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Adsorption

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Adsorption Mr. Mohammed A. jaber Antibody can be removed from a serum sample by adsorption to red cells carrying the corresponding antigen. After the antibody ... – PowerPoint PPT presentation

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Title: Adsorption


1
Adsorption
  • Mr. Mohammed A. jaber

2
Adsorption
  • 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.

3
Adsorption techniques are useful in such
situations as
  • 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.

4
  • 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.

5
Procedure
  • Wash the selected red cells at least three times
    with saline.
  • 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.
  • Mix appropriate volumes of the packed red cells
    and serum and incubate at the desired temperature
    for 30 to 60 minutes.

6
  1. Mix the serum/cell mixture periodically
    throughout the incubation phase.
  2. 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.
  3. 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.
  4. 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.

7
Interpretation
  • If reactivity remains, the antibody has not been
    completely removed. No reactivity signifies that
    antibody has been completely adsorbed.

8
Notes
  1. Adsorption is more effective if the area of
    contact between the red cells and serum is large
    use of a largebore 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.

9
  1. Repeat adsorptions should use a fresh aliquot of
    cells and not the cells from the prior
    adsorption.
  2. Enzyme pretreatment of adsorbing cells can be
    performed to increase antibody uptake for
    enzyme-resistant antigens.
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