Evaluation of methods for quantitating erythrocyte antibodies and description of a new method using horseradish...

Concentrates of anti-D and rabbit anti-human globulin (AHG) prepared by standard elution and ammonium sulphate precipitation methods were labelled with 125I and 131I and horseradish peroxidase (HRP), respectively. The number of anti-D molecules attached per rbc ranged from 12,500 to 21,300 for the various phenotypes studied and the label of D-negative rbc never exceeded 3.4% of these values. With 131I-AHG the uptake by control D-negative cells averaged 13% of the uptake by D-positive cells. It was also found that the average ratio of AHG molecules reacting with each IgG molecule was between 2.6 and 3.3 in free solution regardless of the label but was between 3.4 and 7.5 on rbc and ghosts with 131I-AHG and 3 or lower with HRP-AHG. A colorimetric procedure for quantitating IgG antibodies on rbc ghosts is described using HRP-labelled-AHG and o-dianisidine as the hydrogen donor. The method is very sensitive and useful for the detection of coating antibodies but cannot be used for precise quantitation because about 50% of the IgG molecules on rbc are lost in preparing the ghosts. An AutoAnalyzer method for estimating the number of antibodies attached to red cells is briefly described. Direct measurements of numbers of antigens receptors with radiolabeled specific antibodies gave the most reproducible results. Labelled rabbit AHG was not as good because the ratio of AHG to IgG varied. The AutoAnalyzer method may prove useful because of its convenience.