Figur 10-6 Indirect fluorescent antibody tests for Toxoplasma gondii, IgG antibodies. A, Positive reaction. Br Negative reaction. (Courtesy Meridian, Cincinnati, Ohio.)
The introduction of membrane-bound ELISA components has improved sensitivity and ease of use dramatically. Slot-blot and dot-blot assays force the target antigen through a membrane filter, causing it to become affixed in the shape of the hole (a dot or a slot). Several antigens can be placed on one membrane. When test (patient) serum is layered onto the membrane, specific antibodies, if present, will bind to the corresponding dot or slot of antigen. Addition of a labeled second antibody and subsequent development of the label allows visual detection of the presence of antibodies based on the pattern of antigen sites. Cassette-based membrane-bound ELISA assays, designed for testing a single serum, can be performed rapidly (often within 10 minutes). Commercial kits to detect antibodies to Helicobacter pybri, T. gondii, and some other infectious agents are available. Accuracy of the results of tests using each of these formats is variable, however.
Antibody capture ELISAs are particularly valuable for detecting IgM in the presence of IgG. Anti-IgM antibodies are fixed to the solid phase, and thus only IgM antibodies, if present in the patient's serum, are bound. In a second step, specific antigen is added in a sandwich format and a second antigen-specific labeled antibody is finally added. Toxoplasmosis, rubella, and other infections are diagnosed using this technology, usually in research settings.
Indirect Fluorescent Antibody Tests and Other Immunomicroscopic Methods
A widely applied method to detect diverse antibodies is that of indirect fluorescent antibody determination
(IFA), which is described in detail in Chapter 9. For tests of this type, the antigen against which the patient makes antibody (e.g., whole Toxoplasma organisms or virus-infected tissue culture cells) is affixed to the surface of a microscope slide. The patient's serum to be tested is diluted and placed on the slide, covering the area in which antigen was placed. If present in the serum, antibody will bind to its specific antigen. Unbound antibody is then removed by washing the slide. In the second stage of the procedure, a conjugate of antihuman globulin, which may be directed specifically against IgG or IgM, and a dye that will fluoresce when exposed to ultraviolet light (e.g., fluorescein) is placed on the slide. This labeled marker for human antibody will bind to the antibody already bound to the antigen on the slide and will serve as a detector of binding of the antibody to the antigen when viewed under a fluorescence microscope (Figure 10-6). Commercially available test kits include the slides with the antigens, positive and negative control sera, diluent for the patients' sera, and the properly diluted conjugate. As with other commerdal products, IFA systems should be used as units, without modifying the manufacturer's instructions. Currently, commercially available IFA tests in-dude those for antibodies to Legionella spedes, B. burgdorferi, T. gondii, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus capsid antigen, early antigen and nuclear antigen, herpes simplex viruses types 1 and 2, rubella virus, M. pneumoniae, T. pallidum (the fluorescent treponemal antibody absorption test [FTA-ABS]), and several rickettsiae. Most of these tests, if performed properly, give extremely specific and sensitive results. Proper interpretation of IFA tests requires experienced and technically competent technologists. These tests can be performed rapidly and are cost effective if only a few samples are tested.
Infectious | | Lysate is agent w iysed in solution placed into with sodium dodecyi trough of Polyacrylamide sulphate to release slab gel.
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Anti-human antibody enzyme conjugate is added. Conjugate binds to antigen-antibody complexes.
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