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The detection and identification of saxiphilins using saxitoxin-biotin conjugates

A technology of avidin and toxin protein, applied in biological testing, biomaterial analysis, measuring devices, etc., can solve the problem of difficult separation and purification of saxiphilin

Inactive Publication Date: 2006-05-17
CLEVELAND BIOSENSORS PYT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, saxiphilins have proven to be a difficult group of compounds to isolate and purify, and until now only bullfrog saxiphilins have been well characterized

Method used

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  • The detection and identification of saxiphilins using saxitoxin-biotin conjugates
  • The detection and identification of saxiphilins using saxitoxin-biotin conjugates
  • The detection and identification of saxiphilins using saxitoxin-biotin conjugates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Synthesis of Biotin-Link 11-STX and Synthesis of Biotin-Link 4-STX

[0061] In a sealed, evacuated glass tube, the saxitoxin isolated from crustaceans was converted into decarbamoyl-saxitoxin (dcSTX) by hydrolysis in 6M HCl at 110°C for 4 hours. This solution was freeze-dried. The residue was redissolved in 0.05M acetic acid, and the solution was passed through a C18 solid phase extraction cartridge. DcSTX was purified by gel filter-P2 chromatography and freeze-dried.

[0062] Then, dcSTX was re-dissolved in 0.1M sodium phosphate buffer with pH 6.8, and converted into succinic anhydride (ratio of dcSTX: succinic anhydride=1:20) at 10°C for 2 hours. DcSTX hemisuccinate while maintaining a temperature of 10°C and a pH of 5.7±0.1. Then dcSTX hemisuccinate was separated from dcSTX and purified by anion exchange chromatography using 0.01M sodium phosphate buffer as the elution solvent, and solid phase extraction was performed by Carbograph graphitized carbon black, and Use ult...

Embodiment 2

[0070] Synthesis of Biotin-Link 18-STX

[0071] In a sealed, evacuated glass tube, the saxitoxin isolated from crustaceans was converted into decarbamoyl-saxitoxin (dcSTX) by hydrolysis in 6M HCl at 110°C for 4 hours. This solution was freeze-dried. The residue was redissolved in 0.05M acetic acid, and the solution was passed through a C18 solid phase extraction cartridge. DcSTX was purified by gel filter-P2 chromatography and freeze-dried. The residue was re-dissolved in anhydrous DMF and reacted with excess PMPI (N-(p-maleimidophenyl) isocyanate) at room temperature overnight to produce PMPI-STX. PMPI-STX was purified by reverse phase HPLC-MS. The NHS-LC-Biotin was reacted with 10 mM cysteamine in a pH 7.7 sodium phosphate buffer for 3 minutes at room temperature. The final product (sulfhydryl derivative of biotin) was purified by C18 solid phase extraction and freeze-dried overnight. PMPI-STX was re-dissolved in 10mM sodium phosphate buffer pH 6.8, and reacted with excess sul...

Embodiment 3

[0076] Preparation of avidin. Biotin-link 4-STX, streptavidin. Biotin- Link 4-STX, avidin. Biotin-link 11-STX, and streptavidin Protein. Biotin-Link 11-STX complex

[0077] Streptavidin. Biotin-Link 4-STX and Streptavidin. Biotin-Link 11-STX: 324.8 pmol of Biotin-Link 4-STX and 90.4 pmol of Biotin-Link 11 -STX was each mixed with 30 μg of immunopure streptavidin in 10 mM phosphate buffer (pH 6.5), and incubated at +4°C for 2 hours. 400 μL of 0.1% formic acid was added to the solution, and then the solution was filtered to as little as 30 μL using a 5,000 cutoff microdialysis centrifuge tube. Repeat the same steps once. Then add 200 μL of 0.1% formic acid, and filter the solution again to as little as 30 μL. The final volume of biotin-linked 4-STX was adjusted to 65 μL (final concentration of 5 μM) with water, and the final volume of biotin-linked 11-STX was adjusted to 90 μL (final concentration of 1 μM) with water.

[0078] Avidin.Biotin-Link 4-STX and Avidin.Biotin-Link 11...

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Abstract

The present invention relates to a method for capturing saxiphilin so as to detect, characterize, separate and / or purify said saxiphilin or its ligand, the method comprising: (1) providing a PST conjugate , the PST conjugate includes a PST part, which is directly or indirectly bound to the biotin part through a linker and through a non-binding site of saxitoxin; (2) exposing the PST conjugate for a sample assumed to contain said saxiphilin, to generate a reaction mixture, and expose to (strep)avidin; and (3) allow binding to saxiphilin via the PST moiety, and via This biotin moiety binds to (streptavidin) to form the captured PST complex.

Description

Technical field [0001] The invention relates to a paralytic shellfish toxin conjugate. In particular, the present invention relates to the application of paralytic shellfish toxin conjugates in the detection, characterization, separation and / or purification of molecules of interest, especially saxiphilin and its ligands, although its application is not limited to this . Background technique [0002] The so-called "saxiphilins" are different kinds of polypeptides, which are characterized by their ability to bind to saxiphilins, which is one of the paralytic shellfish toxins (or PSTs). The term "saxiphilin" is a coined term that includes the prefix "saXi" from saxitoxin and the suffix "philic", which means analogy (or pro) saxitoxin. Saxiphilin does not share any specific chemical structure or physiological function, and the physiological function of saxiphilin does not seem to be bound to saxitoxin. For example, the so-called "bullfrog saxiphilin" is a molecule that shares more th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C07D487/14C07D487/20G01N33/543
CPCC07D487/20G01N33/54306G01N2333/8139
Inventor 塞德里克·埃米尔·弗朗索瓦·罗比约
Owner CLEVELAND BIOSENSORS PYT LTD
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