CN104483486A - Kit for identifying deer antlerblood slide - Google Patents
Kit for identifying deer antlerblood slide Download PDFInfo
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- CN104483486A CN104483486A CN201410804555.1A CN201410804555A CN104483486A CN 104483486 A CN104483486 A CN 104483486A CN 201410804555 A CN201410804555 A CN 201410804555A CN 104483486 A CN104483486 A CN 104483486A
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
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- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an application of a deer nerve factor serving as an index for identifying an antlerblood slide, and further discloses a kit for identifying a deerantlerblood slide. Firstly, a rabbit polyclonal antibody of the deer nerve factor is prepared and taken as a capture antibody and well coats an elisa plate; a monoclonal antibodyof the deer nerve factor is further prepared and taken as a detecting antibody; and a corresponding enzyme labeled second antibody and substrate are well prepared, and a kit is prepared for standby application. The kit for identifying the deer antlerblood slide is an ELISA kit and comprises the elisa plate coated with the polyclonal antibody of the deer nerve factor, the detecting antibody, the labeled second antibody, a developing substrate, a terminating agent, a sample dissolving solution, a washing solution and a standard substance; the elisa plate, the labeling second antibody, the developing substrate, the terminating agent and the washing solution are all commercial standard products, and the polyclonal antibody of the deer nerve factor, the detecting antibody (a rat anti-deer nerve factormonoclonal antibody) and the standard product (the deer nerve factor) are specially prepared for the kit.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a kind of kit differentiating antler blood sheet.
Background technology
Current, because velvet product price is higher, especially the highest with XUERONG PIAN price best in quality, velvet product market has occurred a large amount of counterfeit and shoddy goods, all kinds of counterfeit and shoddy goods even occupy the pilose antler piece market of more than 90%.Current main employing is observed, the Genetic identification method of microexamination and PCR-based technology to be to detect all kinds of counterfeit and shoddy goods, for non-deer source product, be easier to identify containing the pilose antler piece of false deer blood, but for the more difficult difference of imitative product of the fine and soft matrix of vacation true deer blood.In addition, also have some to be adopt to make without blood pilose antler matrix coating deer blood really in imitative product, although cannot distinguish with XUERONG PIAN, there is bigger difference in its active component and real blood young pilose antler.Current discriminating means cannot distinguish the pseudo-sheet of this type of XUERONG PIAN.
The regeneration of annual pilose antler, except bone tissue, some supporting tissues such as nerve also can regenerate.The neural speed of growth is about 1cm/d.The mushroom mechanism of current control deer nerve it be unclear that.But the semiquantitative RT-PCR such as Garcia have detected the relative expression of NT-3 (neurotrophin-3) mRNA in the different tissues of pilose antler top, find the highest in the expression of the epidermal area at pilose antler top, and minimum in cartilage layers, this result is just in time consistent with the Substance P situation of these tissues.The extracting and developing from freeze-drying pilose antler of sika such as Huo Yushu obtains the protein and peptide class component of molecular weight more than 10000, through application cell regulatory factor activity determination method, finds that this component has the effect of nerve growth factor increment.Contemporary scientific research confirms the space-time characterisation that nerve growth factor distributes substantially, indicates the deer nerve growth factor of proprietary higher level in pilose antler, and has the trend proportional with quality of pilose antler.Currently there is no any this index that utilizes to carry out the report of pilose antler product quality qualification.
Summary of the invention
In existing detection means, substantially differentiate according to form, chromaticness, smell and hereditary capacity, the counterfeit and shoddy goods of most of type can be detected, but cannot differentiate for the combined false making mode in deer source, especially with really without the XUERONG PIAN that blood pilose antler matrix coating deer blood is made, be real from outward appearance to hereditary capacity, but compared with real XUERONG PIAN, its active component content is very low, does not reach the result of use of expectations of customers.Also there is not effective detection means to differentiate this type of fake products at present.The present invention select XUERONG PIAN a core active factor---deer neural factor is as Testing index, and made identification kit as an effective detection means to true and false XUERONG PIAN using this, in preliminary examination inspection experiment, fake products recall rate reaches 100%.
For solving the discriminating problem of XUERONG PIAN, the present invention have selected a core active factor of XUERONG PIAN: deer neural factor as Testing index, and makes kit as an effective detection means to true and false XUERONG PIAN using this.
The invention discloses deer neural factor as the application differentiating antler blood sheet index.
The invention also discloses a kind of kit differentiating antler blood sheet: how anti-the rabbit source of first having prepared deer neural factor is as capture antibody, and bag is by good ELISA Plate; The monoclonal antibody of further preparation deer neural factor is as detection antibody; Prepare corresponding enzyme labeling two to resist and substrate, be made into kit for subsequent use.
The kit of discriminating antler blood sheet of the present invention is ELISA kit, comprises the how anti-ELISA Plate of quilt deer neural factor, detection antibody, mark two anti-, chromogenic substrate, terminator, sample dissolution liquid, cleansing solution, standard items; Wherein, ELISA Plate material, mark two are anti-, chromogenic substrate, terminator, cleansing solution are commercialization standardized product, deer neural factor many anti-, detect antibody (mouse-anti deer neural factor monoclonal antibody), standard items (deer neural factor) and prepare specially for the present invention.
Kit of the present invention solves the qualification problem that background technology cannot solve the similar counterfeit of genetic background technically, by choosing for the very strong deer neural factor of the fine and soft specificity of blood as Testing index item, and adopt the highly sensitive immunodiagnosis kit mode of current main-stream to detect, detection efficiency also improves greatly, same batch can be detected more than 80 sample simultaneously, and consuming time less, complete every batch of detection and only need 4-5 hour.
In preliminary examination inspection experiment, fake products recall rate reaches 100%.
Embodiment
The present invention have selected the special core active factor of an XUERONG PIAN: deer neural factor is as Testing index, by making conventional ELISA kit, comprise the how anti-ELISA Plate of quilt deer neural factor, detection antibody, mark two anti-, chromogenic substrate, terminator, cleansing solution, standard items etc., achieve the effective detection means differentiating the XUERONG PIAN true and false.
The present invention first separation and purification obtains deer neural factor antigen: dilute fresh antler blood → 2 with 1%HAc solution 1:5, and 000rpm/min low-temperature centrifugation → supernatant Sephadex G-50 post roughing out → Peak Activity is collected liquid and obtained antigen with pH6.0 phosphate buffer dialysis → CM-Sepharose FF post repurity → Sephacryl S-200 column chromatographic isolation and purification.Deer neural factor after part purifying makes standard items through dilution.Then, the rabbit source of having prepared deer neural factor further resists more: animal immune (new zealand white rabbit) → Elisa bioactivity (or WB detectable antigens) → antiserum → recombinant protein A column separating purification; Prepare the mouse source monoclonal antibody of deer neural factor the same period: the batch production of the hybridoma cell clone → monoclonal antibody of the confirmation → secretory antibody of deer neural factor antigen sensibilization bone-marrow-derived lymphocyte (immune animal) → Hybridoma preparation, screening → hybridoma secretory antibody.Finally, the collocation assembly kit of the present invention of ELISA kit routinely.
The embodiment of concrete enforcement kit of the present invention:
1. make by oneself 5 XUERONG PIAN samples, 5 be coated with inferior piece of antler (without blood piece of antler) sample and collection 20 commercial samples with deer blood, ox blood, pig blood, sheep blood, chicken blood respectively, through cryogrinding, add sample lysate (PBST) 5mL/g respectively, hatch 2 hours for 37 DEG C, centrifuging and taking supernatant;
2. the leaching liquor 0.1ml adding measuring samples, in the reacting hole of coated elisa plate, puts 37 DEG C and hatches 1 hour.Then wash, blank well, negative control hole and Positive control wells are set simultaneously.
3., in each reacting hole, hatch 0.5 ~ 1 hour for 37 DEG C after adding detection antibody, washing, then add ELIAS secondary antibody 0.1ml.Hatch 0.5 ~ 1 hour for 37 DEG C, washing.Then add Chromogenic Substrate Solution 0.1ml, 37 DEG C 10 ~ 30 minutes; Finally in each reacting hole, add stop buffer 0.05ml.
6. result judges: in white background, directly detect by an unaided eye result: according to color that is blank and negative control hole, the reacting hole that color is darker, positive degree is stronger, what color was more shallow than blank and negative control hole is then judged to be feminine gender, according to be the depth of color, represent with "+", "-" number.Further employing microplate reader measures, and to survey each hole OD value after blank or negative control hole zeroing, if be greater than 2.1 times of the negative control OD value of regulation, is namely judged to the positive.In these 30 samples detected, homemade 10 sample detection results meet the truth of sample completely, and 5 homemade personation samples are detected as feminine gender entirely; In collected 20 commercial samples, 6 is positive, and all the other are negative, and positive rate is 30%.
Claims (2)
1. deer neural factor is as the application differentiating antler blood sheet index.
2. differentiate the kit of antler blood sheet for one kind, it is characterized in that: be ELISA kit, comprise the how anti-ELISA Plate of quilt deer neural factor, detection antibody, mark two anti-, chromogenic substrate, terminator, sample dissolution liquid, cleansing solution, deer neural factor standard items.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410804555.1A CN104483486A (en) | 2014-12-22 | 2014-12-22 | Kit for identifying deer antlerblood slide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410804555.1A CN104483486A (en) | 2014-12-22 | 2014-12-22 | Kit for identifying deer antlerblood slide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104483486A true CN104483486A (en) | 2015-04-01 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410804555.1A Pending CN104483486A (en) | 2014-12-22 | 2014-12-22 | Kit for identifying deer antlerblood slide |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051589A (en) * | 2017-12-06 | 2018-05-18 | 扬州大学 | A kind of rapid detection card for differentiating pilose antler Blood piece |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003076929A2 (en) * | 2002-03-11 | 2003-09-18 | Ca Minister Agriculture & Food | Method for the evaluation of velvet antler |
| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
| CN101020715A (en) * | 2007-03-15 | 2007-08-22 | 王利忠 | Process of extracting and preparing deer nerve growth factor (DEER NGF) |
| CN101392292A (en) * | 2007-10-30 | 2009-03-25 | 大韩民国食品医药品安全厅 | Method for distinguishing between antler and horn of rein deer using real time pcr |
| CN102645542A (en) * | 2012-04-20 | 2012-08-22 | 苏州大学 | BDNF enzyme-linked immunoassay kit |
| CN103424546A (en) * | 2012-05-18 | 2013-12-04 | 苏州市红冠庄国药饮片有限公司 | Specific identification method for biological active components in deer leather gel |
-
2014
- 2014-12-22 CN CN201410804555.1A patent/CN104483486A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003076929A2 (en) * | 2002-03-11 | 2003-09-18 | Ca Minister Agriculture & Food | Method for the evaluation of velvet antler |
| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
| CN101020715A (en) * | 2007-03-15 | 2007-08-22 | 王利忠 | Process of extracting and preparing deer nerve growth factor (DEER NGF) |
| CN101392292A (en) * | 2007-10-30 | 2009-03-25 | 大韩民国食品医药品安全厅 | Method for distinguishing between antler and horn of rein deer using real time pcr |
| CN102645542A (en) * | 2012-04-20 | 2012-08-22 | 苏州大学 | BDNF enzyme-linked immunoassay kit |
| CN103424546A (en) * | 2012-05-18 | 2013-12-04 | 苏州市红冠庄国药饮片有限公司 | Specific identification method for biological active components in deer leather gel |
Non-Patent Citations (5)
| Title |
|---|
| R L GARCIA, ET AL.: "Expression of neurotrophin-3 in the growing velvet antler of the red deer Cervus elaphus.", 《JOURNAL OF MOLECULAR ENDOCRINOLOGY》 * |
| 潘思羽: "冻鲜马鹿茸蛋白成分的分离与活性初探", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 潘思羽: "冻鲜马鹿茸蛋白成分的分离与活性初探", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 12, 15 December 2010 (2010-12-15) * |
| 郝林琳，等: "鲜马鹿茸不同部位多肽的提取及含量比较", 《吉林农业大学学报》 * |
| 闫永红，主编: "《中药鉴定学》", 31 July 2013, 中国医药科技出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051589A (en) * | 2017-12-06 | 2018-05-18 | 扬州大学 | A kind of rapid detection card for differentiating pilose antler Blood piece |
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Application publication date: 20150401 |