CN102621313B - Colloidal gold chromatography anti-Sp100 antibody detection test paper and preparation method thereof - Google Patents
Colloidal gold chromatography anti-Sp100 antibody detection test paper and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a colloidal gold chromatography anti-Sp100 antibody detection test paper and a preparation method thereof. The colloidal gold chromatography anti-Sp100 antibody detection test paper comprises a sample pad, a combination pad, a cellulose nitrate coated film and a water absorption pad, and the sample pad, the combination pad, the cellulose nitrate coated film and the water absorption pad are pasted on a base plate sequentially from one side of the base plate to the other side of the base plate, wherein the combination pad is coated with a gold-labeled antibody a and a gold-labeled antibody b; and the cellulose nitrate coated film is provided with a detection line and a quality control line, the detection line is coated with an Sp100 antigen protein, and the quality control line is coated with a gold-labeled antibody c. By adopting indirect immunoassay to introduce the Sp100 antigen protein in the invention to carry out process optimization on the combination pad and the sample pad, so high sensitivity, high specificity and high accuracy detection performances of the anti-Sp100 antibody are realized, and a reference basis is provided for the auxiliary diagnosis of primary biliary cirrhosis.
Description
Technical field
The invention belongs to medical immunology application, be specifically related to test paper utilizing the anti-Sp100 antibody of colloidal gold immunochromatographimethod technology for detection and preparation method thereof.
Background technology
In PBC patient 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, and the course of disease just occurs that AMA is this sick feature in early days.
AMA finds first by indirect immunofluorescence by WalRer etc. in nineteen sixty-five in PBC patients serum, studies show that thereafter, and PBC patient's AMA positive rate can be up to 90%, and this detects the main inspection item that has become PBC diagnosis.(Neuberger J, Thomson R.PBC and AMA-what is the connection[J] .Hepatology, 1999,29 (1) 265:271) its AMA-M2 susceptibility in PBC can reach more than 90%, still have amynologic index that the PBC patient of 10%AMA-M2 feminine gender needs other to this part patient detect ([1] Gao Rongkai. primary biliary cirrhosis clinical analysis .[J]. the practical medical .2009 of China, 4 (1): 116-117.).
Except AMA, in a part of patients serum, also there is ANA, the positive PBC patient of serum antinuclear antibodies (ANA) accounts for greatly 50% left and right.The common ANA immunofluorescence of PBC patient has nucleus spot type, multinuclear point type, nuclear membrane type, kinetochore type, nucleolar pattern.Multinuclear point type and nuclear membrane type ANA have higher specificity to PBC patient, can be used as AMA negative patient and diagnose PBC serological index.(Luettig B, Boeker K H, Schoessler W, et al.The antinuclearautoantibodies Sp100 and gp210 persist after orthotopic livertransplantation in patients with primary biliary cirrhosis[J] .JHepatol, 1998,28 (5): 824-828), the wherein immune fluorescent patterns of multinuclear point type is that (size 13～110 μ m) for the point that occurs 3～20 different sizes in the whole nucleus except kernel.
Sp100 and progranulocyte leukemia albumen (PML) all produce multinuclear point type immune fluorescent patterns, and they are positioned in nucleome structure, are the target antigen of PBC specific autoantibody.The detecting of anti-Sp100 antibody can be avoided omission and the delay to the negative PBC patient diagnosis of AMA.(Shao Jie, Wei Lai, Wang Hao, Deng. the detection .[J of multinuclear point type antinuclear antibodies in patients with primary biliary cirrhosis]. world Chinese digests magazine, 2005,13 (12): 1478-1481.) anti-Sp100 antibody is PBC specific autoantibody, and its specificity is almost 100%.(Liu Yan, Yan Huiping, Tan Yufen etc. the detection .[J of the anti-Sp100 antibody of patients with primary biliary cirrhosis and epipole type antinuclear antibodies]. clinical examination magazine 2009,27 (4): 256-258.)
Sp100 albumen and PML albumen are positioned epipole (also claiming nucleome) jointly, are called as epipole albumen, and the two reacts with PBC patients serum and presents similar fluorescence and show an epipole type, but the two is diverse antigen molecule.Their function it be unclear that, and regulates active DNA in conjunction with territory but its structure comprises a zinc fingers with having, and Sp100 is similar to the sequence of MHC-I antigen-binding site conservative region in addition.
The impact of the factors such as the expression of epipole albumen on core is infected by the virus, interferon.Initial western blotting method shows that Sp100 antigen is the molecule of molecular weight 100KD left and right, but what obtain by clonal expression is 480 amino acid compositions of a coding, molecular weight is the molecule of 53KD, because it shows as the unusual mobility of 100KD when the gel electrophoresis, therefore called after Sp100.Be 296-311 and two regions of 332-351 by the main linear epitope of genetic fragment phage display peptide library technical Analysis Sp100, reaction core is respectively SNSKVE/EPLEVFISA.
Anti-Sp100 antibody often shows as multinuclear point type (multiplenuclear dots in immunofluorescence method, MND), be visible more than 3～20 the point-like particles of interval cell, vary in size and be distributed in whole nucleus, endochylema, without fluorescence, is easily mistaken for anti-centromere antibody (ACA), but ACA can make, division cells chromosome is painted presents typical case performance, its numbers of particles is more constant, and size evenly.
Taking restructuring Sp100 as antigen, detect this antibody by ELISA method at present, susceptibility and specificity all improve compared with IIF method.The anti-Sp100 antibody positive rate of the detection PBC patients such as Szostechi is 31%, and in other autoimmunity diseases (as SLE, PSS, MCTD, PM/DM etc.) its positive rate < 3%; In the negative PBC patient of AMA, there is the anti-Sp100 antibody of 48% appearance, point out this antibody to the negative PBC patient's of AMA diagnosis helpful (Szostecki C, et al.Autoantibodies against ' nuclear dots ' in Drimary billary cirrhosis.Seminars LiverDi s.1997; 17 (1): 71-78.)
Z ü chner etc. finds anti-Sp100 antibody positive rate 21% in 170 routine PBC patients, anti-PML antibody positive rate 13%, under fluorescent microscope, multinuclear point type (MND type) ANA positive patient accounts for 25%, this class patient liver histological makes fast progress: after the UDCA treatment of 2 years, the antibody of identification total length Sp100 does not change before and after research, the Sp100 epitope of finding part PBC patient treatment front and back antibody recognition by IBT rule changes, this may be that UDCA regulates (Z ü chner D due to body fluid immunization, et al.Prevalence, kinetics, and therapeutic modulationof autoantibodies against Sp100 and promyelocytic leukemiaprotein in a large cohort of patients with primary billarycirrhosis.Hepatology.1997:26 (5): 1123-1130.).
In view of anti-SP100 antibody can reach 60% at the PBC patient's of AMA feminine gender positive rate, therefore, the diagnosis that anti-Sp100 antibody test is the PBC patient of AMA feminine gender has very important significance.Anti-Sp100 antibody is the specific autoantibody of PBC, and this antibody specificity in PBC diagnosis, up to 97-100%, seldom appears in other autoimmune diseases, and susceptibility is 20%-30%, can be used as an important auxiliary diagnostic index of PBC.
The present invention adopts escherichia coli prokaryotic expression cloned gene to obtain antigen protein Sp100, is the detectable antigens of this product.
At present the method for the conventional anti-soluble acid nucleoprotein of detection (SP100) antibody in laboratory 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.
Indirect immunofluorescence, observations needs professional to operate special instrument and equipment, indirect immunofluorescence exists grow but must have fluorescence microscope result detection time, technical program more complicated, and there is an artificial error in judgement of certain subjectivity, be subject in addition other and disturb generation false positive, standardization difficulty, is also not suitable for the detection of high flux sample.
Enzyme linked immunosorbent assay, needs repeatedly application of sample and washing step, completes whole experimentation and needs about three hours.
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 details, need repeatedly application of sample and washing, need microplate reader, also need professional immunological technique personnel in laboratory, to carry out experimental implementation, be subject to the restriction in personnel and place, be subject to the impact of the environmental baseline factors such as various temperature and incubation time simultaneously, test is brought to inconvenience.
Linear immunoassay is mainly used in the examination of the large class of disease, and specific aim is relatively poor, is also not suitable for the detection of high flux sample simultaneously.
The anti-Sp100 antibody assay kit of commercialization is in the market mainly Western blot, though combine the high resolution of SDS-PAGE and high specific and the susceptibility of ELISA method, but operation relative complex, complete whole experimentation and need about three hours, also need professional immunological technique personnel in laboratory, to carry out experimental implementation, be subject to the impact of the environmental baseline factors such as various temperature and incubation time simultaneously, test is brought to inconvenience.
Colloidal gold chromatography is applied in the detection of anti-Sp100 antibody.Colloidal gold immunity chromatography (gold-immunochromatography assay, GICA) be application colloidal gold-labeled method, using collaurum as tracer, taking fibre strip chromatographic material as solid phase, make sample solution swimming on chromatography strip by capillary effect, make the acceptor (as antigen or antibody) for determinand on determinand in sample and pad that immune response occur, and there is immune response and be trapped with the antigen (or antibody) on fibre strip chromatographic material, and then form macroscopic aubergine band, obtain experimental result intuitively, reach the object (Sikowicz G et al.One-stepchromatographic immunoassay for qualitative determination ofchoricogonadotropin in urine.Clin Chem.1990 (36) 1579-1586.) of fast detecting.When use only by sample pipetting volume on sample pad, within several minutes, just occur that according to aubergine band on detection line situation judges yin and yang attribute result.The advantages such as compared with other detection methods, detection time is short, only needs 5～10 minutes, and operating personnel are without training, easy and simple to handle, quick, preserves without low temperature, and accumulating is convenient.
Aspect autoimmune disease, on foreign market, occurred that at present some detect the test strips product of autoimmune disease, but the colloid gold immune test paper of anti-Sp100 antibody does not still come out at home and abroad on market.
Summary of the invention
Technical matters to be solved by this invention is in order to overcome above methodological deficiency, colloidal gold chromatography is applied in the detection of anti-Sp100 antibody, first Sp100 antigen protein is applied in colloidal gold chromatography simultaneously, adopt indirect method to realize the anti-SP100 antibody test in blood, realize the detection performance of high special, high sensitivity, high accuracy, rapid screening goes out the positive sample of anti-SP100 antibody, can be fast, auxiliary diagnosis primary biliary cirrhosis easily.
One of technical matters to be solved by this invention is to provide a kind of chromatographic colloidal gold anti-Sp 100 antibody test paper.
Two of technical matters to be solved by this invention is to provide a kind of preparation method of chromatographic colloidal gold anti-Sp 100 antibody test paper.
As a kind of chromatographic colloidal gold anti-Sp 100 antibody test paper of first aspect present invention, include sample pad, pad, cellulose nitrate coated film, adsorptive pads, described sample pad, pad, cellulose nitrate coated film, adsorptive pads are sticked on 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 and golden labeling antibody b;
On described cellulose nitrate coated film, be provided with detection line and nature controlling line, described detection line is coated with Sp100 antigen protein, and described nature controlling line is coated with golden labeling antibody c.
Further, the antibody A in described golden labeling antibody a is one or more in anti-human IgG monoclonal antibody, anti-human IgG polyclonal antibody, staphylococcal protein A (SPA), streptococcal protein G (Protein G).
Described anti-human IgG polyclonal antibody is the one in mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source.
Described anti-human IgG monoclonal antibody is the one in Yang Yuan or rabbit source.
Antibody A in described golden labeling antibody a is preferably staphylococcal protein A.
When antibody C while in antibody B and golden labeling antibody c in described golden labeling antibody b or difference, be the one in monoclonal antibody or polyclonal antibody, can there is specific binding formation immune complex in the antibody C in antibody B and the golden labeling antibody c in golden labeling antibody b wherein.
Described polyclonal antibody is the one in mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source, and monoclonal antibody is the one in Huo Tu source, mouse source.
Antibody B in described golden labeling antibody b is preferably rabbit igg, and correspondingly the antibody C in golden labeling antibody c is preferably goat anti-rabbit igg.
On described detection line, coated Sp100 antigen protein is the Sp100 antigen protein obtaining by prokaryotic expression cloned gene.
Described Sp100 antigen protein is the recombinant protein obtaining by the cloned gene of escherichia coli prokaryotic expression.
The sample of described sample pad is from human serum, blood plasma, whole blood sample.
According to the present invention the monoclonal antibody of application can by by Kohler etc. (Continuouscultures of fused cells secreting antibody of predefinedspecificity[J] .Nature, 1975 (256): 495-497) the hybridoma method of first describing is prepared, or can be prepared by recombinant DNA method (seeing United States Patent (USP) 4816567)." monoclonal antibody " by Clackson etc. (Making antibody fragments using phagedisplay libraries[J] .Nature, 1991:624-628) and the described technology of Marks etc. (By-passingimmunization:Human antibodies from V-gene libraries displayed onphage[J] .Journal of Molecular Biology, 1991:581-597) from phage antibody library, separate.
According to the present invention, the polyclonal antibody of application can be prepared by immune animal by Chen Xueqing etc. (immunology common experimental method .[M], 2000:15-26).
SPA of the present invention, Protein G be by prokaryotic expression cloning recombination, the escherichia coli prokaryotic expression cloned gene of being described by J. Pehanorm Brooker etc. (molecular cloning experiment guide .[M], 2002:1228-1232).
As the preparation method of a kind of chromatographic colloidal gold anti-Sp 100 antibody test paper of second aspect present invention, this test paper is made up of jointly sample pad, pad, cellulose nitrate coated film, adsorptive pads and base plate, and its special disease is that the method includes following steps:
(1) preparation of Sp100 antigen protein, obtains Sp100 antigen protein by prokaryotic expression cloned gene;
(2) preparation of golden labeling antibody c: with 0.1M sal tartari adjusting collaurum pH 7.0～9.0, slowly add 4～25 μ g antibody C by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(3) preparation of cellulose nitrate coated film, the Sp100 antigen protein of preparing by coated film damping fluid dilution step (1) to concentration is 0.5～1.5mg/mL, is coated on the detection line of cellulose nitrate coated film; Dilute antibody c is diluted to 0.8～2.0mg/mL with coated film damping fluid, be coated on the nature controlling line of nitrocellulose filter with 1～10 μ l/cm, after cellulose nitrate coated film prepares, be placed in 37 DEG C of dry for standby;
(1) preparation of golden labeling antibody a: with 0.1M sal tartari adjusting collaurum pH 7.0～9.0, slowly add 4～25 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(2) preparation of golden labeling antibody b: with 0.1M sal tartari adjusting collaurum pH 7.0～9.0, slowly add 4～25 μ g antibody B by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(3) preparation of pad: the polyester film of treated liquid dip treating, after oven dry, by after golden labeling antibody a and golden labeling antibody b mixing, be sprayed on pretreated polyester film with the consumption of 0.5～4 μ l/cm, 25 DEG C～37 DEG C after dry pad, pad is placed under the environment of 2 DEG C～8 DEG C for subsequent use;
To after treated glass fibre membrane liquid dip treating, make sample pad, sample is padded on after 37 DEG C of oven dry for subsequent use;
In (2) of described step 1, the preparation of gold labeling antibody c: with 0.1M sal tartari adjusting collaurum pH 8.0～9.0, slowly add 5～15 μ g antibody C by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use; Wherein antibody C is goat anti-rabbit igg.
In (1) of described step 2, the preparation method of gold labeling antibody a, for regulating collaurum pH value to 8.0～9.0 with 0.1M sal tartari damping fluid, slowly adds 5～15 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA 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 DEG C for subsequent use; Wherein antibody A is goat anti-human igg.
In (1) of described step 2, the preparation method of gold labeling antibody a is for regulating collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid, slowly add 8～12 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use.Then gold is marked to SPAOD 30 2～4 μ l/cm and be sprayed at pad, dry rear for subsequent use; Wherein antibody A is mouse-anti human IgG.
In (1) of described step 2, the preparation method of gold labeling antibody a is for regulating collaurum pH value to 5.0～6.5 with 0.1M sal tartari damping fluid, slowly add 10～15 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use.Then gold is marked to SPA OD 20 2～4 μ l/cm and be sprayed at pad, dry rear for subsequent use; Wherein antibody A is staphylococcal protein A.
In (2) of described step 2, the preparation method of gold labeling antibody b is for regulating collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid, slowly add 5～15 μ g antibody B by every milliliter of colloidal gold solution, rabbit igg, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use, wherein antibody B is rabbit igg.
In (3) of described step 2, by the golden labeling antibody a preparing and golden labeling antibody b 20～40: 15～20 ratios mixing by volume, be sprayed on pretreated polyester film with 2.0～4 μ l/cm with BIO-Dot Membrane jetter, 25 DEG C～37 DEG C dry pads that to obtain, pad envelope is placed under the environment of 2 DEG C～8 DEG C for subsequent use; Wherein the antibody A in golden labeling antibody a is staphylococcal protein A, and golden labeling antibody b is gold mark rabbit igg.
The object of described damping fluid is for providing certain pH and ionic strength to make gold chloride collaurum present required state of charge and ionic strength, its albumen being labeled is coated in around the ion of gold chloride collaurum taking Aucl-as core, and forms metastable a kind of state.Every kind of albumen is because its state of charge is different with amino acid composition, and therefore coated required condition is also different, need be by testing the kind, concentration, the pH that determine its damping fluid.In general, the required damping fluid of antibody labeling can be sal tartari, borate buffer, phosphate buffer, carbonic acid buffer etc., and its concentration is 0.1～0.5M, and pH is 5～10.
In described step 3, the width of the test paper cutting into is preferably two kinds of 4mm and 3mm.
The application of chromatographic colloidal gold anti-Sp 100 antibody test paper of the present invention, it is anti-Sp100 antibody in qualitative detection human serum, auxiliary diagnosis early primary bile cirrhosis.
Detection principle of the present invention is specially the recombinant expressed SP100 antigen protein of selecting affinitive layer purification, gold labeling antibody a and gold mark rabbit igg are as colloid gold label compound, be sprayed at pad, utilize indirect method to detect and in blood serum sample, whether contain anti-SP100 antibody.When detection, sample is along with chromatography swimming is to pad and infiltrate colloid gold label compound, human IgG wherein and golden labeling antibody a are in conjunction with forming human IgG-Jin labeling antibody a compound, due to capillary effect, this person IgG-gold labeling antibody a compound along coated film swimming forward, if there is anti-SP100 antibody in blood serum sample, this person IgG-gold labeling antibody a compound be coated in the antigen protein generation specific immunity association reaction on nitrocellulose filter, form golden labeling antibody a-human IgG-Sp100 antigen protein triplet compound and be trapped within on 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 nature controlling line and special immune response occurs be trapped, be enriched in gradually the darker aubergine band of formation on nature controlling line, unnecessary unconjugated material continues chromatography to adsorptive pads, therefore all occurs the positive findings that is judged to of band at detection line and nature controlling line, if do not contain anti-SP100 antibody in blood serum sample, when gold labeling antibody a arrives detection line, not with the antigen protein generation immune response being coated on detection line, therefore there is not aubergine band at detection line place, gold labeling antibody a continues swimming and arrives forward adsorptive pads, and gold mark rabbit igg continue swimming forward be coated in nature controlling line place goat anti-rabbit igg and special immune response occur and be trapped, be enriched in gradually on nature controlling line and form aubergine band, therefore only in Quality Control, occur that band is judged to negative findings.
The antigen protein that the present invention expresses genetic engineering bacterium is incorporated in test strips first, has realized the detection performance of high specific, high sensitivity, pin-point accuracy.Also occurred on the market at present commercialization ELISA kit, but gold mark chromatographic test paper by comparison, still has many differences, is not subject to place personnel's limit, detection time is short is 5-10 minute, and sentence read result is easy.In addition this test paper has high specific, high sensitivity, pin-point accuracy, can meet the rapid screening PBC in market, for patient's diagnoses and treatment early provides condition, meanwhile, also can meet the demand of laboratories, instant detection, bedside detection.
Compared with existing detection method, advantage of the present invention:
1. the Sp100 antigen protein first Application that uniqueness of the present invention is DNA recombinant expression is in colloid gold chromatographic test paper, greatly improve its detection sensitivity, can rapid screening go out all positive sample of anti-SP100 antibody (can detect the sample that is greater than 25RU/ml) by colloid gold test paper.
2. advantage of the present invention is that production cost is low.The required core reagent of Test paper of anti-SP100 antibody provided by the present invention is that antibody A is anti-human IgG or SPA or PROTEING, 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.
With published for compared with the additive method of anti-SP100 antibody test, test paper of the present invention has advantages of that many additive methods can not compare, as short in detection time (5～10min); Without any need for specific apparatus, can realize bedside detection and outpatient service and immediately detect; Easy and simple to handle, only need single step reaction, operating personnel are without training, and testing cost is low; Temperature, without particular/special requirement, without freezing, is stored to convenient transportation, and room temperature can be preserved 24 months.
Brief description of the drawings
Fig. 1 is side structure schematic diagram of the present invention.
Fig. 2 is testing result schematic diagram of the present invention.
Wherein:
Fig. 2 a is depicted as: after application of sample, reaction 3～5min can see on detection zone D and E relevant position, control zone and occurs aubergine band;
Fig. 2 b is depicted as: when aubergine band all appears in control zone E and detection zone D, result is positive, illustrates and in serum, contains anti-Sp100 antibody;
Fig. 2 c is depicted as: as only there is an aubergine band at control zone E, aubergine band does not appear in detection zone D, and result is negative, illustrates in serum not containing anti-Sp100 antibody;
Fig. 2 d, 2e are depicted as: as aubergine band does not appear in control zone E, no matter whether detection zone D has band to occur, all illustrates that test paper lost efficacy.
Embodiment
The anti-Sp100 antibody test of colloidal gold immunity chromatography of the present invention test paper, as shown in Figure 1, this test paper is mutually to paste in turn cellulose nitrate coated film 3, pad 2, sample pad 1, adsorptive pads 6 by a side direction opposite side on base plate 7 with overlapping.
On pad 2, be coated with golden labeling antibody a and golden labeling antibody b, the antibody A of golden labeling antibody a is goat anti-rabbit igg gold labeling antibody or mouse-anti human IgG gold labeling antibody or the golden labeling antibody of staphylococcal protein A (SPA) or streptococcal protein G gold labeling antibody; Antibody B in gold labeling antibody b is rabbit igg.
On cellulose nitrate coated film 3, be provided with detection line 4 and nature controlling line 5, detection line 4 is coated with Cenp-B antigen protein, and nature controlling line 5 is coated with golden labeling antibody c, and the antibody in golden labeling antibody c is goat anti-rabbit igg.
Below in conjunction with specific embodiments and the drawings, further set forth the present invention.
The Sp100 antigen protein that is applied to this test paper is to build recombination by gene clone technology, and then adopting prokaryotic expression technology successful expression to go out is all the Sp100 antigen protein in people source.Wherein, Sp100 antigen protein derives from purifying gene engineering antigen protein.
Antibody A and antibody C, antibody B are prepared by following method.Wherein the anti-human IgG in antibody A, antibody C and antibody B generally can by animal in repeatedly subcutaneous (sc) or peritonaeum immunogene and the adjuvant of (ip) injection purifying produce.
By 0.05mg～1mg immune formulation (respectively for goat or mouse) and Freund ' the s Freund's complete adjuvant of 3 times of volumes are mixed to get to injection solution, by the multiple location injection in Animal Skin of this injection solution, after one month by animal with Freund ' the s Freund's complete adjuvant mixed liquor of 1/5 to 1/10 human IgG of originally measuring through multiple location Animal Skin hemostasis and booster immunization.After 7～14 days, by animal bloodletting, measure the anti-human IgG titre of serum.To animal booster immunization until titre reaches plateau.The method of producing polyclonal antibody has been described in many immunology textbooks, for example, Chen Xueqing etc. " immunology common experimental method ".By from being reclaimed splenocyte and make cellular immortalization by immune animal, for example, by merge or pass through Epstein-Barr virus Transformation with myeloma cell, and screening can be expressed monoclonal antibody (the Kohl er of object antibody, Milstein.Derivation of specificantibody-producing tissue culture and tumor lines by cell fusion[J] .European Journal of Immunology, 1976:501-511).
Can carry out restructuring staphylococcal protein A and the streptococcal protein G in Dispersal risk A by escherichia coli prokaryotic expression cloned gene, concrete operation method referring to J. Pehanorm Brooker etc. (molecular cloning experiment guide .[M], 2002:1228-1232), or buy commercial restructuring staphylococcal protein A or streptococcal protein G.
Colloidal gold solution preparation
By 0.01% HAuCl
4solution is heated to boiling, adds rapidly every 100mL HAuCl
4solution adds appropriate reductant solution, and color is from blueness, then light blue, blue, then heating occurs redly, boils 7～10min and occurs transparent orange red.With ultrafiltration or miillpore filter, (0.45 μ m) filters, to remove polymkeric substance and other impurity that may sneak into wherein again.The collaurum outward appearance preparing should be pure, bright, without precipitation and floating thing, when grease and a large amount of black particle shape sediment appear in liquid level, abandon.
The reductive agent that wherein used can, for trisodium citrate (Frens 1973), tannic acid-trisodium citrate (Slot and Gueeze 1985), white phosphorus, preferably use trisodium citrate, more preferably uses 1% trisodium citrate.
Wherein glass container used should be definitely clean, with before need be through pickling, silication.Its water should be deionization ultrapure water, and resistivity reaches 18.2M Ω.
In 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 DEG C for subsequent use, 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.
The preparation method of the anti-Sp100 antibody test of colloidal gold immunity chromatography of the present invention test paper, specifically comprises the steps:
(1) adopt the method for embodiment 1 to prepare Sp100 antigen protein;
(2) preparation of goat anti-rabbit igg gold labeling antibody:
The collaurum pH 7.0～9.0 that regulates embodiment 3 to prepare 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, then add BSA 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 DEG C for subsequent use;
(3) preparation of cellulose nitrate coated film 3, diluting Sp100 antigen protein to concentration with coated film damping fluid is 1.0～1.5mg/mL, adjust BIO-Dot instrument, be sprayed at the upper detection line of nitrocellulose filter (NC) 4 places, near pad 2 ends, apart from the about 9.5mm of pad 2, Cenp-B antigen protein is coated on the detection line 4 of cellulose nitrate coated film;
Goat anti-rabbit igg is diluted to 0.8～1.5mg/mL with coated damping fluid, is sprayed at upper nature controlling line 5 places near adsorptive pads 6 of nitrocellulose filter (NC) with 1～10 μ l/cm with BIO-Dot instrument, apart from the about 9mm of adsorptive pads 6. Detection line 4 and 5 liang of approximately 5～8mm of linear distance of nature controlling line, spray line is answered even thickness.37 DEG C of oven dry, encapsulate for subsequent use.
The coated damping fluid of its use can be borate, carbonate, phosphate, Tris-HCl or Tris-phosphate, acetate, barbital, etc., the object of its damping fluid is for providing certain pH and ionic strength to make albumen be coated with and firmly be coated in NC film, and its pH of cushioning fluid is generally about in 6～9.5 scopes, be preferably within the scope of 6.5～7.5 neutral buffered, and most preferably the pH value of damping fluid is in 7.0～7.4 scopes.Damping fluid is preferably phosphate.
Nitrocellulose filter (NC) wherein can be any commercialization nitrocellulose filter, S & SAE99, whatman 8 μ m, millipore M135, sartorius CN140 etc.The concrete NC film using is not key of the present invention, but in each mensuration, above-mentioned several NC films can be used as preferably.The film of the different damping fluid processing containing different surfaces activating agent that different manufacturers uses, there is gap in various degree with detection line antibody-solutions affinity used, also can largely cause lines inhomogeneous, traction or the phenomenon of disperse, therefore use assembling test paper to select 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 to prepare 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, then add BSA 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 DEG C for subsequent use;
(2) preparation of rabbit igg gold labeling antibody: collaurum pH value to 7.0～8.0 that regulate embodiment 3 to prepare with 0.1M sal tartari, slowly add 8～12 μ g rabbit iggs by every milliliter of colloidal gold solution, stir 10～30min, then add BSA 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 DEG C for subsequent use.
(3) preparation of pad: polyester film is soaked 30 minutes through damping fluid, 37 DEG C of oven dry, after goat anti-human igg gold labeling antibody and rabbit igg gold labeling antibody are mixed according to a certain percentage, use BIO-Dot instrument, be sprayed on pretreated polyester film with the consumption of 0.5～4 μ l/cm, 25 DEG C～37 DEG C dry, the complete pad 2 that obtains afterwards to be dried, pad 2 vacuum packagings, put 2 DEG C～8 DEG C for subsequent use.Be placed under the environment of 2 DEG C～8 DEG C for subsequent use;
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 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 pretreated polyester film with BIO-Dot discharge rate 1 μ l/cm, with 0.01MPBS be sample-loading buffer, can obtain the band of color intensity of expection, determine 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 to gradient dilution to final concentration OD 100,80,60,40,20, then rabbit igg gold labeling antibody is diluted to final concentration OD 20, with BIO-Dot be that 3 μ l/cm are sprayed on the polyester film of handling well by above goat anti-human igg gold labeling antibody and rabbit igg gold labeling antibody potpourri discharge rate, the cellulose nitrate coated film 3 of preparation, pad 2, sample pad 1, adsorptive pads 6 are pasted on base plate 7 successively, with positive serum, critical reference value serum, negative serum be debugger object.Judgment basis: both band color intensities of the detection line 4 of positive serum and nature controlling line 5 are consistent is foundation, and critical reference value serum can occur, that OD value that negative serum occurs without band, is the application quantity of this batch.Show that by this test OD20～40 comparatively meet the requirements.
Glass fibre membrane is pressed to 45mL/ sheet, evenly spill and be applied on glass fibre membrane with damping fluid, obtain sample pad after 37 DEG C of oven dry, sample pad encapsulates with aluminium foil bag, for subsequent use;
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.
First carry out starting material pre-cut:
Cutting of sample pad: with guillotine, sample pad is cut into the i.e. long 28cm of base plate equal length making with PVC, wide 2.4cm, puts between drying shed for subsequent use.
Cutting of adsorptive pads: with trimmer, thieving paper is cut into the i.e. long 28cm of base plate equal length making with PVC, wide 3cm makes adsorptive pads, puts between drying shed for subsequent use.
Cutting of pad: be cut into the i.e. long 28cm of base plate equal length making with PVC in connection with pad with guillotine, wide 2.4cm, puts between drying shed for subsequent use.
Cutting of cellulose nitrate coated film: with trimmer, cellulose nitrate coated film is cut into the i.e. long 28cm of base plate equal length making with PVC, wide 1cm, puts drying shed for subsequent use.
Cellulose nitrate coated film 3, pad 2, sample pad 1, adsorptive pads 6, by stacking gradually shown in Fig. 1 on the base plate 7 of making at PVC plastics, are formed to large plate.Composing room's temperature should be controlled at 25 DEG C～37 DEG C, humidity 20%～30%.
Slitting: large plate is cut into single part with cutting cutter, every person-portion width is cut into the width of 2.5mm～4mm according to certain requirement, random sampling observation, sensitivity can detect Internal Quality Control sample (i.e. weak positive sample), band colour developing degree reaches the d as Fig. 2, and specific band nothing but, product specifies to become specification product by Quality Control.
Assembling, packaging: the test paper that 1 person-portion has been cut is assembled in the test card of getting ready, make the sample pad of the corresponding test paper of application of sample window, the corresponding detection zone of result display window and control zone, and composing room's temperature should be controlled at 25 DEG C～37 DEG C, humidity 20%～30%.Be encapsulated in outer bag with drying agent, instructions, sample pipetting volume device again, keep in Dark Place in 4～25 DEG C.
In the preparation process of above-mentioned anti-Cenp-B antibody detecting test paper adopting colloidal gold immunochromatography, the collocation method of each solution is as follows:
1.0.1M the preparation of sal tartari: by ultrapure water preparation, 0.22 μ m membrane filtration mistake, put 4 DEG C for subsequent use, the term of validity one month.1000mL 0.1M K
2cO
3solution formula: 13.8g K
2cO
3; Ultrapure 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 DEG C for subsequent use, the term of validity two weeks.1000mL 10%BSA solution formula: 100g BSA, 0.5g NaN
3; Ultrapure water is settled to 1000mL.
3, the preparation of coated damping fluid: 9gNac l, 1.15gNa
2hPO
4, 0.23g NaH
2pO
4, 10gSucrose, 0.5gEDTA be dissolved in 1L ultrapure water, filter be placed in 4 DEG C for subsequent use.
4. cleansing solution also preserves the preparation of liquid:
2%BSA, 0.05% (NaN
3), 0.01M pH 7.2PBS, 0.22 μ m membrane filtration mistake, put 4 DEG C for subsequent use, the term of validity two weeks.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 ultrapure water, adjust pH 8.0, constant volume is to 1000mL.
Gold mark is diluted to after working concentration with dilution, adds the trehalose of 20%Sucrose and 5%.
The preparation method of the anti-Sp100 antibody test of the colloidal gold immunity chromatography test paper of this embodiment, its step is basic identical with embodiment 4, just the preparation process of goat anti-human igg's gold labeling antibody of (1) in the step 2 of this embodiment is replaced with to the preparation of the golden labeling antibody of staphylococcal protein A (SPA), specific as follows:
With pH 9.0 0.2M borate buffers adjusting collaurum pH value to 5.0～6.5, slowly add 10～15 μ gSPA albumen by every milliliter of colloidal gold solution, stir 10～30min, then add BSA 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 DEG C for subsequent use.
Blending ratio between the golden labeling antibody of staphylococcal protein A (SPA) in this embodiment step 2 and rabbit igg gold labeling antibody can be carried out according to the method for embodiment 4.
The preparation method of the anti-Sp100 antibody test of the colloidal gold immunity chromatography test paper of this embodiment, its step is basic identical with embodiment 4, just the preparation process of goat anti-human igg's gold labeling antibody of (1) in the step 2 of this embodiment is replaced with to the preparation of mouse-anti human IgG gold labeling antibody, specific as follows:
With 0.1M sal tartari adjusting collaurum pH value to 7.0～8.0, slowly add 8～12 μ g mouse-anti human IgGs by every milliliter of colloidal gold solution, stir 10～30min, then add BSA 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 DEG C for subsequent use.
Blending ratio between mouse-anti human IgG gold labeling antibody in this embodiment step 2 and rabbit igg gold labeling antibody can be carried out according to the method for embodiment 4.
Embodiment 7
The preparation method of the anti-Sp100 antibody test of the colloidal gold immunity chromatography test paper of this embodiment, its step is basic identical with embodiment 4, just the preparation process of goat anti-human igg's gold labeling antibody of (1) in the step 2 of this embodiment is replaced with to the preparation of streptococcal protein G gold labeling antibody, specific as follows:
With pH 9.0 0.2M borate buffers adjusting collaurum pH value to 5.0～6.5, slowly add 10～15 μ g streptococcal protein Gs by every milliliter of colloidal gold solution, stir 10～30min, then add BSA 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 DEG C for subsequent use.
Blending ratio between streptococcal protein G gold labeling antibody in this embodiment step 2 and rabbit igg gold labeling antibody can be carried out according to the method for embodiment 4.
Recombinant expressed Sp100 antigen protein is incorporated into chromatographic colloidal gold anti-Sp 100 antibody test paper by the present invention, can realize the detection of the anti-Sp100 antibody in blood sample, can be fast, auxiliary diagnosis primary biliary 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 obtain 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.Or serum sample is slowly dripped to 3～5 in sample pad with suction pipe, 5～10min observations, occurs that according to band situation carrys out interpretation yin and yang attribute result.
Embodiment 9
The detection of kit and clinical performance assessment
The antibody A of the golden labeling antibody a of the anti-Sp100 antibody test of collaurum of the present invention test paper is preferably SPA, and the antibody B of antibody b is 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
1.1 37 DEG C of accelerated stabilities
Test paper is placed in to 37 DEG C and accelerates experiment, take out every day and test with Internal Quality Control product, the withinrun precision of the yin and yang attribute coincidence rate by yin and yang attribute reference material (each 10 parts) judges the stability of test paper.After 4 months, result shows, 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%.Yin and yang attribute reference material is 10 parts of anti-SP100 Positive Seras and 10 parts of anti-SP100 negative antibody serum.
1.2 4 DEG C of stability experiments
Test paper is placed in to 4 DEG C and carries out conventional stability experiment, monthly take out with the test of Internal Quality Control product, the withinrun precision of the yin and yang attribute coincidence rate by yin and yang attribute reference material (each 10 parts) judges the stability of test paper equally.After 12 months, result shows, quality-control product testing result meets expection, and the yin and yang attribute coincidence rate of each yin and yang attribute reference material is 100%.After 18 months, result shows, the yin and yang attribute coincidence rate of each yin and yang attribute reference material is still 100%.After 24 months, testing result shows, occurs an official holiday feminine gender.Illustrate that based on the above results test paper, 2～8 DEG C of storages, is stable in 2 years.
2. diagnostic sensitivity
From clinical, collect 100 parts of serum that are diagnosed as primary biliary hepatitis (PBC) patient, the operation steps going up to specifications with homemade colloid gold test paper detects the PBC patients serum who collects above.After 5min, statistics is as follows:
According to the result of adding up above, according to the detection of the PBC patients serum to 100 parts of confirmations, the sample size that can find that there is anti-SP100 antibody positive is 22 examples, and negative sample quantity is 78 examples.Therefore by calculating:
Diagnosis sensitivity (%)=22/ (22+78) × 100%=22%
3. specificity
From clinical, collect each 300 parts of healthy blood donor's serum and non-patient PBC (comprise other autoimmune diseases as rheumatoid arthritis and certainly exempt from anti-Sp100 antibody human) serum, the operation steps going up to specifications with homemade colloid gold test paper detects the healthy blood donor who collects above and non-patient's PBC serum.After 5min, statistics is as follows:
According to the statistics of the detection to non-patient PBC and the each 300 parts of serum of healthy blood donor, the sample size that discovery records anti-SP100 negative antibody in healthy blood donor is 298 examples, but not the sample size of PBC patients serum's anti-SP100 negative antibody is 297 examples.Therefore by calculating:
(healthy blood donor) specificity (%)=298/300 × 100%=99.3%
(non-PBC patients serum) specificity (%)=297/300=99%
600 routine control groups (comprising healthy blood donor and non-PBC patients serum)
Specificity (%)=595/600=99.16%
More than the description of this invention and non-limiting, based on other embodiment of inventive concept, all among protection scope of the present invention.
Claims (13)
1. chromatographic colloidal gold anti-Sp 100 antibody 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 mutually overlapped and stick on described base plate (7) above to the opposite side of described base plate (7) successively by a side of base plate (7), it is characterized in that:
Described pad (2) is coated with golden labeling antibody a and golden labeling antibody b;
On described cellulose nitrate coated film (3), be provided with detection line (4) and nature controlling line (5), described detection line (4) is coated with Sp100 antigen protein, and described nature controlling line (5) is coated with golden labeling antibody c;
The preparation method of described chromatographic colloidal gold anti-Sp 100 antibody test paper, specifically comprises the steps:
Step 1, the preparation of cellulose nitrate coated film
(1) preparation of Sp100 antigen protein, obtains Sp100 antigen protein by prokaryotic expression cloned gene;
(2) preparation of golden labeling antibody c: with 0.1M sal tartari adjusting collaurum pH7.0～9.0, slowly add 4～25 μ g antibody C by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(3) preparation of cellulose nitrate coated film, the Sp100 antigen protein of preparing by coated film damping fluid dilution step (1) to concentration is 0.5～1.5mg/mL, is coated on the detection line of cellulose nitrate coated film; Golden labeling antibody c is diluted to 0.8～2.0mg/mL with coated film damping fluid, is coated on the nature controlling line of nitrocellulose filter with 1～10 μ l/cm, after cellulose nitrate coated film prepares, be placed in 37 DEG C of dry for standby;
Described coated film damping fluid is the one in borate buffer solution, carbonate buffer solution, phosphate buffer, Tris-HCl damping fluid, Tris-phosphate buffer, acetate buffer, barbitol buffer solution, and coated film pH of cushioning fluid is 6～9.5;
Step 2, the preparation of pad
(1) preparation of golden labeling antibody a: with 0.1M sal tartari adjusting collaurum pH7.0～9.0, slowly add 4～25 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(2) preparation of golden labeling antibody b: with 0.1M sal tartari adjusting collaurum pH7.0～9.0, slowly add 4～25 μ g antibody B by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(3) preparation of pad: through the polyester film of damping fluid dip treating, after oven dry, by after golden labeling antibody a and golden labeling antibody b mixing, be sprayed on pretreated polyester film with the consumption of 0.5～4 μ l/cm, 25 DEG C～37 DEG C after dry pad, pad is placed under the environment of 2 DEG C～8 DEG C for subsequent use;
Described damping fluid is one of following:
(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;
Step 3, the preparation of sample pad
Glass fibre membrane is made to sample pad after damping fluid dip treating, and sample is padded on after 37 DEG C of oven dry for subsequent use;
Described damping fluid is one of following:
（a）0.05M?Borax、0.01MPBS、0.1%Sodium?Casein、1%PEG20000、2%BSA、0.05%NaN
3；
（b）0.05M?Borax、0.01MPBS、0.1%Sodium?Casein、1%PEG20000、2%Casein、0.05%NaN
3；
（c）0.05M?Tris-cl、0.01MPBS、0.1%Sodium?Casein、1%PEG20000、2%Casein、0.05%NaN
3；
Step 4, on base plate, overlap joint is pasted the cellulose nitrate coated film, pad, sample pad and the adsorptive pads that complete through abovementioned steps 1-3 made mutually in turn;
Step 5, the material that step 4 made is completed, cuts into test strips;
Described golden labeling antibody a can the IgG in human serum sample be combined and be formed compound, if there is Sp100 antibody in blood serum sample, golden labeling antibody a can be combined with Sp100 antibody and be formed compound and this compound and can the Sp100 antigen on being coated on detection line be combined and then complete the detection of Sp100 antibody, if there is no Sp100 antibody in blood serum sample, gold labeling antibody a-human IgG compound can not be combined by the Sp100 antigen on detection line, be that golden labeling antibody a can the IgG in human serum sample be combined and be formed compound, the antigen capture in the situation that of only having analyte to exist in tested sample on the tested survey line of golden labeling antibody a-human IgG compound ability, gold labeling antibody b is not combined with analyte, but can be combined by the golden labeling antibody c on nature controlling line.
2. chromatographic colloidal gold anti-Sp 100 antibody test paper according to claim 1, is characterized in that: the antibody A in described golden labeling antibody a is one or more in anti-human IgG monoclonal antibody, anti-human IgG polyclonal antibody, staphylococcal protein A (SPA), streptococcal protein G (Protein G).
3. chromatographic colloidal gold anti-Sp 100 antibody test paper according to claim 2, is characterized in that: described anti-human IgG polyclonal antibody is the one in mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source; Described anti-human IgG monoclonal antibody is the one in Huo Tu source, mouse source.
4. chromatographic colloidal gold anti-Sp 100 antibody test paper according to claim 1, is characterized in that: the antibody A in described golden labeling antibody a is staphylococcal protein A.
5. chromatographic colloidal gold anti-Sp 100 antibody test paper according to claim 1, it is characterized in that: when the antibody C while in antibody B and golden labeling antibody c in described golden labeling antibody b or difference, be the one in monoclonal antibody or polyclonal antibody, wherein specific binding formation immune complex can occur the antibody C in antibody B and the golden labeling antibody c in golden labeling antibody b.
6. chromatographic colloidal gold anti-Sp 100 antibody test paper according to claim 5, it is characterized in that: described polyclonal antibody is the one in mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source, described monoclonal antibody is the one in Huo Tu source, mouse source.
7. chromatographic colloidal gold anti-Sp 100 antibody test paper according to claim 1, is characterized in that: on described detection line, coated Sp100 antigen protein is the Sp100 antigen protein obtaining by prokaryotic expression cloned gene.
8. a preparation method for chromatographic colloidal gold anti-Sp 100 antibody test paper, is characterized in that, specifically comprises the steps:
Step 1, the preparation of cellulose nitrate coated film
(1) preparation of Sp100 antigen protein, obtains Sp100 antigen protein by prokaryotic expression cloned gene;
(2) preparation of golden labeling antibody c: with 0.1M sal tartari adjusting collaurum pH7.0～9.0, slowly add 4～25 μ g antibody C by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(3) preparation of cellulose nitrate coated film, the Sp100 antigen protein of preparing by coated film damping fluid dilution step (1) to concentration is 0.5～1.5mg/mL, is coated on the detection line of cellulose nitrate coated film; Golden labeling antibody c is diluted to 0.8～2.0mg/mL with coated film damping fluid, is coated on the nature controlling line of nitrocellulose filter with 1～10 μ l/cm, after cellulose nitrate coated film prepares, be placed in 37 DEG C of dry for standby;
Described coated film damping fluid is the one in borate buffer solution, carbonate buffer solution, phosphate buffer, Tris-HCl damping fluid, Tris-phosphate buffer, acetate buffer, barbitol buffer solution, and coated film pH of cushioning fluid is 6～9.5;
Step 2, the preparation of pad
(1) preparation of golden labeling antibody a: with 0.1M sal tartari adjusting collaurum pH7.0～9.0, slowly add 4～25 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(2) preparation of golden labeling antibody b: with 0.1M sal tartari adjusting collaurum pH7.0～9.0, slowly add 4～25 μ g antibody B by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～5%, 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 DEG C for subsequent use;
(3) preparation of pad: through the polyester film of damping fluid dip treating, after oven dry, by after golden labeling antibody a and golden labeling antibody b mixing, be sprayed on pretreated polyester film with the consumption of 0.5～4 μ l/cm, 25 DEG C～37 DEG C after dry pad, pad is placed under the environment of 2 DEG C～8 DEG C for subsequent use;
Described damping fluid is one of following:
(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;
Step 3, the preparation of sample pad
Glass fibre membrane is made to sample pad after damping fluid dip treating, and sample is padded on after 37 DEG C of oven dry for subsequent use;
Described damping fluid is one of following:
（a）0.05M?Borax、0.01MPBS、0.1%Sodium?Casein、1%PEG20000、2%BSA、0.05%NaN
3；
（b）0.05M?Borax、0.01MPBS、0.1%Sodium?Casein、1%PEG20000、2%Casein、0.05%NaN
3；
（c）0.05M?Tris-cl、0.01MPBS、0.1%Sodium?Casein、1%PEG20000、2%Casein、0.05%NaN
3；
Step 4, on base plate, overlap joint is pasted the cellulose nitrate coated film, pad, sample pad and the adsorptive pads that complete through abovementioned steps 1-3 made mutually in turn;
Step 5, the material that step 4 made is completed, cuts into test strips;
Described golden labeling antibody a can the IgG in human serum sample be combined and be formed compound, if there is Sp100 antibody in blood serum sample, golden labeling antibody a can be combined with Sp100 antibody and be formed compound and this compound and can the Sp100 antigen on being coated on detection line be combined and then complete the detection of Sp100 antibody, if there is no Sp100 antibody in blood serum sample, gold labeling antibody a-human IgG compound can not be combined by the Sp100 antigen on detection line, be that golden labeling antibody a can the IgG in human serum sample be combined and be formed compound, the antigen capture in the situation that of only having analyte to exist in tested sample on the tested survey line of golden labeling antibody a-human IgG compound ability, gold labeling antibody b is not combined with analyte, but can be combined by the golden labeling antibody c on nature controlling line.
9. method as claimed in claim 8, is characterized in that, in (2) of described step 1, the preparation of gold labeling antibody c: with 0.1M sal tartari adjusting collaurum pH8.0～9.0, slowly add 5～15 μ g antibody C by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use; Wherein antibody C is goat anti-rabbit igg.
10. method as claimed in claim 8, it is characterized in that, in (1) of described step 2, the preparation method of gold labeling antibody a is for regulating collaurum pH value to 8.0～9.0 with 0.1M sal tartari damping fluid, slowly add 5～15 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use; Wherein antibody A is goat anti-human igg.
11. methods as claimed in claim 8, it is characterized in that, in (1) of described step 2, the preparation method of gold labeling antibody a is for regulating collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid, slowly add 8～12 μ g antibody A by every milliliter of colloidal gold solution, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use; Wherein antibody A is mouse-anti human IgG.
12. methods as claimed in claim 8, it is characterized in that, in (2) of described step 2, the preparation method of gold labeling antibody b is for regulating collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid, slowly add 5～15 μ g antibody B by every milliliter of colloidal gold solution, rabbit igg, stir 10～30min, then add BSA to final concentration 0.5～1%, stir 10～30min, centrifugal, abandon supernatant, will precipitate with cleansing solution washing 2～3 times, last will precipitate resuspended with the preservation liquid of 1/10th initial volumes, put 4 DEG C for subsequent use, wherein antibody B is rabbit igg.
13. methods as claimed in claim 8, it is characterized in that, in (3) of described step 2, by the golden labeling antibody a preparing and golden labeling antibody b 20～40:15～20 ratio mixing by volume, be sprayed on pretreated polyester film with 2.0～4 μ l/cm with BIO-Dot Membrane jetter, 25 DEG C～37 DEG C dry pads that to obtain, pad envelope is placed under the environment of 2 DEG C～8 DEG C for subsequent use; Wherein the antibody A in golden labeling antibody a is staphylococcal protein A, and golden labeling antibody b is gold mark rabbit igg.
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