Technology - Applications

ELISA is a common method for detecting the presence of an antigen or an antibody in a sample. The DropArray™ technology enables semi-automated miniaturized ELISA through its unique method of well washing, which allows uniform and parallel washing of small assay volumes with no cross-talk between wells and minimal carryover between washing steps. Using DropArray™ technology, ELISAs can be performed with just 2 µL of sample and reagents, enabling cost-effective ELISAs, even when samples are scarce and reagents are expensive.

Reasons for performing ELISA on DropArray™ ELISA Plates

• Savings in assay time (from antibody coating to the end of assay) of at least 60%
• Reduction in the quantity of samples required by 50 times (100 µL to 2 µL)
• Reduction in the quantity of reagents (coating and detection antibodies) by 50 times
• Greater assay sensitivity and dynamic range from the use of a fluorigenic detection substrate
• Reduction in assay cost (including reagents and plate costs) by up to 80% (compared to commercial ELISA kits using 96 well plates).

We compared ELISA performed on a 96-well plate to the same ELISA performed on a DropArray™ ELISA plate using the DuoSet ELISA development systems from R&D Systems.The 96-well plate assay was performed according to instructions from the manufacturer with one minor modification: the colorimetric horseradish peroxidase (HRP) substrate 3,3',5,5'-Tetramethylbenzidine was replaced with the fluorigenic HRP substrate Amplex UltraRed (Invitrogen Inc). The same procedure was followed for assays on the DropArray™ ELISA Plate employing DropArray™ technology to enable washing of wells and to prevent assay evaporation.

Human leptin ELISA

Figure 1: Comparison of human leptin ELISA performed in a 100 µL volume on a 96 well ELISA plate from R&D Systems and in a 2 µL volume on a DropArray™ ELISA Plate

Examination of cross-contamination between wells on DropArray™ ELISA plate

To investigate the reliability and robustness of DropArray™ technology, and whether a sample dispensed in one well will contaminate samples in neighboring wells, we dispensed from well column 2 to well column 11, samples containing 62.5 pg/mL human leptin (•) and 0 pg/mL human leptin (;">•) alternately. In wells from column 12, we dispensed a sample containing a saturating concentration of human leptin (• 4000 pg/mL) and in wells from column 1, the same steps were performed as in other wells, except that no sample (•) was dispensed (Figure 2).

Figure 2: Diagram showing the well contents of a DropArray™ ELISA Plate S48 used to examine cross-contamination between wells.

The result of the contamination test is shown in figure 3. The graph in black shows the raw data while the graph in red shows the same data after subtracting the data from well column 1 (which contains no sample) from all the other wells. The data show very reliable discrimination between samples containing 0 pg/mL human leptin and 62.5 pg/mL human leptin, which happened to be the lowest concentration of human leptin detectible according to the R&D Systems ELISA platform. The greater data variability at 0 pg/mL leptin is due to greater noise when values are very close to zero.

More importantly, when we examine whether wells in column 12 (which contains samples with a saturating 4000 pg/mL leptin), would affect neighboring wells in column 11 (which contains samples with 0 pg/mL leptin), we did not observe any trace of contamination between neighboring wells.

Figure 3: Graphs showing variation in fluorescence intensity across different well columns of a DropArray™ ELISA plate S48. Wells in even columns 2 to 10 contain samples with 62.5 pg/mL leptin while wells in the odd columns contain samples without leptin. Well column 12 has samples containing a saturating 4000 pg/mL leptin. The raw fluorescence signals from the well columns are plotted as a black line while the fluorescence signals after subtracting the signal from a well column containing no sample are plotted using a red line.