6 - Antibody Interaction

6.1 Recommended setup

6.1.1 Capture Kinetic

General Procedure:

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

6.1.2 Direct Kinetic

General Procedure:

Immobilize surface sample
Test Binding
Test regeneration
Perform a Test capture cycle, including surface sample, solution sample and regeneration
Run experiment
1. Include Start-Up Cycles
2. Include zero injections

	Capture Kinetic	Direct Kinetic
Pro	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
Contra	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].

$Rmax_{analyte} = { {Mr_{analyte}\cdot R_{ligand}\cdot Valency_{ligand}} \over {Mr_{ligand}} }[RU]$

(6-1)

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
Strength	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

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.