Enhancement of Two Commercial Insulin ELISAs using a Fluorescent Reporter System

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 834-867-Islet Biology
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-856
Bruce D Gaylinn*, Natalie N Walker, Susanna R Keller and Michael O Thorner
University of Virginia Health System, Charlottesville, VA
A commercial two antibody sandwich assay for mouse insulin (ALPCO # 80-INSMSU-E01) measures insulin levels in small samples (assay range = 0.188 to 6.9 ng/mL for 5 ul plasma). However, insulin levels in fasted mice were often still too low to measure with this assay.  By switching from the colorimetric reporter included in the kit to a fluorescent reporter (Amplex Red fluorogenic peroxidase substrate, Thermo Sci) we were able to improve assay sensitivity and detect insulin in nearly all fasting mouse samples using 5 or 10 ul of plasma.  We encountered similar difficulties in detecting insulin in samples from fasted human subjects using an Immulite 2000 human insulin assay (fasting samples were off-scale low).  When assaying these same samples with the ALPCO ultrasensitive human insulin ELISA (ALPCO # 80-INSHUU-E01.1) most samples were now detectable, but some fasting samples had unidentified interferences causing them to be off scale low while after meal samples were off scale high.  After incorporating a wash step between the sample incubation and the second antisera, and in addition using fluorescent detection (QuantaBlu fluorogenic peroxidase substrate, Thermo Sci) nearly all insulin levels measured within the range of the modified ALPCO ultrasensitive human insulin ELISA.  If samples were diluted their measured insulin levels decreased proportionally.  Although the fluorogenic substrate did not improve sensitivity in this assay, it expanded the dynamic range (avoiding saturation) such that all samples remained on-scale from fasting to fed without requiring dilution of samples (unmodified assay range 0.15 to 20 μIU/mL versus  modified assay range 0.15 to 300 μIU/mL).  Thus, replacing colorimetric with fluorogenic peroxidase substrates in commercially available ELISA kits can improve sensitivity (mouse insulin assay) or extend the range of the standard curve (human insulin assay). In addition, some interferences can easily be removed with an additional wash step.  (300 μIU/mL human insulin standard and additional human insulin assay kits to complete this test were kindly supplied by ALPCO).

Disclosure: MOT: Founder, Ammonett Pharma, Recipient Award, Novo Nordisk, Advisory Group Member, Pfizer, Inc., Advisory Group Member, Ipsen, Advisory Group Member, Chaisma. Nothing to Disclose: BDG, NNW, SRK

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: NIH Grant 1R01DK076037 awarded to MOT; 1R01DK081471 awarded to SRK