CN105259341A - Sudden death disease molecular probe and preparation method thereof - Google Patents
Sudden death disease molecular probe and preparation method thereof Download PDFInfo
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- CN105259341A CN105259341A CN201510711946.3A CN201510711946A CN105259341A CN 105259341 A CN105259341 A CN 105259341A CN 201510711946 A CN201510711946 A CN 201510711946A CN 105259341 A CN105259341 A CN 105259341A
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- pad
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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
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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
- G01N33/531—Production of immunochemical test materials
Abstract
A sudden death disease molecular probe is characterized by being formed through bonding an ABS gasket, a sample pad, a colloidal gold pad, a nitrocellulose pad, a water absorption pad and a protection pad sequentially, wherein the thickness of the part, stacked and attached to the colloidal gold pad, of the sample pad is 1 mn, the thickness of the part, stacked and attached to the nitrocellulose pad, of the colloidal gold pad is 1 mn, and the thickness, of the part stacked and attached to the nitrocellulose pad, of the water absorption pad is 1 mn. The sudden death disease molecular probe has the advantages that the production process is simple, and the probe is small and portable; samples of oral liquid, nose liquid, tear and urine are taken noninvasively; the detection speed is high, and the test is finished in minutes; monoclonal colloidal gold can be realized, and the qualitative result is accurate and stable; a rapid detection method is provided for rescuing a patient suffering from sudden death disease, and the rescue rate of the patient suffering from the sudden death disease is increased.
Description
Technical field
The present invention relates to a kind of Emergency Medicine inspection technology field, particularly relate to a kind of preparation method of die suddenly molecular probe and sudden death molecular probe.
Background technology
Sudden death disease is disease the most fearful in the world, its incidence of disease exceedes the summation of colorectal cancer, breast cancer, prostate cancer, influenza, pneumonia, traffic accident, HIV, gun case and the lethal number of family fire, the whole world has 1,200 ten thousand people's sudden deaths every year, and China has 1,800,000 people's sudden deaths every year.According to clinical medicine statistics, Sudden Death Syndrome morbidity after, within its hour, case fatality rate reaches 2%, and all case fatality rate reach 50%, the moon case fatality rate reach 75%, year case fatality rate reach 90%, within 10 years, case fatality rate reaches 100%.Research finds, before the Sudden Death Syndrome morbidity of 99%, urine is now died suddenly antigen, and its sensitivity reaches 98%, and specificity reaches 100%.
Study discovery, Ca2+ binding parent's sudden death antigenic complex (troponincomplex), make the head of sudden death antigen ride on actin after formation gap bridge, sudden death antigen A TP enzyme hydrolysis releases energy, and produces tension force, and actin is slided.
Actin+sudden death antigen-ATP → flesh dynamic sudden death antigen+ADP+P1.
Myocardial relaxation, required sufficient ATP activates sudden death antigen A TP enzyme, to recover convergent force.
Flesh dynamic sudden death antigen+ATP → actin+sudden death antigen-ATP.
The tired phosphate produced on blood lactase acid, anoxic, infection activation phosphatidase hydrolysis cardiac muscle cell's membrane phospholipid and sudden death antigen molecule, sudden death antigen is lost activity and enters blood, urine, cardiac muscle cell die suddenly antigen stop delivery actin, macroscopically show cardiac muscle not shrink, sudden death antigen molecule motor disappearance life power causes sudden death disease and occurs.
Sudden death antigen is mainly stored in cardiac muscle cell and Skeletal Muscle Cell, and normal person's urine is not containing sudden death antigen.Cardiac muscle cell and Skeletal Muscle Cell major injury, sudden death antigen inactivation enters blood, and humoral immune reaction appears in body.Sudden death antigen enters urine through renal excretion, meets the aobvious blue reaction of TMB-SNE-AMS.Therefore, antigen of dying suddenly becomes blood, the urine biochemical marker of early stage sudden death disease.
The hurried state of an illness of Sudden Death Syndrome onset is dangerous, recovers slow case fatality rate high, in threat, young people, has become stealthy killer maximum on the job markets such as IT industry.
There is no desirable checkout equipment at present and detect reagent.
Summary of the invention
Object of the present invention, is to provide a kind of sudden death disease molecular probe, uses sudden death disease molecular probe of the present invention fast simple to operate, result accurate stable, testing cost is cheap, is suitable for individual, family and clinic, is of value to timely diagnosis and the rescue of sudden death disease patient.
Another object of the present invention, is to provide a kind of preparation method of sudden death disease molecular probe.
The technical scheme adopted is:
A kind of sudden death disease molecular probe, is characterized in that being made up of ABS liner, sample pad, gold mark pad, folded the sticking together of cellulose nitrate pad, adsorptive pads and neonychium successively.Wherein the folded mark mat thickness of gilding of sample pad is 0.9-1.1mn, and adsorptive pads folded subsides cellulose nitrate mat thickness is 0.9-1.1mn.
Described sample pad is through Tris, the bag of casein-sodium saline solution process is by the glass fibre of ProteinA element film, described gold mark pad is through Tris, the bag of bsa solution process is died suddenly by mouse-anti people the glass fibre membrane of antigen monoclonal antibody-colloidal gold composite, the detection zone of described cellulose nitrate pad is that bag is by the T line of specific genetic recombination sudden death antigen, the quality control region of described cellulose nitrate pad is that bag is by the C line of sheep anti-mouse igg polyclonal antibody, the sudden death antigen amount of described T line bag quilt is higher than can cross-linking antibody is combined in gold mark pad sudden death antigen amount in sample liquid.
A preparation method for sudden death disease molecular probe, comprises the steps:
1), prepare mouse-anti people to die suddenly antigen monoclonal antibody: die suddenly antigen for antigen with people, intrasplenic injection immunized mice, get immune mouse spleen cell and myeloma cell fusion prepares hybridoma, then hybridoma is inoculated in mouse abdominal cavity prepares ascites, extract mouse-anti people and to die suddenly antigen monoclonal antibody;
2), sheep anti-mouse igg polyclonal antibody is prepared: to die suddenly antigen monoclonal antibody immune goat with mouse-anti people, get serum through centrifugal obtained sheep anti-mouse igg polyclonal antibody;
3), prepare collaurum: get 0.01%HAuCl4 aqueous solution 100ml, add 1% citric acid three sodium water solution 0.75ml, heating is boiled and is taken on a red color to solution, is cooled to 4 DEG C, adds 0.1Mol/LK2CO30.5ml and make 50nm collaurum;
4), prepare mouse-anti people to die suddenly antigen monoclonal antibody-colloidal gold composite: adjust colloidal gold solution pH value to 8.2 with 0.1M sal tartari, in the ratio of 0.02mgIgM/mL collaurum add mouse-anti people die suddenly antigen monoclonal antibody mixing, high speed centrifugation abandons supernatant, obtained mouse-anti people dies suddenly antigen monoclonal antibody-colloidal gold composite, puts in the bovine serum albumin(BSA) of 5%, 1% polyglycol, 0.02MTris-HCl damping fluid and saves backup;
5), preparation sample pad: by ProteinA pH value be 8.2 2% trehalose and 0.02Mol/LPBS liquid to be diluted to after 1mg/mL even application at glass fibre element film, fully dry obtained sample pad;
6), preparation gold mark pad: antigen monoclonal antibody-colloidal gold composite of mouse-anti people being died suddenly evenly is coated on glass fibre membrane, fully dry obtained gold mark pad;
7), cellulose nitrate pad is prepared: the quality control region and the detection zone that sheep anti-mouse igg polyclonal antibody and restructuring sudden death disease antigen are coated on respectively nitrocellulose filter, fully dry obtained cellulose nitrate pad;
8) sudden death disease molecular probe, is prepared: be attached on ABS liner successively by sample pad, gold mark pad, cellulose nitrate pad and adsorptive pads, sample pad folded gold mark pad 1mn, the folded cellulose nitrate pad 1mn of gold mark pad, adsorptive pads is folded cellulose nitrate pad 1mn and is obtained sudden death disease molecular probe, to obtain final product.
Advantage of the present invention is:
(1) production technology is simple, and probe is small and exquisite portable;
(2) without earning disease sample: oral fluid, nose liquid, tear, urine;
(3) detection speed is fast, within 5 minutes, completes test;
(4) monoclonal gold mark, qualitative results accurate stable;
The present invention provides a kind of method for quickly detecting for rescuing sudden death disease patient, improves the successful rescue probability of sudden death patient.
Accompanying drawing explanation
Fig. 1 is sudden death disease molecular probe decomposition texture schematic diagram of the present invention.
Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 and Fig. 7 are sudden death disease molecular probe overhaul flow chart.
Fig. 8, Fig. 9, Figure 10 and Figure 11 are sudden death molecular probe testing result display figure of the present invention.
Embodiment
Embodiment one
A kind of sudden death disease molecular probe, is characterized in that being folded to stick together formed by ABS liner 1, sample pad 2, gold mark pad 3, cellulose nitrate pad 4, adsorptive pads 5 and neonychium 6 successively.Wherein, the folded mark pad of gilding of sample pad 2 is thick is 1mn, and gold mark pad folded subsides cellulose nitrate pad is thick is 1mn, and it is 1mn that adsorptive pads is folded cellulose nitrate pad thick.
Gold mark pad bag to be died suddenly antigen monoclonal antibody-colloidal gold composite by mouse-anti people, and cellulose nitrate pad detection zone bag is by specific genetic recombination sudden death antigen, and cellulose nitrate pad quality control region bag is by sheep anti-mouse igg polyclonal antibody.
Embodiment two
A kind of sudden death disease molecular probe and preparation method thereof, its structure is substantially identical with embodiment one, its difference be the folded mark pad of gilding of sample pad 2 thick be 0.9mn, gold mark pad is folded, and to paste cellulose nitrate pad thick be 0.9mn, and adsorptive pads folded subsides cellulose nitrate pad is thick is 0.9mn.
Embodiment three
A kind of sudden death disease molecular probe and preparation method thereof, its structure is substantially identical with embodiment one, its difference be the folded mark pad of gilding of sample pad 2 thick be 1.1mn, gold mark pad is folded, and to paste cellulose nitrate pad thick be 1.1mn, and adsorptive pads folded subsides cellulose nitrate pad is thick is 1.1mn.
Fig. 2-Fig. 7 shows sudden death disease molecular probe detection method of the present invention: with cotton swab sampling liquid or hand-held sudden death disease molecular probe, sample pad is dipped vertically into sample liquid 1 second, within 5 minutes, qualitatively judges and detect sudden death disease result.If all aobvious red tape of C line, T line, prompting sudden death disease risk; If C line shows red tape, the not aobvious red tape of T line, then prompting is without sudden death disease risk; If the not aobvious red tape of C line, then point out sudden death disease molecular probe invalid.
Embodiment four
Fig. 8-Figure 11 shows sudden death disease molecular probe Cleaning Principle of the present invention: during detection, sudden death antigen in sample liquid and gold mark mouse-anti people antigen monoclonal antibody of dying suddenly and are combined and form antigen antibody complex, when flowing to the T district of cellulose nitrate pad, the restructuring sudden death antigen specific antigen be fixed on cellulose nitrate pad catches this compound be aggregated into a macroscopic red T line gradually, and unconjugated gold mark mouse-anti people antigen monoclonal antibody of dying suddenly flows through T district and formed macroscopic red C line by many anti-the catching of the sheep anti mouse in C district.Display C line then shows that sudden death disease molecular probe is effective; Display T line then shows in liquid sample containing sudden death disease antigen.
Result judges: if there is sudden death disease antigen in sample liquid, then all aobvious red stripes of nature controlling line, detection line, and result is positive; If there is not sudden death disease antigen in sample liquid, then only nature controlling line shows red stripes; As nature controlling line, place has no band, then sudden death disease molecular probe is invalid.
Claims (5)
1. a sudden death disease molecular probe, is characterized in that being made up of ABS liner (1), sample pad (2), gold mark pad (3), cellulose nitrate pad (4), adsorptive pads (5) and folded the sticking together of neonychium (6) successively; Wherein, the folded mark pad of gilding of sample pad (2) is thick is 1mn, and gold mark pad folded subsides cellulose nitrate pad is thick is 1mn, and it is 1mn that adsorptive pads is folded cellulose nitrate pad thick.
2. a kind of sudden death disease molecular probe according to claim 1 and preparation method thereof, it is characterized in that: the folded mark pad of gilding of sample pad (2) is thick is 0.9mn, it is 0.9mn that gold is marked pad folded subsides cellulose nitrate pad thick, and adsorptive pads folded subsides cellulose nitrate pad is thick is 0.9mn.
3. a kind of sudden death disease molecular probe according to claim 1 and preparation method thereof, it is characterized in that: the folded mark pad of gilding of sample pad (2) is thick is 1.1mn, it is 1.1mn that gold is marked pad folded subsides cellulose nitrate pad thick, and adsorptive pads folded subsides cellulose nitrate pad is thick is 1.1mn.
4. a kind of sudden death disease molecular probe according to claim 1 and preparation method thereof, it is characterized in that: described sample pad is through Tris, the bag of casein-sodium saline solution process is by the glass fibre of ProteinA element film, described gold mark pad is through Tris, the bag of bsa solution process is died suddenly by mouse-anti people the glass fibre membrane of antigen monoclonal antibody-colloidal gold composite, the detection zone of described cellulose nitrate pad is that bag is by the T line of specific genetic recombination sudden death antigen, the quality control region of described cellulose nitrate pad is that bag is by the C line of sheep anti-mouse igg polyclonal antibody, the sudden death antigen amount of described T line bag quilt is higher than can cross-linking antibody is combined in gold mark pad sudden death antigen amount in sample liquid.
5. the preparation method of a kind of sudden death disease molecular probe according to claim 1, is characterized in that: comprise the steps:
1), prepare mouse-anti people to die suddenly antigen monoclonal antibody: die suddenly antigen for antigen with people, intrasplenic injection immunized mice, get immune mouse spleen cell and myeloma cell fusion prepares hybridoma, then hybridoma is inoculated in mouse abdominal cavity prepares ascites, extract mouse-anti people and to die suddenly antigen monoclonal antibody;
2), sheep anti-mouse igg polyclonal antibody is prepared: to die suddenly antigen monoclonal antibody immune goat with mouse-anti people, get serum through centrifugal obtained sheep anti-mouse igg polyclonal antibody;
3), prepare collaurum: get 0.01%HauCl4 aqueous solution 100ml, add 1% citric acid three sodium water solution 0.75ml, heating is boiled and is taken on a red color to solution, is cooled to 4 DEG C, adds 0.1Mol/LK2CO30.5ml and make 50nm collaurum;
4), prepare mouse-anti people to die suddenly antigen monoclonal antibody-colloidal gold composite: adjust colloidal gold solution pH value to 8.2 with 0.1M sal tartari, in the ratio of 0.02mgIgM/mL collaurum add mouse-anti people die suddenly antigen monoclonal antibody mixing, high speed centrifugation abandons supernatant, obtained mouse-anti people dies suddenly antigen monoclonal antibody-colloidal gold composite, puts in the bovine serum albumin(BSA) of 5%, 1% polyglycol, 0.02MTris-HCl damping fluid and saves backup;
5), preparation sample pad: by ProteinA pH value be 8.2 2% trehalose and 0.02Mol/LPBS liquid to be diluted to after 1mg/mL even application at glass fibre element film, fully dry obtained sample pad;
6), preparation gold mark pad: antigen monoclonal antibody-colloidal gold composite of mouse-anti people being died suddenly evenly is coated on glass fibre membrane, fully dry obtained gold mark pad;
7), cellulose nitrate pad is prepared: the quality control region and the detection zone that sheep anti-mouse igg polyclonal antibody and restructuring sudden death disease antigen are coated on respectively nitrocellulose filter, fully dry obtained cellulose nitrate pad;
8) sudden death disease molecular probe, is prepared: be attached on ABS liner successively by sample pad, gold mark pad, cellulose nitrate pad and adsorptive pads, sample pad folded gold mark pad 1mn, the folded cellulose nitrate pad 1mn of gold mark pad, adsorptive pads is folded cellulose nitrate pad 1mn and is obtained sudden death disease molecular probe, to obtain final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510711946.3A CN105259341A (en) | 2015-10-28 | 2015-10-28 | Sudden death disease molecular probe and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510711946.3A CN105259341A (en) | 2015-10-28 | 2015-10-28 | Sudden death disease molecular probe and preparation method thereof |
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| CN105259341A true CN105259341A (en) | 2016-01-20 |
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| CN201510711946.3A Pending CN105259341A (en) | 2015-10-28 | 2015-10-28 | Sudden death disease molecular probe and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175070A (en) * | 2020-10-25 | 2021-01-05 | 巴德生物科技有限公司 | Method for preparing monoclonal antibody for rapid immunization |
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| CN101432626B (en) * | 2006-04-04 | 2013-06-19 | 神谷来克斯公司 | Highly sensitive system and methods for analysis of troponin |
| CN103173420A (en) * | 2013-04-08 | 2013-06-26 | 深圳市菲鹏生物股份有限公司 | Hybridoma cell capable of secreting anti-cardiac troponin I monoclonal antibodies and applications thereof |
| CN203101403U (en) * | 2013-01-22 | 2013-07-31 | 济南卓冠生物技术有限公司 | Kit for quickly and quantitatively detecting troponin I by immunochromatography |
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2015
- 2015-10-28 CN CN201510711946.3A patent/CN105259341A/en active Pending
Patent Citations (5)
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| CN101432626B (en) * | 2006-04-04 | 2013-06-19 | 神谷来克斯公司 | Highly sensitive system and methods for analysis of troponin |
| CN203101403U (en) * | 2013-01-22 | 2013-07-31 | 济南卓冠生物技术有限公司 | Kit for quickly and quantitatively detecting troponin I by immunochromatography |
| CN103173420A (en) * | 2013-04-08 | 2013-06-26 | 深圳市菲鹏生物股份有限公司 | Hybridoma cell capable of secreting anti-cardiac troponin I monoclonal antibodies and applications thereof |
| CN104237537A (en) * | 2014-10-21 | 2014-12-24 | 南京基蛋生物科技有限公司 | Immune chromatography fluorescence reagent strip for detecting cardiac troponin and preparation method thereof |
| CN104991077A (en) * | 2015-07-29 | 2015-10-21 | 安徽省煦棠医疗科技有限公司 | Troponin I competition turbidimetry detecting kit |
Non-Patent Citations (1)
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| 李碧珊: "联合测定血清中的肌钙蛋白I/CK-MB/肌红蛋白的临床意义", 《中国实用医药》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175070A (en) * | 2020-10-25 | 2021-01-05 | 巴德生物科技有限公司 | Method for preparing monoclonal antibody for rapid immunization |
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