Measurement of the affinity of anti-D in the serum of immunized mothers and in immunoglobulin preparations with unlabeled antibodies

Abstract

BACKGROUND: Few data are available on the affinity of maternal anti-D responsible for hemolytic disease of the fetus and the newborn (HDN) and of anti-D used for the prophylaxis of that disease. A method was recently described to measure the affinity (Ka) of untagged anti-D monoclonal antibodies (MoAbs). In this work, the same method was applied to determine the Ka of polyclonal anti-D. STUDY DESIGN AND METHODS: O R1r red blood cells (RBCs) were sensitized with increasing concentrations of native anti-D in serum samples from immunized mothers and donors and in RhIG preparations. At equilibrium, the amount of anti-D bound to RBCs was measured by enzyme-linked immunosorbent assay. Scatchard and Langmuir equations were used to determine Ka. RESULTS: The experimental data fitted well with the Scatchard equation (mean r2 = 0.95) but a better correlation was observed with the Langmuir equation (mean r2 = 0.99). The mean K a of anti-D in 11 maternal serum samples, in 6 immunized donors, and in 5 lots of RhIG were 5.6 × 108 per M (from 2.8 × 108 to 12 × 108/M), 3.9 × 108 per M (from 1.5 × 108 to 6.8 × 108/M), and 3.4 × 108 per M (from 3.1 × 108 to 4.2 × 108/M), respectively. The comparison of anti-D affinity in 5 cases of HDN with fetal anemia and in 6 cases of HDN with postnatal anemia showed no significant difference. CONCLUSION: The method previously described for anti-D MoAbs was applied to polyclonal anti-D present in the serum of immunized subjects and in immunoglobulin preparations. The experimental data fitted well with the Langmuir equation, and the affinity of polyclonal of anti-D was measured with accuracy.