CN101358981A - Immune colloidal gold chromatography method for detecting food allergy to aquatic products - Google Patents
Immune colloidal gold chromatography method for detecting food allergy to aquatic products Download PDFInfo
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- CN101358981A CN101358981A CNA200810071731XA CN200810071731A CN101358981A CN 101358981 A CN101358981 A CN 101358981A CN A200810071731X A CNA200810071731X A CN A200810071731XA CN 200810071731 A CN200810071731 A CN 200810071731A CN 101358981 A CN101358981 A CN 101358981A
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Abstract
The present invention discloses a gold-immunochromatography assay method which is used for detecting the aquatic product food allergy. The method comprises preparing colloidal gold, labeling protein with the colloidal gold, detecting with the gold-immunochromatography assay, and other steps. The invention adopts the gold-immunochromatography assay technology to detect the aquatic product food allergy; the technology not only has the advantages of high specificity and high sensitivity of the enzyme immunoassay, but also has the advantages of rapid and simple detection of the prick test; and the technology can rapidly detect whether the food has aquatic product allergens or aquatic product allergen antibodies.
Description
Technical field
The present invention relates to the detection method of food hypersenstivity, particularly relate to a kind of immune colloidal gold chromatography method that detects food allergy to aquatic products, this method mainly is the immune colloidal gold chromatography detection at shell-fish tropomyosin (TM) and fish parvalbumin (Par) food hypersenstivity.
Background technology
Food hypersenstivity medically also claims allergic reaction.The material that causes the food hypersenstivity reaction is called food allergen, as fish, shrimp, crab, milk, eggs etc.Its reaction mechanism is that anaphylactogen enters after the body, can cause body that normal or excessive immune response, i.e. hypersensitivity take place.A plurality of IgE molecules and antibodies when contacting identical anaphylactogen once more, the Fc end structure that causes IgE changes, cause cell degranulation, discharge biological respiratory activity material such as histamine, serotonin, leukotriene and act on effector organ, show as that smooth muscle contraction, telangiectasis, permeability increase, allergic, respiratory tract anaphylaxis, hemopoietic system allergy even anaphylactic shock (Zhao Junfang, Deng. food allergen detects and application prospect. Chinese laboratory medicine magazine, 2007,8:948-950).
The food hypersenstivity problem has caused consumers in general, food producer and scientific research person's common concern in recent years.According to epidemiology survey, the whole world has the adult of 2%-2.5% to be subjected to the puzzlement of food hypersenstivity disease approximately, and infant's the incidence of disease is higher, reaches 4%-6%, and children are about 2%-3%.In the irritated food of the whole world eight big classes that FAO (Food and Agriculture Organization of the United Nation) announces, fish and shell-fish aquatic products are two wherein important big classes, its main anaphylactogen be respectively shell-fish tropomyosin (TM) and fish parvalbumin (Par) (Food and AgricultureOrganization.Report of the FAO technical consultation on foodallergies.Rome, Italy.1995.).
For the present no special methods of treatment of food hypersenstivity, it is effective method that contact allergy food is avoided in strictness.Detection anaphylactogen method commonly used has anaphylactogen pricking method test in the body, vitro detection specific IgE (RAST suppresses experiment and EAST suppresses experiment), the provocative test of double blinding food and enzyme linked immunosorbent assay etc.In these methods, anaphylactogen pricking method test is prone to false positive in the body; The deficiency that RAST inhibition experiment and EAST suppress to test is the dependence to human serum, and serum is that very difficult assurance is consistent, so this method is difficult to standardization; The provocative test of double blinding food is subject to the other factors influence, and also not easy to operate in the practice, links such as test dose, placebo and double blinding control are still waiting perfect; The enzyme linked immunosorbent assay step is many, the time long (Zhao Junfang, etc. food allergen detects and application prospect. Chinese laboratory medicine magazine, 2007,8:948-950).
The immune colloidal gold chromatography technology is a kind of tachysynthesis analytical approach that immunological technique and chromatography technology are combined that grows up the eighties in 20th century.Its principle is to be solid phase with the fibre strip chromatographic material, make sample solution swimming on chromatography strip by capillary action, make determinand in the sample and the acceptor on the chromatographic material (as antibody or antigen) that the immune response of high special, high-affinity takes place simultaneously, immune complex is by enrichment or be trapped in certain zone (detect band) of chromatographic material in the chromatography process, colored marker by enzyme-catalytic chromogenic reaction or direct use can be estimated just can obtain experimental result intuitively in the 5-10min.It need not carry out separating of binding label and free label, has saved loaded down with trivial details application of sample, washing step.Thereby this analytical technology is simple to operate, and analysis result is clear fast, is easy to judge, need not instrument and equipment, cost is low, can be in market, hospital, family, office space even field carry out various field quick detection, but testing result also long preservation (Wu Gang, etc.The application of colloidal gold immunochromatographimethod technology in food inspection.Food industry science and technology, 2007,12:216-218), but still there is not the immune colloidal gold chromatography detection method of shell-fish tropomyosin (TM) and fish parvalbumin (Par) food hypersenstivity at present.
Summary of the invention
The object of the present invention is to provide a kind of immune colloidal gold chromatography method that detects food allergy to aquatic products.This method utilizes the immune colloidal gold chromatography of animal's antibody to detect former TM of food allergy to aquatic products and Par, and utilize TM and Par antibody in the immune colloidal gold chromatography human body serum of natural aquatic products anaphylactogen, thereby reach fast, purpose that sensitivity detects food allergy to aquatic products.
For achieving the above object, technical solution of the present invention is:
The present invention is a kind of immune colloidal gold chromatography method that detects food allergy to aquatic products, and its concrete steps are as follows:
(1) preparation collaurum: draw the 0.5-2.0mL gold chloride and be added in the 50-200mL ultrapure water, be heated to boiling after, add 2-5mL 1% trisodium citrate rapidly, heated and boiled 5-10min obtains colloidal gold solution transparent, claret;
(2) colloid gold label protein: dropwise add protein solution (anaphylactogen antibody or anaphylactogen) while stirring to colloidal gold solution, the protein final concentration is about 5-15 μ g/mL, continues to stir 0.5-1h; Dropwise add 100-300 μ L 10% bovine serum albumin(BSA) (BSA) while stirring, continue to stir 10-15min, standing over night; The colloidal gold solution of getting labelled protein is in 5000g, 4 ℃ of centrifugal 10-15min down; Get supernatant in 17000g, 4 ℃ of following centrifugal 30-45min; The sucking-off supernatant, precipitation is dissolved in the 1-3mL phosphate buffer, makes the protein of colloid gold label, and it is standby to show usefulness or 4 ℃ of preservations.
(3) immune colloidal gold chromatography detects: glass fibre membrane, nitrocellulose membrane, thieving paper are attached to form immune gold test paper strip on the base plate successively; Drip 1-3 μ L staphylococcal protein A in protein (antibody or antigen) and check plot that the detection zone of the nitrocellulose membrane of immune gold test paper strip drips 1-3 μ L colloid gold label respectively, the protein concentration of colloid gold label is 0.5-5mg/mL, dries; At the protein (antibody or antigen) of gold-marking region dropping 10-20 μ L colloid gold label, the protein concentration of colloid gold label is 0.5-5mg/mL; Drip 20-200 μ L example reaction 5-10min to be checked in the glass fibre district, if punctation all appears in detection zone and check plot, sample detection result is positive; Punctation appears in the check plot if detection zone does not have colour developing, and sample detection result is negative; If do not develop the color in the check plot, sample detection result is invalid.
The present invention is with colloid gold label anaphylactogen antibody or anaphylactogen and prepare immune gold test paper strip, is used to detect the immune colloidal gold chromatography kit of food allergy to aquatic products.
The present invention is an immune colloidal gold chromatography technology of utilizing rabbit anti-mud crab TM antibody and the anti-silver carp Par of rabbit antibody, simultaneously aquatic products anaphylactogen TM and Par is carried out specific detection; Utilize natural shrimp crab class TM and the immune colloidal gold chromatography technology of fish Par, simultaneously TM antibody in the human serum and Par antibody are carried out specific detection.Advantage of the present invention is: only need chromatograph test strip, sample to be checked does not need pre-treatment or only needs simple process, testing result directly shows with color, 5-10min can judge whether have whether contain aquatic products anaphylactogen antibody in aquatic products anaphylactogen or the human serum in the sample, thereby have characteristics such as easy and simple to handle, that cost is low, the result clear can preserve, be not subjected to that extraneous factor influences, can be in market, hospital, family, office space and field etc. carry out various field quick detection.
The present invention utilizes the immune colloidal gold chromatography technology that food allergy to aquatic products is detected, the high specificity of the existing enzyme immunoassay of this technology and highly sensitive advantage, the advantage of quick, the easy detection of pricking method test is arranged again, can detect whether have whether contain aquatic products anaphylactogen antibody in aquatic products anaphylactogen or the human serum in the food fast.
The present invention is further illustrated below in conjunction with the drawings and specific embodiments.
Description of drawings
Fig. 1 colloidal gold immunochromatographitest test strip synoptic diagram;
The colloidal gold chromatographic detection prawn TM (A) of the anti-TM antibody of Fig. 2 rabbit and silver carp Par (B) be figure as a result;
The colloidal gold chromatographic of the anti-TM antibody of Fig. 3 rabbit detects behind prawn TM (A), mud crab TM enzymolysis liquid (B) and the prawn TM irradiation solution (C) figure as a result;
The colloidal gold chromatographic of Fig. 4 shrimp crab class TM detects in the allergy sufferers serum TM antibody (A) and Par antibody (B) figure as a result;
The colloidal gold chromatographic of Fig. 5 silver carp Par detects in the allergy sufferers serum Par antibody (A) and TM antibody (B) figure as a result;
Fig. 6 colloidal gold chromatographic detects in the allergy sufferers serum TM antibody (A), Par antibody (B) and no allergy sufferers serum (C) figure as a result.
Embodiment
Embodiment 1: whether contain TM in the colloidal gold chromatographic test sample of the anti-TM antibody of rabbit
(1) preparation collaurum.Draw the 1.0mL gold chloride and be added in the 100mL ultrapure water, be heated to boiling after, add 4mL 1% trisodium citrate rapidly, heated and boiled 8min obtains colloidal gold solution transparent, claret.
(2) colloid gold label of protein.Dropwise add the anti-TM antibody of rabbit while stirring to colloidal gold solution, the protein final concentration is about 8 μ g/mL, continues to stir 0.5h; Dropwise add 200 μ L, 10% bovine serum albumin(BSA)s (BSA) while stirring, continue to stir 10min, standing over night.The protein solution of mark collaurum is in 5000g, and 4 ℃ are descended centrifugal 10min; Get supernatant in 17000g, 4 ℃ of following centrifugal 30min; The sucking-off supernatant makes the protein of colloid gold label, and precipitation is dissolved in the 2mL phosphate buffer, existing with or 4 ℃ of preservations standby.
(3) immune colloidal gold chromatography detects.
As shown in Figure 2, label 1. staphylococcal protein As; 2. the anti-TM antibody of rabbit; 3. the anti-TM antibody of the rabbit of colloid gold label.
As shown in Figure 3, the anti-TM antibody of label 1. rabbits; 2. the anti-TM antibody of the rabbit of colloid gold label.
Successively glass fibre membrane, nitrocellulose membrane, thieving paper are attached to and form immune gold test paper strip on the base plate.Detection zone at the nitrocellulose membrane of immune gold test paper strip is fixed the anti-TM antibody of 1 μ L rabbit, and 1 μ L staphylococcal protein A solution is fixed in the check plot, adds the anti-TM antibody of rabbit of 12 μ L colloid gold labels at gold-marking region, to whether containing TM in the food samples detects.Paper slip A from Fig. 2 as can be seen, sample to be checked is a prawn TM solution, punctation appears in the detection zone on the film bar; The sample to be checked that adds among the paper slip B among Fig. 2 is a silver carp Par solution, and detection zone does not develop the color, and shows not contain TM in the sample.In order further to verify the specificity of the anti-TM antibody colloidal gold of rabbit chromatography, detected prawn TM, mud crab TM enzymolysis liquid and prawn TM solution behind 10kGy irradiation respectively, as shown in Figure 3, punctation (the paper slip A among Fig. 3) appears in the detection zone of prawn TM solution, and the detection zone of two kinds of solution does not all have colour developing (the paper slip B among Fig. 3 and the paper slip C among Fig. 3) in addition, shows that the TM in these two kinds of solution all degrades.
Embodiment 2: the colloidal gold chromatographic of shrimp crab class TM detects in the allergy sufferers serum whether contain TM antibody
As shown in Figure 4, label 1. prawn TM; 2. mud crab TM; 3. the prawn TM of colloid gold label.
The preparation of colloidal gold immunochromatographitest test strip is with reference to embodiment 1 (wherein the protein of colloid gold label is changed to shrimp crab class TM in the present embodiment by the anti-TM antibody of the rabbit of embodiment 1).Two detection zones on the nitrocellulose membrane of immune gold test paper strip are fixed 1 μ L prawn TM and mud crab TM respectively, and gold-marking region adds the prawn TM of 12 μ L colloid gold labels, to whether containing TM antibody among the human serum sample detect.Paper slip A from Fig. 4 as can be seen, sample to be checked is a TM allergy sufferers serum, punctation all appears in two detection zones of result; The sample to be checked that adds among the paper slip B among Fig. 4 is a Par allergy sufferers serum, and two detection zones of result all do not develop the color, and shows and does not contain TM antibody in the sample.
Embodiment 3: the colloidal gold chromatographic of silver carp Par detects in the allergy sufferers serum whether contain Par antibody
As shown in Figure 5, label 1. silver carp Par; 2. crucian Par; 3. the silver carp Par of colloid gold label.
The preparation of colloidal gold immunochromatographitest test strip is with reference to embodiment 1 (wherein the protein of colloid gold label is changed to silver carp Par in the present embodiment by the anti-TM antibody of the rabbit of embodiment 1).Two detection zones on nitrocellulose membrane are fixed 1 μ L silver carp Par and crucian Par respectively, and gold-marking region adds the silver carp Par of 12 μ L colloid gold labels, to whether containing Par antibody among the human serum sample detect.Paper slip A from Fig. 5 as can be seen, sample to be checked is a Par allergy sufferers serum, punctation all appears in two detection zones of result; The sample to be checked that adds among the paper slip B among Fig. 5 is a TM allergy sufferers serum, and two detection zones of result all do not develop the color, and shows and does not contain Par antibody in the sample.
Embodiment 4: colloidal gold chromatographic detects whether contain TM antibody and Par antibody in the allergy sufferers serum simultaneously
As shown in Figure 6, label 1. silver carp Par; 2. prawn TM; 3. the staphylococcal protein A of colloid gold label.
The preparation of colloidal gold immunochromatographitest test strip is with reference to embodiment 1 (wherein the protein of colloid gold label is changed to prawn TM and silver carp Par in the present embodiment by the anti-TM antibody of the rabbit of embodiment 1).Two detection zones on immune gold test paper strip nitrocellulose membrane are fixed 1 μ L prawn TM and silver carp Par respectively, gold-marking region adds the staphylococcal protein A of 12 μ L colloid gold labels, to whether containing TM antibody among the human serum sample and Par antibody detects simultaneously.From Fig. 6-A as can be seen, sample to be checked is a TM allergy sufferers serum, and punctation appears in prawn TM bearing on the film bar, and silver carp Par bearing does not develop the color; The sample to be checked that adds among Fig. 6-B is a Par allergy sufferers serum, and punctation appears in silver carp Par bearing on the film bar, and prawn TM bearing does not develop the color; The sample to be checked that adds among Fig. 6-C is no allergy sufferers serum, and prawn TM and silver carp Par bearing all do not develop the color on the film bar.
Claims (2)
1, a kind of immune colloidal gold chromatography method that detects food allergy to aquatic products, it is characterized in that: its concrete steps are as follows:
(1) preparation collaurum: draw the 0.5-2.0mL gold chloride and be added in the 50-200mL ultrapure water, be heated to boiling after, add 2-5mL 1% trisodium citrate rapidly, heated and boiled 5-10min obtains colloidal gold solution transparent, claret;
(2) colloid gold label protein: dropwise add protein solution while stirring to colloidal gold solution, the protein final concentration is about 5-15 μ g/mL, continues to stir 0.5-1h; Dropwise add 100-300 μ L 10% bovine serum albumin(BSA) (BSA) while stirring, continue to stir 10-15min, standing over night; The colloidal gold solution of getting labelled protein is in 5000g, 4 ℃ of centrifugal 10-15min down; Get supernatant in 17000g, 4 ℃ of following centrifugal 30-45min; The sucking-off supernatant, precipitation is dissolved in the 1-3mL phosphate buffer, makes the protein of colloid gold label, and it is standby to show usefulness or 4 ℃ of preservations.
(3) immune colloidal gold chromatography detects: glass fibre membrane, nitrocellulose membrane, thieving paper are attached to form immune gold test paper strip on the base plate successively; Drip 1-3 μ L staphylococcal protein A in protein and check plot that the detection zone of the nitrocellulose membrane of immune gold test paper strip drips 1-3 μ L colloid gold label respectively, protein concentration is 0.5-5mg/mL, oven dry; At the protein of gold-marking region dropping 10-20 μ L colloid gold label, the protein concentration of colloid gold label is 0.5-5mg/mL; Drip 20-200 μ L example reaction 5-10min to be checked in the glass fibre district, if punctation all appears in detection zone and check plot, sample detection result is positive; Punctation appears in the check plot if detection zone does not have colour developing, and sample detection result is negative; If do not develop the color in the check plot, sample detection result is invalid.
2, according to immune colloidal gold chromatography method according to the described detection food allergy to aquatic products of claim 1, it is characterized in that: with colloid gold label anaphylactogen antibody or anaphylactogen and prepare immune gold test paper strip, be used to detect the immune colloidal gold chromatography kit of food allergy to aquatic products.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043055A (en) * | 2010-10-27 | 2011-05-04 | 上海海洋大学 | Rapid immunomagnetic bead chromatographic method for main allergens in aquatic products |
| CN102636650A (en) * | 2012-03-23 | 2012-08-15 | 沃克(天津)生物科技有限公司 | Milk allergen test plate and preparation method thereof |
| CN103983749A (en) * | 2014-03-25 | 2014-08-13 | 中国海洋大学 | Capillary immunity-chromatography rapid detection method for parvalbumin in aquatic products |
| CN110308274A (en) * | 2019-07-24 | 2019-10-08 | 南京黎明生物制品有限公司 | A kind of kit and preparation method thereof detecting listeria monocytogenes |
| CN110531089A (en) * | 2019-10-10 | 2019-12-03 | 首都医科大学附属北京同仁医院 | The total IgE colloidal-gold detecting-card of nasal discharge, kit and application |
| CN114217072A (en) * | 2021-12-07 | 2022-03-22 | 江南大学 | Colloidal gold immunochromatographic test strip for detecting freshness of fish and preparation and application thereof |
-
2008
- 2008-09-04 CN CNA200810071731XA patent/CN101358981A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043055A (en) * | 2010-10-27 | 2011-05-04 | 上海海洋大学 | Rapid immunomagnetic bead chromatographic method for main allergens in aquatic products |
| CN102636650A (en) * | 2012-03-23 | 2012-08-15 | 沃克(天津)生物科技有限公司 | Milk allergen test plate and preparation method thereof |
| CN102636650B (en) * | 2012-03-23 | 2014-08-13 | 沃克(天津)生物科技有限公司 | Milk allergen test plate and preparation method thereof |
| CN103983749A (en) * | 2014-03-25 | 2014-08-13 | 中国海洋大学 | Capillary immunity-chromatography rapid detection method for parvalbumin in aquatic products |
| CN110308274A (en) * | 2019-07-24 | 2019-10-08 | 南京黎明生物制品有限公司 | A kind of kit and preparation method thereof detecting listeria monocytogenes |
| CN110531089A (en) * | 2019-10-10 | 2019-12-03 | 首都医科大学附属北京同仁医院 | The total IgE colloidal-gold detecting-card of nasal discharge, kit and application |
| CN114217072A (en) * | 2021-12-07 | 2022-03-22 | 江南大学 | Colloidal gold immunochromatographic test strip for detecting freshness of fish and preparation and application thereof |
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