Topics Covered
Background
Antigens and Antibodies
Dynamic Light Scattering for Measuring Particle
Size
Dealing with Small Particles and Dilute Concentrations
Case Study – Interactions Between Antibodies and a
Virus
Experimental Procedure
Results
Conclusions
Background
Antigens and
Antibodies
Antibodies (Ab) are proteins produced by the
body in response to the presence of a foreign body. Antigens (Ag) are foreign
compounds that instigate the production of antibodies. They may occur free in
solution or they may be bound to the surface of cells or particles such as
viruses or bacteria. An antibody produced in response to a foreign body reacts
with the antigen to form an immune complex (Ab-Ag) and is therefore
inactivated.
Dynamic Light
Scattering for Measuring Particle Size
Dynamic
light scattering (photon correlation spectroscopy) is a technique that can
measure the size of particles down to 1nm in diameter. Particles in suspension
undergo random Brownian motion. If these particles are illuminated with a laser
beam, the intensity of the scattered light fluctuates at a rate that is
dependent upon the size of the particles. Analysis of these intensity
fluctuations yields the velocity of the Brownian motion and hence the particle
size.
Dealing with Small
Particles and Dilute Concentrations
The intensity of scattered light obtained
from small particles or from samples that are very dilute will be insufficient
for repeatable PCS sizing measurements using a standard 10mW He-Ne
laser.
One method of overcoming such problems is to
use a higher efficiency detector. The Malvern Zetasizer HS instrument contains an avalanche photodiode
(APD).
This detector allows measurement of small
particles and samples of low concentration without the need for a more powerful
laser.
Case Study – Interactions Between
Antibodies and a Virus
This application note describes dynamic
light scattering measurements in which the affinity of two different antibodies
for a virus sample can be studied.
Experimental
Procedure
The measurements were made on a Malvern Zetasizer 3000HS at a temperature of 25°C. The scattered light
was detected at an angle of 90°.
Results
Figure 1 shows the intensity size
distributions obtained from 3 repeat measurements of a sample of serum
containing antibodies. The distributions were obtained from analysis of the data
with the CONTIN algorithm. The distributions are monomodal with peak modes at
32nm respectively. The affinity of two different antibodies for a virus was
determined by mixing each of the antibody samples with a sample of the virus and
making rapid sizing measurements (every 30 seconds). Figure 2 shows the
z-average diameters (the mean diameter based on the intensity of scattered
light) obtained for the mixtures of 2 antibodies with virus particles as a
function of elapsed time after mixing. The increase in the z-average diameter
results from the formation of the antibody-virus complex. Antibody 1 shows
higher affinity for the virus indicated by the steeper slope of the
curve.
Figure 1. Intensity size distributions
obtained from 3 repeat measurements of a sample of serum containing
antibodies.
Figure 2.A plot of the z-average
diameters from mixtures of 2 different antibodies with a virus as a function of
the elapsed time after mixing.
Conclusions
Photon correlation spectroscopy can be used
to measure the size of small particles. It is a non-invasive technique that can
be used to assess the affinity of various antibodies for a particular
virus.
Source: The Study of Antibody: Antigen Interactions Using Light
Scattering Techniques”, Application Note by Malvern Instruments.
For more information on this source please visit Malvern
Instruments Ltd (UK) or Malvern Instruments
(USA).