Other Rapid Formats Immunogold Lateral Flow Immunoassay Immunochromatography Optical Immunoassay Endogenous Viral Encoded Enzyme Assay

Membrane EIAs usually involve a series of steps: addition of sample, wash step, addition of conjugate, wash step, addition of substrate, and then stop reagent. The result is read as a colored spot or triangle on a solid surface. By substituting an IgG binding dye (e.g., staphylococcal protein A-gold reagent) for the anti-immunoglobulin conjugate, the procedure can be shortened by one step. Like membrane EIAs, most of these tests include a built-in control; if the test differentiates two different agents (e.g., influenza A and B), two controls are included.

Immunochromatographic or lateral flow assays require the addition of only one or no reagent and thus are extremely simple to perform. These tests use antibodies spotted onto nitrocellulose membranes with lateral or vertical flow of sample or reagents to interact with immobilized antibody (Fig. 3.4). Use of an antibody sandwich increases sensitivity. Specific antibody is adsorbed onto a nitrocellulose membrane in the sample line, and a control antibody is adsorbed onto the same membrane as second line. Both antibodies are conjugated to visualizing particles that are dried onto an inert fibrous support. Conjugate pad and striped membrane are combined to construct the test strip. An extracted sample is added at one end

Capture Colloidal Gold-

antibody V* Antigen y Antibody Conjugate

Sample with antigen added to sample pad

Antigen moves by capillary action and binds to conjugate

Antigen-colloidal gold antibody complex binds to capture antibody

Sample with antigen added to sample pad

Antigen moves by capillary action and binds to conjugate

Antigen-colloidal gold antibody complex binds to capture antibody

Figure 3.4. Lateral flow immunochromatography.

and moves along the membrane by capillary action to reach the immobilized antibody stripes. Alternatively, a test strip can be inserted vertically into a tube containing the extracted sample.

Optical immunoassays allow direct visualization of a physical change in the thickness of molecular thin films (Boivin et al., 2001). The observed physical change is due to antigen-antibody binding on an optical surface of a silicon wafer, on which specific antibodies have been immobilized. When an extracted specimen is placed directly on the optical surface, antigen is captured. After a wash step, substrate is added, and the thickness of the thin film increases. This change in thickness alters the reflected light path and is perceived as a color change.

One rapid test uses a chromogenic substrate that is uniquely recognized by the influenza virus-encoded neuraminidase (NA) enzyme (Hamilton et al., 2002). The unique substrate is coupled to a color-generating molecule, and in the presence of influenza NA, the coupled substrate is cleaved, and a colored product is produced and precipitates. This strategy bypasses the need for the antibody-capture step and wash procedures of other antigen tests.

Disadvantages of rapid membrane assays in general include subjective interpretation, lack of automation, and possible errors if the reader is color-blind. Although simple to perform, lack of attention to technique can lead to errors. Samples must disperse within specified time limits, and pipettes must be held vertically for correct delivery of reagent volumes. Accurate timing of steps can be adversely affected when multiple samples are tested. These formats are useful primarily for small-volume testing. Conventional EIA and similar methods scale up to larger sample volumes more efficiently.

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