Course code;- MBIO-306 B Couse Title:- Immunology - I

1. Antigen-Antibody Interactions, Immune Assays and
Experimental Systems
While reading this chapter, please pay close attention to all terms that are printed in
bold type.
Serology
Serotyping
Precipitation
Agglutination
Complement Activation
Cross-linking
Particulate antigens
Titer
Don�t worry too much about the theoretics of the Primary Interactions between
Antibody and Antigen except for this:
The word affinity is used to describe the strength of binding between one
antibody binding site and an antigenic determinant (epitope or
hapten). The association constant is the mathematical value that is the
measure of the strength of binding.
Anibody molecules are multivalent and antigens are also
often multivalent. This multivalency tends to increase the strength of the
interaction, and this really represents the true state of affairs. This overall
binding energy that results in the binding of a multivalent antibody with a
multivalent antigen is called the functional affinity or the avidity.
Agglutination Reactions
Read over the discussion in the text. Paying attention to the definition
of titer and prozone.
The discussion covering zeta Potential and the Coombs Test (which is also known as
the anti-globulin test or the anti-immunoglobulin test) is very important. What is
the distinction between the direct Coombs test and the indirect Coombs test?

2. Passive agglutination tests involve attaching an antigen to a particle of some sort
(latex beads, tanned red blood cells) and then running an agglutination reaction with
antibody.
Precipitation Reactions
This describes the reaction between soluble antibody and soluble antigen in which an
insoluble product results. Please note the discussion describing the effects of antibody
excess, antigen excess, and the zone of optimal proportions (equivalence zone) on the
production of a precipitate. (Remember that precipitation is a secondary
phenomenon. Ag-Ab reactions may occur and form soluble immune complexes even
without the production of a visible precipitate!)
Precipitation reactions can be done in a variety of ways:
In test tubes
In agarose gels:
As double diffusion
As single diffusion
After electrophoresis
Immunoassays
Solid phase immunoassays;
ELISA � Enzyme linked immunosorbant assay
1. You need reagent antibodies or reagent binding proteins that have
been �tagged� with an enzyme label. This means that the enzyme
has been covalently coupled to the protein reagent.
a. Typical enzymes include: Horseradish peroxidase or Alkaline
phosphatase

3. b. Reagent proteins include antibodies such as goat-anti-human-
IgG. Or, bacterial proteins that bind to antibodies such as
Staphylococcal protein A or Streptococcal protein G. Biotin
and Avidin can also be used ---- avidin has several high-
affinity binding sites for biotin thus it can be used to bridge
molecules that have been �tagged� with biotin.
2. You need some kind of �solid phase� to which proteins can
stick. This would usually be some kind of plastic microtiter
plate. Thus, as is the example in Fig 5.11, antigen can be used to
�naturally� coat the wells of a microtiter plate. After you do this
you would have to �block� the plate with some kind of irrelevant
protein (or detergent). We usually use a milk solution for this that is
called BLOTTO.
3. One then reacts the antigen-coated plate with appropriate enzyme-
tagged antibody. (You can actually sandwich several different
reagents at this point � as will be discussed in class.) Afterwards the
appropriate substrate is added and a color-change will indicate a
�positive� test.
Western Blot
In these assays, antigens are first electrophoresed and then blotted onto a
piece of filter-paper. Thus the filter paper is the solid phase to which the
antigen is bound.

4. The filter is blocked with BLOTTO and then reacted with appropriate
�tagged� antibodies and then substrate. As above, a color reaction
indicates a �positive� test.
Immunofluorescence
In these assays, antibodies or other reagent proteins are �tagged� or labeled
with fluorescent dyes. These fluorescent reagents can then be used to stain
samples mounted onto microscope slides and the slides can be examined using
a fluorescent microscope.

5. Typical fluorescent labels include FITC, TRITC, PE and many others.
Cells in suspension can also be stained fluorescently and then analyzed
by fluorescence-activated cell sorting or FACS analyis.

6. Reagent Antibodies
For many of the serological tests described above, it is essential to have
antibodies of defined specificity which can be used as reagents in the tests.
Traditionally such antibodies were made as polyclonal antibodies. To make
such antibodies, very pure antigen was injected into subject animals and then
after an appropriate amount of time (often after several �booster shots�) the
antibody containing serum was harvested from the animal by bleeding.
Such polyclonal antisera are very valuable, but they do have some
limitations. Particularly, such antisera contain populations of antibodies
that react to all the epitopes of the antigen prep � including any
impurities that might have contaminated the antigen. Of course,
polyclonal antisera also contain multiple isotypes of antibody too.
To get around this problem, one can immunize an animal and then remove and
culture and clone its B-cells. In this technique, one then cultures single clones
of B-cells, each clone producing only one specificity of antibody, reactive with
only one epitope of the immunizing antigen.
This procedure involves a number of sophisticated tricks that we will list
here and discuss in more detail in class.
1. Normal B-cells will not survive very long in culture so one
must immortalize them by giving them genes from cancerous B-
cells.
 Cancerous plasma cells (antibody secreting B-cells) are
called myeloma cells.
 B-cells from immunized animals are mixed with myeloma
cells in the presence of a mild detergent (polyethylene
glycol or PEG). This causes cells to fuse
forming hybridomas or hybrids between normal and myeloma
cells.
2. A selection system is used that preferentially allows the hybridoma
cells to grow while suppressing the growth of any non-hybridized
myeloma cells.
3. Hybridoma cells are fragile, so feeder cells of macrophages are used
to produce growth promoting cytokines.

7. 4. Appropriate immunoassays (usually ELISA�s) have that allow you
detect any antibodies being produced by the hybridoma clones.