TH13664C3 - A polyclonal antibody specific to the genotype. - Google Patents

Abstract

The invention is the creation of polyclonal antibodies (PAbs) that are specific to genotype, with the aim of applying polyclonal antibodies to the G-year analysis method. No Xyrid fortyine and saponins produced in Panchkhan By means of immunological analysis (Immunoassay), such as the Elisa method, which allows the analysis of genesides, fortinines, and steroid saponins, which are Important substances contained in Panchakhan at a glance Which polyclonal antibodies used to detect important substances in herbs This method is highly sensitive and reduces sample preparation time for analysis. Due to the polyclonal The antibodies used for the analysis were created to be specific to the substance to be analyzed. Therefore can be used to Can be analyzed in terms of qualitative, analytical and analytical quantities. Quickly and accurately

Claims (6)

Disclaimer (all) which will not appear on the advertisement page: Revised 15/5/2017 1. The production of polyclonal antibodies specific to the geneside. It consists of the following steps: a) Synthesize the conjugate of GPenosides Fortynine with BSA. By using a standard 5 mg gpenosides fortyynine, add 0.5 mg of methanol, then add a solution of periodate milk (NaIO4) (5 mg in 0.5 mg of water). One drop at a time, mixed with a magnetic stick for stirring at 25 ° C for 1 hour, then add 10.0 mg of BSA solution in 50 mmolar PBS buffer (pH 9.5-9.7). 2.0 ml (using the genetics ratio Sized fortyine and BSA 1: 2) were mixed to 25 ° C for 6 h. The resulting solution was dosed in distilled water 5 times at temperature. 4 ° C and lyophilize, Gpinoside Fotynine conjugated with BSA in the 7-12 mg range. B) Rabbit immunization. At least 3 times every 20 days, with 1: 1 injection of immunomodulators from incomplete frostbite and Gpenosides photonine connected to BS. A 0.4 mg / ml Subcutaneous injection, the second time IFS is used as an incomplete frost stimulator. In place of complete frostbite stimulants, injected intramuscularly, then 3 days, check for specific immune substances. Gepenosides by means of Enzyme Lingetommunoser Ban Essay or Elisa, which uses a specific binding principle. Of antigens and antibodies The third or fourth stimulation was administered with a 0.2 mil g / ml BSA-linked Gpinoside Fortinine, dissolved in 1 mL of 7.0-7.5 phosphate buffered saline pH. After 3 days, the antibody concentration was tested by the eliza method after the 3rd or 4th stimulation. C) Antibody extraction. By taking the blood of the rabbit from the heart blood draw is about 30-50 ml per rabbit. Bring the blood to spin at a speed of 2,000-3,000 rpm, separate the upper part or the serum and purify. Using a G protein column The substance was separated with a low pH buffer, dialized at a temperature in the range of 4-8 ° C and lyophilized to get polyclonal. Purified Antibody 2. A process for producing polyclonal antibodies specific to genotype. According to claim 1, where the conjugate synthesis of GPenosides Fortynine with BSA Can be replaced by the conjugate synthesis of G. Pinosai Forty Nine with HSA It was changed from the addition of a BSA solution to an HSA solution. 3. The production process of polyclonal antibodies specific to the gpenosides. According to claim 1, where the conjugate synthesis of GPenosides Fortynine with BSA Can be replaced by conjugate synthesis Of Gpinosides Fortynine and OVA By changing from adding a BSA solution to an OVA solution 4. The use of polyclonal antibodies that are specific to the genotype. According to any one of the 1 to 3 claims By means of immunohistochemistry for the analysis of anabolic steroid saponins with a structure similar to the Gpinoside Four (original revised). / 2016) 1. Polyclonal antibodies that are specific to the genotype. The production process is as follows: a) Synthesize the conjugate of GPenosides Fortynine with BSA. Or synthesize the conjugate of the gepino Said-Fortinine with HSA Or synthesize the conjugate of gpenosides fortyine and OVA b) stimulating immunity to rabbits at least 3 times every 20 days, with the first time preparing an injection for immunotherapy, ratio 1 : 1 From the incomplete freoid stimulant and the Gpinoside Fortinine connected to BSA 0.4 mg / ml. The second subcutaneous injection uses IFS, an incomplete freoid stimulant. Instead of a complete freoid stimulant, injected intramuscularly, then 3 days, check for specific immune substances. Gepenosides by means of Enzyme lingate immunosorbent essay or Elisa, which uses a specific binding principle. Of antigens and antibodies The third or fourth stimulation was administered with a 0.2 mg / ml BSA-linked Gpinosides of 0,2 mg / mL dissolved in 1 mL of 7.0-7.5 phosphate, baphosphate, intravenous. After 3 days, the antibody concentration was tested by the eliza method after the 3rd or 4th injection. C) The antibody purification. By taking the blood of the rabbit from the heart blood draw will be about 30-50 milliliters of blood per rabbit. Bring the blood to spin at a speed of 2,000-3,000 rpm, separate the upper part or the serum and purify. Using a G protein column The substance was separated with a low pH buffer, dialized at a temperature in the range of 4-8 ° C and lyophilized to get polyclonal. Purified antibodies 2. Polyclonal antibodies that are specific to genome compounds. According to claim 1, where the conjugate synthesis Of G Pinosai Forty Nine with BSA By using the standard 5 mg gpenosides fortyynine, add 0.5 ml of methanol and then add sodium periphase (NaIO4) solution (5 mg in 0.5 ml of water) to the Each drop is mixed with a magnetic stick for stirring at 25 ° C for 1 hour, then add 10.0 mg of BSA solution in 50 mmolar PBS (pH 9.5-9.7) 2.0 buffer. Milliliter (using the ratio of Gpinoside Fortinine with BSA 1 to 2) and mix well at 25 ° C for 6 hrs. In 5 times distilled water at a temperature of 4 ° C and the lyophilize (Iyophilize), the conjugate of Gpinoside Fotynine with BSA in the range of 7-12 mg. A leclonol antibody specific to the genotype. According to one of the claims 1 to 2, where the conjugate synthesis of Gpinosides Fortynine with HSA By changing the addition of BSA solution It is an HSA solution. 4. Polyclonal antibodies that are specific to the genotype. According to one of the claims 1 to 3, where the conjugate synthesis of Gpinosides Fortynine with OVA By changing the addition of BSA solution It is an OVA solution. 5. The use of polyclonal antibodies that are specific to the genes According to any one of the 1 to 4 claims By means of immunohistochemistry for the analysis of anabolic steroids, saponins with a structure close to the genesis four -------------- --------------------------- 1. Polyclonal antibodies that are specific to the genotype. Which has the ability to bind specifically to G-year substances Nozeid four and steroid sapo ninjakhan in the aglycone area. 2. Process for producing polyclonal antibodies that are specific to the genes. Claim 1 consists of the following steps: a) Synthesize the conjugate of Gypenoside fortyine with BSA (Gypenoside XLIX-BSA conjugates) or synthesize the Gypeno conjugate. Xyrotinine with HSA (Gypenoside XLIX-HSA conjugates) or synthesize the conjugate of Gypenoside fortyine and OVA (Oval Albumin: OVA) b. ) Stimulation of the immune system Immunization was performed with rabbits at least 3 times every 20 days. The first time was a 1: 1 immunization agent from CFS (Complete Freund \ 's adjuvant; CFA) and Gpinoside Four. Tinine connected to BSA 0.4 mg / ml. Incomplete Freund \ 's adjuvant; IFA (IFA) instead of CFS, intramuscular injection, then 3 days to check for specific immune substances. GPenosides by Enzyme linked immunosorbant assay (ELISA) method, which uses the method of coagulation. Specificity of antigens and antibodies 3rd or 4th stimulation with Gepenoside fibrinine at It was connected to BSA 0.2 mg / ml dissolved in 1 ml of phosphate, buffersaline, pH 7.0-7.5, intravenously in the auricle, then 3 days. Antibody concentration was tested by eliza after injection. 3rd or 4th time c) Purification of antibodies By taking the blood of the rabbit from the heart blood draw will be about 30-50 milliliters of blood per rabbit. Bring the blood to spin at a speed of 2,000-3,000 rpm, separate the upper part or the serum and purify. Column protein G was used and separated with a low pH buffer. Dialized at temperatures in the range of 4-8 degrees Celsius and lyophilized. Will get a pure polyclonal antibody 3. The production process of polyclonal antibodies that is specific to the genotype. According to claim 2, where the conjugate synthesis of Gpinosides Fortynine with BSA By using the standard 5 mg gepenosides fortyynine, add 0.5 ml of methanol, then add sodium periphase (NaIO4) solution (5 mg in 0.5 ml of water) one by one. The drops are mixed with a magnetic stirrer at 25 ํ C for 1 hour, then add 10.0 mg of BSA solution in 50 mmolar PBS (pH) buffer. 9.5-9.7) 2.0 ml (using the ratio of Gpinoside Fortinine with BSA 1: 2) and mix it into The resulting solution was dialyzed in 5 times of distilled water at 4 ° C and the lyophilized (Iyophilize) was conjugated. Gates of Gpinoside Fotynine with BSA in the range of 7-12 mg. 4. The production process of polyclonal antibodies that is specific to the genotype. According to any of the 2 to 3 claims, where The conjugate synthesis of Gpinosides Fortynine with HSA. By changing from filling BSA solution is a HSA solution. 5. The production process of polyclonal antibodies that is specific to the genotype. According to any of the 2 to 3 claims, where the conjugate synthesis of Gpinosides Fortynine with OVA By changing from filling BSA solution is an OVA solution. 6. The use of polyclonal antibodies that are specific to the genotype. According to any one of the 1 to 5 claims By means of immunohistochemistry for the analysis of anabolic steroid saponins with a structure similar to that of the genus four.