CN102645537B

CN102645537B - Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application

Info

Publication number: CN102645537B
Application number: CN201210127509.3A
Authority: CN
Inventors: 金鑫
Original assignee: BEIJING COMPULIFE BIOTECH RESEARCH CENTER
Current assignee: BEIJING MOKOBIO LIFE SCIENCE CO., LTD.
Priority date: 2012-04-26
Filing date: 2012-04-26
Publication date: 2014-07-30
Anticipated expiration: 2032-04-26
Also published as: CN102645537A

Abstract

The invention discloses a latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, a preparation method thereof and application. Components of the kit include diluent, blank solution and a latex reagent of an antibody of pepsinogen I or an antibody of pepsinogen II and can further include a calibrator and quality control serum. The latex reagent contains nanoparticles coupled with the antibody of the pepsinogen I or the antibody of the pepsinogen II, and the particle sizes of the nanoparticles are different. The examination sensitivity cannot meet requirements when latex with the single particle size lower than 100nm is used for examination, the linear range is small when latex with the single particle size of about 200nm is used for examination, and accordingly the examination sensitivity and the linear range cannot meet requirements when latex with the single particle size is used for examination. After composite latex of the kit is used for marking, the examination sensitivity can be improved, the linear examination range can be broadened, and the latex enhanced turbidimetric immunoassay kit has the advantages of fast examination, high sensitivity and specificity, good accuracy and the like in terms of gastric disease or gastric cancer examination.

Description

Be used for the latex enhancing immune of diagnosis of gastrosis or cancer of the stomach than turbid kit and its preparation method and application

Technical field

The present invention relates to a kind of kit of in-vitro diagnosis, relate in particular to a kind of by measuring the concentration of experimenter's pepsinogen I and pepsinogen I I and relatively coming the latex enhancing immune of diagnosis of gastrosis or early carcinoma of stomach than turbid kit with the cut-off value of setting, the invention still further relates to preparation method and the application of this kit, belong to diagnosis or the detection field of stomach trouble or cancer of the stomach.

Background technology

Propepsin (PG) is a kind of L-aminobutanedioic acid proteinase precursor, is that molecular mass is the single chain polypeptide of 42kDa, comprises 3 two mercapto keys, and isoelectric point is 3.7.Mankind PG can be divided into 7 components according to its electrophoretic mobility, and the immunogenicity of comparatively fast shifting to positive 1-5 component is approximate, is called pepsinogen I (PG I), mainly by chief cell and the mucus neck cell of gastric gland, is secreted; Component 6-7 is called as pepsinogen I I (PG II), except the chief cell secretion of secreting acid gland by mucous membrane at the bottom of body of stomach and stomach, the mucilage cell of pyloric gland and the Brunner gland of duodenum epimere of secreting mucus neck cell, cardiac gland and the stomach hole of acid gland also can produce PG II.On the gene loci of pepsinogen I and pepsinogen I I, immunoreactivity and biochemical characteristic, all there is some difference.The generation of PG not of the same race seems and following several factor analysis: the modification after the quantity of structural gene, individual allelic difference and translation.Propepsin non-activity in stomach under hydrochloric acid effect, or under acid condition, by autocatalysis, changes activated pepsin into from 42 amino acid residues of N end hydrolysis.Pepsin is restriction endonuclease, can decompose most of protein for mark and peptone, and polypeptide or the amino acid of generation are less.At PG, be transformed in pepsic process, molecular weight is reduced to 35kDa by 42kDa, and isoelectric point reduces to-1 by 3.7, PG in chief cell is stored in the secretory granules at cell top, when cell is upset, by exocytosis major part, be released into lumen of gland, only have 1% to enter blood circulation.PG I concentration in normal human serum is 6 times of PGII.Because stomach is almost unique source of PG, and can change in the secretory volume in secretion stage, therefore, serum PG I and PGII have not only reflected the quantity of stomach lining body of gland and cell, have also indirectly reflected the secreting function of stomach lining different parts.The gastritis that the superficial gastritis of gastroxia and H pylori infect, the secretion of PG I and PG II can increase; And in chronic serious atrophic gastritis, when chief cell reduces, PG I content declines; When atrophic gastritis is with intestines, the false gland metaplasia of stomach sinus gland, PG II content can increase thereupon.When intestinal metaplasia (intestinal metaplasia, IM), atypical hyperplasia, PG I secretion can reduce, and PG I/PG II also can change, and therefore, serum PG can be used as the reliable mark that monitoring gastric mucosa changes.The PG I of high concentration, also as a dangerous subclinical indication of duodenal ulcer and complication thereof, also can be used as and observes the index that H pylori infects eradication therapy curative effect.

Atrophy of gastric mucosa and intestinal metaplasia (particularly forme fruste colonization is raw) and dysplasia are the precancerous lesions of cancer of the stomach, and CAG patient is especially carried out to state of illness monitoring with intestines and dysplasia person, great for the Discovery Significance of early carcinoma of stomach.The sum of Japan and Chinese gastric cancer patients almost accounts for half of global patients with gastric cancer sum.The screening of cancer of the stomach starts from the Japan of the High Risk For Gastric Cancer sixties, adopts at that time the stepped screening method of gastrointestinal double contrast radiography-gastroscope-pathologic finding, and manpower and materials cost is larger.From 1991, serum PG I < 50ng/ml PGI/GPII ratio < 3.0 and PGI < 70ng/ml the PGI/PGII ratio < 3.0 primary dcreening operation index as gastric cancer screening is successively usingd in Japan, go through and within 5 years, detect altogether serum sample 25415 examples, detect cancer of the stomach 43 examples (0.17%), wherein cancer 32 examples (74%) early.Another take the Cut-off value that serum GPI < 70ng/ml GPI/GPII ratio < 3.0 be cancer of the stomach screening, its sensitivity 84.6%, specificity 73.5%, positive predictive value 0.81%, negative predictive value 99.9%.Continue after in 10 years existing more than 130,000 people accepted serum PG method gastric cancer screening, detect patients with gastric cancer 123 people, recall rate 0.13%.

China expert group adopts two-wheeled screening method to carry out gastric cancer screening in 1997-1999 Nian the High Risk For Gastric Cancer In China district, the gastroscopy pathology histodiagnosis of the take two kinds of prescreening methods that have been standard comparative evaluation: barium agent-x-ray double-contrast is according to shadow and Serum Pepsinogen I/II content detection.Result shows, in accepting 1750 people of Serum Pepsinogen I/II (PGI/PGII) detection, take PGI≤50+PGI/PGII≤3 and PGI≤70+PGI/GPII ratio≤3 respectively as primary dcreening operation Cut-off value, the latter sifts out and answers nearly 400 people of gastroscope sifter, person's 17 examples that meet diagnosing gastric cancer, detection sensitivity is about 53%.Specificity reaches 78%, the second takes turns gastroscope and scrutinizes and in this group crowd, detect cancer of the stomach 32 examples.Accordingly, Serum Pepsinogen I/II (PGI/PGII) content detection is better than double-contrast according to shadow, and serum PG I content and PGI/GPII value combine and be more suitable in as Gastric Cancer of Chinese primary dcreening operation and stomach major disease definition.

Summary of the invention

Fundamental purpose of the present invention is to provide that a Species sensitivity is high, the latex enhancing immune of high specificity and good diagnosis of gastrosis or the cancer of the stomach, particularly early carcinoma of stomach of accuracy is than turbid kit;

Another object of the present invention is to provide a kind ofly prepares above-mentioned latex enhancing immune than the method for turbid kit;

A further object of the present invention is to provide the application of described kit in diagnosis of gastrosis or cancer of the stomach.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

A kind of latex enhancing immune for diagnosis of gastrosis or cancer of the stomach of the present invention, than turbid kit, is characterized in that comprising: emulsion reagent and the blank solution of the antibody of dilution, the antibody that contains pepsinogen I or pepsinogen I I;

The Properties of Polystyrene Nano Particles that contains the different-grain diameter that has been coupled the antibody of pepsinogen I or the antibody of pepsinogen I I in wherein said emulsion reagent.

In the present invention, preferably, described dilution is the damping fluid that contains increased response agent, described damping fluid is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid, more preferably phosphate buffer; Described increased response agent is any one or several in PEG-300, PEG2000 or PEG6000, is preferably PEG6000; The sodium chloride that also contains 2mol/L in most preferred described dilution.

In the present invention, preferred, in described emulsion reagent, containing the particle diameter that has been coupled the antibody of pepsinogen I or the antibody of pepsinogen I I is the Properties of Polystyrene Nano Particles that 60-90nm and particle diameter are 160-200nm.

Described emulsion reagent can prepare in accordance with the following methods:

(1) latex pre-treatment

Getting the unlabelled Properties of Polystyrene Nano Particles of 1ml adds in 10ml 0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml 0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml 0.02M pH7.4 phosphate buffer is resuspended, 4 ℃ of preservations are stand-by;

(2) latex particle mark

After step (1) is processed, latex is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, then add 1mg EDC, mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02MPH7.4 phosphate buffer, add the antibody of 1mg pepsinogen I or pepsinogen I I in 10mg/ml2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle is used confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1ml/ and props up, 4 ℃ of preservations,

(3) use above-mentioned steps respectively the latex of particle diameter 60-90nm and 160-200nm to be distinguished to mark, after mark finishes, described 60-90nm latex and described 160-200nm latex are mixed, by quality percent by volume, the concentration that makes 60-90nm latex is 0.673-0.720% (w/v), the concentration that makes 160-200nm latex is 0.147-0.323% (w/v), after mixing, preserve, the above-mentioned flow process of preferred use is distinguished mark to the latex of particle diameter 60-80nm and 160-180nm respectively, after mark finishes, described 60-80nm latex and described 160-180nm latex are mixed, by quality percent by volume, making 60-80nm latex concentration is 0.673-0.708% (w/v), making 160-180nm latex concentration is 0.147-0.235% (w/v), after mixing, preserve,

Described confining liquid is the phosphate buffer of the 0.02M PH7.4 that contains 5g/L BSA;

Described dispersion liquid is by BSA, Tween-20, PVP-K30 and biological preservative are formulated by the phosphate buffer of 0.02M PH7.4, wherein BSA concentration is 1g/L, Tween-20 concentration is 0.05% (v/v), PVP-K30 concentration is 2g/L, biological antiseptic agent concentration is 0.05% (v/v), is PC-300, purchased from sigma company at the biological preservative described in a specific embodiment of the present invention.

In order to reach better detection effect, the former I of described stomach cardia or pepsinogen II are the native proteins extracting from people's mucosa tissue; Its extracting method can be the methods such as ion-exchange and gel-filtration chromatography.

As a reference, a kind of method of extracting pepsinogen I or pepsinogen I I from people's mucosa tissue, comprising:

(1) collect the gastric tissue of surgical resection, use phosphate buffer rinsing, separated gastric mucosa, blots, and weighs, add phosphate buffer, smash homogenate to pieces, centrifugal, collect supernatant, precipitate phosphoric acid salt buffer is resuspended, again centrifugal, merges supernatant, obtains propepsin crude extract;

(2) propepsin crude extract is added in counter-balanced DEAE-52 chromatographic column, with PBS buffer solution elution chromatographic column to the light absorption value of eluent at 280nm place, be 0, continue, with PBS buffer solution elution chromatographic column, to merge eluting peak, obtain containing activated propepsin;

(3) get active protease parent peak, be splined in gel chromatography column, with 50mmol, contain 0.05mol/LNa ₂s0 ₄pBS wash-out, flow velocity 3.0ml/min, pressure is 8Kpa, elution curve is for showing 4 albumen eluting peaks, the 3rd protein peak is pepsinogen I, the 4th protein peak is the potpourri of pepsin antigen I and pepsinogen I I;

(4) adopt the potpourri of the separated pepsinogen I of Q-2 anion-exchange chromatography post and pepsin antigen II, the 4th eluting peak after gel chromatography first used 0.05mol/L Tris-HCL, pH7.0 is the rear loading anion chromatography post of folding thoroughly, by concentration, be 0-0.5mol/L NaCL gradient elution, flow velocity is 1mL/min; Occur three protein peaks, wherein the 1st, 2 peaks are pepsinogen I, and the 3rd peak is pepsinogen I I.

Purity >=98% of the pepsinogen I obtaining through said method purifying, specific activity is 13.6U/mg; Purity >=96.8% of pepsinogen I I, specific activity is 19.7U/mg.

Prepared pepsinogen I or pepsinogen I I immune mouse are prepared to fused cell and just can further obtain secreting the monoclonal antibody of antipepsin antigen I or pepsin antigen II, preferably, described monoclonal antibody is the potpourri for the monoclonal antibody of the different antigenic determinants on pepsinogen I or pepsinogen I I surface.

As a reference, the preparation method of described pepsinogen I or pepsinogen I I monoclonal antibody, comprising:

(1) preparation of fused cell: with 50-100 μ g human pepsinogen I or human pepsinogen II and the Fu Shi Freund's complete adjuvant equal-volume mixing hypodermic injection immunity female BALB/C mice in 8 week age, after At intervals of two to three weeks, with the antigen booster immunization of same dosage 2-3 time, after last immunity, within 3-4 days, getting tail vein detects, when tiring, reach 4000 when above, prepare to merge; After mouse through immune is put to death, partly sterilised cuts open the belly, and under aseptic condition, gets spleen, is prepared into splenocyte suspension; 2-3 days before merging, reaches 4 bottles of cells by every bottle of Sp2/0 myeloma cell, gets cell in exponential phase for merging; After splenocyte and SP2/0 cell are counted respectively, in the ratios of 7: 1, add in 50ml centrifuge tube and mix, centrifugal 10 minutes, use up supernatant, make two kinds of cells be mixed into pasty state, add the 50%PEG 4000 of 37 ℃ of pre-temperature, Fusion of Cells has been stirred on dropping limit, limit.

(2) cell after fusion is prepared into cell suspension with the HAT nutrient solution containing 20% calf serum, and graduation is selected to cultivate in 192 porocyte culture plates; In selecting nutrient solution, cultivate after 4 days, other cell fades away, and only has the hybridoma cluster growth of fusion, form little cell colony, after 8 days, stop using HAT nutrient solution, use HT nutrient solution instead, after one week, use instead containing the DMEM nutrient solution of 10% cow's serum and cultivate; Until hybridoma, cover with 1/3 o'clock of culture hole bottom, at this moment draw culture supernatant and screen.By indirect enzyme-linked absorption method (ELISA), carry out detection of specific antibody, have the hybridoma of the anti-ALB antigen-antibody of secretion;

(3) take human pepsinogen I or pepsinogen I I is envelope antigen, with indirect ELISA method, hybridoma is screened, the positive contrast of serum with immune mouse, the supernatant that the Sp2/0 cell of take is cultivated is blank, not grow clone's the negative contrast of culture supernatant in Tissue Culture Plate; Choose the hybridoma colony to pepsinogen I or the reaction of pepsinogen I I antigen positive, with limiting dilution assay, carry out cloning cultivation, selection antibody titer is high, be single clonal growth, the cell hole that form is good, continuation is carried out 2 time clonings and expands cultivating by limiting dilution assay, obtains secreting the hybridoma cell strain of specific antibody;

(4), at BALB/c lumbar injection whiteruss in 6 week age of homology, the hybridoma of secretion specific monoclonal antibody after clone was injected to (1-2 * 10, abdominal cavity in the approximately the 8th day ⁶cell/mouse), after mouse web portion starts to increase, collect ascites; Mouse is collected after ascites 2-3 time, kills the disposable ascites of getting of mouse, after ascites taking-up, after aseptic filtration, after inner wrapping packing in-20 ℃ frozen, also desirable part is further carried out purifying;

(5) centrifugal cell and the cell fragment of removing in ascites, add isopyknic saturated ammonium sulfate in supernatant, solution is placed on magnetic stirring apparatus and is stirred 6 hours, protein is fully precipitated, sediment is dissolved in a small amount of PBS, with 10mmol/LPBS (pH7.4), at 4 ℃, dialyses 24 hours.Then by DEAE-SephadexA52 chromatographic column, collect and merge protein-contg part in eluent, obtaining the former I of antipepsin or the former II monoclonal antibody of antipepsin of purifying.

In specific embodiments of the invention, pepsinogen I or pepsinogen I I monoclonal antibody are except can producing by the mode of hybridoma, also can directly by commercial sources, buy and obtain, monoclonal antibody at the pepsinogen I described in another specific embodiment of the present invention is monoclonal antibody PGI-8003 and the PGI-8015 (Medix for the different antigenic determinants on pepsinogen I surface, lot number is 0021636) potpourri, the monoclonal antibody of pepsinogen I I is for the monoclonal antibody PGII-8103 of the different antigenic determinants on pepsinogen I I surface and PGII-8101 (Medix, lot number is 0023880) potpourri.Pepsinogen I monoclonal antibody and pepsinogen I I monoclonal antibody also can be bought from Applichem company or Abcam company, and pepsinogen I antibody lot number is respectively M5537 and A1476; Pepsinogen I I antibody lot number is respectively M5538 and A1483, can realize object of the present invention equally.

In kit of the present invention, also can contain standard dilution, calibration object and quality-control product.

Preferably, described standard dilution is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid, is preferably phosphate buffer.

Preferably, described calibration object with in quality-control product, contain from the separated pepsinogen I of people's gastric mucosa or pepsinogen I I.

Kit described in above any one is being prepared diagnosis of gastrosis or cancer of the stomach.The particularly application in early carcinoma of stomach reagent.

The present invention has adopted from the pepsinogen I of people's gastric mucosa separation and pepsinogen I I and as immune mouse, has prepared the immunogene of monoclonal antibody, the standard items of using are also pepsinogen I or the pepsinogen I I adopting from the separation of people's gastric mucosa, make up the next defect of pepsin original structure different band that adopts animal propepsin and people, improved susceptibility, specificity and the accuracy of product diagnosis.

This kit adopts immunoturbidimetry, take Immunoturbidimetry as measuring principle.PG I in sample or PGII and mark the latex particle of anti-PG I antibody or anti-PGII antibody there is specific antigen-antibody reaction, form immune complex, thereby cause the variation of absorbance.The variation of this absorbance and the PG I in sample or PGII concentration are proportional.With PG I or the PGII standard items of concentration known, make working curve, from this curve, can calculate PG I or PGII content in sample.

The present invention assesses experimenter's gastric mucosa situation by pepsinogen I (PGI), the pepsinogen I I (PGII) of determination and analysis thing, be specially adapted to diagnose mucous membrane stomach to change, atrophic gastritis for example, and diagnosable early carcinoma of stomach, described method comprises: the concentration value of measuring pepsinogen I and pepsinogen I I from described experimenter's sample; The predetermined critical of analyte concentration measurement value and this analyte is compared, obtain the combination of the specific comparative result of experimenter.Whether analysis measurement value is greater than, is equal to or less than critical value separately, and the ratio of two analytes is assessed experimenter's gastric mucosa situation, and then show that this information that risk that experimenter gets a cancer of the stomach produces is about being made diagnostic comments by the comparative result of gained or to treatment or further observe and/or detect and advise, and point out experimenter to suffer from the risk of early carcinoma of stomach.

Accompanying drawing explanation

The typical curve of Fig. 1 for adopting PG I standard items concentration to make;

The schematic flow sheet of Fig. 2 for using kit of the present invention to detect.

Embodiment

Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence change, but all will fall within the scope of protection of the present invention.

The purchase source of the related main material of the embodiment of the present invention and reagent:

Q-2 anion-exchange chromatography post, DEAE-Sephadex A52 (DEAE-52) chromatographic column are bought from U.S. GE company;

The female BALB/C mice in 8 week age is bought from Beijing Vital River Experimental Animals Technology Co., Ltd.;

Freund's complete adjuvant, biological preservative PC-300,2-(N-morpholine) ethyl sulfonic acid (MES) is bought from SIGMA;

10% (w/w) Properties of Polystyrene Nano Particles solution is bought from U.S. Bangs;

Pepsinogen I antibody PGI-8003 and PGI-8015 buy from Medix lot number: 0021636,

Pepsinogen I I antibody PGII-8103 and PGII-8101 buy from Medix lot number: 0023880

(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, Tween-20 etc. are purchased from traditional Chinese medicines group for PVP-K30 (Polyvinylpyrrolidone), sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride PFG-6000, EDC;

Bovine serum albumin(BSA) (BSA) is purchased from the prosperous Golden Horse of unit.

The extraction purifying of embodiment 1 pepsinogen I, pepsinogen I I

(1) collect the gastric tissue of surgical resection, use phosphate buffer rinsing, separated gastric mucosa, blots, and weighs, add phosphate buffer, smash homogenate to pieces, centrifugal, collect supernatant, precipitation is resuspended, again centrifugal with phosphate buffer, merges supernatant, obtains propepsin crude extract;

(2) propepsin crude extract is added in counter-balanced DEAE-52 chromatographic column, with PBS buffer solution elution chromatographic column, extremely at 280nm place, light absorption value is 0, continues with PBS buffer solution elution chromatographic column, merge eluting peak, obtain containing activated propepsin;

(3) get active protease parent peak, be splined in gel chromatography column, with 50mmol, contain 0.05mol/LNa ₂sO ₄pBS wash-out, flow velocity 3.0ml/min, pressure is 8Kpa, elution curve is for showing 4 albumen eluting peaks, the 3rd protein peak is pepsinogen I, the 4th protein peak is the potpourri of pepsin antigen I and pepsinogen I I;

(4) adopt the potpourri of the separated pepsinogen I of Q-2 anion-exchange chromatography post and pepsin antigen II, the 4th eluting peak after gel chromatography first used 0.05mol/L Tris-HCL, loading anion chromatography post after pH7.0 dialysis, by concentration, be 0.4mol/L NaCL gradient elution, flow velocity is 1mL/min; Occur three protein peaks, wherein the 1st, 2 peaks are pepsinogen I, and the 3rd peak is pepsinogen I I.

The preparation of the embodiment former I of 2 antipepsin or the former II monoclonal antibody of antipepsin

(1) preparation of fused cell: the 50-100 μ g human pepsinogen I preparing with embodiment 1 or human pepsinogen II and the Freund's complete adjuvant equal-volume mixing hypodermic injection immunity female BALB/C mice in 8 week age, after At intervals of two to three weeks, with the antigen booster immunization of same dosage 2-3 time, after last immunity, within 3-4 days, getting tail vein detects, when tiring, reach 4000 when above, prepare to merge; After mouse through immune is put to death, partly sterilised cuts open the belly, and under aseptic condition, gets spleen, is prepared into splenocyte suspension; 2-3 days before merging, reaches 4 bottles of cells by every bottle of Sp2/0 myeloma cell, gets cell in exponential phase for merging; After splenocyte and SP2/0 cell are counted respectively, in the ratios of 7: 1, add in 50ml centrifuge tube and mix, centrifugal 10 minutes, use up supernatant, make two kinds of cells be mixed into pasty state, add the 50%PEG 4000 of 37 ℃ of pre-temperature, Fusion of Cells has been stirred on dropping limit, limit.

The assembling of embodiment 3 kits (take PG II as example)

1 damping fluid configuration

The preparation of 1.1 dilutions (R1)

Reagent in following table is weighed and puts into clean container, add purified water and mix, measuring pH value is 7.4, constant volume 1000ml.

Reagent	Every liter of volume
		Sodium hydrogen phosphate	2.88g
Sodium dihydrogen phosphate	2.4g
		Sodium chloride	116.88g
PEG-6000	2g
		BSA	10.0g
Tween-20	0.5ml

PC-300	0.5ml
		Add purified water extremely	1000ml

1.2 the preparation of standard dilution

Mentioned reagent is weighed and puts into clean container, add purified water dissolving and mix, measuring pH value is 7.4, is settled to 1000ml.

Reagent	Every liter of volume
		Sodium hydrogen phosphate	1.44g
Potassium dihydrogen phosphate	1.36g
		Sodium chloride	8g
Potassium chloride	0.2g
		BSA	10g
Tween-20	0.5mL
		PC-300	0.5ml
Add purified water extremely	1000ml

2 preparations of the emulsion reagent (reagent 2) containing pepsinogen I I antibody

The configuration of 2.1 damping fluids

2.2 latex pre-treatments

2.2.1 get the unmarked latex particle of 1ml, being about to 1ml 10% Properties of Polystyrene Nano Particles solution adds in 10ml0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml 0.02M pH7.4 phosphate buffer is resuspended, 4 ℃ of preservations are stand-by.

2.3 latex particle marks

According to the quality of latex particle, latex after 2.2 processing is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, add 1mg EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02M PH7.4 phosphate buffer, add the monoclonal antibody 0.5mg PGII-8103 of different antigenic determinants and the potpourri of 0.5mg PGII-8101 for pepsinogen I I surface, antibody is in 10mg/ml 2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle is used confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, and precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1ml/ and props up 4 ℃ of preservations.

Use above-mentioned flow process respectively the latex of particle diameter 70nm and 170nm to be distinguished to mark, after mark finishes, described 70nm latex and described 170nm latex are mixed, making 70nm latex concentration is 0.691% (w/v), and making 170nm latex concentration is 0.191% (w/v).

5, determine extension rate

Get antibody labeling residue supernatant liquid and use respectively OD ₂₆₀and OD ₂₈₀measure its absorbance, utilize measurement result and according to following formula, calculate concentration and the labeling effciency that remains antibody in supernatant; Finally according to labelled antibody amount, determine extension rate.

Antibody concentration=1.45*A ₂₈₀-0.74*A ₂₆₀

Protein content in labelled antibody amount=mark total protein concentration-supernatant

Labeling effciency=(protein content in mark total protein concentration-supernatant)/mark total protein concentration * 100%

6, latex particle dilution

According to antibody excess principle, according to fixed required labelled antibody concentration, with dispersion liquid, the antibody of mark is diluted.

Dilution process: the emulsion reagent that mark is completed dilutes, dilution ratio is 1: 8; 1: 9; 1: 10, use respectively the emulsion reagent after dilution to carry out calibration test, choosing linear optimal is best dilution ratio.

7, packing

By detecting qualified present latex particulate working fluid, carry out packing, labelled after, be placed in 2～8 ℃ of preservations.

8, calibration object and quality-control product preparation

Get 6, sizeable container, use standard diluent preparing concentration point is followed successively by the calibration object each point of 0,5.4,15.0,31.0,64.5,107.8 μ g/L, and the quality-control product of 10 μ g/L, and each concentration point will mix with the raw material strong solution adding successively.

9, packing

Through after the assay was approved, the standard items that prepare and quality-control product are divided and installed in 1ml plastic bottle, 1.0ml/ bottle.

The object of quality-control product: after calibration completes, first test quality-control product, its concentration should be within the scope indicating, thinks that as exceeded the unsuccessful or kit of kit calibration lost efficacy.In the present embodiment, quality-control product concentration is 30 μ g/L, if detectable concentration is between 9-12 μ g/L, thinks that kit is effective, exceeds this scope and assert that measurement result is invalid.

The assembling of embodiment 4 kits (take PG I as example)

1 damping fluid configuration

The preparation of 1.1 dilutions (R1)

Reagent	Every liter of volume
		Sodium hydrogen phosphate	2.88g
Sodium dihydrogen phosphate	2.4g
		Sodium chloride	116.88g
PEG-6000	2g
		BSA	10.0g
Tween-20	0.5ml
		PC-300	0.5ml
Add purified water extremely	1000ml

1.2 the preparation of standard dilution

Reagent	Every liter of volume
		Sodium hydrogen phosphate	1.44g
Potassium dihydrogen phosphate	1.36g
		Sodium chloride	8g
Potassium chloride	0.2g
		BSA	10g

Tween-20	0.5mL
		PC-300	0.5ml
Add purified water extremely	1000ml

2 preparations of the emulsion reagent (reagent 2) containing pepsinogen I antibody

The configuration of 2.1 damping fluids

2.2 latex pre-treatments

2.2.1 get the unmarked latex particle of 1ml, be that 1ml10% Properties of Polystyrene Nano Particles solution adds in 10ml0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml 0.02M pH7.4 phosphate buffer is resuspended, 4 ℃ of preservations are stand-by.

2.3 latex particle marks

According to the quality of latex particle, latex after 2.2 processing is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, add 1mg EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02M PH7.4 phosphate buffer, add 0.5mg PGI-8003 antibody and 0.5mgPGI-8015 antibody in 10mg/ml 2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, is separated to other containers for future use by supernatant; Particle is used confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, and precipitation is redissolved in dispersion liquid; The latex particle that mark is completed is distributed into 1ml/ and props up 4 ℃ of preservations.

Use above-mentioned flow process respectively the latex of particle diameter 75nm and 167nm to be distinguished to mark, after mark finishes, described 75nm latex and described 167nm latex are mixed, making 75nm latex concentration is 0.679% (w/v), and making 167nm latex concentration is 0.178w/v% (w/v).After mixing, preserve.

2.4 determine extension rate

Get antibody labeling residue supernatant liquid and use respectively OD ₂₆₀and OD ₂₈₀measure its absorbance, utilize concentration and the labeling effciency of antibody in measurement result basis formula calculating once residue supernatant; Finally according to labelled antibody amount, determine extension rate.

Antibody concentration=1.45*A ₂₈₀-0.74*A ₂₆₀

Through measurement, calculating protein content in supernatant is 0.367mg, and labelled antibody amount is 1.633mg, and labeling effciency is 81.65%.

2.5 latex particle dilutions

2.6 packing

3 calibration objects and quality-control product

3.1 calibration objects and quality-control product compound method

3.1.1 adopt the preparation of pointwise application of sample method;

3.1.2A be standard dilution;

3.1.3 get 6, sizeable container, by standard diluent preparing concentration point, be followed successively by B, C, D, E, the F calibration object each point of 0,13.2,33.3,67.0,150.5,245.0 μ g/L, and the quality-control product of 30 μ g/L, the raw material strong solution that each concentration point will add successively mixes.

3.2 packing

The object of quality-control product: after calibration completes, first test quality-control product, its concentration should be within the scope indicating, thinks that as exceeded the unsuccessful or kit of kit calibration lost efficacy.In the present embodiment, quality-control product concentration is 30 μ g/L, if detectable concentration is between 26-36 μ g/L, thinks that kit is effective, exceeds this scope and assert that measurement result is invalid.

The using method of embodiment 5 kits of the present invention (the PG I latex enhancing immune that the embodiment 4 of take prepares is example than turbid kit)

Inspection principle: adopt immunoturbidimetry, take Immunoturbidimetry as measuring principle.PG I in sample and mark the latex particle of anti-PG I antibody there is specific antigen-antibody reaction, form immune complex, thereby cause the variation of absorbance.The variation of this absorbance and the PG I concentration in sample are proportional.With the PG I standard items of concentration known, make working curve, from this curve, can calculate the PG I content in sample.

The chief component composition of table 1 kit of the present invention

Form	Loading amount	Principal ingredient
			Dilution (reagent R1)	25mL/ bottle	PBS damping fluid
Emulsion reagent (reagent R2)	5mL/ bottle	The latex solution of anti-human PG I monoclonal antibody
			Blank solution	1mL/ bottle	PBS damping fluid
Calibration object	1mL/ bottle * 5	Pepsin antigen I raw material
			Quality-control product	1mL/ bottle	Pepsin antigen I raw material
Instructions	1 part

Condition of storage and the term of validity: kit should be at 2-8 ℃ kept dry, during storage, avoid weight and should protection against the tide, lucifuge, be Protected from Heat; The term of validity: 12 months.

Applicable instrument: the Biochemical Analyzers such as Toshiba (TBA-40FR), Di Rui (CS-T300), Laura (FAITH-1000), Olympus (AU-1000), Mai Rui (BS-200).

Sample requires: patient specimen, without special processing, adopts conventional medical technology to collect whole blood sample, after centrifuging, draw serum for detection of.Test serum, as used within 24 hours, can, in 2～8 ℃ of preservations, if need long-term storage should be kept at below-20 ℃, and be avoided multigelation.Please do not use significant hemolysis, piarhemia or jaundice sample.

The method of inspection:

1. reagent configuration: reagent i.e. use, and damping fluid and emulsion reagent will fully mix before mensuration;

2. test condition:

Serum specimen: 5ul

R1:150ul R2:30ul wavelength: 578nm temperature of reaction: 37 ℃

First add R1 reagent 150ul, then add serum sample 5ul, 37 ℃ of reactions added R2 reagent 30ul to read OD after 5 minutes, reacted after 5 minutes reading out data under the wavelength of 578nm, and process flow diagram as shown in Figure 2.

3. result is calculated: with standard items concentration, do typical curve, as shown in Figure 1, the antigen concentration in sample is read by typical curve.

Test example 1

1, adopt kit of the present invention according to the methods analyst of embodiment 5 without 280 of stomach trouble crowds (confirm without disease of digestive tract after health check-up, liver, kidney disease, without the crowd of stomachache history) and stomach trouble group 329 examples, (all through gastrocopy, pathology is made a definite diagnosis.Be divided into 5 groups: duodenal bulbar ulcer group 75 examples; Gastric ulcer group 55 examples; Atrophic gastritis group 61 examples; Cancer of the stomach group 113 examples; Cardia cancer group 5 examples.The results are shown in Table 4:

Table 4

According to clinical test results, in conjunction with literature research, kit of the present invention adopts following diagnosis index, and this evaluation is usingd 1-10X as evaluating cancer of the stomach degree of risk, and wherein 1X is minimum, and 10X is the highest:

pG I>=67ng/ml, PG I/PG II>=7.5 are without disease of stomach, cancer of the stomach risk index low (1X);

pG I>=67ng/ml, PG I/PGII≤7.5 duodenal bulbar ulcer or gastric ulcer, cancer of the stomach risk index low (1X)

35ng/ml≤PG I≤67ng/ml, PG I/PGII≤3.0 are diagnosed as atrophic gastritis, cancer of the stomach risk index high (7-9X), suggestion endoscopy.

pG I < 35ng/ml, PG I/PGII≤1.5 are diagnosed as cancer of the stomach, cancer of the stomach risk index (9-10X), suggestion endoscopy is to make a definite diagnosis.

2, the result that adopts kit of the present invention to diagnose model case

Case 1

Case 2

Case 3

Case 4

Claims

For the latex enhancing immune of diagnosis of gastrosis or cancer of the stomach than a turbid kit, it is characterized in that comprising: emulsion reagent and the blank solution of the antibody of dilution, the antibody that contains pepsinogen I or PGⅡ;

Wherein, described dilution is the damping fluid that contains increased response agent, and described damping fluid is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid; Described increased response agent is any one or several in PEG-300, PEG2000 or PEG6000;

Wherein, the Properties of Polystyrene Nano Particles that contains the different-grain diameter that has been coupled the antibody of pepsinogen I or the antibody of PGⅡ in described emulsion reagent;

Described emulsion reagent prepares in accordance with the following methods:

(1) latex pre-treatment

Getting the unlabelled Properties of Polystyrene Nano Particles of 1mL adds in 10mL0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10mL0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5mL0.02M pH7.4 phosphate buffer is resuspended, 4 ℃ of preservations are stand-by;

(2) latex particle mark

After step (1) is processed, latex is diluted to 10mg/mL concentration with 0.02M pH6.1MES damping fluid by latex particle, then add 1mg EDC, mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02M pH7.4 phosphate buffer, add the antibody of 1mg pepsinogen I or PGⅡ in 10mg/mL2mL latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle is used confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1mL/ and props up, 4 ℃ of preservations,

(3) use above-mentioned steps respectively the latex of particle diameter 60-90nm and 160-200nm to be distinguished to mark, after mark finishes, described 60-90nm latex and described 160-200nm latex are mixed, by quality percent by volume, the concentration that makes 60-90nm latex is 0.673-0.720%, making the concentration of 160-200nm latex is 0.147-0.323%, after mixing, preserves;

Described confining liquid is the phosphate buffer of the 0.02M pH7.4 that contains 5g/L BSA;

Described dispersion liquid is by BSA, Tween-20, PVP-K30 and biological preservative are formulated by the phosphate buffer of 0.02M pH7.4, wherein BSA concentration is 1g/L, the concentration of volume percent of Tween-20 is 0.05%, PVP-K30 concentration is 2g/L, and the concentration of volume percent of biological preservative is 0.05%.
2. according to kit claimed in claim 1, it is characterized in that: described dilution is the damping fluid that contains increased response agent, wherein, described damping fluid is phosphate buffer, described increased response agent PEG6000; In described dilution, also contain the sodium chloride of 2mol/L.
3. according to kit claimed in claim 1, it is characterized in that: described emulsion reagent prepares in accordance with the following methods:

(1) latex pre-treatment

Getting the unlabelled Properties of Polystyrene Nano Particles of 1mL adds in 10mL0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10mL0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5mL0.02M pH7.4 phosphate buffer is resuspended, 4 ℃ of preservations are stand-by;

(2) latex particle mark

After step (1) is processed, latex is diluted to 10mg/mL concentration with 0.02M pH6.1MES damping fluid by latex particle, then add 1mg EDC, mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02M pH7.4 phosphate buffer, add the antibody of 1mg pepsinogen I or PGⅡ in 10mg/mL2mL latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle is used confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1mL/ and props up, 4 ℃ of preservations,

(3) use above-mentioned flow process respectively the latex of particle diameter 60-80nm and 160-180nm to be distinguished to mark, after mark finishes, described 60-80nm latex and described 160-180nm latex are mixed, by quality percent by volume, making 60-80nm latex concentration is 0.673-0.708%, making 160-180nm latex concentration is 0.147-0.235%, after mixing, preserves;

Described confining liquid is the phosphate buffer of the 0.02M pH7.4 that contains 5g/L BSA;

Described dispersion liquid is by BSA, Tween-20, PVP-K30 and biological preservative are formulated by the phosphate buffer of 0.02M pH7.4, wherein BSA concentration is 1g/L, the concentration of volume percent of Tween-20 is 0.05%, the concentration of volume percent of PVP-K30 is 2g/L, and the concentration of volume percent of biological preservative is 0.05%.
4. according to kit claimed in claim 1, it is characterized in that: described pepsinogen I or PGⅡ are from people's mucosa tissue, to extract the native protein obtaining.
5. according to kit claimed in claim 4, it is characterized in that described pepsinogen I or PGⅡ are from people's mucosa tissue, to extract and obtain in accordance with the following methods:

(1) collect the gastric tissue of surgical resection, use phosphate buffer rinsing, separated gastric mucosa, blots, and weighs, add phosphate buffer, smash homogenate to pieces, centrifugal, collect supernatant, precipitation is resuspended, again centrifugal with phosphate buffer, merges supernatant, obtains propepsin crude extract;

(2) propepsin crude extract is added in counter-balanced DEAE-52 chromatographic column, with PBS buffer solution elution chromatographic column to the light absorption value of eluent at 280nm place, be 0, continue, with PBS buffer solution elution chromatographic column, to merge eluting peak, obtain containing activated propepsin;

(3) get active protease parent peak, be splined in gel chromatography column, with 50mmol, contain 0.05mol/LNa ₂sO ₄pBS wash-out, flow velocity 3.0mL/min, pressure is 8Kpa, elution curve is for showing 4 albumen eluting peaks, the 3rd protein peak is pepsinogen I, the 4th protein peak is the potpourri of pepsin antigen I and PGⅡ;

(4) adopt the potpourri of the separated pepsinogen I of Q-2 anion-exchange chromatography post and pepsin antigen II, the 4th eluting peak after gel chromatography first used 0.05mol/L Tris-HCL, loading anion chromatography post after pH7.0 dialysis, by concentration, be 0-0.5mol/L NaCL gradient elution, flow velocity is 1mL/min; Occur three protein peaks, wherein the 1st, 2 peaks are pepsinogen I, and the 3rd peak is PGⅡ.
6. according to kit claimed in claim 1, it is characterized in that: the antibody of described pepsinogen I or the antibody of PGⅡ are monoclonal antibody.
7. according to kit claimed in claim 6, it is characterized in that: described monoclonal antibody is the potpourri of the monoclonal antibody of the different antigenic determinants for pepsinogen I or PGⅡ surface.
8. according to kit claimed in claim 1, it is characterized in that: also contain standard dilution, calibration object and quality-control product, described standard dilution is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid.
9. according to kit claimed in claim 8, it is characterized in that: described standard dilution is phosphate buffer.
10. according to kit claimed in claim 8, it is characterized in that: described calibration object with in quality-control product, contain from people's gastric mucosa separated propepsin I or PGⅡ.
The application of kit described in 11. claim 1-10 any one in preparing diagnosis of gastrosis or cancer of the stomach reagent.