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Singulex Digital Single Molecule Counting

The Core is developing new assays with the Singulex Erenna Reader. Only a few similar instruments currently are installed in academic labs. The Singulex system is based on digital single molecule counting. It can be thought of as a “smart” ELISA reader with a greatly extended lower analytical measurement range. The purpose of this research is to increase sensitivity of the assay and to improve reproducibility at the limits of measurement. Input into the reader is a standard immunoassay such as a completely conventional two-site insulin ELISA immunoassay. This means that many current assays can be candidates for improvement without materially altering the ELISA itself. However, signal detection and processing is radically different. Typical current ELISAs for insulin are based on principles developed 50 years ago. Exciting light is continuously admitted into the measuring chamber and fluorescent emitted light is continuously detected with a monochrometer in analog format. For example, the optical density of a sample light emitted might be 0.600, 0.603, and 0.594 when measured over time, similar to the varying intensity of an old analog television signal. This process is substantially improved with digital single molecule counting. Fluorescent samples are solubilized and slowly withdrawn from the sample well into a laser detector. This ensures that only a small amount of solubilized sample is analyzed at one time and that the maximum amount of information is obtained from it. Incipient light is pulsed into the sample and the resultant amplified emission photons are counted. A single pulse of incipient light might generate 600 closely-spaced emission photons over microseconds, but this burst is still an analog signal and it will vary over time. The detector electronically filters the output light using several criteria: It must have a higher photon density than when there is no incipient light; it must have a time course consistent with excitation from the previous light pulse; it must have a photon number near that expected from the excitation photons; it must undergo internal consistency checks with respect to amplitude and clustering. After photons pass these criteria they are digitized. For example, if a sample has 600, 603 and 594 emission photons in clusters after three pulses of exciting light, the detector records one molecule of detected labeled material. This is a digital output that can resemble a “needle in a haystack” in its ability to be detected above a large nonspecific background.

Core Lab currently offers mouse, rat and human insulin by the Singulex method. These assays are particularly useful for very small sample volumes encountered in work with recombinant mice and in circumstances where insulin levels are suppressed and conventional insulin immunoassays cannot detect it. The Table below shows the assay plate with output reported either by the conventional ELISA method (total photons at the correct wavelength) and the digitized analysis reporting detected events (single labeled molecules). Typical RIA and automated assays use 100 µl of sample and some instruments require a total volume of 250 µl to account for programmed pipetting. The standard amount for a Singulex insulin assay is only 5 µl, the amount used in the experiment reported in the Table. Singulex mouse insulin demonstrates the increased sensitivity obtainable with detection at 19.5 pg/ml using digital single molecule counting vs 625 pg/ml using total photon output. Thus, digital single molecule counting can be used to detect mouse insulin with high sensitivity on small volumes, as is required to quantify fasting insulin levels in mice. Core Lab uses 384-well plates and has performed 25 Singulex mouse insulin assays for clients.

Table. Singulex Mouse Insulin Assay Vs. Usual ELISA

Insulin Concentration (pg/ml) Detected Events (Single Molecules) Total Photons (Usual Assay Output)

 

0
19.5
39
78
156
312
625
138
212
335
695
1754
4579
9576

26,650,000
27,129,000
26,886,000
27,038,000
27,584,000
29,926,000
38,300,000

Core Lab has also begun studies on the Singulex platform for VEGF-a, a protein that is closely linked to diabetic vascular and eye disease. Since VEGF-a is found in platelets and is present in high concentration in serum, we have validated the use of platelet-poor EDTA plasma and platelet-stabilized citrated plasma for the measurement of circulating VEGF-a. In a group of 117 subjects with metabolic syndrome, VEGF-a was positively correlated with serum insulin and negatively correlated with plasma glucagon levels. These data are among the first to relate VEGF-a levels to components of the metabolic syndrome and suggest that the microbead-based, two-site Singulex VEGF-a assay may be a tool to further investigate the pathogenesis of diabetic microvascular disease and atherosclerosis.

Washington University has an agreement with Singulex to allow collaborative development of trace analytes and biomarkers and we invite your input for future work.