| A | B |
|---|
| Homogeneous EIA | No physical separation step of bound and labeled antigens required. Patient antigen and labeled antigen react with reagent antibody in solution. Enzyme label is inactivated when reagent antigen binds to antibody. |
| Heterogeneous | Requires solid-phase material to allow for antigen or antibody binding. Includes both competitive and noncompetitive immunoassay designs |
| Competitive | Patient antigen (Ag) competes with labeled antigen (Ag*) for limited antibody-binding (Ab) sites. Final detected signal is Ag*Ab (labeled antigen bound to antibody), as shown in the reaction: Ag, Ag*, Ab→Ag*Ab + AgAb |
| Noncompetitive, indirect ELISA for antibody detection | Excess solid-phase antigen binds patient antibody, and a second labeled antibody (anti-human immunoglobulin) is added. |
| Capture or sandwich | Excess solid-phase antibody binds patient antigen, and a second labeled antibody (to the antigen) is added after a wash step. |
| Enzyme-multiplied immunoassay technique (EMIT) | Reagent antigen is bound to an enzyme tag. Change in enzyme activity is observed as specific antigen–antibody interaction occurs in solution. If reagent antibody binds to the enzyme–antigen pair, enzyme activity is blocked. Otherwise, enzyme is active. Active enzyme (commonly glucose-6-phosphate dehydrogenase) catalyzes the reduction of NAD+ to NADH, leading to increased absorbance in the UV-wavelength region. |
| Direct immunofluorescence | Patient sample (e.g., tissue) is attached to a slide. Specific fluorescent-labeled antibody is added. In cell-based immunoassays, patient cells are incubated with fluorescent-labeled antibody to a cell surface antigen. |
| Indirect immunofluorescence | To detect patient antibody, reagent antigen is attached to a slide and incubated with the patient sample. A second fluorescent-labeled antibody (anti-human immunoglobulin) is added. To detect antigen, patient sample is fixed to a slide and incubated with antibody to the antigen (primary antibody), followed by labeled secondary antibody, directed against the primary antibody. |
| Fluorescence polarization | Fluorescent-labeled antigen competes with patient antigen for a limited number of soluble antibody-binding sites. |
| Rapid Immunochromatographic | Patient sample is added to a test strip and migrates through the strip. Patient antigen complexes to antibody-labeled particles or competes with antigen-labeled particles across individual test lanes at unique detection zones. |
