Download Adsorption

Practical
Blood Bank
Adsorption
Adsorption



Antibody can be removed from a serum sample
by adsorption to red cells carrying the
corresponding antigen.
After the antibody attaches to the membranebound antigens and the serum and cells are
separated, the specific antibody remains
attached to the red cells.
It may be possible to harvest the bound
antibody by elution.
Adsorption techniques are useful in such
situations as:
1.
2.
3.
4.
5.
Separating multiple antibodies present in a single serum.
Removing autoantibody activity to permit detection of
coexisting alloantibodies.
Removing unwanted antibody (often anti-A and/or anti-B)
from serum that contains an antibody suitable for reagent
use.
Confirming the presence of specific antigens on red cells
through their ability to remove antibody of corresponding
specificity from previously characterized serum.
Confirming the specificity of an antibody by showing that it
can be adsorbed only to red cells of a particular blood group
phenotype.

Specimen


Serum or plasma containing antibody to be
adsorbed.
Reagents

Red cells (eg, autologous or allogeneic) that carry
the antigen corresponding to the antibody
specificity to be adsorbed.
Procedure
1. Wash the selected red cells at least three times with
saline.
2. After the last wash, centrifuge the red cells at 800 to
1000 × g for at least 5 minutes and remove as much of
the supernatant saline as possible. Additional saline
may be removed by touching the red cell mass with a
narrow piece of filter paper.
3. Mix appropriate volumes of the packed red cells and
serum and incubate at the desired temperature for 30
to 60 minutes.
4. Mix the serum/cell mixture periodically throughout the
incubation phase.
5. Centrifuge the red cells at 800 to 1000 × g for 5 minutes
to pack cells tightly. Centrifuge at the incubation
temperature, if possible, to avoid dissociation of antibody
from the red cell membranes.
6. Transfer the supernatant fluid, which is the adsorbed
serum, to a clean test tube. If an eluate is to be prepared,
save the red cells.
7. Test an aliquot of the adsorbed serum, preferably against
an additional aliquot of the cells used for adsorption, to
see if all antibody has been removed.
Interpretation

If reactivity remains, the antibody has not been
completely removed. No reactivity signifies that
antibody has been completely adsorbed.
Notes
1. Adsorption is more effective if the area of contact
between the red cells and serum is large; use of a large
bore test tube (13 mm or larger) is recommended.
2. Multiple adsorptions may be necessary to completely
remove an antibody, but each successive adsorption
increases the likelihood that the serum will be diluted
and unadsorbed antibodies weakened.
3. Repeat adsorptions should use a fresh aliquot of cells
and not the cells from the prior adsorption.
4. Enzyme pretreatment of adsorbing cells can be
performed to increase antibody uptake for enzymeresistant antigens.