ELISA to evaluate plasma anti-asparaginase IgG concentrations in patients with acute lymphoblastic leukemia

Abstract

The development of antibodies to asparaginase may attenuate the pharmacologic effect of asparaginase treatment, may be associated with hypersensitivity reactions, and may necessitate switching to a different commercial asparaginase preparation for current or future therapy. Thus, development of an ELISA for measurement of anti-asparaginase antibody levels is important in the clinical setting. An anti-asparaginase antibody reference was established by screening 65 plasma samples from six patients with acute lymphoblastic leukemia (ALL) who had recently developed a hypersensitivity reaction to Escherichia coli or Erwinia chrysanthemi asparaginase therapy. Twenty-one plasma samples were selected for the anti-asparaginase antibody reference pool. Five micrograms per milliliter of commercial E. coli and Erwinia asparaginase and 10 μg/ml of E. coli asparaginase conjugated with polyethylene glycol (PEG asparaginase) were found to be optimal as coating antigen concentrations. Anti-asparaginase antibody concentrations were determined using a commercial polyclonal goat anti-human IgG horseradish peroxidase conjugate. The antibody reference curves were linear in a range of absorbance from 0.1 to 1.5 O.D. units for dilutions from 1:1600 to 1:51,200. Inter-assay coefficients of variation were 9.04, 14.7 and 13.0%, and intra-assay coefficients of variation were 1.44, 4.43 and 3.28% for antibodies against E. coli, Erwinia, and PEG l-asparaginase, respectively. The cut-off for positivity in plasma was determined as mean+2 S.D. of the optical density values for plasma from untreated healthy volunteers. Measurement of specific IgG by this ELISA allows for the evaluation of plasma anti-asparaginase antibody concentrations in patients receiving one or more of the multiple commercial l-asparaginase preparations.