Oral Presentations - Federation of Clinical Immunology Societies

S20 Abstracts
may be altered in chronic T1D. However, it is still unclear if
this immune phenotypic variation is a cause or effect of
T1D. Using polychromatic flow cytometry to analyze whole
blood, we have extended to pediatric T1D patients the
findings by others that adult T1D cases have increased
expression of CD11b on monocytes as compared to
apparently healthy non-T1D adults. We examined 37 T1D
patients (age 9.2–18.3 years, median 14.4 years; median
time past diagnosis for non-newly diagnosed cases=4.3
years) and 63 healthy volunteers (age 9.9–60 years, median
34 years). CD11b expression (MFI) in HLA-DR+ monocyte
subsets were as follows: on CD16+ monocytes, 265±20 vs.
218±10 (pediatric T1D cases vs. non-T1D adults); CD16+
CD14+ monocytes, 1108 ±51 vs. 840±27; CD14++ ‘inflammatory’
monocytes, 1479±38 vs. 1146±54. Expression
levels of CCR2 on CD14++ monocytes were lower in T1D
patients than in healthy volunteers (MFI 62±2 vs. 75±2),
consistent with an activated monocyte phenotype. CD11b
and CCR2 expression differences were independent of ages
at diagnosis or at immunophenotyping. In order to help
distinguish between cause and effect and begin to explore
the possibility that extremes of these phenotypes may be
precursors to disease, we will analyze age-matched
subjects and unaffected siblings, and test the association
of alleles of known T1D susceptibility genes (e.g., HLA
haplotypes, INS, PTPN22, CD25, IFIH1, CTLA4) with monocyte-subset
phenotypes.
doi:10.1016/j.clim.2007.03.224
F.12 A High Specificity Competitive Europium Based
Assay for Autoantibodies of APS1 Patients Reacting
with Interferon Alpha
Li Zhang, Fellow, Barbara Davis Center, Denver, CO,
Raffaele Badolato, MD, PhD, Department of Pediatrics,
University of Brescia, Brescia, Jennifer Barker, Assistant
Professor of Pediatrics, Barbara Davis Center for Childhood
Diabetes, Denver, CO, Maureen Su Pra, University of
California, San Francisco Diabetes Center, San Francisco,
CA, Sunanda Babu, Research Associate, Barbara Davis
Center, Denver, CO, Mickie Cheng, MD, PhD, University of
California, San Francisco Diabetes Center, San Francisco,
CA, Tony Shum, MD, University of California, San Francisco
Diabetes Center, San Francisco, CA, Ehud Zamir, MD, The
Royal Victorian Eye and Ear Hospital, Victoria, BC, Canada,
Adam Law, MD, Ithaca Medical School, Ithaca, NY, George
Eisenbarth, Executive Director, Barbar Davis Center, Denver,
CO, Mark Anderson, Assistant Professor, University of
California, San Francisco Diabetes Center, San Francisco, CA
IFN-α autoantibodies have been described in autoimmune
polyglandular syndrome type 1 (APS1) patients. We have
established a competitive Europium time resolved fluorescence
assay for autoantibodies to IFN-α and have tested it in
a cohort of APS1 patients. Methods: The europium-ELISA
method utilizes plate bound IF α incubated with or without
competition with fluid phase IFN-α and sera. Anti-IgG
biotinylated antibody is added, followed by incubation with
streptavidin–europium and detection with time resolved
fluorescence (measured in counts per second, CPS). Seven
APS1 patients, 6 relatives, 71 non-APS1 Addison patients and
141 T1DM patients were tested as well as 100 normal
controls. The APS1 patients had multiple different mutations
in the AIRE gene. Results: Normal control CPS without
competition was 31,237∼17,328 CPS and delta with competition
was −6563∼10,303 CPS. The initial APS1 patient (used
to create the index, index 1.0) gave 394,063 CPS without
competition and a delta of 363,662∼31,587 CPS with
competition. Scatchard plot analysis revealed a high avidity
(Kd of 0.5 nM). The CPS, delta and index for 6/7 APS1
patients were strongly positive and above 3 standard
deviations of controls. Relatives were negative as were 71
Addison disease (non-APS-1) and 141 T1DM patients. The one
negative APS1 patient is presumed APS1 based on clinical
history. The assay has a sensitivity of 86% or greater and
specificity greater than 99.5%. Conclusion: IFN-α autoantibodies
are detected specifically in APS1 patients with
specificity greater than 99% using a competitive Europium
time resolved fluorescence assay.
doi:10.1016/j.clim.2007.03.225
F.13 Validation of Anti-Idiotypic Bridging ELISA to
Quantitate TRX4 (Anti-Human CD3) Mab Levels in
Human Serum
Francis Carmody, Preclinical Scientist III, Preclinical
Development, Cambridge, MA, Michael Slavonic, Preclinical
Scientist II, Tolerx, Preclinical Development, Cambridge, MA,
Adam O’Shea, Research Scientist II, Tolerx, Applied Research,
Cambridge, MA, Michael Paglia, Senior Manager Manufacturing,
Tolerx, Manufacturing, Cambridge, MA, Reema Gulati,
Research Scientist II, Tolerx, Applied Research, Cambridge, MA,
Sharon Li, Former Tolerx Employee, Applied Research,
Cambridge, MA, Herman Waldmann, Professor of Pathology,
Head of Department, University of Oxford, Sir William Dunn
School of Pathology, Cambridge, MA, Michale Rosenzweig,
Director Preclinical Development, Tolerx, Preclinical
Development, Cambridge, MA
TRX4 is an aglycosyl anti-human CD3μ monoclonal antibody
(Mab) that is being evaluated as a therapy for autoimmune
disorders such as new-onset type 1 diabetes. Our goal was to
develop a sensitive, high throughput assay to quantitate TRX4
serum levels that could be used as an alternative to a cell-based
assay that had been used in previous studies. Historically, TRX4
was detected with a polyclonal anti-human IgG after it bound to
the human T cell line, HuT 78. This method was both laborious
and lacked sufficient sensitivity. Two different approaches were
used to raise monoclonal antibodies to TRX4 complementarity
determining regions (CDRs). First, transgenic human CD3 mice
were tolerized to human IgG by two 1 mg IV doses and then
subsequently immunized with 100 μg IP of TRX4. For the second
strategy, BALB/c mice were administered 100 μg SC of TRX4 F
(ab)′2 fragments in incomplete Freund’s adjuvant. These
independent immunization strategies produced two non-competing
anti-idiotypic TRX4 Mabs that satisfied specificity
criteria. Significant serum interference was observed with
both the Mabs when used individually in ELISAs. However,
when used together, these anti-idiotypic antibodies were able to
capture and detect TRX4 in whole sera with no detectable