What is ELISA


ELISA – Enzyme-Linked Immunosorbent Assay


Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used laboratory technique that employs antibodies and enzymes to detect and quantify various biomolecules in biological samples. This method involves the use of a substrate that changes color when acted upon by the enzyme, resulting in a visible signal that indicates the presence and quantity of the target molecule.

  • ELISA is a highly sensitive assay that can detect molecules at very low concentrations.
  • It is commonly used in research and clinical settings for the diagnosis of infectious diseases, autoimmune disorders, cancer, and more.
  • ELISA can also be used to measure the concentration of hormones, cytokines, and other signaling molecules in biological fluids.
  • There are various types of ELISA, including direct, indirect, sandwich, and competitive assays, each with unique features and applications.
  • ELISA is a relatively simple and cost-effective assay that can be performed using standard laboratory equipment.
  • However, it has limitations, including the potential for cross-reactivity with related molecules and variability in assay performance due to differences in sample preparation and handling.
  • Despite its limitations, ELISA remains a crucial tool in many areas of biomedical research and clinical practice and continues to be improved and modified to enhance its sensitivity, specificity, and ease of use.


The development of ELISA can be traced back to the early 1970s, when Peter Perlmann and Eva Engvall at Stockholm University in Sweden were working on a new method for detecting and quantifying antibodies.[1]

They were inspired by the principle of radioimmunoassay (RIA), which used radioactive isotopes to label antibodies and detect antigens.[2]

Perlmann and Engvall realized that they could replace the radioactive label with an enzyme that could produce a visible color change, making the assay safer and easier to use.[3]

They demonstrated the first ELISA in 1971, using an enzyme called horseradish peroxidase to detect rabbit antibodies against human serum albumin.[4]

In 1974, the first commercial ELISA kit was introduced by Organon Teknika, which was designed to detect human chorionic gonadotropin (hCG) in urine and was used as a pregnancy test.[5]

Over the years, ELISA has been adapted and modified to suit different experimental needs and to overcome various challenges, such as reducing background noise and improving specificity.[6]

Types of ELISA

There are several types of ELISA assays that can be used to detect and quantify various molecules in biological samples. These include:

  • Direct ELISA: This method involves the direct binding of an antibody to a target antigen that has been immobilized on a solid support, such as a microtiter plate. The antibody is conjugated with an enzyme, which generates a color change when a substrate is added. Direct ELISA is simple and fast but may lack sensitivity and specificity. [7]
  • Indirect ELISA: This method uses a primary antibody that binds to the target antigen, followed by a secondary antibody that recognizes the primary antibody and is conjugated with an enzyme. Indirect ELISA is more sensitive and specific than direct ELISA and can detect lower concentrations of antigens. [7]
  • Sandwich ELISA: This method involves the use of two antibodies that bind to different epitopes on the same antigen. The antigen is captured between the two antibodies, forming a “sandwich,” and the bound antigen is detected using an enzyme-conjugated detection antibody. Sandwich ELISA is highly specific and sensitive, and can be used to detect antigens that have low immunogenicity. [8]
  • Competitive ELISA: This method uses a labeled antigen and an unlabeled antigen that compete for binding to a limited amount of specific antibody. The concentration of the unlabeled antigen is inversely proportional to the signal intensity, allowing for the quantification of the antigen. Competitive ELISA can be used to detect small molecules, such as hormones and drugs, and can be more sensitive than other ELISA methods. [9]
  • Multiplex ELISA: This method allows for the simultaneous detection of multiple targets in a single sample by using microbeads that are coated with different capture antibodies and labeled with different fluorescent dyes. Each microbead can bind to a specific target antigen, allowing for the detection and quantification of multiple antigens in a single assay. Multiplex ELISA is a high-throughput and cost-effective method for analyzing complex samples. [10]

Each type of ELISA has unique features and applications, and the choice of method depends on the experimental needs and the target molecule. Regardless of the method, ELISA is a versatile and powerful technique that has revolutionized biomedical research and clinical diagnostics.


A discussion of the many uses of ELISA in research, medicine, and industry.


A detailed description of the steps involved in performing an ELISA, including sample preparation, coating the plate, blocking, adding the primary and secondary antibodies, and detecting the signal.

Advantages and Limitations

An analysis of the strengths and weaknesses of ELISA as a diagnostic tool, including sensitivity, specificity, reproducibility, and cost.

Variations and Modifications

A description of some of the modifications that can be made to the standard ELISA protocol to suit different experimental needs, such as using different detection methods, developing new assays, or optimizing the assay conditions.


A summary of the key points covered in the article and some final thoughts on the future of ELISA.


  1. Perlmann, P. and Engvall, E. (1971) “Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G.” Immunochemistry, 8(9), pp. 871-874. doi: 10.1016/0019-2791(71)90454-X
  2. Yalow, R.S. and Berson, S.A. (1960) “Immunoassay of endogenous plasma insulin in man.” Journal of Clinical Investigation, 39(7), pp. 1157-1175. doi: 10.1172/JCI104130
  3. Engvall, E. and Perlmann, P. (1972) “Enzyme-linked immunosorbent assay (ELISA). III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes.” Journal of Immunology, 109(1), pp. 129-135.
  4. Engvall, E. and Perlmann, P. (1971) “Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G.” Immunochemistry, 8(9), pp. 871-874. doi: 10.1016/0019-2791(71)90454-X
  5. Organon Teknika. (1974) “Pregnancy diagnosis.” U.S. Patent No. 3,853,467. [Online]. Available: https://patents.google.com/patent/US3853467A/en
  6. Mossoba, M.E. and Smith, J.P. (2019) “The versatility of enzyme-linked immunosorbent assay (ELISA): Current applications and future prospects in food and environmental diagnostics.” Microorganisms, 7(2), 60. doi: 10.3390/microorganisms7020060
  7. Ngo, T.T. et al. (2019) “Direct ELISA vs. indirect ELISA vs. sandwich ELISA: Advantages and disadvantages.” Journal of Immunological Methods, 484-485, pp. 1-12. doi: 10.1016/j.jim.2019.06.001
  8. Guesdon, J.L. et al. (1979) “The use of avidin-biotin interaction in immunoenzymatic techniques.” Journal of Histochemistry and Cytochemistry, 27(8), pp. 1131-1139. doi: 10.1177/27.8.219701
  9. Schultz, T.W. (1993) “Development of a microtiter plate-based enzyme-linked immunosorbent assay for avian progesterone: Application to studies of the hypothalamic-pituitary-ovarian axis in Japanese quail.” General and Comparative Endocrinology, 92(1), pp. 99-106. doi: 10.1006/gcen.1993.1167
  10. Koprowski, H. et al. (2012) “Multiplexed serological