4.

ANTIGEN-ANTIBODY
INTERACTIONS & COMPLEMENT
4.1 - WHAT IS IMPORTANT ABOUT AN ANTIBODY?
The heavy and light chains. Together, the light and heavy chains dictate
isotype. Antigens are bound in the upper ‘Y’ part of the antibody and the variable
region in this part dictates antigen specificity. The bottom F c region docks the cells
and can promote phagocytosis as well as function.
AFFINITY: the strength of the reaction between a single antigenic determinant and a
single antibody combining site. It is the sum of the attractive and repulsive forces.
AVIDITY: the overall strength of binding between and antigen with many
determinants and multivalent antibodies. Avidity is important in antibodies such as
IgM, which is a pentamer and has ten antigen binding sites, and therefore low affinity
but high avidity.
SPECIFICITY: The ability of an individual antibody combining site or a population of
antibody molecules to react with only one antigenic determinant
CROSS-REACTIVITY: The ability of an individual antibody combining site or
population of antibody molecules to react with more than one antigenic determinant

Why do we study antigen-antibody interactions?

1. To learn more about the nature of the interaction between antigen and antibody
2. To identify presence or quantify antibody to particular antigen present in animal
3. To use antibodies as very specific reagents in basic biological research to
characterize or purify a particular antigen

4.2 - APPLICATIONS USING ANTIGEN-ANTIBODY INTERACTION

Immunoprecipitation
 Antibody can form large, extensive complexes and these complexes become
insoluble and precipitate out of solution
 Immunoprecipitates can be collected using magnetic beads
 After rinsing the antigen-antibody complex can be dissociated and the antigen can
be studied.
 Presence of a particular antigen can be visually confirmed
 Not very quantitative, but indicates presence
 Can be used to determine which specific antigen is present

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ELISA – Enzyme-Linked Immunosorbent Assays

Western Blotting
 A sensitive way to find very small
amounts of a protein of interest in
a diverse mixture of proteins

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Tests for Cell-Associated Antigens
 Fluorochromes are
conjugated to the Fc portion
of the antibody, allowing
them to be detected.
 Immunofluorescence and
Flow Cytometry
 Most important techniques
 Allows you to look at multiple
antibody-antigen reactions on
a cell

Immunofluorescence
 Primary antibody is added to the cell and allowed to bind to the antigen
 If the protein of interest is on the cell membrane, no preparation
 Used if you want to know what’s going in a tissue (if a particular receptor is being
expressed, etc.)

Flow Cytometry
 Technique for counting and examining microscopic particles using antibodies
 Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It
provides a method for sorting a heterogeneous mixture of biological cells into two
or more containers
 FACS sorts cells with particular antigens
1. Cells are labeled by antibody
2. Cells passed through a narrow
nozzle that only allows one cell to
pass through at a time
3. Each droplet given an electrical
charge and scanned by a laser,
which indicates type and amount
of fluorescence the cell carries
4. A computer readout indicates
how many cells are in the
original mixture
5. A laser scan applies a charge to
two metal plates as the droplet
passes between them, deflecting
to cell into a collection tube
based on the amount and type of
fluorescence it expresses

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4.3 – COMPLEMENT
Antibody alone are not very effective at controlling infections so they induce a
‘series of enzymatic reactions that unleashes an extremely deadly and diverse set of
activities by other proteins that are present in the blood’ collectively, the proteins are
called complement

COMPLEMENT: Complex group of >20 different proteins that work to lyse cells
 Has a central role in inflammation as it causes opsonisation and lysis of
pathogens, chemotaxis of phagocytes, and clearance of immune complexes
 Complement eliminates bacteria, viruses, and other pathogens
o Complement operates as a cascade mechanism. When one component is
activated, the next activates, and then the next, etc.
o Sequential activation occurs by proteolysis since many components are
proteases that clip an inactive ‘proenzyme’ and convert it to active form
 There are three types of complement pathway: lectin, classical, and alternative
o The classical pathway uses antibody and is considered part of the
adaptive immunity
o The lectin and alternative pathways are freestanding and are considered
part of the innate immunity
o All cascades lead to common convertase, and ultimately to MAC insertion
 Complement lyses cells through the insertion of a lytic complex into the cell
membrane, known as the ‘membrane attack complex’ (MAC)
 The complement system must get MAC to the right place and prevent it from
forming in the wrong place
o The ‘right place’ is to the surface of foreign substances like bacterial
cells, viruses, protozoa, orhinfected self-cells.
o The wrong place is any healthy normal cell of the body.
 Anywhere you have lymph, you have complement

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5. MAJOR HISTOCOMPATIBILITY
COMPLEX AND T- CELL RECEPTORS
 T cells respond to antigen only if the antigen is ‘presented’ to them by antigen-
presenting cells
 Molecules that serve as carriers of antigenic material on the surface of APCs are
called the MHC proteins
 ‘Histocompatibility’ means ‘tissue compatibility’
 MHC proteins form a complex with fragments of antigenic proteins and that the T-
cell receptor binds to this complex of MHC and foreign proteins

MHC Diversity
 The class I and II proteins are polymorphic unlike most genetic loci which are
monomorphic (more than 99% of the individuals in a species are genetically
identical at that site).
 MHC Class I and II loci have many more than 100 different alleles in mice and in
humans. Although an individual has a limited, fixed number of genes that encode
these proteins, the whole species contains an enormous variety of combinations of
these alleles.
 It’s unlikely that any two individuals of an outbreeding species like Homo sapiens
are identical for all the MHC proteins that cells express
 MHC protein diversity is why transplants are difficult since we’re all different from
each other in our MHC
 MHC proteins vary more from individual to individual than any other protein
 MHC genes are co-dominant, which means that each cell expresses both the
maternal and paternal alleles of the genes

Role of MHC in immune response

 TCR recognizes antigen presented in MHC
o Binding of antigen peptides is non-covalent
 Two types of MHC (Class I and Class II) are recognized by different T cell subsets
o Cytotoxic T cells recognize antigen peptides in MHC Class I
o T-helper cells recognize antigen peptides in MHC Class II

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Important aspects of MHC
 Alleles for MHC genes are co-dominant
o Each MHC gene product is expressed on the surface of each individual cell
 Each MHC has one peptide binding site
o But, each MHC can bind many different peptides (interaction of a protein
with its ‘substrate’ is relatively nonspecific)
o Only one peptide can be bound at a time
 MHC polymorphism is determined in germline (mom and dad)
o NO recombination mechanism for creating diversity in MHC
 Peptide must bind with individual’s MHC to induce immune response
 Class I MHC is expressed on nucleated cells and has an alpha chain and a small
beta chain, but only the alpha chain binds the antigen (only cell without MHC
Class I is red blood cells)
 Class II MHC is expressed on antigen-presenting cells, such as B lymphocytes,
dendritic cells, and macrophages and has two alpha and two beta chains that come
together to make the antibody binding site
 MHC Class II has two independent alpha chains and so is more diverse than MHC
Class I, which has only 1 alpha chain

MHC and Disease susceptibility

 Whether an immune system can recognize and respond to an antigen depends on
whether the antigen can be bound to Class I and/or Class II proteins and
‘presented’ to T Lymphocytes
 Thus, if an individual lacks the appropriate Class I or II protein that binds an
antigen, it will be unable to respond to the antigen. Some individuals with other
combinations of Class I or II alleles will be able to respond and present the
antigens and mount an immune response
 Not all individuals can respond to all possible antigens
 So the species is pretty well protected against almost all pathogens, even when
certain individuals are not