Ryan J. Hansen

An ELISA for quantification of murine IgG in rat plasma: application to the pharmacokinetic characterization of AP-3, a murine anti-glycoprotein IIIa monoclonal antibody, in the rat

An enzyme-linked immunosorbent assay (ELISA) has been developed to determine concentrations of murine IgG in rat plasma. Specifically, the assay was developed to measure a murine anti-glycoprotein IIIa antibody (AP-3) in rat plasma to facilitate future investigations of AP-3 pharmacokinetics and pharmacodynamics in the rat. The working range of the assay is 15-100 ng ml(-1), corresponding to a limit of quantification of 1.5 microg ml(-1) in rat plasma. The assay was validated with respect to accuracy, precision, and cross-reactivity with both pooled rat and mouse IgG. Intra-assay recoveries of AP-3 in rat plasma ranged from 93 to 103%, with CV% values ranging from 5.2 to 8.5%. Inter-assay recoveries of the plasma AP-3 samples ranged from 107 to 119% with CV% values ranging from 17.7 to 25.1%. The assay has no appreciable cross reactivity with pooled rat IgG and full cross reactivity with pooled mouse IgG, making this an ideal assay to determine plasma pharmacokinetics of mouse antibodies in the rat. The assay was used to determine the pharmacokinetics of AP-3 in a Sprague-Dawley rat.

IVIG effects on autoantibody elimination

We have read with interest the paper by Dr Simon and Späth, which reviews mechanisms of intravenous immunoglobulin (IVIG) action (1). In general, the review is very well written, and the authors have provided a thoughtful and concise description of a variety of proposed mechanisms of IVIG action. However, the authors have incorrectly interpreted results recently presented by our laboratory regarding the effects of IVIG on antiplatelet antibody elimination in mice. Dr Simon and Späth commented that our work showed that within ...FcRn knockout mice IVIG did not slower down the clearance of a monoclonal anti-platelet Ab, while in the wildtype animals it did . Our data, in fact, showed the opposite result. The IVIG increased the rate of elimination of antiplatelet antibody within wild-type animals, with clearance increasing from 5.2 ± 0.3 to 14.4 ± 1.4 ml/day/kg (2). This result was consistent with our observations in a rat model of immune thrombocytopenia, where IVIG (2 g/kg) increased the clearance of anti-platelet antibody to 2.4-fold that seen in saline-treated controls (3). However, in FcRn knockout mice, we found that IVIG did not increase the clearance of anti-platelet antibody (2). As such, our results were opposite of the description provided by Dr Simon and Späth; nonetheless, our observations are consistent with the hypothesis that IVIG increases antibody elimination via competitive inhibition of FcRn.

Survival and tumorigenesis in O6-methylguanine DNA methyltransferase-deficient mice following cyclophosphamide exposure

O(6)-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/- background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-proficient or -deficient mice (either Nf1+/+ or Nf1+/-) were given 6 weekly injections of a maximally tolerated dose of CP (250 mg/kg) or vehicle and followed for 15 months. CP-treated mice had more deaths than control mice but there was no difference in the long-term survival between Mgmt+/+ and Mgmt-/- mice (12 of 83 Mgmt+/+ mice died compared to 12 of 80 Mgmt-/- mice, disregarding Nf1 status). Lymphomas and adrenal tumours were the most frequent malignancies. Interestingly, CP-treated, Mgmt-deficient mice developed fewer tumours than controls. Ten of 71 (14%) Mgmt-proficient mice developed tumours after CP treatment compared to only 2 of 68 (3%) Mgmt-deficient mice (P = 0.02). Mgmt-/-, Nf1+/- mice developed fewer tumours (1 of 35, 3%) following CP compared to Mgmt+/+, Nf1+/- mice (7 of 37, 19%) (P = 0.03). Hypoxanthine-guanine phosphoribosyltransferase mutation assays showed no significant increases in mutant frequencies in Mgmt-/- (18.1 x 10(6)) compared to Mgmt+/+ mice (12.9 x 10(6)). These data indicate that MGMT deficiency does not protect against long-term toxicity or mutagenicity from CP and appears to attenuate the occurrence of CP-induced tumours in an Nf1+/- background.