Immuno Lab Midterm

Labs 1-4

What is the function of the primary lymphoid organs?

Site where lymphocytes (B cells and T cells) develop and mature

What are the types of primary lymphoid organs?

Bone marrow and thymus

What is the function of the thymus?

T cell development and maturation

What is the function of the bone marrow?

• Production of B cells and T cells

• B cell development and maturation

What is the function of the secondary lymphoid organs?

Sites where antigen from pathogens is presented and lymphocytes are activated in response

What are the names of the secondary lymphoid organs?

• Spleen

• Lymph Nodes

• Lymphatic Vessels

• Tonsils and Adenoids

• MALT - mucosa associated lymphoid tissue

What are the lymph nodes and what do they do?

• Tiny bean shaped filters at the junctions of lymphatic vessels

• Cleanse lymph as it filters through by clearing out damaged cells and cancer cells

• Regulates immune response

What is the function of the lymphatic vessels?

To transport lymph through the body

What is lymph?

A clear-to-white watery fluid that allows for plasma and lymphocyte movement to and from lymphatic tissues

What is the spleen and what is its function?

• The biggest secondary lymphoid organ

• Important in RBC filtration

• Reservoir of monocytes (macrophages and dendritic cells)

Why is the ear punch used in mice?

To mark specific individual animals in different ways when there are multiple similar animals being used in an experiment

What do the different shapes of the ear punch mean?

Circle = 1,2,3

Slit = 4,5,6

Double Slit = 7,8,9

What do the different sides of the ear punch mean?

Left = # x 1

Right = # x 10

Why is serum free DMEM used for counting cells?

To prevent serum proteins from binding to the Trypan Blue dye

What volumes of cell suspension and trypan blue did we mix for the hemacytometer sample?

100 uL of each

How much sample did we load into each chamber of the hemacytometer?

~10 uL

What magnification ocular and objective lens did we use during cell counting?

10x ocular, 10x objective

What cells are stained by Trypan Blue and what cells are not?

Dead cells are stained blue, live cells remain clear

Why do we use Trypan Blue for cell counting?

It can get through the damaged cell membranes of nonviable cells so they can take up the Trypan Blue dye and stain their cytoplasm blue, but it can not get into the intact cell membranes of viable cells, so the dye is excluded and they have a clear cytoplasm. This helps differentiate between viable and nonviable cells.

What are the rules for the proper method of counting cells on the hemacytometer?

• Do not count cells that are fused together in clumps

• Count cells touching the middle line on the top and left sides of the square

• Exclude cells touching the middle line on the bottom and right sides of the square

What is Trypan Blue?

A dye that is used as a vital stain to selectively color dead tissues or cells blue (live cells or tissues with intact membranes are not colored)

What is the Trypan Blue dye exclusion test used to determine?

The number of viable cells present in a cell suspension

What is the formula for the dilution factor?

Vf/Vi

What is the formula for the total cells/0.1 uL of diluted sample? (Column A)

(Average # Viable Cells) + (Average # Dead Cells)

• Should be the same numbers as the last column in table A

What is the formula for the total cells/0.1 uL of original sample? (Column B)

(Total cells/0.1 uL of diluted sample) x DF

• (Column A) x DF

What is the formula for cells/mL? (Column C)

(Total Cells/0.1 uL of original sample) x (10,000)

• (Column B) x (10,000)

What is the formula for cells/spleen, aka total cells? (Column D)

(cells/mL) x (volume pellet was resuspended in)

• (Column C) x (volume pellet was resuspended in)

What is the formula for cell viability %? (Column E)

(Total # viable cells) / (Total # cells) x 100%

• (Total # unstained) / (Total # stained and unstained) x 100%

What are other ways to count cells besides using a Hemocytometer?

Electronic devices (Coulter Counter, Hema-Counter, Cellometer)

How do you calculate weight/volume solutions?

w/v % = (mass of solute (g)) / (volume of solution (mL)) x 100

How do you calculate volume/volume solutions?

v/v % = (volume of solute) / (total volume of solution) x 100

How do you calculate for serial dilutions of sera?

(C1)(V1) = (C2)(V2)

What is immunodiffusion?

Antigens and antibodies are placed in wells that are cut into a gel and they can diffuse through the gel media

What type of ELISA did we do?

Indirect MicroELISA

What type of immunodiffusion did we do?

Ouchterlony double diffusion

What are the 5 classes of immunoglobulins?

MADGE

• IgM, IgA, IgD, IgG, IgE

What is the percentage breakdown of how much of each antibody is found in the body?

• IgM: 5-15%

• IgA: 15%

• IgD: less than 1%

• IgG: about 75%

• IgE: less than 0.1%

What is the valency (# of binding sites) of each antibody?

• IgM: pentamer

• IgA: monomer or dimer

• IgD: monomer

• IgG: monomer

• IgE: monomer

What is the first antibody to be secreted by plasma cells after initial exposure to any antigen?

IgM

Where is IgM found?

In the blood and lymph

Where is IgA found?

Tears, saliva, mucus, milk, GI secretions, blood, and lymph

Where is IgD found?

Blood, lymph, and on the surface of B cells

Where is IgG found?

Blood, lymph, and in the intestines

Where is IgE found?

On mast cells and basophils

What is the function of IgM?

Present as monomers on the surface of B cells serving as antigen receptors

What is the function of IgA?

Provide localized protection on mucus membranes

What is the function of IgD?

Involved in activation of B cells

What is the function of IgG?

• Protect from bacteria and viruses by enhancing phagocytosis, neutralizing toxins, and triggering the complement system

• The only antibody that can pass the placenta from mother to fetus and confer immune protection in newborns

What is the function of IgE?

Involved in allergic reactions

Why was there a pre-bleed done to collect blood before mouse immunization in the mouse experiments?

• To show that prior to immunization the animal made no preexisting antibodies to the experimental antigen

• This is used for the pre-immunized sera (PS) samples

What was Mouse #1 injected with?

Soluble antigen (rabbit IgG)

• First immunization: rabbit IgG + CFA

• Re-immunization: rabbit IgG + IFA

What was Mouse #2 injected with?

Insoluble antigen (whole sheep erythrocytes, SRBCs)

• No adjuvant needed

What were the 3 samples taken from both Mouse #1 and Mouse #2?

Pre-immunization, after first immunization, and after re-immunization (booster)

What was the soluble antigen that was used in the mice experiment?

Rabbit IgG

What was the insoluble antigen that was used in the mice experiment?

Whole sheep erythrocytes - Sheep Red Blood Cells (SRBCs)

Were the antigens used in the mice experiments T-cell dependent or T-cell independent?

They were both T-cell dependent

What are adjuvants?

Substances that non-specifically enhance the immune response to an antigen

• They are employed in vaccines and injected together with an antigen

• They are like a catalyst and cannot elicit the immune response themselves without an antigen

What is Complete Freund’s Adjuvant (CFA) composed of?

A mixture of mineral oil and detergent containing inactivated (heat killed) and dried Mycobacterium tuberculosis

What is CFA used for?

Primary immunizations

What is the function of the different components of CFA?

• Oil and detergent: allows for slow, prolonged release of the antigen into the animal circulation (prolongs interaction between antigen and APCs, induces local inflammation, and recruits APCs)

• Mycobacteria: stimulates the animal’s immune system (both innate and adaptive) by attracting macrophages to the site of infection

What is Incomplete Freund’s Adjuvant (IFA) composed of?

A mixture of oil and detergent alone

What is IFA used for?

Secondary immunizations

What is the function of IFA?

Ensures that mouse antibody production (both IgM and IgG) is focused on the antigen of choice (T cell dependent antigen - Rabbit IgG in our case)

What is the “Whole Blood”?

Includes all components of blood cell and plasma

What is the blood “plasma”?

Cell-free liquid obtained from whole blood after anticoagulation treatment and centrifugation

• Blood fluid that contains blood clotting agents

• Makes up about 55% of total volume of blood

• Contains WBCs and platelets

What is the blood “serum”?

Liquid collected from whole blood without anticoagulation treatment

• The water fluid from the blood without the clotting factors

• Volume of serum is less in comparison to plasma

• Does not contain WBCs, RBCs, or platelets

What is ELISA used to detect?

The presence of an antibody or antigen in a sample

What are the core steps of ELISA?

1. Coating/Capture: direct or indirect mobilization of antigens to the surface of microplate wells

2. Plate Blocking: addition of irrelevant protein or other molecules to cover all unsaturated surface binding sites of the microplate wells (aka used to prevent unspecific binding to the antibody/antigen)

3. Probing/Detection: incubation with antigen-specific antibodies that affinity-bind to the antigens

4. Detection of the signal via the direct or secondary tag on the specific antibody (often enzymes (enzyme-linked antibodies))

What are the different types of ELISAs we talked about?

Direct, Indirect, Sandwich, Competitive

What is a Direct ELISA?

• Uses one antibody

• Sample of known antigen is applied to the well, primary antibody with conjugated enzyme is added

What is the application of a Direct ELISA?

Analyze immune response to antigens

What is an Indirect ELISA?

• Uses multiple antibodies

• Sample of known antigen is applied, unknown antigen (primary antibody) is added, secondary antibody conjugated to enzyme is added

What is the application of an Indirect ELISA?

Detects antibodies to determine concentration

What is a Sandwich ELISA?

• Uses two antibodies that bind to different epitopes on the same antigen

• Monoclonal antibody is added first, then the unknown amounts of antigen is added, then the secondary antibody conjugated with the enzyme is added

What is the application of a Sandwich ELISA?

Detects and measures antigens

What is a Competitive ELISA?

• Constant amount of antibodies are incubated in a solution containing variable amounts of antigen

• Ag-Ab mixture is added to antigen-coated microtiter well

• The more antigens in the original solution, the less free antibodies there are to bind to the antigens in the wells

• Secondary antibody conjugated to enzyme is added (specific to isotype of primary antibody)

What is the application of a Competitive ELISA?

Measures antigen levels in pre-incubation

• Antigen needs to be small, having only one epitope

What is added at the end for all ELISAs

Substrate that the enzyme converts into a chromogenic signal (detection)

Samples from which mouse were used in the MicroELISA?

Mouse #1

What antibodies were produced by Mouse #1?

Mouse anti-rabbit antibodies

What did we determine with our Indirect MicroELISA?

• If mouse anti-rabbit IgG was present in the blood of pre-immunized, immunized, and re-immunized mice

• The antibody titer, which tells us the effectiveness of immunizations

What blocking buffer did we use in our MicroELISA, and what does it do?

1% BSA in PBS

• Covers all unsaturated surface-binding sites of wells to prevent non-specific binding of proteins to the surface of wells during following steps

What wash buffer did we use in our MicroELISA and what does it do?

PBS + Tween 20

• Used to wash plates and remove unbound antibody (also prevents non-specific binding) leaving the antibody-antigen complexes

What was the positive control in our MicroELISA and why?

Commercial mouse anti-rabbit antibody

• We used this because this is the type of antibody we expect to be produced from the immunization, therefore this will show us the expected reaction to compare our experimental samples to

What was the negative control in our MicroELISA and why?

PBS + Tween 20

• It does not contain antibodies, therefore any wells containing the negative control should not produce a reaction

What was the antigen used in our MicroELISA?

Rabbit IgG

What was the primary antibody used in our Micro ELISA?

Mouse anti-rabbit antibody (IgG)

What was the secondary antibody used in our Micro ELISA?

Goat anti-mouse antibody (HRP conjugated)

What was the enzyme used in our Micro ELISA?

Horseradish Peroxidase (HRP)

What was the substrate and activator used in our Micro ELISA?

ABTS activated by H₂O₂

What was the color changed to in our Micro ELISA?

Blue/violet read at 414 nm

What is the qualitative determination of antibody titer?

Visual detection - are the antibodies/antigen present

What is the quantitative determination of antibody titer?

Absorbance measurements at 414 nm - determines the antibody titer

What were the expected antibody titers for our sera samples?

PS: technically a negative control and should produce no result

IS: 100

ISR: 100

What were the antibody titer results from our sera samples?

• Sam: IS was 100, ISR was 1000

How are color intensity and antibody titer related?

The intensity of the color change is directly related to the amount of primary antibody present (mouse anti-rabbit antibody (IgG)), therefore, the more intense the color change, the higher the amount of antibody in the sera

How is absorbance related to antibody titer?

Absorbance will measure the intensity of the color change —> the more intense the color, the more antibodies there are, the higher the absorbance value will be

What are the different types of mouse blood collection?

Cardiac, retro-orbital, tail vein, saphenous vein (from leg), submandibular vein (cheek)

What are the specifics of cardiac blood collection?

• Takes 0.5-1 mL

• Needs anesthesia and kills mouse (terminal)

What are the specifics of retro-orbital blood collection?

• From orbital sinus

• Takes 0.2 mL

• Requires anesthesia