capture approach / pre steady state approach

Hello,
I'm going to measure several kinetics of protein-protein interactions using the ligand capture approach.

I am using:

-Biacore 3000,

-CM5 chip,

-Capturing molecule: Anti-Human IgG (Fc) antibody (GE Healthcare) - immobilized by amine coupling-Ligand: Fc-Receptor (Disulfide-linked homodimer)

-4 Analytes: 21,6kDa; 19,7kD; 25,7kDa ; 23,3kDa; They are trimers or unfortunately mixture of monomer and trimer. They interact with Fc-Receptor (initial experiments on Biacore 3000 in 50 mM TRIS pH 7.4, 150 mM NaCl, 0.005% P20 , 0.02% NaN3).


I have no experience with capture approach so I have several questions.

1. What kind of reference surface should I use? What is better: only activation and deactivation of carboxymethylated dextran or immobilization Anti-Human IgG (Fc) antibody just like an active flow cell.

2. How much RU of Anti-Human IgG (Fc) should I immobilized to the chip surface of active flow path? Are there any recommendations for the density of capturing molecule?

3. Is it correct to use an equation RL= Rmax/n × MWL/MWA to calculate appropriate ligand (Fc-Receptor) density in case of captured ligand?

4. What is the proper way to determine Rmax? Is this better: to inject high analyte concentrations or get the value from fitting parameters (from kinetic or equilibrium<?> analysis).

Probably, every of my analyte forms symmetric trimer complex with ligand, which is composed of three noninteracting receptor monomers held together by the trimeric analyte. But I have dimeric receptor-Fc fusions… I realize, that this complexity is a big problem… I need Rmax to find condition (ligand dencity) where one monomer of Fc-receptor interacts with trimeric analyte (1 binding site). It would be possible to analyze the data with a Langmuir 1:1 binding model.

5. What do you mean about SPR pre-steady state approach?

“Due to the very slowdissociation of the TRAIL–receptor complex, only pre-steady statebinding data could be obtained. Furthermore, a fast initial dissociationwas observed directly after the end of injection, pointing atsome heterogeneity in complex formation. To obtain data thatrepresent proper high-affinity complex formation, the response ateach concentration was recorded 30 s after the end of the injections(contact time, 30 s). The response data as a function of TRAILconcentration were fitted by using a four-parameter equation to give an apparent affinity constant.”

van der Sloot, A. M., Tur, V., Szegezdi, E., Mullally, M. M., Cool, R. H.,Samali, A., Serrano, L., and Quax, W. J. (2006) Designed tumor necrosisfactor-related apoptosis-inducing ligand variants initiating apoptosis exclusivelyvia the DR5 receptor. Proc. Natl. Acad. Sci. U.S.A. 103, 8634–8639

Thank you very much,

Dorothy

Hi Dorothy:
below some answers for you.

I have no experience with capture approach so I have several questions.

1. What kind of reference surface should I use? What is better: only activation and deactivation of carboxymethylated dextran or immobilization Anti-Human IgG (Fc) antibody just like an active flow cell.
In this case I should choose for immobilizing the reference channel with anti-Human IgG (Fc). If you have the same amount on the reference as on the other channels, you have a better compensation for drift and bulk effects.

2. How much RU of Anti-Human IgG (Fc) should I immobilized to the chip surface of active flow path? Are there any recommendations for the density of capturing molecule?
It is hard to say. It depends for instance on the interaction between the antibody and your Fc-receptor. In the end you aim for enough Fc-receptor to have a proper signal when you inject the analytes (~50-150RU). The trimere nature of the analytes makes it hard to forecast the amount of anti human IgG (Fc) and FC-receptor to be immobilzed/captured.

3. Is it correct to use an equation RL= Rmax/n × MWL/MWA to calculate appropriate ligand (Fc-Receptor) density in case of captured ligand?
Use RL= Rmax * MwL / MwA * n. The trimere nature of the analyte makes it more difficult.

4. What is the proper way to determine Rmax? Is this better: to inject high analyte concentrations or get the value from fitting parameters (from kinetic or equilibrium<?> analysis).
Use the values from the fittings. They are fairly accurate en you don't spoil analyte to try to determine the Rmax. You don't need to saturate the ligand to get proper kinetic values. See: Rich RL, Myszka DG. Survey of the year 2007 commercial optical biosensor literature. J.Mol.Recognit. 2008;21:355-400.
Probably, every of my analyte forms symmetric trimer complex with ligand, which is composed of three noninteracting receptor monomers held together by the trimeric analyte. But I have dimeric receptor-Fc fusions… I realize, that this complexity is a big problem… I need Rmax to find condition (ligand density) where one monomer of Fc-receptor interacts with trimeric analyte (1 binding site). It would be possible to analyze the data with a Langmuir 1:1 binding model.

This can be of help: Schasfoort RBM, de Lau W, van der Kooi A, Clevers H, Engbers GHM. Method for estimating the single molecular affinity. Anal.Biochem. 2012;421:794-796.

5. What do you mean about SPR pre-steady state approach?
“Due to the very slow dissociation of the TRAIL–receptor complex, only pre-steady state binding data could be obtained. Furthermore, a fast initial dissociation was observed directly after the end of injection, pointing at some heterogeneity in complex formation. To obtain data that represent proper high-affinity complex formation, the response at each concentration was recorded 30 s after the end of the injections (contact time, 30 s). The response data as a function of TRAIL concentration were fitted by using a four-parameter equation to give an apparent affinity constant.”

van der Sloot, A. M., Tur, V., Szegezdi, E., Mullally, M. M., Cool, R. H.,Samali, A., Serrano, L., and Quax, W. J. (2006) Designed tumor necrosisfactor-related apoptosis-inducing ligand variants initiating apoptosis exclusivelyvia the DR5 receptor. Proc. Natl. Acad. Sci. U.S.A. 103, 8634–8639

As far as I understand (they show no sensorgrams!), they had also difficulties with analysis of the curves. They tried to get steady state but did not succeed. Therefore they took the end of injection response and plotted the values. This is not a valid method to determine kinetic parameters. See also the first publication!

Kind regards

Arnoud

Thanks a lot for your help,

Dorothy