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 microg/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.