Medical Fun Facts Podcast

MFF0062: Western blots and protein immunoblots

It’s Thursday 4 May 2017.

Protein immunoblots are probably better known as Western blots. The name is derived sort of from knowing that Northern blots detect RNA and Southern blots detect DNA, so the name Western blot was assigned to immunoblots that detect protein.

Western blots or protein immunoblots are used to detect antibodies in serum specimens. They were commonly used in diagnosing infections caused by the Human Immunodeficiency virus and the Hepatitis C virus.

Proteins from pathogens like HIV and HCV can be applied to membrane strips. These proteins are often important antigens or conserved proteins that are specific for the pathogen.

The number of proteins is usually kept to a number sufficient to make a diagnosis and to ensure specificity. In most types of assays, not all the proteins need to be demonstrated for a result to be regarded as the detection of a pathogen’s antibodies. There is usually a threshold number, for example, out of ten proteins, at least five may have to be reactive to say the test detected the specific antibody. In some situations, there might also be proteins that must react for a result to be reported as antibody detected. For example, if you have proteins named 1 through to 10, you may need five of those proteins to react and proteins 1 and 4 must be reactive for the result to be reported as antibody detected. In this way, protein immunoblots can be specific.

To perform the test, it is usually a matter of incubating the patient’s serum with a membrane strip containing the test proteins. After incubation, the patient’s serum is washed off and an antihuman antibody tagged with a signal like an enzyme (this is known as a conjugate reagent) can be added and incubated. After the incubation period, the conjugate is washed off and the substrate is added for enzymatic reactions so that after a short period of time, the reaction can be visualised and read.

Protein immunoblots by nature are specific and therefore get used commonly to confirm assays which may be sensitive but lack specificity. This two-step approach provides greater certainty for diagnosing certain infections. For example, diagnosing HIV infection serologically usually involved a very sensitive enzyme immunoassay that can be performed on a large high throughput random access instrument and any reactive specimens then undergo confirmatory testing via Western blot or protein immunoblot.

The protein immunoblot is a good, accurate and robust approach but it takes time and significant operator skill and experience. These tests require good training and external proficiency testing is vital. Rest assured, in Australia, under our medical testing accreditation framework, when Western blots are performed, the operators are skilled and systems are required to undergo external proficiency testing.

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