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Biochemistry-Sandwich ELISA

IASYS-binding cuvette and getting KD

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 coated cuvette

  1. 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.
  2. 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.
  3. Thaw one vial of each NHS and EDC
  4. 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.
  5. 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.
  6. Remove unreacted activation mixture and wash with PBS/T buffer for 2 minutes.
  7. Add ligate solution at optimized concentration and pH for 10 minutes.
  8. 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.
  9. Add 1 M ethanolamine, pH 8.5, for 2 minutes to block the activated site of the cuvette.
  10. 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.

  1. 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).
  2. Aspirate and sodium phosphate buffer. Add 200 µl of BS3 solution (1mM, 0.57 mg/ml) and wait for 15 minutes.
  3. Aspirate the BS3 and add 200 µl sodium phosphate buffer to wash the cuvette three times.
  4. Once a stable baseline was achieved, Add ligate solution at optimized concentration and wait for 20 minutes.
  5. 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.
  6. 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.
  7. 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).

 

Interaction analysis

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).

  1. Establish a PBST buffer baseline for 5 minutes.
  2. Add the ligate by spiking the PBST buffer with calculated volumes of the stock ligate solution. The association interaction was monitored for 9 minutes.
  3. Wash the cuvette with 10 mM PBST for 3 minutes to remove the unbound antibody.
  4. Add the regenerate solution to the cuvette for 2 minutes to removed the ligate and regenerate the immobilized ligand.
  5. Wash the cuvette with PBS/T buffer and re-establish the baseline.
  6. Repeat the binding cycle with further concentrations of the ligate or different ligate.

 

Data analysis

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).



Welch 4.264
The University of Texas at Austin
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