CN104764881A - Preparation method and application of anti-MPO-ANCA antibody immunity chromatography test paper - Google Patents
Preparation method and application of anti-MPO-ANCA antibody immunity chromatography test paper Download PDFInfo
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Abstract
The invention discloses an anti-MPO-ANCA antibody immunity chromatography test paper, and a preparation method and application thereof. The test strip includes a sample pad (1), a combination pad (2), an analysis membrane (3), a water absorption pad (6), a base plate (7), a test zone T line (4) and a quality control C line (5). The upper part of the base plate (7) is adhered to the analysis membrane (3), and the the upper two ends of the analysis membrane (3) are respectively adhered to the combination pad (2) and the water absorption pad (6); one upper end of the combination pad (2) is adhered to the sample pad (1); the test zone T line (4) and the quality control C line (5) are arranged on the analysis membrane (3). The invention employs indirect immunodetection and MPO-ANCA antigen protein to realize high sensitivity, high specificity and rapid detection of MPO-ANCA antibody protein and provide the basis for auxiliary diagnosis of clinical autoimmune diseases such as inflammatory bowel disease.
Description
Technical field
The present invention relates to field of immunoassay detection.Specifically, the present invention relates to a kind of preparation method and the application that detect the immunity chromatography detection test paper of MPO-ANCA.
Background technology
Inflammatory bowel disease (inflammatory bowel disease, IBD) be a kind of cause of disease chronic nonspecific inflammatory bowel disease still not fully aware of, comprise ulcerative colitis (Ulcerative colitis, and Crohn disease (Crohn ' s disease, CD) UC).Crohn disease was described in 1932 the earliest by Crohn, Ginzterg and Oppenheime, therefore obtained this name.1973, this disease was named as Crohn is sick by the council of medical science international organization of the World Health Organization (WHO).Its feature is that the cause of disease is not bright, is more common in young people, shows as granulomatous inflammation pathology, merges fiberization and ulcer.Can invade and complete GI any position, comprise oral cavity, anus, pathology is segmental or jumping characteristic distribution, and can to invade and beyond enteron aisle, particularly skin.Clinical manifestation is diversified because diseased region, scope and degree are different, and the course of disease is slow, easily recurs.
IBD is the common disease in North America and Europe, and over nearly 30 years, the Japanese IBD incidence of disease is also in progressively increasing trend, though China there is no the epidemiologic data of general population, the medical number of this disease is in progressively increasing trend then clearly during the nearly last ten years.The complicated clinical manifestation of IBD is various, not only has symptom of digestive tract, and intestines also can be had outward to show.Because symptom is non-specific enteritis performances such as suffering from abdominal pain, suffer from diarrhoea, have blood in stool, CD and UC and other enteron aisle chronic diseases such as IBD and tuberculous enteritis are difficult to antidiastole, special in inorganizable evidence, need have more heterogeneous is that diagnosis is offered help to specific mark.Research finds, the bioactive marker of multiple Noninvasive to the diagnosis of IBD and the evaluation of course inflammatory activity significant.
Research in recent years about serum specific antibody label is more, comprise the anti-Antineutrophil antibody of core week type (Perinuclear anti-neutrophil cytoplasmic antibody, pANCA), anti-cerevisiae antibody (anti-Saccharomyces cerevisiae immunoglobulin A, anti-ASCA), anti-pancreatic acini antibody (PAB), anti-cell outer membrane protein composite antibody (anti-OmpC), I2 antibody etc.The autoantibody that pANCA is is antigen with neutrophil leucocyte and monocyte cytoplasmic components, the pANCA relevant to IBD is atypia ANCA, the current report of positive rate in IBD is different, and the pANCA positive rate of UC is between 27 ~ 83%, and CD is then 0 ~ 24%.Cause each laboratory detection result difference reason may with the race of research object, crowd select and the difference of research sample size relevant, different relevant more importantly from detection method.The pANCA target antigen relevant to UC mainly myeloperoxidase (myeloperoxidase, and permeability sterilization protein (bactericidal/permeability-increasing protein MPO), BPI-ANCA), in bibliographical information UC patient, the positive rate of anti-MPO-ANCA is 10 ~ 25%, based on IgG type.The detection method of the anti-MPO-ANCA antibody of bibliographical information has two kinds, enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence (Indirect immunofluorescence, IFA) detection technique.ELISA method detects and can be used for high flux sample mensuration, sensitivity is also higher, also extensively approved at present, but its operation is more loaded down with trivial details, need repeatedly application of sample to wash and incubation step, completing whole experimentation needs 3 hours, need microplate reader, require higher for experimenter's professional technique, be subject to the impact of various environmental baseline simultaneously, inconvenience is brought to experiment.
Indirect immunofluorescene assay technology is that cell antigen is fixed on microslide, and fluorescent dye fluorescent microscope is observed, and this technology has high specific, but because microscopic examination needs subjective judgement, high to the requirement of reviewer, and depend on fluorescent microscope, use inconvenience.
Immunochromatographic method (Immunochromatography) is a kind of quick diagnosis technology of rising in recent years, its principle is a certain zone special antibody being first fixed on nitrocellulose filter or cellulose acetate membrane, after sample (urine or serum) is immersed in cellulose one end of this drying, due to capillarity, sample will move forward along this film, when moving to the region being fixed with antibody, in sample corresponding antigen namely with this antibody generation specific binding, form macroscopic detection zone.The trace labelling particle that existing immuno-chromatographic test paper strip product is conventional has nm of gold, nanometer selenium, colored latex etc., and be wherein most widely used with nm of gold, nm of gold is also referred to as collaurum.
Immune colloidal gold technique (Immune colloidal gold technique, GICT) is called using collaurum as the immunochromatography technique spy of missing label.Collaurum be by gold chloride (HAuCl4) at reductive agent as under the effect such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, polymerization becomes the gold grain of specific size, and gains the name because electrostatic interaction becomes a kind of stable colloidal state.Collaurum is electronegative under mild alkaline conditions, can be formed firmly be combined, because this combination is electrostatical binding, so do not affect the biological nature of protein with the positive charge group of protein molecule.Collaurum except with protein bound except, can also be combined, as SPA, PHA, ConA etc. with other biomacromolecules many.According to some physical behaviors of collaurum, as high electron density, grain size, shape and color reaction, add immunity and the biological characteristics of bond, thus make collaurum be widely used in the fields such as immunology, histology, pathology and cell biology.
The colored latex microsphere of surface band reactive group also can as the tracer agent of immunochromatography, the same with colloidal gold technique, colored latex mark immunity-chromatography technology also have easy and simple to handle, stable reagent, fast go out result, can the advantage such as room temperature preservation.Colored latex mark is by different-diameter scope 0.1 μM ~ 1 μM colored latex microsphere and containing ester class or other macromolecular substances covalent bond such as carboxyl, amino, hydroxyls, make color on these macromolecular marker, when enrichment forms macroscopic color stripes after stopping on detection line or nature controlling line.
Compare with IFA with prior art ELISA, the advantage of immunochromatographyassay assay mainly comprises: (1) is fast easy to use, and be convenient to basic unit and use and onsite application, institute responds and can complete in 20 minutes; (2) cost is low, does not need special instrument and equipment; (3) applied range, can adapt to multiple testing conditions; (4) can carry out multinomial detection, if the more difficult acquisition of positive sample, multinomial detection can save sample, reduces costs; (5) label is stablized, and mark sample stores more than 2 year year at 4 DEG C, no signal relaxation phenomenon; (6) collaurum is originally as redness, does not need to add chromogenic agents, eliminates the step of enzyme target carcinogenicity substrate and stop buffer, to human non-toxic's evil.
In autoimmune disease diagnosis, there is more immune chromatography test paper, but at home and abroad still do not come out in market based on the immune chromatography test paper of anti-MPO-ANCA antibody test.
Summary of the invention
Technical matters to be solved by this invention is to overcome now methodical deficiency, immunochromatographic method is applied in the detection of anti-MPO-ANCA antibody, by MPO-ANCA antigen protein being coated in the detection zone of pad or analyzing film, realize special to the height of MPO-ANCA antibody anti-in sample, high sensitivity, high accuracy detection, rapid screening anti-MPO-ANCA antibody positive sample, for the autoimmune diseases such as clinical UC provide auxiliary diagnosis fast.
An object of the present invention is the immunochromatographydetecting detecting test strip and the preparation method that provide a kind of anti-MPO-ANCA antibody fast and accurately, and two of object is to provide a kind of anti-MPO-ANCA antibody detection method based on immunochromatography technique.
As the immunochromatographydetecting detecting test strip of a kind of anti-MPO-ANCA antibody of the present invention first aspect, comprise sample pad, pad, analyzing film, adsorptive pads and base plate, analyzing film is arranged detection zone and quality control band, the top of described base plate is fitted with analyzing film, the two ends on analyzing film top are fitted with pad and adsorptive pads respectively, and the one end on pad top is fitted with sample pad.
Described pad combining nano gold label or colored latex label, pad is 1 layer (Fig. 1) or 2 layers (Fig. 2), wherein 2 layers time stragglyly to overlap with analyzing film.
The preparation method of the immunochromatographydetecting detecting test strip of described anti-MPO-ANCA antibody, comprises the following steps:
The preparation of step 1.MPO-ANCA antigen protein
Prepared by step 2. pad (2)
(1) preparation of nano gold mark thing: with the nano-Au solution of 20 ~ 40nm particle diameter mark MPO-ANCA antigen protein, anti-human IgG antibodies, staphylococcal protein A (SPA), streptococcal protein G (Protein G), and other and quality control band C line can form the albumen of immune complex.
(2) preparation of colored latex label: with the colored latex mark MPO-ANCA antigen protein of band active amino or carboxylic group, anti-human IgG antibodies, staphylococcal protein A (SPA), streptococcal protein G (Protein G), and other and quality control band C line can form the albumen of immune complex.
(3) pad (2) preparation: glass fibre membrane or polymer PET are as pad, first use containing BSA and sugared damping fluid pre-service and dry, the nano gold mark thing that step (1) is prepared or latex label, be sprayed on pretreated glass fibre membrane or polymer PET with spraying instrument, drying for standby.
Prepared by step 3. analyzing film (3)
(1) detection zone T line (4) preparation: according to pad labelled protein type selecting detection zone T line coating protein, the coating protein containing MPO-ANCA, detection zone T line as the associated proteins on pad is anti-human igg.If containing anti-human igg on pad, detection zone T line coating protein is MPO-ANCA.
(2) quality control band C line (5) preparation: according to the coating protein of the labelled protein type selecting quality control band of pad, if pad labelled protein is single MPO-ANCA, the coating protein of quality control band C line is the antibody of anti-MPO-ANCA; If pad labelled protein is single anti-human IgG antibodies, the labelled protein of quality control band C line is the antibody of corresponding anti-human IgG antibodies; Or other pad labelled proteins form the albumen of immune complex.
Prepared by step 4. sample pad
Glass fibre membrane or polyester film are flooded dry for standby through damping fluid, or directly uses.
Step 5. test strips is assembled
The material completed made by step 1 ~ 4, by Fig. 1 or Fig. 2 overlap joint, cut into certain length and width according to the specification of base plate, in being arranged on base plate and getting stuck.Preferably, test strips length is 6.5cm, and width is 3mm or 4mm.
Described MPO-ANCA antigen protein is total length MPO-ANCA gene or the gene order containing important epitope, and be cloned in protokaryon or carrier for expression of eukaryon, expression and purification obtains.
Described MPO-ANCA antigen protein is with Peptide synthesizer synthesis containing the MPO-ANCA polypeptide of the important epitope of MPO-ANCA, and is coupled on carrier protein and obtains.
Described anti-human IgG antibodies is one or more monoclonal antibodies in mouse source, rabbit source, Yang Yuan, Ma Yuan, camel source or cavy source or polyclonal antibody.
The antibody of described anti-MPO-ANCA take MPO-ANCA as immunogenic, one or more monoclonal antibodies in mouse source, rabbit source, Yang Yuan, Ma Yuan, camel source or cavy source or polyclonal antibody.
The antibody of described anti-human IgG antibodies refers to and can form the antibody of immune complex with this antibody, and such as this anti-human IgG antibodies is mouse-anti human IgG, and its antibody is the antibody of sheep anti-mouse igg or the mouse IgG antibody in other non-mouse sources.
In another aspect of this invention, provide a kind of anti-MPO-ANCA antibody detection method, by the anti-MPO-ANCA antibody immune chromatography test strips prepared by first aspect present invention method and step, measuring samples detected, concrete steps:
(1) sample process: get serum, blood plasma or whole blood sample, with sample loading buffer dilution 0 ~ 100 times;
(2) sample drop of above-mentioned process is added in the sample pad of test strips, leaves standstill 5 ~ 20 minutes, observe T line and C line;
(3) result interpretation: T line and C line all manifest, result is positive; T line does not manifest, and C line manifests, and result is negative; T line and C line all do not manifest, and prompting test paper lost efficacy.
Cleaning Principle of the present invention:
Select gold mark/latex etc. to mark MPO-ANCA antigen protein or other can object is combined in measuring samples albumen, be sprayed on pad, make pad, can be there is immunoreactive albumen with labelled protein-object albumen composition in bag in detection zone, when after in sample application to be checked to sample pad, MPO-ANCA antibody in sample and the labelled protein on pad form immune complex, due to capillary effect, this compound is to the swimming of adsorptive pads direction, this compound and the antigen protein generation specific immunity association reaction be coated on detection zone T line, form gold mark/latex protein-anti-MPO-ANCA antibody-MPO-ANCA antigen protein triplet compound and be trapped within detection line, enrichment forms darker aubergine band or latex colors gradually, because capillary effect continues swimming forward, special immune response is there is and is trapped in gold mark/latex rabbit igg with the goat anti-rabbit igg be coated on nature controlling line, be enriched in gradually on nature controlling line and form darker aubergine band or latex colors, unnecessary unconjugated material continues chromatography on adsorptive pads, and what therefore all occur band at detection line and nature controlling line is judged to positive findings, if not containing anti-MPO-ANCA antibody in blood serum sample, when labelled protein arrives detection line, not with the corresponding protein generation immune response be coated on detection line, therefore not there is developed band at detection line place, and there is special immune response to the corresponding coating protein being coated in nature controlling line place forward and be trapped in gold mark/latex rabbit igg continuation swimming, be enriched in gradually on nature controlling line and form aubergine band or latex colors, in Quality Control, therefore only occur that band is judged to negative findings.
Immunochromatography technique is applied to anti-MPO-ANCA antibody test by the present invention first, achieves high specific, highly sensitive detection perform.Compare with chemical luminescence reagent kit with the ELISA of existing bibliographical information, advantage of the present invention mainly comprises: detection time short (5 ~ 20min); Without any need for specific apparatus, can realize bedside detect and outpatient service immediately detect; Easy and simple to handle, only need single step reaction, operating personnel are without the need to training, and testing cost is low; To temperature without particular/special requirement, without the need to freezing, store convenient transportation, room temperature can preserve 24 months.
Accompanying drawing explanation
Fig. 1 side structure schematic diagram 1 of the present invention.
Fig. 2 side structure schematic diagram 2 of the present invention.
Positive and negative findings schematic diagram 3 when Fig. 3 detection zone of the present invention is 1.
Wherein 3a is band schematic diagram; 3b is positive findings; 3c is negative findings; 3d and 3e represents that test strips lost efficacy.
Embodiment
MPO-ANCA antibody test immune chromatography test paper of the present invention, as shown in Figure 1, this test paper mutually pastes analyzing film (3), pad (2), sample pad (1), adsorptive pads (6) by side in turn with overlapping to opposite side on base plate (7).
Pad (2) is coated with gold mark/latex labelled protein, analyzing film (3) is arranged detection zone (4) and quality control band (5), according to the difference of labelled protein on pad, select corresponding detection zone coating protein and quality control band coating protein.Preferably, the labelled protein that pad sprays is MPO-ANCA antigen protein and mouse IgG, then the coating protein in detection zone is anti-human IgG antibodies, and the coating protein on quality control band is against murine IgG.Another is preferred, and the labelled protein that pad sprays is anti-human IgG antibodies, then the coating protein in detection zone is MPO-ANCA antigen protein.
Below in conjunction with specific embodiments and the drawings, set forth the present invention further.
Prepared by embodiment 1.MPO-ANCA antigen protein
Antigen protein preparation method one: recombinant protein
1) reagent
E. coli host bacteria DH5 α, BL21 (DE3), cloning vector pCR2.1T-vector, expression plasmid pET28a (+), archaeal dna polymerase rTaq, T4DNA ligase, archaeal dna polymerase rTaq, LATaq and restriction enzyme BamH I, Hind III and EcoR I, BamH I, DL2000DNA Marker, T4DNA ligase, low-molecular-weight standard protein, DNA glue reclaims kit, IPTG etc.
2) instrument
Common shaking table SCS-24; Water isolation type constant temperature electric heating incubator; Biophotometer spectrophotometer, tabletop refrigerated centrifuge Centrifuge5810R, desk centrifuge MiniSpin; High speed freezing centrifuge; Protein electrophorese instrument and gel imaging system; PCR instrument; Ultrasonic degradation instrument; Constant-temperature metal bath; HIS protein purification post etc.
3) experimental technique
A. vector construction:
Design primer CGC CAT ATG ATG GGG GTT CCC TTC TTC TCT and CCG CTC GAG GGC CTC TAG GAG GCTTCC CTC pcr amplification from template DNA go out MPO-ANCA fragment, and glue is connected to pCR2.1 cloning vector after reclaiming kit recovery fragment and carries out order-checking qualification.By sequence clone correct for qualification in expression vector pET28a (+), restriction enzyme site is EcoR I, BamH I, carrier has 6 × HIS label simultaneously, is convenient to follow-up protein purification.
B. express
By the Plastid transformation that obtains to e. coli bl21 (DE3), by resistance screening positive expression bacterial strain.By the positive bacteria liquid screened in 1: 1000 ratio inoculation be added with in the LB nutrient culture media of Kan resistance, each bacterium inoculation two pipe, a pipe is used for induction, and another pipe is used for non-induced contrast, will inoculate a pipe empty plasmid bacterium simultaneously and compare.When 37 DEG C of incubated overnight to OD600 values are about 0.4-0.6, it is 1mmol/L that induction pipe and empty control plasmid pipe add IPTG to final concentration, and non-induced contrast does not add, two bacterium that final selection ability to express is high, for the expression of a large amount of albumen.And SDS-PAGE method is conveniently identified expression product.
C. purifying
By the product HIS protein purification post purifying protein of expressing, and dialysis desalting, the protein concentration after measured after purifying is 509mg/L.
MPO-ANCA antigen protein preparation method 2:
Screening MPO-ANCA important antigen epitope polypeptide 2-6, carry out from end (aminoterminal) synthesis from C end (c-terminus) to N with Peptide synthesizer, then with carrier protein couplet, molecular weight is increased, be convenient to be combined on pad and analyzing film.Conventional carrier protein includes but not limited to the poly-D-lysine (PLL) etc. of bovine serum albumin(BSA) (BSA), ovalbumin (OA), keyhole limpet hemocyanin (KLH), human serum albumins (HSA) and Prof. Du Yucang.
Prepared by embodiment 2 antibody
Pad and detection zone coated antibody anti-human igg monoclonal antibody and polyclonal antibody, and for the monoclonal antibody of quality control band and other animal origins of pad and polyclonal antibody, obtain by immune animal.
1, the concrete operation method of monoclonal antibody preparation is:
By 0.05mg ~ 5mg immune formulation (respectively for goat, mouse, rabbit, horse and cavy) and the Freund's complete adjuvant of 1 times of volume are mixed to get injection solution, by the multiple location injection in Animal Skin of this injection solution;
After one month by Freund's complete adjuvant mixed liquor through multiple location Animal Skin hemostasis booster immunization.By animal bloodletting after 7 ~ 14 days, measure antiserum titre.
To animal booster immunization until titre reaches plateau.By merging from by the animal of immunity recovery splenocyte and murine myeloma cell SP2/0, the hybridoma of energy stably express object antibody after screening, can be obtained.
The hybridoma of in vitro culture, Mice Inoculated or rat abdominal cavity, get ascites purifying and obtain monoclonal antibody; Or purifying obtains monoclonal antibody from culture supernatant.
2, polyclonal antibody preparation:
With monoclonal antibody step 1) and 2), after acquisition antiserum, purifying antiserum obtains polyclonal antibody.
3, staphylococcal protein A and streptococcal protein G preparation
According to the gene order of staphylococcal protein A and streptococcal protein G, prepare by escherichia coli prokaryotic expression cloned gene, concrete operation method can see (Molecular Cloning: A Laboratory guide), or the step of embodiment 1MPO-ANCA.
The each component preparation of embodiment 3 gold test strip
1, collaurum liquid preparation
By the HAuCl of 0.01% (w/v)
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 red, boils 7 ~ 10min and occurs transparent orange red.Filter with ultrafiltration or miillpore filter (0.45 μM) again, to remove polymkeric substance wherein and other impurity that may be mixed into.The collaurum outward appearance prepared should pure, bright, without precipitation and floating thing, abandon when grease and a large amount of black particle shape sediment impurity appear in liquid level.
Wherein used reductive agent can be trisodium citrate, tannic acid-trisodium citrate, white phosphorus, preferably uses trisodium citrate, more preferably uses 1% (w/v) trisodium citrate.
Wherein absolute cleanliness answered by glass container used, with before need through pickling.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: 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 three months; 1000mL1%HAuCl
4solution formula: 10g HAuCl
4, ultrapure water is settled to 1000mL; The preparation of 1% trisodium citrate (Sodium Citrate): dissolve Sodium Citrate with ultrapure water, be made into 1% solution, 0.22 μM of membrane filtration mistake, now with the current.
2, gold mark albumen preparation
The present invention's gold mark to be marked albumen includes but not limited to mouse-anti human IgG, MPO-ANCA antigen protein, mouse IgG, sheep IgA, SPA, goat anti-human igg, horse anti-human igg, rabbit IgM, mouse IgA.
Collaurum pH7.0 ~ 8.5 are regulated with 0.1M sodium carbonate, 5 ~ 25 μ G albumen to be marked is slowly added by every milliliter of colloidal gold solution, mixing, leave standstill 10 ~ 30min, then add BSA to final concentration 0.5 ~ 1%, mixing, leave standstill 5 ~ 10min, the centrifugal 5min of 3000rpm, go precipitation, upper solution is gone to new pipe, the centrifugal 30min of 9000rpm, remove supernatant, add re-suspension liquid to commercial weight, solution is moved to new pipe, 9000rpm is centrifugal 30min again, add and will precipitate resuspended with the conserving liquid of 1/10th initial volumes, put 4 DEG C for subsequent use.
3, analyzing film preparation
Analyzing film is selected: the optional nitrocellulose filter of material (NC) of analyzing film or cellulose acetate membrane, commercial nitrocellulose filter comprises S & SAE99, whatman8 μm, millipore M135, sartorius CN140 etc.The concrete NC film or the cellulose acetate membrane that use which kind of specification are not keys of the present invention, but in each mensuration, above-mentioned several NC film can as preferably.The film of the different damping fluid process containing different surfaces activating agent that different manufacturers uses, gap is in various degree had with detection line antibody-solutions affinity used, also can largely cause lines uneven, traction or the phenomenon of disperse, therefore use assembling test paper to select preferred NC film or cellulose acetate membrane.
Prepared by analyzing film: be buffered liquid dilution detection zone coating protein to 0.5 ~ 1.5mG/mL concentration with bag, with drawing film gold spraying instrument HM3035, distance pad 1cm place, be sprayed on film with 0.5 ~ 1.5 μ L/cm, form detection zone (4), distance detection zone is about 5mm place, is sprayed on same film with 0.5 ~ 1.5 μ L/cm with drawing film gold spraying instrument instrument, form quality control band (5), quality control band simultaneously distance adsorptive pads is about 1cm.Analyzing film 37 DEG C oven dry, encapsulates for subsequent use.
It can be borate, carbonate, phosphate, Tris-HCl or Tris-phosphate etc. that the bag used is buffered liquid, the effect of damping fluid is to provide certain pH and ionic strength makes albumen firmly be coated in NC film, pH of cushioning fluid is generally about in 6 ~ 9.5 scopes, be preferably within the scope of the neutral buffered of 6.5 ~ 7.5, and most preferably the pH value of damping fluid is in 7.0 ~ 7.4 scopes.
A. analyzing film 1
Detection zone T line: by rabbit anti-human igg and pH be 7.2 10mM PB buffer solution mixed preparing become the mixed solution that concentration is 0.4mG/mL, be sprayed on nitrocellulose membrane, coating weight is 1 μ L/cm.
Quality control band C line: by sheep anti-mouse igg and pH be 7.5 10mM PB buffer solution mixed preparing to become concentration be 0.5mG/mL mixed solution, be sprayed on nitrocellulose membrane, coating weight is 1 μ L/cm.
B. analyzing film 2
Detection zone T line: by goat anti-human igg and pH be 7.3 10mM carbonate buffer solution mixed preparing become the mixed solution that concentration is 0.3mG/mL, be sprayed on cellulose acetate film, coating weight is 1 μ L/cm.
Quality control band C line: be the 10mM carbonate buffer solution preparation mouse-anti MPO-ANCA monoclonal antibody of 7.5 with pH, concentration is 0.3mG/mL, is sprayed on cellulose acetate film, and coating weight is 1 μ L/cm.
C. analyzing film 3
Detection zone T line: be the 10mM PB buffer preparation guinea pig anti-human IgG of 7.5 with pH, concentration is 0.5mg/mL, is sprayed on nitrocellulose membrane, and coating weight is 1.2 μ L/cm.
Quality control band C line: be 10mM PB buffer preparation Protein G and the mouse-anti sheep IgA of 7.6 with pH, concentration is respectively 0.8mG/mL and 0.6mG/mL, 1: 1 mixing, be sprayed on nitrocellulose membrane, coating weight is 1 μ L/cm.
D. analyzing film 4
Detection zone T line: be the 10mM PB buffer preparation MPO-ANCA antigen protein of 6.7 with pH, concentration is 0.6mG/mL, is sprayed on cellulose acetate film, and coating weight is 1.0 μ L/cm.
Quality control band C line: be the anti-sheep IgG of 10mM PB buffer preparation rabbit of 6.8 with pH, concentration is 0.3mG/mL, is sprayed on cellulose acetate film, and coating weight is 1.0 μ L/cm.
4, the preparation of pad
The process of pad: pad material is all-glass paper or polyester film, pad is soaked with the pad treating fluid prepared, after at 37 DEG C, 1h is dried, antibody or albumen are sprayed on pretreated polymer PET with the consumption of 4uL/cm, obtain pad after 37 DEG C of dry 1h, pad is for subsequent use under being placed in the environment of 2 ~ 8 DEG C; Consisting of of pad treating fluid: pH is the 10mM PB buffer solution of 7.0 ~ 9.0, containing 1%BSA, and 10 ~ 20% sucrose, 0.1%Tween-20.Pad also can not do pre-service, directly uses.
A. pad 1 glass fibre membrane, double-deck, 2a layer is MPO-ANCA antigen protein, and 2b layer is mouse IgG.
B. pad 2 polyester film, individual layer, MPO-ANCA antigen protein.
C. pad 3 glass fibre membrane, individual layer, MPO-ANCA and the mixing of sheep IgA equal proportion.
D. pad 4 glass fibre membrane, individual layer, goat anti-human igg.
5, the preparation of sample pad
Sample pad material is all-glass paper or polyester film, soaks sample pad with the sample pad treating fluid prepared, and at 37 DEG C, 1h is dried, consisting of of sample pad damping fluid: pH is the 10Mm PB buffer solution of 7.0 ~ 9.0, containing 1%BSA, 10% sucrose, 0.1%Tween-20.Sample pad also can not be done pre-service and directly use.
Prepared by embodiment 4 colloid gold label anti-MPO-ANCA antibody test test paper 1 ~ 4
The wide fillet of 1.7cm, 0.8cm, 2.5cm and 1.5cm sheared respectively by the sample pad of also drying embodiment 3 prepared with guillotine, pad, analyzing film, thieving paper, large plate is overlapped to form by Fig. 1 or Fig. 2 mode, with cutting cutter, large plate is cut into single part, the difference that every person-portion width gets stuck according to base plate and different, preferred 3mm and the 4mm width of the present invention.Be assembled in the test card got ready by the test paper that single part has cut, make the sample pad of the corresponding test paper of application of sample window, the corresponding detection zone of result display window and quality control region, during assembling, temperature controls at 25 ~ 37 DEG C, humidity 20 ~ 30%.
Each component of gold label test strip 1 ~ 4 is as following table:
The colored latex labelled protein preparation of embodiment 5.
1, the preparation of colored latex marking fluid
Latex covalent activated: ultrasound wave process latex microsphere body 30 seconds, regulates latex microsphere bulk concentration to be 1.0 × 10
12centrifugal 10 minutes of/mL, 15000rpm, collected after centrifugation sediment distilled water dissolves, and disperses 30 seconds with 200W ultrasound wave; First add the 50mg/mL EDC of 50 μ L, vibration mixing, then add the 50mg/mL N-hydroxy thiosuccinimide of 50 μ L, vibration mixing, equilibrate at room temperature after 30 minutes at 15000rpm centrifugal 10 minutes, precipitation 50mM citrate buffer solution dissolves, and is statically placed in 4 DEG C of refrigerators.
2, the preparation of colored latex labelled protein
The present invention's albumen to be marked includes but not limited to: horse anti-human igg, SPA, MPO-ANCA antigen protein, mouse IgA.
By the latex after activation in 200W ultrasound wave process 30 seconds, antibody to be marked or albumen is added according to the ratio of the colored latex of 100 ~ 150 μ G-protein/100 μ L, mixing stirring at room temperature reaction 1 ~ 2 hour, centrifuge washing, under each 15000rpm centrifugal 10 minutes, wash 3 times altogether, precipitate and to dissolve with PBS-TBN and 100W ultrasound wave process 30 seconds, then be diluted to centrifugal front volume with PBS-TBN, 4 DEG C save backup.
3, analyzing film preparation
Analyzing film material, bag are buffered formula of liquid and method for coating with embodiment 3, and the present embodiment is following 4 kinds of analyzing films preferably:
A. analyzing film 1 cellulose acetate film
T line: MPO-ANCA antigen protein, it is pH7.0 10mM PB, protein concentration 0.8mG/ μ L, coating weight 1 μ L/cm that bag is buffered liquid.
C line: with the 10mM PB of pH value 7.2, dilution rabbit anti-horse IgG and Protein G, concentration is respectively 0.6mG/ μ L and 1.0mG/ μ L, and 1: 1 mixing coating, coating weight is 1 μ L/cm.
B. analyzing film 2 nitrocellulose membrane
T line: guinea pig anti-human IgG, it is pH7.210mM PB that bag is buffered liquid, protein concentration 0.4mG/ μ L, coating weight 1 μ L/cm.
C line: with the 10mM PB of pH value 7.2, dilution mouse-anti rabbit IgM, concentration 0.4mG/ μ L, coating weight is 0.8 μ L/cm.
C. analyzing film 3 nitrocellulose membrane
T line: camel anti-human igg, bag is buffered the 10mM PB that liquid is pH7.8, protein concentration 0.8mG/ μ L, coating weight 1 μ L/cm.
C line: with the 10mM PB of pH value 7.6, dilution sheep anti mouse IgA, concentration 0.5mG/ μ L, coating weight is 1 μ L/cm.
D. analyzing film 4 cellulose acetate film
T line: MPO-ANCA antigen protein (polypeptide coupling BSA), bag is buffered the 10mM PB that liquid is pH6.8, protein concentration 0.5mG/ μ L, coating weight 1 μ L/cm.
C line: with the 10mM PB of pH value 7.6, dilution mouse-anti rabbit igg, concentration 0.3mG/ μ L, coating weight is 1 μ L/cm.
4, pad preparation
Pad pre-service: polymer PET or glass fibre membrane are soaked 30 minutes with pad damping fluid, 37 DEG C of oven dry, the albumen stroke film gold spraying instrument that colored latex marks, are sprayed on pretreated film with the consumption of 2 ~ 6 μ L/cm, 37 DEG C of dryings, put 2 ~ 8 DEG C for subsequent use.Pad buffer formulation is with embodiment 3, and the present embodiment is following 4 kinds of pads preferably:
A. pad 1: polyester film, 1 layer, the SPA that the horse anti-human igg of red latex mark and blue latex mark, 1: 1 mixing, 4 μ L/cm spray.
B. pad 2: polyester film, 2 layers, 2a layer is the MPO-ANCA recombinant protein of red latex mark, and 2b layer is the rabbit IgM of blue latex mark, and 3 μ L/cm and 5 μ L/cm spray respectively.
C. pad 3: glass fibre membrane, 1 layer, purple latex mark MPO-ANCA (coupling KLH) mixes by 1: 1 with the mouse IgA that red latex marks, and 3 μ L/cm spray.
D. pad 4: glass fibre membrane, 2 layers, 2a layer is the mouse-anti human IgG of red latex mark, and 2b layer is the rabbit igg of blue latex mark.
5, the preparation of sample pad
Sample pad material is all-glass paper or polyester film, soaks sample pad with the sample pad treating fluid prepared, and at 37 DEG C, 1h is dried, consisting of of sample pad damping fluid: pH is the 10Mm PB buffer solution of 7.0 ~ 9.0, containing 1%BSA, 10 ~ 20% sucrose, 0.1%Tween-20.Sample pad also can not be done pre-service and directly use.
The colored latex of embodiment 6 anti-MPO-ANCA antibody test test paper 1 ~ 4 is assembled
The wide fillet of 1.7cm, 0.8cm, 2.5cm and 1.5cm sheared respectively by the sample pad of also drying embodiment 3 prepared with guillotine, pad, analyzing film, thieving paper, large plate is overlapped to form by Fig. 1 or Fig. 2 mode, with cutting cutter, large plate is cut into single part, the difference that every person-portion width gets stuck according to base plate and different, preferred 3mm and the 4mm width of the present embodiment.Be assembled in the test card got ready by the test paper that single part has cut, make the sample pad of the corresponding test paper of application of sample window, the corresponding detection zone of result display window and quality control region, during assembling, temperature controls at 25 ~ 37 DEG C, humidity 20 ~ 30%.
Each component of colored latex test strips 1 ~ 4 is as following table:
Embodiment 7 sample process
Serum sample: get whole blood 1 ~ 5mL in serum collection pipe, leaves standstill the centrifugal 5 ~ 10min of 30min ~ 2h, 3000 ~ 5000g, gets supernatant and get final product.According to the accuracy of detection of test strips, sample sample loading buffer dilution 0-100 times, get 50-100 μ L and be added drop-wise in the well of test strips, leave standstill observations after 5 ~ 20 minutes.
Plasma sample: get whole blood 1 ~ 5mL and mix in sodium citrate or liquaemin anticoagulant tube, the centrifugal 5 ~ 10min of 1000 ~ 3000g, gets supernatant and namely obtain plasma sample.According to the accuracy of detection of test strips, sample sample loading buffer dilution 0-100 times, get 50-100 μ L and be added drop-wise in the well of test strips, leave standstill observations after 5 ~ 20 minutes.
Whole blood sample: fetching point or ear-lobe fresh blood about 50 μ L, be added drop-wise in well, is added drop-wise in well with 50 μ L sample loading buffers at once and dilutes, leave standstill observations after 5 ~ 20 minutes.
Embodiment 8 test strips Performance Evaluation
The performance of reagent strip of the present invention comprise stability, batch in inaccuracy and batch between inaccuracy assessment.
1,4 DEG C of stability tests
Test strips is placed 2 ~ 8 DEG C of freezers, and monthly the withinrun precision of the yin and yang attribute coincidence rate of each 10 parts of taking-up quality-control product harmonizing yinyang reference material judges the stability of test paper.Result display after 12 months, 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%.Result display after 18 months, the yin and yang attribute coincidence rate of each yin and yang attribute reference material is still 100%.After 24 months, there is not false negative in testing result display.Illustrating that test paper is 2 ~ 8 DEG C of storages based on the above results, was stable in 2 years.
2, interior inaccuracy is criticized
Get gold label test strip and the latex test strips of each one batch, respectively with high, medium and low three kinds of positive serums and each portion of negative serum, every part of serum uses gold label test strip and latex test strips duplicate detection 10 times respectively, result is as shown in the table, the testing result of all positive serums is the positive, the testing result of all negative serums is feminine gender, points out colloidal gold strip of the present invention and latex test strips to criticize interior inaccuracy and meets standard.
3, criticize between inaccuracy
Test strips 1 and each three batches of test strips 5, detect with high, medium and low three kinds of positive serums and negative serum, and every part of Virus monitory 10, observations after 10 minutes also inserts table 2.As shown in the table, test strips 1 is consistent with the testing result of each three different batches of test strips 5, illustrates that between criticizing, inaccuracy meets sample.
Embodiment 9 ELISA test strip and clinical performance assessment
The gold label test strip prepared by embodiment 4 and embodiment 6 prepare the UC patient and normal human serum or each 100 examples of blood plasma that latex ELISA test strip makes a definite diagnosis.Wherein detection sensitivity is in all positive clinical case loads, the number percent shared by ELISA test strip number positive, and detection specificity is the number percent in all clinical negative case numbers shared by the negative number of ELISA test strip.
As can be seen from the result of following table, MPO-ANCA antibody test gold label test strip prepared by the present invention and latex test strips are for the sensitivity about 5 ~ 12% of UC patient diagnosis, consistent with bibliographical information.
Claims (10)
1. an anti-MPO-ANCA antibody immune chromatography test strips, comprise sample pad (1), pad (2), analyzing film (3), adsorptive pads (6), base plate (7), detection zone T line (4) and quality control band C line (5), the top of described base plate (7) is fitted with analyzing film (3), the two ends on described analyzing film (3) top are fitted with pad (2) and adsorptive pads (6) respectively, the one end on described pad (2) top is fitted with sample pad (1), described detection zone T line (4) and quality control band C line (5) are arranged on analyzing film (3), it is characterized in that, MPO-ANCA antigen protein is combined on pad (2) or is coated on detection zone T line (4).
2. immuno-chromatographic test paper strip according to claim 1, is characterized in that: described pad (2) bag is by colloid gold label thing or latex (latex) label.
3. MPO-ANCA antibody test test strips according to claim 1, it is characterized in that, described pad is 1 layer, overlaps as shown in Figure 1 with analyzing film, or point upper (2a), under (2b) two-layer with analyzing film overlap joint straggly (Fig. 2).
4. a preparation method for anti-MPO-ANCA antibody immune chromatography test strips, comprises the steps:
The preparation of step 1.MPO-ANCA antigen protein
Prepared by step 2. pad (2)
(1) preparation of nano gold mark thing: with nano-Au solution mark MPO-ANCA antigen protein, anti-human IgG antibodies, staphylococcal protein A (SPA), streptococcal protein G (Protein G), and other and detection zone T line and quality control band C line can form the albumen of immune complex.
(2) preparation of colored latex label: with the colored latex mark MPO-ANCA antigen protein of band reactive group, anti-human IgG antibodies, staphylococcal protein A (SPA), streptococcal protein G (Protein G), and other and detection zone T line and quality control band C line can form the albumen of immune complex.
(3) pad (2) preparation: glass fibre membrane or polymer PET are as pad, the nano gold mark thing that step (1) is prepared or latex label, be sprayed on pretreated glass fibre membrane or polymer PET with spraying instrument, drying for standby.
Prepared by step 3. analyzing film (3)
A. detection zone T line (4) preparation: according to pad labelled protein type selecting detection zone T line coating protein, if the associated proteins on pad is containing MPO-ANCA antigen protein, the coating protein of detection zone T line is anti-human IgG antibodies.If containing anti-human IgG antibodies on pad, detection zone T line coating protein is MPO-ANCA antigen protein.
B. quality control band C line (5) preparation: according to the coating protein of the labelled protein type selecting quality control band of pad, if pad labelled protein is single MPO-ANCA, the coating protein of quality control band C line is the antibody of anti-MPO-ANCA; If pad labelled protein is single anti-human IgG antibodies, the labelled protein of quality control band C line is the antibody of corresponding anti-human IgG antibodies; When pad labelled protein has 2 layers, the coating protein of quality control band C line can form the albumen of immune complex with pad respective layer associated proteins.
Prepared by step 4. sample pad
Glass fibre membrane or polyester film are flooded dry for standby through damping fluid, or directly uses.
Step 5. test strips is assembled
The material completed made by step 1 ~ 4, by Fig. 1 or Fig. 2 overlap joint, cut into certain length and width according to the specification of base plate, in being arranged on base plate and getting stuck.
5. immuno-chromatographic test paper strip according to claim 4, it is characterized in that: described MPO-ANCA antigen protein is total length MPO-ANCA gene or the gene order containing important epitope, be cloned in protokaryon or carrier for expression of eukaryon, expression and purification obtains.
6. immuno-chromatographic test paper strip according to claim 4, is characterized in that: described MPO-ANCA antigen protein is with Peptide synthesizer synthesis containing the MPO-ANCA polypeptide of the important epitope of MPO-ANCA, and is coupled on carrier protein and obtains.
7. immuno-chromatographic test paper strip according to claim 6, is characterized in that: described carrier protein includes but not limited to the poly-D-lysine (PLL) etc. of bovine serum albumin(BSA) (BSA), ovalbumin (OA), keyhole limpet hemocyanin (KLH), human serum albumins (HSA) and Prof. Du Yucang.
8. immuno-chromatographic test paper strip according to claim 4, is characterized in that, described anti-human IgG antibodies is one or more monoclonal antibodies in mouse source, rabbit source, Yang Yuan, Ma Yuan, camel source or cavy source or polyclonal antibody.
9. immuno-chromatographic test paper strip according to claim 4, is characterized in that, the antibody of described anti-human IgG antibodies refers to and can form the albumen of immune complex with this antibody.
10. an anti-MPO-ANCA antibody detection method, is characterized in that, with the immuno-chromatographic test paper strip described in claim 1 ~ 9, detect measuring samples, concrete steps comprise:
(1) sample process: get serum, blood plasma or whole blood sample, with sample loading buffer dilution 0 ~ 100 times;
(2) sample drop of above-mentioned process is added in the sample pad of test strips, leaves standstill 5 ~ 30 minutes, observe T line and C line;
(3) result interpretation: T line and C line all manifest, result is positive; T line does not manifest, and C line manifests, and result is negative; T line and C line all do not manifest or other phenomenons, and prompting test paper lost efficacy.
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