Fixation of Antigen–Antibody Reaction by Glutaraldehyde

Abstract

GLUTARALDEHYDE, a di-aldehyde fixative, has been found to bind antibodies rapidly and permanently to antigens of red blood cells. The system investigated and described in this report was similar to that used by Evans et al.¹ in which purified cold agglutinins (anti-I), labelled with iodine-131 or iodine-125, were incubated with human adult (I-positive) or umbilical cord (I-negative) erythrocytes. Cold agglutinins react strongly with I-positive cells at low temperatures, but at 37° C they have almost no avidity and are rapidly released into the fluid medium. Even at low temperatures the avidity of cold agglutinin for I-positive erythrocytes is not sufficient to prevent elution of significant amounts of antibody as the cells are washed in large volumes of buffer. During kinetic and equilibrium investigations of cold agglutinins it became apparent that ordinary techniques for “instantaneous sampling”, that is, rapid centrifugation of samples, collecting of supernatant, and washing cell buttons one to three times, did not prevent continuing shift of antibody from cells to supernatant nor allow measurement of extremely rapid change effected by variations in temperature. The use of glutaraldehyde to circumvent these difficulties was considered because this fixative has two reactive aldehyde groups which might attach simultaneously to antibody and antigen and bind them firmly together.