ANTIBODY TESTING

• SALINE METHOD FOR IgM ANTIBODIES TESTING

• • Saline method is the simplest serological
technique for determining the reaction of IgM
antibodies against ABO, H, Ii, MN, Lewis (Lea ,Leb),
Lutheran (Lu), and P blood groups antigens
• • The IgM antibodies react at lower temperature
(4-7 oC).
• • IgM antibodies are capable of agglutination red
cells suspended in 0.85% saline solution.
• Method
• 1. Put 2 drops of test serum or known serum in properly labeled tube.
• 2. Add 1 drop of 2-4% suspension of appropriate red cells in saline and
mix.
• 3. Incubate at R.T. for 30-45 min. In urgent cases, centrifuge the tubes at
1000 rpm for1 min. after 5-10 min incubation at room temperature
(Spin method)
• 4. Observe the supernatant fluid for the presence of hemolysis against
well lighted background.
• 5. Gently disperse the cells button and check for agglutination against a
well lighted background.
• 6. Where no agglutination is seen visually, examine the contents under
microscope.
• 7. Record the result immediately.
• Interpretation
• Agglutination or haemolysis is a positive result
• METHOD FOR IgG ANTIBODY TESTING
• • IgG antibodies are acquired (immune) antibodies and are
clinically significant.
• • They are responsible for hemolytic transfusion reactions
and hemolytic diseases of the new born.
• • To detect the presence of IgG antibodies several media
are used to enhance and potentiate the antibody-antigen
reaction. e.g. 22 % albumin, enzymes, low ionic strength
solution (LISS), and antihuman globulin sera.
• • Several enhancement media/ reagents are aimed at
reducing the net negative charge (zeta potential) on the
surface of red cells,
• ALBUMIN TESTS FOR IgG
• Albumin is used in blood group serology as
• i) 22% concentration for enhancing the
agglutination of red cells coated with IgG antibodies
• ii) concentration of 1-7% as a stabilizer in other
reagents, specially those to be stored at 4 oC.
• Mostly 22% bovine albumin is used to enhance the
agglutination of red cells coated with IgG antibodies
for detection and identification of IgG antibodies,
cross-matching and cell typing.
• Method
• A One Stage Method (Albumin used as additive)
• 1. Add two drops test serum or known serum to a labeled tube.
• 2. Add one drop of 2-4% suspension of appropriate RBCs in saline
• 3. Add two drops of bovine albumin 22%
• 4. Mix and incubate at 37 oC for 30-60 min.
• 5. Centrifuge at 1000 rpm for 1 min.
• 6. Gently resuspend the cell button and observe for
agglutination/ haemolysis
• 7. Confirm all negative results under microscope
• 8. Agglutination or hemolysis is a positive result
• Two Stage Albumin Method (Albumin layering)
• 1. Add 2-3 drops of serum to a labeled tube
• 2. Add 1 drop of 2-4 % red cell suspension in saline in the tube
• 3. Mix and incubate at 37 oC for 30 minutes
• 4. Centrifuge at 1000 rpm for 1-2 minutes
• 5. Without disturbing the cell button, gently add two drops of
22% albumin to run down the inside of the tube. Albumin will
form a layer on the top of cells. Do not mix
• 6. Incubate at 37 oC for 10-20 minutes
• 7. Gently resuspend the cells and observe for agglutination/
hemolysis
• 8. Confirm all negative results under microscope
• • One-stage technique (additive method)
takes less time and is more frequently used,
but the two-stage technique (layering
method) is more sensitive.
• • Manufacturer’s instructions should be
followed:
• • Agglutination or hemolysis is a positive
result.
 ANTIBODY ELUTION
• Elution is a technique use to dissociate
antibody from sensitized red cells and to
recover antibody in an inert diluent such as
normal saline or 6% albumin, which is known
as elute. This elute can be tested, like serum,
to determine the antibody specificity.
• Elution can be performed by a number of
following physical and chemical procedures:
• Heat Elution(Landsteiner and Miller 1925)
• This is useful in eluting anti-A and/ or anti-B from
red cells in the diagnosis of ABO HDN or for
eluting other antibodies which bound more
strongly at low temperature.
• It is not muchefficient in recovering IgG
antibodies.
• This method has the advantage of speed and
simplicity.
• Materials
• • 6% Bovine albumin, prepared by diluting 22% bovine
albumin with saline (1.4 ml 22% bovine albumin + 3.6 ml
normal saline) or AB serum.
• • Antibody coated red cells, DAT positive (2 ml)
• • Supernatant saline from last wash from antibody coated
cells.
• Methods
• 1. Preparation of supernatant from last wash
• Wash the antibody coated red cells (from which the elute
is to be made) six times in large volumes of normal saline.
• The final wash should be performed by adding
saline equal to the volume of washed packed
red cells. Centrifuge and recover the
supernatant of the last wash and test it in
parallel with the elute.
• This last wash is an important negative control
that demonstrates that the residual serum
antibody has been removed before the elute is
prepared from the washed red cells.
• 2. Preparation of Elute
• • Add saline (or AB serum or 6% albumin) equal to the
volume of washed packed cells.
• • Place the tube in a 56 oC water bath for 5-10 minutes.
Agitate frequently during incubation.
• • Immediately centrifuge at 1000 rpm for 2 minutes. A
heated centrifuge maintained at 56 oC is ideal, and
centrifuge quickly.
• • Immediately transfer the supernatant elute into another
test tube and test against a panelof cells for the presence
of antibody in parallel with the last wash supernatant.
• • If elute is used same day, it can be prepared
in saline, if storage is anticipated elute is
prepared in AB serum or in 6% albumin.
• • Last wash supernatant is tested for antibody
in parallel with elute to ensure that any eluted
antibody is not contaminants from residual
serum.
• ELUTION BY THE USE OF ORGANIC SOLVENT
(ETHER, XYLENE)
• Organic solvent disrupt antibody-antigen
bonds by lowering the surface tension of the
liquid media,thereby reversing forces needed
to hold antigen and antibody together. Organic
solvent are more sensitive because they
remove more antibodies from RBCs in elute
for testing antibodies.
• ELUTION BY ETHER
• It is useful in separation of warm-reactive auto-and
alloantibodies (IgG) from red cells which are positive to
direct antiglobulin test (DAT) viz. in Rh- HDN, immune
hemolytic anemias andsuspected transfusion reaction etc.
• Materials
• • Diethyl ether (reagent grade or anesthesiologic grade).
• • Packed DAT positive red cells (2 ml), washed 6 times in
saline.
• • Supernatant from last wash from antibody coated red
cells.
• Method
• 1. Preparation of supernatant from last wash
• • Wash the antibody red cells from which the elute
is to made, six times in a large volume of saline.
• • Recover the last wsh as describe above in step 1
in Heat Elution method.
• 2. Preparation of elute
• 1. Add an equal volume of saline to the washed
packed red cells and mix.
• 2. Add ether in an amount equal to the total volume of red
cells plus saline. The
• ether must be be fresh; the appearance of brown colour of
the ether indicates that the ether is oxidized and the elute
will be useless.
• 3. Stopper and mix the tube by repeated inversion for 1 min.
• 4. Carefully remove the stopper to release the volatile ether.
• 5. Incubate at 37 oC for 30 min. This step is optional, but it
will result in a more potent elute.
• 6. Centrifuge the tube at 1000x g (3400 rpm) for 10 min. The
tube will then contain
• 3 layers-clear ether on the top, red cell stroma in the middle
and haemoglobin strained elute at the bottom.
• 7. Aspirate and discard the top layer.
• 8. Carefully pierce the middle stroma layer with a pipette
and aspirate the bottom hemoglobin stained layer below the
stromal layer into a clean tube.
• 9. Incubate the elute in the unstoppered tube at 37 oC for 15
min and periodically bubble air through elute by pipette to
drive off residual ether.
• 10. Test the elute for the presence of antibody in parallel
with the last wash supernatant
• • Last wash supernatant is tested in parallel with elute to
ensure that any eluted antibody is not contaminant from
residual serum.