Practical Blood Bank Lab 8 Adsorption Adsorption q

Practical Blood Bank Lab 8 Adsorption

Adsorption q q q Antibody can be removed from a serum sample by adsorption to red cells carrying the corresponding antigen. After the antibody attaches to the membrane-bound 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.

n Specimen q n Serum or plasma containing antibody to be adsorbed. Reagents q 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 n n 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.