Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2025 Feb;53(2):3000605251315913.
doi: 10.1177/03000605251315913.

An overview of ELISA: a review and update on best laboratory practices for quantifying peptides and proteins in biological fluids

Suleyman Aydin  1 , Elif Emre  2 , Kader Ugur  3 , Mustafa Ata Aydin  4 , İbrahim Sahin  1   5 , Vedat Cinar  6 , Taner Akbulut  6
Affiliations
Review

An overview of ELISA: a review and update on best laboratory practices for quantifying peptides and proteins in biological fluids

Suleyman Aydin et al. J Int Med Res. 2025 Feb.

Abstract

The enzyme-linked immunosorbent assay (ELISA) detects antigen-antibody interactions by using enzyme-labelled conjugates and enzyme substrates that generate colour changes. This review aims to provide an overview of ELISA, its various types, and its applications in detecting metabolites in biological fluids. The article discusses the history of the assay, its underlying principles and procedures, common ELISA protocols, and the most accurate and reliable techniques for measuring peptide molecules in biological fluids. Additionally, we emphasize best laboratory practices to achieve consistent, high-quality results and outline the essential materials for setting up an ELISA laboratory, drawing from our over 30 years of experience in the field.

Keywords: Biological fluids, ELISA, antibody, antigen; enzyme-linked immunosorbent assay.

PubMed Disclaimer

Conflict of interest statement

Declaration of conflicting interestThe authors declare there are no conflicts of interest.

Figures

Figure 1.
Figure 1.
ELISA kit contents. (a) Microplate; (b) Microplate loaded with analytes; (c) Yellow colour occurs when stopping solution is added and (d) Yellow colour occurs when stopping solution is added.
Figure 2.
Figure 2.
Schematic drawings of: (a) Direct ELISA; (b) Indirect ELISA; (c) Competitive ELISA; (d) Sandwich ELISA; (e) Direct cellular ELISA and (f) Indirect cellular ELISA.
See this image and copyright information in PMC

References

    1. Espe S. Malacards: The Human Disease Database. J Med Libr Assoc 2018; 106: 140–141.
    1. Marth JD. A unified vision of the building blocks of life. Nat Cell Biol 2008; 10: 1015–1016. 10.1038/ncb0908-1015. - DOI - PMC - PubMed
    1. Jacob KD, Noren Hooten N, Trzeciak AR, et al.. Markers of oxidant stress that are clinically relevant in aging and age-related disease. Mech Ageing Dev 2013; 134: 139–157. - PMC - PubMed
    1. Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 2015; 72: 4–15. - PubMed
    1. Schöfl C, Horn R, Schill T, et al.. Circulating ghrelin levels in patients with polycystic ovary syndrome. J Clin Endocrinol Metab 2002; 87: 4607–4610. - PubMed

MeSH terms

LinkOut - more resources