Use of horseradish peroxidase-labelled antiglobulin for the colorimetric quantitation of erythrocyte antibodies.

Horseradish peroxidase (HRP)-labelled antiglobulin has been used extensively as a histochemical marker. In the method described the stable reaction product formed by using 3,3'-diaminobenzidine (DAB) as the hydrogen donor in the peroxidase reaction was dissolved in a fluoro alcohol, 1,1,1,3,3,3-hexafluoroisopropanol (HPF). Reproducible standardisation curves resulted with HRP. Based on these observations a colorimetric assay procedure was developed to quantitate the amount of antibody coating red cells. This was achieved by treating the coated red blood cells with HRP-labelled rabbit antihuman IgG followed by hypotonic lysis separating the ghosts for colour development with the DAB reagent, solubilisation in HFP, and reading at 450 nm. Preliminary estimates of the number of anti-D molecules on Rhesus positive red cells were found to approximate the results reported using radioiodinated antibodies. The smallest number of anti-D molecules detected was 350 per red cell. Qualitative studies indicate that this procedure can be applied to other blood group systems.