***"

chapter 6
Antigen-Antibody
Interactions: Principles
and Applications

HE ANTIGEN-ANTIBODY INTERACTION IS A BIOMOLECULAR

association similar to an
enzyme-substrate interac- Fluorescent antibody staining reveals intracellular
tion, with an important distinction: it does not lead
to an irreversible chemical alteration in either the
immunoglobulin. [H.A. Schreuder et al, 1997, Nature
or the antigen. The association between an
antibody 386:196; courtesy H. Schreuder, Hoechst Marion
Roussel.]
antibody and an
antigen involves various noncovalent interactions between
the antigenic determinant, or epitope, of the Strength of Antigen-Antibody Interactions
antigen and the
variable-region (V/V) domain of the antibody molecule,
particularly the hypervariable regions, or complementarity- Cross-Reactivity
determining regions (CDRs). The exquisite specificity of Surface Plasmon Resonance
(SPR)
antigen-antibody interactions has led to the development of
a variety of
immunologic assays, which can be used to detect Precipitation Reactions
the presence of either antibody or
have played vital roles in
antigen. Immunoassays AgglutinationReactions
diagnosing diseases, monitoring the
level of the humoral immune
response, and identifying
molecules of biological or medical interest. These
Radioimmunoassay
assays
differ in their speed and sensitivity; some are
strictly qualita- Enzyme-Linked Immunosorbent Assay
tive, others are quantitative. This chapter examines the
nature of the
antigen-antibody interaction and various
Western Blotting
immunologic assays that measure or exploit this interaction. Immunoprecipitation
Immunofluorescence
Strength of Antigen-Antibody FlowCytometry and Fluorescence
Interactions Alternatives to Antigen-Antibody Reactions
The noncovalent interactions that form the basis of
antigen- Immunoelectron Microscopy
antibody (Ag-Ab) binding include hydrogen bonds, ionic
bonds, hydrophobic interactions, and van der Waals interac-
tions (Figure 6-1). Because these interactions are
individu-
ally weak (compared with a covalent bond), a large number that underlies the
of such exquisite specificity that characterizes
interactions are required to form a strong Ag-Ab antigen-antibody interactions.
interaction. Furthermore, each of these noncovalent interac-
tions operates over a
very short distance, generally about
1X10- mm (1 angstrom, A); consequently, a strong Antibody affinity is a quantitative measure
Ag-Ab interaction depends on a very close fit between the of binding strength
antigen and antibody. Such fits require a high degree of com- The combined strength of the noncovalent interactions
plementarity between antigen and antibody, a requirement between a single antigen-binding site on an antibody and a
145
 146 PART GENERATION OF B-CELL AND T-CELL RESPONSES

const.int
the
associatiOn
K
ANTIGEN ANTIBODY terms, the
ratio k,/k_, is In i m m u n i "
K), a measure of affinity. the
iogy. i
equil
NH2
k/k
called the affinity
constant.
Because K, Is

binding Site of tie

ibri
CH-OH.O=C-CH2-CH2-Hydrogenbond c o n s t a n t for

with antigen and

the reaction of a
since the atration kantib
molar concentration o otf a d,
single
concentration,
- CH2 -CH2-NH3* O to the antibody
binding site is equal ratio of the molar concentrath be
O
C-CH2-CH2-lonic bond calculated from the
m o l a r concentrations
of
to the
bournd Ag-Ab complex
equilibrium as un
antibody at ows:
CH bound antigen and

CH-CH Hydrophobic
[Ag-Ab]
CH CH3 interactions
K Ab][Ag
CH-CH-CH2 The value of K, varies for different Ag-Ab complexes and
van der Waals which is expressed in unite
CHCH CH CH depends on both k, of
interactions and k_, Which is expresse
(L/mol/s),
liters/mole/second
CH
CH, units of l/second. For small haptens,
the torward rate const
high; in some cases, high 4 x Ink
as as
k can be extremely
CH, -C L/mol/s, approaching the theoretical upper limit of diffusio
H3N-CH2 lonic bondd limited reactions (10 L/mol/s). For larger protein antigens
however, k, is smaller, with values in the range of 10* L/moll
FIGURE 6-1 The interaction between The rate at which bound antigen leaves an antibody's
gen depends on four of
an
antibody and an anti- binding site (i.e., the dissociation rate constant, k_,) is a
types noncovalent forces: (1)
bonds, in which hydrogen
a
hydrogen atom is
shared between two elec- major determinant of the antibody's affinity for an anti.
tronegativeatoms; (2) ionic bonds between gen. Table 6-1 illustrates the role of k-, in determining the
residues; (3) hydrophobic interactions, in oppositely charged association constant K, for several Ag-Ab interactions. For
which water forces
hydrophobic groups together; and (4) van der Waals interactions example, the k, for the DNP--lysine system is about one
between the outer electron louds of fifth that for the fluorescein
two or more atoms. In an
aqueous environment, noncovalent interactions are system, but its k_ is 200
times greater;
and extremely weak
depend on close complementarity of the shapes of consequently, the affinity of the antifluores-
cein antibody K, for the fluorescein
antigen. antibody and system is about 1000-
fold higher than that of anti-DNP
Ag-Ab complexes have
antibody.
Low-affinity
K, values between 10 and 10
L/mol; high-affinity complexes can have
single epitope is the affinity of the antibody for that as 10 L/mol. K, values as high
Low-affinity antibodies bind antigen epitope.
weakly and tend to For some
purposes, the
dissociate readily, whereas
high-affinity antibodies bind antibody complex is of interest:dissociation of the
antigen-
antigen tightly and remain bound longer. The associa-
more
tion between a
binding site on an antibody (Ab) with a Ag-Ab Ab Ag +
monovalent antigen (Ag) can be described The
by the equation equilibrium
the dissociation constant for this dissociation
constant, is equal to reaction, Ka
Ag + Ab Ag-Ab the
k_ reciprocal of Ka
where
K= Ab][Ag
k,
is the reverse
is the forward(association) rate constant and k_ Very stable [Ab-Ag
=
1/K,
(dissociation) rate constant. In biochemical ones have complexes have very low values of
higher values. Even
though K, is notKa;the affinity
less stabie
Forward and
TABLE 6-1
(K and K)
reverse rate
for three constants(, and
k 1) and association
ligand-antibody interactions and
Antibody dissociation constants
Ligand
k
Anti-DNP k_1
Anti-fluorescein e-DNP-1-lysine 8X 107 Ka
1
Fluorescein 4X 108
Anti-bovine serum albumin 1X 108
(BSA) Dansyl-BSA 5X 10-3
SOURCE: Adapted from
3x 105
1X 1011 1x 10-8
H. N. Eisen, 1990,
Immunology, 3rd ed.,
Harper &
2 10-3
Row, Publishers. 1x 10-1
1.7x 108
S9 x 10
 ANTIGEN-ANTIBODYINTERACT:ONS:PPINCIPLES AND APPLICATIONS CHAPTER6 147

(a) (b)
Control: No antibody present Control
(ligand cquilibrates on buth sides cquaily)

A
100R
B

50

Initial state
Equilibrium

Experimental: Antibody in A Experimental

(at equilibrium more ligand in A due to Ab binding)

100
B

Antibody
Radiolabeled 30 Ligand bound
ligand
by antibody
B

Initial state 6 8
Equilibrium
Time, h

FIGURE 6-2 Determination of antibody affinity by equilibrium measured. (6) Plot of concentration of ligand in each compartment
with time. At equilibrium, the difference in the concentration of
dialysis. (a) The dialysis chamber contains two compartments (A and
radioactive ligand in the two compartments represents the amount
B) separated by a semipermeable membrane. Antibody is added to
one compartment and a radiolabeled ligand to another. At equilib- of ligand bound to antibody
rium, the concentration of radioactivity in both compartments is

constant, the affinity constant, Ka, can of course be readily ments represents the concentration of ligand bound to the
calculated from Ka as follows: K = 1/Ka. antibody (i.e., the concentration of Ag-Ab complex). The
The affinity constant, K, formerly determined by equi higher the affinity of the antibody, the more ligand is
librium dialysis, is now derived by newer methods, espe- bound.
Since the concentration of antibody in the equilibrium
cially surface plasmon resonance (SPR), which is discussed of
in a later section. Because equilibrium dialysis illustrates dialysis chamber is known, the concentration antigen-
determined by [Ab-Agl/n, where
important principles and remains for some immunologists antibody complexes can be
n is the number of binding
sites per antibody molecule. The
the standardagainst which other methods are evaluated, it for a single
is described here. This procedure uses a dialysis chamber equilibrium constant or affinity constant, K,
binding site is:
Containing two equal compartments separated by
a
K, = [Ab-Agl/lAb][Agl = «iu=
in
Semipermeable membrane. Antibody is placed one com- - r)
c(n
partment, and a radioactively labeled ligand small enough the ratio of the
of bound lig-
concentration
in where r equals
o pass through the semipermeable Suitable placedin-
membrane is c is the concentration of
and to total antibody concentration,
the other compartment (Figure 6-2). ligands remains the number of binding sites per
In the free ligand, and n
be rearranged to
Clude haptens, oligosaccharides, and oligopeptides. molecule. This expression can
B will antibody
absence of antibody, ligand added to compartment Scatchard equation:
6-2a), In give the
on both sides of the membrane (Figure
cquilibrate
the presence of antibody, however, part of the labeled ligand K-K
the equilib-
antibody at equilibrium, trapping
the the and c can be
obtained by repeating
will be bound to
vessel, wlhereas unbound Values for r
concentration of antibody but
gand on the antibody side of the in both compartments. dialysis with the
same
rium If R, is a constant, that
concentrations ofligand.
gand will be equally distributed will be greater in the
with different the
antibodies within
chamber have the
dialysis
hus, the total concentration of ligand The dif- is, if all the then a Scatchard plot of r/c
(Figure 6-2b). for the ligand,
Compartment containing antibody in the two compart- same affinity

concentration
Lerence in the ligand
148 PAR T GENERATION OF B-CELL AND T-CELL. RESPONsES

(a) Homogeneous antibody ( Heterogencous antibody

#3
4.0 4.0
#4

3.0 Slope = -Ka 3.0 S l o p e a t r o f 1/2 n = -Ko

x 108
108
2.0
2.0

1.0
1.0
Intercept = n
Intercept = n

2.0
1.0
2.0

dimeric gA,n= 4. In thic

n 10, and for
=

FIGURE 6-3 Scatchard plots are based on repeated equilibrium gM, which is pentameric, than antibody #2. (b) If the
#1 has a higher affinity range of affinities,
dialyses with a antibody and varying
constant concentration of graph, antibody and has a
a

preparation is
polyclonal
concentration of ligand. In these plots, r equals moles
of bound antibody is constantly changing
a curved
line whose slope
antibody and c is the concentration of free ligand. From a
Scatchard plot yields be calculated by determining the
ligand/mole The average affinity
constant Ko can
both the equilibrium constant
(K) and the number of are occupied (i.e., whenr= 1
Scatchard plot, when half of the binding sites
binding sites per antibody molecule (n), or its valency,
can be ob- value of K this graph,
antiserum #3
has a higher affinity

If all antibodies have the same affinity, then

a Scatchard in this example). In
#4 (K%=
1.25 X 105). Note that the
tained. (a) 10s) than antiserum divalent antibodies such as lgG.
x
of The x intercept is n, the K=2.4
plot yields a straight line with slope -K.
a for
Ccurves shown in (a)
and (b) are
which is 2 for divalent and other lgs. For
valency of the antibody, lgG

the bind-
better measure
than the affinity for quantifying
is
straight line with a slope of -K (Fig-
a
within biological systems (e.g.,
v e r s u s r will yield a of a n antibody
concentration of unbound ligand cincreases,
ing capacity with antigenic
determinants on
ure 6-3a). As the the reaction of a n antibody

0 and r approaches n, the valency, equal

to
bacterial cell). Avidity
is the equilibrium constant,
r/c approaches a virus o r and the
molecule. the intact antibody
the number of binding sites per antibody interaction between

Most antibody preparations

are polyclonal-a heteroge- Keg for the the equilibrium
c o n s t a n t for the reaction
antigen; affinity is the
antibodies with a range of
affinities is pre- site of the antibody and
n e o u s mixture of between a n individual binding the
be
of heterogeneous antibody yields
a
avidity
the should
sent. A Scatchard plot antigen. Under ideal conditions
changing, reflecting individual binding sites of an
curved line whose slope is constantly the affinities of the
6-3b). With this type
of product of which has two binding sites
(Figure
this antibody heterogeneity affin- antibody. For an IgG molecule, conditions
to determine the average the avidity under ideal
Scatchard plot, it is possible half of with identical affinities,
determining the value of K when
ity constant, Ko, by are filled. This is conveniently done would be:
the antigen-binding sites where half
at the point Avidity K, X K, = (K,)3 = K e q A b l[Ag
the slope of the c u r v e
by determining filled.
sites are
of the antigen binding
This equation represents a theoretical maximum for avidity;
affinity actual avidities are always several orders of magnitude lower
Antibody avidity incorporates than the product of the affinities. The difference is due pri-
of multiple binding sites site
always reflect the marily to the geometry of Ab-Ag binding. The binding
binding site does
not
The affinity at one
When
of an antibody in contact with a single epitope of an antigen
interaction.
true strength of the antibody-antigen may be optimally oriented for the best fit, whereas binding
repeating antigenic
Complex antigens containing multiple, containing multiple to multiple epitopes on the target antigen may require
with antibodies
mixed
determinants are
molecule with somewhat strained geometry and less optimal bindinginter
interaction of antibody
binding sites, the
an
actions. Despite geometrical barriers to the achievement or
site will increase the probability
an antigen molecule at one
site. maximum theoretical values of avidity, the avidity is higher
molecules at a second
o reaction between those interactions between a multi-
two
than the affinity. Thus, high avidity can often compensate
The strength of such multiple
Valent antibody and antigen is called the avidity. The avidity for low affinity. For example, the binding sites of secreted
 ANTIGEN-ANTI2O9Y INTERACTIONS: PRINCIPLES AND APPLICATIONS cHAPTE R 6
149

A number of viruses and bacteria have antigenic determi-

pentameric IgM molecules often have significantly lower
a
of nants identical or similar to normal host cell components.
affinity than those of monomeric IgG, but the high avidity In some cases, these microbial antigens have been shown to
enables it to bind
IgM, resulting from its higher valence, elicit antibody that cross-Teacts with the host cell
Indeed, in the case of an antigen with compo-
antigen effectively. nents, resulting in a tissue-damaging autoimmune reaction.
such as those found
many closely spaced, repcating epitopes,
on the surface of bacteria and other pathogens, an IgM with The bacterium Streptococcus pyogenes, for example, expresses
cell wall proteins called M antigens. Antibodies produced to
lower-affinity binding sites may bind more tightly than an

IgG with sites of higher affinity.
Cross-Reactivity
Although Ag-Ab reactions are highly specific, in some cases
antibody elicited by one antigen can cross-react with an un-
related antigen. Such cross-reactivity occurs if two different
antigens share an identical or very similar epitope. In the lat-
ter case, the antibody's affinity for the cross-reacting epitope
is usually less than that for the original epitope.
streptococcal M antigens have been shown to cross-react
with several myocardial and skeletal muscle proteins and
have been implicated in heart and kidney damage following
streptococcal infections. The role of other cross-reacting
antigens in the development of autoimmune diseases is
discussed in Chapter 16.
Some vaccines also exhibit cross-reactivity. For instance,
vaccinia virus, which causes cowpox, expresses cross-reacting
epitopes with variola virus, the causative agent of smallpox.
This cross-reactivity was the basis of Jenner's methodof
using vaccinia virus to induce immunity to smallpox, as

Cross-reactivity is often observed among polysaccharide mentioned in Chapter 1.

that contain similar oligosaccharide residues. The

antigens
ABO blood-group antigens, for example, are glycolipids
expressed red blood cells. Subtle differences in the ter-
on

minal residues of the sugars attached to these cell surface

glycolipids distinguish the A and B blood-group antigens.
Although the mechanisms of tolerance prevent formation
of antibodies against one's own blood group antigens,
an
have
individual lacking one or both of these antigens will
serum antibodies to the missing antigen(s), induced not by
Surface Plasmon Resonance (SPR)
Equilibrium dialysis methods for measuring antibody affin-
ity have been superseded since the mid-1990s by surface
plasmon resonance (SPR), which is far more sensitive, con-
venient, and rapid (Figure 6-4a). In addition to allowing mea-
surements of affinity constants, SPR can be used to determine

exposure to red blood cell antigens but by exposure to rates of antigen-antibody reactions and can even be adapted
cross-reacting microbial antigens present on common in- to measure concentrations of antibody. SPR works by
de-
for-
testinal bacteria. These microbial antigens induce the tecting changes in the reflectance properties of the surface of
mation of antibodies in individuals lacking the similar an antigen-coated sensor v hen it binds antibody. Although
blood-group antigens on their red blood cells. (In
individu-
the physics underlying SPR is rather sophisticated and best
antigens, complementary antibodies reference (Rich and
alspossessing these explored by consulting the reading
would be eliminated during the developmental stage,
in site listed at the end of the chapter,
Myszka, 2003) and Web
which B cells making antibodies that recognize self epitopes the method is straightforward and ingenious. A beam of
are weeded out; see Chapter 16.) The blood-group antibodies, onto a thin gold
polarized light is directed through prism and reflected
a

elicited by microbial antigens, will cross-react film (coate with antigen

on the opposite side)
although
with similar oligosaccharides on foreign red
blood cells, off the gold film toward a light-collecting sensor. However,
for at a unique angle, some incident light
is absorbed by the gold
providing the basis for blood-typing tests and accounting
the necessity of compatible blood types during blood
trans- into charge waves called surface
layer, its energy transformed
B reflected light intensity can be
fusions. A type A individual has anti-B antibodies, a type plasmons. A sharp dip in the
individual has anti-A, and a type O individual has anti-A
and measured at that called the resonant angle. The angle
angle,
the thickness and conduc-
anti-B (Table 6-2). depends the color of the light,
on
of the
and the optical properties
tivity of the metal flm, surfaces. The SPR method
material close to the gold layer's
factors: the binding of
an-

TABLE6-2 ABO blood types takes advantage of the last of these

to the chip has
a strong
tibodies to the antigen attached
in the resonant
Serum antibodies a detectable change
Blood type Antigens on RBCs enough effect to produce to the
has been found to be proportional
angle. The change the rate at
Anti-B antibodies. By measuring
A number of bound
an antigen-
Anti-A which the resonant angle
changes during reaction
rate of the antigen-antibody
A and B Neither antibody reaction, the this is done
AB
can be determined (Figure
6-4b). Operationally,
concentration of antibody
Anti-A and anti-B a known
Neither a solution with
by passing
 150 PAR T I GENERATION OF B-CELL AND T-CELL RESPONSES

(a)

Resonance angle
Polarizcd sensitive to binding Detcctor
lighi source of antibody

Stages
Prism
Ab added to Formation Chamber
No Ab flow. Ag-Ab
of i flushed with
No Ag-Ab complexes
icomplexes butrer solution.

Gold complexesj levels i Ag:Ab Complexes

formed. form.
layer off. dissociate.

Dissociation
Association

Ag+AbAg-Ab{
Ag-AbAg+Ab
Flow Immobilized
Time
chamber antigen
is introduced into
a Stage ll: Antibody
baseline.
FIGURE 6-4 Surface plasmon resonance (SPR). (a) A buffer present, establishing
form. The ascending slope of this
solution containing antibody is passed through a flow chamber, one the flow and Ag-Ab complexes reaction. Stage 1: The
forward rate of the
wall of which contains a layer of immobilized antigen. As explained in curve is proportional to the
bound at the prevailing
that can be
the text, formation of antigen-antibody complexes on this layer Curve plateaus when all sites the plateau is directly
filled. The height of
causes a change in the resonant angle of a beam of polarized light antibody concentration are concentration. Stage V: The flow cell is
against the back face of the layer.A sensitive detector records changes proportional to the antibody
no antibody
and the Ag-Ab complexes
in the resonant angle as antigen-antibody complexes form. (b) Inter- flushed with buffer containing
to the slope of the
dissociation is proportional
dissociate. The rate of
pretation of a sensorgram. There are four stages in the plot of the ascending over descending,
dissociation c u r v e . The ratio of
the slopes,
detector response (expressed as resonance units, which represent a
change of 0.0001 degree in the r e s o n a n c e angle)
versus time. Stage : equalsk,/k2 =ka
Buffer is passedthrough the flow chamber. No Ag-Ab complexes are

over the antigen-coated chip. A plot of the changes

measured SPR be used to characterize the epitope
can
antibodies
during a n SPR experiment
versus time is called a sensorgram. specificities of collections of
reaction, the s e n s o r -
In the c o u r s e of an antigen-antibody SPR has uses beyond measuring affinity
or concentration of
until all of the sites capable of binding anti- anti-
gram plot rises that antibodies. Suppose one has two different monoclonal
given concentration) have done so. Beyond bodies against an antigen such as gp120, the envelope protein
body (at a
from these mea-

point the sensorgram plateaus. The data of HIV. Do these antibodies bind to the same or different epi-
the association rate
surements can be used to calculate ki, topes of this key protein? Surface plasmon resonance provides
constant for the reaction: a powerful approach to answering this question. Antibodies
Ab +Ag Ab-Ag that bind to different epitopes on an antigen give a character
istic sensorgram plot when added to the chamber serially
no
Once the plateau has been reached, solution containing (Figure 6-5a). Antibodies that bind to the same epitope com-
chamber. Under these
antibody c a n be passed through the pete with each other for binding sites, each blocking the bind-
complexes dissociate, al-
conditions, the antigen-antibody Mea- ing of the other, demonstrated by the plots in Figure 6-5b.
dissociation rate constant, k.
Jowing calculation of the the affinity In a variation of this procedure, one can use chemical
s u r e m e n t of k, and k,
allows determination of synthesis or judicious enzymatic hydrolysis to generate a Set
constant, K, since K, = k , / k
of fragments of an antigen and then immobilize each of
also be used to
measure

plasmon r e s o n a n c e can
Surface these fragments on a chip. Assuming that the antigen frag
The a m o u n t of
the concentration of antibody in a sample. ments have the same three-dimensional conformation as the
formed on the chip is represented
antigen-antibodycomplex native antigen, one can determine the reactivities (or lack
and this m e a s u r e is directly
by the height of the sensorgram, in the solution thereof) of antibodies and the locations of the epitopes
proportional to the amount of antibody within the original antigen molecule can be isolated, a pro-
to reference
flowing through the chamber. By comparison cedure is known as epitope mapping.
be determined.
data, antibody concentrations can
 ANTIGEN-ANTIBODYINTERACTIONS PRINGIPLES AND APPLICATIONS cHAPTtR
151

(a) Abj and Ab, bind different epitopes of the same antigen.

Ab
added added
Ab Ab,

Epitopes

Time

(b) Ab, and Ab2 bind to the same epitope

Ab Ab2 Ab2 Ab
added added added added
AD AD2

Epitopes

Time Time

FIGURE 6-5 SPR can be used to determine if different antibod-
ies bind to the same or different epitopes. (a) Ab, and Ab, recog-
nize different epitopes of the antigen. Initial injection of Ab, causes a
rise from baseline to a plateau; subsequent addition of Ab2 causes a
rise to a second, higher plateau. This indicates that Ab, and Ab, react
with different epitopes on the antigen, for example, two spatially dis-
tinct regions of a protein. (b) Ab, and Ab, recognize the same epitope
of the antigen. In the first sensorgram, addition of Ab, reveals a
response, and subsequent injection of Abz generates no additional
response. In the second sensorgram, addition of Ab, first reveals a re-
sponse, but subsequent addition of Ab, generates no additional
response. This pattern indicates that Ab, and Ab, bind competitively,
both recognizing the same epitope on the antigen; binding of one
blocks subsequent binding of the other.

produce a precipitate. Table 6-3 presents a comparison of the

Precipitation Reactions sensitivity, or minimum amount of antibody detectable, ofa
number of immunoassays.
Antibody and soluble antigen interacting in aqueous solution
form a lattice that eventually develops into a visible precipi-
tate. Antibodies that aggregate soluble antigens are called Precipitation reactions in gels yield visible
precipitins. Although formation of the soluble Ag-Ab complex precipitin lines
occurs within minutes, formation of visible precipitate ocurs in
more slowly, often taking a day or two to reach completion.
Immune precipitates can form not only in solution but also
an agar matrix. When antigen
and antibody diffuse toward one
Formation of an Ag-Ab lattice depends on the valency of another in agar or when antibody is incorporated into the agar
both the antibody and antigen: matrix, a
and antigen diffuses into the antibody-containing
The antibody must be bivalent; a precipitate will not visible line of precipitation will form. As in a precipitation
occurs in the region of
form with monovalent Fab fragments reaction in fluid, visible precipitation
forms in regions of
equivalence, whereas no visible precipitate
The antigen must be either bivalent or polyvalent; that is, Two types of immunodiffiusion
revac

it must have at least two copies of the same epitope or antibody or antigen excess. relative concentrations of
anti-
tions can be used to determine the
have different epitopes that react with different or to determine

antibodies present in polyclonal antisera.

bodies antigens, to compare antigens,
or
are radial
preparation. They
relative purity of an antigen double im-
immunodiffusion (the
Mancini method) and
Although various modifications of the precipitalion reaction carried
munodiffusion (the Ouchterlony
method); both are
were at one time the major types of assay usecd in immunology,
radial immunodif-
they have been largely replaced by methods that are faster out in a
agar. In
semisolid mecdium such
as
well and allowed to
is placed in a
and, because they are far more sensitive, require only very fusion, an antigen sample dilution of an anti-
a suitable
small quantities of antigen or antibody. Also, modern assay dilfuse into agar containing the region of
diffuses into the agar,
methods are not limited to antigen-antibody reactions that serum. As the antigen
152 ART
GENERATION OF B-CELL AND T-CELL RESPONSES

RADIAL IMMUNODIFFUSION
TABLE 6-3 Sensitivity of various immunoassays
Antigen
Sensitivity* diffusion
Assay (g antibody/ml)|
Precipitation reaction in fluids 20-200
Precipitation reactions in gels Antibody
incorporated Antigen
Mancini radial immunodiffusion 10-50 in agar

Ouchterlony double immunodiffusion 20-200

Immunoelectrophoresis 20-200
Precipitate
Rocket electrophoresis 2 forms ring

Agglutination reactions
Direct 0.3 DOUBLE IMMUNODIFFUSION

Antigen
Passive agglutination 0.006-0.06 Antibody-

Agglutination inhibition 0.006-0.06

Radioimmunoassay (RIA) 0.0006-0.006

Enzyme-linked immunosorbent
assay (ELISA) -0.0001-0.01
ELISA using chemiluminescence -0.00001-0.011
Immunofluorescence 1.0

Flow cytometry 0.006-0.06 Agar matrix Precipitate

The sensitivity depends on the affinity of the antibody used for the assay as
well as the epitope density and distribution on the antigen.
Note that the sensitivity of chemiluminescence-based ELISA assays can be
made to match that of RIA.
sOURCE: Updated and adapted from N. R. Rose et al, eds., 1997, Manual of
Clinical Laboratory Immunology, 5th ed., American Society for Microbiology,
Washington, DC.
FIGURE 6-6 Diagrammatic representation of radial immuno-
diffusion (Mancini method) and double immunodiffusion
(Ouchter
lony method) in a gel. In both cases, large insoluble complexes form
in the agar in the zone of
equivalence, visible as lines of precipitation
(purple regions). Only the antigen (red) diffuses in radial immunodif-
fusion, whereas both the antibody
double immunodiffusion.
(blue) and antigen (red) diffuse in

equivalence is established and a ring of precipitation, a pre-

proportions (Figure 6-7).
cipitin ring, forms around the well (Pigure 6-6, upper panel). Immunoelectrophoresis
clinical laboratories to detect
the presence or
is used in
The arca of the precipitin ring is proportional to the concen- teins in the serum. A absence of pro-
the individual serum sample serum is
of
tration of antigen. By comparing the area of the precipitin ring
with a standard curve (obtained by measuring the precipitin components electrophoresed,
are identified
and
specific for a
given with antisera
arcas of known concentrations of the antigen), the concentra-
technique is useful inprotein or immunoglobulin class. This
abnormally low determining
tion of the antigen sample can be determined. In the Ouchter- duces whether a patient pro
lony method, both antigen and antibody diffuse radially from
characteristic of certain amounts of one or more
isotypes,
wells toward each other, thereby establishing a concentration
yadicnt. As equivalence is reached, a visible line of precipita-
also show
whether a
protein, such as
immunodeficiency diseases.
patient overproduces some It can
tion, a precipitin line, forms (Figure 6-6, lower panel). albumin, serun
The
with immunoelectrophoreticimmunoglobulin,
patterns of
or
transferrin.
multiple myeloma show serum from
Immunoelectrophoresis combines protein. Because a
large amount of patients
electrophoresis and double immunodiffusion tive
immunoelectrophoresis
technique that detects
centrations (greater only
is a
strictly
myeloma
qualita
In immunoelectrophoresis, the antigen mixture is first elec-
limited to the than several relatively high antibody
trophorescd to separate its components by charge. "Troughs
detection of hundred con
ate then cut into the ayar gel parallel to the direction ofthe
electric ficld, and antiserum is aded to the trouglhs. Anti-
when the
deficiency departure
states and
quantitative wg/ml), its utility 1s
from normal
is abnormalities only
striking,
body and antigen then dilfuse toward cach other and pro-
duce lines of precipitation wlhere they e e t in
appropriate
A related
does immunoprol
permit quantitative
measurement
iferatrocket
technique, ive disorders.immuno
as in

trophoresis, negatively of antigen levels.electrophoresis

a

charged antigen is elect

In
rocket elec
 ANTIGEN-ANTIBODY INTERACTIONS: PRINCIPLES AND APPLICATIONS CHAPTER 6 153

they depend on the cross-linking of polyvalent antigens. Just as

an excess of
Antigens antibody inhibits precipitation reactions, such
excess can also inhibit
agglutination reactions; this inhibition is
O called the prozone effect. Because prozone effects are encoun-
tered in many types of immunoassays,
understanding the basis
of this
phenomenon is of general importance.
Several mechanisms can cause the prozone effect. First, at
high antibody concentrations, the number of antibody bind-
ing 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
O cross-link one antigen to another. Prozone effects are readily
diagnosed by performing the assay at a variety of antibody
(or antigen) concentrations. As dilutes sample to
one a an
optimum antibody concentration, one sees higher levels of
Antlbody agglutination or whatever parameter is measured in the
assay being used. When polyclonal antibodies are used, the
prozone effect can occur for another reason. The antiserum
may contain high concentrations of antibodies that bind to
the antigen but do not induce
agglutination; these antibod-
ies, called incomplete antibodies, are often of the IgG class.
At high concentrations of
IgG, incomplete antibodies may
occupy most of the antigenic sites, thus blocking access by
IgM, which is a good agglutinin. This effect is not seen with
. agglutinating monoclonal antibodies. The lack of
aggluti-
nating activity of an incomplete antibody may be due to re-
stricted flexibility in the hinge region, making it difficult for
the antibody to assume the
required angle for optimal cross-
linking of epitopes on two or more particulate antigens. Al-
ternatively, the density of epitope distribution or the
FIGURE 6-7 location of
Immunoelectrophoresis of an antigen mixture. An some epitopes in deep pockets of a
particulate
antigen preparation (orange) is first
electrophoresed, which separates antigen may make it difficult for the antibodies specific for
the component these epitopes to agglutinate certain
antigens on the basis of charge. Antiserum (blue) is particulate antigens.
then added to troughs on one or both sides of the When feasible, the solution to both of these
and allowed to diffuse; in time, lines of
separated antigens problems is to
precipitation (colored arcs try different antibodies that may react with other epitopes of
form where
specific antibody and antigen
interact. the antigen that do not present these limitations.

in a
Hemagglutination is used in blood typing
gel
containing antibody. The precipitate formed between
antigen and antibody has the shape of a rocket, the height of Agglutination reactions (Figure 6-8) are routinely per-
which is formed to type red blood cells (RBCs). With tens of millions
well. One
proportional to the concentration of antigen in the
limitation of rocket electrophoresis is the need for of blood-typing determinations run each year, this is one of
the antigen to be
for electrophorcticnegatively charged, which is
m ont u i t h n t l
a
requirement
 154 PART IGENERATION OF B-CELL ANDT-CELL RESPONSES
cells on the bottos
red blood
tom
the world's most frequently used immunoassays. In typing of agglutinated reaction.
spread pattern seen in
agglutination
ns
for the ABO antigens, RBCs are mixed on a slide with anti- like the pattern
of the well,
(see Figure 6-8). shift away from red
blona
sera to the A or B
blood-group antigens. If the antigen is pre- there has
been a lood
sent on the cells, In r e c e n t years, beads, as matrices f
they agglutinate, forming a visible clump particles,
such as latex
been coupled
on the slide. As an additional check, the donor's blood is ex- cells to synthetic the antigen has
reactions. Once
amined for the presence of antibodies against ABO antigens. agglutination c a n be
used immediately o
preparation of
If a donor does not contain antibody against his or her own to latex beads, the beads offers advantages
the
The use of synthetic aggluti
ABO antigens, the cell and antibody determinations are con- stored.
and stability. Furthermore,
cordant, and the cell typing is confirmed as correct. If the re- consistency, uniformity, Deads
can be read

nation reactions
employing synthetic the beads with
sults are discordant and agglutination assays indicate the minutes of mixng
within 3 to 5
donor has antibodies that react with their own ABO anti- rapidly, often on red blood cells or the
Whether based
gens, then there is either an assay error (the cell or the anti- the test sample. beads, agglutination
convenient and versatile synthetic
body assay is incorrect) or the donor is making antibodies to more
do not require expensive
simple to perform,
self, a manifestation of autoimmunity (see Chapter 16). reactions are amounts of antibody (con
detect small
equipment, and c a n
Samples that produce discordant results on retesting are not
c e n t r a t i o n s as low
as nanograms per
milliliter).

used for transfusion.

inhibition, absence
of
Bacterial agglutination is used In agglutination
of antigen
to diagnose infection agglutination is diagnostic
agglutination reaction, called aggluti-
A bacterial infection often elicits the production
of serum A modification of the
antibodies specific for surface antigens on the
bacterial cells. nation inhibition, provides a highly sensitive assay for small
inhibition assays can
The presence of such antibodies can be detected by bacterial quantities of an antigen. Agglutination
an individual is using c e r
from patient thought to be also be used to determine whether
agglutination reactions. Serum
a
heroin. A urine o r blood
infected with a given bacterium is serially diluted in an array tain illicit drugs, such as cocaine o r

for the sus-

of tubes to which the bacteria is added. The last tube show- sample is first incubated with antibody specific
added. If the
e r u m antibody titer
pected drug. Then antigen-coated particles
are
will reflect the s
ing visible agglutination defined as the recipro- are not agglutinated by the antibody, it indicates the
of the patient. The agglutinin
titer is particles
dilution that elicits a positive ag- sample contained an antigen recognized by the antibody
cal of the greatest s e r u m
For example, if serial
twofold dilutions suggesting that the individual was using the drug. One prob-
glutination reaction. 1/640 shows ag- lem with these tests is that legal drugs having chemical struc-
and if the dilution of
of s e r u m a r e prepared then the ag-
dilution of 1/1280 does not, tures similar to those of illicit drugs may cross-react with the
glutination but the is 640. In s o m e cases, antibody, giving a false-positive reaction. For this reason,
the patient's s e r u m
glutination titer of and still show aggluti- positive reactions are confirmed by a nonimmunologic
diluted up to 1/50,000
s e r u m c a n be
method.
nation of bacteria.
antiserum c a n be used to diag- Agglutination inhibition assays are widely used in clinical
of an
The agglutinin titer typhoid fever, for
Patients with laboratories to determine whether
an individual has been
bacterial infectíon. titer to
nose a
significant rise in
the agglutination exposed to certain types of viruses that
eanple, show a
Agglutination
reactions also provide
a way
of red blood cells. If an
individual's
cause
agglutination
Salmonella typhi. different species of the
bac-
antiviral antibodies, then the contains specific
serum

1ype bacteria.For instane, re-

virus and interfere with antibodies will bind to the
t distinguished by agglutination
technique is commonly hemagglutination
Salmonella c a n be
1eTiun by the virus. This
of typing antisera. used in
ations with a panel mine the immune status of premarital testing to deter-
virus. The reciprocal of women with respect to rubella
is useful the last serum
Passive agglutination bition of rubella dilution to
show inhi
with soluble antigens A titer
greater hemagglutination
than 10
(1:10
is the titer of the
seru
of agglutinatíon
reactions can
is immune to dilution) indicates that a woma
rubella, whereas
Tir tensitivity and simplicity of passive a titer
aiiuble antigens by 1he techniquc dicative of
lack of
a of less than 10 is
in
antigen-coated red
tion with the rubella immunity
extepdrd uy
te
ia this technigic,
and the need for
tetnaphjiutifiatitosn.
a soluble
antigen with red vaccine. immuniata-
prepased by mixing
tirad i l s are
been trestrd with lnnic tcid or
that have
ril dsorption of

fs
cironinhtitir,

a t i l i i t4

1ialiy
inth of whih promote

the sisrlsse uf
the els, e m 1 COntaining

dilteed into m e
rotiter plate wells,
and
an-

the Radioimmunoassay
One of the most
sensitive
sty i, 14
a r e thhetn
alded to cah well;
antibody is techniques for detecting
developedradioimmunoassay
tated red blruf telis
ttigri

3gpiutisiafsnn 1s
itd ry the s/ oltur
hacteristic first in 1960
by two (RIA). The technique
antigen
endocrinologists,
S.
wa* A. n
5er
 CHAPTE R 6 155
APPLICATIONS
ANTIGENANTIBODY INTERACTIONS: PRINCIPLES AND

If the
and Rosalyn Yalow, of Staphylococcus aureus has high affinity for IgG.
to determinc ievek ofinsolin-anti-insulin contains an IgG antibody, the complex
can

complexes in diahetics. Although their technique encoun Ag-Ab compiex formalin-killed S.

aureus.

tered some skepticism, it soon proved its value for measur- be precipitatedby mixing with
either of these methods,
ing hormoncs, scrum protcins, drugs, and vitamins at After removal of the complex by
labeled antigen remaining in the su-
concentrations of 0.001 miurOgrams per milliliter or less. In the amount of free sub-
measured in a radiation counter;
1977, sonne ycars after Berson's death, thc significance of the pernatant c a n be of labeled
from the total a m o u n t
technique was acknowledged by the award of a Nobel prize tracting this value
the a m o u n t of labeled antigen
to Yalo. antigen added yields
The principle of RlA involves competitive binding of bound.
RIAs have been developed that
radiolabeled antigen and unlabeled antigen to a high- Various solid-phase
the Ag-Ab complex from the
un-
affinity antibody. The labeled antigen is mixed with anti- make it casier to separate
the antibody is covalently
body at a concentration that saturates the antigen-binding bound antigen. In s o m e cases,
The a m o u n t of radiola-
sites of the antibody. Then test samples of unlabeled anti- cross-linked to Sepharose beads.
c a n be measured after
the
gen of unknown concentration are added in progressively beled antigen bound to the beads
and washed. Alternatively, the
larger amounts. The antibody does not distinguish labeled beads have been centrifuged
from unlabeled antigen, so the two kinds of antigen com- antibody can be
immobilized o n polystyrene or
poly
amount of free labeled antigen
pete for available binding sites on the antibody. As the vinylchloride wells and the
be determined in a radiation
concentration of unlabeled antigen increases, more la- in the supernatant can

counter. In another approach,

beled antigen will be displaced from the binding sites. The
decrease in the amount of radiolabeled antigen bound to
specific antibody in the presence of the test sample is mea-
sured in order to determine the amount of antigen present
in the test sample.
The antigen is generally labeled with a gamma-emitting
isotope such as 1251, but beta-emitting isotopes such as tri-
tium (H) are also routinely used as labels. The radiola-
beled antigen is part of the assay mixture; the test sample
may be a complex mixture, such as serum or other body
fluids, that contains the unlabeled antigen. The first step in
setting up an RIA is to determine the amount of antibody
needed to bind 50% to 70% of a fixed quantity of radioac-
tive antigen (Ag") in the assay mixture. This ratio of anti-
body to Ag' is chosen to ensure that the number of epitopes
presented by the labeled antigen always exceeds the total
number of antibody binding sites. Consequently, unlabeled
antigen added to the sample mixture will compete with ra-
diolabeled antigen for the limited supply of antibody. Even
a small amount of unlabeled
antigen added to the assay
mixture of labeled antigen and antibody will cause a de-
crease in the amount of radioactive antigen bound, and
this decrease will be proportional to the amount of unla-
beled antigen added. To determine the amount of labeled
antigen bound, the Ag-Ab complex is precipitated to sepa-
rate it from free antigen (antigen bound to antibody),
not
and the
radioactivity in the precipitate is measured. A stan-
dard curve can be
generated using unlabeled antigen sanm-
ples of known concentration (in
and
place of the test sample)
from this plot the amount of in the test mix-
ture may be
antigen
precisely determined.
Several mnethods have been
developed for separating
DOund antigen fron free antigen in RIA. One method in-
precipitating Ag-Ab complexes with a secondary
-150type antiserum. For example, if the Ag-Ab com-
Pcx contains rabbit Ig antibody, then goat anti-rabbil
g will bind to the rabbit IgCi and precipitate the com-
PCX. Another
method makes use of the fact that proten A
the antibody is immobilized
o n the walls of microtiter
wells and the amount of bound
antigen determined. Because the procedure requires small only
conducted in
small amounts of sample and c a n be
96-well microtiter plates (slightly larger than a 3 x 5 card),
this procedure is well suited for determining the c o n c e n -
tration of a particular antigen in large numbers of samples.
For example, a microtiter RIA has been widely used to
screen for the presence of the hepatitis B virus (Figure 6-9).
RIA screening of donor blood has sharply reduced the in-
cidence of hepatitis B infections in recipients of blood
transfusions.
Enzyme-Linked
Immunosorbent Assay
Enzyme-linked immunosorbent assay, commonly known
as ELISA (or ELA), is similar in principle to RIA but depends
on an enzyme rather than a radioactive label. An enzyme
conjugated with an antibody reacts with a colorless substrate
to generate a colored reaction product. Such a substrate is
called a chromogenic substrate. A number of enzymes have
been employed for ELISA, including alkaline phosphatase,
horseradish peroxidase, and B-galactosidase. These assays
match the sensitivity of RIAs and have the advantage of
being safer and less costly.
There are numerous variants of ELISA
A number of variations of ELISA have been developed, al
lowing qualitative detection or quantitative measurement of
either antigen or antibody. Each type of ELISA can be used
qualitatively to detect the presence otf antibody or antigen.
Alternatively, a standard curve based on known concentra-
tions of antibody or antigen is prepared, from which the
unknowin concentration ofa sample can be determined.
156 PART GENERATION OF B-CELL AND T-CELL RESPONSES

(b)
(a) 70
Uninfected (a) Indirect ESISA
Infected serum 1251)HBsAg 1251]HBsAg
Serum

60
Unlabclcd
HBsAg
Approximately linear pa.
part of cuve
Anti-HBsAg
TI251 bound
J1251 bound Ant
Coate

(b) Sandwich ELIS

FIGURE 6-9 A solid-phase radioimmunoassay (RIA) detect to
coated 30
Microtiter wells are
hepatitis B virus in blood samples. (a) the surtace
With a constant amount of antibody specific for HBsAg. are
and [231]HBsAg
on hepatitis B virions. A serum sample
antigen removed and
the rd- 20
then added. After incubation,
the supernatant is
if tne
complexes is measured.
dioactivity of the antigen-antibody will be less than in con-
a m o u n t of label
bound
sample is infected, the is obtained by adding
10
A standard curve
trols with uninfected s e r u m . (b)
a fixed quantityor
c o n c e n t r a t i o n s of
unlabeled HBsAg to or
4
increasing percentage
From the plotofthe HBSAg, ng/m
lHBsAg and specific antibody. concentration of unlabeled antigen,
0 of
unlabeled

Concentration (C) Comppet

bound v e r s u s the deter
labeled antigen samples can
be
concentration of HBsAg in unknown serum
the
of the curve.
oved by washin
mined by using the linear part antibody
is remov
sub
free
second
reaction
is meae
product is measured.
After any colored
and the
strate is added,
Indirect ELISA
determined with
quantitatively Competitive ELISA
can be detected or
other samn- antigen is com.
of
Antibody Serum o r some amounts
6-10a). measuring
indirect ELISA (Figure antigen- variation for
is added to this technique, antibody is
an an
Another
antibody (Ab,) 6-10c). In
ple containing primaryand allowed to with the antigen ELISA (Figure antigen.
petitive with a sample containing
react
well
coated microtiter is washed away, the first incubated
in solution
well. After any free Ab, added to an antigen-
attached to the detected by mixture is then
bound to the antigen is The antigen-antibody present in the sam-
of antibody that wel. The m o r e antigen FIGURE
secondary antibody (Ab,)
presence
coated microtiter
adding an enzyme-conjugated
is then washed will be available to bind to the assay (
free Ab, the less free antibody
binds to the primary antibody. Any added. The amount of ple, Addition of enzyme-conjugated
an gen. Ea-
for the enzyme
a substrate
is antigen-coated well.
and
is measured by special- for the isotype of the parison

secondary antibody (Ab,) specificdetermine the amount of

away,
colored reaction product that forms m e a s u r e the antiboc
readers, which can used to
ized spectrophotometric plate
in seconds. primary antibody can be
96-well plate as in an indirect ELISA. In
absorbance of all of the wells of a primary antibody bound to the well,
method of choice to detect the pres- concentration of
Indirect ElLISA is the the competitive assay, however, the higher the
human immunodeficiency
ence of serum antibodies against antigen in the original sample, the lower the absorbance. meas

virus (HIV),
the causative agent of AlDS. In this assay, recom- minc
of HIV are adsorbed as
binant envelope and core proteins Chemiluminescence chrc
to microtiter wells. Individuals infected
solid-phase antigens tect
with HIV will produce serum antibodies to epitopes on these Light produced by chemiluminescence during certain chem-
viral protcins, Generally, serum antibodies to HIV can be de- ical reactions provides a convenient and fro
highly sensitive a adc
tected by indirect ELSA within 6 weeks of infection. ternative to absorbance measurements in the ELISA.
versions of the ELISA
Sandwich ELISA
using chemiluminescence, a luxog
ight-generating) substrate takes the place of the chro
Antigen can be detected or measured by a sandwich ELISA mogenic substrate in conventional ELISA reactions. ro
For ex
(Figure 6-10b). Jn this technique, the antibody (rather than ample, oxidation of the the
the antigen) is immobilized on a microtiter well. A compound luminol by H,0,anu
sample
ontaining antigen is added and allowed to react with the enzyme horseradish
peroxidase (HRP) produces lign.
mobilized antibody. After the well is washed, a ond im
enzyme-linked antibody specific for a dilferent second Ab-HRP + Ag Ab-HRP-Ag luminol+H.0: light
antigen is added and allowed to react with the epitope on the The light produced during
bound antigen. d
tected by its ability to exposeluxogenic reactions may
itati
photographic film.
im. Quantitatie
 GENERATION OF B-CELL AND T-CELL RESPONSES
158 PAR T IT

Western Blottin8 comp mplex mixtu

a of
in
as
Well coated with
protelna West-
rotein known
techntgue

of a
specific a rn blott
anticytokine by Southern

g
l d e n t i f i c a t i o n

accomplished
similarity to blotting
antibodyy be Norther
proteins ca for its mivich

and ture is
WwYYYJ
named protein

ern
blotting,
DNA
f r a g m e n t s ,

b l o t t i n g s

SDS-polyacryl
a
which
detects
W e s t e r n
d o d e c v i . e
ge
( s ) Secretor m R N A s .
In
separated
on an
with
sodium sulfate
NS) Nonsecretor
etects
ectrophoretically infused
6-12). The
prote
bands
gel be
Add test cell a
slab
(Figure m e m b r a n e

elec
population
(SDS- -PAGE),
dissociating
agent
n i t r o c e l l u l o s e
bands are ident
.
a protein
DS), a
transferred
to radiolabeled or en:
fe
i n d i v i d u a l

the
are
and
membrane
with
a n t i b o d y
specific or the
r o p h o r e s i s ,

the monoclonal
that form on he
flooding c o m p l e x e s

antibod..
Incubate at 37°C by p o l y c l o n a l
or

The
Ag-Ab by the dy
protein of inter
nked r e c o g n i z e d

interest.

protein
of
the
protein If the
If
o n the
containing o f ways. position

its
band
be visualized in
variety
antibody,
to a sheet
eet of
r a d i o a c t i v e

imembrane
the
bound bya e x p o s ia
nugt o r a d i o g r a p h y . . However t
Discard cells
was
d e t e r m i n e d by called employ
enzu
1zyme-
be
Wash plate
can

a
procedure
p r o c e d u r e s

binding ofsthe
X-ray film,
d e t e c t i o n

After
used
the
protein. chromogenic
s.
ub-
generally
most

a n t i b o d i e s
against addition
ofa insoluble
produes
linked conjugate,

colored and the tar

t h e site of
enzyme-antibody

a highly b a n d at
produces
that colored
achieved if a

Add enzyme-linked
strate

the
appearance
ofa sensitivity
can
be
enhancina
causes suitable g
anticytokine
Even greater with
antigen. compound site.
get antigen
at the
chemiluminescent
antibody
light in a
used to
produce specific antibody
is also identity molecular
Side view agents
blotting
can
of
well-defined

E = enzyme
known antigens
Western
blotted onto
nitro.
C C S =c h r o m o g e n i c
In this case, and
substrate
mixture.
by
SDS-PAGE

antigens
are then
separated known
bands of
are
CP= colored weight antibody
cellulose.
The separated of containing
product suspected
R e a c t i o n of a n
an-
with the sample
probed of these antigens.
more radiolabeled
one or either
specific for detected by using for the
band is is specific
tibody with
a that
antibody
Site o f
enzyme-linked
secondary
The most widely
secreting cell or sample. in the test
the antibodies
testing
species of is in confirmatory
T o p view
application of this procedure determine
used
Western blotting
is used to
for HIV, where react with
one or
has antibodies that
whether the patient
more viral proteins.

coated with anti-

a well is
ELISPOT assay,
FIGURE
6-11 In
the
of interest,
a cytokine ampl
in this example, Immunoprecipitation
the antigen to contain of al-
population thought
body against of a cell The immunoprecipitation technique has the advantage
suspension the cytokine is lay-
interest for further ana-
a
and then and secreting
members
synthesizing
and incubated. Most of the lowing the isolation of the antigen of of a
It also provides a sensitive assay for the presence o
some
bottom of
the well
ered onto
the
particular cell
react with nearby ysis.
secreted by a
washed
particular antigen in a given cell or tissue type. An extraCt P
molecules the well is
cytokine the incubation period, duced by disruption of cells or tissues is mixed with an ai
antibodies. After
vwell-bound is added After washing
anticytokine antibody
and an
enzyme-labeled substrate that forms an body against the antigen of interest in order to for
a chromogenic
unbound antibody, ver,
avway is added. The
colored product (purple) pre- antigen-antibody complex that will precipitate. Howev
insoluble colored product on the areas of the well where the antigen concentration is low (often the case in cell and
only
and forrns a spot
cytokine-secreting
cipitates cells were deposited. By counting the number of Sue extracts), the assembly of antigen-antibody compie
determine how many cytokine-secreting into precipitates can take
colored spots, it
is possible to hours, even days, and it is difficut
added cell suspension. isolate the small amount
cells were present
in the
of immunoprecipitate that of
 ANTIGEN-ANTIBODY INTERACTIONS: PRINCIPLES AND APPLICATIONS HAP TER 6 159

(a) Add SiDS-treated

(b) Electrophores* FIGURE 6-12 in Western blotting, a protein mixture is (a)
protein nmixture to in SDS-polyacrylamide treated with SDS, a strong denaturing detergent, (b) then sepa
well of gel Zel rated by electrophoresis in an SDS polyacrylamide gel (SDS-
to their
PAGE). which separates the components according
molecular weight; lower-molecular-weight components migrate
removed
farther than higher-molecular-weight ones. (c) The gel is
sheet of nitro-
from the apparatus and applied to a protein-binding
cellulose or nylon, and the proteins in the gel are transferred to the
sheet by the passage of an electric current. (d) Addition of enzyme-
interest, and (e) the position
linked antibodies detects the antigen of
Dircction means of an ELISA
reaction that
of the antibodies is visualized by
at the
of colored insoluble product that is deposited
generates highly
a
migration a chemiluminescent ELISA can be
site of the reaction. Alternatively,
Protcin antigens detected by exposure of the blot
denatured in SDS used to generate light that is readily
film.
to a piece of photographic

these limitations.
() Remove gel and
There are a number of ways to avoid
perform electrotransfer solid support, such as a
antibody to a
One is to attach the
allows the antigen-antibody complex
synthetic bead, which is to add a sec-
to be collected by
centrifugation. Another
to bind
for the primary antibody
ondary antibody specific
If the secondary antibody
the antigen-antibody complexes. be col-
bead, the immune complexes can
is attached to a
version
particularly ingenious
lected by centrifugation. A
the coupling of the secondary
of this procedure involves
magnetic
to beads. After the secondary antibody
antibody immunoprecipitates are
binds to the primary antibody,
Electric the side of the tube
collected by placing a magnet against
current
(Figure 6-13). radioiso-
When used in conjunction with biosynthetic
Porous also be used to de-
immunoprecipitation can
membrane
tope labeling, synthesized
sheet termine whether a particular
antigen is actually
of proteins synthesized by
tissue. Radiolabeling
by a cell or the cells in cell
cul-
of interest be done by growing
(d) Bind antigen cells of interest can r a d i o l a b e l e d amino
more
one or
with enzyme-linked containing
ture medium for this application
antibodies the amino acids used
acids. Generally, metabolic
modification, such as
resistant to
those most
growth in the
ra-
are After
methionine.
or to a
leucine, cysteine, and subjected
the cells are lysed
dioactive medium, interest. The
the antigen of
specific for
primary antibody immunoprecipitation,

is c o l l e c t e d by amino acid
complex
Ag-Ab unincorporated
radiolabeled

washed free of complex c a n The

and then
analyzed.
impurities, obtain a quantita-
and other c o u n t e r to
scintillation

counted in
a protein synthe- of the
be amount
determination
ot the
disruption
of the
tive involves
often iden-
substrate to Further analysis so that the
(c) Add sized. and heat,
use of SDS confirmed
activale c o l o r reaction
usually by antigen
can be
complex, tor
that expected
i m m u n o p r e c i p i t a t e d

of the
tity molecular weight is of the
that its separation
by checking of interest. This is done by autora
subsequent
and
the antigen SIDS-PAGE radiolabeled

complex by of the
disrupted deternmine
the position
to
diography
on the gel.
antigen
160 PAR T I GENERATION OF B-CELL AND T-CELL RESPONSES
(a) b) ( (d
Specific
antibody Magnetic
bead
Antigen
A
Apply magnet
Add secondary and rinse to
Add specific
antibody remove
antibody to
coupled unbound allows the rapid c
cell extract tube
to magnetic material
the side
of the
to remove An
against A f t e r rinsing
beads
a
magnet complexes.
can be dissociated
() Placing a n t i g e n - a n t i b o d y
complexes
using of the cell with
can be collected lection
a n t i g e n - a n t i b o d y
showing a
Immunoprecipitates Treatmentof material,
the micrograph
electron [Part
6-13 (a) unbound
(d) An
antibodies.
secondary antibody.
FIGURE studied. via
anti-A antibody
surface
beads coupled to a and the
antigen to its
magnetic with a m o u s e
antigen A (red)
attached
Addi- Zurich-Irchel.]
complexes. (b)
beads
extract containing magnetic University of
a cell of
antigen-antibody
linked ofAnatomy,
formation
antibody is
Institute
results in the antimouse Groscurth,
(blue) which a rabbit mouse lg).
beads to unreacted
tion of magnetic complexes (and any f l u o r e s c e n c e

at a longer
antigen-antibody
binds the Because it emits used in two-color
be
(546 nm). it can
specific to
fluorescein,
than
wavelength An antibody
immunofluorescence assays. and an
fluorescein,
with
Immunofluorescence is labeled labeled with
antigen is
determinant
antibodies
could be labeled one
recognizing
a
different
s h o w e d that
fluorescein-tagged
Fluores- antibody
Coons of the
1944,
Albert fuorescence.
of and The location color, easy
in the property rhodamine.
its yellow-green
that have (excitation)
molecules wavelength be visible by where the
with
absorb light ofone If antibody antibody will color emitted
molecules (emission). from the red
cent wavelength fluorochrome, todistinguish bound. By conjugating
antibody has
another
emit light of with a
fuorescent dye, o r labeled an- rhodamine-tagged rhodamine to another,
tagged
molecules
are
containing
these fluorescently
emission when fluorescein antibody and
to one
two
complexes colored light simultaneously
visualize
immune
c a n be
detected by Antibody one can, for example, cell.
(FA) wavelength. on the same
tibodies
of the
appropriate
can sim- different cell
membrane antigens
tissue sections
cxcited by light in cells o r (~30-fold
efficient absorber of light
bound to antigens viewed with a flu-
molecules
emitted
The light can be UV light Phycoerythrin is an emitter of red
visualized. with a and a brilliant
ilarly be which is equipped flu- greater than fluorescein) for
as a label
stimulating its wide use
microscope, immunofluorescence,
orescence
known as fluorescence,
technique, rhodamine are in
sourcc.
In this fluorescein and
immunofluorescence.
such as also
compounds substances are
fluorescent
membrane molecules
arescent
but other highly
use,
phycoerythrin,
an intensely col- Fluorescent-antibody staining of
cell
(Figure 6-14). In
annmon
such as
routinely employed, obtained from algae. tissue sections can be direct or indirect
fluorescent pigment
or
oned and highly to the Pc region
be conjugated
of an anti- direct staining, the specific antibody (the primary antiboay
directly conjugated with fluorescein; in indirect staining
molccules can antibody.
affecting the specificity ofthe
Thesc is
without
nolecule one wave- the primary antibody is unlabeled and is detected with an a
dy luorochromes
below absorbs light at
Each of the ditional fluorochrome-labeled reagent. A number of reagel
cmits light
at a longer wavelength:
Jeygtiy asnd have been developed for indirect staining. The most commo
that is the most widely used in
Fuorescein, an organic dye is a fluorochrome-labeled secondary antibody raiseu
immuohuorescence procedures, absorbs blue one species against antibodies of another species, sucn
latel for
nmj and emits
an intense yellow-green
igt 49 fluorescein-labeled goat antimouse immunoglobulin.
fuoresue1nte (517 nm) an-
Indirect immunofluorescence
absorbs in the yellow
staining has two adv oes
hodamine, another organic dye, tagesover direct staining. First,
the primary antibody d
nn) and emits a deep red fluorescence
(515 not need to be
gIecn ange conjugated with a fluorochrome.
the
Because
 f ptor

NAs prectptuf

(D

JQ
tacb druAy
D
O

D H
ound
a9gwtnah'o
an
tyn ypc
btad
tln twqdde
O

D
3
162 PART I
GENERATION OF B-CELL ANDT-CELL RESPONSES
ould be p
ossible
woul
it
cells,
all T the total
for an antigen
p r e s e n t

on
or T cells in c e l l - s o r t i n . " t e
percentage the
the using
determine
Then, be
Cells stained with: to
cell
population.
cytometer, it
would po
ossibl to
Anti-A + anti-B
Dlood
of the
flow leukocyte population,
leukocyte
antibody capabilities fraction ofthe ar
Ultasonic
nozzle vibrator
Anti-A antibody
Anti-B antibody
Isolate
distribution

the
T-cell
ofcells
in a sample
by
population
fluoresce cence
ditens
ccording
ofthe
d e t e r m i n e d
The
Unstained densities
as
a
m e a s u r e
cell.Ution
obtain
to antigen of
possible
to
the
p o p u l a t i o n
that
It is thus within
powerful rful feature ofthe
density is a
ofantigen This
Fluorescence>
LAser
the
antigen.
type
o f cell may
devolS
possess
instrument,
since thesame depending
on its
on its developmental
Computer screen different
levels of antigen
A-Bt cells A*B* cells or physiological state.
i n t o r m a t i o n

is derived fron from analys

The size of cells.
This
properties
of members nbers of
of the
cell
A"B- cells| A*B cells light-scattering
Deflection plates
p
of the
o p u l a t i o n u n d e r e x a m i n a t i o n .
to analyze cell
it pOSSible
or even three popu-
makes
Flow cytometry
also with two
Anti-A antibody >> been
labeled
differ.
fluorescence lations that have
antibodies. For
example, if a blood
sample is
ent
fluorescent
fluorescein-tagged ntibody specifio
antib
reacted with
a
antibod0r
also with a phycoerythrin-tagged
T cells and
the percentages ot B and T cells may be d
O cific for B cells,
| termined simultaneously
with a
Single analysis. Numero.
variations of such multicolor analyses are common, incllud
"colors" at once.
A-B and AB- A*B+ experiments employing many
ingFlow
AB cells cells cells cytometry now oCcupies a key position in in
munology and cell biologY, and it has become an indispens.
FIGURE 6-15 Separation of fluorochrome-labeled cells with
the flow cytometer. In the example shown, a mixed cell population able clinical tool as well. In many medical centers, the flow
is stained with two antibodies, one specific for surface antigenA and the cytometer is one ofthe essential tools for the detection and
other specific for surface antigen B. The anti-A antibodies are labeled classification of leukemias (see Clinical Focus). The choice of
with fluorescein (green) and the anti-B antibodies with rhodamine treatment for leukemia depends heavily on the cell types
(red).The stained cells are loaded into the sample chamber of the cy- involved, and identification
tometer.The cells are expelled, one at a time, from a small precise of the neoplastic cells is
vibrating noz- an essential
zle that generates part of clinical practice. Likewise, the
microdroplets, each containing no more than a single surement ofT-cell rapid mea-
cellL As it leaves the nozzle, each
and the computer that controls the
droplet receives a small electric charge, indicator in AIDS, is
subpopulations, an important prognostic
when a
flow cytometer can detect
exactly routinely done by flow-cytometric
analysis. In this procedure, labeled
drop generated by the nozle
ught that excites the fluorochrome, The through beam of laser
passes the
against the major T-cell monoclonal antibodies
enitted by each droplet that
contains a
intensity
cell is
of the fluorescence antigens are used to determine subtypes bearing the CD4 and CD8
arnd displayed on a monitored by a detector their ratios in the
computer screen. Because the blood. When the patients
orsition of each droplet, it is computer tracks the number of CD4 T cells
falls below a certan
droplet vill arive between the possible to determine when a level, the patient is at
particular high risk for
2ry charge to the deflection
deflection plates.
plates when
By applying a momen- opportunistic infections
them, t is
aother
possible to deflect the path of a a droplet is passing between ween Alternatives to
tn
collecting vessel. This allows
the
particular droplet into one or
upuplations sorting of a
of surfacepopulation clls
in
into the
and are

the
computer
ower left-hand
having diferent profiles
display,
judged not to panel have
e. Those that
each dot markers.
of
represents a cell. Cells that
have reacted background levels of fall
Antigen-Antibody Reactions
As a
defense
evolved the against host
fluorescence to make antibodies, Some
with
appear
in the either
antibody anti-A of ability bacteri have
proteins that sometothe Fcreg
the upper-left panel reacted
arnti-A, and tho0se or IgG molecules pro
in anti- bind
arti-8. The lower-right panel
with anti-B but not molecule, with high
and anti-8. upper-right panel known affinity
contains reacted with (K
e shown cells that react withanti-A but not G,some strains of protein A, is found 103). One Such
in as
the
subpopulanti-Aations haveexampl
with each been here, the A"B"-and both anti-A appears in theStaphylococcusaureus,
in the cell walls
tions and ant-8 sorted Into the
A*Bt- cloning walls of aureu and another, prote
to be fluorescent a
the
antibodies separate tube.
distin gulshed proteingrouj
a genes for group C and G
A" allows four Staining hybrid Streptococ
87,A *B*, A "B *, and A*®. subpopula- known:as both, one
of A
one can and proteinG and genera
:
molecules protein
are A/G, that make features
combi
combines
recombinant
ofboth. Thes
a protein
species. useful
terent
Thus, because
nus, thev can theyey bind IgG from n they can be ahi
 I GENERATION OF B-CELL AND T-CELL RESPONSES
PART
164
fo.
on ur
depend Rich, R
molecules in the SUMMARY
types of
interactions
used to detect IgG bond ds, ionic bonds.
or radioactivity and or h y d r o g e n
Trends
formed ELISA, RIA, Antigen-antibody
complexes during interactions: der
an d Waals
e r Waals interactione
fluores- van
antigen-antibody "
Rse, N.
as flow cytometry or and
fluorescence-based
n o n c o v a l e n t

such assays can i n t e r a c t i o n s ,

d e t e r m i n e d
bv Ogy Ar
IgG-binding proteins be
h y d r o p h o b i c
These bacterial
by Scatchard
cence microscopy. isolation of IgG. which can measure ure of the stren Wild, D
columns for the constant, of
q u a n t i t a t i v e

also be used to make affinity biotin, of the antigen and a single Amster
called avidin that binds Theaffinity a o f the:
contain a protein epitope
provides
Egg whites believed to an reflects
Avidin is analysis, between
avidity
Ihe
a that is essential for fat synthesis.
vitamin interaction multivalent
verall
rodents that rob antibody. b e t w e e n

defense a mus:bog
have evolved as a against marauding and the site i n t e r a c t i o n s
between avidin binding
ofan molecule at
nests and eat the stolen eggs. The binding antigen
much higher affinity of the m u l t i v a l e n t
http://p
strength
biotin is extremely specific and of allows thecharach
the characterizatic
reaction. A bac molecule anda
known antigen-antibody r e s o n a n c e
(SPR) Explo
(K- 10) than any antigen-antibc

made by Streptomycesavidin plasmon

ibody interactions, kinds
terial protein called streptavidin, Surface kinetics
of
extraordinary and used
has similarly high affinity
and specificity. The affinity and precipitatino
these pro- ofthe soluble antigen g anti
of the interaction of forms an Ag-Ab prec
afinity and exquisite specificity interaction of a medium http://jc
proce The
teins with biotin is widely used in many immunological an be combined with precipitation
liquid o r gel combined
with biotin
is labeled be llon Attth
bodyin a can
dures. The primary or secondary antibody Electrophoresis i m m u n o e l e c t r o p h o r

unbound Web
with the target antigen, and the tate. called
and allowed to react or technique
streptavidin a
antibody is then washed away. Subsequently,
in gels in
antigen and agglu:
guide
fluorochrome, or radioac- between a particulate clinic
avidin conjugated with an enzyme, The interaction
tive label is used to detect the bound antibody. produces viSible clumping.o.
nating antibody (agglutinin) basis of simple, rapid, and http://w-
that forms the instrumee
agglutination,
sensitive immunoassays.
Immunoelectron Microscopy Excell
The fine specificity of antibodies has made them powerful tools Radioimmunoassay (RIA) is a highly sensitive and a 1 a n s :
tative procedure that utilizes radioactively labeled antigen surfa
for visualizing specific intracellular tissue components by im-
munoclectron microscopy. In this tedhnique, an electron- or antibody.
dense label is either conjugated to the Fc portion of a The enzyme-linked immunosorbent assay (ELISA) de-
specific antibody for direct staining or conjugated to an anti-
pends on an enzyme-substrate reaction that generates CLINICAL
immunoglobulin reagent for indirect staining. A number of
a colored reaction detect
electron-dense labels have been employed, including feritin and product. ELISA assays that employ
colloidal gold. The electron-dense label can be includ
visualized with chemiluminescence instead of a chromogenic reaction are
the electron microscope as small black dots. the most sensitive monoc
In the case of im- immunoassays available.
munogold labeling, different antibodies can be In Western 1. Indica
conjugated with
gold particles of different sizes, allowing identification blotting, a protein mixture is
antigens within a cell by the different sizes of the of several
trophoresis; then the protein bands are separated by elec false.
goid particles attached to the antibodies electron-dense transferred onto electrophoretically a. Ir
(Figure 6-16). nitrocellulose and identified with labeled
antibody or labeled antigen.
Fluorescence b.

C.
microscopy using antibodies labeled with
fluorescent molecules
or within cells. can be used
to visualize
antigen on d.
Flowcytometry provides an
o8y tor the quantitative
e.
unusually powerful
I
tions labeled
with one oranalysis and sorting of celltechno
more popula"
References fluorescent antibodies. f. F
IGUPE 6-16 B., et al. m
An BiNew
erer, York. 2005.
as
abell yehoma A
MHC was stained of the
Current Protocols
in
tet modetudes immunoelectronmicrograph
with twO Harlow, E., m
I
and D. Immunology Wiley tri
Tte
MHC days labeled ith 30-nm antlbodles: surface Manual.
tles. ayirot one Lane.
a ot td clats, mol I CO
ten
ecules labeled gold partlcles andagainst Harbor, NY.Cold Spring 1999.
pi
f o' fnanA rnwolet udes with 15-nm an- Harbor Using Anti
ntibodies: A
jnelet al, exreeds
that gold Labooratory h. EL
Lsityarvnhe, Uiersty Mede
1997,PNAS 94:7 of class IW on par-this Herzenberg, L. A., ed. Laboratory Press, oCold Sp tiv
al School of Debi269-recen,7274; courtesy mmunology, 5th ed. 1996. Weir's}
Ilungary MalAndmqyist, M. 1993. Oxford, BlackwellHandbook of Experimental
Caurrentmeasurement ofSurface Scientific Publications. Us=
Opinion in plasmon
plasmon resonance Fu
resonance
anmtibody-antigenfor detectio
Immunology 5:282. affinity anu etics