis instrument.
When using particular solution first, the luminous value in each hole is shown in Table 6.
Table 6
When using particular solution second, the luminous value in each hole is shown in Table 7.
Table 7
Data processing is carried out using log-logit linear fit mode.It is calculated in particular solution second according to standard curve
G-17 concentration.It the results are shown in Table 8.Two Quality Controls of kit measurement high concentration and low concentration of three batches, variation within batch coefficient is all
Less than 10%, show that the homogeneity of kit is good, as a result there is repeatability.The two kinds of packing specification measurements of comprehensive three batches of kits
Two quality-control products of high concentration and low concentration as a result, interassay coefficient of variation shows between the kit of different batches less than 15%
Make a variation very little, and measurement result has repeatability.
Table 8
In conclusion the standard of the main performance index of kit provided by the invention is as follows:
Minimum detection limit: it is not higher than 15pg/ml；
Analysis specificity: PG I (200ng/ml), PG II (50ng/ml) cross reaction value are not more than 15pg/ml；
Repeatability: variation within batch coefficient is not higher than 10%；
Difference between batch: interassay coefficient of variation is not higher than 15.0%.
Claims (4)
1. a kind of for detecting the kit of people's gastrin 17, including component 1, component 2, component 3, component 4 and component 5；
The component 1 is the microwell plate for being coated with G-17-BSA；The G-17-BSA is polypeptide, is successively contained from N-terminal to C-terminal
There are following two sections: G-17 polypeptide and bovine serum albumin(BSA) mature peptide；The G-17 polypeptide are as follows: in 1 institute of sequence of sequence table
Show that first amino acid residue end of N-terminal of polypeptide connects the polypeptide that a pyroglutamic acid obtains；
The component 2 is enzyme conjugates solution；The enzyme conjugates solution is made of solute and solvent；The solvent be pH7.4,
The PBS buffer solution of 0.02M；The solute and its concentration in enzyme conjugates solution are as follows: the anti-G-17 of mouse of HRP label is mono-
The volumn concentration of clonal antibody is 0.02%, the volumn concentration of casein 0.1g/100ml, Proclin-300 are
0.02%, BSA2g/100ml；
The component 3 is 20 × concentration washing lotion；20 × concentration washing lotion is made of solute and solvent；Solvent is pH7.4,0.2mol/L
PBS buffer solution；Concentration in the solute and its 20 × concentration washing lotion is as follows: the volumn concentration of Tween 20 is
1%；
The component 4 is luminescent solution A and luminescent solution B；Luminescent solution A is made of solute and solvent；The solvent be pH8.0,
The Tris-HCl buffer of 0.2mol/L；The solute and its concentration in luminescent solution A are as follows: luminol 3mg/ml, to iodine
Phenol 0.25mg/ml, tetraphenylboron sodium 0.5mg/ml；Luminescent solution B is made of solute and solvent；The solvent be pH8.0,
The Tris-HCl buffer of 0.2mol/L；The solute and its concentration in luminescent solution B are as follows: urea peroxide 0.5mg/ml；
The component 5 is G-17 standard solution；G-17 standard solution is by S0Solution, S1Solution, S2Solution, S3Solution, S4It is molten
Liquid and S5Solution composition；S0Solution is the Proclin-300 that BSA containing 1g/100ml and volumn concentration are 0.02%
The PBS buffer solution of pH7.4,0.02M；S1Solution, S2Solution, S3Solution, S4Solution and S5Solution is containing G-17,1g/100ml
The PBS buffer solution of pH7.4,0.02M of the Proclin-300 that BSA and volumn concentration are 0.02%；S1In solution, G-17
Concentration be 20pg/ml；S2In solution, the concentration of G-17 is 100pg/ml；S3In solution, the concentration of G-17 is 200pg/ml；S4
In solution, the concentration of G-17 is 500pg/ml；S5In solution, the concentration of G-17 is 1000pg/ml；
The microwell plate for being coated with G-17-BSA the preparation method is as follows:
(1) G-17-BSA is diluted with the CB buffer of pH9.6,0.05mol/L, obtains the coating buffer that protein concentration is 2 μ g/ml；
(2) microwell plate is taken, the coating buffer of 100 μ l steps (1) preparation, 2-8 DEG C of standing 18-24h, then in reject hole is added in every hole
Liquid；
(3) after completing step (2), the microwell plate is taken, 200 μ l confining liquids are added in every hole, then 2-8 DEG C of standing 18-24h is abandoned
Except liquid in hole；Confining liquid is made of solute and solvent；The solvent is the PBS buffer solution of pH7.4,0.02M；The solute and
Its concentration in confining liquid is as follows: sucrose 8g/100ml, BSA0.5g/100ml, casein 0.5g/100ml；
(4) after completing step (3), the microwell plate is taken, the dry 18-24h in 20-30 DEG C, the environment of humidity < 40%.
2. kit as described in claim 1, it is characterised in that: the kit further includes recording following operating method
Carrier:
(1) normal equation is made
1. taking the microwell plate for being coated with G-17-BSA, 50 μ lG-17 standard solutions and 100 μ l enzyme conjugates solution are added in every hole,
Oscillation mixes, and then stands 60 minutes for 37 DEG C；
2. being washed 5 times after completing step 1. with cleaning solution, button is dry；The cleaning solution be with distilled water or deionized water by 20 ×
Concentration washing lotion is diluted to the solution that 20 times of volumes obtain；
3. after completing step 2., 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, oscillation mixes, then exempts from chemiluminescence
Luminous value is detected on epidemic disease analyzer；
Data processing is carried out using log-logit linear fit mode, makes normal equation: ln [percentage Percentage bound/(1- percentage
Percentage bound)]=Blog10Concentration+A；A, B is straight line parameter；S1Solution is to S5The corresponding luminous value of any solution is divided by S in solution0
The corresponding luminous value of solution is percentage Percentage bound；
(2) solution to be measured is detected
Solution to be measured is the lysate of testing liquid sample, the dilution of testing liquid sample or solid sample to be measured；
1. taking the microwell plate for being coated with G-17-BSA, 50 μ l solution to be measured and 100 μ l enzyme conjugates solution are added in every hole, and oscillation is mixed
It is even, then stand 60 minutes for 37 DEG C；
2. being washed 5 times after completing step 1. with the cleaning solution, button is dry；
3. after completing step 2., after, 50 μ l luminescent solution A and 50 μ l luminescent solution B are added in every hole, and oscillation mixes, and then send out in chemistry
Luminous value is detected on light immunity analysis instrument；
According to the normal equation that step (1) obtains, people's gastrin 17 concentration in solution to be measured is calculated.
3. whether a kind of auxiliary diagnosis person under test suffers from the chronic gastritis that corpus atrophy occurs or the chronic stomach of antrum atrophy occurs
Scorching kit, including kit of any of claims 1 or 2.
4. kit as claimed in claim 3, it is characterised in that: the kit further includes recording following judgment criteria
Carrier: if the concentration of gastrin 17 is higher than 120pg/ml in serum, person under test is doubtful with the chronic stomach that corpus atrophy occurs
It is scorching；If the concentration of gastrin 17 is lower than 40pg/ml in serum, person under test is doubtful with the chronic gastritis that antrum atrophy occurs；
If the concentration of gastrin 17 is 50-100pg/ml in serum, person under test is doubtful not with the chronic gastritis that corpus atrophy occurs
And not with the chronic gastritis that antrum atrophy occurs.
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| CN109307776A (en) * | 2018-11-16 | 2019-02-05 | 郑州安图生物工程股份有限公司 | A kind of gastrin 17 detection kit of improvement |
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| FI97304C (en) * | 1994-11-16 | 1996-11-25 | Locus Genex Oy | A method for screening for the risk of gastric cancer |
| CA2520010C (en) * | 2003-03-28 | 2012-07-10 | Aphton Corporation | Gastrin hormone immunoassays |
| CN1687751A (en) * | 2005-05-25 | 2005-10-26 | 段虎 | High stable enhanced chemiluminescence substrate |
| CN101750497A (en) * | 2008-12-17 | 2010-06-23 | 北京科美东雅生物技术有限公司 | Enzymatic chemical luminous immunoassay quantitative determination reagent kit for simultaneously detecting various tumor markers |
| CN104614536B (en) * | 2015-02-10 | 2017-06-27 | 深圳市新产业生物医学工程股份有限公司 | A kind of kit for detecting G17 and its preparation method and application |
| CN104914251A (en) * | 2015-05-22 | 2015-09-16 | 必欧瀚生物技术（合肥）有限公司 | Gastrin-17 enzymatic chemiluminescence immunoassay kit |
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Inventor after: Fu Jie Inventor after: Zhao Jingchao Inventor after: Li Huabai Inventor after: Gao Jiang Inventor after: Liu Weixue Inventor after: Lan Xin Inventor after: Zhou Fen Inventor before: Zhao Jingchao Inventor before: Li Huabai Inventor before: Gao Jiang Inventor before: Liu Weixue Inventor before: Lan Xin Inventor before: Zhou Fen |