Immunoblotting labelling techniques

Figure 10.13 shows a comparison of different labeling techniques, after an identical mouse monoclonal antibody was used in the primary immunoblotting step.15 Lane A shows the indirect immunoperoxidase method, whereby the primary monoclonal is detected using a peroxidase-conjugated goat anti-mouse IgG antibody, followed by a peroxidase activity stain. Lane B shows the peroxidase anti-peroxidase... 

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

Precise controls for the specificity of antibody reactions reside with other techniques, such as immunoprecipitation or immunoblotting. Controls for the labeling... 

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ( blotted ) onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds. 

Colorimetric development of immunoblots probed with phosphatase and peroxidase-labeled antibodies is possible. However, these procedures are relatively insensitive, the color can fade, and they are generally less versatile than other techniques. Electrochemi-iUuminescence (ECL) is just as easy to perform and gives a... 

Immunoprecipitation is a sensitive technique, especially when it is combined with detection by I-labeled antibodies. Labeling the first antibody with produces a more specific reagent and, therefore, cleaner immunoblot, although it may be less sensitive, since it does not have the amplification of signal (and background), which occurs with methods incorporating another layer in the detection system. 

Labelled monoclonal antibodies are employed in detecting, assaying, and locating their complementary epitopes and are the key reagent in the techniques of immunoassay, immunoc3rtochemistiy, immunoblotting, and antibody-based cell sorting. 

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