CN102645537A - Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application - Google Patents
Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application Download PDFInfo
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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
Technical field
The present invention relates to a kind of kit of in-vitro diagnosis; Relate in particular to a kind of concentration and latex enhancing immune of relatively diagnosing stomach trouble or early carcinoma of stomach with the cut-off value of setting through measuring experimenter's pepsinogen I and pepsinogen I I than turbid kit; The invention still further relates to the preparation method and the application of this kit, belong to the diagnosis or the detection range 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.Human 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), main chief cell and the secretion of mucus neck cell by gastric gland; Component 6-7 is called as pepsinogen I I (PG II); Except that the chief cell secretion of secreting acid gland by mucous membrane at the bottom of body of stomach and the stomach, mucilage cell and the Brunner gland of duodenum epimere of pyloric gland of secreting mucus neck cell, cardiac gland and the stomach hole of acid gland also can produce PG II.All there is some difference on the gene loci of pepsinogen I and pepsinogen I I, immunoreactivity and the biochemical characteristic.As if the generation of PG not of the same race is relevant with following several kinds of factors: the modification after the quantity of structural gene, individual allelic difference and the translation.The propepsin non-activity is in stomach under the hydrochloric acid effect, or under acid condition, through autocatalysis, behind 42 amino acid residues of N end hydrolysis and change activated pepsin into.Pepsin is a restriction endonuclease, can decompose most of protein and be mark and peptone, and the polypeptide or the amino acid of generation are less.Be transformed in the pepsic process at PG, molecular weight is reduced to 35kDa by 42kDa, and isoelectric point reduces to-1 by 3.7; PG in the chief cell is stored in the secretory granules at cell top; When cell is upset, be released into lumen of gland through the exocytosis major part, have only 1% to get into blood circulation.PG I concentration in normal human serum is 6 times of PGII.Because stomach almost is 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 reflected the secreting function of stomach lining different parts indirectly.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, PG I content descends when chief cell reduces; When atrophic gastritis with the false pylorus glandular metaplasia of intestinesization, stomach sinus gland, PG II content can increase thereupon.When intestinal metaplasia (intestinal metaplasia, IM), during atypical hyperplasia, PG I secretion can reduce, PG I/PG II also can change, therefore, serum PG can be used as the reliable mark that the monitoring gastric mucosa changes.The PG I of high concentration also can be used as and observes the index that H pylori infects the eradication therapy curative effect also as a subclinical indication of the danger of duodenal ulcer and complication thereof.
Atrophy of gastric mucosa and intestinal metaplasia (particularly the forme fruste colonization is given birth to) and dysplasia are the precancerous lesions of cancer of the stomach, and CAG patient is especially carried out state of illness monitoring with intestinesization with the dysplasia person, and be significant for the discovery of early carcinoma of stomach.Sum Japanese and Chinese patients with gastric cancer almost accounts for the half the of global patients with gastric cancer sum.The screening of cancer of the stomach starts from cancer of the stomach sixties Japan occurred frequently, adopts the stepped screening method of gastrointestinal double contrast radiography-gastroscope-pathologic finding at that time, and the manpower and materials cost is bigger.From beginning in 1991; Japan is successively with serum PG I＜50ng/ml and PGI/GPII ratio＜3.0 and PGI＜70ng/ml and the PGI/PGII ratio＜3.0 primary dcreening operation index as gastric cancer screening; Go through and detected serum sample 25415 examples in 5 years altogether; Detect cancer of the stomach 43 examples (0.17%), wherein cancer 32 examples (74%) early.Be the Cut-off value of cancer of the stomach screening with serum GPI＜70ng/ml and GPI/GPII ratio＜3.0 in addition, its sensitivity 84.6%, specificity 73.5%, positive predictive value 0.81%, negative predictive value 99.9%.Continue after in 10 years surplus in the of existing 130,000 the people accepted serum PG method gastric cancer screening, detect patients with gastric cancer 123 people, recall rate 0.13%.
China expert group adopts the two-wheeled screening method to carry out gastric cancer screening in 1997-1999 in Chinese cancer of the stomach district occurred frequently, with the gastroscopy pathology histodiagnosis two kinds of prescreening methods that have been the standard comparative evaluation: barium agent-X line biconjugate is according to shadow and Serum Pepsinogen I/II content detection.The result shows; Accepting 1750 philtrums that Serum Pepsinogen I/II (PGI/PGII) detects; Be primary dcreening operation Cut-off value with PGI≤50+PGI/PGII≤3 and PGI≤70+PGI/GPII ratio≤3 respectively; The latter sifts out and answers nearly 400 people of gastroscope sifter, person's 17 examples that meet the diagnosing gastric cancer, and 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.In view of the above, Serum Pepsinogen I/II (PGI/PGII) content detection is superior to biconjugate according to shadow, and serum PG I content and PGI/GPII value combine and be more suitable in generally investigating standard as Chinese's cancer of the stomach primary dcreening operation and stomach major disease.
Summary of the invention
Fundamental purpose of the present invention provides that a kind of susceptibility is high, the latex enhancing immune of the diagnosis stomach trouble or the cancer of the stomach, particularly early carcinoma of stomach of high specificity and accuracy good is than turbid kit;
Another object of the present invention provides a kind of method for preparing above-mentioned latex enhancing immune than turbid kit;
A further object of the present invention provides the application of said kit in diagnosis stomach trouble or cancer of the stomach.
Above-mentioned purpose of the present invention realizes through following technical scheme:
A kind of latex enhancing immune that is used to diagnose stomach trouble or cancer of the stomach of the present invention is characterized in that comprising: the emulsion reagent and the blank solution of the antibody of dilution, the antibody that contains pepsinogen I or pepsinogen I I than turbid kit;
Contain the pipe/polyhenylethylene nano microballoon of different-grain diameter of antibody that coupling has joined antibody or the pepsinogen I I of pepsinogen I in the wherein said emulsion reagent.
In the present invention; Preferably; Described dilution is the damping fluid that contains the 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 the HEPPS damping fluid, more preferably phosphate buffer; Described increased response agent is any or several kinds among PEG-300, PEG2000 or the PEG6000, is preferably PEG6000; The sodium chloride that also contains 2mol/L in the most preferred described dilution.
In the present invention, preferred, containing the particle diameter of antibody that coupling joined antibody or the pepsinogen I I of pepsinogen I in the described emulsion reagent is that 60-90nm and particle diameter are the pipe/polyhenylethylene nano microballoon of 160-200nm.
Described emulsion reagent can prepare according to following method:
(1) latex pre-treatment
Getting the unlabelled pipe/polyhenylethylene nano microballoon of 1ml adds in the 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, remove supernatant and resuspended with 5ml 0.02M pH7.4 phosphate buffer, 4 ℃ of preservations are for use;
(2) latex particle mark
Step (1) is handled back latex latex particle is diluted to 10mg/ml concentration, add 1mg EDC then, mixing activation 15 minutes with 0.02M pH6.1MES damping fluid; Centrifugal 8000rpm supernatant discarded, and resuspended with the 0.02MPH7.4 phosphate buffer, and the antibody that adds 1mg pepsinogen I or pepsinogen I I is in the 10mg/ml2ml latex fluid; Room temperature mixing 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm is separated to other containers for future use with supernatant; Particle uses confining liquid resuspended, and room temperature mixing 1 hour, the centrifugal 15min of hydro-extractor 8000rpm; Deposition is redissolved in dispersion liquid, and the latex particle that mark is accomplished 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 mark; Mark after finishing mixes said 60-90nm latex and said 160-200nm latex, and by mass and size number percent, 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); Preserve after the mixing, preferably use above-mentioned flow process respectively the latex of particle diameter 60-80nm and 160-180nm to be distinguished mark, mark after finishing mixes said 60-80nm latex and said 160-180nm latex; By mass and size number percent; Making the 60-80nm latex concentration is 0.673-0.708% (w/v), and making the 160-180nm latex concentration is 0.147-0.235% (w/v), preserves after the mixing;
Described confining liquid is the phosphate buffer that contains the 0.02M PH7.4 of 5g/L BSA;
Described dispersion liquid is by BSA; Tween-20, PVP-K30 and biological preservative are formulated through the phosphate buffer of 0.02M PH7.4, and wherein BSA concentration is 1g/L, and Tween-20 concentration is 0.05% (v/v); PVP-K30 concentration is 2g/L; The biological antiseptic agent concentration is 0.05% (v/v), is PC-300 at the biological preservative described in the specific embodiment of the present invention, available from sigma company.
In order to reach better detection effect, former I of described stomach cardia or pepsinogen II are the native proteins that from people's mucosa tissue, extracts; Its method for distilling can be methods such as ion-exchange and gel-filtration chromatography.
As a reference, a kind of method of from people's mucosa tissue, extracting pepsinogen I or pepsinogen I I comprises:
(1) gastric tissue of collection surgical resection is used the phosphate buffer rinsing, separates gastric mucosa, blots, and weighs; Add phosphate buffer, smash homogenate to pieces, centrifugal, collect supernatant; The precipitate phosphoric acid salt buffer is resuspended, and is centrifugal once more, merges supernatant, obtains the propepsin crude extract;
(2) the propepsin crude extract is added in the DEAE-52 chromatographic column of balance; Using PBS buffer solution elution chromatographic column to the light absorption value of eluent at the 280nm place is 0; Continue to merge eluting peak, obtain containing active propepsin with PBS buffer solution elution chromatographic column;
(3) get the active protease parent peak, be splined in the gel chromatography column, contain 0.05mol/LNa with 50mmol
2S0
4The PBS wash-out, flow velocity 3.0ml/min, pressure are 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 Q-2 anion-exchange chromatography post to separate the potpourri of pepsinogen I and pepsin antigen II; The 4th eluting peak behind the gel chromatography used earlier 0.05mol/L Tris-HCL; PH7.0 passes through the folding back and goes up appearance anion chromatography post; Use concentration to be 0-0.5mol/L NaCL gradient elution, flow velocity is 1mL/min; Three protein peaks occur, wherein the 1st, 2 peaks are pepsinogen I, and the 3rd peak is pepsinogen I I.
Purity >=98% of the pepsinogen I that process said method purifying obtains, specific activity is 13.6U/mg; The purity of pepsinogen I I >=96.8%, specific activity are 19.7U/mg.
Prepared pepsinogen I or pepsinogen I I immune mouse are prepared the monoclonal antibody that fused cell just can further obtain secreting antipepsin antigen I or pepsin antigen II; Preferably, described monoclonal antibody is the potpourri to the monoclonal antibodies of the different antigenic determinants on pepsinogen I or pepsinogen I I surface.
As a reference, said pepsinogen I or pepsinogen I I MONOCLONAL ANTIBODIES SPECIFIC FOR method comprise:
(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 ages in week; Behind the At intervals of two to three weeks; With the antigen booster immunization of same dosage 2-3 time; The back was got tail vein in 3-4 days and is detected after the last immunity, reached 4000 when tiring when above, promptly prepared fusion; Will be after mice immunized be put to death, partly sterilised cuts open the belly, and gets spleen under the aseptic condition, is prepared into splenocyte suspension; Merged preceding 2-3 days, every bottle of Sp2/0 myeloma cell is reached 4 bottles of cells, get the cell that is in exponential phase and be used for merging; After splenocyte and SP2/0 cell counted respectively, add in the 50ml centrifuge tube in 7: 1 ratios and to mix centrifugal 10 minutes; Use up supernatant; Make two kinds of cells be mixed into pasty state, add 37 ℃ of 50%PEG 4000 of temperature in advance, stir while dripping and accomplish Fusion of Cells.
(2) cell after the fusion becomes cell suspension with the HAT inoculum preparation that contains 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 has only the hybridoma cluster growth of fusion; Form little cell colony, after 8 days, stop using the HAT nutrient solution; Use the HT nutrient solution instead, use the DMEM nutrient solution that contains 10% cow's serum after the week instead and cultivate; Treat that hybridoma covers with 1/3 o'clock of culture hole bottom, at this moment draws culture supernatant and screens.Carry out specific antibody with indirect enzyme-linked absorption method (ELISA) and detect, the hybridoma of the anti-ALB antigen-antibody of secretion is arranged;
(3) be envelope antigen with human pepsinogen I or pepsinogen I I; With indirect ELISA method hybridoma is screened; With the positive contrast of the serum of immune mouse; Supernatant with the Sp2/0 cellular incubation is a blank, with the negative contrast of the culture supernatant that does not grow the clone in the Tissue Culture Plate; Choose hybridoma colony to pepsinogen I or the reaction of pepsinogen I I antigen positive; Carrying out cloning with limiting dilution assay cultivates; The selection antibody titer is high, is single clonal growth, form good cell hole; Continuation is carried out 2 time clonings and enlarged culture by limiting dilution assay, obtains secreting the hybridoma cell strain of specific antibody;
(4) homology 6 age in week BALB/c lumbar injection whiteruss, will clone hybridoma injection (1-2 * 10, abdominal cavity of back secretion specific monoclonal antibody in about the 8th day
6Cell/mouse), treat that mouse web portion begins to increase after, collect ascites; Mouse kills the disposable ascites of getting of mouse after collecting ascites 2-3 time, after the ascites taking-up, after the aseptic filtration, after the inner wrapping packing in-20 ℃ frozen, also desirable part is further carried out purifying;
(5) centrifugal cell and the cell fragment of removing in the ascites; Add isopyknic saturated ammonium sulfate in supernatant, solution is placed on the magnetic stirring apparatus stirred 6 hours, protein is fully precipitated; Sediment is dissolved among the little P BS, dialysed 24 hours at 4 ℃ with 10mmol/LPBS (pH7.4).Through the DEAE-SephadexA52 chromatographic column, collection also merges protein-contg part in the eluent, promptly obtains the former I of antipepsin or the former II monoclonal antibody of antipepsin of purifying then.
In specific embodiment of the present invention; Pepsinogen I or pepsinogen I I monoclonal antibody are except can producing through the mode of hybridoma; Also can directly buy and obtain through commercial sources; Monoclonal antibody at the pepsinogen I described in another specific embodiment of the present invention is monoclonal antibody PGI-8003 and PGI-8015 (Medix to the surperficial different antigenic determinants of pepsinogen I; Lot number is 0021636) potpourri, the monoclonal antibody of pepsinogen I I is for to monoclonal antibody PGII-8103 of the different antigenic determinants on pepsinogen I I surface and the potpourri of PGII-8101 (Medix, lot number are 0023880).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 the object of the invention equally.
Also can contain standard dilution, calibration object and quality-control product in the kit of the present invention.
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 the HEPPS damping fluid, is preferably phosphate buffer.
Preferably, contain pepsinogen I or the pepsinogen I I that separates from people's gastric mucosa in said calibration object and the quality-control product.
More than each described kit in preparation diagnosis stomach trouble or cancer of the stomach.The particularly application in the early carcinoma of stomach reagent.
The immunogene that the present invention has adopted the pepsinogen I that separates from people's gastric mucosa and pepsinogen I I to prepare monoclonal antibody as immune mouse; The standard items of using also are to adopt pepsinogen I or the pepsinogen I I that separates from people's gastric mucosa; Remedy the next defective of pepsin original structure different band that adopts animal propepsin and people, improved product diagnostic sensitivity, specificity and accuracy.
This kit adopts immunoturbidimetry, is measuring principle with latex enhance immunity turbidimetry.PG I in the sample or PGII and mark the latex particle of anti-PG I antibody or anti-PGII antibody specific antigen-antibody reaction takes place, form immune complex, thereby cause the variation of absorbance.The variation of this absorbance and the PG I in the sample or PGII concentration are proportional.PG I or PGII standard items with concentration known are made working curve, from this curve, can calculate PG I or PGII content the sample.
The present invention assesses experimenter's gastric mucosa situation through pepsinogen I (PGI), the pepsinogen I I (PGII) of determination and analysis thing; Be specially adapted to diagnose the mucous membrane stomach to change; Atrophic gastritis for example; And diagnosable early carcinoma of stomach, said method comprises: the concentration value of from said experimenter's sample, measuring pepsinogen I and pepsinogen I I; The predetermined critical of analyte concentration measurement value and this analyte is compared, obtain the combination of the specific comparative result of experimenter.The analysis measurement value whether greater than, be equal to or less than critical value separately; And the ratio of two analytes is assessed experimenter's gastric mucosa situation; And then to draw this information that risk that the experimenter gets a cancer of the stomach produces be about being made diagnostic comments by the comparative result of gained or to treatment or further observe and/or detect and advise, and the prompting experimenter suffers from the risk of early carcinoma of stomach.
Description of drawings
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
Through experiment and combination embodiment the present invention is further specified below, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.Those of ordinary skills understand, and in spirit that claim of the present invention limited and scope, can carry out many changes to it, revise, even equivalence change, but all will fall in protection scope of the present invention.
The purchase source of related main material of the embodiment of the 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 ages in week is bought from Beijing Vital River Experimental Animals Technology Co., Ltd.;
Freund's complete adjuvant, biological preservative PC-300,2-(N-morphine quinoline) ethyl sulfonic acid (MES) are bought from SIGMA;
10% (w/w) pipe/polyhenylethylene nano microspheres solution is bought from U.S. Bangs;
Pepsinogen I antibody PGI-8003 and PGI-8015 buy from the Medix lot number: 0021636,
Pepsinogen I I antibody PGII-8103 and PGII-8101 buy from the Medix lot number: 0023880
(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, Tween-20 etc. are available 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 available from the prosperous Golden Horse of unit.
The extraction purifying of embodiment 1 pepsinogen I, pepsinogen I I
(1) gastric tissue of collection surgical resection is used the phosphate buffer rinsing, separates gastric mucosa, blots, and weighs; Add phosphate buffer, smash homogenate to pieces, centrifugal, collect supernatant; Precipitate resuspendedly with phosphate buffer, centrifugal once more, merge supernatant, obtain the propepsin crude extract;
(2) the propepsin crude extract is added in the DEAE-52 chromatographic column of balance, at 280nm place, light absorption value is 0 with PBS buffer solution elution chromatographic column, continues with PBS buffer solution elution chromatographic column, and the merging eluting peak obtains containing the propepsin of activity;
(3) get the active protease parent peak, be splined in the gel chromatography column, contain 0.05mol/LNa with 50mmol
2SO
4The PBS wash-out, flow velocity 3.0ml/min, pressure are 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 Q-2 anion-exchange chromatography post to separate the potpourri of pepsinogen I and pepsin antigen II; The 4th eluting peak behind the gel chromatography used earlier 0.05mol/L Tris-HCL; Appearance anion chromatography post is gone up in pH7.0 dialysis back; Use concentration to be 0.4mol/L NaCL gradient elution, flow velocity is 1mL/min; Three protein peaks occur, wherein the 1st, 2 peaks are pepsinogen I, and the 3rd peak is pepsinogen I I.
Purity >=98% of the pepsinogen I that process said method purifying obtains, specific activity is 13.6U/mg; The purity of pepsinogen I I >=96.8%, specific activity are 19.7U/mg.
The former II MONOCLONAL ANTIBODIES SPECIFIC FOR of former I of embodiment 2 antipepsins or antipepsin
(1) preparation of fused cell: the female BALB/C mice in 50-100 μ g human pepsinogen I for preparing with embodiment 1 or human pepsinogen II and 8 ages in week of Freund's complete adjuvant equal-volume mixing hypodermic injection immunity; Behind the At intervals of two to three weeks; With the antigen booster immunization of same dosage 2-3 time; The back was got tail vein in 3-4 days and is detected after the last immunity, reached 4000 when tiring when above, promptly prepared fusion; Will be after mice immunized be put to death, partly sterilised cuts open the belly, and gets spleen under the aseptic condition, is prepared into splenocyte suspension; Merged preceding 2-3 days, every bottle of Sp2/0 myeloma cell is reached 4 bottles of cells, get the cell that is in exponential phase and be used for merging; After splenocyte and SP2/0 cell counted respectively, add in the 50ml centrifuge tube in 7: 1 ratios and to mix centrifugal 10 minutes; Use up supernatant; Make two kinds of cells be mixed into pasty state, add 37 ℃ of 50%PEG 4000 of temperature in advance, stir while dripping and accomplish Fusion of Cells.
(2) cell after the fusion becomes cell suspension with the HAT inoculum preparation that contains 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 has only the hybridoma cluster growth of fusion; Form little cell colony, after 8 days, stop using the HAT nutrient solution; Use the HT nutrient solution instead, use the DMEM nutrient solution that contains 10% cow's serum after the week instead and cultivate; Treat that hybridoma covers with 1/3 o'clock of culture hole bottom, at this moment draws culture supernatant and screens.Carry out specific antibody with indirect enzyme-linked absorption method (ELISA) and detect, the hybridoma of the anti-ALB antigen-antibody of secretion is arranged;
(3) be envelope antigen with human pepsinogen I or pepsinogen I I; With indirect ELISA method hybridoma is screened; With the positive contrast of the serum of immune mouse; Supernatant with the Sp2/0 cellular incubation is a blank, with the negative contrast of the culture supernatant that does not grow the clone in the Tissue Culture Plate; Choose hybridoma colony to pepsinogen I or the reaction of pepsinogen I I antigen positive; Carrying out cloning with limiting dilution assay cultivates; The selection antibody titer is high, is single clonal growth, form good cell hole; Continuation is carried out 2 time clonings and enlarged culture by limiting dilution assay, obtains secreting the hybridoma cell strain of specific antibody;
(4) homology 6 age in week BALB/c lumbar injection whiteruss, will clone hybridoma injection (1-2 * 10, abdominal cavity of back secretion specific monoclonal antibody in about the 8th day
6Cell/mouse), treat that mouse web portion begins to increase after, collect ascites; Mouse kills the disposable ascites of getting of mouse after collecting ascites 2-3 time, after the ascites taking-up, after the aseptic filtration, after the inner wrapping packing in-20 ℃ frozen, also desirable part is further carried out purifying;
(5) centrifugal cell and the cell fragment of removing in the ascites; Add isopyknic saturated ammonium sulfate in supernatant, solution is placed on the magnetic stirring apparatus stirred 6 hours, protein is fully precipitated; Sediment is dissolved among the little P BS, dialysed 24 hours at 4 ℃ with 10mmol/LPBS (pH7.4).Through the DEAE-SephadexA52 chromatographic column, collection also merges protein-contg part in the eluent, promptly obtains the former I of antipepsin or the former II monoclonal antibody of antipepsin of purifying then.
The assembling of embodiment 3 kits (is example with PG II)
The configuration of 1 damping fluid
1.1 the preparation of dilution (R1)
Reagent weighing in the following table is put into clean container well, add the purified water mixing, measuring the pH value is 7.4, constant volume 1000ml.
| Reagent | Every liter of liquid consumption |
| Sodium hydrogen phosphate | 2.88g |
| Sodium dihydrogen phosphate | 2.4g |
| Sodium chloride | 116.88g |
| PEG-6000 | 2g |
| BSA | 10.0g |
| Tween-20 | 0.5ml |
[0072]
| PC-300 | 0.5ml |
| Add purified water extremely | 1000ml |
1.2 the preparation of standard dilution
The mentioned reagent weighing is put into clean container well, add purified water dissolving mixing, measuring the pH value is 7.4, is settled to 1000ml.
| Reagent | Every liter of liquid consumption |
| 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 contain emulsion reagent (reagent 2) preparation of pepsinogen I I antibody
2.1 the configuration of damping fluid
2.2 latex pre-treatment
2.2.1 get the unmarked latex particle of 1ml; Being about to 1ml 10% pipe/polyhenylethylene nano microspheres solution adds in the 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, remove supernatant and resuspended with 5ml 0.02M pH7.4 phosphate buffer, 4 ℃ of preservations are for use.
2.3 latex particle mark
Handle back latex according to the quality of latex particle with 2.2 and latex particle is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid; Add 1mg EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), mixing activation 15 minutes, centrifugal 8000rpm supernatant discarded; And it is resuspended with 0.02M PH7.4 phosphate buffer; Add to monoclonal antibody 0.5mg PGII-8103 of the different antigenic determinants on pepsinogen I I surface and the potpourri of 0.5mg PGII-8101, antibody is in 10mg/ml 2ml latex fluid, room temperature mixing 2-4 hour; Centrifugal 15 minutes of hydro-extractor 8000rpm is separated to other containers for future use with supernatant; Particle uses confining liquid resuspended, and room temperature mixing 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, and deposition is redissolved in dispersion liquid; The latex particle that mark is accomplished 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 mark; Mark after finishing mixes said 70nm latex and said 170nm latex; Making the 70nm latex concentration is 0.691% (w/v), and making the 170nm latex concentration is 0.191% (w/v).
5, confirm extension rate
Get antibody labeling residue supernatant liquid and use OD respectively
260And OD
280Measure its absorbance, utilize measurement result and calculate the concentration and the labeling effciency of antibody in the residue supernatant according to following formula; Confirm extension rate according to the labelled antibody amount at last.
AC=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 the antibody excess principle, dilute with the antibody of dispersion liquid mark according to fixed required labelled antibody concentration.
Dilution process: the emulsion reagent that mark is accomplished dilutes, and dilution ratio is 1: 8; 1: 9; 1: 10, use the emulsion reagent after diluting to carry out calibration test respectively, choosing linear optimal is best dilution ratio.
7, packing
Carry out packing with detecting qualified present latex particulate working fluid, labelled after, place 2～8 ℃ of preservations.
8, calibration object and quality-control product preparation
Get 6 in sizeable container, use standard diluent preparing concentration point to be 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, each concentration point will with the raw material strong solution mixing that adds successively.
9, packing
Through after the assay was approved, standard items that prepare and quality-control product branch are installed in the 1ml plastic bottle 1.0ml/ bottle.
The purpose of quality-control product: after calibration is accomplished, at first test quality-control product, its concentration should be within the scope that indicates, as exceeds and think that then unsuccessful the or kit of kit calibration lost efficacy.Quality-control product concentration is 30 μ g/L in the present embodiment, if detectable concentration is between the 9-12 μ g/L, thinks that then kit is effective, exceeds this scope and assert that then the mensuration result is invalid.
The assembling of embodiment 4 kits (is example with PG I)
The configuration of 1 damping fluid
1.1 the preparation of dilution (R1)
Reagent weighing in the following table is put into clean container well, add the purified water mixing, measuring the pH value is 7.4, constant volume 1000ml.
| Reagent | Every liter of liquid consumption |
| 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
The mentioned reagent weighing is put into clean container well, add purified water dissolving mixing, measuring the pH value is 7.4, is settled to 1000ml.
| Reagent | Every liter of liquid consumption |
| Sodium hydrogen phosphate | 1.44g |
| Potassium dihydrogen phosphate | 1.36g |
| Sodium chloride | 8g |
| Potassium chloride | 0.2g |
| BSA | 10g |
[0107]
| Tween-20 | 0.5mL |
| PC-300 | 0.5ml |
| Add purified water extremely | 1000ml |
2 contain emulsion reagent (reagent 2) preparation of pepsinogen I antibody
2.1 the configuration of damping fluid
2.2 latex pre-treatment
2.2.1 get the unmarked latex particle of 1ml; Be that 1ml10% pipe/polyhenylethylene nano microspheres solution adds in the 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, remove supernatant and resuspended with 5ml 0.02M pH7.4 phosphate buffer, 4 ℃ of preservations are for use.
2.3 latex particle mark
Handle back latex according to the quality of latex particle with 2.2 and latex particle is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid; Add 1mg EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride); Mixing 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 mixing 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm is separated to other containers for future use with supernatant; Particle uses confining liquid resuspended, and room temperature mixing 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, and deposition is redissolved in dispersion liquid; The latex particle that mark is accomplished 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 mark; Mark after finishing mixes said 75nm latex and said 167nm latex; Making the 75nm latex concentration is 0.679% (w/v), and making the 167nm latex concentration is 0.178w/v% (w/v).Preserve after the mixing.
2.4 confirm extension rate
Get antibody labeling residue supernatant liquid and use OD respectively
260And OD
280Measure its absorbance, utilize the concentration and the labeling effciency of antibody in measurement result and the basis formula calculating once residue supernatant; Confirm extension rate according to the labelled antibody amount at last.
AC=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%
Calculate through measuring that protein content is 0.367mg in the supernatant, the labelled antibody amount is 1.633mg, and labeling effciency is 81.65%.
2.5 latex particle dilution
According to the antibody excess principle, dilute with the antibody of dispersion liquid mark according to fixed required labelled antibody concentration.
Dilution process: the emulsion reagent that mark is accomplished dilutes, and dilution ratio is 1: 8; 1: 9; 1: 10, use the emulsion reagent after diluting to carry out calibration test respectively, choosing linear optimal is best dilution ratio.
2.6 packing
Carry out packing with detecting qualified present latex particulate working fluid, labelled after, place 2～8 ℃ of preservations.
3 calibration objects and quality-control product
3.1 calibration object and quality-control product compound method
3.1.1 adopt the preparation of pointwise application of sample method;
3.1.2A be the standard dilution;
3.1.3 get 6 in sizeable container; 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 with standard diluent preparing concentration point; And the quality-control product of 30 μ g/L, the raw material strong solution mixing that each concentration point will add successively.
3.2 packing
Through after the assay was approved, standard items that prepare and quality-control product branch are installed in the 1ml plastic bottle 1.0ml/ bottle.
The purpose of quality-control product: after calibration is accomplished, at first test quality-control product, its concentration should be within the scope that indicates, as exceeds and think that then unsuccessful the or kit of kit calibration lost efficacy.Quality-control product concentration is 30 μ g/L in the present embodiment, if detectable concentration is between the 26-36 μ g/L, thinks that then kit is effective, exceeds this scope and assert that then the mensuration result is invalid.
The method of application of embodiment 5 kits of the present invention (the PG I latex enhancing immune for preparing with embodiment 4 is an example than turbid kit)
Inspection principle: adopting immunoturbidimetry, is measuring principle with latex enhance immunity turbidimetry.PG I in the sample and mark the latex particle of anti-PG I antibody specific antigen-antibody reaction takes place, form immune complex, thereby cause the variation of absorbance.The variation of this absorbance and the PG I concentration in the sample are proportional.PG I standard items with concentration known are made working curve, from this curve, can calculate the PG I content the sample.
The main composition of table 1 kit of the present invention
| Form | Loading amount | Principal ingredient |
| Dilution (reagent R1) | The 25mL/ bottle | The PBS damping fluid |
| Emulsion reagent (reagent R2) | The 5mL/ bottle | The latex solution of anti-people PG I monoclonal antibody |
| Blank solution | The 1mL/ bottle | The PBS damping fluid |
| Calibration object | 1mL/ bottle * 5 | Pepsin antigen I raw material |
| Quality-control product | The 1mL/ bottle | Pepsin antigen I raw material |
| Instructions | 1 part | ? |
The condition of storage and the term of validity: kit should avoid weight also to answer protection against the tide, lucifuge, be Protected from Heat 2-8 ℃ of following kept dry during storage; The term of validity: 12 months.
Be suitable for instrument: Toshiba (TBA-40FR), Di Rui (CS-T300), Laura (FAITH-1000), Olympus (AU-1000), Mai Rui Biochemical Analyzers such as (BS-200).
Sample requires: patient specimen need not special processing, adopts conventional medical technology to collect whole blood sample, draws serum after the centrifuging and is used for detecting.Test serum can if need long-term storage should be kept at below-20 ℃, and be avoided multigelation in 2～8 ℃ of preservations as within 24 hours, using.Please do not use significant hemolysis, piarhemia or jaundice sample.
The method of inspection:
1. reagent configuration: the reagent uncork i.e. usefulness, and damping fluid and emulsion reagent are wanted abundant mixing before mensuration;
2. test condition:
Serum specimen: 5ul
R1:150ul R2:30ul wavelength: 578nm temperature of reaction: 37 ℃
Add earlier R1 reagent 150ul, add serum sample 5ul again, 37 ℃ of reactions add R2 reagent 30ul and read OD after 5 minutes, react after 5 minutes reading of data under the wavelength of 578nm, and process flow diagram is as shown in Figure 2.
3. the result calculates: do typical curve with standard items concentration, as shown in Figure 1, the antigen concentration in the sample is read through typical curve.
Test Example 1
1, adopt kit of the present invention according to the methods analyst of embodiment 5 280 of no stomach trouble crowds (confirm no disease of digestive tract after the health check-up, liver, kidney disease do not have the crowd of the history of having a stomachache) 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 result sees table 4:
Table 4
According to clinical test results, in conjunction with literature research, the diagnosis index below kit of the present invention adopts, as estimating the cancer of the stomach degree of risk, wherein 1X is minimum with 1-10X in this evaluation, and 10X is the highest:
2, the result who adopts kit of the present invention that model case is diagnosed
Case 1
Case 2
Case 3
Case 4
Claims (10)
1. a latex enhancing immune that is used to diagnose stomach trouble or cancer of the stomach is characterized in that comprising than turbid kit: the emulsion reagent and the blank solution of the antibody of dilution, the antibody that contains pepsinogen I or pepsinogen I I;
Contain the pipe/polyhenylethylene nano microballoon of different-grain diameter of antibody that coupling has joined antibody or the pepsinogen I I of pepsinogen I in the wherein said emulsion reagent.
2. according to the described kit of claim 1; It is characterized in that: described dilution is the damping fluid that contains the 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 the HEPPS damping fluid, is preferably phosphate buffer; Described increased response agent is any or several kinds among PEG-300, PEG2000 or the PEG6000, is preferably PEG6000; The sodium chloride that also contains 2mol/L in the preferred described dilution.
3. according to the described kit of claim 1, it is characterized in that: containing the particle diameter of antibody that coupling joined antibody or the pepsinogen I I of pepsinogen I in the described emulsion reagent is that 60-90nm and particle diameter are the pipe/polyhenylethylene nano microballoon of 160-200nm.
4. according to the described kit of claim 1, it is characterized in that: described emulsion reagent prepares according to following method:
(1) latex pre-treatment
Getting the unlabelled pipe/polyhenylethylene nano microballoon of 1ml adds in the 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, remove supernatant and resuspended with 5ml 0.02M pH7.4 phosphate buffer, 4 ℃ of preservations are for use;
(2) latex particle mark
Step (1) is handled back latex latex particle is diluted to 10mg/ml concentration, add 1mg EDC then, mixing activation 15 minutes with 0.02M pH6.1MES damping fluid; Centrifugal 8000rpm supernatant discarded, and resuspended with the 0.02MPH7.4 phosphate buffer, and the antibody that adds 1mg pepsinogen I or pepsinogen I I is in the 10mg/ml2ml latex fluid; Room temperature mixing 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm is separated to other containers for future use with supernatant; Particle uses confining liquid resuspended, and room temperature mixing 1 hour, the centrifugal 15min of hydro-extractor 8000rpm; Deposition is redissolved in dispersion liquid, and the latex particle that mark is accomplished 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 mark; Mark after finishing mixes said 60-90nm latex and said 160-200nm latex, and by mass and size number percent, the concentration that makes 60-90nm latex is 0.673-0.720%; The concentration that makes 160-200nm latex is 0.147-0.323%; Preserve after the mixing, preferably use above-mentioned flow process respectively the latex of particle diameter 60-80nm and 160-180nm to be distinguished mark, mark after finishing mixes said 60-80nm latex and said 160-180nm latex; By mass and size number percent; Making the 60-80nm latex concentration is 0.673-0.708%, and making the 160-180nm latex concentration is 0.147-0.235%, preserves after the mixing;
Described confining liquid is the phosphate buffer that contains the 0.02M PH7.4 of 5g/L BSA;
Described dispersion liquid is by BSA; Tween-20, PVP-K30 and biological preservative are formulated through the phosphate buffer of 0.02M PH7.4, and wherein BSA concentration is 1g/L, and Tween-20 concentration is 0.05% (v/v); PVP-K30 concentration is 2g/L, and the biological antiseptic agent concentration is 0.05% (v/v).
5. according to the described kit of claim 1; It is characterized in that: described pepsinogen I or pepsinogen I I extract the native protein that obtains from people's mucosa tissue; Preferably, described pepsinogen I or pepsinogen I I extract from people's mucosa tissue according to following method to obtain:
(1) gastric tissue of collection surgical resection is used the phosphate buffer rinsing, separates gastric mucosa, blots, and weighs; Add phosphate buffer, smash homogenate to pieces, centrifugal, collect supernatant; The precipitate phosphoric acid salt buffer is resuspended, and is centrifugal once more, merges supernatant, obtains the propepsin crude extract;
(2) the propepsin crude extract is added in the DEAE-52 chromatographic column of balance; Using PBS buffer solution elution chromatographic column to the light absorption value of eluent at the 280nm place is 0; Continue to merge eluting peak, obtain containing active propepsin with PBS buffer solution elution chromatographic column;
(3) get the active protease parent peak, be splined in the gel chromatography column, contain 0.05mol/LNa with 50mmol
2SO
4The PBS wash-out, flow velocity 3.0ml/min, pressure are 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 Q-2 anion-exchange chromatography post to separate the potpourri of pepsinogen I and pepsin antigen II; The 4th eluting peak behind the gel chromatography used earlier 0.05mol/L Tris-HCL; PH7.0 passes through the folding back and goes up appearance anion chromatography post; Use concentration to be 0-0.5mol/L NaCL gradient elution, flow velocity is 1mL/min; Three protein peaks occur, wherein the 1st, 2 peaks are pepsinogen I, and the 3rd peak is pepsinogen I I.
6. according to the described kit of claim 1; It is characterized in that: the antibody of the antibody of described pepsinogen I or pepsinogen I I is monoclonal antibody; Preferably, described monoclonal antibody is the potpourri to the monoclonal antibodies of the different antigenic determinants on pepsinogen I or pepsinogen I I surface.
7. according to the described kit of claim 1, it is characterized in that: also contain standard dilution, calibration object and quality-control product.
8. according to the described kit of claim 7; It is characterized in that: 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 the HEPPS damping fluid, is preferably phosphate buffer.
9. according to the described kit of claim 7, it is characterized in that: contain pepsinogen I or the pepsinogen I I that separates from people's gastric mucosa in said calibration object and the quality-control product.
10. the application of each described kit of claim 1-9 in preparation diagnosis stomach trouble or cancer of the stomach reagent.
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