IASYS-binding cuvette and
Immobilization of ligands on cuvette surfaces and measure
the interactions of ligand which is immobilized on the cuvette and the ligate
which is added to the immobilized cuvette.
Immobilization of ligands to the cuvette.
Immobilization of ligands to the carboxymethylated dextran
- Dissolve 0.2 g N-Hydroxysuccinimide (NHS) in 15 ml of
deionized H2O to get 0.0133 g/ml
(0.116 mol/L) concentration and aliquot into 250 µl volume and store
in a -20 °C freezer.
- Dissolve 1.15 g 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) in 15 ml of deionized H2O
to get 0.077 g/ml (0.400 mol/L) concentration and aliquot into 250 µL
volume and store in a -20 °C freezer.
- Thaw one vial of each NHS and EDC
- Incubate the cuvette to thermally equilibrate by pipetting
200 µl of PBS/T buffer (10 mM PBST + 0.05% Tween-20) and wait until
a stable baseline is obtained ( the changes of the signal response were
smaller than 3 arc seconds in 10 minutes) and obtain a PBS/T buffer base
line for 7 minutes.
- Immediately prior to use, thoroughly mix equal volumes
of NHS and EDC solutions. Add 200 µl of the mixture to the cuvette
while sip up the PBS/T buffer and activate the cuvette for 7 minutes.
- Remove unreacted activation mixture and wash with PBS/T
buffer for 2 minutes.
- Add ligate solution at optimized concentration and pH
for 10 minutes.
- Remove non-coupled ligand with a PBS/T buffer and wash
for 2 minutes. If the arcsec of the immobilized ligand is less than expected,
repeat step 7 and 8.
- Add 1 M ethanolamine, pH 8.5, for 2 minutes to block
the activated site of the cuvette.
- Wash with PBS/T buffer for 5 minutes and calculate the
amount of immobilized ligand.
Immobilization of ligands to aminosilane cuvette
All solutions for immobilization were prepared in 10 mM
sodium phosphate buffer (10 mM, pH 7.7) which was made with ultrapure water
and filtered by 0.2 mm polyethersulfone sterilized membrane.
- Add 200 µl of sodium phosphate buffer (10 mM, pH
7.7) to the cuvette and obtain a stable baseline (the changes of the signal
response were smaller than 3 arc seconds in 10 minutes).
- Aspirate and sodium phosphate buffer. Add 200 µl
of BS3 solution (1mM, 0.57 mg/ml) and wait for 15 minutes.
- Aspirate the BS3 and add 200 µl sodium phosphate
buffer to wash the cuvette three times.
- Once a stable baseline was achieved, Add ligate solution
at optimized concentration and wait for 20 minutes.
- Wash the cuvette three times with 200 µl sodium
phosphate buffer. The difference in the pre- and post-immobilization responses
was used to calculate the amount of ligand immobilized.
- Block any remaining sites on the activated surface of
the cuvette by addition of 200 µl of 2 mg/ml bovine serum albumin
for 5 minutes.
- Aspirate the cuvette and wash three times with 10 mM
sodium phosphate buffer and the sodium phosphate buffer was then changed
to PBST buffer (10 mM phosphate-buffer saline, 0.05% v/v Tween-20).
After the ligand was immobilized to the hydrogel surface
of the cuvette, the IAsys system was used to test a series of different
samples or different concentrations of the same sample.
Each binding cycle was performed with constant instrument
parameters (temperature: 23 °C, stirring speed: 50 revolutions/minute,
sampling interval: 2 seconds, smoothing: 5).
- Establish a PBST buffer baseline for 5 minutes.
- Add the ligate by spiking the PBST buffer with calculated
volumes of the stock ligate solution. The association interaction was monitored
for 9 minutes.
- Wash the cuvette with 10 mM PBST for 3 minutes to remove
the unbound antibody.
- Add the regenerate solution to the cuvette for 2 minutes
to removed the ligate and regenerate the immobilized ligand.
- Wash the cuvette with PBS/T buffer and re-establish the
- Repeat the binding cycle with further concentrations
of the ligate or different ligate.
Kinetic parameters of an interaction were determined by
IAsys FASTfit software which can be used to analyze data obtained using
the IAsys system. The on-rate constant was determined by analyzing the initial
90% of the binding of varying concentrations of ligate to immobilized ligand.
If the association curve had two phases, the measured on-rate constant obtained
from first phase of biphasic association analysis was used. A plot of on-rate
constants versus the concentrations of the antibody was obtained. The slope
of the plot is the association rate constant (Ka) and the intercept value
on the y-axis is the dissociation rate (Kd). The equilibrium constant can
then be calculated from association and dissociation rate constants (KD = Kd/Ka).
Kd can also be obtained by analysis of the dissociation
curve. The off-rate constant was determined by analyzing the initial 90%
of the binding of varying concentrations of ligate to immobilized ligand.
The average of the off-rate is Kd. The equilibrium constant can also be
calculated from association rate constant obtained by on-rate analysis and
dissociation rate constant obtained by off-rate analysis (KD = Kd/Ka).
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