spacer
home > ebr > autumn 2015 > method of choice
PUBLICATIONS
European Biopharmaceutical Review

Method of Choice

Access to basic binding parameters is essential for the design and development of drugs, antibodies, aptamers, and other diagnostic or therapeutic molecules. Currently, various methods are used to determine these parameters – for example, binding affinity, stoichiometry, thermodynamics and kinetics. Assays range from semi-quantitative approaches – including pull-down assays or electromobility shift assays – to quantitative methods – such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), fluorescence anisotrophy, biolayer interferometry and fluorescence correlation spectroscopy.

SPR is a commonly-used method that measures changes in the index of refraction at the surface where a binding event occurs between an immobilised molecule on a thin metal film and its ligand in solution. This sensitive technology allows for time-resolved measurements of binding events to the surface-immobilised molecule. It can be used to establish kinetics, stoichiometry and affinities from sub nM to mM (dissociation constant KD). Despite drawbacks such as intense laboratory set-up, necessary modification of one interaction partner by immobilisation and influence of mass transport limitations on data quality, SPR is the method of choice if Kon and Koff rates are to be determined.

Label-free ITC enables direct access to thermodynamics, binding affinity (nM to sub mM range) and stoichiometry, by measuring the dissipated and absorbed heat of a binding reaction between a target molecule and ligand. However, to be able to detect these changes in heat, larger amounts of sample are necessary. In addition, the technology is limited in buffer usage, is laboratory intense, and only interaction with a higher change in enthalpy can be measured. Nonetheless, as thermodynamics can be directly measured, ITC is the method of choice when determining these parameters.

Novel Approach

MicroScale Thermophoresis (MST) is a novel technology that can be used to characterise molecular interactions and it has enjoyed considerable attention in scientific literature (1-3). The basic principle behind this technique is a physical phenomenon called thermophoresis, which describes the motion of molecules in temperature fields (3,4). The thermophoretic effect is highly sensitive to the interface between a solvent and molecule; binding of a ligand to a target molecule will alter at least one essential parameter – either size, charge or hydration shell – which determines the thermophoretic movement of a molecule. Consequently, the movement of the molecule-ligand complex can be compared against the action of single molecules. Furthermore, MST can monitor the movement of molecules in temperature gradients, interpret this movement as binding events, and use this information to access basic binding parameters.

Based on an optical system, MST detects the intrinsic fluorescence of tryptophanes within proteins or the fluorescence signal of a fluorophore attached to one of the interaction partners (5). The movement of the fluorescent molecules is monitored in a μm-sized temperature gradient, with respect to increasing concentrations of the potential binding partner.

With MST, the optics focus on the centre of thin glass capillaries holding 4-6μl of sample, in which a microscopic temperature gradient with a diameter of ~50μm and a temperature difference of ΔT of 2-6°C is established by an infrared (IR) laser. On activation of the laser, thermophoretic depletion or accumulation of molecules can be observed in the region of elevated temperature, which is quantified by the Soret coefficient.

Time Trace


Size, charge and hydration shell determine the thermophoretic behaviour of a molecule. The binding of a ligand to the target molecule will alter at least one of these parameters, resulting in changed thermophoretic movement of the complex, compared to the single molecules. This effect can be used to study equilibrium constants, such as the dissociation constant KD. To obtain this information, a serial dilution of the ligand is first prepared, mixed with a constant concentration of labelled target molecule, loaded into capillaries, and then analysed in the instrument by subsequent scanning of each capillary.

Technique Advantages

MST allows for fast and flexible assay set-up and optimisation, and a KD value can be obtained within just minutes or hours. MST – as an in-solution method – offers free choice of buffers, making close to native measurements in sera or lysates possible (6,7). To obtain high-quality data, the technique provides integrated quality controls to detect sticking and aggregation/precipitation in real time. Upon detection of these effects, technical conditions and buffers can easily be optimised to ensure an ideal binding environment. Furthermore, as the thermophoresis of molecules depends on the charge and hydration shell, there are no limitations in the size of measured interaction partners. Finally, the dynamic affinity range from pM to mM, together with low sample consumption, add to the strengths of MST.

MST Applications

Besides binding affinity, thermodynamics and stoichiometry, MST offers a broad application range. It can be used for screening assays, competition assays, and assays with multiple binding partners. In addition, the technology can gauge the serum stability of interactions. All of these provide high information content.

MST is able to perform binding assays in a fluorescently labelled version or in a label-free assay format. In the fluorescent assay, the optically visible molecule is detected via the fluorescent dye attached to it. This assay is highly sensitive, allowing for pM interactions to be detected in crude extracts or sera. In contrast, the label-free assay uses the intrinsic fluorescence of tryptophanes within a target protein. This approach enables the study of protein-target interaction in a label-free manner without modification of the target protein (5).

Scientists have used MST to study different interactions between various molecular classes, such as nucleic acid, protein-nucleic acid, protein-protein, protein-small molecule and proteincarbohydrate (8-15). Even challenging binding events like protein-liposome interactions can be investigated (16,17). Literature also indicates that data derived from MST measurements have a high overlap with data from ITC and SPR studies, making this technology perfectly suited for early-phase drug development, antibody characterisation and aptamer design. Over 600 publications using MST could be found in June 2015.

Innovation Technology


MST is a novel technique that can be used to characterise the essential binding parameters of a range of molecular interactions, such as binding affinity (pM to mM range), stoichiometry and thermodynamics. It is a fast and flexible approach that allows researchers to study interactions under close to native conditions – in sera or lysates – while using low sample material and producing quality controlled data. It has wide application across drug development, antibody and aptamer characterisation, epigenetics or basic science.

References


1. Duhr S and Baaske P, GIT Laboratory Journal, 2013
2. Jerabek-Willemsen M et al, Journal of Molecular Structure, 2014
3. Jerabek-Willemsen M et al, Assay Drug Dev Technol 9: p342, 2011
4. Baaske P et al, Angew Chem Int Ed Engl 49: p2,238, 2010
5. Seidel SA et al, Angew Chem Int Ed Engl 51: p10,656, 2012
6. Seidel SA et al, Methods 59: p301, 2013
7. Wienken CJ et al, Nat Commun 1: p100, 2010
8. Zillner K et al, Methods in Molecular Biology 815: p241, 2012
9. Schubert T et al, Mol Cell 48: p434, 2012
10. Pham TH et al, Nucleic Acids Res, 2013
11. Josling GA et al, Cell Host Microbe 17: p741, 2010
12. Xiong X et al, Nature 497: p392, 2013
13. Immekus F et al, ACS Chem Biol 8: p1,163, 2013
14. Patnaik S et al, J Med Chem 55: p5,734, 2012
15. Wong JE et al, Acta Crystallogr D Biol Crystallogr 71: p592, 2015
16. van den Bogaart G et al, J Biol Chem 287: p16,447, 2012
17. van den Bogaart G et al, Nat Struct Mol Biol 18: p805, 2011


Read full article from PDF >>

Rate this article You must be a member of the site to make a vote.  
Average rating:
0
     

There are no comments in regards to this article.

 You must be a member of the site to make a comment.
spacer
Dr Thomas Schubert studied Biology in Regensburg, Germany, where he specialised in Biochemistry. His PhD research focused on the use of MST technology to detect and quantify molecular interactions in the field of epigenetic regulation. Thomas originally joined 2bind as a MST Application Specialist and, in 2013, was nominated as Chief Executive Officer of the company.
spacer
Thomas Schubert
spacer
spacer
Print this page
Send to a friend
Privacy statement
News and Press Releases

Innovative Micro-Infusion Pump for Gerresheimer Subsidiary Sensile Medical Developed and Successfully Launched for EVER Pharma

Düsseldorf/Olten, July 03, 2019 – Developed specially by Sensile Medical for EVER Pharma under the brand name D-mine®, this wearable microinfusion pump recently received European CE certification and has already been launched in several European countries. The compact, patient-friendly infusion pump is used for the continuous subcutaneous administration of drugs to treat Parkinson’s disease. The first micro pump from Gerresheimer subsidiary Sensile Medical to be available on the market, it gives Parkinson’s patients greater independence in their day-today lives.
More info >>

White Papers

Clinical Trial Labelling – More Than Just Labels

Faubel & Co. Nachfolger GmbH

Deciding which label is best for a particular trial project is not always easy. Labels have become multifunctional tools which are able to convey variable data in different languages, indicate first opening, product originality, support ease of use or blind study drugs. They are no longer used as mere carriers of specified contents.
More info >>

 
Industry Events

CPhI & P-MEC India

26-28 November 2019, India Expo Centre, Greater Noida, Delhi NCR, India

As the pharma industry looks increasingly towards India forhigh quality, low   cost pharma solutions, CPhI & P-MEC Indiais the ideal event for companies wanting to pick up on the latest trends and innovations the market has to offer. At CPhI & P-MEC India, you will meet the movers and shakers from India’s pharma machinery, technology and ingredients industries, giving you a competitive advantage that will help grow your business.
More info >>

 

 

©2000-2011 Samedan Ltd.
Add to favourites

Print this page

Send to a friend
Privacy statement