CN104597251A - Chemiluminescent quantitative detection kit of pepsinogen I and preparation method thereof - Google Patents

Chemiluminescent quantitative detection kit of pepsinogen I and preparation method thereof Download PDF

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Publication number
CN104597251A
CN104597251A CN201510034183.3A CN201510034183A CN104597251A CN 104597251 A CN104597251 A CN 104597251A CN 201510034183 A CN201510034183 A CN 201510034183A CN 104597251 A CN104597251 A CN 104597251A
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pepsinogen
antibody
preparation
cgene
pepsinogen cgene
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赵焕朝
俞宁
胡锰鸽
翟秋菊
张喜娟
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HENAN MAINCARE BIOLOGICAL TECHNOLOGY Co Ltd
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HENAN MAINCARE BIOLOGICAL TECHNOLOGY Co Ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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Abstract

The invention relates to the field of immunodetection and particularly relates to a chemiluminescent quantitative detection kit of pepsinogen I and a preparation method thereof. The chemiluminescent quantitative detection kit of pepsinogen I comprises 1) a pepsinogen I antibody coated microplate; 2) an HRP labeled pepsinogen I antibody; 3) a series of pepsinogen I calibrating materials diluted by a standard diluting liquid; 4) a luminous substrate A; 5) a luminous substrate B; and 6) a solid lotion. The kit provided by the invention can be used for detecting the content of pepsinogen I in blood serum, wherein the level of pepsinogen I in blood serum is positively associated with atrophic gastritis and peptic ulcer; in treatment of helicobacter pylori, the curative effect can be judged by monitoring change of pepsinogen after sterilization. The kit has the advantages of being noninvasive, simple and rapid in detection method, high in sensitivity, wide in detection range and the like.

Description

A kind of pepsinogen Cgene chemical luminescent analysis reagent kid and preparation method thereof
Technical field
The present invention relates to field of immunodetection, be specifically related to a kind of pepsinogen Cgene chemical luminescent analysis reagent kid and preparation method thereof.
Background technology
Propepsin (pepsinogen, PG) is a kind of digestive ferment precursor of stomachial secretion, is pepsic inactive precursor in gastric juice, it is a polypeptide chain be made up of 375 amino acid, average molecular mass is 42,000, has 7 groups of stomach cardia isozymogens in people's gastric mucosa.PG synthesizes on ribosomes, secretes cell by golgiosome, is become pepsin by after salt acid active.Can be divided into PG I, PG II two subgroups according to distribution in biochemical property, immunogenicity, cell derived and tissue, wherein 1 ~ 5 component immunogenicity is similar to, and be called PG I, the mucous neck cells primarily of the chief cell of gastric gland is secreted.
Pepsinogen Cgene is become micromolecular pepsin by after salt acid active, pepsin only plays a role in sour environment, namely pH>6 loses activity, and acts preferentially on native protein during pH=2, can digest some specific peptide during pH=4.In vitro, pepsic continuous action can make the peptide bond of protein molecular about have 30% division.Pepsin does not act on keratin and glutinous albumen, does not also act on the protein derivatives of low relative molecular mass.In B B-complex and protein bound with discharge, pepsin also plays a major role.
Serum PG concentration can be used as the Testing index of gastric acid secretion, in Helicobacter pylori body, PG I level and PG I/PG II ratio and gastric acid output significant correlation, wherein the latter and gastric acid output correlativity stronger.In Helicobacter pylori-Negative body, the level of PG I and gastric acid output significant correlation, but PG I/PG II ratio and gastric acid output non-correlation.By serum PG concentration sealing gastric acid output, there is value for clinical application.
PG content is relevant with discriminating that is good, malignant gastric ulcer, serum PG I level and atrophic gastritis and peptic ulcer are proportionate, serum PG I level rising person, should notice whether it exists the possibility of ulcer, and the discriminating for asymptomatic ulcer clinically has meaning.Serum PG I is relevant to oxyntic gland cell function, and PG II is comparatively large with the correlativity of gastric mucosa pathology, and serum PG reflection stomach totally secretes PG level.The superficial gastritis that gastroxia causes and the atrophic gastritis that helicobacter pylori infections causes all can cause the secretion of PG I and PG II to increase; Chronic severe atrophy gastritis PG I content when chief cell reduces declines; Atrophic gastritis is when with intestines, stomach Dou Xian Pseudopyloric gland metaplasia, and PG II content can increase thereupon.
Propepsin, to the development course of disease of stomach, generally can be expressed as: superficial gastritis → gastric mucosal erosion ulcer → atrophic gastritis, and Other diseases has good diagnosis and screening effect.Pepsinogen Cgene/II detection kit, for detecting the content of pepsinogen Cgene/II in serum, has easy, advantage fast, avoids the infringement of X-ray to human body and the inconvenience of gastroscope.
20 century 70 Arakawe in mid-term reported first luminous signals carry out analysis and detect, namely luminous chemical reaction (chemiluminescence is utilized, and biological respinse (bioluminescence CL), BL) ultramicron material is analyzed, especially for checking ultramicron active substance in clinical immunoassays.This method is transitioned into clinical medical conventional sense means from the rare technology in laboratory, obtains very large development nearly ten years.Chemiluminescence immune assay (chemiluminescence immunoassay, CLIA) highly sensitive (10 ~ 18mol), fast (interior generation in a few second chemiluminescence signal, sustainable several hours of signal in some situations), easy, test in do not use harmful reagent, therefore become one of the most promising method in on-radiation immunoassay.
The chemiluminescence immune analysis method that my company adopts, is utilize enzymatic luminous substrate to make it that chemical reaction occur and discharge a large amount of energy, produces excited state intermediate.This excited state intermediate, when it gets back to stable ground state, photon can be launched simultaneously, luminous signal surveying instrument is utilized to measure quantum yield of luminscence, the amount of the test substance in this quantum yield of luminscence and sample is proportional, thus can Criterion curve the content of test substance in calculation sample.With several traditional immunological detection method contrastingly, no matter from the stability of lowest detectable limit, detection time, the range of linearity, reagent, have the aspect contrasts such as non-environmental-pollution, the advantage of chemoluminescence method all seems very outstanding.
Summary of the invention
The object of this invention is to provide a kind of pepsinogen Cgene chemical luminescent analysis reagent kid and preparation method thereof.
Pepsinogen Cgene chemical luminescent analysis reagent kid of the present invention, comprising: 1) pepsinogen Cgene antibody bag is by microwell plate; 2) HRP marks pepsinogen Cgene antibody; 3) the serial pepsinogen I calibrator after calibration object diluted; 4) luminous substrate A; 5) luminous substrate B; 6) solid washing lotion.
Described pepsinogen Cgene antibody is mouse source or rabbit source human pepsinogen I monoclonal antibody.
The microwell plate of described bag quilt is the micropore lath in 48 holes or 96 holes.
The formula of described calibration object dilution is:
Deionized water 1L
Na 2HPO 42.9g
NaH 2PO 40.2g
NaCl 8.2g
Compound protein protective agent PHD 0.5g
5% casein 10g
Tween-20 10mL
0.1% Sodium azide 1mL.
The dilution pH regulating pepsinogen I calibrator is 7.4.
Described luminous substrate A is luminol and sodium hydroxide solution.
Described luminous substrate B is hydrogen peroxide and to iodophenol solution.
Described solid washing lotion is PBST, PBST solid lotion prescription
KH 2PO 44.8g
Na 2HPO 4·12H 2O 28.8g
NaCl 155g
Tween-20 10mL。
Pepsinogen Cgene chemical luminescent analysis reagent kid preparation method of the present invention, comprises the following steps: 1) prepare PBST solid washing lotion and dilution thereof; 2) pepsinogen Cgene antibody bag is by the preparation of microwell plate; 3) HRP marks the preparation of pepsinogen Cgene antibody; 4) preparation of pepsinogen I calibrator; 5) luminous substrate A, luminous substrate B is prepared; 6) packing solid washing lotion, enzyme labelled antibody, calibration object, luminous substrate A and luminous substrate B be assembled into finished product.Wherein:
Described preparation PBST solid washing lotion and the step of dilution thereof are: the PBST solid washing lotion after mixing reduces pressure low temperature drying under air pressure 500Pa, temperature 10 DEG C, humidity 30% state, use aluminium foil bag packing after 1h, during use, the PBST solid washing lotion of every 5g is dissolved in 500mL deionized water and mixes.
Described pepsinogen Cgene antibody bag of preparing by the step of microwell plate is: adopting bag to be buffered liquid, pepsinogen Cgene monoclonal antibody to be diluted to concentration be 1 ~ 5 μ g/mL; be adsorbed on microwell plate; wash plate twice with solid washing lotion dilution again, adopt confining liquid to bag by after microwell plate carry out closing and protecting.
It is carbonate buffer solution, phosphate buffer or Tris-HCl damping fluid that described bag is buffered liquid.
The step of the preparation of the pepsinogen Cgene antibody of described HRP mark is: preparation enzyme labelled antibody dilution, by periodates method, HRP is marked in monoclonal antibody, prove through test, the working concentration scope of pepsinogen Cgene enzyme labelled antibody is more than 50ng/mL.
The step of the preparation of described pepsinogen I calibrator is: preparation pepsinogen I calibrator dilution, become content to be respectively the series concentration of 5 μ g/mL, 20 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL the pepsinogen Cgene calibration object diluted of Prof. Du Yucang, set up quantitative calibration product.
The formula that described HRP marks pepsinogen Cgene antibody enzyme labelled antibody dilution used is:
Deionized water 1L
Na 2HPO 42.9g
NaH 2PO 40.2g
NaCl 8.2g
25%BSA 10g
Enzyme stabilizers 1g
0.1% Sodium azide 1mL.
Technical scheme of the present invention: a kind of Serum Pepsinogen I chemiluminescence quantitative detecting method, based on chemiluminescence immunoassay.Pepsinogen Cgene monoclonal antibody bag, by chemiluminescence microwell plate, forms insolubilized antibody.Added blood serum sample or pepsinogen I calibrator in micropore to bag during detection, then the pepsinogen Cgene monoclonal antibody adding HRP mark is reacted, form antibody-antigen-antibody-HRP compound.Wash away unconjugated HRP-pepsinogen Cgene monoclonal antibody with solid washing lotion dilution after reaction, then add luminous substrate, detect the intensity of luminous signal.
According to kit of the present invention, the antibody that the pepsinogen Cgene monoclonal antibody of HRP mark and solid phase carrier wrap quilt forms the sandwich complex structure of " coated antibody-antigen-enzyme labelled antibody " with the pepsinogen Cgene in sample, therefore " double antibody sandwich method " reaction pattern of the present invention's employing, both the high specific of antigen-antibody reaction had been effectively utilized, combine again the high sensitivity of chemiluminescence immunoassay technology, can be clinical diagnosis and more special, quick, reliable foundation is provided.
According to kit of the present invention, reaction pattern is: first add 20 μ L pepsinogen I calibrator or serum in micropore, then the pepsinogen Cgene monoclonal antibody of the HRP mark of 50 μ L is added, micro oscillator mixes 30s, 37 DEG C of incubation 1h, then wash plate 5 times with solid washing lotion dilution, thieving paper pats dry gently, add each 50 μ L of luminous substrate A and B and mix 5s, room temperature lucifuge places 5 minutes, detects luminous value with chemical illumination immunity analysis instrument.
According to kit of the present invention, linear fit mode is taked to process to the luminous value read, optional content logarithm and four parameters, two kinds of fit approach being calculated to pepsinogen Cgene in sample.
Pepsinogen Cgene of the present invention (PG I) chemical luminescent analysis reagent kid can detect pepsinogen Cgene (PG I) content in serum.Serum-concentration PG can be used as the Testing index of gastric acid secretion, in Helicobacter pylori body, PG I level and PG I/PG II ratio and gastric acid output significant correlation, wherein the latter and gastric acid output correlativity stronger.In Helicobacter pylori-Negative body, the level of PG I and gastric acid output significant correlation, but PG I/PG II ratio and gastric acid output non-correlation.Therefore by serum PG concentration sealing gastric acid output, there is value for clinical application.PG content is also relevant with discriminating that is good, malignant gastric ulcer; Serum PG I level and atrophic gastritis, peptic ulcer are proportionate; In the treatment of helicobacter pylori, by monitor degerming after PG change can judge result for the treatment of.The advantages such as it has nothing wound, detection method is simple and quick, highly sensitive, sensing range is wide.The indices of this pepsinogen Cgene (PG I) chemical luminescent analysis reagent kid reaches technical requirement all simultaneously.
Accompanying drawing explanation
The calibration object linear graph of the kit of Fig. 1 prepared by embodiment 1.
Embodiment
embodiment 1application carbonate bag is buffered liquid and prepares pepsinogen Cgene chemical luminescent analysis reagent kid of the present invention
One, the preparation of PBST solid washing lotion and dilution:
1) PBST solid lotion prescription is
KH 2PO 44.8g
Na 2HPO 4·12H 2O 28.8g
NaCl 155g
Tween-20 10mL；
2) the PBST solid washing lotion after mixing reduces pressure low temperature drying under air pressure 500Pa, temperature 10 DEG C, humidity 30% state, aluminium foil bag packing is used after 1h, during use, the PBST solid washing lotion of every 5g is dissolved in 500mL deionized water and mixes, obtained PBST solid washing lotion dilution.
Two, wrap by the preparation of microwell plate:
1) selection of antibody: adopt mouse resource monoclonal antibody;
2) the bag quilt of microwell plate: by the pH value of 0.02mol/L be 9.6 carbonate buffer solution pepsinogen Cgene monoclonal antibody to be diluted to concentration be 5 μ g/mL, be mixed with coating buffer, and be adsorbed on microwell plate with 100 μ L/ holes.Described pH value be 9.6 the carbonate bag formula that is buffered liquid be:
Deionized water 1L
Na 2CO 30.636g
NaHCO 31.172g
After dissolving mixing, adjust pH to 9.6;
3) closed protective: wash plate twice with solid washing lotion dilution, adds on the microwell plate of confining liquid after washing with 150 μ L/ holes, at 21 DEG C ~ 25 DEG C closed 3h, gets rid of confining liquid, thieving paper pats dry.Room temperature removal moisture drying 24h envelope.Then labeling is put 2 DEG C ~ 8 DEG C and is saved backup.The formula of confining liquid is:
Deionized water 1L
Na 2CO 31.59g
NaHCO 32.93g
NaCl 8g
BSA 10g
0.1% Sodium azide 1mL
Sucrose 25g.
Three, HRP marks the preparation of pepsinogen Cgene antibody:
Be marked in monoclonal antibody by periodates method by HRP, the formula of enzyme labelled antibody dilution is:
Deionized water 1L
Na 2HPO 42.9g
NaH 2PO 40.2g
NaCl 8.2g
25%BSA 10g
Enzyme stabilizers 1g
0.1% Sodium azide 1mL.
Four, enzyme labelled antibody concentration is selected:
Prove through test, the working concentration scope of pepsinogen Cgene enzyme labelled antibody is more than 50ng/mL.
Five, the preparation of calibration object:
Become content to be respectively the series concentration of 5 μ g/mL, 20 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL the pepsinogen Cgene calibration object diluted of Prof. Du Yucang, set up quantitative calibration product.The formula of calibration object dilution is:
Deionized water 1L
Na 2HPO 42.9g
NaH 2PO 40.2g
NaCl 8.2g
Compound protein protective agent PHD 0.5g
5% casein 10g
Tween-20 10mL
0.1% Sodium azide 1mL
The dilution pH regulating pepsinogen I calibrator is 7.4.
Six, the preparation of luminous substrate A, luminous substrate B
The step of described preparation luminous substrate A is: 10mg luminol adds 1000mL deionized water, then adjusts pH to 8.6 with NaOH.
The step of described preparation luminous substrate B is: add hydrogen peroxide in deionized water and to iodophenol, making hydrogen peroxide final concentration in solution be 3mmol/L, is 5mg/mL to iodophenol final concentration.
Seven, the assembling of semi-manufacture and finished product
The packing of above-mentioned steps products obtained therefrom is semi-manufacture, randomly draws three parts and be assembled into kit after specificity, accuracy, sensitivity and Detection of Stability are qualified.
embodiment 2application phosphate bag is buffered liquid and prepares pepsinogen Cgene chemical luminescent analysis reagent kid of the present invention
Bag quilt: being buffered liquid with the phosphate bag that the pH value of 0.2mol/L is 6.5, pepsinogen Cgene monoclonal antibody is diluted to concentration is 3 μ g/mL, is mixed with coating buffer, and is adsorbed on microwell plate with 100 μ L/ holes, wrap by 4h under 37 DEG C of conditions.Particularly, to be the phosphate bag preparation method that is buffered liquid of 6.5 be described pH value: the NaH of the 0.2mol/L of 68.5mL 2pO 4the Na of the 0.2mol/L of solution and 31.5mL 2hPO 4solution mixes.
Antibody selects rabbit source human pepsinogen I monoclonal antibody, and other reagent are consistent with the compound method of component and the method for embodiment 1.
embodiment 3application Tris-HCl bag is buffered liquid and prepares pepsinogen Cgene chemical luminescent analysis reagent kid of the present invention
Bag quilt: being buffered liquid with the Tris-HCl bag that the pH value of 0.05mol/L is 7.4, pepsinogen Cgene monoclonal antibody is diluted to concentration is 1 μ g/mL, is mixed with coating buffer, and is adsorbed on microwell plate with 100 μ L/ holes, wrap by 4h under 37 DEG C of conditions.Particularly, to be the Tris-HCl bag preparation method that is buffered liquid of 7.4 be described pH value: after the HCl solution mixing of the Tris aqueous slkali of the 0.1mol/L of 50.0mL and the 0.1mol/L of 42.0mL, add water and be settled to 100mL.
Other reagent are consistent with the compound method of component and the method for embodiment 1.
the using method of kit of the present invention
One, prepare before experiment:
1) kit is taken out from cold storage environment, place room temperature 18 DEG C ~ 25 DEG C and balance at least 30min;
2) constant temperature oven or water-bath are transferred to 37 DEG C, use after temperature stabilization;
3) for subsequent use after getting solid washing lotion 5g 500mL deionized water dissolving.
Two, the requirement of sample:
1) correct medical technology is adopted to collect serum sample;
2) sediment in sample may affect test findings, should centrifugal removing, and determines that sample is rotten and can use;
3) sample of significant hemolysis or piarhemia can not be used for measuring;
4) sample measures in 24h, then should deposit in 2 DEG C ~ 8 DEG C refrigerators, if Long-Time Service, then should be frozen in less than-20 DEG C, and avoid multigelation; Return to room temperature before using, shake mixing gently.
Three, detection method:
The concrete operation step using embodiment 1 made pepsinogen Cgene chemical luminescent analysis reagent kid to carry out detecting is as follows:
1) take out a certain amount of bag by hole, every hole adds 20 μ L calibration object or blood serum samples respectively;
2) every hole adds enzyme conjugates 50 μ L respectively;
3) 30s that vibrates on microoscillator makes liquid in hole mix, and puts 37 DEG C of incubation 60min;
4) wash plate 5 times with solid washing lotion dilution, finally pat dry on thieving paper;
5) every hole adds each 50 μ L of luminous substrate A and B respectively, room temperature (18 DEG C ~ 25 DEG C) lucifuge reaction 5min;
6) chemiluminescence detector detects luminous intensity;
7) result calculates: select suitable curve mode, this kit recommendation linear regression fit equation Criterion curve, but also other can be adopted to fit mode according to different situations.With the logarithm value of series of calibration product concentration for horizontal ordinate (X-axis), with the logarithm value of series of calibration product luminous intensity values for ordinate (Y-axis) Criterion curve (log-log) (Fig. 1), the luminous intensity values of the blood serum sample recorded is substituted in typical curve (log-log), calculates the pepsinogen Cgene content in blood serum sample.
Four, reference value (term of reference):
With this kit measurement 1000 parts of normal person's Diagnostic Value of Fasting Serum samples, obtain normal person's fasted conditions term of reference for being greater than 65 μ g/L.With PGⅡ joint-detection, the reference value of normal person's sample pepsinogen Cgene and PGⅡ ratio is greater than 7.5.Normal reference value should be set up according to oneself physical condition and exposed population group in each laboratory.This kit only makes one of supplementary means diagnosed, for reference for clinicians.
the qualification of pepsinogen Cgene chemical luminescent analysis reagent kid of the present invention in methodology
Identify the kit manufactured in embodiment 1 according to manufacture conventional in this area and vertification regulation, prove through a large amount of experiments, kit of the present invention is when measuring for pepsinogen Cgene concentration level, and the party's science of law index is as follows:
1) accuracy: pepsinogen Cgene high level sample A is joined in low value sample B, the volume ratio added between sample A and sample B be 1:9, according to the formulae discovery recovery (R) below.The recovery is 99%, in 85% ~ 115% scope.
In formula:
The R-recovery;
V-adds A liquid and amasss;
V 0the volume of-serum or corresponding matrix B;
C-blood serum sample or corresponding matrix add the detectable concentration after A liquid;
C 0the detectable concentration of-blood serum sample or corresponding matrix B;
The concentration of Cs-A liquid.
2) lowest detectable limit: detect as sample with zero calibration object or Sample dilution, replication 20 times, draw the RLU value (relative light unit) of 20 measurement results, calculate its mean value (M) and standard deviation (SD), draw M+2SD, obtain corresponding concentration value, be lowest detectable limit.Lowest detection is limited to 0.6 μ g/L, is less than 1 μ g/L.
3) linear: diluted by a certain percentage by the high level sample close to the range of linearity upper limit at least 5 kinds of concentration, wherein low value concentration of specimens must close to the lower limit of the range of linearity.Each concentration duplicate detection 2 times, calculating mean value, carries out fitting a straight line by result mean value and dilution ratio least square method, and calculates linearly dependent coefficient r.In the range of linearity (1 ~ 200) the μ g/L of defined, the correlation coefficient r of kit is 0.9985, r >=0.9900.
4) repeatability: each duplicate detection of sample 10 times being respectively 50 ± 10 μ g/L and 100 ± 20 μ g/L by concentration, calculates its concentration value, calculates mean value M and the standard deviation SD of 10 measurement results, draws coefficient of variation CV according to formula CV=SD/M × 100%.The coefficient of variation (CV) is 8.62%, is less than 15%.
5) specificity: the PG II sample duplicate detection 2 times with content being 200 μ g/L, calculates its concentration value.Result is 0.3 μ g/L, is less than 1 μ g/L.
6) difference between batch: detect same increment respectively originally with three lot number kits, concentration is 100 ± 20 μ g/L, and each repetition 10 times, calculates its concentration value, calculate mean value M and the standard deviation SD of 30 concentration values, draw coefficient of variation CV according to formula CV=SD/M × 100%.Interassay coefficient of variation (CV) between three lot number kits is 10.21%, is less than 15%.
7) heat stabilization test: get kit in the term of validity and place 7 days in 37 DEG C, accuracy in detection, lowest detectable limit, linear and repeatability still meet the requirements.
This kit range of linearity is 1.0 ~ 200.0 μ g/L.Measured value more than the sample of 200.0 μ g/L, consequently by result of calculation that calibration object curve extension draws.If obtain its result more accurately, need dilute sample.Pepsinogen Cgene chemical luminescent analysis reagent kid of the present invention sample size used is few, originates simple and convenient, vitro detection to tested object without any toxic and side effect.The chemiluminescence immune analysis method that the present invention simultaneously adopts, does not produce radioactive contamination.The inventive method is utilized to detect, with several traditional immunological detection method contrastingly, no matter from the stability of lowest detectable limit, detection time, the range of linearity, reagent, have the aspect contrasts such as non-environmental-pollution, the advantage of chemoluminescence method all seems very outstanding.Kit of the present invention is except having the simple and quick advantage of detection method, and also have the advantages such as highly sensitive, sensing range is wide, therefore more and more receiving the concern of clinical diagnostic laboratories, is the study hotspot in this field in recent years.Therefore the present invention is pepsinogen Cgene content in clinical detection serum, thus examination cancer of the stomach and evaluation helicobacter pylori (HP) eradication therapy effect, for after judgement recurrent peptic ulcer and resection of gastric carcinoma, recurrence provides a kind of simple, quick, sensitive method.

Claims (4)

1. a pepsinogen Cgene chemical luminescent analysis reagent kid, comprising: 1) pepsinogen Cgene antibody bag is by microwell plate; 2) HRP marks pepsinogen Cgene antibody; 3) the serial pepsinogen I calibrator after calibration object diluted; 4) luminous substrate A; 5) luminous substrate B; 6) solid washing lotion;
Described pepsinogen Cgene antibody is mouse source or rabbit source human pepsinogen I monoclonal antibody;
The microwell plate of described bag quilt is the micropore lath in 48 holes or 96 holes;
The formula of described calibration object dilution is:
Deionized water 1L
Na 2HPO 42.9g
NaH 2PO 40.2g
NaCl 8.2g
Compound protein protective agent PHD 0.5g
5% casein 10g
Tween-20 10mL
0.1% Sodium azide 1mL
The dilution pH regulating pepsinogen I calibrator is 7.4;
Described luminous substrate A is luminol and sodium hydroxide solution;
Described luminous substrate B is hydrogen peroxide and to iodophenol solution;
Described solid washing lotion is phosphate Tween buffer (PBST), and PBST solid lotion prescription is
KH 2PO 44.8g
Na 2HPO 4·12H 2O 28.8g
NaCl 155g
Tween-20 10mL。
2. the preparation method of pepsinogen Cgene chemical luminescent analysis reagent kid described in claim 1, comprises the following steps: 1) prepare PBST solid washing lotion and dilution thereof; 2) pepsinogen Cgene antibody bag is by the preparation of microwell plate; 3) HRP marks the preparation of pepsinogen Cgene antibody; 4) preparation of pepsinogen I calibrator; 5) luminous substrate A, luminous substrate B is prepared; 6) packing solid washing lotion, enzyme labelled antibody, calibration object, luminous substrate A and luminous substrate B be assembled into finished product;
Wherein:
1) described preparation PBST solid washing lotion and the step of dilution thereof are: the PBST solid washing lotion after mixing reduces pressure low temperature drying under air pressure 500Pa, temperature 10 DEG C, humidity 30% state, use aluminium foil bag packing after 1h, during use, the PBST solid washing lotion of every 5g is dissolved in 500mL deionized water and mixes;
2) described pepsinogen Cgene antibody bag of preparing by the step of microwell plate is: adopting bag to be buffered liquid, pepsinogen Cgene monoclonal antibody to be diluted to concentration be 1 ~ 5 μ g/mL, be adsorbed on microwell plate, wash plate twice with solid washing lotion dilution again, adopt confining liquid to bag by after microwell plate carry out closing and protecting;
3) step of the preparation of the pepsinogen Cgene antibody of described HRP mark is: preparation enzyme labelled antibody dilution, by periodates method, HRP is marked in monoclonal antibody, prove through test, the working concentration scope of pepsinogen Cgene enzyme labelled antibody is more than 50ng/mL;
4) step of the preparation of described pepsinogen I calibrator is: preparation pepsinogen I calibrator dilution, become content to be respectively the series concentration of 5 μ g/mL, 20 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL the pepsinogen Cgene calibration object diluted of Prof. Du Yucang, set up quantitative calibration product.
3. the preparation method of pepsinogen Cgene chemical luminescent analysis reagent kid described in claim 2, is characterized in that the formula of the enzyme labelled antibody dilution that described HRP mark pepsinogen Cgene antibody is used is:
Deionized water 1L
Na 2HPO 42.9g
NaH 2PO 40.2g
NaCl 8.2g
25%BSA 10g
Enzyme stabilizers 1g
0.1% Sodium azide 1mL.
4. the preparation method of pepsinogen Cgene chemical luminescent analysis reagent kid described in claim 2, it is characterized in that described bag is buffered liquid is carbonate buffer solution, phosphate buffer or Tris-HCl damping fluid.
CN201510034183.3A 2015-01-23 2015-01-23 Chemiluminescent quantitative detection kit of pepsinogen I and preparation method thereof Pending CN104597251A (en)

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