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Chapter 7 : Enzyme Immunoassays and Immunochromatography

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Abstract:

This chapter deals with principles of enzyme immunoassay (EIA) and immunochromatography (ICR) and contemporary applications of both methods in viral antibody and antigen detection in the diagnostic virology laboratory. Immunoperoxidase staining, also called histochemical EIA or immunohistologic staining, is a type of EIA. “Immunoperoxidase staining” is the term used to describe the assays because most involve the enzyme horseradish peroxidase. Traditional EIA-type steps involving solid-phase reactants and enzyme-labeled detection complexes are performed. The most prominent application of membrane EIAs in diagnostic virology is detection of viral antigens, most commonly either influenza A and B or respiratory syncytial virus (RSV), in patients’ samples collected from the respiratory tract. Like the membrane-based rapid EIAs, optical immunoassays (OIAs) have several steps involved in testing and are classified as moderately complex according to Clinical Laboratory Improvement Act (CLIA). EIA and ICR methods for detection of rotavirus antigen in fecal and other types of samples are especially popular because this virus does not proliferate in standard cell cultures, and immunofluorescence techniques are not useful in detecting rotavirus antigen. EIAs are sometimes named by their detection system. Chemiluminescence and biotin-avidin EIAs are two of these. Membrane-based EIAs, OIAs, and ICRs, are predictably qualitative in nature, with results reported as positive or negative. The application of membrane EIAs, OIAs, and ICRs to viral antigen and antibody detection have opened an avenue for rapid viral diagnosis that allows testing to be conducted in more venues such as point-of-care and physician's office laboratories.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Figures

Image of FIGURE 1
FIGURE 1

Tube-based noncompetitive EIA for antibody detection. (Step 1) The patient’s serum is added to a tube or microwell coated with known antigen. (Step 2) Patient’s antibodies bind to the antigen. Unbound components are washed away. (Step 3) Enzyme-labeled anti-human IgG is added. This binds to antibodies that bound in step 2. Unbound components are washed away. (Step 4) A substrate solution is added. (Step 5) The enzymes that are part of the enzyme-labeled anti-human IgG act on the substrate to produce a color change.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Image of FIGURE 2
FIGURE 2

Bead-based noncompetitive EIA for antibody detection. (Step 1) An antigen-coated bead is incubated in a dilution of the patient’s serum. (Step 2) Patient’s antibodies bind to the antigen. Unbound components are washed away. (Step 3) Enzyme-labeled anti-human IgG is added. This binds to antibodies that bound in step 2. Unbound components are washed away. (Step 4) A substrate solution is added. (Step 5) The enzymes that are part of the enzyme-labeled anti-human IgG act on the substrate to produce a color change.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Image of FIGURE 3
FIGURE 3

Noncompetitive tube-based EIA for antigen detection. (Step 1) The patient’s sample (feces, throat or nasal wash, etc.) is added to a tube or microwell coated with antiviral antibodies of known specificity. (Step 2) The antibodies coating the tube recognize and bind (or capture) the antigen in the patient’s sample. Unbound components are washed away. (Step 3) Enzyme-labeled antiviral antibodies are added. They bind to the viral antigens captured in step 2. Unbound components are washed away. (Step 4) A substrate solution is added. (Step 5) The enzymes that are part of the bound enzyme-labeled antiviral antibodies act on the substrate to produce a color change.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Image of FIGURE 4
FIGURE 4

Competitive tube-based EIA for antibody detection. (Step 1) The patient’s serum and enzyme-labeled antibodies are added to an antigen-coated tube or microwell. (Step 2) The patient’s antibodies compete with the enzyme-labeled antibodies for binding to the antigen. If a sufficient quantity of the patient’s antibodies are present, they bind to the antigen, and few, if any, of the enzyme-labeled antibodies succeed in binding. Unbound components are washed away. (Step 3) A substrate solution is added. (Step 4) There is no color change because unlabeled antibodies from the patient’s serum have bound and few, if any, enzyme-labeled antibodies have bound.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Image of FIGURE 5
FIGURE 5

Membrane EIA testing mechanism and gross appearance. Testing mechanism: (A) viral antigen in specimen is nonspecifically adhered to the membrane by filtration through a focusing device; (B) enzyme-labeled antiviral antibody is added and binds to viral antigen present on the membrane; (C) a substrate solution is added and changes color when acted upon by the enzyme. Gross appearance: (D) a test area is defined within the cassette—this is colorless at the beginning of the assay; (E) the test area changes color due to the action of the enzyme on the substrate solution. The colored dot in the center of the test area is a built-in control to ensure that the device is functioning properly.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Image of FIGURE 6
FIGURE 6

OIA testing mechanism and gross appearance. Testing mechanism: (A) viral antigen in the specimen is added to a silicon wafer coated with antiviral antibody; (B) enzyme-labeled antiviral antibody is added and binds to viral antigen bound in the previous step; (C) a substrate solution is added and changes color when acted upon by the enzyme—this is enhanced by mass enhancement due to the change in thickness of the silicon wafer produced by antigen/antibody complexes and precipitated substrate, thus changing the reflectance of light off the wafer. Gross appearance: (D) the surface of the silicon wafer is mirror-like; (E) a change in the color of the test area and of light reflected from the wafer is seen due to color change and mass enhancement. The colored dot in the center of the test area is a built-in control to ensure that the device is functioning.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Image of FIGURE 7
FIGURE 7

ICR (lateral flow) testing mechanism. (A) Viral antigen in the specimen is added to the sample port of a nitrocellulose strip; nonspecifically bound labeled antiviral antibodies are present in the specimen area. The nitrocellulose strip also includes a test area of unlabeled antiviral antibodies and a control area of unlabeled animal antihuman IgG. (B) Viral antigen in the sample binds to labeled antiviral antibodies in the test area, and the complexes begin to migrate along the strip. (C) Migration continues. (D) Viral antigen, in complex with labeled antiviral antibodies, is recognized by the unlabeled antiviral antibodies in the test area of the strip and captured to form a visible line; excess labeled antiviral antibodies continue to migrate and are captured at the control line by anti-IgG. This control ensures that the specimen migrated the entire length of the strip and that the strip is functioning properly.

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7
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Tables

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TABLE 1

Rapid EIA, OIA, and ICR methods for RSV or influenza A and B antigen detection a

Citation: Leland D. 2009. Enzyme Immunoassays and Immunochromatography, p 89-102. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch7

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