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Join experts Russell Neuner, PhD, and Daniel Schwartz as they explain how the SimpleStep ELISA® kit can decrease your assay time to just 90 minutes. Learn how the SimpleStep ELISA® kit provides specific and sensitive quantification of target protein with one simple antibody and sample addition step.
The term 'ELISA' stands for Enzyme-Linked Immunosorbent Assay. This technique is used to detect in quantity a specific analyte from a complex mixture or solution. Compared to other immunoassay techniques, such as western blotting, an ELISA provides better specificity and dynamic range.
The most common type of ELISA is the sandwich ELISA where two antibodies are used to measure a specific target protein in a biological sample, such as human serum or cell culture extract. In a sandwich ELISA, one antibody is immobilized to the bottom of every well in a microtiter plate. This antibody is called the capture antibody and, upon sample incubation, binds the target protein. An additional step incorporates a second antibody that can be labeled with a reporter enzyme, such as horseradish peroxidase or HRP. This antibody is called the detector antibody and completes the sandwich ELISA
How is SimpleStep ELISA different from a traditional ELISA?
The SimpleStep ELISA provides improved and differentiated performance characteristics, while still retaining the familiar process and standard data outputs of a traditional ELISA.
A SimpleStep ELISA utilizes a capture antibody that is conjugated to an affinity tag and a detector antibody that is conjugated to HRP. Together, these two antibodies complete the sandwich ELISA. In order for the sandwich complex to remain bound to the microtiter plate, every well in a SimpleStep ELISA is precoated with a highly-specific anti-tag monoclonal antibody. This antibody binds to the affinity tag conjugated to the capture antibody and is essential for this technology.
What are the benefits of a SimpleStep ELISA?
The SimpleStep ELISA technology has many great benefits for use in the lab: first, the SimpleStep ELISA protocol is, as the name infers, simple! Additionally, the SimpleStep ELISA has a shortened protocol time, having reduced complexity and a shorter assay duration allows for easier and faster data generation.
The SimpleStep ELISA retains the familiar process and standard data outputs of a traditional ELISA. This requires no special training or costly instrumentation. The liquid phase incubation kinetics of both the capture and detector antibody drives the assay to superior sensitivity. In addition, allowing both the capture and detector antibody to interact with the target analyte in solution, imparts a more efficient binding process. This is compared to having an immobilized capture antibody in a microtiter plate common to the traditional ELISA.
The SimpleStep ELISA has a single incubation step and a single wash step, having reduced the number of sample handling steps can lead to a greater reproducibility and accuracy in an assay. Finally, the SimpleStep ELISA technology allows for concurrent, parallel target analysis in a single microplate.
What is included in a SimpleStep ELISA kit?
Every SimpleStep ELISA kit from Abcam will contain the following reagents for use in your research:
• A SimpleStep ELISA microplate capable of performing 96 tests; this arrives pre-coated with a highly specific monoclonal antibody.
• A 5X cell extraction buffer; this is specially formulated for cell culture and tissue extraction samples.
• A concentrated 10X capture antibody, and concentrated 10X detector antibody, 10X wash buffer for use in the single wash step,
• TMB detection reagent and stop solution.
Additionally, every SimpleStep ELISA will include a protein standard. This is a highly purified protein used to generate a standard curve. Each standard curve is plotted using a 4-parameter logistic, non-linear regression model.
A protein standard is also very useful in an assay to measure or interpolate the amount of target analyte in an unknown sample. Every SimpleStep ELISA will include a sample diluent, and this is alternatively referred to as a standard diluent or calibrator diluent. A sample diluent is a specially formulated buffer that is optimized to correct for non-specific interactions, such as a matrix effect. SimpleStep sample diluents generate maximum recovery of target analyte in a variety of sample types.
Lastly, every SimpleStep ELISA will include an antibody diluent, this is alternatively referred to as an assay diluent or blocking buffer. The antibody diluent is a specially formulated buffer that is optimized to reduce non-specific antibody conjugate interactions. It eliminates any interference due to heterophilic antibodiesdy. Heterophilic antibodies are present in complex serum or plasma sample types and are capable of generating strong false/positive results in immunoassay techniques.