Lab 01: Antigen-Antibody Reactions

What do antigens bind to on T cells

TCR

What do antigens bind to on B cells

BCR (membrane-bound antibody)

Immunogen

Something that binds and produces an immune response

Are all antigens immunogens

No, not everything recognized by the immune system will produce a response

Regions/parts of an antibody

• Variable region

• Constant region

• Heavy chain

• Light chain

Which part of the antibody binds to the antigen

Variable region (F_ab)

Which part of the antibody interacts with other immune components to produce the effector response

Constant region

Types of antibody classes/isoforms

• IgA

• IgD

• IgE

• IgG

• IgM

Which type of antibody only exists as a BCR (is not secreted as a free antibody)

IgD

Which type of antibody is the first type to be secreted in a response

IgM

Which type of antibody signals phagocytes

IgG

Which type of antibody forms a dimer and neutralizes antigens

IgA

Which type of antibody is key for parasite infections

IgE

What allows antigens to bind to multiple types of antibodies

Multiple epitopes (binding sites)

What types of molecules can T cells bind

Proteins only

What types of molecules can B cells bind

Proteins, CHOs, NA, and lipids

Native antigens are bound by what immune cell

T cells

Processed antigens are bound by what immune cell

B cells

For the antigens that are bound by BCRs, where are the epitopes

On the outside

Why are proteins good activating antigens

They are long and complex molecules with lots of epitopes

List the 4 functions of antibodies

• Neutralization

• Opsonization

• Agglutination

• Complement fixation

Neutralization

Antibody binds to antigen, and doesn’t cause an effector response

Opsonization

Antibody binds to an antigen and flags it for phagocytosis by a macrophage

Agglutination

Antibody binds to antigens and clumps together with other Ag:Ab complexes

Complement fixation

Activating complement proteins to lyse cells

Types of immunological tests that use antibodies

• SNAP tests

• ELISA

• Precipitation

• Agglutination

Polyclonal antibodies

Solution with multiple different types of antibodies from multiple B cells that can recognize multiple epitopes for one infection

Monoclonal antibodies

Solution with one specific type of antibody that recognizes one epitope

How are polyclonal antibodies made

Antigen injected into a rabbit → serum collected → distill out the multiple types of antibodies

How are monoclonal antibodies made

Antigen injected into mice → isolate plasma cell of interest → fuse plasma cell with myeloma → culture for specific hybridomas → harvest antibodies

Why are myelomas used to make monoclonal antibodies

They are immortalized cancerous cells that don’t produce any of their own antibodies

Precipitation test

Soluble antigens are bound by antibodies and the complex comes out of solution when the reaction occurs and can be quantified

Key feature of antibodies that is needed for precipitation

They need to be able to bind 2+ Ags at the same time so that cross linking can occur

Agglutination test

Antibodies clump particulate antigens

Two important structures/features in antibodies that facilitates precipitation and agglutination

• They are divalent molecules that have two antigen binding sites

• They have a hinge region that gives them flexibility when cross linking antigens

What happens in a precipitation if there is antibody excess

Clumps don’t form

What happens in a precipitation reaction if there are roughly equal amounts of antibody and antigen

Precipitation will happen

What happens in a precipitation reaction if there is an antigen excess

No clumping

Purpose of an immunodiffusion test

Uses agarose gel with wells containing soluble antigen and antibodies to find the point of equivalence

Purpose of a slide agglutination test

To determine if animals have antibodies to certain antigens

Purpose of Reporter Systems that use antibodies

Qualitative or quantitative tests that make it easier to see reactions

ELISA process

Wells lined with Abs → inoculate with serum → wash → incubate with Ab:enzyme complex → observe for color change

Other types of reporter system tests

• ELISpot

• Immunochromatography (SNAP)

• Immunofluorescence

Immunofluorescence testing

Uses antibodies tagged with a fluorescent dye to detect antigens in a tissue or cells

Purpose of flow cytometry

Uses antibodies tagged with fluorescent dye, a laser to activate the dye, and a sensor to quantify how many things are tagged

Purpose of a virus neutralization assay

Evaluates how effective antibodies in the serum can protect the animal

Common clinical use of an agglutination test

Blood typing cards

Major RBC antigen in dogs

DEA1

Major RBC antigens in cats

AB

Which small animal species has preformed anti-RBC antibodies even before a transfusion

Cats

Chemical class of RBC surface antigens

Proteins and CHOs

Types of tests for blood typing

• Agglutination card

• Major/minor cross match

Major cross match

Does the patient serum attack the donor RBCs

Minor cross match

Does the donor serum attack patient RBCs

Which type of canine blood type is most common

DEA1 positive

Which type of feline blood type is most common

A

Why can’t you give an unmatched first transfusion to a cat

They have preformed antibodies for their opposite blood type, and will have a severe reaction

Which cat blood type will have a less severe reaction if given a mismatched blood transfusion

A cats have a milder reaction to type B blood than the inverse

Secondary RBC antigen in cats that may contribute to a reaction even if the A/B type is matched

Mik antigen