Immunochemical characterization of a polyclonal human antibody to factor IX

Abstract

Inhibitors of clotting factors occuring in humans are often antibody molecules synthesized in response to exogeneous proteins used in replacement therapy. Extensive studies of inhibitors to factor VIII indicate such antibodies may be monoclonal or polyclonal in nature. To date, only one factor IX inhibitor has been subjected to detailed immunochemical analysis and it appears to be a monoclonal IgGA lambda antibody. We have discovered a second inhibitor of factor IX in a patient with severe hemophilia B and have subjected it to immunochemical analysis. Studies on this second inhibitor have been carried out before and after an anamnestic response. Column chromatography, preparative zone electrophoresis, and specific inhibitor neutralization assays using monospecific heterologous antisera to human immunoglobulin classes, subclasses, and light-chain types indicate that the antibody is of the IgG class and contains both kappa and lambda light chains and probably all four IgG subclasses. Thus, the inhibitor appears to be polyclonal by immunochemical and structural criteria. In addition, preparative isoelectric focusing of pre- and postanamnestic inhibitor samples indicates that recruitment of new clones of IgG antibody occurs as a result of anamnesis. It is conceivable that an antibody initially restricted in immunoglobulin subclass became polyclonal following an anamnestic response.

Human factor IX inhibitors: immunochemical characteristics and treatment with activated concentrate

“…This factor IX antibody was described as monoclonal. A second report (Reisner et al 1977) presented the results of a similar analysis suggesting that the factor IX antibody in that case was polyclonal. This inhibitor was neutralized by specific antiserum to IgG, by antiserum to kappa light chains and by antiserum to lambda light chains.…”

Alloantibodies to Factor IX in Haemophilia B Characterized by Crossed Immunoelectrophoresis and Enzyme‐conjugated Antisera to Human Immunoglobulins

“…The inhibitors from patients P.B.W. and R.J. have previously been characterized by preparative zone electrophoresis and immunoneutralization (Pike et al, 1972;Reisner et al, 1977). Plasma from a patient with severe haemophlia B and no inhibitor to factor IX was used as control plasma.…”

“…predominantly IgG antibodies with K light chains (Lechner, 1971), one patient had a polyclonal IgG4A antibody (Pike et al, 1972) and one patient had a polyclonal IgG antibody containing both K and 1 light chains (Reisner et al, 1977). The antibody nature of these inhibitors was determined by neutralization of inhibitor activity with antisera to Ig classes, subclasses and light chain types.…”

“…The antibody nature of these inhibitors was determined by neutralization of inhibitor activity with antisera to Ig classes, subclasses and light chain types. This technique is not suitable for the study of low-titre inhibitors (Reisner et al, 1977).…”

Results in three Australian haemophilia B patients with high‐responding inhibitors treated with the Malmö model

“…The incidence of inhibitor development is lower in haemophilia B than in haemophilia A, and figures ranging from 2.5% to 16% have been reported for severe cases of haemophilia B [l-31. Like F VIII inhibitors, F IX inhibitors are antibodies of IgC class, and usually of subclass 4 [3][4][5][6]. Clinically, patients with inhibitors can be divided into 'high-responders', who manifest a strong anamnestic response after exposure to F VIIMX, and 'low-responders', who manifest little or no anamnestic response.…”