Enzyme-Linked Immunosorbent Assay (ELISA) is an immunological assay that detects various targets such as antibodies, antigens, proteins, and glycoproteins. It is a quick and simple method to perform. Hence, it is more often used for research and diagnostic purposes. ELISA involves the use of enzymes and the specific binding of antibody and antigen. Based on how the reactions occur, there are four types of ELISA: direct ELISA, indirect ELISA, sandwich ELISA, and inhibition ELISA. Direct, indirect and sandwich ELISA are types of noncompetitive ELISA while inhibition ELISA is a type of completive ELISA. The main aim of this article is to discuss the difference between competitive and noncompetitive ELISA.
1. Overview and Key Difference
2. What is Competitive ELISA
3. What is Noncompetitive ELISA
4. Similarities Between Competitive and Noncompetitive ELISA
5. Side by Side Comparison – Competitive vs Noncompetitive ELISA in Tabular Form
What is Competitive ELISA?
Competitive ELISA measures the antigen concentration in a sample through the detection of signal interference. Here, the assay uses an inhibitor antigen. Hence, it is a type of inhibition ELISA. During this procedure, the antigens present in the sample compete with the selected reference antigen for binding to a specific amount of labeled antibody. Furthermore, this procedure initiates with the incubation of the sample, with the excess amount of labeled antibody. Also, the reference antigen should be pre-coated on a multiple well assay plate. Then the sample mixture should be added to the assay plate which contains the reference antigen. Free antibodies will bind to the reference antigen depending on the amount of antigen in the sample. Hence, if more sample antigen is present, less reference antigen will be detected. Thus, it creates a weaker signal.
In contrast, when the sample contains less amount of antigen, more and more reference antigen will occupy the antibodies and give a strong signal. Since competitive ELISA gives a stronger signal when the sample contains a low amount of antigens, competitive ELISA is a very sensitive assay, even for samples with a small number of antigens.
In some competitive ELISA kits, a labeled antigen is used instead of a labeled antibody. Here, the labeled antigen and the sample antigen will compete for binding to the primary antibody. Similarly, when the amount of antigen in the sample is low, the amount of labeled antigen that binds to antibodies will be higher and create a stronger signal.
What is Noncompetitive ELISA?
Among the four types of ELISA, three types are noncompetitive ELISA. They are direct ELISA, indirect ELISA, and sandwich ELISA. All three formats work under the common principle of ELISA with slight differences in their methodologies.
Direct ELISA uses an enzyme labeled primary antibody. Hence, it does not require a secondary antibody. Therefore, direct ELISA is faster than other types of ELISA. The primary antibody will directly bind to the immobilized target antigen that is present in the plate. Hence, the enzyme-substrate reaction will take place, producing a visible signal.
Unlike direct ELISA, indirect ELISA uses an unlabeled primary antibody and an enzyme labeled secondary antibody. Once the primary antibody binds to the immobilized antigen, the secondary antibody binds to the primary antibody. The enzyme in the secondary antibody will react with the antigen and produce a visible signal. Sandwich ELISA is different to both direct and indirect ELISA. Here, the antibody is immobilized to the wall of the assay plate.
What are the Similarities Between Competitive and Noncompetitive ELISA?
- Competitive and noncompetitive ELISA are two types of immunosorbent assay techniques.
- They are highly flexible and sensitive techniques.
- Furthermore, both competitive and non competitive ELISA are enzyme-linked immunoassays.
- Also, both methods generate a signal due to an enzyme-substrate reaction.
What is the Difference Between Competitive and Noncompetitive ELISA?
Competitive ELISA is a type of ELISA that relies on the completion between analyte antigen and labeled antigen for a limited amount of specific antibody. On the other hand, no such competition exists between antigen of interest and reference antigen in noncompetitive ELISA. Thus, this is the key difference between competitive and noncompetitive ELISA. Similarly, competitive ELISA is an inhibition ELISA while noncompetitive ELISA is not an inhibition ELISA. So, this is also a difference between competitive and noncompetitive ELISA.
Moreover, competitive ELISA is more suitable to measure a low amount of antigen of interest. It is a sensitive method. On the other hand, noncompetitive ELISA does not depend on the number of antigens present in a sample. Therefore, this is also a difference between competitive and noncompetitive ELISA.
Below infographic explains the difference between competitive and noncompetitive ELISA comparatively.
Summary – Competitive vs Noncompetitive ELISA
Competitive and noncompetitive ELISA are the two major types of ELISA or enzyme-linked immunosorbent assays. In summarizing the difference between competitive and noncompetitive ELISA, the competitive ELISA relies on the competition between antigen of interest and reference antigen towards the limited amount of antibodies. Therefore, competitive ELISA is also named as inhibition ELISA. On the other hand, noncompetitive ELISA does not rely on the competition between target antigens and reference antigens. It uses an excess of labeled specific antibody toward the analyte of interest. Thus, this is the key difference between competitive and noncompetitive ELISA. Direct ELISA, indirect ELISA, and sandwich ELISA are the three main types of noncompetitive ELISA.