The enzyme-linked immunosorbent assay (ELISA) is a critical tool for researchers aiming to detect and quantify peptides, proteins, antibodies, and hormones. This assay can be performed using several different methods—direct, indirect, competitive, and sandwich—each involving the immobilization of the antigen of interest, either by direct adsorption to the assay plate or indirectly via a capture antibody.
While the sandwich ELISA assay is the most commonly used format, it is not a one-size-fits-all solution. Each method is uniquely suited to different research needs, based on the required sensitivity, antigen of interest, available resources, training of lab staff, and time constraints. Moreover, selecting an inappropriate ELISA technique can lead to skewed data, wasted resources, and other inefficiencies that compromise research outcomes.
A clear understanding of how each method works, along with its pros and cons, is essential for navigating these complexities. Our ELISA Flowchart offers a detailed step-by-step guide for each method, with insights into which format to use and when. It also serves as a visual reference during experimentation, illustrating the molecular interactions within the assay wells. Understanding these interactions can aid in troubleshooting and results interpretation, ultimately enhancing the accuracy and reproducibility of your experiments.
Enhance your ELISA experiments with our ELISA Flowchart, and gain insights into:
- Step-by-step procedural guidance for each type of ELISA
- What is happening at a molecular level within the wells of your ELISA
- The benefits and drawbacks of all ELISA techniques
59425_LMFlowChart_AV (5) (1)
ELISA FlOWCHART
1° Ab
Ag
2° Ab
1° Ab
Ag
2° Ab
1° Ab
Reference Ag
2° Ab
Ag
1° Ab
DIReCT ELISA
Directly detects the antigen
INDIReCT ELISA
Detects primary antibody-antigen complex
COMPeTITIVe ELISA
Competition between antigen & reference antigen for the labeled antibody
SANDWICH ELISA
Antigen is sandwiched between antibodies (capture & detection)
Prepare samples
Add sample
Coat wells with reference antigen
Coat wells with primary antibody
Wash and block microplate
Add primary antibody
Add antigen-antibody mixture
Add sample
Wash microplate
Add substrate
Read microplate
Calculate results
Add conjugated secondary antibody
Wash microplate
Lab safety is a critical responsibility for all lab managers. With the Lab Safety Management Certificate program from Lab Manager Academy, you will learn how to mitigate risks, improve safety culture, and manage your lab's EHS systems. Take the first steps towards a safer lab today by visiting www.academy.labmanager.com
 (1)_files/Image_001.png)
 (1)_files/Image_002.png){kind=small}
HOW IT WORks
The enzyme-linked immunosorbent assay (ELISA) detects and quantifies peptides, proteins, antibodies, and hormones. The first step in an ELISA is immobilizing the target molecule (antigen) directly to the microplate or indirectly with a capture antibody. Detection antibodies, which bind to the antigen, are added next. The antibodies are either directly conjugated to a reporter enzyme or provide a binding site for a labeled secondary antibody. Consistency and standardization are the most important factors when performing an ELISA since they contribute to the reproducibility and accuracy of results.
WHAT TyPe Of ELISA SHOulD I CHOOse?
ELISA TyPe | ADVANTAGES | DISADVANTAGES |
Direct ELISA | Short protocol and no cross-reactivity from a secondary antibody. | High background, no signal amplification, and low flexibility since the primary antibody must be conjugated. |
Indirect ELISA | Signal amplification, high flexibility, and high sensitivity. | Long protocol and potential cross-reactivity from the secondary antibody. |
Competitive ELISA | High flexibility since it can be based on direct, indirect, or sandwich ELISA. | Depends on if it is direct or indirect. |
Sandwich ELISA | High specificity, flexibility, and sensitivity. | Requires two antibodies against the target at different epitopes. |
HOW CONFIDENT ARE YOU IN YOUR LAB LEADERSHIP SKILLS?
The IACET-accredited Lab Management Certificate delivers the essential knowledge and training that every lab manager needs to succeed.
Visit academy.labmanager.com to learn more and get certified!
As an IACET Accredited Provider, Lab Manager Academy offers IACET CEUs for its learning events that comply with the ANSI/IACET Continuing Education and Training Standard.
 (1)_files/Image_003.png)