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Hapten compounds and their polyclonal antibodies, kits and applications

A hapten and compound technology, applied in the field of bioengineering, can solve problems such as inconvenience, long analysis time, and complexity, and achieve the effects of reduced cost, strong specificity and high sensitivity

Active Publication Date: 2018-03-02
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the polarity of aconitine alkaloids is small, the control of chromatographic conditions for detection is more complicated, and the analysis time is too long (65min), which is extremely inconvenient; at the same time, due to the low content of medicinal and toxic components detected , and the total content is only 2 / 10,000. Slight differences or fluctuations in sample processing, condition control or personnel operation during analysis can bring about obvious deviations in the measurement results
The Institute of Forensic Science and Technology of the Ministry of Law of the People's Republic of China has established a method and standard for detecting the content of aconitine, neoaconitine and hypoaconitine in biological samples by LC-MS method. High requirements and large investment are difficult to popularize and apply, and the pretreatment of test samples is also extremely cumbersome

Method used

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  • Hapten compounds and their polyclonal antibodies, kits and applications
  • Hapten compounds and their polyclonal antibodies, kits and applications
  • Hapten compounds and their polyclonal antibodies, kits and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation and identification of hapten—3-glutaryl aconitine (AC-GS)

[0034] 1. Weigh 50 mg of aconitine, 8.8 mg of glutaric anhydride, and 2 mL of pyridine and react for 20 hours. After the reaction is terminated with 3 ml of ice water, extract 3 times with 3 times the amount of ethyl acetate, and evaporate to dryness under reduced pressure.

[0035] 2. The reactant was purified by Sephadex LH-20 column chromatography, using methanol / dichloromethane as the eluent, the effluent was collected, and evaporated to dryness under reduced pressure to obtain the pure product.

[0036] 3. After ESI-MS, 13 C-NMR, 1 H-NMR, HMBC, HSQC two-dimensional spectra identified the chemical structure of the compound of formula Ⅰ. ESI-MS measuring instrument: MSD-Trap-XCT, Q-Tof micro (ESI-MS) type mass spectrometer (Agilent Company), 1 H-NMR, 13 C-NMR, HMBC, and HSQC were all measured using an AVANCE III-400 nuclear magnetic resonance instrument (Bruker Company)

[0037] 3....

Embodiment 2

[0046] The preparation of embodiment 2 immune antigen (AC-GS-BSA) and coating antigen (AC-GS-OVA)

[0047] 1. Accurately weigh 10 mg of 3-glutaryl aconitine, 1.5 mg of N-hydroxysuccinimide, and 30 mg of N,N′-dicycloethylcarboimide, add them to 1 mL of DMF, and place them on a magnetic stirrer to avoid Stir at room temperature for 4h. Dissolve 20mg BSA in 2mL0.01mol L -1 PBS (pH7.4), then added dropwise to the above reaction solution under stirring, and stirred at 4°C overnight.

[0048] 2. After the reaction was completed, the reactant was dialyzed with double distilled water for 72 hours, freeze-dried to obtain a white powder, and stored at -20°C.

[0049] 3. Use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to identify the coupling status of the immunogen conjugate (AC-GS-BSA):

[0050] MALDI-TOFMs can directly display the hapten number (Hapten Number) of the immune operation, and the method is simple and fast.

[0051] Figu...

Embodiment 3

[0056] Example 3 Immunization of New Zealand rabbits with immune antigens to obtain diester-type aconitine polyclonal antibodies

[0057] 1. Configuration of immune antigen: Add Freund's complete adjuvant to AC-GS-BSA solution to form a white water-in-oil emulsion. For booster immunization, the same concentration of AC-GS-BSA was added to an equal volume of Freund's incomplete adjuvant.

[0058] 2. Immunized animals: Rabbits were reared in a single cage for 7-10 days before immunization, and immunized after observing their health status. Before immunization, 1.5-2 mL of blood was taken from the ear vein as negative serum. At the first immunization, each rabbit was injected subcutaneously at 6 points on both sides of the spine, and a booster immunization was performed every 14 days. On the 7th day after the last immunization, blood was collected from the carotid artery of the rabbits, and the polyclonal antibody serum was separated.

[0059] 3. Immune monitoring: take the ra...

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Abstract

The invention relates to the technical field of bioengineering, and especially relates to a semiantigen compound 3-glutaryl aconitine. The compound is represented by structural formula (I) shown in the specification. The invention also discloses a polyclonal antibody obtained through immunizing New Zealand rabbit with a conjugate of the semiantigen compound and bovine serum albumin, and a kit thereof. The polyclonal antibody and the kit thereof can be used to detect diester aconitine.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a hapten compound and its polyclonal antibody, kit and application. Background technique [0002] Aconitine compounds are diterpene-type alkaloids, which can be divided into three types according to the different substituents on the core of their structures: ① diester type, including hypoaconitine, aconitine, neoaconitine, etc., with certain It has anti-inflammatory and analgesic effects, but it is also the main source of the toxicity of Chinese herbal medicine aconitine, and its toxic dose is extremely small, such as 1-2mg aconitine can cause death. ②Monoester type, including benzoyl aconitine, benzoyl mesoaconitine, benzoyl hypoconitine, etc., are relatively less toxic and have analgesic, antiarrhythmic and antiepileptic effects. ③No The ester type, including aconitine, neoaconitine, etc., can resist arrhythmia and has lower toxicity. It can be seen that different acon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D221/22C07K16/44G01N21/31G01N33/53
Inventor 华茉莉许玉黄磊徐云辉袁帅
Owner SHANGHAI INST OF PHARMA IND CO LTD
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