What Is the Principle of a Sandwich ELISA?

The Sandwich ELISA (Enzyme-Linked Immunosorbent Assay) is a powerful and widely used technique in biomedical research and clinical diagnostics for detecting and quantifying specific proteins or antigens in a sample. This method offers both sensitivity and specificity, making it ideal for applications where precise measurement of low-abundance proteins is necessary. To understand the principle of a Sandwich ELISA, it's essential to delve into its components and how they interact to produce measurable results.

At its core, the Sandwich ELISA involves the use of two antibodies that bind to different epitopes on the target antigen. The first antibody, known as the capture antibody, is immobilized on a solid surface, such as a microplate well. This antibody is specifically chosen to have a high affinity for the target antigen, ensuring that the antigen is effectively “captured” from the sample when it is added to the well.

Once the sample containing the antigen is introduced, the antigen binds to the capture antibody. This forms the first part of the "sandwich." Following an incubation period that allows for optimal binding, the well is washed to remove unbound substances, preventing interference in the subsequent steps.

The next step in the Sandwich ELISA involves the addition of a second antibody, referred to as the detection antibody. This antibody is also specific to the target antigen, but it binds to a different epitope than the capture antibody. The detection antibody is typically linked to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase. This enzyme acts as a reporter, transforming a substrate into a detectable signal, usually a colorimetric change, when added in the final stages of the assay.

After the detection antibody binds to the antigen, forming the complete "sandwich," the well is washed once more to remove any unbound detection antibodies. This ensures that the signal generated in the final step correlates directly with the amount of antigen present in the sample.

Finally, a substrate specific to the linked enzyme is added to the well. The enzyme catalyzes a reaction with the substrate, resulting in a color change that can be measured using a spectrophotometer or other optical detection systems. The intensity of the color is proportional to the concentration of the target antigen in the sample, allowing for quantification.

The Sandwich ELISA is renowned for its specificity, due to the dual antibody recognition, and its sensitivity, enabled by the amplification of the signal through enzymatic reactions. This assay is employed in various fields, from detecting biomarkers in disease states to verifying the presence of allergens in food products. Additionally, the method can be easily adapted for high-throughput screening, enhancing its utility in large-scale studies.

In conclusion, the Sandwich ELISA is a robust and versatile assay that plays an integral role in scientific research and clinical diagnostics. Its combination of precision, reliability, and adaptability makes it an indispensable tool for detecting and quantifying proteins across a myriad of applications.

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