The main difference between direct and indirect immunofluorescence test is that direct immunofluorescence test uses conjugated antibodies to stain the target protein. In contrast, an indirect immunofluorescence test uses a primary antibody to the target that binds to the conjugated secondary antibody to generate a color.
Direct and indirect immunofluorescence tests are two immunofluorescence tests used to detect specific antigens in samples. They use fluorescent dyes and antibodies. They allow the visualization of the distribution of target molecules in a sample.
Key Areas Covered
1. What is Direct Immunofluorescence Test
– Definition, Features, Importance
2. What is Indirect Immunofluorescence Test
– Definition, Features, Importance
3. Similarities Between Direct and Indirect Immunofluorescence Tests
– Outline of Common Features
4. Difference Between Direct and Indirect Immunofluorescence Tests
– Comparison of Key Differences
Key Terms
Direct Immunofluorescence Test, Indirect Immunofluorescence Test
What is Direct Immunofluorescence Test
Direct immunofluorescence test is a type of immunofluorescence test in which a conjugated antibody is used to detect an antigen in the sample. Therefore, only one type of antibody can bind to the antigen. Here, the conjugated antibody recognizes the antigen and binds with the epitope of the antigen. Moreover, monoclonal antibodies are used in the direct immunofluorescence test. This process manipulates the immune response of adaptive immunity. Then, fluorescent microscopy can detect the antigen. However, direct immunofluorescence test is somewhat less common.
Figure 1: Type of Immunofluorescence
Furthermore, direct fluorescence antibody allows the direct attachment of antibodies to the antigen. This reduces the number of steps and saves time. Also, it reduces the non-specific background signal. Additionally, it reduces the possibility of cross-reactivity. But the number of antibody molecules that can bind to the antigen is low. Therefore, this method is less sensitive and can result in false negatives.
What is an Indirect Immunofluorescence Test
An indirect immunofluorescence test is another type of immunofluorescence test in which the primary antibody binds with the antigen in the sample. The binding of the conjugated secondary antibody to the primary antibody allows antigen detection. Here, multiple secondary antibodies can bind to the same primary antibody. Importantly, this enables the amplification of the signal. Also, the method is more flexible than the direct immunofluorescence test. However, the protocol is more complex and more time-consuming.
Figure 2: Immunofluorescence
Moreover, structure-wise, an antibody molecule consists of two parts: constant and variable regions. Although the variable region is different, the constant region of the antibodies can be the same. Primary antibodies can be designed with different constant regions when raised in different species. Therefore, different primary and secondary antibody designs are possible for a particular reaction.
Similarities Between Direct and Indirect Immunofluorescence Test
- Direct and indirect immunofluorescence tests are two types of immunofluorescence tests used to detect the distribution of target antigens in a sample.
- They use antibodies and dyes for the visualization of antigens.
Difference Between Direct and Indirect Immunofluorescence Test
Definition
Direct immunofluorescence test refers to a one-step procedure that involves the application of fluorescence antibodies to a sample, while indirect immunofluorescence test refers to a standard virologic technique to identify the presence of antibodies by their specific ability to react with viral antigens expressed in infected cells; bound antibodies are visualized by incubation with fluorescently labeled antihuman antibody.
Antigens
Antigens bind with the conjugated antibodies in the direct immunofluorescence test, while antigens bind with the primary antibody in the indirect immunofluorescence test.
Protocol
Direct immunofluorescence test is a shorter and simple protocol, while indirect immunofluorescence test is a long and complex protocol.
Background Fluorescence
Direct immunofluorescence test produces improved background, while indirect immunofluorescence test produces high background.
Signal
Direct immunofluorescence test produces a weaker signal, while indirect immunofluorescence test produces an amplified signal.
Flexibility
Direct immunofluorescence test has limited flexibility, while indirect immunofluorescence test has greater flexibility.
Conclusion
In brief, direct and indirect immunofluorescence tests are two types of immunofluorescence tests used to visualize a particular antigen in a sample. Direct immunofluorescence test involves binding a conjugated antibody to the antigen in the sample. Therefore, it is a shorter protocol that produces a weaker signal and improved background. Also, it has limited flexibility. In comparison, the indirect immunofluorescence test involves binding a primary antibody to the antigen in the sample. A conjugated secondary antibody binding allows the detection of the antigen. Hence, it is a long and complex protocol that produces an amplified signal and high background. It has greater flexibility. Therefore, the main difference between direct and indirect immunofluorescence tests is their binding to antibodies.
References:
- Tech tip: Combined direct and indirect if using primary antibodies from the same host. Biotium. (2021, December 30).
Image Courtesy:
- “Immunofluorescence” By Westhayl618 – Own work (CC-BY SA 4.0) via Commons Wikimedia
- “Main antinuclear antibody patterns on immunofluorescence” By Al-Mughales JA – Own Work (CC-BY 4.0) via Commons Wikimedia
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