Immunology Lab Final

Describe the theory and procedure of ELISA

Ag samples (HIV) added first into wells Add primary ab specific to HIV ag; wash Add secondary ab specific to primary ab with ENZYME to detect HIV Add substrate to interact with enzyme to make CHROMOGENIC PRODUCT; color change to blue

Why is the block needed in western blot?

Binds to all spots on membrane where no proteins are found so primary ab does not bind here

What are the applications of IF?

Clinically - can be used to identify the presence of pathogen in a patient sample Uses ANTIBODIES to identify a specific protein/cellular structure within cell

What is a fluorochrome?

Compound that absorbs light at one wavelength and emits light at a second wavelength

Applications for SDS/PAGE Western blot?

Identify/quantify a SPECIFIC protein in a sample using an ANTIBODY

How do we analyze western blot results?

Light is visualized as a band which means the patient has been exposed to HIV and has produced antibodies

Purpose/name of wash used in Dot Blot?

MEM wash buffer; wash done after every step to ensure no cross contamination occurs/all ab that did not bind is gone

What was the block used in western blot?

Milk block/protein

Primary antibodies used in western blot?

Mouse anti-HIV IgG

Purpose/name of block used in Dot Blot?

NAP; adds nonspecific protein to bind to all spots on nitrocellulose that protein of interest did not bind to

What did we test for in western blot?

Presence of HIV antibodies

Direct immunofluorescence?

Primary antibody attached with a fluorochrome

What is the theory behind western blot?

Probing samples that tested positive for HIV and CONFIRMING that these patients are positive

What is the transfer membrane in western blot? Why cant it be touched without gloves? COME BACKKKK

Proteins from student's hands can alter results and stick to transfer membrane

Purpose/name of primary ab in Dot Blot?

Rabbit anti-HBsAg; ab that binds to ag HBsAg in sample Took antibody to HBsAg from rabbit

Explain false negatives

Results state test is negative for an antigen, but when tested again is positive

Explain false positive

Results state test is positive for an antigen, but when tested again is negative

What is in the sample (loading) buffer? What is each component used for?

SDS - makes all proteins negatively charged Glycerol - gives the protein sample density so it can sink to the bottom; VERTICAL buffer EDTA - minimizes protein degradation and maintains stability DTT - reduces disulfide bonds and LINEARIZES proteins to based amino acids Bromophenol blue - visualizes the sample by making it blue

Electrophorisis

Separation of macromolecules in an electric field

Know how to graph standard and analyze data for ELISA COME BACKS

X axis - concentration of antigen y axis - absorbance @450nm R2 value close to 1 to be accurate

What is the transfer process?

- Starting 5 minutes before the gel is done running, soak PVDF in methanol -When gel is done running, make a transfer sandwich -PVDF/nitrocellulose needs to be on RED side and gel is on BLACK - RED is positive cathode so negative charged proteins will travel here under electric current

What cells were used within the immunofluorescence lab?

2 patient nasal epithelial cells were used as samples

Sandwich ELISA procedure

Add ANTIBODY Add antigen specific for ab Add primary ab specific to ag Add secondary ab specific to primary ab; enzyme linked Add substate Observe product

Non-competitive ELISA

Add ANTIGEN Add primary ANTIBODY specific to ag Add secondary ab specific to primary ab; enzyme linked Add substrate Observe product

Why is a block used in immunofluorescence?

After cells are fixed to the slide, cells are then blocked to ensure all surfaces are covered either by cells or an inert protein

Indirect immunofluorescence?

An immunofluorescent diagnostic technique in which the fluorochrome is not attached to the primary antibody that recognizes the target antigen, but to a secondary antibody that binds the primary antibody More common - improves visualization because multiple secondary antibodies can bind to first antibody

How are the antibodies made in Dot blot? What does rabbit anti-goat IgG mean?

Antibodies taken from animals like rabbits/goats that have been exposed to Hep B Rabbit anti-goat IgG means we have taken antibody from a rabbit that has antibodies against goat antibodies

Immunofluorescence

Antibody with attached fluorescent tag used to identify specific cellular structures - Antibody binds to specific protein

What was the block used in immunofluorescence?

BSA/milk

What does the primary antibody bind to in western blot?

Binds to HIV immunoglobulin in body; binds to antibodies made against HIV

What is in the mounting media for immunofluorescence?

DAPI

What is the purpose of the immunofluorescence lab?

Determine if 2 patients are positive for COVID-19, searching for COVID SPIKE PROTEN

What are we testing for in Dot Blot?

Determine if a patient is infected with Hepatitis B; HBsAg

What was the secondary antibody used in western blot?

Goat anti-mouse IgG

Purpose/name of secondary ab in Dot Blot?

Goat anti-rabbit IgG; ab that binds specifically to primary ab with enzyme to detect HBsAg Goat that has antibody to rabbit antibody

What results should we expect to see with the immunofluorescence lab?

Green around the cell if the patient is positive for infection Due to Alexa 488 immunofluorescence DAPI is used to identify the nucleus of the patients cells

What did we test for in ELISA

HIV antigen

What was the enzyme-substrate system? How was it used for detection?

HRP (enzyme) + ECL (substrate) Used to produce light; chemiluminescence

What antibody is made if patients were infected with HIV in western blot?

IgG

Do we use a substrate in Immunofluorescence?

No sir

Why do we need controls?

Positive and negative controls ensure that the results in the sample are validated

How does the gel work in western blot?

SDS page - separates proteins in polyacrylamide matrix using electric field based molecular weight and gives protein a negative charge Stacking layer 4% - has bigger pore, UPPER layer, designed to sweep up sample so they can STACK Resolving layer 10% - has smaller pores, LOWER layer that is responsible for separating proteins by size

What were the secondary antibodies used and fluorochromes attached in your experiment? (Immunofluorescence)

Secondary antibody, goat-anti mouse IgG Alexa 488, has fluorochrome attached Alexa fluorochromes used - Alexa 488 - absorbs at 488 and emits at 519

Purpose/name of substrate used in Dot Blot?

TMB; reacts with enzyme HRP to produce detectable product which is chromogenic

Emission wavelength

The wavelength of light emitted by a fluorescent molecule

Excitation wavelength

The wavelength of light that must be absorbed by a molecule in order for the molecule to fluoresce

How does the fluorescent microscope aid in the process of immunofluorescence?

Use the microscopes specific filter to excite the fluorochrome and then observe the emission wavelength

What are applications of Dot Blot?

Used in both research/clinical applications Test for the presence of antibody or antigen Similar to ELISA/western blot but NO separation of gel

Why is DAPI important in immunofluorescence?

Way to locate/visualize the nucleus of the cells Intercalates into the cells DNA and fluoresce as a blue color

Home applications of ELISA?

Detect presence of an antigen in system Pregnancy tests, disease detection, detecting illegal drugs, testing indoor air quality, and determining if food is labeled accurately

Clinical/Industrial applications of ELISA?

Detect the presence of an antibody in system Examples - HIV or COVID-19