CN102128928A - Pepsase chemiluminescent immunoassay kit and preparation method thereof - Google Patents
Pepsase chemiluminescent immunoassay kit and preparation method thereof Download PDFInfo
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- CN102128928A CN102128928A CN2010105742461A CN201010574246A CN102128928A CN 102128928 A CN102128928 A CN 102128928A CN 2010105742461 A CN2010105742461 A CN 2010105742461A CN 201010574246 A CN201010574246 A CN 201010574246A CN 102128928 A CN102128928 A CN 102128928A
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Abstract
The invention relates to the field of immunological detection, in particular to a pepsase chemiluminescent immunoassay kit and a method thereof. The pepsase chemiluminescent immunoassay kit provided by the invention comprises: 1) a pepsase antigen calibrator; 2) sample collecting solution at a pH value of 2 to 3; 3) sample diluents at a pH value of 6 to 8; 4) a microporous plate coated by a pepsase antibody; 5) a pepsase antibody marker; 6) a chemical luminescence substrate liquid; and 7) concentrated cleaning solution. The kit provided by the invention can detect the pepsase content in gastric juice, esophageal content, throat secretion and tracheal bronchus secretion, judge if gastroesophageal reflux happens according to the result of the pepsase detection and judge the treatment effect of stomach diseases and change of state of illness according to the detected pepsase content. The kit has the advantages of noninvasiveness, simplicity, convenience, quickness, sensitivity, stability and the like.
Description
Technical field
The present invention relates to field of immunodetection, relate to a kind of pepsin chemiluminescence immune detection reagent kit and method thereof particularly.
Background technology
Pepsin (pepsin) belongs to the aspartic acid enzyme of proteolysis, it is the peptic digest proteolytic enzyme, relative molecular weight is 31-36kd, its precursor is a propepsin, propepsin is synthetic in adult vertebrate stomach lining, by the chief cell secretion, under the acid condition of gastric juice, convert pepsin to.Its function is that the protein in the food is decomposed into little fragments of peptides.
GERD (GERD) is meant that gastrointestinal contents backflows and causes symptoms of digestive tract such as returning acid, heartburn, pectoralgia into oesophagus, and then has a strong impact on quality of life and can cause the disease of esophageal precancerous lesion.Be a kind of very common oesophagus illness that influences quality of life, its pathogenic factor mainly is that the anti-reflux barrier of oesophagus weakens or the oesophagus mucous membrane is exposed under the gastric juice for a long time, causes hydrochloric acid in gastric juice and pepsin infringement mucous membrane of esophagus.Part patient need take all the life and press down acid supplement, and part patient then need go under the laparoscope fundoplication and treat and control.
Generally, inferior esophageal sphincter (LES) is open when swallowing, and closes subsequently and prevents that hydrochloric acid in gastric juice from backflowing into oesophagus.LES pressure is enough to prevent that food from backflowing during tranquillization.The improper lax of LES can cause hydrochloric acid in gastric juice to backflow into oesophagus, causes irritant reaction.The pathogenesis of gastroesophageal reflux disease (GERD) is relevant with gastric and esophageal power, comprises oesophagus removing ability drop and delayed gastric emptying.50% patient has the forfeiture of esophageal peristalsis function.Hiatal hernia infringement oesophagus air emptying function, along with the hernia hole increases, problem also increases, and causes hydrochloric acid in gastric juice to be removed and slows down.
Sings and symptoms GERD symptom is brought out after entering in the stomach with fat after the meal usually.Bend over, lie on the back and anxiety can increase the weight of symptom, obesity, gestation, ascites, abdominal belt tension etc. also can cause sx.A series of symptoms of GERD comprise stomach burn feeling (83%), gastric disorder causing nausea (70%), dyscatabrosis (37%), respiratory symptom (30%), stomachache (10%), pectoralgia (10%), feel sick (8%), belch (7%) and hemorrhage (4%).Patient main suit has classical symptoms such as stomach burn feeling and gastric disorder causing nausea after the meal.The serious pectoralgia of patient main suit is also arranged, often be attributed to heart attack mistakenly.In addition, also has the SOA that trachyphonia or typical hydrochloric acid in gastric juice such as stridulate cause.Some patients do not have classical symptom, after oesophagus is badly damaged, and patient main suit's dysphagia, even hemorrhage, this is noted behind be everlasting spitting blood or black tarry stools.Night, esophageal reflux increased the danger of esophagitis because night swallowing activity and salivary secretion reduce can not in and hydrochloric acid in gastric juice to cause it to rest in the oesophagus time longer.
Check that the patient has trachyphonia, upper abdomen tenderness, hunchback, enamel destruction or auscultation of lung bruit de craquement (companion's aspiration pneumonia).The clothes that are in tights also can make these symptoms highlight owing to can increase abdominal pressure.Medical history and physical examination often are enough to diagnose GERD.There have report to think that continuous 7d takes heavy dose of Omeprazole recently to be effectively sick to this, because it can be secreted by gastric acid inhibitory, stops the generation of GERD symptom.If medicinal treatment is invalid behind the patient diagnosis, or their main suit's dyscatabrosis, endoscopy then is the best means of diagnosis esophagitis.The upper gastrointestinal barivm meal fluoroscopy (screem) is (98.7%) quite accurately to the diagnosis of severe esophagitis, moderate and slight esophagitis take second place (being respectively 81.6% and 24.6%).The pH value is monitored in the oesophagus, promptly measures the acidity of stomach and esophageal contents, is helpful to the endoscopy negative patients.Esophageal manometry is promptly measured LES pressure and oesophagus function by nasal meatus to gastric intubation, also helps to make a definite diagnosis.Should be noted that all inspections can produce false negative result.
Basically use the denatured hemoglobin method to measure its activity at present to pepsic detection, and for the detection of pepsin content, Zhou Sufang, Deng Yong, Zhou Deyi etc. reported once in " different gastric juice pepsin contents and active mensuration " literary composition that utilizing the red cassia tree agglutinin to set up the ELISA method detected, and its principle is to utilize agglutinin to have the character that combines with the glycoprotein specificity.But this method can't be distinguished some glycoprotein of food in other enzyme in the sample and the gastric juice, thereby can cause the false positive of detection.
Wang Jianrong, Cheng Yanshuan, Ma Yanlan mentioned in " variation of different positions and mode nasal feeding patient's oropharynx and bronchial secretion pepsin content " once that the method with radiommunoassay detected pepsin content; But its radioimmunoassay running time is long, and label has radioactive contamination, is eliminated by market gradually.
Inspection method to sum up, endoscope and barium meal examination bring uncomfortable and certain injury to patient, and to slight esophagitis accuracy low (24.6%); And pH value testing process is loaded down with trivial details in the oesophagus, and influenced factor is many, can not satisfy clinical requirement.The active detecting operation of stomach cardia is loaded down with trivial details, is not easy to clinical examination; The intersection factor that the agglutinin reaction exists is many, causes false positive easily; Radio-immunity react and the running time long, environment is had pollution.
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) came out in 1977, last century the nineties, the production of the development of chemical luminescence immune analysis reagent box and automatic measurement instrument has obtained breakthrough, thereby enters the high speed development stage.Chemiluminescence immune assay is a new immuno analytical method that grows up after fluorescence, radioactive isotope and EIA enzyme immunoassay, according to lot of experiment results and clinical practice data, from practicality, stability, accuracy and development prospect, chemiluminescence immune assay maintains the leading position in nonradioactive labeling's analytical technology, the direction and the trend of world today's development have been represented, it not only has immunoreactive specificity, and (detection limit can reach 10 to have the high sensitivity of chemiluminescence reaction
-15-10
-18Mol/L).Advantages such as that chemiluminescence immunoassay technology has is highly sensitive, quick, accurate, good reproducibility, the effect phase is long and safety non-toxic is pollution-free become the first-selection that replaces radiommunoassay and EIA enzyme immunoassay technology.
Because pepsin can be sheared albumen or polypeptide, the optimum pH of its enzymatic activity is about 3, still has activity when pH value 6.So when utilizing the method for immunoassay to carry out specific detection, should keep pepsic immunocompetence, avoid the capacity of decomposition of its enzymatic activity again to albumen, this also is one of difficult point of immunoassay kits exploitation, and chemiluminescence immune assay is as a kind of emerging analytical technology, and it applies to pepsin and detects report is not arranged yet.Backflow when we utilize GER and contain gastric juice in the thing, and contain pepsin in the gastric juice, thereby estimate whether there is GER by detecting pepsin.The pepsin chemiluminescence immune detection reagent kit arises at the historic moment.
Summary of the invention
Develop and finished the present invention in order to address the above problem, soon chemiluminescence is learned effectively with pepsic immunoassay and is combined, used simultaneously that luminous signal is strong, longer duration, the self-control chemical luminous substrate liquid of high s/n ratio more, provide a kind of and can easier, quick, sensitive, stably detect pepsic kit.The method that kit simultaneously of the present invention adopts is than other analytic approach is highly sensitive at present, and therefore the reaction height specificity makes this kit be more suitable for carrying out effectively promotion and application on industry.
Therefore, the purpose of this invention is to provide a kind of pepsin chemiluminescence immune detection reagent kit.
A further object of the present invention provides a kind of method for preparing above-mentioned pepsin chemiluminescence immune detection reagent kit.
According to pepsin chemiluminescence immune detection reagent kit of the present invention, comprising:
1) pepsin antigen calibration object; 2) sample collection liquid, pH 2-3; 3) sample diluting liquid, pH 6-8; 4) microwell plate of pepsin antibody sandwich; 5) pepsin antibody labeling thing; 6) chemical luminous substrate liquid; And 7) concentrated cleaning solution.
Because pepsin can be sheared albumen or polypeptide, the optimum pH of its enzymatic activity is about 3, still has activity when pH value 6.So when utilizing the method for immunoassay to carry out specific detection, should keep pepsic immunocompetence, avoid again its enzymatic activity to the capacity of decomposition of albumen according to kit of the present invention, behind the sample collecting, be kept in the sample collection liquid (pH2-3) that hangs down the pH value and can keep pepsic activity, before sample detection, sample diluting liquid with pH 6-8 dilutes, make the neutrality that rises to of its solution to be measured, avoided pepsin enzyme segmentation ability under sour environment like this, but to the not influence of pepsic immunocompetence, moreover neutral pH value environment also helps the reaction of antigen-antibody.
According to kit of the present invention, the antibody of bag quilt forms the sandwich complex structure of " coated antibody-Ag-Ab-label " on the antibody of label mark and the solid phase carrier with the pepsin antigen in the sample, so " double antibody sandwich method " reaction pattern of the present invention's employing, both effectively utilized the high specific of antigen-antibody reaction, combine the high sensitivity of chemiluminescence immunoassay technology again, and can make reaction sensitivity obtain higher level by the biotin-avidin system, can be clinical diagnosis and provide more special, fast, reliable foundation.
According to kit of the present invention can be used for detecting pharynx nasalis, bottleneck throat secretion thing, whether esophageal contents contains pepsin, thereby judges whether to exist GERD; Kit of the present invention can also be used for detecting the gastric juice pepsin concn, thus the auxiliary diagnosis Gastric Diseases by Spraying.
According to kit of the present invention, wherein, described label is horseradish peroxidase, alkaline phosphatase, biotin and derivant thereof.
According to kit of the present invention, wherein, when described label biotin and derivant thereof, the spike enzyme is horseradish peroxidase-labeled streptavidin (HRP-SA).
According to kit of the present invention, wherein, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
According to kit of the present invention, wherein, described sample collection liquid is the damping fluid of pH 2～3, and according to concrete enforcement of the present invention, the prescription of described sample collection liquid is:
Citric Acid Mono 21g
Proclin300 1mL
Distilled water is settled to 1000mL.
According to kit of the present invention, wherein, described sample diluting liquid is the protein-contg damping fluid of pH6～8, and according to a particular embodiment of the invention, the prescription of described sample diluting liquid is:
NaH
2PO
4·2H
2O 0.6g
Na
2HPO
4·12H
2O 5.8g
NaCl 8.766g
Bovine serum albumin(BSA) 10g
Proclin300 1mL
Distilled water is settled to 1000mL.
According to kit of the present invention, wherein, described pepsin antibody can be monoclonal antibody or polyclonal antibody.
According to kit of the present invention, wherein, described antibody, its source can be mouse, rabbit, goat, sheep, cavy, ox, donkey, horse, chicken, monkey.
The invention provides a kind of method for preparing the mentioned reagent box, may further comprise the steps: 1) pepsin antigen calibration object; 2) sample collection liquid; 3) sample diluting liquid; 4) microwell plate of pepsin antibody sandwich; 5) mark pepsin antibody; 6) preparation chemical luminous substrate liquid; 7) preparation concentrated cleaning solution; 8) the pepsin antibody of the above-mentioned pepsin antigen of packing calibration object, mark, chemical luminous substrate liquid and concentrated cleaning solution; And 9) be assembled into finished product.
The method according to this invention, described bag be may further comprise the steps by the step 4) of microwell plate:
I) preparation of antibody
Synthetic pepsin polypeptide, with immune rabbit behind the synthetic crosslinked BSA of polypeptide, the anti-people's pepsin of preparation rabbit antibody.
II) bag quilt
With 0.05mol/L pH value is that 9.6 the carbonate buffer solution and the pepsin antibody of debita spissitudo are mixed with coating buffer, and it is carried on the microwell plate;
III) wash above-mentioned microwell plate with physiological saline; And
IV) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10g bovine serum albumin(BSA) and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.6, then the gained confining liquid is carried on the microwell plate after the above-mentioned washing.
According to the method for preparing the mentioned reagent box of the present invention, wherein pepsin abzyme mark thing is a horseradish peroxidase, adopts the sodium periodate method to carry out mark; Pepsin abzyme mark thing is an alkaline phosphatase, adopts the glutaraldehyde cross-linking method to carry out mark; And pepsin antibody labeling biotin adopts the coupling of EDC/NHS method, and adopts the sodium periodate method to carry out the horseradish peroxidase streptavidin.
Described pepsin antigen calibration object is an artificial synthetic polypeptide, purity must not be lower than 90%, the microwell plate of bag quilt is the micropore lath in 48 or 96 holes, the enzyme of mark is coupling alkaline phosphatase (ALP) or horseradish peroxidase, effective luminescent substance in the chemical luminous substrate liquid is luminol or AMPPD, and concentrated cleaning solution is PBST or the Tris-HCl damping fluid that contains Tween20.
The present invention's " pepsin chemiluminescence immune detection reagent kit " can detect in the gastric juice, the pepsin content in esophageal contents, the bottleneck throat secretion thing, pharynx nasalis secretion, tracheal bronchus secretion, and judge whether to exist gastroesophageal reflux according to whether detecting pepsin, can judge the effect of Gastric Diseases by Spraying treatment and the variation of the state of an illness thereof according to detecting pepsin content.Advantages such as it has and do not have wound, easy, quick, sensitive, stable.Every index of this pepsin chemiluminescence immune detection reagent kit has all reached technical requirement simultaneously.
Description of drawings
Fig. 1 is the calibration object linear graph in the prepared kit of embodiment 1.
Embodiment
One, the preparation of horseradish peroxidase-labeled antibody
Dissolving 5mg HRP adds 0.5mL sodium periodate (50mmol/L) stirring at room 30min in 0.5mL distilled water, be 4.4 sodium-acetate buffers through 10mM pH value, and the dialysis back adds 5mg pepsin antibody, stirs 2h, uses 200mM NaBH at last
4Reduce, after the dialysis of 0.02M PBS damping fluid, add equal-volume glycerine, preserve below-20 ℃.
Two, enzyme labelled antibody dilution
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
NaCl 8.766g
Enzyme stabilizers 1g
BSA 10g
Proclin 1mL
Distilled water 1000mL
Three, enzyme labelled antibody concentration is selected
Through evidence, the working concentration scope of enzyme labelled antibody is more than 1: 500.
Four, the preparation of pepsin antigen calibration object
The pepsin polypeptide antigen of synthetic become with the calibration object diluted content is respectively 0,2,6,20,60, the 200ng/mL series concentration, set up quantitative calibration object.
Five, the preparation of the solid phase carrier of pepsin antibody sandwich.
(1) bag quilt
Adopting 0.05mol/L pH value is that 9.6 the carbonate buffer solution and the pepsin antibody of debita spissitudo are mixed and made into coating buffer, and it is carried on the microwell plate;
Particularly, the prescription of described 0.05mol/L carbonate buffer solution is:
Natrium carbonicum calcinatum 1.59g
Sodium bicarbonate 2.93g
Deionized water 1000mL
Behind the dissolving mixing, adjust pH value to 9.6, add 5.0mg pepsin antibody mixing, in the amount adding microwell plate by 100 μ L/ holes, 4 ℃ are spent the night.
(2) washing: wash above-mentioned bag by good microwell plate with physiological saline.
(3) sealing
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
Bovine serum albumin(BSA) 10g
Proclin300 1mL
Distilled water is settled to 1000mL
The mentioned reagent weighing is put into clean container well, add the distilled water constant volume, the dissolving mixing, adjusting the pH value is 7.0～7.6.
Amount by 200 μ L/ holes adds in the microwell plate, and room temperature was placed 2-4 hour.Get rid of confining liquid, on thieving paper, pat dry.Vacuum sealing bag is carried out in the room temperature removal moisture drying immediately after 24 hours.Place behind the envelope and checked whether leak gas in 15 minutes, then need envelope again if any gas leakage, as 2～8 ℃ of preservations of no gas leak phenomenon labeling postposition, standby.
Six, sample collection liquid
The 100mM citrate buffer solution is transferred pH to 2.5 with NaOH solution; Particularly, described prescription is as follows
Citric Acid Mono 21g
Proclin300 1mL
Distilled water is settled to 1000mL
Adjust pH to 2.5 with NaOH solution.
Seven, sample diluting liquid
Adopt protein-contg 20mM pH7.4PBS damping fluid;
Particularly, described diluted sample formula of liquid is as follows:
NaH
2PO
4·2H
2O 0.6g
Na
2HPO
4·12H
2O 5.8g
NaCl 8.766g
Bovine serum albumin(BSA) 10g
Proclin300 1mL
Distilled water is settled to 1000mL
Adjust pH to 7.4
Eight, chemical luminous substrate
A liquid: in the 100ml distilled water, add the Tris-HCl damping fluid that 1.21g Tris and the dense HCl of 295 μ L are made into 0.1M pH8.5.The Tween20 of Luminol, the 20 μ L of adding 0.5g and 0.1g mix iodophenol in this damping fluid.
B liquid: in the 100ml distilled water, add trisodium citrate 0.73g and citric acid 0.44g, be mixed with the citrate buffer solution of 0.1M pH5.0, in this solution, add the 0.1g urea peroxide.
Using method: before using A liquid is mixed the back with B liquid in 1: 1 ratio and use
Nine, concentrated cleaning solution
NaH2PO4·2H2O 4g
Na2HPO4·12H2O 58g
NaCl 155g
Tween-20 10mL
Add deionized water and be settled to 1000mL
Adjust pH to 7.2～7.4
Ten, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Randomly draw three parts through being assembled into the Pepsin chemiluminescence immune analysis quantitative determination reagent kit behind specificity, accuracy, sensitivity and the stable assay approval.
Embodiment 2 using basic phosphatase systems produce pepsin chemiluminescence immune detection reagent kit of the present invention
One, the preparation of alkali phosphatase enzyme mark antibody
Because this kit adopts to such an extent that be alkaline phosphatase, therefore generally all adopt the glutaraldehyde cross-linking method to prepare the enzyme labeling thing.In this cross-linking method, adopt two-step approach promptly earlier with enzyme and glutaraldehyde effect, remove unnecessary glutaraldehyde after the dialysis, forming the enzyme labeling thing with the antibody effect.Owing to enzyme and antibody in the enzyme labelled antibody are bioactivator, therefore inactivation needs to add isopyknic glycerine and preservation in low temperature refrigerator (20 ℃) in bond solution easily.
Two, chemical luminous substrate
The compound method of the chemical luminous substrate liquid of alkaline phosphatase used in the present invention (ALP):
Tris 24.22g
NaCl 175g
KCl 4g
HCl 15mL
Distilled water 1000mL
Proclin?300 1mL
AMPPD 200mL
Three, concentrated cleaning solution
Tris 24.22g
NaCl 175g
Tween20 10ml
HCl 15mL
Add deionized water and be settled to 1000mL
Adjust pH value to 7.4.
The collocation method of other reagent and component and the method for horseradish peroxidase systems produce pepsin chemiluminescence immune detection reagent kit are consistent.
Embodiment 3 applicating biotin streptavidin systems produce pepsin chemiluminescence immune detection reagent kit of the present invention
One, the preparation of biotin labeling antibody
Biotin labeling pepsin antibody with the biotin succinimide ester under the alkalescence condition with antibody generation coupling reaction, PBS is fully dialysed, the biotin labeling thing dilutes with NBCS, adds Proclin300, preserves below-20 ℃.
Two, the preparation of horseradish peroxidase-labeled streptavidin
Dissolving 5mg HRP adds 0.5mL sodium periodate (50mmol/L) stirring at room 30min in 0.5mL distilled water, be 4.4 sodium-acetate buffers through 10mM pH value, and the dialysis back adds 2mg pepsin antibody, stirs 2h, uses 200mM NaBH at last
4Reduce, after the dialysis of 0.02M PBS damping fluid, add equal-volume glycerine, preserve below-20 ℃.
The collocation method of other reagent and component and the method for embodiment 1 are consistent.
In sum, in research process of the present invention, at first used starting material shaker test and Quality Identification have been carried out, accuracy with warranty test, then method for coating is studied, select optimal bag and be cushioned liquid and confining liquid, find best concentration conditions, and made and to make the enzyme labeling thing keep active dilution for a long time.
Utilize kit of the present invention to detect, highly sensitive, high specificity, sensing range is wide, simple to operate, "dead" pollution, the kit cost is low, clinical applicability is strong, can be used for detecting the variation of pepsin content in the gastric juice, more is applicable to clinically the monitoring to GERD.
The using method of embodiment 4 kits of the present invention
Before using this kit, should take out room temperature earlier and place 15～30 minutes, make them equilibrate to room temperature; Afterwards, be ready to suitable micro sample adding appliance and corresponding suction nozzle and check Chemiluminescence Apparatus and supplementary instrument, as wash plate machine etc., whether operate as normal.
One, the preparation of sample
1, gastric juice: get 1ml gastric juice, join in the sample collection liquid that contains 0.5ml, centrifugal 5 minutes of 4000rcp/min gets supernatant, carries out dilution in 1: 500 (as adding 2 μ l samples in the 1ml sample diluting liquid) with sample diluting liquid before detecting.
2, GER thing: wipe away paper with pharynx and scrape and get pharynx nasalis, bottleneck throat secretion thing, or under the condition of negative pressure, aspirate tracheae or bronchial secretion with disposable sputum gatherer, to immerse in the sample collection liquid, centrifugal 5 minutes of 4000rcp/min, get supernatant, carry out dilution in 1: 5 (as adding 100 μ l samples in the 500 μ l sample diluting liquids) with sample diluting liquid before detecting.
3, sputum: bring up the deep part of throat sputum, spit in the pipe that contains sample collection liquid, centrifugal 5 minutes of 4000rcp/min gets supernatant, carries out dilution in 1: 5 (as adding 100 μ l samples in the 500 μ l sample diluting liquids) with sample diluting liquid before detecting.
Two, detection method
Use this kit as follows according to the concrete operations step that the method for embodiment 1 experimentizes:
1) will test the microwell plate (microwell plate has wrapped by good antibody, 96/48 hole) that needs is placed on the grillage;
2) the testing sample 100 μ l after adding is diluted respectively in the reacting hole add each concentration calibration object 0,2,6,20,60,200ng/mL again, and every hole adds 100 μ L, and blank 1 hole is established in each test;
3) the 30 seconds mixings that vibrate gently are with shrouding film shrouding, 37 ℃ of incubation 60min;
4) take out reaction plate, discard solution in the hole, on automatic washer or by hand, wash plate 5 times, on thieving paper, pat dry with the PBST washing lotion;
5) except the blank well, every hole adds the bond 100 μ l that contain biotin labeling pepsin antibody;
6) the 30 seconds mixings that vibrate gently are with shrouding film shrouding, 37 ℃ of incubation 30min;
7) wash plate 5 times as step 4 method, on thieving paper, pat dry;
Except 8 blank well, every hole adds enzyme working fluid 100 μ l;
9) mixing that vibrates gently is with shrouding film shrouding, 37 ℃ of incubation 30min;
10) wash plate 5 times as step 4 method, on thieving paper, pat dry;
11) every hole adds the HRP luminous substrate liquid A 50 μ L that contain 1uminol, adds the HRP luminous substrate liquid B 50 μ L that contain urea peroxide again; Perhaps earlier the A liquid and the B liquid of luminol chemiluminescence substrate solution are pressed mixing in 1: 1, every hole adds 100 μ L;
12) mixing that vibrates gently, room temperature (20～27 ℃) lucifuge reaction 5min;
13) on the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn, Measuring Time 0.1-1 second/hole;
14) read calibration object concentration value and its corresponding RLU value respectively, on the double logarithmic curve of setting up, set up typical curve, on typical curve, find pepsic concentration in this sample, calculate testing result (referring to accompanying drawing 1) with each test serum RLU value;
15) print test results report.
Embodiment 5 evaluations of kit of the present invention on methodology
The kit of preparation among the embodiment 1 is identified according to manufacturing conventional in this area and vertification regulation experiment showed, kit of the present invention when being used for the pepsin concn level determination through a large amount of, this methodology index is as follows:
1) sensing range: 0.3～250ng/mL;
2) limit of identification replicate determination 20 hole null value calibration object serum, calculate null value calibration object RLU mean value (X) and standard deviation (S), with X+2S is in the definite value substitution typical curve equation, obtains its corresponding concentration value, and it is 0.3ng/mL that pairing concentration value is limit of identification.
3) precision: measure the concentration of pepsin antigen quality-control product, duplicate detection 3 times repeats 20 holes at every turn, carry out precision and measure, precision is 4.6% in the computational analysis, less than 10%, precision between analysis (high, medium and low three quality control clearances) is 8.3%, less than 15%;
4) specificity: marks such as mensuration and pepsinogen I, pepsinogen I I, ptyalin, lysozyme carry out cross reaction respectively, and its cross reacting rate is respectively 0.03%, 0.02%, 0.04%, 0.04% all less than 0.1%;
5) stability: each reagent set placed 37 ℃ of baking ovens respectively 3 days, 7 days and 10 days, and experiment finds that each component is still stable.
6) determining of normal person's reference serum: according to the clinical trial result, the range of normal value in anti-stream thing, secretion, this detection method should be less than 5ng/ml.
Kit specimen in use amount of the present invention is few, and required sample source is simple and convenient, vitro detection to tested object without any toxic and side effect.The chemiluminescence enzyme immunoassay method that the present invention simultaneously adopts does not produce radioactive contamination.Utilize the inventive method to detect, highly sensitive, high specificity, sensing range is wide, and is simple to operate, "dead" pollution, the kit cost is low, and clinical applicability is strong, more is applicable to China clinical detection examination laboratory.Therefore the present invention is clinical detection pepsin (Pepsin), and it is a kind of more accurate, special that the auxiliary diagnosis GERD provides, and is convenient, efficiently method.
Claims (8)
1. a pepsin chemiluminescence immune detection reagent kit is characterized in that, described kit comprises:
1) pepsin antigen calibration object; 2) sample collection liquid, pH 2-3; 3) sample diluting liquid, pH 6-8; 4) microwell plate of pepsin antibody sandwich; 5) pepsin antibody labeling thing; 6) chemical luminous substrate liquid; And 7) concentrated cleaning solution.
2. pepsin chemiluminescence immune detection reagent kit according to claim 1 is characterized in that the prescription of described sample collection liquid is
Citric Acid Mono 21g
Proclin300 1mL
Distilled water is settled to 1000mL.
3. pepsin chemiluminescence immune detection reagent kit according to claim 1 is characterized in that the prescription of described sample diluting liquid is
NaH
2PO
4·2H
2O 0.6g
Na
2HPO
4·12H
2O 5.8g
NaCl 8.766g
Bovine serum albumin(BSA) 10g
Proclin300 1mL
Distilled water is settled to 1000mL.
4. pepsin chemiluminescence immune detection reagent kit according to claim 1 is characterized in that, described label is horseradish peroxidase, alkaline phosphatase, biotin and derivant thereof.
5. pepsin chemiluminescence immune detection reagent kit according to claim 1 is characterized in that, described chemical luminous substrate liquid is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
6. pepsin chemiluminescence immune detection reagent kit according to claim 1 is characterized in that, described pepsin antibody is monoclonal antibody or polyclonal antibody.
7. pepsin chemiluminescence immune detection reagent kit according to claim 1 is characterized in that described antibody is derived from mouse, rabbit, goat, sheep, cavy, ox, donkey, horse, chicken, monkey.
8. a method for preparing the described kit of claim 1 is characterized in that, said method comprising the steps of:
1) preparation pepsin antigen calibration object;
2) with pepsin antibody sandwich microwell plate;
3) mark pepsin antibody gets pepsin antibody labeling thing;
4) preparation sample collection liquid, its pH is 2-3;
5) preparation sample diluting liquid, its pH is 6-8;
6) the preparation chemistry substrate solution of giving out light;
7) preparation concentrated cleaning solution;
8) packing antigen calibration object, pepsin antibody labeling thing, chemical luminous substrate liquid and concentrated cleaning solution; And
9) be assembled into finished product.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105742461A CN102128928B (en) | 2010-11-30 | 2010-11-30 | Pepsase chemiluminescent immunoassay kit and preparation method thereof |
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| US20190324018A1 (en) * | 2018-04-19 | 2019-10-24 | Mark D. Noar | Saliva reagent strip for detecting concentration of pepsin or pepsinogen |
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