Enzyme Immunoassay Principle

ENZYME IMMUNOASSAYS (EIA)

Enzyme immunoassay (EIA)

INTRODUCTION

Enzyme immunoassay (EIA) or enzyme linked immunosorbant assay (ELISA) is a powerful technique used for detecting and quantifying antigens and antibodies in clinical samples. It is widely used in clinical laboratories for diagnoses, prognoses and for monitoring immune responses.

The aim of this essay is to summarise the principle of this technique and to outline the main types of enzyme linked immunosorbant assay (ELISA) and its application in clinical laboratories. Subsequently, it will discuss the application of this system in the haematology field.

PRINCIPLES

The general principle of this technique is based on the binding of conjugated enzyme molecule with specific antibodies to detect and quantify the presence of either antigens or antibodies in the test sample. This is followed by adding appropriate colourless substrate which catalyses the interaction complex to produce a visible coloured product. There are many chromogenic substrates used in ELISA technique but the most common are alkaline phosphatase (AP) and horseradish peroxidase (HRP). The end product can be determined by using spectrophotometer and the intensity of colour is directly proportional to the presence of either antigens or antibodies in the test samples.

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TYPES

There are several types of ELISA which include: indirect, sandwich, competitive, chemiluminescence and elispot assay. Indirect ELISA can be used to detect the antibodies that are present in patient’s serum. With this method, patient’s serum containing antibodies is added to the microtitre wells which are coated with the antigen and the intensity of the end colour product is directly proportional to the amount of antibodies that are present in the patient’s sample. (KUBY) Figure1. Illustrate the different types of ELISA to detect either antigen or antibody.

The second type of enzyme linked immunosorbant assay is called sandwich ELISA, in which the target antigen that is to be detected must be located between two antibodies. The antigen of interest that is present in the clinical sample is added to a microtitre wells which is coated with the antibody and the end product is determined by spectrophotometer to measure the amount of antigen in the clinical sample.(DIAGNOSTIC MICRO. BOOK) Figure1.

The development of biotechnology has become widespread and one example of its progress is the modification of ELISA by using chemiluminescence substrate rather than chromogenic substrate to increase the sensitivity of the reaction. With this method, chemilumescence (CL) substrate and some enhancing agents, which have the ability to bind an antibody or antigen in the reaction, are used to create light emissions which can be measured by specific spectrometry. This approach has been used to diagnose several types of tumour disease, and quantify numerous compounds at low concentration (Baeyens et al. 1998).

Another example of ELISA modification is enzyme linked immunospot (ELISPOT) assay. It is a quantitative technique often used to detect cytokines that are secreted from single cell in response to the antigen. During the incubation period, the cytokines of interest is released and captured by immobilised anti-cytokines on the microtitre plate. After washing them and adding labelled anti-cytokines antibody and substrate, the end colour product displays as a “footprint” around each of the secreting cells, which can be estimated. This technique has great potential for monitoring immune responses (Cox, Ferrari and Janetzki 2006). In addition, sensitive ELISPOT assay has the ability to detect low level of secreted IFN-? compared with other IFN-? assays (Lion et al. 2009).

APPLICATION

There are many applications of enzyme immunoassays in clinical laboratories and the largest application is related to the detections of viruses. For example, the screening and diagnosis of hepatitis virus is routinely used in the lab. These include the detection of HBsAg and HBeAg by direct ELISA, anti-HBc antibody and anti-HBe antibody by competitive ELISA and anti-HBs antibody by indirect ELISA (Tsitsilonis et al. 2004).What is more, ELISA can detect HCV antibodies in serum samples as screening tests and the positive samples are conformed by molecular HCV RNA (Erensoy 2001).

The beneficial effect of using an enzyme immunoassay in clinical hematology is that it has the ability to identify alloantibodies in patients who frequently receive platelet transfusions for therapeutic purposes. It has been demonstrated that ELISA is more sensitive to the detection of alloantibodies than other immunoassays, such as lymphocytotoxic test (LCT) and the platelet immunofluorescence test (PIFT), and it is performed in a short period of time (Levin et al. 2003). Furthermore, enzyme immunoassays are able to detect anti-platelet antibodies, platelet associated IgG (PAIgG) , HLA and circulating antibodies in patients with Idiopathic thrombocytopenic purpura (ITP) (Schiffer and Young 1983).

There are several pieces of evidence which suggest that a ubiquitous herpes virus, in particular Epstein-Barr virus (EBV), is implicated in many types of lymphoma such as Hodgkin’s disease and Burkitt’s lymphoma, so the early detection of EBV antigen specific T cells is essential and therefore ELISPOT assay can be used to analyse T cell responses (Yang et al. 2000).

Another of its feature is related to acquired von Willebrand syndrome, which is caused by autoantibodies against von Willebrand factor (vWF). This plays an essential role in hemostasis and is characterised by prolonged bleeding due to the defect in vWF activity. Detection of anti-vWF antibody can be performed by ELISA (Mohri 2003), and (Stewart et al. 1997) mention that competitive ELISA is a useful technique in the detection of anti-vWFA antibodies in a clinical samples.

Despite the benefits of ELISA in the detection of HLA class I specific antibodies, it is less sensitive than flow cytometry, so before platelet transplantation, flow cytometry must be used (Worthington et al. 2001). Although chemiluminescence immunoassay has great sensitivity in detecting HBsAg in blood donor samples, it has less sensitivity than the polymerase chain reaction (PCR)(Sato et al. 2001), so to increase the safety of blood from HIV-1 , HCV, and HBV, the nucleic acid amplification technique (NAT) has been applied for the screening of blood donor samples (Weber, Mühlbacher and Melchior 2005).

CONCLUSION

Enzyme Immunoassay is one of the most significant techniques to have been used in clinical laboratories to analyse the antigen-antibody reaction in a short period of time. It is sensitive to screening a large number of clinical samples and therefore has been applied in many clinical laboratories for diagnoses, prognoses and for monitoring immune responses.

REFRENCES

BAEYENS, W. R. G., et al. (1998). Chemiluminescence-based detection: Principles and analytical applications in flowing streams and in immunoassays. Journal of pharmaceutical and biomedical analysis, 17 (6-7), 941-953.

COX, Josephine H., FERRARI, Guido and JANETZKI, Sylvia (2006). Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods, 38 (4), 274-282.

ERENSOY, Selda (2001). Diagnosis of hepatitis C virus (HCV) infection and laboratory monitoring of its therapy. Journal of clinical virology, 21 (3), 271-281.

LEVIN, M. D., et al. (2003). Screening for alloantibodies in the serum of patients receiving platelet transfusions: A comparison of the ELISA, lymphocytotoxicity, and the indirect immunofluorescence method. Transfusion, 43 (1), 72-77.

LION, E., et al. (2009). Quantification of IFN-? produced by human purified NK cells following tumor cell stimulation: Comparison of three IFN-? assays. Journal of immunological methods, 350 (1-2), 89-96.

MOHRI, H. (2003). Acquired von willebrand syndrome: Its pathophysiology, laboratory features and management. [online]. Journal of thrombosis and thrombolysis, 15 (3), 141-149.

SATO, S., et al. (2001). Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay. Transfusion, 41 (9), 1107-1113.

SCHIFFER, CA and YOUNG, V. (1983). Detection of platelet antibodies using a micro-enzyme-linked immunosorbent assay (ELISA). [online]. Blood, 61 (2), 311.

STEWART, M. W., et al. (1997). vWf inhibitor detection by competitive ELISA. Journal of immunological methods, 200 (1-2), 113-119.

TSITSILONIS, Ourania E., et al. (2004). Serological detection of hepatitis B viral infection by a panel of solid-phase enzyme-linked immunosorbent assays (ELISA). Journal of pharmaceutical and biomedical analysis, 34 (4), 811-822.

WEBER, Bernard, MÜHLBACHER, Annelies and MELCHIOR, Walter (2005). Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing. Journal of clinical virology, 32 (1), 67-70.

WORTHINGTON, J. E., et al. (2001). A comparison of enzyme-linked immunoabsorbent assays and flow cytometry techniques for the detection of HLA specific antibodies. Human immunology, 62 (10), 1178-1184.

YANG, J., et al. (2000). Application of the ELISPOT assay to the characterization of CD8(+) responses to epstein-barr virus antigens. Blood, 95 (1), 241-248.