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Western Blot Detection Methods

As the number of HIV and AIDS cases rose worldwide, the need for accurate HIV testing became essential. Now after positive ELISA HIV tests, medical professionals use a Western blot test to confirm a positive HIV infection. Doctors diagnose Lyme disease through Western blot testing as well. The test utilizes intricate detection methods: medical workers must transfer blood samples from a gel to a membrane to facilitate the detection process. To actually identify antibodies in the membrane, workers use specific enzymes, substrates and/or flurophores.

  1. AP Enzyme

    • Alkaline phosphatase (AP) constitutes one of the primary enzymes in Western blot testing. AP has a linear reaction time, which gives it a detection advantage over other enzymes. The more time an AP reaction has to occur, the more accurate the test. However, the longer period can pick up other chemical compounds and "background" noise, which can skew the results of the test.

    HRP Enzyme

    • Medical workers consider horseradish peroxidase (HRP) superior to AP, due to HRP's smaller size, high activity rate, compatibility with the reaction membrane, reliability, cheap price and widespread availability.

    Chromogenic Substrates

    • Medical professionals consider chromogenic substrates best for viewing the inner-membrane reaction of blood and enzyme. Chromogenic substrates are not as sensitive as other substrates and tend to disappear as the blot dries. But for a quick visual test confirmation, chromogenic substrates make an effective choice.

    Chemiluminescent Blotting Substrates

    • Chemiluminescent blotting substrates require a constant reaction for a successful test. If the enzyme ceases activity or the substrate is depleted, the testing result becomes lost. However, in an optimal test, the testing result becomes available in one to 24 hours. Medical workers use digital imaging equipment or X-rays to actually see the test result, because the naked eye cannot detect it.

    Flurophores

    • If medical workers do not test with enzymes and their respective substrates, then they utilize flurophore-conjugated antibodies. The flurophore test requires less steps but needs special equipment. The test requires an excitation light source, which causes the proper reaction in the membrane. Flurophores and the lighting equipment are expensive, but medical workers have the benefit of using multiple flurophores in the same experiment.

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