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ELISA Introduction

> ELISA Introduction

What are Sandwich ELISAs?

        The ELISA (Enzyme-linked immunosorbent assay is the most popular and sensitive immunoassay used in research laboratories nowadays and it is first presented by Engvall and Perlman in 1971. It is a potent plate-based biochemical method used to detect and quantify proteins, antigens, antibodies, and biomolecules in a complex mixture. As this immunoassay is highly sensitive, its detection range is 0.01 ng to 0.1 ng or 0.1 to 1 fmole. An ELISA, like other types of immunoassays, majorly depends on antibodies to detect a target antigen by using extremely particular antibody-antigen interactions (Engvall and Perlmann,1971).

 

        A Sandwich ELISA is the most commonly used format of ELISA and it measures antigen between two layers of antibodies (capture and detection antibody). The Sandwich ELISA needs two antibodies and they are stated as matched antibody pairs. The target antigen which needs to be detected in a sample must have at least two antigenic sites proficient in binding to antibodies. One of the antibodies is named a capture antibody and it is used to facilitate the immobilization of the antigen.  While the other antibody is a detection antibody (conjugated to fluorophore label or enzyme) that helps to detect the target antigen. This format is called Sandwich ELISA because the target antigen is Sandwiched between antibodies (Crowther,2001).

 

        In Sandwich ELISA kits, monoclonal or polyclonal antibodies can be used as the detection and capture antibodies. Monoclonal antibodies identify a single epitope that permits the quantification of minor variations in antigen. A polyclonal antibody is usually used as the capture antibody to catch as much of the antigen as probable (Hutchings and Ferris ,2006).

Direct and Indirect Sandwich ELISA:

        There are two types of Sandwich ELISA. One of them is direct Sandwich ELISA in which the detection antibody can be enzyme-conjugated. But if the detection antibody is unlabeled, then a secondary enzyme-conjugated detection antibody is necessary. This type of Sandwich ELISA is known as an indirect Sandwich ELISA (Hutchings and Ferris ,2006).

Basic Procedure:

        The basic steps of this immunoassay include the coating of the microtiter plate with capture antibody and incubating the plate overnight. After that coating solution is removed and blocking buffer is added to block the remaining protein-binding sites in the coated wells. Then plate is washed with PBS. Then samples are added to each well and microtiter plate is again incubated for some time. After that plate is washed again with PBS solution. Then detection antibody is added to each well and the plate is incubated again for about 2 hours at room temperature. After that plate is washed about four times with PBS solution. Lastly, enzyme-conjugated secondary antibody is added that will bind to the primary detection antibody. The substrate will react with the enzyme-conjugated secondary antibody and gives the signal that will indicate the presence of the target antigen in the sample (Gonzalez et al.,2008).

Detection Methods of Sandwich ELISAs

        The detection antibody will bind to the target antigen if it is present in the sample and then the conjugated enzyme will react with the substrate and produce a signal. This signal can be light-producing (chemiluminescence) or simple chromogenic, typically colorimetric 450 nm. The signal that is created is directly proportional to the enzyme-substrate pair used for detection purpose. But if the target antigen is absent in the sample, then the enzyme-conjugated detection antibody will not do binding and thus no signal will be produced. The detection antibody will be marked by a fluorophore instead of an enzyme in case of a fluorescence ELISA. A fluorophore is a substance that emits light after it is agitated by incident light. The incident light excites the electrons of the fluorophore into a higher energy level and thus it will emit light and release energy. An ELISA reader is a multipurpose apparatus that can be used to identify any of these labels, hence a range of labels are accessible and easy to use in Sandwich ELISAs (TIP ,2010).

Why Sandwich ELISAs are more reliable

        This format of ELISA is more reliable as compared to other formats as it is 2-5 times more sensitive than direct and indirect ELISA. Moreover, the Sandwich ELISA is highly specific as it uses two antibodies to detect different epitopes which can produce more accurate results by preventing interference. Lastly, it can analyze complex samples easily and can also measure impure antigens in the specific sample (Gonzalez et al.,2008). However, a competitive ELISA only needs one binding site on the antigen, making it less sensitive to experimental mistakes as compared to Sandwich ELISA.

References:

 

  • Crowther, J.R., 2001. Systems in ELISA. In The ELISA Guidebook (pp. 9-44). Humana Press.

  • Chiu, M.L., Lawi, W., Snyder, S.T., Wong, P.K., Liao, J.C. and Gau, V., 2010. Matrix effects—a challenge toward automation of molecular analysis. JALA: Journal of the Association for Laboratory Automation, 15(3), pp.233-242.

 

  • Engvall, E. and Perlmann, P., 1971. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry, 8(9), pp.871-874.

 

  • Gonzalez, R.M., Seurynck-Servoss, S.L., Crowley, S.A., Brown, M., Omenn, G.S., Hayes, D.F. and Zangar, R.C., 2008. Development and validation of Sandwich ELISA microarrays with minimal assay interference. Journal of proteome research, 7(6), pp.2406-2414.

 

  • Hutchings, G.H. and Ferris, N.P., 2006. Indirect Sandwich ELISA for antigen detection of African swine fever virus: comparison of polyclonal and monoclonal antibodies. Journal of virological methods, 131(2), pp.213-217.

 

  • Hosseini, S., Vázquez-Villegas, P., Rito-Palomares, M. and Martinez-Chapa, S.O., 2018. General overviews on applications of ELISA. In Enzyme-Linked Immunosorbent Assay (ELISA) (pp. 19-29). Springer, Singapore.

 

  • TIP, T., 2010. ELISA technical guide and protocols. Thermo Fisher Scientific Inc USA, Bartlesville, OK.

 

  • Burkin, M.A., Nuriev, R.I., Wang, Z. and Galvidis, I.A., 2018. Development of Sandwich double-competitive ELISA for sulfonamides. Comparative analytical characteristics and matrix effect resistance. Food analytical methods, 11(3), pp.663-674.

  • Lugos, M.D., Damulak, O.D., Perikala, V., Davou, G.I., Obeta, U.M., Banda, J.M., Oluwatayo, B.O. and Okwori, J.A., 2019. Assay linearity and spike-recovery assessment in optimization protocol for the analysis of serum cytokines by Sandwich ELISA platform. Am J Biomed Sci, 3(2).

  • Miller, J.J. and LEVINSON, S.S., 1996. Interferences in immunoassays. In Immunoassay (pp. 165-190). Academic Press.

  • Monaci, L., Brohée, M., Tregoat, V. and van Hengel, A., 2011. Influence of baking time and matrix effects on the detection of milk allergens in cookie model food system by ELISA. Food Chemistry, 127(2), pp.669-675.

  • Rosenberger, C.M. and Finlay, B.B., 2003. Phagocyte sabotage: disruption of macrophage signalling by bacterial pathogens. Nature reviews Molecular cell biology, 4(5), pp.385-396.

  • Scientific, T.F., 2007. Spike-and-recovery and linearity-of-dilution assessment. Thermo Scientific Tech Tip, 58.

  • Selby, C., 1999. Interference in immunoassay. Annals of clinical biochemistry, 36(6), pp.704-721.

  • Zhou, W., Yang, S. and Wang, P.G., 2017. Matrix effects and application of matrix effect factor. Bioanalysis, 9(23), pp.1839-1844.

  • Marks, M.A., Wenzel, T., Whitehouse, M.J., Loose, M., Zack, T., Barth, M., Worgard, L., Krasz, V., Eby, G.N., Stosnach, H. and Markl, G., 2012. The volatile inventory (F, Cl, Br, S, C) of magmatic apatite: An integrated analytical approach. Chemical Geology, 291, pp.241-255.

  • Zhang, Z., Lin, H., Sui, J., Han, X., Wang, L., Sun, X. and Cao, L., 2022. The effect of chlorophyll on the enzyme‐linked immunosorbent assay (ELISA) of procymidone in vegetables and the way to overcome the matrix interference. Journal of the Science of Food and Agriculture, 102(8), pp.3393-3399.

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