Requirements for the detection of very specific antibodies have driven the development of the Western blot immunoassay (Figure 1). The method is based on the electrophoretic separation of major proteins of an infectious agent in a two-dimensional agarose (first dimension) and acrylamide (second dimension) matrix. A suspension of the organism is mechanically or chemically disrupted, and the solubilized antigen suspension is placed at one end of a polyacrylamide (polymer) gel. Under the influence of an electrical current, the proteins migrate through the gel. Most bacteria or viruses contain several major proteins that can be recognized based on their position in the gel after electrophoresis. Smaller proteins travel faster and migrate farther in the lanes of the gel. The protein bands are transferred from the gel to a nitrocellulose or other type of thin membrane, and the membrane is treated to immobilize the proteins. The membrane is then cut into many thin strips, each carrying the pattern of protein bands. When patient serum is layered over the strip, antibodies bind to each of the protein components represented by a band on the strip. The pattern of antibodies present can be used to deter mine whether the patient has a current infection or is immune to the agent (Figure 2). Antibodies against microbes with numerous cross-reacting antibodies, such as T. pallidum, B. burgdorferi, herpes simplex virus types 1 and 2, and HIV, are identified more specifically using this technology than a single method that is used to identify a single antibody type. For example, the CDC defines an ELISA or immunofluorescence assay as a first-line test for Lyme disease antibody, but positive or equivocal results must be confirmed by a Western blot test.

Fig1. Diagram of Western blot immunoassay system. 

Fig2.  Human immunodeficiency virus type 1 (HIV-1) Western  blot immunoassay. Samples are characterized as positive, indeterminate, or negative based on the bands found to be present in  significant intensity. A positive blot has any two or more of the following bands: p24, gp41, and/or gp120/160. An indeterminate blot  contains some bands but not the definitive ones. A negative blot  has no bands present. Lane 16 shows antibodies from a control  serum binding to the virus-specific proteins (p) and glycoproteins  (gp) transferred onto the nitrocellulose paper. (Courtesy Calypte  Biomedical Corp., Pleasanton, Calif.)

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مواضيع ذات صلة

Blood Coagulation Tests

Indirect Fluorescent Antibody Tests and Other Immunomicroscopic Methods

Enzyme-Linked Immunosorbent Assays

Laboratory Diagnosis of Thyrotoxicosis

Laboratory Findings of Beta-Thalassemia Syndromes

Complement Fixation Assays

Flocculation Tests

Particle Agglutination Tests

Direct Whole Pathogen Agglutination Assays

Separating IgM From IgG For Serologic Testing

Serodiagnosis of Infectious Diseases

TSH- Receptor Antibodies