Antigen-

Antibody Reactions
antigens such cts enzymes and in screening the
General features of antigen-antibody reactions population for a particular infection.
Measurement of antigen and antibody u,---ln ge~eral, these reactions can be used for the

SEROLOGICAL REACTIONS detection and q_yantitation of either anti en o.r

antibodies.
PRECIPITATION REACTION
~ -antibody reactions in vitro are known as
Mechanism of precipitation
_serological reactions.
Applications
AGGLUTINATION REACTION Stages
COMPLEMENT FIXATION TEST (CFT) The reactions between antigens and antibodies occur
in three stages:
NEUTRALISATION TESTS
• Primary stage: This is the initial interaction
OPSONISATION between anti ens and antibodies, without any visible
RADIOIMMUNOASSAY (RIA) ~ffects. This reaction is rapid, occurs even at low
ENZYME IMMUNOASSAY (EIA) CD temperatures and obeys the general laws of physi~

Enzyme-linked immunosorbent assay (ELISA) cal chemistry and thermodynamics. The reaction
is reversible, the combination 1;,etween antigen and
CHEMILUMINESCENCE IMMUNOASSAY (CUA)
antibody molecules bei affected b weaker int r-
IMMUNOELECTROBLOT/WESTERN BLOT molecular s such a an der Waal's force , ionic
TECHNIQUES Q.Qlliis and dro en bondin , rather than Q):' the
IMMUNOCHROMATOGRAPHIC TESTS firmer covalent bonding. '<fne 12rimary_ reaction can
IMMUNOELECTRON MICROSCOPIC TESTS b~t!_!!lating fr-¥e and bound antigens
or antibodies se12arately in the reaction mixture by_1l
IMMUNOFLUORESCENCE number of physical and chemical methods, includ-
in1g the use of markers such as radioac.gve isotopes,
9 flu9rescent dyes or furritin.
• Secondary stage: In most, but not all, instances, the
INTRODUCTION primary_ stage is followed by the secondary stage,
~ing to den onstra le ev ts such as P.recipitation, ©
Antigens and antibodies, by definition, combine
@agglutination, xsis of cells, "Uing of !i've antigens,
with each other., specifically and in an observahle
neutralisation of ~ and other biologicaliy active
.!!!.illlli-er .
a.ntigens, fixation of complement, immobilisation Qf
Uses : The reactions between antigens and antibodies motile organisms and enhancement of phagocytosis.
se_!Y.e several purposes: When such reactions were discovered one by one,
.-.. In the body, they form the basis of a.2tibody-medi- it was believed that a different type of antibody ~s
ated i_mmunity in infectious diseases, or of tissue responsible for each type of reaction, and the anti-
injur1 in some types of hypersensitivity and autoim- bodies came to be designated by the reactions th9
mune diseases. were thought to produce)·TI11:i's, the antibody ~s-
.-e;-- In the laboratory, they help in the diagnosis of ing agglutination was called a/glutinin, that causing
infections. precipitation precipitin, and so on, and the cor -
• In e£idemiological surveys, they assist in the iden- responding ant igen, ag lutino en, erecipitinogen,
tification of infectious agents and non-infectious and so on. It is true that a single antibody can cause
, 106 Part II IMMUNOLOGY

precipitation, agglutination and most of the other whereas high-affinity antibodies bind antigens
serological reactions and an antigen can stimulate more tightly and remain bound longer.
the production of different classes of immunoglobu- • Avidity is the strength of the bond afru tb~JQt.-
lins which djffer in their reaction capacities as~l mat~on of the antigen-antibody complexes J!!Jd
as i11,. other properties (Table 12.1 ). is a better measure of its bindin ca acit within
• Tertiary stage: Some antigen-antibody reactions ~iological systems (for example, the reaction of an
occurring in vivo initiate chain reactions that lead to
neutralisation or destruction of injurious antigens,
antibody with antigenic
, -
determinants on a virus
or bacterial cell) than the affinit of its individual
o.LJ_o tissue damage. These are tertia_cy_r:eactions binding sites.\Seereted pentameric ~ often has
and include burooraJ immunity against 'in_fu.ctious lower af~nity than 1.gQ, but the high avidity _.2f
diseases as well .as clinical allergy and other immu- IgM, resulting from its hi her valence, enables it
nological diseases. · f?bind antigens effectively. --
- ~
6. Both antigens and antibodies participate in the for -
General features of antigen-antibody mation of agglutinates or precipitates.
reaction 7. Antigens and antibodies can combine in_y_arying E!:9·
portions, unlike chemicals with fixed valencies . Both
1. The reaction is s_pecific, an antigen combining only
antigens and antibodies are multivalent. AnJj_.hQgies
with its homologous antibody and vice versa. The
are gener~lly bivalent, though IgM m~ul~ may
specificity, however, is not absolute and 'cross-
have five or ten combining sites . Antigens may have
~actions' may occur &To a ~ i c similarity Qr
valen1:,ies up to the hundreds .
relatedness .
2. Entire molecules, and not fra ments, react. When Measurement of antigen and antibody
an antigenic determinant present in a large mol~e
Many methods are available for the measurement of
or o~ 'carrier' particle reacts with its antibody,
the antigens and antibodies participating in the pri -
whole molecules or particles are agglutinat~
here is ·no denaturation of the antigen or the anti- 1,!!MY, s~ary and t~rtiary re~ns. Measurement
may be in terms of mass (for example, mg nitrogen)
body during the reaction.
or mo~mmonly as units or titre. The antibody
4. The combination occurs at the surface. Therefore,
titre of a serum is the hi hest dilution of the ser m
it is the surface antigens that are immunologically
that shows an observable reaction · the anti en.in
relevan ."ruitibodies to the surface antigens of infec-
the pa~ticular test. The titre of a serum is influenced
tious agents are generally protective.
by the nature and quantity of the antigen and the
5. The combinatfon is fir.in but reversible. The firmness
type and conditions of the test. Antigens may also be
of the u!!!Q!Lis influenced by the affini and ~
titrated against sera.
of the reaction:
Two important parameters of serological tests are
• Affinity refers to the intensity of attraction
sensitivity and specificity:
between the antigen and antibody molecules. It
• Sensitivity refers to the ability of the test to detect
--
is a function of the closeness of fit between an
epit~d the anti en-combinin region of its
even very minute quantities of antigen or antibody.
When a test is highly sensitive, false negative results
antibod (paratope). Affinity is a quant~ e
will be absent or minimal.
measure of bindin stren th between an antibo y
• Specificity refers to the ability of the test to detect
and an epitope. Low-affinity antibodies bind
ly, reactions between homologous antigens and anti-
antigens w:eakly and tend to dissociate ~
bodies only, and with no other. In a highly specific
Table 12.1 Comparative efficiency of the immunoglobu- test, false positive reactions are absent or minimal.
In general, the sensitivity and specificity of a test are
Un classes in different serological reactions
,_ in inverse proportion.
Originally, reagents for serological tests were pre-
Precipitation Strong Weak Variable
Agglutination Weak Strong Moderate pared by individual laboratories, leading to batch varia-
Complement fixation Strong Wea k Negative tion, and lack of reproducibility and comparability. The
Lysis Weak Strong Negative commercial availability of readymade standardised test
 Antigen-Antibody Reactions

kits has simplified test procedures, improved quality clinical serology, as sera rich in antibody IllilY ~me-
and greatly enlarged their scope and use. times give a false negative precipitation or agglutiw-
-
tion result, unless several dilutions are ~ d .
.
SEROLOGICAL REACTIONS Mechanism of precipitation
Marrack (1934) proposed the ~ttice hypothesis to
PRECIPITATION REACTION explain the mechanism of precipitation. According
Precipitation: When a soluble antigen combines with to this concept, which is stipported gysonsiderable
its antibod~ in the presence of electrolytes (NaCl) ~t experimental evidence and is now widely accepted,
a suitable temperature and £1!, the antigen-antibody ~ltivalent antigens combine with bivalent antibodies
com lex for s an insolub e ec· ·tate. in varying proportions, depending on the antigen-anti-
body ratio in the reacting ·!T¼ixture. Precipitation results
Flocculation: When, instead of sedimenting, the Ee-
when a large lattice is formed consisting of alternating
cipitate remains suspended as floccules, the reaction
antig_en and antibody molecules: Thi~ is possible only
is known as flocculation. Precipitation can take place
in the zone of equival_ence. In_-the Z(?nes of a.?tigen ~
in liquid media or i~ _gels §!!ch as ~ar, agarose Q!
antibody excess, the lattice does · not enlarge, M_ the
polyacrylamide.
valencies of the·antibody and .the antigen, respectively,
Phases: The amount of precipitate formed is greatly are fully satisfied (Fig. i 2.1). The lattice 'hypothesis
influenced by the relative proportions of antigens and hofds good for a~giutination also. · ·
antibodies~creasing quantities of antigens are
added to the same amount of antise indiffe~t Applications
tubes, precipitation will be found to occur most rapidly
The precipitation test may be carried out a__La qualita-
a~ abundantly in one of the middle tubes in which the
tive or quantitative test. It is sensitive in the detection of
antigen and antibody are present in optimal or equiva-
anti~ns and as little as 1~ of protein can be detected.
lent proportions. In the preceding tubes in w_hichJlle
It is relatively less sensitive for the detection of antibod-
antibody ~sin excess andin the later tubes in which the
ies. Precipitation tests have several applica~
antigen is in excess, the reci itation will be weak-9r
-~..> -forensic application in the identification of J;tlQQd
even absent. For a given antigen-antibody system, the
and seminal stains
optimal or equivalent ratio will be constant, irres ective
~ esting for food adulterants
of the quantity of the reactants. If the amounts of pre-
v-Grouping of streptococci QY the Lancefield
cipitate in the different tubes are plotted on a graph,
technique
the resulting curve will have three phases:
he VD RL test for syphilis
. rozone phenomenon: This is caused by excess
• To standardise toxins and toxoids
antibody in the test system. Failure of a visible~-
• To test toxigenicity· in diphtheria bacilli
tion is due to inhibition of lattice formation b the
The following types of precipitation and flocculation
excess antibody.
tests are in common use:
~ one of equivalence: Here, the antigen and antibody
are in optimum proportions. Lattice formation and Antibody Antigen
visible reactions are enhanced. I
3 Post-zone henome n: This is caused b:y the pres-
I
ence of excess antigen in the test system. No visible
t'
-\
reaction will occur.
The prozone and post-zone phenomena may be
corrected by making serial dilutions of ~m, thereby
reducing the concentration of antigen or ~ d yJ!i
the test serum, and optimising the concentrations of Antibody in Lattice formation Antigen in
antigen andantibody. excess Zone of equivalence excess
prozone post-zone
Zoning occurs in agglutination and some other
serological reactions. The prozone is of i~portance in Fig. 12.1 Lattice hypothesis
 Part II IMMUNOLOGY

Ring test: This, the simplest type of precipitation test, form a band of precipitate where they meet at opti-
consists of l~yering the antigen solution over a column mum proportion (Fig. 12.2a).
of antiserum in a narrow tube. A .12recipitate for.ms at 3. Single diffusion in two dimensions (Radial immu-
the junction of the two Ii uids. Rin tests have only nodiffusion): Here, the antiserum is i~orporated
a few clinical a plications n__Q__w. Examples are Ascoli's in agar gel poured on a flat surface (slide or petri
thermoprecipitin test and theilgfouping of streptococci dish). The anti&en is added t<Lthe_ wells S!!.t _Q_n
by the Lanc;efie)d techniqu.e. the surface of the ~- It diffuses radially .fr.Q..m.Jhe
Slide test: When a drop ~ch of...!ruLll~n and the well ar.id forms ring-shaped bands of reci itation
antiserum are J_?laced on a slide and mixed b)' shak- (halos) concentrically around the well. The diameter
ing, Qoccules a~ar~ VDRL test for syphilis js._an of the halo_gives a~ est_imate_of the e:oncentr~tion
exam le of slide flocc~ion. of the ~ntigen. This method has been used for the
Tube test: estimation of the immunoglobulin classes in sera
ahn test for s hilis is an exam le
of a tube flocculatio_n test. A quantitative tube floc- and for screening sera for antibodies tQ_influenza
culation t~ is µsed for the standardisatio n of ~oxins viruses, among others (Fig. 12.2b).
and toxoids. Serial dilutions of the toxin/ toxoid are 4. Double diffusion in two dimensions (Ouchterlony
procedure): This method is most widely employed
added to the tubes· conta~ning a fixe~aniliy of the and helps to compare different antigens and antisera
ai:iJitoxirt. The amount of toxin or toxoid that floccu-
lates optimally with one unit of the antitoxin is defined directly. Agar gel is poured on a slide and wells are
'
as an Lf dose.
-- cut using a template. The antiserum is placed in the
central well, and different antigens in the surround-
Immunodiffusion (precipitation in gel): There are
ing wells. If two adjacent antigens are identical, the
several advantages in allowing precipitation to occur lines of precipitate formed by them will fuse . If they
in a gel rather than in a liquid medium. The reaction are unrelated, the lines will cross each other. Cross-
is visible as a distinct band of precipitation, which is reaction or partial identity is indicated by spur for-
stable aR_d can be stained for preservation, if neces-
mation. A special variety of double diffusion in two
sary. Asq ach antigen-antS° dy reactjon gives .ri§tlo
dimensions is the Elek test for toxigenicity in diph-
a line of precipitation, the number of different anti- theria bacilli. When diphtheria bacilli are streaked
gens in the reacting mixture can be readily observed. at right angles to a filter paper strip carrying the
Immunodiffusion also indicates identity, cross-reac-
antitoxin implanted on a plate of suitable medium,
tion and non-identity between different antigens.
arrowhead-sha ped lines of precipitation appear on
t...-Immunodiffusion is usually performed in a..sclt (1 %)
incubation, if the bacillus is toxigenic (Fig. 12.2c) .
~r or ~arose gel. b. Immunoelectrophoresis: The resolving power
Modifications of the immunodiffusion test: of immunodiffusion was greatly enhanced when
1 Single diffusion in one dimension (Oudin proce- Grabar and Williams devised the technique of immu-
dure): The antibody is incor_porat~ in agar gel ,in noelectrophoresis. This involves the electrophoretic
a test tube and the antigen solution is layered o~r separation of a composite antigen (such as serum)
J!. The antigen diffuses downward through th~ar into its constitu_e nt proteins, followed by immunod-
gel, forming a line of precipitation that appears to iffusion against its antiserum, resulting in separate
move downwards. This is due to the precipitation precipitin lines, indicating reaction between each
formed at the advancing fro!).t of the antigen, and individual protein with its antibody. This enables
is dissolved as the concentration of antigenJ!Lthe identification and approximate quantitation of the
site increases due to diffusion. The number of bands various proteins present in the serum. The tech-
indicates the number of different antigens present. nique is performed on agar or agarose gel on a slide,
2. Double diffusion in one dimension (Oakley- with an antigen well and an antibody trough cut on
Fulthorpe procedure): Here, the antibody is incor- it. The test serum is placed in the antigen well and
porated in gel, above which is placed a column of electrophoresed for about an hour. Antibody against
P,lain agar. The antigen is layered on top of this. human serum is then placed in the trough and dif-
The antigen and antibody move towards each. other fusion allowed to proceed for 18-24 hours. The
through the intervening column of plain agar and resulting precipitin lines can be photographed and
 l
Antigen-Antibody Reactions

Antigen

Precipitin
band

Antibody in
agar gel
ttt
Plain
agar @ @@ © Antigen in well

Ring of
precipitation
Antibody in
agar gel
@
0

©@@
@
o@
@©@
@

Single diffusion Double diffusion

(a) (b)

Agar gel on a slide 0 0

Reaction of partial identity Reaction of identity

Antigen in well 0 Antiserum in a well

Unrelated

0 0
(c)

Fig. 12.2 (a) Single and double diffusion in one dimension; (b) Single diffusion in two dimensions; (c) Double diffusion
in two dimensions

the slides dried, stained and preserved for record. • One-dimensional single electroimmunodiffusion
Over 30 different proteins can be identified by this (rocket electrophoresis): The main application of
method in human serum. This is useful for testing this technique is for quantitative estimation of anti-
for normal and abnormal proteins in serum and urine gens. The antiserum to the antigen to be quantitated
(Fig. 12.3). is incorporated in agarose and gelled on the glass
Electroimmunodiffusion: The development of slide. The antigen, in increasing concentrations, is
precipitin lines can be speeded up by electrically placed in wells punched in the set gel. The antigen
driving the antigen and antibody with diffusion using is then electrophoresed into the antibody containing
various methods in the clinical laboratory as given agarose (Fig. 12 .5). The pattern of immunoprecipi-
below. tation resembles a rocket (hence, the name).
• Counterimmunoelectrophoresis (CIE, counter- • Two-dimensional electrophoresis: In Laurell's two-
current immunoelectrophoresis): This involves dimensional electrophoresis, the antigen mixture is
simultaneous electrophoresis of the antigen and first electrophoretically separated in a direction per-
antibody in gel in opposite directions, resulting in pendicular to that of the final rocket stage. By this
precipitation at a point between them (Fig. 12.4). method, one can quantitate each of several antigens
This method produces visible precipitation lines in a mixture (Fig. 12.6).
within 30 minutes and is ten times more sensitive
than the standard double diffusion techniques. The
AGGLUTINATION REACTION
clinical applications comprise detecting various
antigens such as alpha fetoprotein in serum and When a particulate antigen is mixed with its antibody
specific antigens of cryptococcus and meningococ- in the presence of electrolytes at a suitable temperature
cus in cerebrospinal fluid. and pH, the particles are clumped or agglutinated.
 Part II IMMUNOLOGY

Antiserum
(antibody)
in agarose gel

Precipitin
areas
(rockets)

Q------1-- Antigen placed in

well

l~Aotigec
'-------------___J
components
separated by
electrophoresis
Antigen
wells
• •
Increasing concentration of antigen

Fig. 12.5 Rocket electrophoresis

Precipitin arc
.---+-----~+
Antibody in
gel
Antibody diffuses
towards separated
antigen components O o () C>
+ Ag2 Ag1
Second stage
First stage Ant_igen (Ag)
Precipitin bands form in well
where antibody and
antigen meet at Fig. 12.6 Laurell's two-dimensional electrophoresis
0 optimal proportions

Fig. 12.3 lmmunoelectrophoresis Slide agglutination:

Procedure:
Electric current
1. When a drop of the appropriate antiserum is added
+
to a smooth, uniform suspension of a particulate
~ ,...,.E=- ...,A,,,,b---+- ii~:cipitation
antigen in a drop of saline on a slide or tile, aggluti-

0 0 Slide
nation takes place.
2. A positive result is indicated by the clumping together
of the particles and the clearing of the drop.
Wells containing antigen and antibody 3. The reaction is facilitated by mixing the antigen and
the antiserum with a loop or by gently rocking the
Fig. 12.4 Counterimmunoelectrophoresis slide. Depending on the titre of the serum, aggluti-
nation may occur instantly or within seconds.
Agglutination is more sensitive than precipitation 4. Clumping occurring after a minute may be due to
for the detection of antibodies. The same principles drying of the fluid and should be disregarded.
govern agglutination and precipitation. Agglutination 5. It is essential to have on the same slide a control
occurs optimally when antigens and antibodies react consisting of the antigen suspension in saline, with-
in equivalent proportions. The zone phenomenon may out the antiserum, to ensure that the antigen is not
be seen when either an antibody or an antigen is in autoagglutinable. Agglutination is usually visible to
excess. 'Incomplete' or 'monovalent' antibodies do the unaided eye but may sometimes require confir-
not cause agglutination, though they combine with the mation under the microscope.
antigen. They may act as 'blocking' antibodies, inhib- Uses:
iting agglutination by the complete antibody added • It is a routine procedure for the identification of
subsequently. many bacterial isolates from clinical specimens.
 Antigen-Antibody Reactions

• It is also the method used for blood grouping and Principle: When sera containing incomplete anti-Rh
cross-matching. antibodies are mixed with Rh-positive red cells, the
Tube agglutination: This is a standard quantitative antibody globulin coats the surface of the erythrocytes,
method for the measurement of antibodies. When though they are not agglutinated. When such eryth-
a fixed volume of a particulate antigen suspension rocytes coated with the antibody globulin are washed
is added to an equal volume of serial dilutions of an free of all unattached protein and treated with a rabbit
antiserum in test tubes, the agglutination titre of the antiserum against human gamma globulin (anti-
serum can be estimated. globulin or Coombs serum), the cells are agglutinated
(Fig. 12. 7).
Uses: It is routinely used for the serological diagnosis
of typhoid, brucellosis and typhus fever. In the Widal Types:
test used in typhoid, two types of antigens are used. • Direct Coombs test: The sensitisation of the eryth-
The 'H' or flagellar antigen on combining with its rocytes with incomplete antibodies takes place in
antibody forms large, loose, fluffy clumps resembling vivo, as in hemolytic disease of the newborn due
wisps of cotton wool. The 'O' or somatic antigen forms to Rh incompatibility. When the red cells of eryth-
tight, compact deposits resembling chalk powder. roblastotic infants are washed free of unattached
Agglutinated bacilli spread out in a disc-like pattern at protein and then mixed with a drop of Coombs
the bottom of the tubes . serum, agglutination results. The direct Coombs
test is often negative in hemolytic disease due to
Complications: The tube agglutination test for brucel- ABO incompatibility.
losis may be complicated by the prozone phenom- • Indirect Coombs test: Sensitisation of red cells with
enon. Several dilutions of the serum should be tested the antibody globulin is performed in vitro.
to prevent false negative results due to the prozone of
Uses: Originally employed for the detection of anti-Rh
'blocking' antibodies. Incomplete or blocking anti-
antibodies, the Coombs test is useful for demonstrating
bodies may be detected by doing the test in hypertonic
any type of incomplete or non-agglutinating antibody,
(5%) saline or albumin saline, or more reliably by the
as, for example, in brucellosis.
antiglobulin (Coombs) test.
Passive agglutination test: The only difference
Heterophile agglutination test: between the requirements for the precipitation and
• The Weil-Felix reaction for serodiagnosis of agglutination tests is the physical nature of the antigen.
typhus fevers is a heterophile agglutination test By attaching soluble antigens to the surface of carrier
and is based on the sharing of a common antigen particles, it is possible to convert precipitation tests into
between typhus rickettsiae and some strains of agglutination tests, which are more convenient and
proteus bacilli. more sensitive for the detection of antibodies. Such
• The Streptococcus MG agglutination test for the tests are known as passive agglutination tests.
diagnosis of primary atypical pneumonia.
• Examples of agglutination tests using red cells as Anti-red cell antibody (incomplete)

antigens are the Paul-Bunnell test and the cold

agglutination test. The former is based on the pres-
ence of sheep cell agglutinins in the sera of infectious
mononucleosis patients, which are adsorbed by ox t
red cells but not by guinea pig kidney extract. The
cold agglutination test is positive in mycoplasmal
Antiglobulin --Y
(primary atypical) pneumonia. The patient's sera
agglutinate human O group erythrocytes at 4°C, the
agglutination being reversible at 3 7°C.
Antiglobulin (Coombs) test: This was devised by
Coombs, Mourant and Race (1945) for the detection
Agglutination
of anti-Rh antibodies that do not agglutinate Rh-posi-
tive erythrocytes in saline. Fig. 12.7 Coombs test
 Part II IMMUNOLOGY

The commonly used carrier particles are red cells, sive agglutination. This method is used to diagnose
latex particles or bentonite. Human or sheep eryth- bacterial antigens like Legionella, Streptococcus
rocytes adsorb a variety of antigens. Polysaccharide pyogenes and N.gonorrhoea in clinical samples.
antigens may be adsorbed by simple mixing with the • Co-agglutination test: It is based on agglutination
cells. For adsorption of protein antigens, tanned red of a specific antibody-sensitised protein A-bearing
cells are used. Staphylococcus aureus agglutinating with the solu-
• Hemagglutination: A special type of passive hemag- ble bacterial (e.g., Legionella) antigen in the clinical
glutination test is the Rose- Waaler test. In rheuma- specimen.
toid arthritis, an autoantibody (RA factor) appears
in the serum, which acts as an antibody to gamma COMPLEMENT FIXATION TEST (CFT)
globulin. The RA factor is able to agglutinate red
cells coated with globulins. The antigen used for the Complement takes part in many immunological
test is a suspension of sheep erythrocytes sensitised reactions and is absorbed during the combination of
with a subagglutinating dose of rabbit anti-sheep antigens with their antibodies. In the presence of the
erythrocyte antibody (amboceptor). appropriate antibodies, complement lyses erythrocytes,
• Latex agglutination test: Polystyrene latex, which kills and, in some cases, lyses bacteria, immobilises
can be manufactured as uniform spherical parti- motile organisms, promotes phagocytosis and immune
cles, 0.8-1.0 µm in diameter, can adsorb several adherence and contributes to tissue damage in certain
types of antigens. Latex agglutination tests (latex types of hypersensitivity.
fixation tests) are widely employed in the clinical Principle: The ability of antigen-antibody complexes
laboratory for the detection of anti-streptolysin to 'fix' complement is made use of in the CFT. This is a
0 (ASO), C-reactive protein (CRP), RA factor , very versatile and sensitive test, applicable with various
human chorionic gonadotrophin (HCG) and many types of antigens and antibodies and capable of detect-
other antigens. ing as little as 0.04 mg of antibody nitrogen and 0.1 mg
• Passive agglutination tests are very sensitive and yield of antigen. CFT is a complex procedure consisting of
high titres, but may give false positive results. When, two steps and five reagents-antigen, antibody, com-
instead of the antigen, the antibody is adsorbed plement, sheep erythrocytes and amboceptor (rabbit
to carrier particles in tests for the estimation of antibody to sheep red cells). Each of these reagents has
antigens, the technique is known as reversed pas- to be separately standardised (Fig. 12.8).

INegative CFT I
+

Complement Sheep erythrocytes coated with Lysis

amboceptor (Indicator system)

I Positive CFT I

Antigen Antibody
+
-
Complement Complement is fixed
+

Indicator system
in Ag-Ab reaction

No lysis
(as complement is not free to act
on indicator system)

Fig. 12.8 Complement fixation test

 Antigen-Antibody Reactions

Procedure: serum did not have the antibody (negative CFT).

1. The antigen may be soluble or particulate. The 2. Absence of erythrocyte lysis indicates that the com-
antiserum should be heated at 56°C (inactivated) for plement was used up in the first step and, therefore,
half an hour before the test to destroy any comple- the serum contained the antibody (positive CFT)
ment activity the serum may have and also to remove (Fig. 12.9).
some non-specific inhibitors of complement present Appropriate controls should be used, including the
in some sera (anti-complementary activity). following:
2. The source of the complement is guinea pig serum. 1 . Antigen and serum controls to ensure that they are
As complement activity is heat labile, the serum not anti-complementary
should be freshly drawn, or preserved either in the 2. Complement control to ensure that the desired
lyophilised or frozen state or with special preserva- amount of complement is add,ed
tives, as in Richardson's method. 3. Cell control to observe that sensitised erythrocytes
Standardisation: The guinea pig serum should be do not undergo lysis in the absence of complement
titrated for complement activity. One unit or minimum Indirect complement fixation test: Certain avian
hemolytic dose (MHD) of complement is defined as (for example, duck, turkey, parrot) and mammalian
the highest dilution of the guinea pig serum that lyses (for example, horse, cat) sera do not fix guinea pig
one unit volume of washed sheep erythrocytes in the complement. When such sera are to be tested, the
presence of excess hemolysin (amboceptor) within a indirect complement fixation test may be employed.
fixed time (usually 30 or 60 minutes) at a fixed tem- Here, the test is set up in duplicate and after the first
perature (3 7°C). The amboceptor should be titrated step, the standard antiserum known to fix the comple-
for hemolytic activity. One MHD of amboceptor is ment is added to one set. If the test serum contained
defined as the least amount (or highest dilution) of the antibody, the antigen would have been used up in the
inactivated amboceptor that lyses one unit volume of first step and, therefore, the standard antiserum added
washed sheep erythrocytes in the presence of excess subsequently would not be able to fix the complement.
complement within a fixed time (usually 30 or 60 min- Therefore, in the indirect test, hemolysis indicates a
utes) at a fixed temperature (3 7°C). The diluent used positive result.
for the titrations and for CFT is physiological saline Conglutinating complement absorption test: For
with added calcium and magnesium ions. systems which do not fix guinea pig complement, an
Wasserman reaction: The classical example of CFT alternative method is the conglutinating complement
is the Wassermann reaction, formerly the routine absorption test. This uses horse complement which is
method for the serodiagnosis of syphilis. non-hemolytic. The indicator system is sensitised sheep
Procedure: erythrocytes mixed with bovine serum. Bovine serum
1. The inactivated serum of the patient is incubated
at 3 7°C for one hour with the Wassermann antigen I. Antigen + Test serum }
and a fixed amount (two units) of guinea pig com- (Contains antibody) Complement fixed
+ Complement
plement. If the serum contains syphilitic antibody,
the complement will be utilised during antigen-anti-
body interaction. If the serum does not contain the + Hemolytic system Result - no hemolysis
antibody, no antigen-antibody reaction occurs and Positive CF test
the complement will therefore be left intact.
2. Testing for complement in the post-incubation II. Antigen + Test serum }
mixture will thus indicate whether the serum had (Contains no antibody) Complement not fixed
+ Complement
antibodies or not. This consists of adding sensitised
cells (sheep erythrocytes coated with 4 MHD hemo-
lysin) , and incubating at 3 7°C for 30 minutes. + Hemolytic system Result - hemolysis
Negative CF test
Interpretation of results:
1. Lysis of the erythrocytes indicates that the comple- Fig. 12.9 Complement fixation test-Wassermann
ment was not fixed in the first step and, therefore, the reaction
 Part II IMMUNOLOGY

contains a beta globulin component called conglutinin, the animals. With the diphtheria toxin, which, in small
which acts as antibody to the complement. Therefore, doses, causes a cutaneous reaction, neutralisation tests
conglutinin causes agglutination of sensitised sheep can be done on rabbit skin.
erythrocytes (conglutination) if they have combined The Schick test is based on the ability of circulat-
with the complement. If the horse complement ing antitoxin to neutralise the diphtheria toxin given
had been used up by the antigen-antibody interaction intradermally, and indicates immunity or susceptibility
in the first step, agglutination of sensitised cells will to the disease. Toxin neutralisation in vitro depends on
not occur. the inhibition of some demonstrable toxic effect.
Other complement-dependent serological tests: When Anti-streptolysin O (ASO) test demonstrates that
some bacteria (for example, Vibrio cholerae, Treponema antitoxin present in patient sera neutralises the hemo-
pallidum) react with ,the specific antibody in the pres- lytic activity of the streptococcal O hemolysin (0, an
ence of complement and particulate materials such as immunogenic, oxygen-labile hemolytic toxin). When
erythrocytes or platelets, the bacteria are aggregated the body is infected with streptococci, it produces an-
and adhere to the cells. This is known as immune tibodies against the various antigens that the strepto-
adherence. The immobilisation test is another com- cocci produce. ASO is one such antibody. Raised or
plement-dependent reaction. In the Treponema palli- rising levels can indicate past or present infection.
dum immobilisation test, a highly specific test formerly Nagler's reaction is a test for the identification of
considered the 'gold standard' for the serodiagnosis alpha toxin of Clostridium perfringens in clinical speci-
of syphilis, the test serum is mixed with a live motile mens. This toxin, on addition of antitoxin to cultures
suspension of T.pallidum in the presence of the com- grown on agar medium containing egg yolk (as a source
plement. On incubation, the specific antibody inhibits of lecithin), prevents visible opacity due to lecithinase
the motility of treponemes. Cytolytic or cytocidal tests action which is normally observed around colonies.
are also complement-dependent. When a suitable live
bacterium, such as the cholera vibrio, is mixed with OPSONISATION
its antibody in the presence of the complement, the
bacterium is killed and lysed. This forms the basis of The name 'opsonin' was originally given by Wright
the vibriocidal antibody test for the measurement of ( 1903) to a heat labile substance present in fresh
anti-cholera antibodies. normal sera, which facilitated phagocytosis. This fac-
tor was subsequently identified as a complement. A
heat-stable serum factor with similar activity was called
NEUTRALISATION TESTS 'bacteriotropin'. This appears to be a specific antibody.
Virus neutralisation tests : Neutralisation of viruses The term opsonin is now generally used to refer to
by their antibodies can be demonstrated in various both these factors.
systems. Neutralisation of bacteriophages can be Wright used the 'opsonic index' to study the progress
demonstrated by the plaque inhibition test. When of resistance during the course of diseases. The opsonic
bacteriophages are seeded in appropriate dilution on index was defined as the ratio of the phagocytic activ-
lawn cultures of susceptible bacteria, plaques of lysis ity of the patient's blood for a given bacterium, to the
are produced. Specific antiphage serum inhibits plaque phagocytic activity of blood from a normal individual.
formation. It was measured by incubating fresh citrated blood
Toxin neutralisation: Bacterial exotoxins are good with the bacterial suspension at 3 7°C for 15 minutes
antigens and induce the formation of neutralising and estimating the average number of phagocytosed
antibodies (antitoxins) which are important clinically, bacteria per polymorphonuclear leucocyte (phagocytic
in protection against and recovery from diseases such index) from stained blood films.
as diphtheria and tetanus. The toxicity of endotoxins is
not neutralised by antisera. Toxin neutralisation can be RADIOIMMUNOASSAY (RIA)
tested in vivo or in vitro.
Neutralisation tests in animals consist of injecting Besides fluorescent dyes, many other distinctive 'labels'
toxin-antitoxin mixtures and estimating the least can also be conjugated to antigens and antibodies. The
amount of antitoxin that prevents death or disease in most commonly used labels are radioisotopes and
 Antigen-Antibody Reactions

enzymes. A variety of tests have been devised for the curve. The concentration of antigen in the test sample is
measurement of antigens and antibodies using such computed from the B:T ratio of the test by interpolation
labelled reactants . The term binder-ligand assay has from the calibration curve. RIA and its modifications
been used for these reactions . The substance (antigen) have versatile applications in various areas of biology
whose concentration is to be determined is termed the and medicine, including the quantitation of hormones,
analyte or ligand. The binding protein (ordinarily, drugs, tumour markers, lgE and viral antigens
the antibody) which binds to the ligand is called the (Fig. 12.1 1).
binder. The first reaction of this type was radioim-
munoassay (RIA) described by Berson and Yallow in
ENZYME IMMUNOASSAY (EIA)
1959. RIA permits the measurement of analytes up to
picogram (10- 12 g) quantities. The importance of RIA Enzyme-labelled conjugates were first introduced
was acknowledged when the Nobel Prize was awarded in 1966 for localisation of antigens in tissues, as an
to Yallow for his discovery in 1977. alternative to fluorescent conjugates. In 1971, enzyme-
RIA is a competitive binding assay in which fixed labelled antigens and antibodies were developed as
amounts of antibody and radiolabelled antigen react in serological reagents for the assay of antibodies and
the presence of unlabelled antigen. The labelled and antigens. Their versatility, sensitivity, simplicity, econ-
unlabelled antigens compete for the limited binding omy and absence of radiation hazard have made EIA
sites on the antibody. This competition is determined the most widely used procedure in clinical serology.
by the level of the unlabelled (test) antigen present in The availability of test kits and facility for automation
the patient's serum samples. After the reaction, the has added to their popularity.
antigen is separated into 'free' and 'bound' fractions The term enzyme immunoassay includes all assays
and their radioactive counts measured. The concentra- based on the measurement of enzyme-labelled antigen,
tion of the test antigen can be calculated from the ratio hapten or antibody. EIAs are of two basic types :
of the bound and total antigen labels, using a standard • Homogeneous EIA does not require the bound and
dose-response curve (Fig. 12.10). free fractions to be separated; the test can thus be
For any reacting system, the standard dose-response completed in one step, with all reagents added simul-
or calibrating curve has to be prepared first. This is taneously. This type of EIA can be used only for the
done by running the reaction with fJXed amounts of assay of haptens such as drugs and not for microbial
antibody and labelled antigen, and varying known antigens and antibodies. An example of homoge-
amounts of unlabelled antigen. The ratios of bound neous EIA is enzyme-multiplied immunoassay
antigen to total antigen (B:T ratio) plotted against the technique (EMIT), which is a simple assay method
analyte concentrations give the standard calibration for small-molecule drugs such as opiates, cocaine,

1. Labelled antigen and

antibody at saturation
concentration _
Q*
*-,
barbiturates or amphetamine in serum.
• Heterogeneous EIA requires the separation of the
free and bound fractions either by centrifugation
or by absorption on solid surfaces and washing. It
* is therefore a multistep procedure, with reagents

2. Patients' C:
serum added Ql
Cl
"O .:: Ratio in 'unknown'
C: C:
~ Cll
Presence of antigen Absence of antigen o-
in patient's serum in patient's serum ..0 2
a.s
o.2
.:; c::
Cll Ql
3. Protein A added . ** 0::: .!2>
Precipitation of - * cCll
Ag.Ab complex

Radioactivity in the supernatant and precipitate measured Concentration of unlabelled antigen

Fig. 12.10 Rad ioim munoassay procedure Fig. 12 .11 Radioimmunoassay standard curve
 Part II IMMUNOLOGY

added sequentially. The main type of heterogeneous added and incubated at 37°C for one hour.
EIA is ELISA. 3. After washing, a suitable substrate (para-nitrophenyl
phosphate) is added and held at room temperature
Enzyme-linked immunosorbent assay (ELISA) till the positive controls show the development of a
ELISA is so named because the technique involves yellow colour. The phosphatase enzyme splits the
the use of an immunosorbent, an absorbing material substrate to yield a yellow compound.
specific for one of the components of the reaction: 4 . If the test sample contains rotavirus, it is fixed to
the antigen or antibody. This may be a particulate, for the antibody coating the wells. When the enzyme-
example, cellulose or agarose, or a solid phase such labelled antibody is added subsequently, it is in
as polystyrene, polyvinyl or polycarbonate tubes or turn fixed. The presence of residual enzyme activ-
microwells, or membranes or discs of polyacrylamide, ity, indicated by the development of yellow colour,
paper or plastic. ELISA is usually done using 96-well therefore denotes a positive test (Fig. 12.12).
microtitre plates suitable for automation. The principle 5. If the sample is negative, there is no significant
of the test can be illustrated by outlining its application colour change. An ELISA reader provides quan-
for the detection of rotavirus antigen in feces . titative colour recordings which are directly pro-
portional to the quantity of analyte present in the
Procedure test sample.
Sandwich ELISA:
1. The wells of a microtitre plate are coated with goat Types
antirotavirus antibody. After thorough washing, the • Indirect ELISA: The detection of antibody by
fecal samples to be tested are added and incubated ELISA can be illustrated by the anti-HIV antibody
overnight at 4 °C or for two hours at 3 7°C. Suitable test. Purified inactivated HIV antigen is adsorbed
positive and negative controls are also set up. onto microassay plate wells. Test serum diluted in
2. The wells are washed and guinea pig antirotavirus buffer is added to the well and incubated at 3 7°C
antiserum, labelled with alkaline phosphatase, is for 30 minutes . The well is then thoroughly washed.

Sandwich ELISA Indirect ELISA Competitive ELISA

LuJ LJ LJ
LuJ luJ ~~
LW lW ~~ffi ~ Conjugate is washed out
as antigen is not free to
bind the conjugate

lilJ lDJ ~ stcate Enzyme (tagged to conjugate)

is not there to act on substrate

Colour (Positive) Colour (Positive) No colour (Positive)

• Antigen '1 Conjugate [Antibody to antigen (• )l

~ Antibody
Colour product
,l Conjugate [Antibody to antibody (~ )]
Fig. 12.12 Enzyme-linked immunosorbent assay (ELISA)
 Antigen-Antibody Reactions

If the serum contains anti-HIV antibody, it will is added. After additional washing to remove the
form a stable complex with the HIV antigen on the unbound conjugate, a substrate yielding a coloured
plate. A goat anti-human immunoglobulin antibody product is added .
conjugated with horseradish peroxidase enzyme is Result: A positive result is indicated by a coloured
added and incubated for 30 minutes. After thor- spot developing at the site of the antigen against which
ough washing, the substrate 0-phenylene diamine the antibody is present in the serum. Human immu-
dihydrochloride is added and after 30 minutes, the noglobulin immobilised at a spot on the membrane acts
colour that develops is read using a microassay plate as a control for the test procedure, as shown by the
reader. Positive and negative controls should invari-
development of colour at the site.
ably be used with test sera.
• Competitive ELISA: Similar to RIA, both the Uses of ELISA: ELISA plays a major role in the
unknown antigen (sample) and the known antigen diagnosis of innumerable diseases. Some examples are
(standard) compete with each other for a fixed given below:
amount of antibody. Competitive ELISA yields • HIV detection
an inverse curve, where higher values of antigen • Infectious diseases like hepatitis, EBY, cytomega-
in the samples/ standards yield a lower amount lovirus IgM/IgG, dengue IgG, influenza, TORCH
of colour change. It is normally used for hapten panel, etc.
detection. • Rotavirus detection in fecal specimens and entero-
• Capture ELISA and immunometric tests are even toxin of E.coli in feces
more specific. Several variations of the ELISA • Syphilis IgG/lgM, H.pylori IgG and antigen
technique have been developed to provide simple detection
diagnostic tests, including the card and dipstick • Food toxins like chloramphenicol, streptomycin,
methods suitable for clinical laboratory and bedside penicillin, aflatoxins, etc.
applications. • Food adulterants including E.coli, Campylobacter
• Sandwich ELISA: It is used for antigen detection in and Salmonella antigens
patient sample. The antigen is sandwiched between • Mycobacterial antibody detection in tuberculosis
two layers of antibodies (i.e., capture and detection • Human allergen-specific IgE and IgA ELISA
antibodies).
• Cylinder or cassette ELISA: A simple modifica-
CHEMILUMINESCENCE IMMUNOASSAY (CUA)
tion of ELISA which has found wide application
for testing one or a few samples of sera at a time Chemiluminescence refers to a chemical reaction
is the cylinder or cassette ELISA. Here, each emitting energy in the form of light. Just as radioactive
specimen is tested in a separate disposable cas- conjugates are employed in RIA, fluorescent conju-
sette. The test is rapid (10-15 minutes). There is gates in IFA and enzymes in ELISA, chemiluminescent
no need for microplate washers or readers. The compounds (such as luminol or acridinium esters) are
result is read visually. In-built positive and nega- used in CUA as the label to provide the signal during
tive controls are usually provided for validation of the antigen-antibody reaction. The signal (light) can
the test procedure. be amplified, measured and the concentration of the
An example of cassette ELISA is the Dot Blot analyte calculated. The method has been fully auto-
Assay used for the detection of HIV type 1 and 2 mated and is being increasingly used in laboratories
antibodies. Specific type 1 and 2 antigens are immo- where the volume of work is large.
bilised at separate fixed sites on the nitrocellulose
membrane in the cassette.
IMMUNOELECTROBLOT/WESTERN
Procedure: Test serum is added on the membrane
BLOT TECHNIQUES
and allowed to filter into absorbent material placed
below it in the cassette base. Antibody, if present in Immunoelectroblot or western blot techniques com-
the serum, will bind to the appropriate antigen. After bine the sensitivity of enzyme immunoassay with much
washing to remove the unbound antibody, enzyme- greater specificity. The technique is a combination of
labelled anti-human immunoglobulin antibody three separate procedures:
 Part II IMMUNOLOGY

1. Separation of ligand-antigen components by poly- Immunoenzyme test: Some stable enzymes, such
acrylamide gel electrophoresis as peroxidase, can be conjugated with antibodies.
2. Blotting of the electrophoresed ligand fraction on Tissue sections carrying the corresponding antigens
nitrocellulose membrane strips are treated with peroxidase-labelled antisera. The per-
3. Enzyme immunoassay (or radioimmunoassay) to: oxidase bound to the antigen can be visualised under
• detect antibody in test sera against the various the electron microscope, by microhistochemical meth-
ligand fraction bands ods. Some other enzymes, such as glucose oxidase,
• probe with known antisera against specific anti- phosphatases and tyrosinase, may also be included in
gen bands immunoenzyme tests.
The western blot test, considered to be the defini-
tive/ confirmatory test for the serodiagnosis of HIV
IMMUNOFLUORESCENCE
infection, is an example of the immunoelectroblot
technique. Fluorescence is the property of absorbing light rays
of one particular wavelength and emitting rays with
a different wavelength. Fluorescent dyes show up
IMMUNOCHROMATOGRAPHIC TESTS brightly under ultraviolet light as they convert ultravio-
A one-step, qualitative immunochromatographic let into visible light. Coons and his colleagues (1942)
technique has found wide application in serodiagno- showed that fluorescent dyes can be conjugated to
sis due to its simplicity, economy and reliability. A antibodies and that such labelled antibodies can be
description of its use for HBsAg detection illustrates used to locate and identify antigens in tissues. This
the method. 'fluorescent antibody' or immunofluorescence tech-
The test system is a small cassette containing a mem- nique has several diagnostic and research applications
brane impregnated with anti-HBsAg antibody colloidal (Fig. t 2. t 3).
gold dye conjugate. The membrane is exposed at three
Direct immunofluorescence test: This can be used
windows on the cassette. The test serum is dropped
for the identification of bacteria, viruses or other anti-
into the first window. As the serum travels upstream
gens, using the specific antiserum labelled with a fluo-
by capillary action, a coloured band appears at the sec-
rescent dye. For example, direct immunofluorescence
ond window (test site) if the serum contains HBsAg,
is routinely used as a sensitive method of diagnosing
due to the formation of an HBsAg antibody conjugate
rabies, by detection of the rabies virus antigens in brain
complex. This is the positive reaction. Absence of a
smears. A disadvantage of this method is that separate
coloured band at the test site indicates a negative reac-
fluorescent conjugates have to be prepared against
tion. Simultaneously, a coloured band should appear in
each antigen to be tested.
every case at the third window, which forms an in-built
control, in the absence of which the test is invalid. The Indirect immunofluorescence test: This test over-
test is claimed to be nearly as sensitive and specific as comes the difficulty mentioned above by using an
EIA tests. antiglobulin fluorescent conjugate. An example is the
fluorescent treponemal antibody test for the diagnosis
IMMUNOELECTRON MICROSCOPIC TESTS of syphilis. Here, a drop of the test serum is placed on a
smear of Tpallidum on a slide and after incubation, the
Immunoelectron microscopy: When viral particles slide is washed well to remove all free serum, leaving
mixed with specific antisera are observed under the behind only antibody globulin, if present, coated on the
electron microscope, they are seen to be clumped. This surface of the treponemes. The smear is then treated
finds application in the study of some viruses such as with a fluorescent-labelled antiserum to human gamma
the hepatitis A virus and the viruses causing diarrhea. globulin. The fluorescent conjugate reacts with the anti-
lmmunoferritin test: Ferritin (an electron-dense body globulin bound to the treponemes . After washing
substance from horse spleen) can be conjugated away all the unbound fluorescent conjugate, when
with antibody, and such labelled antibody reacting the slide is examined under ultraviolet illumination,
with an antigen can be viewed under the electron if the test is positive, the treponemes will be seen as
microscope. bright objects against a dark background. If the serum
 Antigen-Antibody Reactions

!Direct immunofluorescence test I

Unknown antigen

I
db +

Fluorescein-labelled Fluorescence under UV

specific antibody light (positive test)

IIndirect immunofluorescence test I

Known antigen

~ +
Patient serum Antigen
+
Fluorescein- Fluorescence under
containing antibody + labelled UV light
Antibody antiglobulin (positive test)
Example:
Treponema pallidum + Serum of syphilis patient + Fluorescein- - Fluorescence
(containing anti-treponemal labelled (positive)
antibodies which is globulin antiglobulin
in nature)

Fig. 12.13 Direct and indirect immunofluorescence tests

does not have anti-treponemal antibody, there will be disadvantage of the technique is the frequent occur-
no globulin coating on the treponemes and therefore rence of non -specific fluorescence in tissues and other
they will not take on the fluorescent conjugates. A materials. The fluorescent dyes commonly used are
single anti-human globulin fluorescent conjugate can fluorescein isothiocynate and rhodamine, exhibiting
be employed for detecting human antibodies to any blue-green and orange-red fluorescence, respectively.
antigen. Flow cytometry: This is the fluorescence technique
Fluorescent dyes may also be conjugated with the used to identify and enumerate cells bearing a particu-
complement. Labelled complement is a versatile tool lar antigen(s) or the surface markers by suspending
and can be employed for the detection of antigen or them in a stream of fluid and passing them through an
antibody. Antigens also take fluorescent labelling but electronic detection apparatus. It allows simultaneous
not as well as antibodies do. For detection of antibod- multiparametric analysis of the physical and/ or chemi-
ies by immunofluorescence, the sandwich technique cal characteristics of up to thousands of particles per
can be used. The antibody is first allowed to react with second. Different populations of molecules, cells or
unlabelled antigen, which is then treated with fluo- particles can be differentiated by size and shape using
rescent-labelled antibody. A sandwich is thus formed , forward and right-angle light scatter. These cells,
the antigen being in the middle and the labelled and particles or molecules, can be labelled with different
unlabelled antibodies on either side. fluorescent labels or with dye-labelled monoclonal
Immunohistochemical technique: By combining the antibodies. In this way, we can measure the amounts
specificity of serology with the localising capacity of or isolated individual cells or populations of particular
histology, immunofluorescence helps in the visualisa- cells from a mixed population.
tion of antigen-antibody reactions in situ and is thus Here, cells are made to flow in a single cell stream
called an immunohistochemical technique. The main in a flow cell by hydrodynamic focussing. In this, the
 Part II IMMUNOLOGY

sample stream containing the cells are focussed by a sample

{stained
1 cells in suspension)
surrounding layer of sheath fluid. An Argon laser (at
488 nm) is focussed on the cells . Various sensors pick ,
up the laser light reflected by the cells.
The parameters measured by the flow cytometer are
'
forward scatter (FS), a measure of cellular size, side
scatter (SS) , a measure of the granularity of the cell Nozzle s, Hydrodynamic focussing
Cells pass through in

,
..,
and fluorescence sensors (FL1-FL8), which measure 'single file'

light emitted by various dyes bound to the cells . The

emitted light is directed into various sensors by a com- Fluorescence emitted
bination of filters (Fig. 12. 14) . from stained cells

,',, 1-=--
detected
Applications: Multiple parameters-the size,
granularity, DNA or RNA content, cellular antigens, Forward and side
receptor levels, etc.,-can be measured using flow
cytometry. It is widely used in research and diag-
(_~() • scattered light from
all cells detected
Laser light source

',
nostics, for example, to count blood cells including
differential leucocyte count (DLC), to isolate T cell
subsets (CD4 and CD8 counts in HIV patients),
for diagnosis, treatment and prognosis in cancer
(especially leukemias) , to study the cell cycle and
apoptosis, etc. Fig. 12.14 Schematic diagram of a flow cytometer

RECAP
• Antigen- antibody reactions enable us to detect, identify and quantify (measure the concentration) of
antigens and antibodies. Since antibodies are present in serum, their study is known as serology.
• Antibodies may be demonstrated by various types of reactions:
❖ In agglutination reactions, specific antibodies (agglutinins) formed in response to the occurrence of
particulate antigens in host tissues combine with a homologous antiserum . Agglutination reactions
are used for the diagnosis of infections due to salmonellae (Widal test), brucellae (brucella agglutina-
tion test), and rickettsiae (Weil-Felix test) and other toxic products elaborated by microorganisms.
❖ In precipitation reactions, the antigen is in a soluble form and, on combination with the antibody,
sediments or remains suspended in the form of floccules . Examples of diagnostic precipitation tests
include the Kahn and VDRL tests.
❖ In the complement fixation reaction, an antigen combines with its (specific) antibody in the presence
of complement, and the antigen-antibody complex adsorbs the complement. This reaction is not
visible and requires the use of an indicator (sheep erythrocytes and the specific anti-erythrocyte
antibody). Examples include the Wassermann test for syphilis and some tests for the detection of
viruses.
❖ In neutralisation tests, the effect of the antigen, toxin or virus is neutralised on mixing with its anti-
body. Examples include the Schick test for diphtheria toxin, antistreptolysin O (ASO) test for stre p-
tococcal O infection, Nagler's reaction for the identification of alpha toxin of Clostridium perfringens,
Treponema pallidum immobilisation (TPI) test for T. pallidum in clinical specimens.
❖ In certain viral diseases, the antibody produced prevents the agglutination of certain red blood cells by
the specific virus; this hemagglutination inhibition test can be used for the diagnosis of influenza.
4