Lab 2

Antibody precipitation reactions based

assay

 Antibody and soluble antigen interacting in aqueous solution form a

lattice that eventually develops into a visible precipitate.
Antibodies that aggregate soluble antigens are called Precipitins.
Although formation of the soluble Ag-Ab complex occurs within
minutes, formation of the visible precipitate occurs more slowly and
often takes a day or two to reach completion. The antibody must be
bivalent; a precipitate will not form with monovalent Fab fragments.
The antigen must be either bivalent or polyvalent; that is, it must
have at least two copies of the same epitope, or have different
epitopes that react with different antibodies present in polyclonal
antisera.

Polyclonal antibodies can form lattices, or large aggregates, that precipitate out of
solution.
However, if each antigen molecule contains only a single epitope recognized by a
given monoclonal antibody, the antibody can link only
two molecules of antigen and no precipitate is formed.

( Lattices or
large aggregates )

( no precipitate is formed
if an Ag contains only a
single copy of each epitope )

1-Precipitation Reactions in Fluids

A quantitative precipitation reaction can be performed by placing a
constant amount of antibody in a series of tubes and adding increasing
amounts of antigen to the tubes.
This method was used to measure the amount of antigen or antibody
present in a sample of interest. After the precipitate forms, each tube is
centrifuged to pellet the precipitate, the supernatant is poured off, and
the amount of precipitate is measured

Precipitation Reactions in Fluids yield a precipitation curve

Plotting the amount of precipitate against increasing antigen concentrations yields
a precipitin curve It reveals 3 zones:
Zone of antibody excess - precipitation is inhibited and antibody not bound to
antigen can be detected in the supernatant;
Zone equivalence - maximal precipitation in which antibody and antigen form
large insoluble complexes and neither antibody nor antigen can be detected in
the supernatant;
Zone of antigen excess - precipitation is inhibited & Ag. not bound to Ab. can be
detected in the supernatant

Precipitation Curve

Precipitation in Solution

Precipitation reactions in
fluids follow the same roles
of precipitation curve zones.

Precipitation in Solution

a)Bottom Precipitate
Occurs when Soluble Ag interact
with soluble Ab and form a
visible precipitate that give
bottom ppt after centrifugation.

Precipitation in Solution (cont.)

b)Ring Precipitate (Ring Test) (Tube
Precipitation test)
Involve soluble antigens with
antibodies in tubes (test or
Capillary tubes).
Layer Ag over Ab
Precipitate occurs at the interface
of the two reagents, forming a
ring.
Simplest test
Qualitative

Test Tube
reaction

Capillary
Tube
reaction

Figure 18.4

2-Precipitation Reactions in Gels

Simple Immunodiffusion (ID)

Immunodiffusion procedures are carried out in an agar gel medium.
The precipitate is easily seen in gels

visible precipitin lines

But no visible precipitate forms in regions of Ab or Ag excess.

a) Single Radial ID (Mancini Reaction)

Ab is put into a gel and Ag is put in a well cut into the gel and
a precipitin ring formed when Ag diffuses out in all
directions.

b) Double ID (Ouchterlony Reaction)

Both Ab and Ag diffuse from wells into a gel medium

Simple Immunodiffusion Reactions

The region of equivalence

Diagrammatic representation of radial & double immunodiffusion.

(Precipitation reactions in gels yield visible precipitin lines;
no visible precipitate forms in regions of Ab or Ag excess.)

Simple Immunodiffusion (ID)

a) Single Radial ID
The technique is quantitative
is based upon the reaction
between an Ag, and a specific Ab
during a diffusion period.
Ag placed in a well diffuses into an
agar containing the Ag (anti-IgG
looking for serum IgG).
The Ag-Ab interaction is
manifested by a well-defined ring
of precipitation around the Ag
well.
Interpretation : Diameter of ring is
proportional to the Antigen
concentration.

Simple Immunodiffusion (ID) (cont.)

b) Double ID (Ouchterlony)
Both antigen and antibody can diffuse independently
It is based upon the simultaneous application of Ag and Ab in separate but adjacent wells of
an agar plate.
As the materials diffuse toward one another, ppt. lines form resulting from the Ag-Ab
interactions (i.e. it is Qualitative).
If multiple wells of Ag are positioned around an Ab well on the same plate, several patterns
of reactivity may be observed.

Agglutination. Antibody based assays

Lab3

Agglutination Reactions
Interaction between antibody and a particulate antigen results in
visible clumping called agglutination. Antibodies that produce such
reactions are called agglutinins.
Agglutination reactions are similar in principle to precipitation
reactions; they depend on the crosslinking of polyvalent antigens.
As an excess of antibody inhibits precipitation reactions, such excess
can also inhibit agglutination reactions; this inhibition is called the
prozone effect.

Possible reasons for Prozon effect

Several mechanisms can cause the prozone effect.
First, at high antibody concentrations, the number of antibody
binding sites may greatly exceed the number of epitopes. As a result,
most antibodies bind antigen only univalently instead of
multivalently.
Antibodies that bind univalently cannot crosslink one antigen to
another. Prozone effects are readily diagnosed by performing the
assay at a variety of antibody (or antigen) concentrations

1-Bacterial Agglutination Assay

(Used to Diagnose Infection)
A bacterial infection often elicits the production of serum antibodies
specific for surface antigens on the bacterial cells
Presence of such antibodies can be detected by bacterial
agglutination reactions
Serum from a patient thought to be infected with a given bacterium is
serially diluted in a the last tube showing visible agglutination will
reflect the serum antibody titer of the patient.

For understanding
The agglutinin titer is defined as the reciprocal of the greatest serum
dilution that elicits a positive agglutination reaction.
For example, if serial twofold dilutions of serum are prepared and if
the dilution of 1/640 shows agglutination but the dilution of 1/1280
does not, then the agglutination titer of the patients serum is 640.n
array of tubes to which the bacteria is added

2-Hemagglutination Assay
Used in Blood Typing
Agglutination reactions are routinely performed to type red blood cells
(RBCs).
For example, if antibodies that bind the A blood group are added and
agglutination occurs, the blood is either type A or type AB.
To determine between type A or type AB, antibodies that bind the B group
are added and if agglutination does not occur, the blood is type A.
A classification system for human blood which identifies four major blood
types/groups based on the presence or absence of two antigens A and B
Four blood types/groups (A, B, AB, and O, in which O designates blood that
lacks both antigens) are important in determining the compatibility of
blood for transfusion.