CN104330568A - Colloidal gold immunity chromatography kit for detecting cyanophycean toxin - Google Patents
Colloidal gold immunity chromatography kit for detecting cyanophycean toxin Download PDFInfo
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- CN104330568A CN104330568A CN201410545656.1A CN201410545656A CN104330568A CN 104330568 A CN104330568 A CN 104330568A CN 201410545656 A CN201410545656 A CN 201410545656A CN 104330568 A CN104330568 A CN 104330568A
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- cyanophycean toxin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/405—Assays involving biological materials from specific organisms or of a specific nature from algae
Abstract
The invention relates to a colloidal gold immunity chromatography kit for detecting cyanophycean toxin, which comprises an adhesive rack, a sample pad, a gold bonder pad, a cellulose nitrate membrane and a water absorption pad are arranged over the adhesive rack from left to right in order; a detection line and a control line are arranged at an intermediate part on the cellulose nitrate membrane from left to right; the sample pad material is glass fibre membrane, is immersed in a treatment fluid containing Tris, casein sodium salt and PVP-10 for processing overnight; the gold bonder pad is immersed in a treatment fluid containing Tris, S-17 and bovine serum albumin for processing, the detection line on the cellulose nitrate membrane is a T line coated by immobilized cyanophycean toxin semiantigen; and the control line on the cellulose nitrate membrane is a C line coated with a goat anti mouse IgG polyclonal antibody.
Description
Technical field
The invention belongs to biomedicine field, relate to the quick detection kit and colloidal gold immunochromatographimethod method of testing that detect cyanophycean toxin, specifically a kind of colloidal gold immunity chromatography applying Competitive assays principle realizes kit cyanophycean toxin in sample being carried out to quantitatively detection.
Background technology
Blue-green algae has another name called " blue-green algae ", and in the water body that some are nutritious, some blue-green algae is often amount reproduction formation in summer " wawter bloom ".While causing water quality deterioration, some " wawter bloom " kind also can produce the secondary metabolite with bio-toxicity, is called " cyanophycean toxin ".The release of these cyanophycean toxins directly produces harm to fish, people and animals.Such as, to the health hazard of the public greatly, serious even can cause death for hepatotoxin and Microcystin (microcystin).Therefore, around how to detect, control and eliminate cyanophycean toxin, the scientific worker of various countries has carried out a large amount of research work.
Whether for the detection of cyanophycean toxin, carrying out toxicity tests by the corresponding living model of structure in early days has cyanophycean toxin to exist to detect in water body.But a series of ethics problems that lower sensitivity, higher experiment expend and toxicity tests brings, make people have to select method for distinguishing to carry out contratoxin and diagnose.Along with the development of micro-example analytical instrument (e.g., HPLC, MALDI-TOF), these instrument developments are relied on to go out the multiple method for detecting cyanophycean toxin in natural water.These methods analyst process simple and fasts and there is very high sensitivity.But because the pre-treatment of analytical instrument to sample has very high requirement, therefore in practice, the process of sample becomes not only time-consuming but also require great effort, and the analytical instrument of costliness and standard items also make many laboratories cannot carry out diagnosis to cyanophycean toxin.Meanwhile, the diagnostic means relying on immunology and biochemical method to develop also applies to the detection of cyanophycean toxin in laboratory or physical environment water body gradually.As, to ELISA method, colourimetry (protein phosphatase inhibitor) and the cytotoxicity diagnosis that hepatotoxin (microcystin and nodularin) detects.But aforementioned multiple diagnostic method, still there is experiment cost cost higher, the time is longer, cannot the drawback of widespread use.
In recent years, in the exposure of Algae toxins and effect relation are studied, utilize molecular biology, immunological technique sets up rapid sensitive, specific detection method, become the hot and difficult topics problem in this field.Colloidal gold immunolabeling detection technique (colloidal-gold immunolabeled technique, CGIT) be cutting edge technology in current field of fast detection it.Developed very rapid since this technology occurs, application is also more and more extensive, almost can be applicable to biomedical each research field at present, especially uses more general in medical test.This technology, through use for many years, shows numerous advantages: operation is simple, without the need to specific installation, and nontoxic pollution-free etc.
Summary of the invention
the technical matters solved:for the convenient test cyanophycean toxin level mentioned in solution background technology is so that the demand of the method for the various harm caused by cyanophycean toxin of stronger control, the invention provides a kind of colloidal gold immunochromatographykit kit of simple, reliable, with low cost, highly sensitive quick detection cyanophycean toxin.
technical scheme:detect the colloidal gold immunochromatographykit kit of cyanophycean toxin, comprise and paste support, be from left to right provided with sample pad, golden bond pad, nitrocellulose filter and adsorptive pads successively pasting above support; On nitrocellulose filter middle part be from left to right provided with one detection line with together with control line; Described sample pad material is glass fibre membrane, spends the night through carrying out immersion treatment containing the treating fluid of Tris, casein sodium salt and PVP-10; Described golden bond pad by the treating fluid immersion treatment containing Tris, S-17 and bovine serum albumin(BSA), containing the antibody linked thing of the anti-cyanophycean toxin of collaurum; Detection line on described nitrocellulose filter is for being coated with the haptenic T line of immobilised cyanophycean toxin; Control line on described nitrocellulose filter is the C line being coated with sheep anti-mouse igg polyclonal antibody.
The amount of the immobilised cyanophycean toxin of described T line bag quilt higher than in sample can with the amount of the cyanophycean toxin of collaurum cross-linking antibody in golden bond pad.
Described support of pasting is strip.
The pH of collaurum is adjusted to 7.4 to add cyanophycean toxin monoclonal antibody more afterwards by the preparation method of the haptenic T line of immobilised cyanophycean toxin, the minimum protein concentration making collaurum stable is minimum is 0.03mg/mL, 2.5mL is diluted in the 0.01mol/l PBS of 0.04mg/mL, the cyanophycean toxin monoclonal antibody of pH=7.4, dropwise add in 2.5mL colloidal gold solution, gained mixed liquor hatches 2 hours and by centrifugal unconjugated monoclonal antibody of leaving away at 4 DEG C, the sediment of gained is resuspended in the PBS of 5mL pH=7.4, is stored in 4 DEG C.Cyanophycean toxin haptens PBS is diluted to 1.0mg/mL, and with discharge rate 1.0 μ L/mm by the T line of antigen point on nitrocellulose membrane, 37 degrees Celsius of baking ovens dry 24h.
The preparation method being coated with the C line of sheep anti-mouse igg polyclonal antibody is the C line that sheep anti-mouse antibody 1.0mg/mL is assigned on nitrocellulose membrane, and 37 degrees Celsius of baking ovens dry 24h.
beneficial effect:the present invention uses colloid gold label mouse monoclonal anti cyanophycean toxin monoclonal antibody, realizes the qualitative detection whether exceeded standard to cyanophycean toxin in lake water with this technology of A competitive inhibition method.Whether the method exceedes normal level of control for the content measuring cyanophycean toxin in lake water with assessment.The level of cyanophycean toxin in the water quality of safety, for Microcystin, is normally lower than 2500ng/L, when cyanophycean toxin level is higher than 3000ng/L, is namely considered to exceed standard, and whether colloidal gold immunity chromatography is adapted to detect cyanophycean toxin in lake water and exceeds standard; The present invention can detect the level of cyanophycean toxin in lake water easily.Its detection sensitivity reaches 10ng/mL level; Fill up domestic and international blank.Whether cost of the present invention is low, easy to use, highly sensitive, detection time is short, can detect cyanophycean toxin in lake water fast and exceed standard.
Accompanying drawing explanation
Fig. 1 is the structural representation of the colloidal gold immunochromatographykit kit detecting cyanophycean toxin;
In figure, 1-pastes support, 2-sample pad, 3-gold bond pad, 4-nitrocellulose filter, 5-adsorptive pads, 6-detection line, 7-control line.
Embodiment
As shown in the figure, the colloidal gold immunochromatographykit kit of detection cyanophycean toxin of the present invention, strip pastes support, is from left to right adhesive with sample pad, golden bond pad, nitrocellulose filter, adsorptive pads successively pasting above support; On nitrocellulose filter middle part be from left to right provided with one detection line with together with control line; It is characterized in that: described sample pad material is glass fibre membrane, carry out process through the main treating fluid containing Tris, casein sodium salt, PVP-10 and spend the night; Described golden bond pad is made up of, containing the antibody linked thing of the anti-cyanophycean toxin of collaurum the glass fibre membrane through the dedicated treating liquid process containing Tris, S-17, bovine serum albumin(BSA) etc.; Detection line on described nitrocellulose filter is for being coated with the haptenic T line of immobilised cyanophycean toxin; Control line on described nitrocellulose filter is the C line being coated with sheep anti-mouse igg polyclonal antibody.
The colloidal gold immunochromatographimethod detection method of cyanophycean toxin in quick detection lake water of the present invention: detected sample is lake water, adopt the colloidal gold immunochromatographykit kit of cyanophycean toxin in above-mentioned quick detection lake water, detected sample is lake water, adopts the colloidal gold immunochromatographykit kit of above-mentioned quick detection cyanophycean toxin; 1) add 100 μ L with nozzle or add 3 lake water samples in sample pad with buret; 2) standing about 2 minutes are kept flat; 3) go out result according to the color developing detection of test section (T line) or quality control region (C line) in 10 minutes, do not develop the color without result or C line in 10 minutes and think that this time operation is invalid.
When sample being added dropwise in test paper sample pad, sample arrives golden bond pad by sample pad, cyanophycean toxin in sample can mark anti-cyanophycean toxin antibody in conjunction with special gold, this antigen antibody complex is towards detection T line district and the migration of control C line district, and unnecessary golden labelled antibody is combined with cyanophycean toxin in T line district and produces claret color.Antigen-antibody complex continues to move forward to the right, reacts and form Article 2 claret band with the sheep anti-mouse antibody being fixed on control C line district; Aobvious positive or negative according to the T line district judgement sample that whether develops the color, wherein T line district produces claret and represents negative, and colourless display is positive, C line district do not develop the color represent this time operate invalid.
The cyanophycean toxin monoclonal anti physical efficiency of colloid gold label identifies specifically and catches the cyanophycean toxin haptens in sample to be checked, then moves to reach detection zone by capillary action.There, the ability of the cyanophycean toxin of the collaurum-antibody of having caught cyanophycean toxin in sample again in Acquisition Detection T line district was lost already, therefore can not form collaurum claret band reflected sample in detection zone and be positive.If cyanophycean toxin content is few or do not contain in sample, arriving the exposed collaurum-antibody detecting T line district will be many, they can in conjunction with being positioned at the cyanophycean toxin antigen detecting T district, and therefore form collaurum claret band in detection T line district, reflected sample is negative.No matter sample is positive or feminine gender quality control region C line, all should develop the color, just represent that this detection is invalid if do not developed the color.The colloidal gold immunochromatographimethod detection method of employing A competitive inhibition method principle that Here it is.
embodiment 1
The preparation of 1 collaurum
Main employing citric acid is reductive agent, and with sodium citrate preparation, colloid gold particle diameter control is in 30-40nm collaurum (Frens, 1973).The chlorauride of 1% of 5mL is added in the distilled water of 500mL, ebuillition of heated.And add the sodium citrate solution of 5mL 1%, constantly boiling is until solution reddens.After continuation keeps boiling 5min, burned Jinsui River is kept at 4 DEG C.
2 colloid gold label cyanophycean toxin antibody
The best combination concentration of titration measuring collaurum and cyanophycean toxin antibody.The phosphate buffered saline of purified IgG antibody 0.002M pH=9.0 is diluted to 0.1mg/mL.The K of collaurum and the pH value 0.1M through the antibody of dilution
2cO
3salt is diluted to pH=7.4.The IgG antibody (0.01-0.1mg/mL) of the variable concentrations be diluted in 0.002M phosphate buffer is added 1mL colloidal gold solution respectively.Hatch 10min, add the NaCl solution of 0.1mL 10% and go out to measure absorbance at 520nm.The horizontal ordinate of the curve that the minimum protein concentration making collaurum stable is drawn by concentration and absorbance is determined.Add 2mL 2% PEG 20000 respectively, room temperature places 5min.The centrifugal 20min of 10000rpm, absorbs supernatant gently.Be the loose collaurum precipitations of 7.4 phosphate buffer resuspensions with 2mL pH, and focus in new pipe.Save backup under 2-8 DEG C of condition.
The preparation of 3 colloid gold reagent bars
Cyanophycean toxin monoclonal antibody is added again after the pH of collaurum being adjusted to 7.4.The minimum protein concentration making collaurum stable is minimum is 0.03mg/mL.2.5mL cyanophycean toxin monoclonal antibody (being diluted in the 0.01mol/L PBS of 0.04mg/mL, pH=7.4) is dropwise added in 2.5mL colloidal gold solution.Gained mixed liquor hatches 2 hours at 4 DEG C and by centrifugal unconjugated monoclonal antibody of leaving away, the sediment of gained is resuspended in the PBS of 5mL pH=7.4, is stored in 4 DEG C.Whether success detects mainly through UV double beam spectrophotometer monoclonal antibody-colloid gold label.The method of immunochromatography bar application Xu and Verheijen makes.Cyanophycean toxin haptens PBS is diluted to 1.0mg/mL, and with discharge rate 1.0 μ L/mm by antigen point (T line) on nitrocellulose membrane, sheep anti-mouse antibody (1.0mg/mL) is assigned to the C line on nitrocellulose membrane, and 37 DEG C of baking ovens dry 24h.Cyanophycean toxin monoclonal antibody-the colloidal gold solution of concentration 1.0mg/mL, with discharge rate 1.0 μ L/mm, spray is with on polyester film, and 37 DEG C of baking ovens dry 12-24h.The glass fibre various process completed has been assembled with selected material.
The basic structure of this cyanophycean toxin Rapid detection test strip as shown in Figure 1.Detect the colloidal gold immunochromatographykit kit of cyanophycean toxin, comprise and paste support 1, be from left to right provided with sample pad 2, golden bond pad 3, nitrocellulose filter 4 and adsorptive pads 5 successively pasting above support; On nitrocellulose filter middle part be from left to right provided with one detection line 6 with together with control line 7; Described sample pad material is glass fibre membrane, spends the night through carrying out immersion treatment containing the treating fluid of Tris, casein sodium salt and PVP-10; Described golden bond pad by the treating fluid immersion treatment containing Tris, S-17 and bovine serum albumin(BSA), containing the antibody linked thing of the anti-cyanophycean toxin of collaurum; Detection line on described nitrocellulose filter is for being coated with the haptenic T line of immobilised cyanophycean toxin; Control line on described nitrocellulose filter is the C line being coated with sheep anti-mouse igg polyclonal antibody.The amount of the immobilised cyanophycean toxin of described T line bag quilt higher than in sample can with the amount of the cyanophycean toxin of collaurum cross-linking antibody in golden bond pad.Described support of pasting is strip.The pH of collaurum is adjusted to 7.4 to add cyanophycean toxin monoclonal antibody more afterwards by the preparation method of the haptenic T line of immobilised cyanophycean toxin, the minimum protein concentration making collaurum stable is minimum is 0.03mg/mL, 2.5mL is diluted in the 0.01mol/l PBS of 0.04mg/mL, the cyanophycean toxin monoclonal antibody of pH=7.4, dropwise add in 2.5mL colloidal gold solution, gained mixed liquor hatches 2 hours and by centrifugal unconjugated monoclonal antibody of leaving away at 4 DEG C, the sediment of gained is resuspended in the PBS of 5mL pH=7.4, is stored in 4 DEG C.Cyanophycean toxin haptens PBS is diluted to 1.0mg/mL, and with discharge rate 1.0 μ L/mm by antigen point (T line) on nitrocellulose membrane, 37 degrees Celsius of baking ovens dry 24h.The preparation method being coated with the C line of sheep anti-mouse igg polyclonal antibody is the C line that sheep anti-mouse antibody 1.0mg/mL is assigned on nitrocellulose membrane, and 37 degrees Celsius of baking ovens dry 24h.
When test strips immerses sample, as lake water, this sample may move through sample pad and arrives gold conjugation pad.In any sample, cyanophycean toxin specifically in conjunction with golden labelled antibody, can be moved to by capillarity and detects T line district and control C line district.According to cyanophycean toxin concentration different in sample, the developing the color or do not develop the color of T line.Unnecessary crosslinked gel gold or free crosslinked gel gold are combined with the immobilization cyanophycean toxin at T line, and form claret band display negative findings.Otherwise, be then positive findings.
The quick detection kit that we make comprises test strips, and be made up of following components (see figure 1): sample pad, golden bond pad, nitrocellulose filter (NC film), adsorptive pads, pastes support, outer box.
1) sample pad, material is glass fibre membrane, spend the night through sample pad treating fluid (mainly containing Tris, casein sodium salt, PVP-10) process, sample pad for remove bulky grain in sample and buffering detection system crosslinked with cyanophycean toxin haptens in guarantee environment system and specific antibody.
2) golden bond pad, through the glass fibre membrane that dedicated treating liquid (containing Tris, S-17, bovine serum albumin(BSA) etc.) processed, containing the antibody linked thing of collaurum cyanophycean toxin.Whether colloidal gold labeled monoclonal antibody really point of contact sample amount needs strict test, to make to be exceeded standard rationalization by the T line district judgement sample cyanophycean toxin content that whether develops the color.
3) nitrocellulose filter (NC film).First district of ruling at nitrocellulose filter is T line detection zone, is C line traffic control district in second district of nitrocellulose filter line.T line is become wire with C line at this film bag.T line contains immobilised cyanophycean toxin, to catch any unnecessary collaurum cross-linking antibody.The colour developing of T line district shows that in sample, cyanophycean toxin content is lower than 2500ng/L, still has unnecessary golden labeling antibody, and unnecessary golden labeling antibody arrives T line district and is combined aobvious claret with immobilised cyanophycean toxin haptens, and result is negative.Otherwise be positive.The immobilised cyanophycean toxin of T line is higher than the amount of collaurum cross-linking antibody in gold conjugation pad.When cyanophycean toxin-collaurum cross-linked composite arrives C line from crosslinked pad with capillary moving, this compound will be caught by sheep anti-mouse igg antibody, and form visible claret band.C line is control line, so that the validity of inspection and detection system.As the not outlet of C line, show that inspection is invalid.
4) adsorptive pads is Whatman filter paper, promotes that sample chromatography is through sample pad, collaurum-antibody conjugates pad, nitrocellulose filter, arrives adsorptive pads district, completes detection to absorb unnecessary sample.
the advantage of quick detection kit
1. economical, applicable: this product, without any need for instrument, equipment, is applicable to any medical institutions and uses.
2. easy and simple to handle: because a large amount of middle work is completed in advance by the producer, operating personnel only need instillation 3 (about 100 μ L) sample to get final product observations.
3. going out result fast: within 10 minutes, go out result, is the other diagnostic method of bed truly.
4. highly sensitive, high specificity: concentrations is 10ng/mL, and rate of accuracy reached is to more than 99%.
5. Quality Control is strict: each brassboard, all with automatic control system, automatically carries out Quality Control while each detection.
6. convenient storage, long shelf-life: kit can preserve 24 months under room temperature (15-28 DEG C) condition.
Claims (5)
1. detect the colloidal gold immunochromatographykit kit of cyanophycean toxin, comprise and paste support (1), be from left to right provided with sample pad (2), golden bond pad (3), nitrocellulose filter (4) and adsorptive pads (5) successively pasting above support; On nitrocellulose filter, middle part is from left to right provided with one detection line (6) and control line (7) together; It is characterized in that: described sample pad material is glass fibre membrane, spending the night through carrying out immersion treatment containing the treating fluid of Tris, casein sodium salt and PVP-10; Described golden bond pad by the treating fluid immersion treatment containing Tris, S-17 and bovine serum albumin(BSA), containing the antibody linked thing of the anti-cyanophycean toxin of collaurum; Detection line on described nitrocellulose filter is for being coated with the haptenic T line of immobilised cyanophycean toxin; Control line on described nitrocellulose filter is the C line being coated with sheep anti-mouse igg polyclonal antibody.
2. the colloidal gold immunochromatographykit kit of detection cyanophycean toxin according to claim 1, is characterized in that: the amount of the immobilised cyanophycean toxin of described T line bag quilt higher than in sample can with the amount of the cyanophycean toxin of collaurum cross-linking antibody in golden bond pad.
3. the colloidal gold immunochromatographykit kit of detection cyanophycean toxin according to claim 1, is characterized in that: described support of pasting is strip.
4. the colloidal gold immunochromatographykit kit of detection cyanophycean toxin according to claim 1, it is characterized in that: the pH of collaurum is adjusted to 7.4 to add cyanophycean toxin monoclonal antibody more afterwards by the preparation method of the haptenic T line of immobilised cyanophycean toxin, the minimum protein concentration making collaurum stable is minimum is 0.03mg/mL, 2.5mL is diluted in the 0.01mol/l PBS of 0.04mg/mL, the cyanophycean toxin monoclonal antibody of pH=7.4, dropwise add in 2.5mL colloidal gold solution, gained mixed liquor hatches 2 hours and by centrifugal unconjugated monoclonal antibody of leaving away at 4 DEG C, the sediment of gained is resuspended in the PBS of 5mL pH=7.4, be stored in 4 DEG C, cyanophycean toxin haptens PBS is diluted to 1.0mg/mL, and with discharge rate 1.0 μ L/mm by the T line of antigen point on nitrocellulose membrane, 37 degrees Celsius of baking ovens dry 24h.
5. the colloidal gold immunochromatographykit kit of detection cyanophycean toxin according to claim 1, it is characterized in that: the preparation method being coated with the C line of sheep anti-mouse igg polyclonal antibody is the C line that sheep anti-mouse antibody 1.0mg/mL is assigned on nitrocellulose membrane, 37 degrees Celsius of baking ovens dry 24h.
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