title: An ELISA method for detection of human antibodies to an immunotoxin.
abstract: BACKGROUND
  The use of biological molecules, such as immunotoxins, as pharmaceuticals is limited by the presence and development of human antibodies to these agents. This immune response can cause significant inflammatory-related toxicities and can interfere with the efficacy of the biological agent. Therefore, a clinically applicable method to detect these human antibodies is needed for screening patients prior to enrollment and for monitoring patients during treatment. The SS1(dsFv)-PE38 immunotoxin currently in clinical trials is a hybrid molecule targeted against mesothelin-expressing cancer cells via the Fv portion of a murine antibody linked to the Pseudomonas exotoxin (PE), which can inhibit protein synthesis leading to cell death. The objective of this study was to determine if an enzyme-linked immunosorbent assay (ELISA)-based method could be used to detect human anti-SS1(dsFv)-PE38 antibodies in patient serum.
  
  
  METHODS
  Human antibodies to the immunotoxin in serially diluted serum specimens were captured on immunotoxin-coated ELISA plates, and detected using a secondary goat antihuman antibody linked to biotin in combination with horseradish peroxidase linked to avidin D (HRP-Avidin). The color was developed with tetramethyl benzidine (TMB). Curves of optical density (OD(630)) versus dilution for 44 serum specimens were compared with positive and negative control serum specimens to classify the serum as positive or negative for anti-immunotoxin antibodies.
  
  
  RESULTS
  Ten out of the 40 patients screened were positive for anti-immunotoxin antibodies. Repeated testing of seven samples produced the same results in two independent experiments. The first two patients treated with the immunotoxin developed anti-immunotoxin antibodies during treatment. The results were in perfect concordance with a tissue culture-based neutralization assay performed by an independent laboratory.
  
  
  CONCLUSIONS
  An ELISA-based strategy using an immunotoxin to capture human anti-immunotoxin antibodies provides a consistently accurate technology for screening and monitoring patient serum specimens in clinical trials.
authors.affiliation: Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA. Doris-Benbrook@ouhsc.edu
authors.forename: Doris M
authors.initials: DM
authors.lastname: Benbrook
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006041:Goats; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D000922:Immunotoxins / Q000276:immunology*; D009500:Neutralization Tests; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D000922:Immunotoxins; C474515:SS1(dsFv)PE38