6 - Antibody Interaction

6.1 Recommended setup

Figure 6-1: Schematic Illustration of the two recommended setup. A Capture Kinetic requires a pre- immobilized capture molecule and the target will be freshly captured each cycle, whereas the Direct Kinetic uses a direct immobilized target.

6.1.1 Capture Kinetic

General Procedure:

  1. Immobilize capture molecule or use ready-to-use sensor (e.g IgG Capture Sensor)
  2. Calculate Rmax and perform a Test capture cycle, including surface sample, solution
    sample and regeneration
  3. Run experiment
    1. Include Start-Up Cycles
    2. Include zero injections

6.1.2 Direct Kinetic

General Procedure:

  1. Immobilize surface sample
  2. Test Binding
  3. Test regeneration
  4. Perform a Test capture cycle, including surface sample, solution sample and regeneration
  5. Run experiment
    1. Include Start-Up Cycles
    2. Include zero injections
Capture Kinetic Direct Kinetic
  • Control of mAb capture level and Rmax (antigen)
  • Surface sample remains active
  • Oriented surface sample
  • Regenerable surface, Regeneration conditions always known
  • Crude samples
  • Simple and fast
  • Lower amount of anti-Antigen mAb needed
  • [Regenerable surface], if solution sample stays active
  • Lower density surface
  • Surface sample may dissociate
  • Higher amount of anti-Antigen mAb needed
  • Regeneration condition maybe unknown
  • High purity of surface sample needed
  • Less control of mAb capture level and Rmax (antigen)
  • Random oriented surface sample
  • Surface sample can be inactivated

6.2 Experimental hints

6.2.1 Hints for response levels

In order to prevent mass transport limitation as influencing parameter on the kinetics, a low-medium response level for the solution sample should be aimed for. As a good starting point, try to achieve max. 20-60 RU for your solution sample or antigen. Calculate your maximum solution sample response using equation (6-1) [https://www.sprpages.nl/sensorgram-tutorial/a-curve].


Especially doing capture kinetics the surface sample density can be controlled efficiently, by performing test capture/binding and find an optimum. Start with initial capture injection times of 15-45 s and start with 0.25-1 ug/mL of your molecule to be captured.

6.2.2 Hints for finding optimal regeneration conditions

In general start with gentle conditions and short contact times, if a new regeneration condition has to be evaluated. After a successful regeneration retest the binding and proof the activity of your capture molecule.

Proper regeneration conditions:

  • Proteins / Antibodies: acid; 5-150 mM
  • Peptides / nucleic acids: SDS; 0.01-0.5%
  • Nucleic acids: 10 mM NaOH
  • Lipids: isoPrOH:HCl 1:1
  • See Table 6-1 for possible regeneration solutions
Table 6-1: Possible regeneration solutions [source: https://www.sprpages.nl/kinetics/regeneration#].
(Click on the table to switch it's size.)
Strength Type of bond
Acidic Basic Hydrophobic Ionic
Weak ph > 2.5 ph < 9 ph < 9
10mM glycine/HCl 10mM HEPES/NaOH 25–50% ethylene glycol 0.5–1 M NaCl
1–10 mM HCl 0.02% SDS
0.5M formic acid
Intermediate ph 2 – 2.5 ph 9 – 10 ph 9 – 10
0.5M formic acid 10–100mM NaOH 50% ethylene glycol 1–2M MgCl2
10mM Glycine/HCl 10mM Glycine/NaOH 0.5–0.5% SDS 1–2M NaCl
10mM Glycine/HCl
0.85% H3PO4
Strong ph < 2 ph > 10 ph > 10
1M formic acid 50–100mM NaOH 25–50% ethylene glycol 2–4M MgCl2
10–100mM HCl 6M guanidinechloride 0.5% SDS
10–50mM Glycine/HCl 1M ethanolamine
0.85% H3PO4
0.1% trifluoracetic acid

6.2.3 Hints for the investigation of tight interactions (SensorCon-2)

Raising temperature, may speed up the off-rate

Figure 6-2: Effect of increasing the temperature on the off-rate

Increasing the dissociation time

In order to get qualified information from an off-rate, a certain decay needs to be detected. At a dissociation time of 180-600s it is difficult to see differences for off-rates at 10-4 and 10-5 s-1 . Thus a longer dissociation time should be chosen.

Figure 6-3: Different dissociation times and off-rates. The same off-rates are displayed using a dissociation time of 180s, 600s, 1800s and 7200s.