TH13664U - Polyclonal antibodies specific to gypenoside. - Google Patents

Abstract

This invention involves the creation of polyclonal antibodies (PAbs) specific to gypenosides. The objective is to utilize these polyclonal antibodies for the immunoassay (e.g., ELISA) of gypenoside fortenine and saponins produced in *Panchakhant* (a traditional Thai herbal medicine). This allows for rapid analysis of gypenoside fortenine and steroid saponins, key compounds in *Panchakhant*. The polyclonal antibodies used for this analysis offer high sensitivity and accuracy, significantly reducing sample preparation time. Because the polyclonal antibodies are specifically formulated for each compound, they can be used for both rapid and accurate qualitative and quantitative analysis.

Claims (6)

Revised 15/5/2017 1. Procedure for producing polyclonal antibodies specific to gypenoside, consisting of the following steps: a) Synthesize conjugate of gypenoside fortenine with BSA by using 5 mg of gypenoside fortenine standard, adding 0.5 mg of methanol, then adding sodium meriodate (NaIO4) solution (5 mg in 0.5 mg water) drop by drop, mixing with a magnetic stirrer at 25°C for 1 hour. Then add 10.0 mg of BSA solution in 2.0 ml of 50 mmol PBS buffer (pH 9.5-9.7) (using the ratio between gypenoside fortenine standard and BSA solution). b) Immunostimulation of rabbits was performed at least three times every 20 days. The first dose involved a 1:1 ratio of incomplete frothed IFA to gypenoside 49 linked with BSA (0.4 mg/ml), injected subcutaneously. The second dose used an incomplete frothed IFA instead of a complete frothed IFA, injected intramuscularly. After three days, specific antibodies against gypenoside were detected using the Enzyme Linked Ammonosorbate Sesame (ELISA) method, which utilizes specific binding principles. For the antigen and antibody, a third or fourth booster injection is administered intravenously via the ear intravenously using 1 ml of 0.2 mg/ml gypenoside 49 bound to BSA dissolved in phosphate-buffered saline (pH 7.0-7.5). After 3 days, the antibody concentration is tested using the ELISA method. c) Antibody purification: Approximately 30-50 ml of rabbit blood (obtained from the heart) is collected per rabbit. The blood is centrifuged at 2,000-3,000 rpm, separating the supernatant (serum). This serum is then purified using a protein G column and separated with a low-pH buffer. Diallysis is performed at a temperature of 4-8°C, and lyophilization is performed to obtain purified polyclonal antibodies. 2. Method for producing polyclonal antibodies specific to gypenoside. According to Patent 1, the synthesis of gypenoside fortenine conjugate with BSA can be replaced by the synthesis of gypenoside fortenine conjugate with HSA by changing the addition of BSA solution to HSA solution. 3. The method for producing polyclonal antibodies specific to gypenosides, according to Patent 1, where the synthesis of gypenoside fortenine conjugate with BSA can be replaced by the synthesis of gypenoside fortenine conjugate with OVA by changing the addition of BSA solution to OVA solution. 4. The use of polyclonal antibodies specific to gypenosides according to any one of Patents 1 through 3. Immunological methods for analyzing steroid saponins with structural similarities to gypenoside 4 (originally revised) Revised 6/09/2016 (30/08/2016) 1. Polyclonal antibodies specific to gypenosides are produced as follows: a) Synthesize conjugates of gypenoside 49 with BSA, or conjugates of gypenoside 49 with HSA, or conjugates of gypenoside 49 with OVA. b) Immunostimulation in rabbits at least 3 times every 20 days. For the first time, prepare a 1:1 ratio immunoglobulin from incomplete foil-coated stimulant and gypenoside 49 bound to BSA 0.4 mg/ml, injected subcutaneously. For the second time, use IFA, an incomplete foil-coated stimulant. Instead of a complete freutic stimulant, inject intramuscularly. After 3 days, check for specific antibodies against gypenoside using the Enzyme-Linked Immunosorban Essay (ELISA) method, which uses the principle of specific binding of antigen and antibody. A 3rd or 4th booster injection is given with 0.2 mg/ml gypenoside fritenin linked to BSA dissolved in phosphate-buffered saline, pH 7.0-7.5, 1 ml, intravenously in the ear. After 3 days, test the antibody concentration using the ELISA method after the 3rd or 4th booster injection. c) Purification of the antibody: Take rabbit blood obtained by drawing blood from the heart (approximately 30-50 ml per rabbit). Centrifuge the blood at 2,000-3,000 rpm, separate the supernatant or serum, and purify it using a G protein column. The substance is then separated using a low pH buffer, dialyzed at a temperature of 4-8 °C, and lyophilized to obtain purified polyclonal antibodies. 2. Polyclonal antibodies specific to gypenoside, according to claim 1, where the conjugate synthesis of gypenoside forty-nine with BSA is performed. Using 5 mg of gypenoside fortenine standard, add 0.5 ml of methanol. Then, add sodium periodicate (NaIO4) solution (5 mg in 0.5 ml of water) drop by drop, stirring with a magnetic stirrer at 25°C for 1 hour. Next, add 10.0 mg of BSA solution in 2.0 ml of 50 mmol PBS buffer (pH 9.5-9.7) (using a ratio of gypenoside fortenine to BSA of 1:2) and stir at 25°C for 6 hours. The resulting solution is then diallyzed five times in distilled water at 4°C and lyophilized to obtain conjugates of gypenoside fortenine and BSA in the range of 7-12 mg. 3. Polyclonal antibodies specific to gypenoside. Under any one of Reservices 1 to 2, whereby the synthesis of a conjugate of gypenoside fortenine with HSA is achieved by replacing the addition of a BSA solution with an HSA solution. 4. A polyclonal antibody specific to gypenoside, under any one of Reservices 1 to 3, whereby the synthesis of a conjugate of gypenoside fortenine with OVA is achieved by replacing the addition of a BSA solution with an OVA solution. 5. The use of a polyclonal antibody specific to gypenoside, under any one of Reservices 1 to 4, with immunological methods, to analyze steroid saponins structurally similar to gypenoside 4. 1. Polyclonal antibodies specific to gypenosides, capable of specifically binding to gypenoside 4 and steroid saponins from Panax quinquefolius in the aglycone part. 2. The procedure for producing polyclonal antibodies specific to gypenoside, as per claim 1, consists of the following steps: a) Synthesize gypenoside XLIX-BSA conjugates, or gypenoside XLIX-HSA conjugates, or gypenoside XLIX-HSA conjugates, or gypenoside XLIX-OVA conjugates; b) Immunization of rabbits at least 3 times every 20 days. For the first dose, prepare an immunogenic solution in a 1:1 ratio of Complete Freund's adjuvant (CFA) and 0.4 mg/ml of BSA-linked gypenoside fortenine. The first injection was administered subcutaneously. The second injection used incomplete Freund's adjuvant (IFA) instead of CFA, administered intramuscularly. After 3 days, the presence of specific antibodies against gypenoside was checked using the Enzyme-linked immunosorbent assay (ELISA), which uses the principle of specific binding between antigen and antibody. A third or fourth booster injection was administered intravenously into the ear using 0.2 mg/ml gypenoside friteine linked to BSA dissolved in 1 ml of phosphate-buffered saline pH 7.0-7.5. After 3 days, the antibody concentration was tested using ELISA after the third or fourth booster injection. c) Antibody purification was achieved by taking approximately 30-50 ml of rabbit blood obtained from the heart of each rabbit. Blood is centrifuged at 2,000-3,000 rpm, separating the supernatant or serum for purification. A protein G column is used, and the substance is separated using a low pH buffer. Diallysis is performed at a temperature of 4-8°C, and lyophilization is performed to obtain purified polyclonal antibodies. 3. Procedure for the production of polyclonal antibodies specific to gypenoside, as per claim 2, in which a conjugate of gypenoside fortenine with BSA is synthesized using 5 mg of gypenoside fortenine standard, 0.5 ml of methanol, followed by the addition of sodium periodoid (NaIO4) solution (5 mg in 0.5 ml of water) drop by drop, stirred with a magnetic stirrer at 25 °C for 1 hour. Next, 10.0 mg of BSA solution in 2.0 ml of 50 mmol PBS buffer (pH 9.5-9.7) is added (using a gypenoside fortenine to BSA ratio of 1:2), and the mixture is thoroughly stirred. The solution was incubated at 25°C for 6 hours. The resulting solution was then diallyzed five times in distilled water at 4°C and lyophilized to obtain a conjugate of gypenoside photonine and BSA in the range of 7-12 mg. 4. A procedure for producing polyclonal antibodies specific to gypenoside, according to either claim 2 or 3, where the synthesis of gypenoside forty-nine conjugate with HSA is achieved by replacing the addition of BSA solution with HSA solution. 5. A procedure for producing polyclonal antibodies specific to gypenoside, according to either claim 2 or 3, where the synthesis of gypenoside forty-nine conjugate with OVA is achieved by replacing the addition of BSA solution with OVA solution. 6. The use of polyclonal antibodies specific to gypenosides, as per any one of claims 1 through 5, with immunological methods to analyze steroid saponins structurally similar to gypenoside 4.