Lab 12: HIV- 1 Detection

using ELISA
 Notebook

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
 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
 Notebook

ELISA
• Enzyme-linked immunosorbent assay
• Measures the concentration of antibodies or
antigens in a solution
• Used primarily as a diagnostic tool in medicine
 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
 Notebook

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
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)
 Notebook

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
 Notebook

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
 12-Well Strip
• To be shared between two students
 Notebook

Results
• Draw the results of the ELISA Assay
• Use black pen and colored pencils
 Notebook

Disease Transmission Map

 Notebook

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?