Guide to Sandwich ELISA Protocol

Guide to Sandwich ELISA Protocol

Struggling to understand the fundamentals of a sandwich ELISA? You’re not alone. This powerful biochemical technique, used for quantifying antigens in biological samples, can seem complex at first glance.

But don't fret - this guide breaks down everything you need to know about detecting antigens using sandwich ELISAs, from underlying principles to step-by-step procedures.

What is a Sandwich ELISA?

A sandwich ELISA, short for Enzyme-Linked Immunosorbent Assay, is a detection method that involves the attachment of a target antigen that is “sandwiched” between two layers of antibodies (capture and detection antibodies).

How it works

This technique functions by using an unlabeled capture antibody immobilized on the ELISA plate to bind specifically to the target antigen from the test sample.

Then a detection antibody solution, linked with an enzyme like alkaline phosphatase or horseradish peroxidase, binds specifically to another region of the same antigen forming a sandwich around it.

Post binding, any unbound substances get washed away using a wash buffer.

Finally, the addition of a substrate solution brings out color changes which essentially signifies successful detection of the target proteins or antigens.

Why utilize a Sandwhich ELISA?

Scientists across different fields like research, healthcare, and food safety widely use this reliable method due to its high specificity since it uses two antibodies targeting different regions of the same antigen.

Besides being well-regarded for their sensitivity in comparing to other types of ELISAs, sandwich ELISAs hold an excellent reputation as they can directly measure antigens even within complex samples.

Advantages and Challenges of Sandwich ELISAs

As with any application, sandwich ELISA’s come with advantages and challenges. It is ultimately up to the immunoassay developer to weigh the pros and cons based on their objectives. Below are key advantages and challenges of sandwich ELISA tests:

Performing a Sandwhich ELISA

In this section, we delve into the intricacies of performing a sandwich ELISA, exploring critical steps and protocols, and offering troubleshooting tips for common obstacles faced during the process.

Steps and protocol

Performing a sandwich ELISA involves a series of carefully executed steps. Here is the protocol:

1. Start by preparing the reagents, including the capture antibody solution, buffers, and antigen samples.
   • Recommended products for outstanding assay performance:
     • DIARECT™ Antigens and Antibodies
       • Surmodics IVD is the exclusive North American representative of DIARECT™ products
2. Immobilize the capture antibody onto the surface of an ELISA plate by adding diluted capture antibody to each well and incubating at room temperature.
   • Recommended reagent for plate coating:
     • BioFX™ ELISA Plate Coating Solution - 10X Concentrate (Product Code:COAT)
3. Remove the capture antibody solution and wash the plate with wash buffer to remove any unbound antibodies.
4. Block non-specific sites by adding a blocking buffer solution to each well.
   • Recommended reagents for unmatched blocking and long-term stability:
     • Surmodics IVD’s Dried Protein Stabilizers & Blockers
5. Add standards, samples and controls to the wells and incubate at room temperature allowing the target protein to bind to the immobilized capture antibodies.
6. Wash away any unbound proteins with wash buffer.
7. Introduce a conjugated detection antibody which is specific for a different epitope on the target antigen. 
   • Recommended reagents for unparalleled long term in-solution stability of the detection antibody:
     • Surmodics IVD’s Protein & Conjugate Stabilizers
8. Incubate at room temperature before washing again to remove any unbound detection antibody.
9. Finally, add substrate solution which will react with the enzyme linked on detection antibody forming a readable signal, along with stop solution to stabilize color change.
   • Recommended reagents for unsurpassed sensitivity and stability:
     • Surmodics IVD’s TMB Microwell Substrates
     • Surmodics IVD’s TMB Stop Solutions

Visit the link below for additional information regarding Surmodics IVD’s Sandwich ELISA Protocol:

Surmodics IVD’s Sandwich ELISA Protocol

Troubleshooting tips

Troubleshooting often becomes necessary in the process of developing a sandwich ELISA. There are many ways to approach challenges within a sandwich ELISA, including the utilization of different immunoassay reagents, choosing the most effective antibodies, and employing different protocols and techniques.

No matter what challenge arises, Surmodics IVD is here to help. Below are common challenges developers face, along with the Surmodics IVD solutions that can help:

What are the types of ELISA formats?

ELISA tests use one of four main formats: Sandwich, Direct, Indirect, and Competitive.

1. A sandwich ELISA measures antigen between two layers of antibodies (capture and detection antibody). The target antigen must contain at least two antigenic sites capable of binding to antibodies. Monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems.
2. A direct ELISA format utilizes a detection antibody labeled with an enzyme or another signal-generating system directly. This detection antibody binds to the immobilized antigen on the surface. The advantage of this format is its simplicity and speed due to fewer steps and reagents. However, it is less versatile than indirect ELISAs because the label is directly conjugated to the primary antibody.
3. An indirect ELISA format involves attachment of an unlabeled primary antibody prior to the labeled detection antibody. The secondary antibody recognizes the primary instead of the antigen itself.  One advantage of indirect ELISAs is enhanced sensitivity.
4. Lastly, in a competitive ELISA format the concentration of the target antigen is determined by detection of signal interference. The target antigen in the sample competes with a labeled detection antibody for binding to a limited amount of antibodies immobilized on the plate.

How accurate is a Sandwich ELISA?

Sandwich ELISA’s perform remarkably well in isolating desired antigens from impure samples since they 'trap' the proteins between two layers of antibodies catering to optimal concentration levels, which leads to more accurate results.

Along with selecting the most effective ELISA type, Surmodics IVD’s gold standard reagents also give immunoassay developers the reassurance needed to build accurate, sensitive, reproducible assays.

Visit the link below to learn more and connect directly with one of our R&D scientists today!

Contact Us