Lab 12: HIV- 1 Detection using ELISA

Objectives

• To understand the molecular biology of the human immunodeficiency virus (HIV) and the pathogenesis of acquired immune deficiency syndrome

• To understand the concepts and methodology involved with enzyme-linked immunosorbent (ELISA) assays

• To simulate the clinical screening of serum samples for antibodies to the HIV virus

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HIV (Human Immunodeficiency Virus)

• Example of a retrovirus

• Infects CD4+ helper T cells, macrophages, and dendritic cells

Macrophages and Dendritic Cells

• Two types of antigen presenting cells

CD4+ Helper T Cells

HIV Infection

HIV

• Spread through the exchange of body fluids, sharing of needles, mother to child, blood transfusion

• Incubation period is 2 months to over 10 years

• Initial symptoms are flu-like

AIDS (Acquired Immune Deficiency

Syndrome)

• Overtime HIV leads to the development of AIDS

• Progressive failure of the immune system

• Defined in terms of a CD4+ T cell count below 200 cells per µL

• Pneumonia, cachexia (muscle wasting), respiratory illnesses, viral-induced cancers

Antigen-Antibody Interaction

• Antibodies are produced by B cells of the immune system

ELISA • Enzyme-linked immunosorbent assay

• Measures the concentration of antibodies or antigens in a solution

• Used primarily as a diagnostic tool in medicine

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Other Uses for ELISA

• Diagnose disease – human and veterinary medicine

• Agriculture – detect viruses and GMOs

• Environmental – indoor air quality (mold toxins)

• Food Safety and Quality – allergic antigens

• Drug Testing – performance enhancing, etc.

• Pregnancy Tests – hCG hormone

How does ELISA work?

• Uses antibodies and color change to identify a substance

• Microtiter plates

Indirect ELISA Steps 1. Antigens are added to the wells of the

microtiter plate and binding occurs

2. Primary antibody solution is added to the wells, allows the antibody to bind to the antigen

3. Enzyme-labeled secondary antibody solution is added to the wells, secondary binds to primary

4. Chromogenic (color-producing) enzyme substrate is added to the wells to allow color to develop

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Indirect ELISA Assay

PBS “Washes”

• Wells are “washed” with PBS (phosphate buffered saline)

• Removes unabsorbed antigens and antibodies

Alternative Approach

Microplate Strips

• Made of polystyrene which absorbs (binds) proteins by hydrophobic interaction

• 96 wells (8 removable rows of 12 wells)

Materials

• Yellow Tube – Your “body” fluids

• Violet Tube (+) – Positive Control

• Blue Tube (-) – Negative Control

• Green Tube (PA) – Primary Antibody

• Orange Tube (SA) – Secondary Antibody

• Brown Tube (SUB) – Enzyme Substrate

• Beaker of Wash Buffer

• Disposable Transfer Pipettes

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Tracking Disease Outbreaks

• Part 1: Share Body Fluids

• Part 2: Detecting p24 HIV-1 Capsid Protein Using ELISA

Sharing Partner #1

Sharing Partner #2

Sharing Partner #3

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12-Well Strip

• To be shared between two students

Results

• Draw the results of the ELISA Assay

• Use black pen and colored pencils

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Disease Transmission Map

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Analysis Questions

1. Are you infected with HIV? Explain how you know.

2. Why did you assay your samples in triplicate?

3. If you tested positive for disease exposure, did you have direct contact with one of the original infected students? If not, what conclusions can you reach about transmissibility of disease in a population?

Notebook