- Single-wash protocol reduces assay time but maintains high sensitivity, speciﬁcity and reproducibility
- Using the SPECTROstar Nano and MARS data reduction software, simpliﬁes assay detection and analysis
Table of contents
Since its introduction in the early 1970’s the enzyme-linked immunosorbent assay (ELISA) has become one of the most reliable and relied upon biochemical assays1. Detecting the presence (and quantity) of an analyte has proved an invaluable tool as a diagnostic in medicine or in basic research spanning nearly every biological discipline.
The main drawbacks of the ELISA approach have always been the relatively low throughput and the tedium of multiple wash steps that normally take 3-5 h to perform. Abcam’s SimpleStep ELISA® technology addresses these issues by streamlining the process to a semi-homogeneous format that results in a simple 90-minute, single wash protocol.
Here, we describe the detection of this innovative ELISA by the SPECTROstar Nano. When combined with MARS data analysis you will get your great results in the fastest, easiest way possible.
Examplary data are shown for three important targets:
- Human Pro-Collagen I alpha: The protein is a component of type I collagen that is found in bones and cartilage. Hence, it is implicated in diseases characterized by fragile bones such as osteogenesis imperfecta or osteoporosis.
- Human IL6: Interleukin 6 (IL6) is an inﬂammatory cytokine that is capable of inducing maturation of B-cells and fever. Therefore, it is often used as an inﬂammation marker.
- GFP: Green ﬂuorescent protein (GFP) is characterized by its ability to ﬂuoresce in the green spectrum when excited by blue light. It is a popular protein, when coupled to a protein of interest.
The sandwich ELISA uses an antibody pair that bind to ing settings: distinct parts of the analyte. One antibody coats the bottom of the well and serves to capture the analyte. Following capture, and requisite washing, the detector antibody is added. Finally, after additional washing, the detection can be performed.
Figure 1 shows how SimpleStep ELISA® technology enables the sandwich ELISA to be performed semi-homogeneously by the addition of an immobilization antibody.
Materials & Methods
- SPECTROstar Nano
- SimpleStep ELISA® kits
- Human Pro-Collagen I alpha 1 ELISA kit (ab210966)
- Human IL-6 ELISA kit (ab178013)
- GFP ELISA kit (ab171581)
For all kits, samples were handled in accordance with instructions2. For all tests, standard curves were prepared based on an 8-point dilution series using duplicate samples. Pro-Collagen 1 alpha and IL-6 used a 2-fold dilution series with starting concentrations of 2000 and 500 pg/mL respectively. GFP used a 3-fold dilution series with a starting concentration of 2000 pg/mL. Assay reproducibility was determined using biological replicates, which included 2 different dilutions of human serum for Pro-Collagen I alpha, 2 dilutions of stimulated peripheral mononuclear blood cells (PMBC) supernatant for IL-6, and 143B cells spiked with 500 and 250 pg/mL of GFP.
Plates were read on a SPECTROstar Nano with the following settings:
|Optic settings||Absorbance spectrum, Endpoint test|
|Wavelength range (step width)||400-700 (2) nm|
|General settings||Number of flashes||45|
|Settling time||0.2 s|
Results & Discussion
BMG LABTECH microplate readers are equipped with a spectrometer for absorbance detection. Collection of spectral data can be done in the same time as reading a single wavelength. This allows easy evaluation of data quality (Figure 2). MARS data analysis templates allow automatic wavelength selection including peak, off-peak and reference wavelengths, for additional calculations that are also performed automatically by the system.
Figure 3 demonstrates how data from the SimpleStep ELISA® kit conforms to the suggested 4-parameter ﬁt. An R2 of at least 0.9995 was seen for all three kits used.
Table 1 shows the results of sensitivity and reproducibility metrics calculated for all 3 kits. These results indicate that SimpleStep ELISA® kits do not sacriﬁce performance while improving ease-of-use taking only one-third of the time to complete than a typical ELISA.
Table 1: SimpleStep ELISA® Sensitivity and Assay Precision
MDD (minimum detectable dose) = recalculated concentration of AvgBlank+2*SDBlank. Blank (n=15), bio-replicates (n = 8).
|MDD (pg/mL)||%CV of Blanks||%CV of bio-replicates Dil. 1||%CV of bio-replicates Dil. 2|
SimpleStep ELISA® kits offer excellent sensitivity and performance while saving signiﬁcant time and effort. Reading microplates with the SPECTROstar Nano provides notable data quality while offering the convenience of linking data analysis to MARS templates, for immediate and accurate results.
1. Lequin, R.M. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clin. Chem.(2005) 51: 2415-2418.
2. SimpleStep ELISA® kits: www.abcam.com/kits/simplestep-elisa-kits