Indirect ELISA Test - Animated Video

Summary

This video explains the indirect enzyme-linked immunosorbent assay (ELISA) used to detect HIV antibodies in patients. The process involves blood collection, centrifugation, and serum preparation. The 96-well plate is coated with HIV antigens, followed by blocking and incubating with the patient's serum. A secondary antibody conjugated with an enzyme is used to detect antibody-antigen complexes. The presence of HIV antibodies is indicated by a color change from blue to yellow after substrate addition and acid treatment. Finally, the spectrometer confirms the result by measuring absorbance, providing a reliable HIV detection method.

Takeaways

• 😀 The Liza (ELISA) test is a plate-based technique used to detect and quantify substances like antibodies, antigens, proteins, peptides, and hormones.
• 😀 The indirect ELISA is a specific method used to detect antibodies, such as those produced in response to an HIV infection.
• 😀 When a person is infected with HIV, their immune system produces antibodies to combat the virus, which can be detected months or years later.
• 😀 The testing process starts with blood collection through venipuncture, as blood contains a higher concentration of HIV antibodies than oral fluid.
• 😀 After blood collection, the sample is processed by centrifugation to separate components based on density, followed by isolation of the serum layer.
• 😀 The next step involves coating the 96-well plate with an HIV antigen (like GP120, GP41, or P24) to detect antibodies.
• 😀 After antigen addition, the plate is incubated, and excess antigen is removed, leaving only the bound antigen on the plate surface.
• 😀 A blocking solution (containing proteins like BSA, casein, or milk) is added to prevent non-specific binding during subsequent antibody detection steps.
• 😀 After blocking, the serum samples from patients are added to the wells to allow antibodies to bind to the antigen, with controls included for accuracy.
• 😀 A secondary antibody, conjugated with an enzyme (usually horseradish peroxidase), is used to detect the bound antibodies, leading to a color change if the target antibody is present.
• 😀 The color change, typically from blue to yellow, is measured using a spectrophotometer, with a positive result indicating the presence of HIV antibodies.

Q & A

What is the purpose of an indirect ELISA test?

An indirect ELISA test is used to detect the presence of antibodies in a sample, particularly to determine if a person has been exposed to a specific pathogen, such as HIV.

How does the immune system respond to HIV infection?

When a person is infected with HIV, their immune system creates antibodies as part of the body's natural defense to fight and neutralize the virus.

Why is blood the preferred specimen for HIV testing?

Blood is preferred for HIV testing because it contains a higher concentration of HIV antibodies compared to oral fluid, making the results more accurate.

What happens during the centrifugation process in the HIV testing procedure?

During centrifugation, the blood components are separated based on their density, allowing the serum to be isolated for further testing.

What is the role of the antigen in the indirect ELISA test?

In indirect ELISA, the antigen (such as proteins from the HIV virus) is immobilized onto the plate to which the antibodies from the serum sample will bind if present.

Why is a blocking solution used in the indirect ELISA process?

The blocking solution, which contains proteins like BSA or casein, is used to cover unoccupied sites on the plate, preventing non-specific binding during subsequent steps.

What is the purpose of incubating the serum samples with the antigen?

The incubation allows the antibodies in the serum samples to specifically bind to the corresponding antigens immobilized on the plate, facilitating the detection of HIV antibodies.

What is the function of the conjugated antibody in the indirect ELISA test?

The conjugated antibody, typically labeled with an enzyme, binds to the antibody-antigen complexes, allowing for detection and quantification of the targeted antibodies.

What does the color change in the wells indicate during the detection step?

A color change from blue to yellow in the wells indicates a positive result, showing the presence of antibodies against HIV in the sample.

How is the final color development in the indirect ELISA test stabilized?

The final color development is stabilized by adding a strong acid, such as sulfuric acid, which halts the enzymatic reaction and ensures accurate measurement of the results.