Question

Which of the following laboratory techniques would be most useful for distinguishing exogenous from endogenous EPO? A. Northern blot B. Peripheral smear C. Spectrophotometry D. Enzyme-linked immunoabsorbant assay (ELISA)

Short answer

The most useful technique is D. Enzyme-linked immunosorbent assay (ELISA).

Step-by-step solution

Understand the Question

The question asks which laboratory technique can distinguish exogenous (externally administered) from endogenous (naturally occurring) EPO (erythropoietin).

Review Options

Examine each option: (A) Northern blot, (B) Peripheral smear, (C) Spectrophotometry, and (D) Enzyme-linked immunosorbent assay (ELISA).

Evaluate Northern Blot

Northern blot is used to detect RNA expression levels. It is not typically used to distinguish different sources of the same protein like EPO.

Evaluate Peripheral Smear

A peripheral smear involves examining blood cells under a microscope. It cannot distinguish the source of EPO.

Evaluate Spectrophotometry

Spectrophotometry measures absorbance of light in a sample, which might differentiate compounds, but not the source of EPO.

Evaluate ELISA

ELISA can use specific antibodies to detect and quantify proteins such as EPO. It can differentiate between exogenous and endogenous EPO based on structural differences recognized by antibodies.

Conclusion

Among the given techniques, ELISA (Option D) is best suited to distinguish between exogenous and endogenous EPO.

Key concepts

Northern Blot

Northern blot is a powerful technique used to detect specific RNA molecules among a mixture of RNA. This method helps understand gene expression and can tell which genes are turned on or off in a given tissue. The process includes:

• Extracting RNA from tissues or cells.
• Running the RNA on a gel electrophoresis to separate it by size.
• Transferring the RNA onto a membrane.
• Hybridizing the membrane with a labeled probe that binds to the target RNA.

While Northern blot is useful for studying RNA, it does not provide information about the origin of proteins like erythropoietin (EPO). Therefore, it can't distinguish between exogenous (externally administered) and endogenous (naturally occurring) EPO. The focus of this technique is purely on levels and types of RNA, not on the source of proteins.

Peripheral Smear

A peripheral smear is a simple yet essential laboratory technique in hematology used to examine blood cells under a microscope. This involves:

• Spreading a drop of blood thinly across a glass slide.
• Staining the blood smear to make the different types of blood cells more visible.
• Examining the slide under a microscope to assess the number, shape, and size of cells.

This technique can diagnose various blood disorders by observing abnormalities in blood cells. However, a peripheral smear cannot determine the source of EPO in the bloodstream. Its role is primarily the identification and characterization of blood cell types and counts, making it unfit for the purpose of distinguishing exogenous from endogenous EPO.

Spectrophotometry

Spectrophotometry is a method used to measure how much light a chemical substance absorbs. It helps in identifying and quantifying substances in a sample based on light absorbance at specific wavelengths. The basic steps include:

• Preparing the sample to be analyzed.
• Passing a beam of light through the sample.
• Recording the amount of light absorbed at different wavelengths.

This technique is highly useful in various chemical and biochemical analyses. However, spectrophotometry cannot differentiate the source of proteins like EPO because it measures only the absorbance properties of a sample, not the origin. It tells us about the presence and concentration of a substance, but not if the substance is internally or externally sourced.

Enzyme-linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is a widely used technique to detect and quantify soluble substances such as proteins, hormones, and antibodies. ELISA's sensitivity and specificity come from using antibodies to recognize and bind to the target molecule. The process involves:

• Coating a microplate with a capture antibody specific to the target protein.
• Adding the sample where the target protein, if present, will bind to the capture antibody.
• Adding a detection antibody that binds to the target protein, forming a 'sandwich'.
• Introducing an enzyme-linked secondary antibody that binds to the detection antibody.
• Adding a substrate that the enzyme converts to a detectable signal, usually a color change.

ELISA is particularly powerful because its antibodies can distinguish between minor structural differences in similar proteins. This technique can differentiate exogenous from endogenous EPO by recognizing specific epitopes unique to each form. Thus, ELISA is the most suitable method to answer the question of distinguishing the source of EPO.