The Western Blot

Both methods have been validated according to the Japanese validation protocol (Sakai et al., 2008), and both primers are commercially available. All the Western blotting and PCR kits are shown in Table 4.11. 

The diagnosis of HIV infection is made either by a baseline serologic screening test such as the ELISA or a rapid test. If reactive, then a confirmatory test is performed. The Western blot (WB) is the gold standard confirmatory test and is commonly used. The WB is considered reactive if two of the three major... 

Western blot A method to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. Some diagnostic applications for the Western blot include Lyme disease, bovine spongiform encephalopathy, and human immunodeficiency virus (HIV) (it is considered the gold standard for HIV diagnostic testing). 

Third Step The Western blotting technique, applied to cell extracts, was used to confirm the pRB immunostaining results in two bladder cancer cell lines of T24 and J82, giving quantitative results for pRB in the two cell lines, comparable with that demonstrated by IHC (Fig. 1.3). 

Figure 1.3 Western blotting of pRB protein extracted from two fresh cell lines, T24 and J82. The pRB proteins in fresh T24 cell line showed a stronger band than that obtained from J82 cell line. The Western blotting results correlated well with IHC staining intensity (Table 1.3 and Fig. 1.1). Reproduced with permission from Shi et al., Biotech. Histochem. 2007 82 301-309.

If the gel separates DNA and the DNA is detected with a DNA probe, it is called a Southern blot. If RNA is separated on the gel and then detected by a DNA probe, it is a Northern. A Western uses specific antibodies to detect specific protein molecules on a blot of a protein gel. In the Western blot, the role of the DNA probe is filled by an antibody that recognizes a specific protein. 

Because of the significant false positive rate for the ELISA test, a second, more specific test for HIV antibodies is also used the Western blot test. This technique has a lower incidence of false positives than the ELISA assay. In practice, serum samples that score antibody positive by the ELISA test are generally retested by the Western blot procedure. Serum samples are considered positive if they are found to contain HIV-specific antibodies by both tests. 

As mentioned before, because of false positives with ELISA, the necessity for the Western blot test arises. 

Some patients will have repeatedly nonspecific reactivity in an ELISA assay, since the HIV-1 antigen used for the antibody assays is produced in cultured human T cells. It is not unexpected that occasional false positive assays occur in human sera from individuals with autoimmune diseases a history of multiple pregnancies or multiple transfusions or antibodies to certain class II histocompatibility antigens (especially HLA-DR4). Block reagents have been added to specimen diluents to minimize cross-reactions in these sera. This necessitates the use of confirmatory tests, especially the Western blot. With the use of both ELISA and Western blots, false positives decrease to less than 1 per 100,000. 

Most HIV-1 antibody positive sera react with all nine bands, in which case interpretation is not difficult. However, some HIV-infected patients do not have detectable antibody to some of the viral antigens, especially very early and very late in HIV infections. The Western blotting pattern can be used to follow HIV disease progression for example, the disappearance of the p 24 band signals increasing replication of virus. 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

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%,... 

Fig. 2. Illustration of the setup of the sandwich cassette in the transfer tank observed from the top for the western blotting experiment.

From the western blot image (Fig. 4), the GAPDH protein band of the untreated sample and the negative control sample (in which the Silencer Select negative control siRNA is used)... 

Open the Image J software and the western blot image (scanned from the film or captured by a camera) (see Notes 16 and 17). 

The Western blot procedure (Experiment 7) is now used to test human serum for the presence of antibodies to the AIDS virus. Find two publications that describe procedures for this assay. 

E3 5. Design a diagnostic test based on the Western blot that would give an indication of infection by the AIDS virus. Assume that a blood serum sample is available from the patient. 

Amott D, Kishiyama A, Luis EA, Ludlum SG, Marsters JC Jr, Stults JT. Selective detection of membrane proteins without antibodies a mass spectro-metric version of the Western blot. Mol Cell Proteomics 2002 1 148-156. 

Fig. 2. Western blotting using a radiolabeled antibody (a) SDS polyacrylamide gel electrophoresis, yielding polypeptides separated into discrete bands (b) nitrocellulose or nylon membrane onto which the protein bands have been blotted (i.e. a Western blot) (c) autoradiograph after incubating the Western blot with radiolabeled antibody, washing away unbound antibody and placing the membrane against X-ray film.

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