(2) The intended purpose of absorption dictates, to some extent, the
necessary techniques. When interfering autoantibodies are to be removed, several
absorptions may be necessary, using a fresh aliquot of cells each time. If the goal is to
separate mixed antibodies, the cells must be selected to contain only one of the
antigenic determinants in question, preferably, that giving the strongest possible
reaction with the antibody.
b. Serologic Considerations.
(1) The temperature should be optimal for the activity of the antibody to be
removed. If both cold and warm antibodies are present, separation is enhanced by
absorbing the cold activity at 4C, or the warm activity, at 37C.
(2) The usual volume is one part undiluted serum to one volume, washed,
hard-packed, cells. For complete absorption of high-titered antibodies, diluted serum
may be more efficient. With weak antibodies, the volume of undiluted serum should be
greater than the volume of RBCs.
(3) Specific techniques for warm and cold absorption of autoantibodies have
been given in appropriate sections.
(1) STEP 1. Wash selected RBCs, at least three times, with isotonic saline.
The cells may be heterologous for antibody separation, and identification, or
autologous, for autoantibody removal. Completely remove supernatant saline after last
washing to avoid dilution of the serum. If the RBCs were in contact with serum or
plasma containing cold antibodies, washing at 37 may be indicated.
(2) STEP 2. The larger the area of contact, the better the absorption. This
is particularly true in prolonged absorption, when the RBCs settle. Use a
large-bore tube. Stopper well and lay tube on side. For volumes of cells and serum,
see b (2) above.
(3) STEP 3. Incubate the packed RBCs and serum at optimum
temperature, for the reaction, for 30 to 60 minutes, agitating frequently.
(4) STEP 4. Centrifuge. If absorption is at 4C, use prechilled centrifuge
cups, or a refrigerated centrifuge. Centrifugation at higher temperatures will result in
elution of antibody from the RBCs.
(5) STEP 5. Remove the serum immediately after centrifugation. When this
serum is used for additional tests, identify it as absorbed serum. If one serum is to
undergo absorption with more than one cell, the absorbed serum should also be
identified by the cell specificity, (for example, absorbed DccEE).