CN102621316A - Colloidal gold chromatography anti-nucleosome antibody detection test paper and preparation method thereof - Google Patents
Colloidal gold chromatography anti-nucleosome antibody detection test paper and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a colloidal gold chromatography anti-nucleosome antibody detection test paper and a preparation method thereof. The colloidal gold chromatography anti-nucleosome 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 a nucleosome antigen protein, and the quality control line is coated with a gold-labeled antibody c. By adopting indirect immunoassay to introduce the nucleosome 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-nucleosome antibody are realized, and a reference basis is provided for the auxiliary diagnosis of systemic lupus erythematosus.
Description
Technical field
The invention belongs to the medical immunology application, be specifically related to test paper that utilizes the anti-nucleosome antibody of colloidal gold immunochromatographimethod technology for detection and preparation method thereof.
Background technology
Nucleosome (nucleosome) is chromosomal fundamental structural unit, is to be made up of jointly DNA and histone.The histone molecule has 5 kinds, is called H1, H2A, H2B, H3 and H4 respectively.Each two molecule of histone H2A, H2B, H3 and H4 have constituted the core of nucleosome jointly, are called octameric histone, claim core histones again.
The unique source of nucleosome in biological living is apoptotic cell; When cell generation apoptosis; DNA in the apoptotic nucleus separates mode with distinctive example under the effect of endogenous endonuclease, make the bonding pad of nucleosome be cut into the oligonucleotide fragment of 180～200bp or its multiple: these dna fragmentations and oligomerization nucleosome formation apoptotic body; Under the normal physiological situation, apoptotic body can be engulfed by phagocyte or adjacent cells rapidly; Accelerate when apoptosis rate, apoptotic body can not be by timely removing, and its nucleosome that includes is released into blood, stimulates body to produce the existing report of multiple autoantibody.(the progress of anti-nucleosome antibody of Gong Baoqi and systemic loupus erythematosus correlativity.[J] foreign medical science internal medicine science fascicle, 2006 (4), 33 (4): 154-157)
Anti-ds-DNA antibody and anti-Sm antibody are the labelled antibodies of SLE, because of its positive rate is low, so susceptibility is relatively poor; Anti-ds-DNA antibody positive rate is 30%～40% (Su Yin, Han Lei, Li Zhanguo; Deng. [J]. Chinese rheumatology magazine .2003,7 (8): 474477.), anti-Sm antibody is 20%～40%; And the positive rate of AnuA is 55%～75%, H is significantly higher than anti-ds.DNA antibody and anti-Sm antibody.Because of it has higher specificity, so can think a kind of new specific antibody of the SLE of being used for diagnosis.Bibliographical information, AnuA have very high positive rate (can reach 60%～65%) in the SLE patient of anti-ds-DNA negative antibody, so the SLE patient of AnuA antagonism ds-DNA negative antibody more has diagnostic value.(Bruns A, Blass S, Hausdorf G, et al. [J] Arthritis Rheum, 2000,43:2307-2315.) nucleosome antibody is the diagnostic flag of SLE, its diagnostic sensitivity is 70-90%.In the active phase of SLE, the recall rate of this antibody is almost 100%, is 62% (corresponding dsDNA antibody recall rate is merely 3.3%) (Min DJ in the SLE of nonmobile phase; Kim SJ, Pank SH, et al. [J] .Clin Exp Rheunlatol; 2002,20 (1): 13-18.).Therefore, nucleosome antibody is higher than dsDNA antibody to the diagnostic sensitivity of SLE, anti-nucleosome antibody even can in the patient of dsDNA negative antibody, detect.However, also maybe the dsDNA antibody positive among the patient of anti-nucleosome negative antibody.(Chairns?Ap，McMillanSA，Crockard?AD.et?al[J]Ann?Rheum?Dis，2003，62(3)：272-273.)
Nucleosome antibody also can detect at drug-induced lupus philtrum.They are early stage marks that SLE worsens, because they are than the more Zao appearance of dsDNA antibody.
The positive rate of anti-nucleosome antibody in Patients with SLE serum is 50-95%, and specificity is almost 100%.Nucleosome is the function subunit of cell chromosome, is made up of with special mode DNA and histone.Anti-nucleosome antibody is than more Zao the early stage of systemic loupus erythematosus that come across of anti-dsDNA antibody, histonic antibody, systemic loupus erythematosus induce and cause a disease in play an important role.Clinical data shows, anti-nucleosome antibody positive among the SLE patient of anti-dsDNA negative antibody of 15-19% is arranged.Therefore joint-detection anti-dsDNA and anti-nucleosome antibody can improve SLE serology recall rate.AnuA is high to the susceptibility of SLE diagnosis, and high specificity possibly be one of mark property antibody of SLE diagnosis; The diagnosis of SI E that particularly resists ds.DNA antibody, anti-Snl negative antibody is significant
The autoimmune response that nucleosome is induced in SLE mainly combines with AnnA and produces.The AnuA that is produced by the B cell of activation can combine to play a role with the nucleosome specificity, not with its composition in DNA or histone react, and prior to the formation of ds.DNA antibody and histonic antibody.。
The present invention adopts escherichia coli prokaryotic expression human source gene engineering method nucleosome, is detection antigen of the present invention.
The method of the anti-nucleosome antibody of detection that present laboratory is commonly used mainly contains enzyme-linked immunosorbent test (enzyme linked immunosorbent assay; ELISA), IIF (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, the operation relative complex, and reagent has stronger toxicity and contaminative.
IIF can be made semiquantitative determination, but the fluorescence microscope result need be arranged, and the objectivity that the result judges is not enough, the standardization difficulty, and the technical program more complicated also is not suitable for the detection of high flux sample.
Linear immunoassay is mainly used in the examination of big type 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, and sensitivity is also higher, at present by extensive approval; But operate more loaded down with trivial detailsly, need repeatedly application of sample and washing, and be subject to the influence of temperature and incubation conditions; In specialized laboratory, operate, bring inconvenience to experiment.In addition, ELISA, the method requires pure antigen, otherwise is prone to false positive or false negative, and each detection all should be provided with the positive and negative standard compares.
The anti-nucleosome antibody assay kit of commercialization in the market is mainly Western blot; Running program is loaded down with trivial details; Accomplish 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.
Colloidal gold chromatography is applied in the anti-nucleosome detection of antibodies.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, makes sample solution swimming on chromatography strip through capillary effect, makes that immune response takes place the acceptor (like antigen or antibody) to determinand on determinand and the pad in the sample; And immune response takes place and be trapped with antigen (or antibody) on the fibre strip chromatographic material; And then form macroscopic aubergine band, and obtain experimental result intuitively, reach the purpose (Sikowicz G et al.One-stepchromatographic immunoassay for qualitative determination ofchoricogonadotropin 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-nucleosome antibody does not at home and abroad still come out on the 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 anti-nucleosome detection of antibodies; First the nucleosome antigen protein is applied in the colloidal gold chromatography simultaneously, adopts indirect method to realize the anti-nucleosome 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-nucleosome antibody, can be fast, assistant diagnosis system lupus erythematosus easily.
One of technical matters to be solved by this invention is to provide the anti-nucleosome antibody test of a kind of colloidal gold chromatography test paper.
Two of technical matters to be solved by this invention is to provide the preparation method of the anti-nucleosome antibody test of a kind of colloidal gold chromatography test paper.
The anti-nucleosome antibody test of a kind of colloidal gold chromatography test paper as first aspect present invention; Include sample pad, pad, cellulose nitrate coated film, adsorptive pads; Said sample pad, pad, cellulose nitrate coated film, adsorptive pads are sticked on the said base plate to the opposite side of said base plate by a side of base plate successively, and its special disease is:
Described pad is coated with golden labeling antibody a and golden labeling antibody b;
Described cellulose nitrate coated film is provided with detection line and nature controlling line, and described detection line is coated with the nucleosome antigen protein, and described nature controlling line is coated with golden labeling antibody c.
Further, the antibody A among the described golden labeling antibody a 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 among the said golden labeling antibody a is preferably staphylococcal protein A.
Antibody B among the described golden labeling antibody b and the antibody C among the golden labeling antibody c simultaneously or be a kind of in monoclonal antibody or the polyclonal antibody simultaneously, specificity can take place and combine the formation immune complex in the antibody C among antibody B among the wherein golden labeling antibody b and the golden labeling antibody c.
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 among the described golden labeling antibody b is preferably rabbit igg, and the antibody C among the correspondingly golden labeling antibody c is preferably goat anti-rabbit igg.
The nucleosome antigen protein of the nucleosome antigen protein that encapsulates on the described detection line for obtaining through the prokaryotic expression cloned gene.
Said nucleosome antigen protein is the recombinant protein that cloned gene obtained through escherichia coli prokaryotic expression.
The sample of said sample pad is from human serum, blood plasma, whole blood sample.
The monoclonal antibody of using according to the present invention can be passed through by (Continuous cultures of fused cells secreting antibody of predefined specificity [J] .Nature such as Kohler; 1975 (256): the hybridoma method of 495-497) at first describing prepares, and perhaps can prepare (seeing United States Patent (USP) 4816567) through the recombinant DNA method." monoclonal antibody " is by (Making antibody fragments using phage display libraries [J] .Nature such as Clackson; 1991:624-628) (By-passing immunization:Human antibodies from V-gene libraries displayed on phage [J] .Journal of Molecular Biology, 1991:581-597) said technology is separated from phage antibody library with Marks etc.
The polyclonal antibody of using according to the present invention can through Chen Xueqing etc. (immunology common experimental method. [M] 2000:15-26) prepares through immune animal.
SPA of the present invention, Protein G be through prokaryotic expression cloning recombination, by J. Sa nurse Brooker etc. (the molecular cloning experiment guide. [M], the escherichia coli prokaryotic expression cloned gene of 2002:1228-1232) describing.
Preparation method as the anti-nucleosome antibody test of a kind of colloidal gold chromatography test paper of second aspect present invention; This test paper is made up of sample pad, pad, cellulose nitrate coated film, adsorptive pads and base plate jointly, and its special disease is that this method includes following steps:
(1) preparation of anti-nucleosome antigen protein obtains anti-nucleosome antigen protein through the prokaryotic expression cloned gene;
(2) preparation of golden labeling antibody c: regulate collaurum pH 7.0～9.0 with 0.1M sal tartari, slowly add 4～25 μ g antibody C, stir 10～30min by every milliliter of colloidal gold solution; Add BSA then 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 ℃ subsequent use;
(3) preparation of cellulose nitrate coated film, anti-nucleosome antigen protein to the concentration for preparing with coated film damping fluid dilution step (1) is 0.5～1.5mg/mL, encapsulates on the detection line of cellulose nitrate coated film; With the dilution of coated film damping fluid antibody c is diluted to 0.8～2.0mg/mL, encapsulates on the nature controlling line of nitrocellulose filter, be placed in 37 ℃ of dry for standby after the cellulose nitrate coated film prepares with 1～10 μ l/cm;
(1) preparation of golden labeling antibody a: regulate collaurum pH 7.0～9.0 with 0.1M sal tartari, slowly add 4～25 μ g antibody A, stir 10～30min by every milliliter of colloidal gold solution; Add BSA then 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 ℃ subsequent use;
(2) preparation of golden labeling antibody b: regulate collaurum pH 7.0～9.0 with 0.1M sal tartari, slowly add 4～25 μ g antibody B, stir 10～30min by every milliliter of colloidal gold solution; Add BSA then 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 ℃ subsequent use;
(3) preparation of pad: the polyester film of treated liquid dip treating; After the oven dry, after golden labeling antibody a and golden labeling antibody b mixed, be sprayed on the pretreated polyester film with the consumption of 0.5～4 μ l/cm; After 25 ℃～37 ℃ dryings pad, pad places under 2 ℃～8 ℃ the environment subsequent use;
With processing sample pad after the treated liquid dip treating of glass fibre membrane, sample pad is subsequent use after 37 ℃ of oven dry;
In (2) of described step 1, the preparation of golden labeling antibody c: regulate collaurum pH 8.0～9.0 with 0.1M sal tartari, slowly add 5～15 μ g antibody C by every milliliter of colloidal gold solution; Stir 10～30min, add BSA then, stir 10～30min to final concentration 0.5～1%; 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 ℃ subsequent use; Wherein antibody C is a goat anti-rabbit igg.
In (1) of described step 2, the preparation method of golden labeling antibody a slowly adds 5～15 μ g antibody A for regulate collaurum pH value to 8.0～9.0 with 0.1M sal tartari damping fluid 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 ℃ subsequent use; Wherein antibody A is the goat anti-human igg.
In (1) of described step 2, the preparation method of golden labeling antibody a slowly adds 8～12 μ g antibody A for regulate collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid by every milliliter of colloidal gold solution; Stir 10～30min, add BSA then, stir 10～30min to final concentration 0.5～1%; 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 ℃ subsequent use.Then gold is marked SPAOD 302～4 μ l/cm and be sprayed at pad, dry back is subsequent use; Wherein antibody A is the mouse-anti human IgG.
In (1) of described step 2, the preparation method of golden labeling antibody a slowly adds 10～15 μ g antibody A for regulate collaurum pH value to 5.0～6.5 with 0.1M sal tartari damping fluid by every milliliter of colloidal gold solution; Stir 10～30min, add BSA then, stir 10～30min to final concentration 0.5～1%; 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 ℃ subsequent use.Then gold is marked SPA OD 202～4 μ l/cm and be sprayed at pad, dry back is subsequent use; Wherein antibody A is a staphylococcal protein A.
In (2) of described step 2, the preparation method of golden labeling antibody b slowly adds 5～15 μ g antibody B, rabbit igg for regulate collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid by every milliliter of colloidal gold solution; Stir 10～30min, add BSA then, stir 10～30min to final concentration 0.5～1%; 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 ℃ subsequent use, wherein antibody B is a rabbit igg.
In (3) of described step 2; With the golden labeling antibody a for preparing and golden labeling antibody b 20～40: 15～20 mixed by volume; Be sprayed on the pretreated polyester film with 2.0～4 μ l/cm with the BIO-Dot Membrane jetter; 25 ℃～37 ℃ dry pads, pad envelope are placed under 2 ℃～8 ℃ the environment subsequent use; Antibody A among the wherein golden labeling antibody a is a staphylococcal protein A, and golden labeling antibody b is a gold mark rabbit igg.
The purpose of described damping fluid is to provide certain pH and ionic strength to make the gold chloride collaurum present required state of charge and ionic strength; Its albumen that is labeled is encapsulated around the gold chloride collaurum with Aucl-is the ion of core, and form metastable a kind of state.Therefore every kind of albumen encapsulate also difference of required condition because its state of charge is different with the amino acid composition, needs confirmed by test kind, concentration, the pH of 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 the described step 3, the width of the test paper that cuts into is preferably two kinds of 4mm and 3mm.
The application of the anti-nucleosome antibody test of colloidal gold chromatography of the present invention test paper, it is an anti-nucleosome antibody in the qualitative detection human serum, the assistant diagnosis system lupus erythematosus.
Detection principle of the present invention is specially the recombinant expressed nucleosome antigen protein of selecting affinitive layer purification for use; Gold labeling antibody a marks rabbit igg as the colloid gold label compound with gold; Be sprayed at pad, utilize indirect method to detect and whether contain anti-nucleosome antibody in the blood serum sample.During detection; Sample along with the chromatography swimming to pad and soak into the colloid gold label compound; Human IgG wherein and golden labeling antibody a combine to form 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-nucleosome antibody is arranged; This compound with encapsulate the antigen protein generation specific immunity association reaction on nitrocellulose filter, form golden labeling antibody a-human IgG-nucleosome 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 is trapped with the special immune response of goat anti-rabbit igg generation that is coated on the nature controlling line; 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-nucleosome 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 the aubergine band do not occur at the detection line place, golden labeling antibody a continues swimming and arrives adsorptive pads forward, and gold mark rabbit igg continue swimming forward with encapsulate in the nature controlling line place that goat anti-rabbit igg special immune response takes place and is trapped; Be enriched in gradually and form the aubergine band on the nature controlling line, therefore band only in Quality Control, occurring is judged to negative findings.
The antigen protein that the present invention expresses genetic engineering bacterium is incorporated in the test strips first, realized the detection performance of high specific, high sensitivity, pin-point accuracy.Also occurred commercialization ELISA kit at present on the market, but gold mark chromatographic test paper by comparison, still has many differences, do not receive place personnel's limit, detection time, weak point was 5-10 minute, and sentence read result is easy.This test paper has high specific, high sensitivity, pin-point accuracy in addition, can satisfy the rapid screening SLE 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 advantage of the present invention with conventional detection:
1. uniqueness of the present invention is that the nucleosome 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 nucleosome antibody (can detect sample) greater than 25RU/ml through the colloid gold test paper rapid screening.
2. advantage of the present invention is that production cost is low.The required core reagent of anti-nucleosome detection of antibodies test paper provided by the present invention 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 through buying commercialization reagent or self-control, antigen protein derives from homemade purifying gene engineering antigen protein.
3. with the disclosed data by MoM and MEI that is used for anti-nucleosome antibody test, test paper of the present invention have many additive methods the advantage that can not compare, like 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 invention.
Fig. 2 is a testing result synoptic diagram of the present invention.
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, explains to contain anti-nucleosome antibody in the serum;
Fig. 2 c is depicted as: as only an aubergine band appears in E in the control zone, the aubergine band does not appear in detection zone D, and the result is negative, explains not contain anti-nucleosome antibody in the serum;
Fig. 2 d, 2e are depicted as: the aubergine band do not occur like control zone E, no matter whether detection zone D has band to occur, and explains that all test paper lost efficacy.
Embodiment
The anti-nucleosome antibody test of colloidal gold immunity chromatography of the present invention test paper, as shown in Figure 1, this test paper is on base plate 7, to paste cellulose nitrate coated film 3, pad 2, sample pad 1, adsorptive pads 6 in order each other by a side direction opposite side overlap joint.
Be coated with golden labeling antibody a and golden labeling antibody b on the pad 2, 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 golden labeling antibody of staphylococcal protein A (SPA) or streptococcal protein G gold labeling antibody; Antibody B among the gold labeling antibody b 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 the nucleosome antigen protein, and nature controlling line 5 is coated with golden labeling antibody c, and the antibody among the golden labeling antibody c is goat anti-rabbit igg.
Below in conjunction with specific embodiment and accompanying drawing, further set forth the present invention.
The preparation of embodiment 1 nucleosome antigen protein
The nucleosome antigen protein that is applied to this test paper is to make up recombination through gene clone technology, and adopting prokaryotic expression technology successful expression to go out then all is the nucleosome antigen protein in people source.Wherein, the nucleosome 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 produce through immunogene and the adjuvant that (ip) injects purifying in repeatedly subcutaneous (sc) or peritonaeum on animal.
Through being mixed with Freund ' the s Freund's complete adjuvant of 3 times of volumes, 0.05mg～1mg immune formulation (respectively to 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 originally measured through the multi-section position animal skins injected and booster immunization.With the animal bloodletting, measure serum anti human IgG titre 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 ".Through reclaiming splenocyte and make cell immortalityization from the animal of quilt immunity; For example through merging with the myeloma cell or transforming through 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 through the escherichia coli prokaryotic expression cloned gene; Concrete operation method referring to J. Sa nurse Brooker etc. (the 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 possibly sneak into wherein.The collaurum outward appearance for preparing should be pure, bright, do not have deposition and floating thing, abandons when liquid level occurs grease with a large amount of black particle shape sediment.
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.
Colloidal gold solution prepares in the process, and the compound method of each solution is following:
1.HAuCl
4Preparation: with ultrapure water dissolved chlorine auric acid, be made into 1% solution, put 4 ℃ 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 is joined existing usefulness at present.
The preparation method of the anti-nucleosome 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 the nucleosome antigen protein;
(2) preparation of goat anti-rabbit igg gold labeling antibody:
Regulate the collaurum pH 7.0～9.0 that embodiment 3 prepares with 0.1M sal tartari, slowly add 4～25 μ g goat anti-rabbit iggs, stir 10～30min by every milliliter of colloidal gold solution; 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 ℃ subsequent use;
(3) preparation of cellulose nitrate coated film 3; Using coated film damping fluid dilution nucleosome antigen protein to concentration is 1.0～1.5mg/mL; Adjustment BIO-Dot instrument is sprayed at nitrocellulose filter (NC) and goes up detection line 4 places, near pad 2 ends; Apart from pad 2 about 9.5mm, the Cenp-B antigen protein is encapsulated on the detection line 4 of cellulose nitrate coated film;
With encapsulating damping fluid goat anti-rabbit igg is diluted to 0.8～1.5mg/mL, is sprayed at nitrocellulose filter (NC) with 1～10 μ l/cm with the BIO-Dot instrument and goes up nature controlling line 5 places, apart from adsorptive pads 6 about 9mm near adsorptive pads 6. 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 subsequent use.
The damping fluid that encapsulates of its use can be a borate, carbonate, phosphate; Tris-HCl or Tris-phosphate, acetate, barbital; Or the like, the purpose of its damping fluid is for providing certain pH and ionic strength albumen is encapsulated and firmly encapsulating the film in NC, 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&SAE99, whatman 8 μ m, millipore M135, sartorius CN140 etc.The concrete NC film that uses is not a key of the present invention, but in each mensuration, above-mentioned several kinds of 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 gap is in various degree 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: regulate the collaurum pH 8.0～9.0 that embodiment 3 prepares with 0.1M sal tartari, slowly add 8～12 μ g goat anti-human iggs, stir 10～30min by every milliliter of colloidal gold solution; 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 ℃ subsequent use;
(2) preparation of rabbit igg gold labeling antibody: regulate collaurum pH value to 7.0～8.0 that embodiment 3 prepares with 0.1M sal tartari, slowly add 8～12 μ g rabbit iggs, stir 10～30min by every milliliter of colloidal gold solution; 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 ℃ subsequent use.
(3) preparation of pad: through damping fluid the polyester film was soaked 30 minutes, 37 ℃ of oven dry are after goat anti-human igg's gold labeling antibody and the golden labeling antibody of rabbit igg 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 ℃ subsequent use.Place under 2 ℃～8 ℃ the environment subsequent use;
In the preparation process of pad, operable damping fluid comprises following several kinds:
(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:
Basically confirm the OD20 of rabbit igg gold labeling antibody through preliminary experiment; Be sprayed on the pretreated polyester film with BIO-Dot discharge rate 1 μ l/cm; Use 0.01MPBS to be sample-loading buffer, the band of the color intensity that can obtain expecting confirms 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; Using BIO-Dot is that 3 μ l/cm are sprayed on the polyester film of handling well 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, use positive serum, critical reference value serum, negative serum to be debugger object.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 through 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 subsequent use;
This step can with damping fluid comprise following several kinds:
(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 of processing with PVC, wide 2.4cm puts between drying shed subsequent use.
Cutting of adsorptive pads: with trimmer thieving paper is cut into the promptly long 28cm of the base plate equal length of processing with PVC, wide 3cm processes adsorptive pads, puts between drying shed subsequent use.
Cutting of pad: with guillotine pad is cut into the promptly long 28cm of the base plate equal length of processing with PVC, wide 2.4cm puts between drying shed subsequent use.
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 of processing with PVC, wide 1cm, it is subsequent use 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 processed 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), and band colour developing degree reaches the d like Fig. 2; And specific band nothing but, then product becomes specification product through 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 following:
1.0.1M the preparation of sal tartari: with ultrapure water preparation, 0.22 μ m membrane filtration mistake, put 4 ℃ subsequent use, 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 ℃ subsequent use, two weeks of the term of validity.1000mL 10%BSA solution formula: 100g BSA, 0.5g NaN
3Ultrapure water is settled to 1000mL.
3, encapsulate the preparation of damping fluid: 9gNacl, 1.15gNa
2HPO
4, 0.23g NaH
2PO
4, 10gSucrose, 0.5gEDTA be dissolved in the 1L ultrapure water, filter place 4 ℃ subsequent use.
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 ℃ subsequent use, 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 preparation method of the anti-nucleosome antibody test of the colloidal gold immunity chromatography of this embodiment 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, stir 10～30min by every milliliter of colloidal gold solution; 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 ℃ subsequent use.
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 preparation method of the anti-nucleosome antibody test of the colloidal gold immunity chromatography of this embodiment 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, stir 10～30min by every milliliter of colloidal gold solution; 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 ℃ subsequent use.
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 preparation method of the anti-nucleosome antibody test of the colloidal gold immunity chromatography of this embodiment 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, stir 10～30min by every milliliter of colloidal gold solution; 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 ℃ subsequent use.
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 present invention is incorporated into the anti-nucleosome antibody test of colloidal gold chromatography test paper with recombinant expressed nucleosome antigen protein, can realize the anti-nucleosome detection of antibodies in the blood sample, can be fast, assistant diagnosis system lupus erythematosus 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 of the golden labeling antibody a of the anti-nucleosome antibody test of collaurum of the present invention 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
1.1 37 ℃ of accelerated stabilities
Place 37 ℃ to carry out accelerated tests on test paper, every day, taking-up was tested with indoor quality-control product, judged the stability of test paper with reference to the withinrun precision of the yin and yang attribute coincidence rate of article (each 10 parts) through yin and yang attribute.The result shows after 4 months, the testing result accord with expectation of quality-control product, and each yin and yang attribute is 100% with reference to the yin and yang attribute coincidence rate of article.Yin and yang attribute is 10 parts of anti-nucleosome antibody positive serum and 10 parts of anti-nucleosome negative antibody serum with reference to article.
1.24 ℃ stability experiment
2. diagnostic sensitivity
From clinical, collect 100 parts of serum that are diagnosed as primary bile hepatitis (SLE) patient, the operation steps that goes up to specifications with homemade colloid gold test paper detects the top SLE patients serum who collects.Statistics is following behind the 5min:
According to the result of top statistics, according to the detection to the SLE patients serum of 100 parts of affirmations, the sample size that can find anti-nucleosome antibody positive is 81 examples, and negatives quantity is 19 examples.Therefore through calculating:
Diagnosis sensitivity (%)=81/ (81+19) * 100%=81%
3. specificity
From clinical, collect each 300 parts of healthy blood donor's serum and serum, the operation steps that goes up to specifications with homemade colloid gold test paper detects top healthy blood donor who collects and non-patient's SLE serum.Statistics is following behind the 5min:
According to the statistics to the detection of each 300 parts of serum of healthy blood donor, the sample size of finding in the healthy blood donor, to record anti-nucleosome negative antibody is 291 examples, therefore through calculating:
(healthy blood donor) specificity (%)=291/300 * 100%=97%
More than be the description of this invention and non-limiting, based on other embodiment of inventive concept, all among protection scope of the present invention.
Claims (14)
1. the anti-nucleosome antibody test of colloidal gold chromatography test paper; Include sample pad (1), pad (2), cellulose nitrate coated film (3), adsorptive pads (6); Said sample pad (1), pad (2), cellulose nitrate coated film (3), adsorptive pads (6) are overlapped each other to the opposite side of said base plate (7) successively by a side of base plate (7) and stick on the said base plate (7), it is characterized in that:
Described pad (2) is coated with golden labeling antibody a and golden labeling antibody b;
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 the nucleosome antigen protein, and described nature controlling line (5) is coated with golden labeling antibody c.
2. the anti-nucleosome antibody test of colloidal gold chromatography according to claim 1 test paper is characterized in that: the antibody A among the said golden labeling antibody a 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. the anti-nucleosome antibody test of colloidal gold chromatography according to claim 2 test paper 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. the anti-nucleosome antibody test of colloidal gold chromatography according to claim 1 test paper is characterized in that: the antibody A among the said golden labeling antibody a is a staphylococcal protein A.
5. the anti-nucleosome antibody test of colloidal gold chromatography according to claim 1 test paper; It is characterized in that: the antibody B among the described golden labeling antibody b and the antibody C among the golden labeling antibody c be a kind of in monoclonal antibody or the polyclonal antibody simultaneously simultaneously or, and the antibody C among antibody B among the wherein golden labeling antibody b and the golden labeling antibody c specificity can take place combines to form immune complex.
6. the anti-nucleosome antibody test of colloidal gold chromatography according to claim 5 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. the anti-nucleosome antibody test of colloidal gold chromatography according to claim 1 test paper is characterized in that: the nucleosome antigen protein of the nucleosome antigen protein that encapsulates on the described detection line for obtaining through the prokaryotic expression cloned gene.
8. the preparation method of the anti-nucleosome antibody test of a colloidal gold chromatography test paper is characterized in that, specifically comprises the steps:
Step 1, the preparation of cellulose nitrate coated film
(1) preparation of nucleosome antigen protein obtains the nucleosome antigen protein through the prokaryotic expression cloned gene;
(2) preparation of golden labeling antibody c: regulate collaurum pH 7.0～9.0 with 0.1M sal tartari, slowly add 4～25 μ g antibody C, stir 10～30min by every milliliter of colloidal gold solution; Add BSA then 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 ℃ subsequent use;
(3) preparation of cellulose nitrate coated film, nucleosome antigen protein to the concentration for preparing with coated film damping fluid dilution step (1) is 0.5～1.5mg/mL, encapsulates on the detection line of cellulose nitrate coated film; With the dilution of coated film damping fluid antibody c is diluted to 0.8～2.Omg/mL, encapsulates on the nature controlling line of nitrocellulose filter, be placed in 37 ℃ of dry for standby after the cellulose nitrate coated film prepares with 1～10 μ l/cm;
Step 2, the preparation of pad
(1) preparation of golden labeling antibody a: regulate collaurum pH 7.0～9.0 with 0.1M sal tartari, slowly add 4～25 μ g antibody A, stir 10～30min by every milliliter of colloidal gold solution; Add BSA then 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 ℃ subsequent use;
(2) preparation of golden labeling antibody b: regulate collaurum pH 7.0～9.0 with 0.1M sal tartari, slowly add 4～25 μ g antibody B, stir 10～30min by every milliliter of colloidal gold solution; Add BSA then 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 ℃ subsequent use;
(3) preparation of pad: the polyester film of treated liquid dip treating; After the oven dry, after golden labeling antibody a and golden labeling antibody b mixed, be sprayed on the pretreated polyester film with the consumption of 0.5～4 μ l/cm; After 25 ℃～37 ℃ dryings pad, pad places under 2 ℃～8 ℃ the environment subsequent use;
Step 3, the preparation of sample pad
With processing sample pad after the treated liquid dip treating of glass fibre membrane, sample pad is subsequent use after 37 ℃ of oven dry;
Step 4 overlaps each other in order on base plate and pastes cellulose nitrate coated film, pad, sample pad and the adsorptive pads of accomplishing through abovementioned steps 1-3 made;
Step 5, the material to step 4 made is accomplished cuts into test strips.
9. method as claimed in claim 8 is characterized in that, in (2) of described step 1, and the preparation of golden labeling antibody c: regulate collaurum pH 8.0～9.0 with 0.1M sal tartari; Slowly add 5～15 μ g antibody C 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 ℃ subsequent use; Wherein antibody C is a goat anti-rabbit igg.
10. method as claimed in claim 8 is characterized in that, in (1) of described step 2; The preparation method of gold labeling antibody a slowly adds 5～15 μ g antibody A for regulate collaurum pH value to 8.0～9.0 with 0.1M sal tartari damping fluid by every milliliter of colloidal gold solution; Stir 10～30min, add BSA then, stir 10～30min to final concentration 0.5～1%; 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 ℃ subsequent use; Wherein antibody A is the goat anti-human igg.
11. method as claimed in claim 8 is characterized in that, in (1) of described step 2; The preparation method of gold labeling antibody a slowly adds 8～12 μ g antibody A for regulate collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid by every milliliter of colloidal gold solution, stirs 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 ℃ subsequent use.Then gold is marked SPAOD 302～4 μ l/cm and be sprayed at pad, dry back is subsequent use; Wherein antibody A is the mouse-anti human IgG.
12. method as claimed in claim 8 is characterized in that, in (1) of described step 2; The preparation method of gold labeling antibody a slowly adds 10～15 μ g antibody A for regulate collaurum pH value to 5.0～6.5 with 0.1M sal tartari damping fluid by every milliliter of colloidal gold solution, stirs 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 ℃ subsequent use.Then gold is marked SPA OD 202～4 μ l/cm and be sprayed at pad, dry back is subsequent use; Wherein antibody A is a staphylococcal protein A.
13. method as claimed in claim 8 is characterized in that, in (2) of described step 2; The preparation method of gold labeling antibody b slowly adds 5～15 μ g antibody B, rabbit igg for regulate collaurum pH value to 7.0～8.0 with 0.1M sal tartari damping fluid by every milliliter of colloidal gold solution; Stir 10～30min, add BSA then, stir 10～30min to final concentration 0.5～1%; 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 ℃ subsequent use, wherein antibody B is a rabbit igg.
14. method as claimed in claim 8; It is characterized in that; In (3) of described step 2,, be sprayed on the pretreated polyester film with 2.0～4 μ l/cm with the BIO-Dot Membrane jetter with the golden labeling antibody a for preparing and golden labeling antibody b 20～40: 15～20 mixed by volume; 25 ℃～37 ℃ dry pads, pad envelope are placed under 2 ℃～8 ℃ the environment subsequent use; Antibody A among the wherein golden labeling antibody a is a staphylococcal protein A, and golden labeling antibody b is a gold mark rabbit igg.
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