What is the Difference Between ELISA and ELFA

The main difference between ELISA and ELFA is that, in ELISA, color development is the detection criteria for the positive samples but, in ELFA, emitting fluorescence is the detection criteria

ELISA and ELFA are two immunological methods used in the detection of proteins in biological samples especially, antibodies and antigens. Although ELISA is a sensitive method, ELFA is more sensitive than ELISA.

Key Areas Covered

1. What is ELISA
     – Definition, Types, Importance
2. What is ELFA
     – Definition, Method, Importance
3. What are the Similarities Between ELISA and ELFA
     – Outline of Common features
4. What is the Difference Between ELISA and ELFA
     – Comparison of Key Differences

Key Terms

Chromogenic Substrate, ELFA, ELISA, Fluorogenic Substrate, Immunological Assays, Sensitivity

Difference Between ELISA and ELFA- Comparison Summary

What is ELISA

ELISA (enzyme-linked immunosorbent assay) is a type of solid phase enzyme immunoassay used in the detection of specific proteins in biological samples with the help of color development through an enzymatic reaction. Based on the methodology, there are three main types of ELISA; direct ELISA, indirect ELISA, and sandwich ELISA.

Direct ELISA

Direct ELISA is the first-developed method of ELISA, which is quite simple. Here, the surface of the microtiter plate (solid phase) is coated with the sample. Then, the enzyme-linked antibodies bind with the specific protein on the plate. With the addition of the chromogenic substrate, it is possible to detect protein-bound antibodies.

Indirect ELISA

Indirect ELISA is a somewhat complex method of ELISA, which uses a two-step process for the detection of a specific protein in a sample. Here, the first step is to coat the microtiter plate with the sample and incubate it with a specific type of primary antibody, which binds to the protein of interest. The next step is to incubate this plate with an enzyme-linked secondary antibody, which binds to the primary antibody. Then, with the addition of the chromogenic substrate, we can detect the specific protein on the plate due to the color development.

What is the Difference Between ELISA and ELFA

Figure 1: ELISA Types

Immunometric/Sandwich ELISA

Sandwich ELISA is also a two-step process. Here, the first step is to sandwich the concerned protein in the sample between the primary and secondary antibody. Therefore, in the first step, the microtiter plate is first coated with the primary antibody, but not with the sample. Secondly, the plate is incubated with the sample. Thirdly, an enzyme-linked secondary antibody is added to the plate. This secondary antibody binds to the specific protein bound to the primary antibody. Finally, color development can detect protein-bound antibody complexes on the plate.

What is ELFA

ELFA (enzyme-linked fluorescence assay) is another type of solid phase enzyme immunoassays, involved in the development of fluorescence instead of a color as in ELISA. However, the general experimental procedure of ELFA is similar to that of the ELISA. The only difference in ELFA is the use of a chromogenic substrate instead of a chromogenic substrate. For example, the chromogenic substrate used for the enzyme alkaline phosphatase in ELISA is p-nitrophenyl phosphate (PNPP). However, in ELFA, the fluorogenic substrate used for the same enzyme alkaline phosphatase is 4-methylumbelliferyl phosphate (4MUP).

More importantly, ELFA is more sensitive than ELISA. Therefore, it can detect the developing antibodies within a shorter time when compared to the time taken by the detection of antibodies by ELISA.

Similarities Between ELISA and ELFA

  • ELISA and ELFA are two types of immunological assays that help to detect and quantify specific proteins in biological samples.
  • The basic design of both experiments is the same.
  • Also, both are solid phase enzyme immunoassays (EIA).
  • Besides, they are high-throughput, high-sensitive, faster, and reproducible methods.

Difference Between ELISA and ELFA


ELISA (enzyme-linked immunosorbent assay) refers to a sensitive technique for detecting and measuring antigens or antibodies in a solution with the use of chromogenic substrates while ELFA (enzyme-linked fluorescence assay) refers to an immunological method in which the enzyme catalyzes a fluorescence, not a color reaction. Thus, this is the main difference between ELISA and ELFA.

Detection Method

Moreover, ELISA detects the development of color on the solid phase but, ELFA detects the development of fluorescence on the solid phase.


The substrate used in ELISA is chromogenic while the substrate used in ELFA is fluorogenic.


Another difference between ELISA and ELFA is that ELFA is 100 times more sensitive than ELISA.

Length of the Window Period

The window period is longer in ELISA while the window period is about five days shorter than ELISA in ELFA. Hence, this is another difference between ELISA and ELFA.


ELISA is a type of immunological assay which detects proteins in a biological sample with the binding of specific antibodies. Here, the detection method is the development of color through an enzymatic reaction. On the other hand, ELFA is another type of immunoassay which detects the presence of a specific protein in the sample with the development of fluorescence through an enzymatic reaction. Therefore, the main difference between ELISA and ELFA is the type of substrate in the enzymatic reaction.


1. Shekarchi, I C et al. “Comparison of enzyme-linked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus” Journal of clinical microbiologyvol. 21,1 (1985): 92-6. Available Here

Image Courtesy:

1. “ELISA” By Jiver – Own work (CC BY-SA 4.0) via Commons Wikimedia 

About the Author: Lakna

Lakna, a graduate in Molecular Biology and Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things. She has a keen interest in writing articles regarding science.

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