Sarsasapogenin monoclonal antibody and application

A monoclonal antibody and cloning antibody technology, applied in the field of bioengineering, can solve problems such as low sensitivity, long detection time, and difficult analysis, and achieve the effect of good correlation, strong specificity, and high sensitivity

Inactive Publication Date: 2012-11-21
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Smilagenin does not have a conjugated polyene or π-bond system in its structure, so that its spectral absorption has only weak ultraviolet terminal absorption, which makes analysis difficult. Existing analysis methods such as TLC, HPLC-ELSD and LC-MS, among which Although thin-layer chromatography has low requirements, the detection sensitivity is too low. Although high-performance liquid chromatography and mass spectrometry have high sensitivity and can carry out large-scale detection, their detection time is long, the analysis sample preparation is complicated, and because smilax saponins Trace detection of smilagenin in biological samples with no UV absorption and low mass spectrometry response, which is difficult to study drug metabolism

Method used

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  • Sarsasapogenin monoclonal antibody and application
  • Sarsasapogenin monoclonal antibody and application
  • Sarsasapogenin monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Production and purification of monoclonal antibodies

[0025] 1. Preparation of immunogen-smilagenin derivatives and bovine serum albumin conjugate (SAR-HS-BSA)

[0026] (1) Weigh 50 mg of smilagenin, 1 g of succinic anhydride, and 1 ml of pyridine for 9 hours under reflux in a boiling water bath. After the reaction product was extracted 3 times with chloroform, the chloroform layer was collected and concentrated, using chloroform:methanol (1:1) as the mobile phase, and separated and purified by Sephadex LH-20 gel column to obtain the smilagenin succinyl derivative (SAR-HS).

[0027] (2) Weigh 9.8 mg of SAR-HS, 13 mg of NHS, and 19 mg of DCC and mix them in 1 mL of DMF, place on a magnetic stirrer and stir at room temperature for 4 hours, then add 10 mL of PBS containing 30 mg of BSA, and stir at 4°C overnight.

[0028] (3) The reaction product was put into a dialysis bag, dialyzed in double distilled water for 72 hours, freeze-dried to obtain a white powder, and stor...

Embodiment 2

[0059] Identification of monoclonal antibody against smilax saponin

[0060] In order to verify the effectiveness of the present invention, the following assays were performed.

[0061] 1. Coupling identification of immunogen and coating agent

[0062] Immunogen (SAR-HS-BSA) by infrared spectroscopy see figure 1 and envelope former (SAR-HS-OVA) see figure 2 for identification. Its infrared spectrum except at 985.91, 926.51, 898.14, 866.29cm -1 It has the characteristics of spirosterane-type steroidal saponins, outside the absorption peak, at 3600-3200cm -1 and 1700-1600cm -1 The region has increased the characteristic absorption peak of amino acid

[0063] 2. Identification of positive clonal cell lines and their antibody production

[0064] After three times of subcloning, a monoclonal antibody that can recognize SAR was obtained

[0065] (1) Monoclonal antibody subtype structure identification: use the monoclonal antibody subtype detection kit to identify the subtyp...

Embodiment 3

[0075] Application of monoclonal antibody to smilax saponin

[0076] 1. Sample preparation method:

[0077] (1) Preparation of reference substance solution

[0078] Accurately weigh 1 mg of the SAR reference substance and make a 1 mg / mL solution with methanol.

[0079] (2) preparation of test solution

[0080] Take Anemarrhena medicinal material, pulverize it, pass through a 60-mesh sieve, accurately weigh 0.5g of medicinal material powder, put it in a stoppered Erlenmeyer flask, add 25mL of methanol, weigh it, let it stand overnight, ultrasonically treat it for 40min, make up the weight, filter, and accurately measure Take 10 mL of the filtrate, evaporate to dryness, add 10% hydrochloric acid and 11 mL of boiling water bath and heat to reflux for 2 hours, and add 40% NaOH to the extract to neutralize. Chloroform extraction twice, each 30mL. The chloroform layers were combined, the solvent was recovered, the residue was dissolved in methanol and the volume was adjusted to ...

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Abstract

The invention discloses a preparation method and an application of a Sarsasapogenin (SAR) monoclonal antibody. On the basis of Sarsasapogenin artificial antigen preparation, the anti-Sarsasapogenin monoclonal antibody is prepared by using a hybridomas technology, the cross reaction rate of the monoclonal antibody with liriope muscari baily saponins C, diosgenin and ruscogenin are respectively detected as 29%, 5.7% and 33%, no obvious cross reaction is generated by monoclonal antibody with diammonium glycyrrhizinate, pseudo-ginseng saponins R1 and oleanolic acid. The invention establishes an ELISA analysis method based on the Sarsasapogenin monoclonal antibody, the correlation of the ELISA analysis method and the HPLC method detection result is good, compared with the HPLC method, the ELISA analysis method has characteristic of rapid microscale detection. The Sarsasapogenin monoclonal antibody is suitable for Sarsasapogenin microscale detection in Sarsasapogenin-containing traditional Chinese medicinal materials and biology samples like blood plasma, and can be used for medicine quality control of active components-containing medicines and pharmacokinetics research.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a smilagenin monoclonal antibody and an application thereof. Background technique [0002] Sarsaparogenin (sarsa sapogenin) is the main steroidal nucleus of steroidal saponins, the main active ingredient of Anemarrhena anemarrhena, and one of the main active ingredients of anemarrhena and other traditional Chinese medicines. The treatment of cough, tuberculosis fever, diabetes, dry stool, etc. can enhance the function of the cholinergic system in the brain of aged animals, improve the metabolism of free radicals in the brain, improve the ability of learning and memory, and the symptoms of senile dementia and brain function decline. Saponins have broad application prospects. Smilagenin does not have a conjugated polyene or π-bond system in its structure, so that its spectral absorption has only weak ultraviolet terminal absorption, which makes analysis difficult. Existing an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/16C07K14/765G01N33/577
Inventor 余伯阳许玉刘吉华
Owner CHINA PHARM UNIV
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