20.5 Fluorescent antibody techniques  (Page 2/8)

The IFA test for syphilis provides an important complement to the VDRL test discussed in Detecting Antigen-Antibody Complexes . The VDRL is more likely to generate false-positive reactions than the IFA test; however, the VDRL is a better test for determining whether an infection is currently active.

IFA tests are also useful for the diagnosis of autoimmune diseases. For example, systemic lupus erythematosus (SLE) (see Autoimmune Disorders ) is characterized by elevated expression levels of antinuclear antibodies (ANA). These autoantibodies can be expressed against a variety of DNA-binding proteins and even against DNA itself. Because autoimmunity is often difficult to diagnose, especially early in disease progression, testing for ANA can be a valuable clue in making a diagnosis and starting appropriate treatment.

The IFA for ANA begins by fixing cells grown in culture to a glass slide and making them permeable to antibody. The slides are then incubated with serial dilutions of serum from the patient. After incubation, the slide is washed to remove unbound proteins, and the fluorescent antibody (antihuman IgG conjugated to a fluorogen) added. After an incubation and wash, the cells can be examined for fluorescence evident around the nucleus ( [link] ). The titer of ANA in the serum is determined by the highest dilution showing fluorescence. Because many healthy people express ANA, the American College of Rheumatology recommends that the titer must be at least 1:40 in the presence of symptoms involving two or more organ systems to be considered indicative of SLE. Gill, James M., ANNA M. Quisel, PETER V. Rocca, and DENE T. Walters. “Diagnosis of systemic lupus erythematosus.” American family physician 68, no. 11 (2003): 2179-2186.

Flow cytometry

Fluorescently labeled antibodies can be used to quantify cells of a specific type in a complex mixture using flow cytometry ( [link] ), an automated, cell-counting system that detects fluorescing cells as they pass through a narrow tube one cell at a time. For example, in HIV infections, it is important to know the level of CD4 T cells in the patient’s blood; if the numbers fall below 500 per μL of blood, the patient becomes more likely to acquire opportunistic infections; below 200 per μL, the patient can no longer mount a useful adaptive immune response at all. The analysis begins by incubating a mixed-cell population (e.g., white blood cells from a donor) with a fluorescently labeled mAb specific for a subpopulation of cells (e.g., anti-CD4). Some experiments look at two cell markers simultaneously by adding a different fluorogen to the appropriate mAb. The cells are then introduced to the flow cytometer through a narrow capillary that forces the cells to pass in single file. A laser is used to activate the fluorogen. The fluorescent light radiates out in all directions, so the fluorescence detector can be positioned at an angle from the incident laser light.