ELISA Overview and Interpretation

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Questions and Answers

What does ELISA stand for?

Enzyme-Linked Immunosorbent Assay.

What are ELISA assays used for in labs?

To determine if a particular antibody is present in a patient's blood sample.

What test can be used to determine whether a patient has an infectious or autoimmune disease?

An ELISA test.

What does a positive result indicate?

An antibody is present and the person has the disease. Signup and view all the answers

The watery fluid of the blood is called?

Serum. Signup and view all the answers

Once isolated, the secondary antibody can be?

Chemically linked to a system that can produce a detectable signal. Signup and view all the answers

What happens when the appropriate chemical (substrate) is added?

The enzyme converts to a colored substance that can be measured. Signup and view all the answers

How is the test quantified?

By the amount of color produced. Signup and view all the answers

What does the amount of color reflect?

The amount of antigen initially present. Signup and view all the answers

What is systemic lupus erythematosus (SLE)?

An autoimmune disease where antibodies fight the body's own cells. Signup and view all the answers

What are the four steps of an ELISA protocol?

Step 1: Bind sample to support. Step 2: Add primary antibody; wash. Step 3: Add secondary antibody-enzyme conjugate; wash. Step 4: Add substrate. Signup and view all the answers

What does a centrifuge do?

It spins tubes to separate solutions into different phases. Signup and view all the answers

What are you preparing in step 2 and why are there three different solutions?

Different dilutions and solutions are needed to determine the level of the antibody in the sample. Signup and view all the answers

What has the ELISA plate been pretreated with and why?

The ELISA plate has been pretreated with SLE antigen to facilitate binding and color formation. Signup and view all the answers

What is the positive control?

The anti-DNA primary antibody. Signup and view all the answers

What is a primary antibody?

The 1st antibody used in an immunoassay to detect the foreign particle. Signup and view all the answers

What is the negative control?

The buffer. Signup and view all the answers

Why is it necessary to have a positive and a negative control?

To rule out problems like reagents changing over time and ensure appropriate testing conditions. Signup and view all the answers

Why incubate the plate in step 5?

To ensure that the antibody present in the sample will interact correctly with the antigen. Signup and view all the answers

Why wash the plate?

To remove any antibody that did not react with the SLE antigen in the well. Signup and view all the answers

What is a secondary antibody?

The 2nd antibody used to recognize the primary antibody as foreign. Signup and view all the answers

What is the attached enzyme in this assay?

HRP (Horseradish Peroxidase). Signup and view all the answers

What is the specific substrate for HRP and what color does it produce?

The substrate is ABTS, which produces a yellow color. Signup and view all the answers

How can the yellow color be quantitatively measured, and at what wavelength?

The color yellow is measured by eye or in a spectrometer at 414 nanometers. Signup and view all the answers

What do the results indicate?

Patient A is likely to have SLE; Patient C probably does not; Patient B might have it, but further testing is required. Signup and view all the answers

How is this same test used to test for the presence of HIV?

ELISA procedure remains the same for HIV, requiring a fresh blood sample and a different enzyme. Signup and view all the answers

Study Notes

ELISA Overview

Stands for Enzyme-Linked Immunosorbent Assay, a common laboratory technique.
Used to determine the presence of specific antibodies in a blood sample.
Can diagnose infectious or autoimmune diseases.

ELISA Results Interpretation

A positive result indicates an antibody is present, suggesting the person has the disease.
The color intensity produced in the test correlates with the concentration of the antigen.

Serum and Controls

Serum is the watery fluid extracted from blood after centrifugation.
Positive control: anti-DNA primary antibody.
Negative control: buffer to ensure reliability of results.

ELISA Steps

Step 1: Samples are bound to a support and centrifuged to separate phases.
Step 2: Different dilutions of samples are prepared for accurate antibody level determination.
Step 3: Primary antibody is added to bind to the antigen; excess is washed away.
Step 4: Secondary antibody-conjugate is added, allowing for detection.
Step 5: Incubation ensures proper interaction between antibodies and antigens.
Step 6: Plate washing removes unbound antibodies.
Step 7: Secondary antibody, from a different species, is added along with HRP enzyme.

Significance of Color Change

Substrate for HRP is ABTS, producing a yellow color which can be measured.
Color is quantitatively analyzed at a wavelength of 414 nanometers.

Application for Disease Testing

Systemic lupus erythematosus (SLE) is an autoimmune condition detected via ELISA.
For HIV testing, a similar ELISA protocol can be followed with different reagents.

Patient Results Interpretation

Results for hypothetical patients indicate likely disease presence:
- Patient A: Likely has SLE/HIV.
- Patient B: Unlikely to have SLE/HIV, further testing needed.
- Patient C: Probably does not have SLE/HIV.

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Description

This quiz covers the fundamentals of the Enzyme-Linked Immunosorbent Assay (ELISA), a vital lab technique for detecting antibodies in blood samples. It includes the interpretation of results, understanding serum controls, and the step-by-step procedure of the ELISA process. Test your knowledge of this important diagnostic tool!