CN201945593U - Test paper for detecting liver diseases by adopting colloidal gold chromatography - Google Patents
Test paper for detecting liver diseases by adopting colloidal gold chromatography Download PDFInfo
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- CN201945593U CN201945593U CN2011200310940U CN201120031094U CN201945593U CN 201945593 U CN201945593 U CN 201945593U CN 2011200310940 U CN2011200310940 U CN 2011200310940U CN 201120031094 U CN201120031094 U CN 201120031094U CN 201945593 U CN201945593 U CN 201945593U
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Abstract
The utility model discloses test paper for detecting liver diseases by adopting colloidal gold chromatography. The test paper comprises a sample pad, a combined pad, a nitrated cellulose coated film and a water-absorbing pad, which are sequentially stuck on a base plate from one side of the base plate to the other side of the base plate; the combined pad is coated with a gold-labelled antibody layer a and a gold-labelled antibody layer b; a detection line and a quality control line are arranged on the nitrated cellulose coated film; the detection line is coated with a Gp210 antigen protein layer; and the quality control line is coated with a gold-labelled antibody layer c. In the test paper, by using indirect immunoassays and introducing the Gp210 antigen protein, the process optimization is carried out on the combined pad and the sample pad, so that the detection performances including the high sensitivity, high specificity and high accuracy in the antibody for resisting Gp210 can be achieved; and the test paper provides reference for assisting in diagnosing the primary biliary cirrhosis of liver.
Description
Technical field
The utility model belongs to the medical immunology application, is specifically related to test paper that utilizes colloidal gold immunochromatographimethod technology for detection hepatopathy and preparation method thereof.
Background technology
Primary biliary cirrhosis of liver (Primary biliary cirrhosis, PBC) be a kind of agnogenic, chronic progressivity liver diseases by immunity infringement mediation, its main pathology shows as the struvite change of carrying out property of little bile duct destruction companion's portal vein in the liver, finally causes liver fibrosis and cirrhosis.Clinical manifestation is chronic obstruction jaundice and hepatosplenomegaly, and the later stage diseases such as liver failure and portal hypertension can occur and resemble.Unless effectively treat or liver transfer operation, portal hypertension will finally cause death with other complication of hepatopathy in latter stage at end.PBC is mainly between 30 to 60 years old, and female patient is comparatively common, and M-F can reach 1: 9.The progress of early stage utilization drug therapy may command disease, and the treatment key of PBC is early treatment, and the prerequisite of early treatment is early diagnosis, early diagnosis just becomes the focus that everybody paid close attention to.
In PBC patient's body, can detect multiple autoantibody, as antinuclear antibodies (ANA), anti-mitochondrial antibody (AMA), smooth muscle antibody (SMA), the anti-soluble acid nucleoprotein of anti-liver kidney microsomal antibody (LKM-1) (Sp100) antibody, anti-nuclear membrane glycoprotein (Gp210) antibody, anti-progranulocyte leukemia albumen (PML), anti-soluble liver antigen/liver pancreas antigen (SLA/LP) antibody etc., wherein topmost antibody is AMA, PBC is often with the AMA of high titre, it is these sick characteristics that AMA just appears in the course of disease in early days, but still therefore some PBC patient's AMA feminine gender needs other amynologic index to realize detecting of this part patient.
(antinuclear antibody in the antinuclear antibodies, ANA) anti-nuclear membrane glycoprotein (GP210) antibody is the high specific autoantibody of PBC, it can occur simultaneously with anti-mitochondrial antibody, also can appear among the PBC patient of anti-mitochondrial antibody feminine gender, specificity can detect this antibody up to 99% among the PBC patients serum of nearly 10%-40%.Anti-GP210 antibody is rarely found other patients in as oneself immunity hepatitis, rheumatic disease, polymyositis and dry syndrome, so can be used as the important auxiliary diagnosis index of PBC.And anti-GP210 antibody can be used as the prognostic indicator of PBC.Studies show that the survival rate that anti-GP210 antibody continues high titre positive patient is starkly lower than the patient who changes patient and the anti-GP210 negative antibody of initial survey through anti-GP210 antibody the moon behind the clinical drug therapy.
1985, Ruffati etc. find to exist among the PBC patients serum at being ring-like antinuclear antibodies (Ruffacti A, et al.Nuclearmembrane-staining antinuclear antibody in patients with primarybillary cirrhosis.J Clin Immnol.1985 under nuclear envelope, the immunofluorescence first; 5 (5): 357-61.).
1988, Lozano, Lassoued etc. detect the PBC patients serum with ring-like ANA under this immunofluorescence with the method for Western blotting and immunoprecipitation and discern the polypeptide about molecular weight 200KD in the nuclear envelope (Lassoued K, et al.Autoantibodies to 200kDpolypeptide (s) of the nuclear envelope:a new serologic marker ofprimary billiary cirrbosis.Clin Exp Immunol.1988; 74 (2): 283-8.Lozano F.et al.Autoantibodies against nuclearenvelope-associated proteins in primary billary cirrbosis.Hepatology.1988; 8 (4): 930-8.).
Nineteen ninety Courvalin etc. extract from liver tissues of rats and purifying obtain a kind of 210KD the complete glycoprotein that is positioned at nucleopore and can with the PBC patients serum reaction with ring-like ANA, thereby determined this kind antibody at antigenic component, be Gp210, its antigenic determinant is the amino acid fragment of aminoterminal 15 linear amino acid fragments and carboxyl terminal.(Courvalin?JC，Worman?HJ.Neclear?envelope?protein?autoantibodies?in?primarybiliary?cirrhosis.Semin?Liver?Dis?1997；(17)：79-90.)。
Anti-Gp210 antibody is 10%-42% to the susceptibility of PBC diagnosis, on average be about 25%, and specificity can reach more than 99%; Anti-Gp210 antibody positive rate 20%-75% in the PBC patient of AMA feminine gender, average about 50%, certain diagnostic value is arranged.Nakamura thinks that the lasting reaction at Gp210 is relevant with the order of severity of interface hepatitis, and anti-Gp210-C terminal peptide can be used as monitoring of UDCA result of treatment and the early stage index of finding to have the PBC patient who develops into hepatic failure in latter stage at end.(Nakamura?M，et?al.Increased?expression?of?nuclear?envelope?GP210?antigen?insmall?bile?ducts?in?primary?billary?cirrhosis.J?Autoimmun.2006；26(2)：138-45.)。
There is the scholar to use the expression of SABC method research Gp210 on the liver puncture sample recently, studies show that on PBC patient's bile duct epithelial cell (BEC) nuclear envelope tangible Gp210 antigen presentation is arranged, the normal control group does not have expression, oneself immunity hepatitis, chronic hepatitis B, chronic hepatitis C only has faint expression: and in PBC patient expression intensity and the portal area inflammation of Gp210, interface hepatitis and the positive correlation of leaflet inflammation, this shows that the expression of Gp210 on the little bile duct of PBC patient may be relevant with the inflammatory damage of BEC, may cause the progress of PBC patient to the hepatic failure in latter stage to the autoimmune response of Gp210.
Wherein anti-nuclear membrane glycoprotein gp210 antibody is the high degree of specificity antibody of PBC, and its specificity seldom comes across among oneself immunity hepatitis, rheumatoid arthritis, dry syndrome and the polymyositis patient up to more than 96%.It is negative or lack the PBC patient of general clinical special disease and its laboratory, pathological examination, unusual PBC patient's have great importance (Nickowitz RE, the Worman HJ. Autoantibodies againstintegral membrane protein of the nuclear envelope in patients withprimary bili of making a definite diagnosis for AMA to detect anti-gp210 antibody
In addition, anti-nuclear membrane glycoprotein gp210 antibody also can be used as the pernicious prediction index that lapses to of disease.The anti-nuclear membrane glycoprotein gp210 antibody positive rate of external report is 9.5%～41%, and its specificity is 96%～99%, and is present among the PBC patient of 20%～47% AMA feminine gender.Its susceptibility of the anti-nuclear membrane glycoprotein of reports such as Zhu Ye gp210 antibody reaches 16.9%, specificity reaches 96% (Zhu Ye, Tu Xiaoqing, Zhou Lin etc. the clinical value [J] of the joint-detection of anti-mitochondrial antibody and hypotype and antinuclear antibodies in the primary bile liver cirrhosis patient serum. modern immunology .2006,26 (2): 136-138.).
Gp210 is that a kind of relative molecular mass is 210000 complete transmembrane proteins in the nucleopore compound, the specific nucleopore district that is positioned at, it is a kind of complete memebrane protein in the nucleopore compound, specificity partly is positioned at the nucleopore district, therefore during immunofluorescence detects, nuclear membrane type fluorescence reaction (the Wozniak RW in the form of a ring at the nucleopore place, Bartnik E, Blobel G.Primary structure analysis of anintegral membrane glycoprotein of the nuclear pore[J] .J CellBiol.1989 (108): 2083-2087.).The major antigen epi-position is positioned at 15 amino acid of albumen endochylema section c-terminus, not only can strengthen understanding to the further research of this antibody to the PBC mechanism of causing a disease, and help the diagnosis PBC that is not true to type, solve some difficulties make a definite diagnosis clinically among the PBC, especially for the AMA feminine gender or laboratory, unusual the making a definite diagnosis of PBC patient of pathological examination acquire a special sense.
Therefore, anti-Gp210 antibody test is the very important serodiagnosis foundation of the negative PBC of diagnosis AMA, has great importance for the PBC patient's of AMA feminine gender diagnosis.Anti-Gp210 antibody is the specific autoantibody of PBC, and this antibody specificity in the PBC diagnosis seldom appears in other autoimmune diseases up to 97-100%, and susceptibility is 20%-30%, can be used as the important auxiliary diagnosis index of PBC.
The method of the anti-nuclear membrane glycoprotein of detection (GP210) antibody that present laboratory is commonly used mainly contains enzyme-linked immunosorbent test (enzyme linked immunosorbent assay, ELISA), indirect immunofluorescence (indirect immunoflurescence, IFF), Western blot (immunoblotting test, IBT), linear immunoassay (Line immuno assay, LIA) etc.
Though Western blot combines the high resolution of SDS-PAGE and the high specific and the susceptibility of ELISA method, but operation relative complex, finish whole experiment and need about three hours, also need the professional immunological technique personnel operation that in the laboratory, experimentizes, be subject to the influence of environmental baseline factors such as all temps and incubation time simultaneously, test is brought inconvenience.And reagent has stronger toxicity and contaminative.The anti-Gp210 antibody assay kit of commercialization in the market is mainly Western blot.
Indirect immunofluorescence can be made semiquantitative determination, but needs the professional to operate special fluorescence microscope result, and the result judges certain subjective artificial error in judgement, objectivity deficiency; Be subject to other in addition and disturb the generation false positive, the standardization difficulty, the technical program more complicated, detection time is long, also is not suitable for the detection of high flux sample.
Linear immunoassay is mainly used in the examination of the big class of disease, and specific aim is relatively poor relatively, also is not suitable for the detection of high flux sample simultaneously.
The ELISA method detects and can be used for high flux sample mensuration, sensitivity is also higher, extensively approved at present, but operate more loaded down with trivial detailsly, need repeatedly application of sample and washing, finish whole experiment and need about three hours, and be subject to the influence of temperature and incubation conditions, need microplate reader, also need the professional immunological technique personnel operation that in specialized laboratory, experimentizes, bring inconvenience to experiment.
Colloidal gold immunity chromatography (gold-immunochromatography assay, GICA) be to use colloidal gold-labeled method, with collaurum as tracer, with the fibre strip chromatographic material is solid phase, make sample solution swimming on chromatography strip by capillary effect, make that immune response takes place the acceptor (as antigen or antibody) at determinand on determinand in the sample and the pad, and immune response takes place and be trapped with antigen (or antibody) on the fibre strip chromatographic material, and then form macroscopic aubergine band, obtain experimental result intuitively, reach the purpose (Sikowicz G et al.One-step chromatographic immunoassay for qualitativedetermination of choricogonadotropin in urine.Clin Chem.1990 (36) 1579-1586.) of fast detecting.During use only with sample pipetting volume on sample pad, just situation occurred and judge the yin and yang attribute result in several minutes according to aubergine band on the detection line.Advantages such as compare with other detection methods, detection time is short, only needs 5～10 minutes, and operating personnel need not training, and are easy and simple to handle, quick, need not cryopreservation, and accumulating is convenient.
Aspect autoimmune disease, occurred some at present on the foreign market and detected the test strips product of autoimmune disease, but the colloid gold immune test paper of anti-Gp210 antibody does not at home and abroad still come out on the market.
The utility model content
Technical problem to be solved in the utility model is in order to overcome above methodological deficiency, colloidal gold chromatography is applied in the anti-Gp210 detection of antibodies, first the Gp210 antigen protein is applied in the colloidal gold chromatography simultaneously, the employing indirect method realizes the anti-GP210 antibody test in the blood, realize the detection performance of high special, high sensitivity, high accuracy, rapid screening goes out the positive sample of anti-GP210 antibody, can be fast, auxiliary diagnosis primary bile cirrhosis easily.
Technical problem to be solved in the utility model is to provide a kind of colloidal gold chromatography hepatopathy to detect test paper.
Technical problem to be solved in the utility model can be achieved through the following technical solutions:
A kind of colloidal gold chromatography hepatopathy detects test paper, include sample pad, pad, cellulose nitrate coated film, adsorptive pads, described sample pad, pad, cellulose nitrate coated film, adsorptive pads are sticked on the described base plate to the opposite side of described base plate successively by a side of base plate, and its special disease is:
Described pad is coated with golden labeling antibody a layer and golden labeling antibody b layer;
Described cellulose nitrate coated film is provided with detection line and nature controlling line, and described detection line is coated with Gp210 antigen protein layer, and described nature controlling line is coated with golden labeling antibody c layer.
Further, the antibody A in the described golden labeling antibody a layer is one or more in anti-human IgG monoclonal antibody, anti-human IgG polyclonal antibody, staphylococcal protein A (SPA), the streptococcal protein G (Protein G).
Described anti-human IgG polyclonal antibody is a kind of in mouse source, Ma Yuan, Yang Yuan, rabbit source or the cavy source.
Described anti-human IgG monoclonal antibody is a kind of in Yang Yuan or the rabbit source.
Antibody A in the described golden labeling antibody a layer is preferably staphylococcal protein A.
Antibody B in the described golden labeling antibody b layer and the antibody C in the golden labeling antibody c layer simultaneously or be a kind of in monoclonal antibody or the polyclonal antibody simultaneously, specificity can take place in conjunction with the formation immune complex in the antibody C in antibody B in the wherein golden labeling antibody b layer and the golden labeling antibody c layer.
Described polyclonal antibody is a kind of in mouse source, Ma Yuan, Yang Yuan, rabbit source or the cavy source, and monoclonal antibody is a kind of in mouse source or the rabbit source.
Antibody B in the described golden labeling antibody b layer is preferably rabbit igg, and the antibody C among the correspondingly golden labeling antibody c is preferably goat anti-rabbit igg.
The Gp210 antigen protein layer of Gp210 antigen protein layer for obtaining of bag quilt on the described detection line by the prokaryotic expression cloned gene.
Described detection is an anti-GP210 antibody in the qualitative detection human serum, auxiliary diagnosis early primary bile hepatitis.
Described Gp210 antigen protein is the recombinant protein that cloned gene obtained by escherichia coli prokaryotic expression.
Described sample is from human serum, blood plasma, whole blood sample.
The monoclonal antibody of using according to the utility model can by by Kohler etc. (Continuouscultures of fused cells secreting antibody of predefinedspecificity[J] .Nature, 1975 (256): 495-497) the hybridoma method of at first describing is prepared, and perhaps can be prepared (seeing United States Patent (USP) 4816567) by the recombinant DNA method." monoclonal antibody " by Clackson etc. (Making antibody fragments using phagedisplay libraries[J] .Nature, 1991:624-628) and Marks etc. (By-passingimmunization:Human antibodies from V-gene libraries displayed onphage[J] .Journal of Molecular Biology, 1991:581-597) described technology is separated from phage antibody library.
The polyclonal antibody of using according to the utility model can by Chen Xueqing etc. (immunology common experimental method .[M], 2000:15-26) prepare by immune animal.
SPA of the present utility model, Protein G be by prokaryotic expression cloning recombination, by J. Sa nurse Brooker etc. (molecular cloning experiment guide .[M], 2002:1228-1232) the escherichia coli prokaryotic expression cloned gene of Miao Shuing.
Detection principle of the present utility model is specially the recombinant expressed GP210 antigen protein of selecting affinitive layer purification for use, gold labeling antibody a and gold mark rabbit igg are as the colloid gold label compound, be sprayed at pad, utilize indirect method to detect and whether contain anti-GP210 antibody in the blood serum sample.During detection, sample is along with the chromatography swimming is marked compound to pad and infiltration gold, human IgG wherein and golden labeling antibody a are in conjunction with forming human IgG-Jin labeling antibody a compound, because capillary effect, this compound along the coated film swimming forward, if in the blood serum sample anti-GP210 antibody is arranged, this compound and the antigen protein generation specific immunity association reaction of bag quilt on nitrocellulose filter, form golden labeling antibody a-human IgG-Gp210 antigen protein triplet compound and be trapped within on the detection line, enrichment forms darker aubergine band gradually; Because capillary effect continues swimming forward, gold mark rabbit igg be coated on goat anti-rabbit igg on the nature controlling line and special immune response takes place be trapped, be enriched in the darker aubergine band of formation on the nature controlling line gradually, unnecessary unconjugated material continues chromatography to adsorptive pads, the positive findings that is judged to of band therefore all occurs at detection line and nature controlling line; If do not contain anti-GP210 antibody in the blood serum sample, when gold labeling antibody a arrives detection line, not with the antigen protein generation immune response that is coated on the detection line, therefore at the detection line place aubergine band does not appear, gold labeling antibody a continues swimming and arrives adsorptive pads forward, and special immune response is taken place with bag in nature controlling line place goat anti-rabbit igg forward and is trapped in gold mark rabbit igg continuation swimming, be enriched in gradually and form the aubergine band on the nature controlling line, therefore only occurring band in Quality Control is judged to negative findings.
The antigen protein that the utility model is expressed genetic engineering bacterium is incorporated in the test strips first, realized the detection performance of high specific, high sensitivity, pin-point accuracy.Compare with the commercialization ELISA kit that present market occurs, still have many differences, it is not subjected to place personnel's limit, and detection time, weak point was 5-10 minute, and sentence read result is easy.Test paper of the present utility model in addition has high specific, high sensitivity, pin-point accuracy, can satisfy the rapid screening PBC in market, for patient's diagnoses and treatment early provides condition, simultaneously, also can satisfy the demand of basic unit laboratory, instant detection, bedside detection.
Compare the utility model advantage with conventional detection:
1. uniqueness of the present utility model is that the Gp210 antigen protein first Application of dna recombinant expression is in colloid gold chromatographic test paper, improved its detection sensitivity greatly, but gone out anti-all positive sample of GP210 antibody (can detect sample) greater than 25RU/ml by the colloid gold test paper rapid screening.
2. the utility model has the advantages that production cost is low.The required core reagent of anti-GP210 detection of antibodies test paper provided by the utility model is the promptly anti-human IgG of antibody A or SPA or PROTEIN G, antibody C, antibody B, antigen protein, its wall scroll test paper agents useful for same amount is few, and can be by buying commercialization reagent or self-control, antigen protein derives from homemade purifying gene engineering antigen protein.
3. compare with the disclosed additive method that is used for anti-GP210 antibody test, test paper of the present utility model have many additive methods the advantage that can not compare, as detection time short (5～10min); Without any need for specific apparatus, can realize that bedside detects and outpatient service detects immediately; Easy and simple to handle, only need single step reaction, operating personnel need not training, and it is low to detect cost; Temperature is not had specific (special) requirements, need not freezingly, store convenient transportation, room temperature can be preserved 24 months.
Description of drawings
Fig. 1 is a side structure synoptic diagram of the present utility model.
Fig. 2 is a testing result synoptic diagram of the present utility model.
Wherein:
Fig. 2 a is depicted as: behind the application of sample, reaction 3～5min can see on detection zone D and the E relevant position, control zone and the aubergine band occurs;
Fig. 2 b is depicted as: when the aubergine band all appears in control zone E and detection zone D, the result is positive, illustrates to contain anti-Gp210 antigen protein in the serum;
Fig. 2 c is depicted as: as only an aubergine band occurring at control zone E, the aubergine band does not appear in detection zone D, and the result is negative, illustrates not contain anti-Gp210 antibody in the serum;
Fig. 2 d, 2e are depicted as: the aubergine band do not occur as control zone E, no matter whether detection zone D has band to occur, and illustrates that all test paper lost efficacy.
Embodiment
Colloidal gold immunity chromatography hepatopathy described in the utility model detects test paper, and as shown in Figure 1, this test paper is to paste cellulose nitrate coated film 3, pad 2, sample pad 1, adsorptive pads 6 in turn mutually by a side direction opposite side on base plate 7 overlap joint.
Be coated with golden labeling antibody a layer and golden labeling antibody b layer on the pad 2, the antibody A of golden labeling antibody a layer is goat anti-rabbit igg gold labeling antibody or mouse-anti human IgG gold labeling antibody or golden labeling antibody of staphylococcal protein A (SPA) or streptococcal protein G gold labeling antibody; Antibody B in the gold labeling antibody b layer is a rabbit igg.
Cellulose nitrate coated film 3 is provided with detection line 4 and nature controlling line 5, and detection line 4 is coated with Gp210 antigen protein layer, and nature controlling line 5 is coated with golden labeling antibody c layer, and the antibody in the golden labeling antibody c layer is goat anti-rabbit igg.
Below in conjunction with specific embodiments and the drawings, further set forth the utility model.
The preparation of embodiment 1Gp210 antigen protein
The Gp210 antigen protein that is applied to this test paper is to make up recombination by gene clone technology, and adopting prokaryotic expression technology successful expression to go out then all is the Gp210 antigen protein in people source.Wherein, the Gp210 antigen protein derives from the purifying gene engineering antigen protein.
Antibody A and antibody C, antibody B prepare with following method.Wherein the anti-human IgG in the antibody A, antibody C and antibody B generally can by on the animal in repeatedly subcutaneous (sc) or peritonaeum the immunogene and the adjuvant of (ip) injection purifying produce.
By being mixed with Freund ' the s Freund's complete adjuvant of 3 times of volumes, 0.05mg～1mg immune formulation (respectively at goat or mouse) obtains injection solution, with the multi-section position injection in animal skins of this injection solution, after one month with animal with Freund ' the s Freund's complete adjuvant mixed liquor of 1/5 to 1/10 human IgG of originally measuring through the animal hypodermic injection of multi-section position and booster immunization.With the animal bloodletting, measure the anti-human IgG titre of serum after 7～14 days.The animal booster immunization is reached plateau until titre.The method of producing polyclonal antibody has been described in many immunology textbooks, for example, Chen Xueqing etc. " immunology common experimental method ".By reclaiming splenocyte and make cell immortalityization from the animal of quilt immunity, for example by merging with the myeloma cell or transforming by Epstein-Barr virus, and screening can be expressed the monoclonal antibody (Kohler of purpose antibody, Milstein.Derivation of specificantibody-producing tissue culture and tumor lines by cell fusion[J] .European Journal of Immunology, 1976:501-511).
Can prepare reorganization staphylococcal protein A and streptococcal protein G in the antibody A by the escherichia coli prokaryotic expression cloned gene, concrete operation method referring to J. Sa nurse Brooker etc. (molecular cloning experiment guide .[M], 2002:1228-1232), or buy commercial reorganization staphylococcal protein A or streptococcal protein G.
The colloidal gold solution preparation
HAuCl with 0.01%
4Solution is heated to boiling, adds every 100mL HAuCl rapidly
4Solution adds an amount of reductant solution, and color is from blueness, and is light blue then, blue, and heating occurs redly again, boils 7～10min and occurs transparent orange red.Filter with ultrafiltration or miillpore filter (0.45 μ m) again, to remove polymkeric substance and other impurity that may sneak into wherein.The collaurum outward appearance for preparing should be pure, bright, do not have precipitation and floating thing, abandons when grease and a large amount of black particle shape sediment appear in liquid level.
Wherein employed reductive agent can preferably use trisodium citrate for trisodium citrate (Frens 1973), tannic acid-trisodium citrate (Slot and Gueeze 1985), white phosphorus, more preferably uses 1% trisodium citrate.
Wherein used glass container should definitely clean, with preceding need through pickling, silication.Its water should be the deionization ultrapure water, and resistivity reaches 18.2M Ω.
In the colloidal gold solution preparation process, the compound method of each solution is as follows:
1.HAuCl
4Preparation: with ultrapure water dissolved chlorine auric acid, be made into 1% solution, put 4 ℃ standby, the term of validity four months.1000mL 1%HAuCl
4Solution formula: 10g HAuCl
4, ultrapure water is settled to 1000mL.
The preparation of 2.1% trisodium citrate: the preparation of 1% trisodium citrate (Sodium Citrate): with ultrapure water dissolving Sodium Citrate, be made into 1% solution, 0.22 μ m membrane filtration mistake, now with the current.
Colloidal gold immunity chromatography hepatopathy of the present utility model detects the preparation method of test paper, specifically comprises the steps:
(1) adopt the method for embodiment 1 to prepare the Gp210 antigen protein;
(2) preparation of goat anti-rabbit igg gold labeling antibody:
Regulate the collaurum pH 7.0～9.0 of embodiment 3 preparations with 0.1M sal tartari, slowly add 4～25 μ g goat anti-rabbit iggs by every milliliter of colloidal gold solution, stir 10～30min, add BSA then to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, to precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 ℃ standby;
(3) preparation of cellulose nitrate coated film 3, with coated film damping fluid dilution Gp210 antigen protein to concentration is 1.0～1.5mg/mL, adjust the BIO-Dot instrument, be sprayed at nitrocellulose filter (NC) and go up detection line 4 places, near pad 2 ends, apart from pad 2 about 9.5mm, make Gp210 antigen protein bag by on the detection line 4 of cellulose nitrate coated film;
Be cushioned liquid with bag goat anti-rabbit igg is diluted to 0.8～1.5mg/mL, be sprayed at nature controlling line 5 places that nitrocellulose filter (NC) is gone up close adsorptive pads 6 with 1～10 μ l/cm with the BIO-Dot instrument, apart from adsorptive pads 6 about 9mm.Detection line 4 and 5 liang of about 5～8mm of linear distance of nature controlling line, the spray line is answered even thickness.37 ℃ of oven dry encapsulate standby.
It can be borate that the bag of its use is cushioned liquid, carbonate, phosphate, Tris-HCl or Tri s-phosphate, acetate, barbital, or the like, the purpose of its damping fluid makes the albumen bag also firmly be wrapped quilt in the NC film for certain pH and ionic strength are provided, and its pH of buffer value generally is about in 6～9.5 scopes, be preferably in 6.5～7.5 the neutral buffered scope, and most preferably the pH value of damping fluid is in 7.0～7.4 scopes.Damping fluid is preferably phosphate.
Nitrocellulose filter wherein (NC) can be any commercialization nitrocellulose filter, S﹠amp; SAE99, whatman 8 μ m, millipore M135, sartorius CN140 etc.The concrete NC film that uses is not a key of the present utility model, but in each mensuration, above-mentioned several NC films can be used as preferably.The film that the different damping fluids that contain the different surfaces activating agent that different manufacturers uses are handled, with used detection line antibody-solutions affinity in various degree gap is arranged, also can largely cause lines inhomogeneous, the traction or the phenomenon of disperse, therefore utilization assembling test paper is selected preferred NC film.
(1) preparation of goat anti-human igg's gold labeling antibody: the collaurum pH 8.0～9.0 that regulates embodiment 3 preparations with 0.1M sal tartari, slowly add 8～12 μ g goat anti-human iggs by every milliliter of colloidal gold solution, stir 10～30min, add BSA then to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, to precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 ℃ standby;
(2) preparation of rabbit igg gold labeling antibody: collaurum pH value to 7.0～8.0 of regulating embodiment 3 preparations with 0.1M sal tartari, slowly add 8～12 μ g rabbit iggs by every milliliter of colloidal gold solution, stir 10～30min, add BSA then to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, to precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 ℃ standby.
(3) preparation of pad: the polyester film was soaked 30 minutes through damping fluid, 37 ℃ of oven dry, after goat anti-human igg gold labeling antibody and rabbit igg gold labeling antibody mixed according to a certain percentage, use the BIO-Dot instrument, consumption with 0.5～4 μ l/cm is sprayed on the pretreated polyester film, and 25 ℃～37 ℃ dryings get pad 2 after to be dried the finishing, pad 2 vacuum packagings, put 2 ℃～8 ℃ standby.Place under 2 ℃～8 ℃ the environment standby;
In the preparation process of pad, operable damping fluid comprises following several:
(a) contain 1%PVA, 0.71%Na
3PO
4, 1%BSA, 0.05%NaN
3, 0.1%TritonX-100;
(b)1％PVA、1％BSA、0.05％PROCLIN
TM300、0.1％TritonX-100，pH7.0PBS；
(c)1％PVA、1％BSA、0.05％PROCLIN
TM300，pH7.0PBS；
(d) contain 1%PVA, 0.71%Na
3PO
4, 1%BSA, 0.05%NaN
3, 0.1%Tween-20;
(e) contain 1%PVA, 0.71%Na
3PO
4, 1%BSA, 0.05%NaN
3, 0.1%Tween-20, pH7.0PBS;
Its preferred damping fluid is damping fluid (d), because it has the ultimate resolution of difference yin and yang attribute sample.PROCLIN
TM300 and NaN
3Play antisepsis, and Tween-20 have decontamination and hydrophilic interaction.
Blending ratio between goat anti-human igg's gold labeling antibody and the rabbit igg gold labeling antibody can be carried out according to following method:
Substantially determine the OD20 of rabbit igg gold labeling antibody by preliminary experiment, be sprayed on the pretreated polyester film with BIO-Dot discharge rate 1 μ l/cm, with 0.01MPBS is sample-loading buffer, and the band of the color intensity that can obtain expecting determines that the application OD discharge rate of rabbit igg gold labeling antibody is 1 μ l.
Subsequently goat anti-human igg's gold labeling antibody is carried out gradient dilution to final concentration OD 100,80,60,40,20, then rabbit igg gold labeling antibody is diluted to final concentration OD 20, is that 3 μ l/cm are sprayed on the polyester film of handling well with BIO-Dot with above goat anti-human igg's gold labeling antibody and rabbit igg gold labeling antibody potpourri discharge rate, cellulose nitrate coated film 3, pad 2, sample pad 1, the adsorptive pads 6 of preparation are pasted on the base plate 7 successively, are debugger object with positive serum, critical reference value serum, negative serum.Judgment basis: both band color intensities of the detection line 4 of positive serum and nature controlling line 5 are consistent to be foundation, and critical reference value serum can occur, and negative serum does not have that OD value that band occurs, and is the application quantity of this batch.Drawing OD20～40 by this test comparatively meets the requirements.
Glass fibre membrane is pressed the 45mL/ sheet, evenly spills with damping fluid and be applied on the glass fibre membrane, after 37 ℃ of oven dry sample pad, sample pad encapsulates with aluminium foil bag, and is standby;
This step can with damping fluid comprise following several:
(a)0.05M?Borax、0.01MPBS(pH?7.0)、0.1％Sodium?Casein、1％PEG20000、2％BSA、0.05％NaN
3；
(b)0.05M?Borax、0.01MPBS(pH?7.0)、0.1％Sodium?Casein、1％PEG20000、2％Casein、0.05％NaN
3；
(c)0.05M?Tris-cl(pH?7.0)、0.01MPBS、0.1％Sodium?Casein、1％PEG20000、2％Casein、0.05％NaN
3。
Preferred damping fluid is damping fluid (a), because it has the ultimate resolution of difference yin and yang attribute sample, NaN
3Play antisepsis.
At first carry out the starting material pre-cut:
Cutting of sample pad: with guillotine sample pad is cut into the promptly long 28cm of the base plate equal length made from PVC, wide 2.4cm puts between drying shed standby.
Cutting of adsorptive pads: with trimmer thieving paper is cut into the promptly long 28cm of the base plate equal length made from PVC, wide 3cm makes adsorptive pads, puts between drying shed standby.
Cutting of pad: with guillotine pad is cut into the promptly long 28cm of the base plate equal length made from PVC, wide 2.4cm puts between drying shed standby.
Cutting of cellulose nitrate coated film: with trimmer the cellulose nitrate coated film is cut into the promptly long 28cm of the base plate equal length made from PVC, wide 1cm, it is standby to put drying shed.
Cellulose nitrate coated film 3, pad 2, sample pad 1, adsorptive pads 6 are stacked gradually on the base plate 7 that the PVC plastics are made by shown in Figure 1, form big plate.Composing room's temperature should be controlled at 25 ℃～37 ℃, humidity 20%～30%.
Slitting: big plate is cut into single part with cutting cutter, everyone part width is cut into the width of 2.5mm～4mm according to certain requirement, sampling observation at random, sensitivity can detect indoor quality-control sample (promptly weak positive sample), band colour developing degree reaches the d as Fig. 2, and specific band nothing but, then product becomes specification product by the Quality Control regulation.
Assembling, packing: the test paper that 1 person-portion has been cut is assembled in the test card of getting ready, makes the sample pad of the corresponding test paper of application of sample window, corresponding detection zone of display window and control zone as a result, and composing room's temperature should be controlled at 25 ℃～37 ℃, humidity 20%～30%.Be encapsulated in the outer bag with drying agent, instructions, sample pipetting volume device again, keep in Dark Place in 4～25 ℃.
In the preparation process of the anti-Cenp-B antibody test of above-mentioned colloidal gold chromatography test paper, the collocation method of each solution is as follows:
1.0.1M the preparation of sal tartari: with ultrapure water preparation, 0.22 μ m membrane filtration mistake, put 4 ℃ standby, the term of validity one month.1000mL 0.1M K
2CO
3Solution formula: 13.8g K
2CO
3Ultrapure water is settled to 1000mL.
The preparation of 2.10%BSA: with ultrapure water preparation, 0.05% Sodium azide (NaN
3), 0.22 μ m membrane filtration mistake, put 4 ℃ standby, two weeks of the term of validity.1000mL 10%BSA solution formula: 100g BSA, 0.5g NaN
3Ultrapure water is settled to 1000mL.
3, bag is cushioned the preparation of liquid: 9gNacl, 1.15gNa
2HPO
4, 0.23g NaH
2PO
4, 10gSucrose, 0.5gEDTA be dissolved in the 1L ultrapure water, filter place 4 ℃ standby.
4. cleansing solution is also promptly preserved the preparation of liquid:
2%BSA, 0.05% (NaN
3), 0.01M pH 7.2PBS, 0.22 μ m membrane filtration mistake, put 4 ℃ standby, two weeks of the term of validity.Formula of liquid is preserved in the washing of 1000mL mark: 20g BSA, 0.5g NaN
3, 0.01MpH 7.2PBS is settled to 1000mL.
5. the preparation of gold mark dilution:
The preparation of 1000mL dilution: 2.423g tris, 10gBSA, 0.2gNaN
3Be dissolved in the ultrapure water, transfer pH 8.0, constant volume is to 1000mL.
After with dilution gold mark being diluted to working concentration, add the trehalose of 20%Sucrose and 5%.
The colloidal gold immunity chromatography hepatopathy of this embodiment detects the preparation method of test paper, its step is basic identical with embodiment 4, just the preparation process of the goat anti-human igg of (1) gold labeling antibody replaces with the preparation of the golden labeling antibody of staphylococcal protein A (SPA) in the step 2 with this embodiment, and is specific as follows:
Regulate collaurum pH value to 5.0～6.5 with pH 9.00.2M borate buffer, slowly add 10～15 μ gSPA albumen by every milliliter of colloidal gold solution, stir 10～30min, add BSA then to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, to precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 ℃ standby.
Blending ratio between golden labeling antibody of staphylococcal protein A (SPA) in this embodiment step 2 and the rabbit igg gold labeling antibody can be carried out according to the method for embodiment 4.
The colloidal gold immunity chromatography hepatopathy of this embodiment detects the preparation method of test paper, its step is basic identical with embodiment 4, just the preparation process of the goat anti-human igg of (1) gold labeling antibody replaces with the preparation of mouse-anti human IgG gold labeling antibody in the step 2 with this embodiment, and is specific as follows:
Regulate collaurum pH value to 7.0～8.0 with 0.1M sal tartari, slowly add 8～12 μ g mouse-anti human IgGs by every milliliter of colloidal gold solution, stir 10～30min, add BSA then to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, to precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 ℃ standby.
Blending ratio between mouse-anti human IgG gold labeling antibody in this embodiment step 2 and the rabbit igg gold labeling antibody can be carried out according to the method for embodiment 4.
The colloidal gold immunity chromatography hepatopathy of this embodiment detects the preparation method of test paper, its step is basic identical with embodiment 4, just the preparation process of the goat anti-human igg of (1) gold labeling antibody replaces with the preparation of streptococcal protein G gold labeling antibody in the step 2 with this embodiment, and is specific as follows:
Regulate collaurum pH value to 5.0～6.5 with pH 9.00.2M borate buffer, slowly add 10～15 μ g streptococcal protein Gs by every milliliter of colloidal gold solution, stir 10～30min, add BSA then to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, to precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 ℃ standby.
Blending ratio between streptococcal protein G gold labeling antibody in this embodiment step 2 and the rabbit igg gold labeling antibody can be carried out according to the method for embodiment 4.
The utility model is incorporated into the anti-Gp210 antibody test of colloidal gold chromatography test paper with recombinant expressed Gp210 antigen protein, can realize the Gp210 detection of antibodies in the blood sample, can be fast, auxiliary diagnosis primary bile cirrhosis easily.
Embodiment 8
Sample process
Get whole blood 1～5ml, natural aggegation is after 5 minutes, and 3000～5000g/5min～10min gets supernatant and promptly obtains testing sample solution, has the above testing sample solution of 100 μ l at least.
Draw above serum 50～70 μ l samples in sample pad with micro sample adding appliance, slowly application of sample.Perhaps with suction pipe serum sample is slowly dripped 3～5 on sample pad, 5～10min observations situation occurs according to band and comes interpretation yin and yang attribute result.
Embodiment 9
The detection of kit and clinical performance assessment
The antibody A that collaurum hepatopathy of the present utility model detects the golden labeling antibody a of test paper is preferably SPA, and the antibody B of antibody b is a rabbit igg, and the antibody of antibody c is goat anti-rabbit igg, and the clinical performance that its test paper is carried out carries out following assessment.
1. stability test
1.137 ℃ accelerated stability
Place 37 ℃ to carry out accelerated tests on test paper, every day, taking-up was tested with indoor quality-control product, and the withinrun precision of the yin and yang attribute coincidence rate by yin and yang attribute reference material (each 10 parts) is judged the stability of test paper.The result shows after 4 months, and the testing result of quality-control product meets expection, and the yin and yang attribute coincidence rate of each yin and yang attribute reference material is 100%.The yin and yang attribute reference material is 10 parts of anti-GP210 antibody positive serum and 10 parts of anti-GP210 negative antibody serum.
1.24 ℃ stability experiment
2. diagnostic sensitivity
Collect 100 parts of serum that are diagnosed as primary bile hepatitis (PBC) patient from clinical, the operation steps that goes up to specifications with homemade colloid gold test paper detects the PBC patients serum who collects above.Statistics is as follows behind the 5min:
According to the result who adds up above, according to the detection to the PBC patients serum of 100 parts of affirmations, the sample size that can find anti-GP210 antibody positive is 29 examples, and negative sample quantity is 71 examples.Therefore by calculating:
Diagnosis sensitivity (%)=22/ (29+71) * 100%=29%
3. specificity
Collect each 300 parts of healthy blood donor's serum and non-patient PBC (comprise other autoimmune diseases such as rheumatoid arthritis and exempt from the hepatopathy people certainly) serum from clinical, the operation steps that goes up to specifications with homemade colloid gold test paper detects the healthy blood donor that collects above and non-patient's PBC serum.Statistics is as follows behind the 5min:
According to statistics to the detection of each 300 parts of serum of non-patient PBC and healthy blood donor, discovery records anti-GP210 negative antibody in the healthy blood donor sample size is 295 examples, but not the sample size of PBC patients serum's anti-GP210 negative antibody is 296 examples.Therefore by calculating:
(healthy blood donor) specificity (%)=295/300 * 100%=98.33%
(non-PBC patients serum) specificity (%)=296/300=98.66%
600 routine control groups (comprising healthy blood donor and non-PBC patients serum)
Specificity (%)=591/600=98.5%
4. diagnostic accuracy
According to the statistics of top specificity and diagnostic sensitivity, can obtain following this summary table:
Can see that according to top statistical form for the patients serum who confirms PBC, detecting the positive sample number that obtains is 80 examples, in 600 routine healthy blood donors and non-PBC patients serum, detecting negative sample is 591 examples.
That is: diagnostic accuracy=(81+591)/700=96%
5. inaccuracy in criticizing
Select certain a collection of test paper of production for use, select each portion of serum (strong, in, weak, feminine gender) of four kinds of variable concentrations simultaneously, every part of serum is done 10 repetitions, observations behind the 5min.
Different seronegativity number of results positive findings numbers
Strong positive serum 0 10
Middle-jiao yang, function of the spleen and stomach serum 0 10
Weak positive serum 0 10
Negative serum 10 0
Learn that according to top result the same a collection of colloidal gold chromatography hepatopathy of production detects test paper, the serum of the variable concentrations of four parts of dilutions detected that each sample repeats 10 times, all can tell yin and yang attribute accurately, obtains consistent yin and yang attribute result.
Therefore, draw to draw a conclusion: the colloidal gold chromatography hepatopathy detects test paper and does not have batch interior inaccurate phenomenon.
6. inaccuracy between criticizing
Select for use the different colloidal gold chromatography hepatopathys of three batches of production to detect test paper, select each portion of serum (strong, in, weak, feminine gender) of four kinds of variable concentrations simultaneously, every part of serum repeats observations behind the 5min 10 times.
Learn that according to top result three batches of colloidal gold chromatography hepatopathys of the different batches of production detect test paper, four parts of different serum detected that each detects and repeats 10 times, all can tell yin and yang attribute accurately, obtains consistent yin and yang attribute result.
Therefore, draw to draw a conclusion: inaccurate phenomenon between colloidal gold chromatography hepatopathy detection test paper is criticized less than existence.
7. the contrast test of the IMTEC-Gp210 Antibodies kit of the product-IMTECAutoimmundiagnostika GmbH that learns with similar test item distinct methods.From clinical collection 100 parts of patient's sample serum at random, detect test paper with the IMTEC-Gp210 Antibodies kit of IMTECAutoimmundiagnostika GmbH and self-control colloidal gold chromatography hepatopathy respectively and detect, obtain following result:
Positive coincidence rate: 45/46 * 100%=97.82%
Negative match-rate: 52/54 * 100%=96.29%
Total coincidence rate: (45+52)/100=97%
According to top statistics, the total coincidence rate of IMTEC-Gp210 Antibodies kit that self-control colloidal gold chromatography hepatopathy detects test paper and IMTECAutoimmundiagnostika GmbH can reach 97%.
More than be to description of the present utility model and non-limiting, based on other embodiment of the utility model thought, all among protection domain of the present utility model.
Claims (7)
1. the colloidal gold chromatography hepatopathy detects test paper, include sample pad (1), pad (2), cellulose nitrate coated film (3), adsorptive pads (6), described sample pad (1), pad (2), cellulose nitrate coated film (3), adsorptive pads (6) are overlapped mutually to the opposite side of described base plate (7) successively by a side of base plate (7) and stick on the described base plate (7), it is characterized in that:
Described pad (2) is coated with golden labeling antibody a layer and golden labeling antibody b layer;
Described cellulose nitrate coated film (3) is provided with detection line (4) and nature controlling line (5), and described detection line (4) is coated with Gp210 antigen protein layer, and described nature controlling line (5) is coated with golden labeling antibody c layer.
2. colloidal gold chromatography hepatopathy according to claim 1 detects test paper, and it is characterized in that: the antibody A in the described golden labeling antibody a layer is one or more in anti-human IgG monoclonal antibody, anti-human IgG polyclonal antibody, staphylococcal protein A (SPA), the streptococcal protein G (Protein G).
3. colloidal gold chromatography hepatopathy according to claim 2 detects test paper, it is characterized in that: described anti-human IgG polyclonal antibody is a kind of in mouse source, Ma Yuan, Yang Yuan, rabbit source or the cavy source; Described anti-human IgG monoclonal antibody is a kind of in mouse source or the rabbit source.
4. colloidal gold chromatography hepatopathy according to claim 1 detects test paper, and it is characterized in that: the antibody A in the described golden labeling antibody a layer is a staphylococcal protein A.
5. colloidal gold chromatography hepatopathy according to claim 1 detects test paper, it is characterized in that: the antibody B in the described golden labeling antibody b layer and the antibody C in the golden labeling antibody c layer be a kind of in monoclonal antibody or the polyclonal antibody simultaneously simultaneously or, and the antibody C in antibody B in the wherein golden labeling antibody b layer and the golden labeling antibody c layer specificity can take place in conjunction with forming immune complex.
6. colloidal gold chromatography hepatopathy according to claim 5 detects test paper, it is characterized in that: described polyclonal antibody is a kind of in mouse source, Ma Yuan, Yang Yuan, rabbit source or the cavy source, and described monoclonal antibody is a kind of in mouse source or the rabbit source.
7. colloidal gold chromatography hepatopathy according to claim 1 detects test paper, it is characterized in that: the Gp210 antigen protein layer of Gp210 antigen protein layer for obtaining by the prokaryotic expression cloned gene of bag quilt on the described detection line.
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| CN102621312A (en) * | 2011-01-28 | 2012-08-01 | 上海科新生物技术股份有限公司 | Colloidal gold chromatography hepatopathy detection test paper and preparation method thereof |
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| CN102621312A (en) * | 2011-01-28 | 2012-08-01 | 上海科新生物技术股份有限公司 | Colloidal gold chromatography hepatopathy detection test paper and preparation method thereof |
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