3. 1. Belong to class of medicines known as
protein therapeutics
2. Effective in treating some forms of cancers
and tumours
3. Abnormal behaviour has adverse effects
Significance of IgG Antibodies
and Platelets
1. Normal functioning/clumping of platelets
produces clots in order to prevent
bleeding.
2. However, their abnormal behaviour could
lead to strokes in heart/brain
5. 09/02/2016 5
I. The clumping together of two or more than two activated platelets.
II
Abnormal Activated Cells
Clumping
Cellular aggregate / clot
Unstable Antibody Molecules
Clumping
Aggregated Antibody Molecules
6. 09/02/2016 6
I. Conventional Microscopy:
a. Overlooks imaging single molecules or small molecular aggregates
with nano-dimensions
b. There is no emphasis on which cells are more prone to aggregation and
why?
c. Lacks the ability to measure the strength of the inter-molecular/inter-cellular
interaction forces that lead to aggregation.
II. Where is the room to improve our understanding?
a. Identification of morphologically different molecules and platelets (activated)
that participate in aggregation
b. Measurement and strength of the inter-molecular and inter-platelet interaction
forces that exist between different molecules/morphologically different platelets
(activated).
7. 09/02/2016 7
I. Imaging of Molecules and Different Cell
Types With Atomic Force Microscopy.
Instead of using an incident beam to visualize
a sample, as would be the case in classical
Microscopy, AFM senses the small forces
(in the piconewton range, ,10-12 N) that act
on the sample surface.
a. Molecules and Cells have been imaged with
AFM.
b. Can provide high-resolution images of
molecules cell surfaces under physiological
conditions.
8. AFM Study of the Effect of
Multiple freeze drying on IgG
- AFM images reveal both crystalline and amorphous features. Globular, protein
like features can also be observed.
- The size of the smaller globular features is similar to the size of monomeric
IgG (~15-20nm) reported in previous AFM studies (Ultramicroscopy 105:103-
110). The larger globular features likely represent aggregates of IgG
250nm 250nm 250nm
Cycle 1 Cycle 2 Cycle 3
AFM images of freeze dried IgG samples, prepared without
excipients (starting concentration 2mg/ml in 0.01M PBS)
Globular
features
Crystalline
9. 09/02/2016 9
AFM study of IgG Freeze
Dried with 20mM Mannitol
- AFM images show both amorphous and
crystalline features.
- Globular features appear to be associated
with distinct regions on the sample surface e.g.
some crystalline features do not appear to be
coated with a protein like layer.
250nm250nm 250nm
A B C
150 nm
D
Crystalline
Amorhous
10. 09/02/2016 10
IgG Freeze Dried with Sucrose
and Mannitol in Combination [1]
- Images of once freeze dried IgG with Sucrose
(20mM) and Mannitol (40mM) in combination
- An increase in the molar concentration of
mannitol reveals distinct crystalline features in
some areas.
-The globular features of IgG profoundly appear to
be associated with the amorphous material.
250nm 250nm 250nm
A B C
150nm
D
Crystalline
Amorhous
11. 09/02/2016 11
This image, taken with atomic force
microscopy, shows E. coli (2-6µm)
bacteria after they have been
exposed to the antimicrobial peptide
CM15. The peptides have begun
destroying the bacteria’s cell walls.
(http://web.mit.edu/newsoffice/2010/micropepti
des-0315.html)
Topographical rearrangements shown by AFM images of
myoblasts fusing due to cytoskeletal dynamics during
myogenesis.
(http://www.mechano-biology.ethz.ch)
13. 09/02/2016 13
I. Characterization of Activated Cells On The Basis of Their Binding
Strength With Bio-membrane Force Probe (BFP).
A B C D
Aggregation receptor is glycoprotein IIb/IIIa (gpIIb/IIIa); calcium-
dependent for fibrinogen. Other receptors areGPIb-V-IX complex (vWF)
and GPVI (collagen)
Connecting agent such as fibrinogen, fibronectin, vitronectin,
thrombospondin, and vWF (von Willebrand factor)
A
14. (a) Specific, (b) non-specific, (c) no adhesion (d)
multiple force displacement curves of the interaction
Plot of adhesion force or binding interaction
strength.
15. I. Determination of molecular/cellular size and identification
of different cell types and those more prone to
aggregation is possible with AFM imaging.
II. Measurement of ligand (fibronectin/fibrinogen) – cell
surface receptor interactions would be ideal to determine
the protein-protein interactions
III. Strength of platelet interactions that contribute maximum
to the aggregation is possible with AFM, and hence,
would be useful for the quantification of cells showing
high propensity to aggregate.
