Erenna® Mouse (Plate-based) Insulin Immunoassay Kit from Singulex®

In 2010, the U. S. population rate of diagnosed diabetes was 8.3% of the population (or 27 million Americans) and has been steadily increasing [1]. The estimated direct medical costs of diabetes were $116 billion in 2007, while indirect costs due to lost work time, disability and premature death, added another estimated $58 billion. The National Institutes of Health will distribute just over one billion dollars for diabetes research in 2011 [2], with some part of this funding being applied to animal studies. Critical to the success of studies using rats and mice are sensitive assays to measure insulin in plasma or serum.

The Erenna® Mouse, Plate-based Insulin Immunoassay Kit from Singulex® combines the advantages of a small sample volume requirement (5 ul of plasma or serum per assay), together with a broad standard curve range (20 to 5000 pg/mL, see note below *). The lower limit of quantification is 20 pg/ml. The assay shows good precision over the entire dynamic range (CV < 10%), and inter-assay CV’s of 7%, 5% and 11% for high (mean level = 954 pg/mL), mid-range (mean = 563 pg/mL) and low (153 pg/mL) control EDTA plasma samples, respectively.

Additional advantages include a 1-step capture and detection step followed by a single wash step. In addition, as many as 280 samples can be assayed on one plate, with remaining wells reserved for standards and controls. The assay also detects rat insulin.

The kit uses a quantitative fluorescent sandwich immunoassay technique. A capture antibody specific for mouse insulin has been pre-coated onto a 384-well assay plate. The user pre-mixes 1 part standard or sample with 3 parts fluorescent dye–labeled detection antibody reagent and transfers 20 ul of the mixture to the assay plate well. (To expedite the loading, the mixing is done in a V-well 96 well plate, followed by transfer of premixed standards, controls and samples to the assay plate, using an 8-channel pipette.) During incubation, the insulin present in the sample binds to the solid phase capture antibody. The detection antibody will recognize and bind to insulin captured onto the plate surface. Unbound components are washed away during the subsequent wash step. Elution buffer is added, and the sample is incubated to dissociate the dye-labeled detection antibodies from the plate. The plate is loaded into the Erenna Immunoassay System, which counts the freely circulating fluorescent molecules. The number of labeled detection antibodies counted is directly proportional to the amount of insulin present in the sample.

The concentration of insulin in unknown samples is interpolated off the standard curve using the Sgx Link™ software. Curve-fitting options include logistic 4- and 5-parameter regression methods. The curve fitting algorithm combines data from single molecule “detected events” as well as total photon fluorescent emission. The contribution of the different signal types are weighted depending on the intensity of the signal. The power of this system is that it achieves a 3- or 4-log dynamic range of detection, which is especially useful for measuring insulin levels that vary broadly depending on whether animals are fasting or fed.

In summary, the advantages for this assay are its sensitivity, low sample volume requirement, broad standard curve range, and 1-step capture and detection step.

Figure 1:

Fig 1A shows accuracy of curve fit for full range of mouse insulin standard curve.

Fig 1B shows low end accuracy of curve fit (< 312 pg/mL).

*Note: This user extends curve with a 10,000 pg/mL standards with excellent accuracy (expected vs observed concentration).

References:
[1] National Diabetes Information Clearinghouse (NDIC), U.S. Department of Health and Human Services.
[2] Research Portfolio Online Reporting Tools (RePORT), U.S. Department of Health and Human Services.