Serological reactions

Serological reactions = antigen (Ag) + antibody (Ab) reactions

The Ag + Ab reaction is highly specific. 

An unknown antigen can be determined with the help of antibodies with known
specificity.
An unknown antibody can be determined with the help of antigens with known
specificity.
Antibody functions
The serological reactions are able to detect specific
antibodies, as well as antigens both qualitatively and
quantitatively.

Detection of serological reactions

I. Ag + Ab specific reaction is directly visible

II. Ag + Ab specific reaction is shown indirectly by means of

an indicator system
I. Reactions utilising sedimentation of Ag+Ab complex

Ag + Ab reaction ends up in the visible sedimentation of Ag-Ab complex.

(1) Agglutination (Ag = cellular or Ag is bound to a particle of cellular size)

~ utilize a particulate, insoluble antigen => visible clumping

- sensitivity: agglutination=0,1; passive agglutination=0,01 ml Ab/mL

• qualitative: slide agglutinations
 isolated microbe (Ag) + test serum (Ab) on a glass slide
(e.g.: serotyping of E. coli , Salmonella isolates )

positive negative
Serological identification: slide agglutination with specific
antibodies against the 'O'-, 'K'-, and 'H'-antigens.
'O'-ag = outermost part of the bacterial LPS, consisting of
polysaccharide, resistant to heat and alcohol, and usually
detected by bacterial agglutination. anti-O antibodies are mainly
IgM in nature.
'K'-ag= external to 'O'-ag, polysaccharide in E.coli, may be
associated with virulence (K1 - meningitis in infancy, some other
types - attachment to epithelial cells prior urinary tract invasion)
'H'-ag = located on the flagella and can be denatured or
removed by heat and alcohol. Anti-H antibodies are mainly IgG
in nature.
 Indirect or passive agglutination assays utilize antigen coupled to particles or
beads composed of an inert material (e.g. latex, colloidal gold,..)

=> serotyping of bacteria

e.g. serotyping of streptococci
/Lancefield/ (A=Streptococcus pyogenes)

 mecA – PBP2a latex-agglutination

MRSA!

=> cerebrospinal /other body fluid : N.

meningitidis/E.coli, H. influenzae B, S.
agalactiae, S. pneumoniae
Direct agglutination of RBCs
/ABO blood group typing/
- Monospot test
Viral serology: hemagglutination and hemagglutination inhibition assays

e.g. Influenza virus, rubeola,

morbilli
 B) Quantitative: tube agglutinations
 serum (Ab) + standard amount of Ag (e.g.: Widal-type
reactions)
Titre : the highest dilution (the lowest content) of Ab which still can
cause agglutination
(~ is the inverse of the greatest dilution, or lowest cc.)
Gruber-Widal:
Ag: Salmonella typhi, paratyphi A,B,C; O, H antigén
Ab:patient serum
Diagnostic cut-off titer: 1:200

Weil-Felix: Rickettsia
Ag: Proteus OX19, OX2, OXK
Ab:patient serum
Diagnostic cut-off titer: 1:800

Wright: Brucella / Francisella

Ag: Brucella/Francisella
Ab:patient serum
Cut-off: 1:80
Paul-Bunnel: EBV
Ag: sheep RBC
Ab: patient serum:heterophile Ab-s
Titer! With
Proteus
OX19
Ma ELISA (és PCR, WB) alapú antigen!
diagnosztika
2. Precipitation (Ag =soluble, dissolved)
a) Precipitation in a liquid phase
- qualitative(“ring”)

- quantitative: serial dilution of Ag + standard amount of Ab (e.g.: flocculation

for toxin + antitoxin reaction; lues serology)
The soluble antigen and the specific antibody precipitate only in the zone of equivalence,
where the amount of the antigen and the antibody is equal. There is no precipitation if there
are antigen or antibody in excess.
(b) Precipitation in a "semi-solid" phase (agar gel):
The precipitation reactions can be visualised by allowing the Ag & the Ab to
diffuse towards each other through agar gels
Immune diffusion
- Elek-test  diphtheria toxin (Ag) + antitoxin (Ab) reaction

=> strip of filter paper

saturated with antitoxin is
placed on an agar plate
(20%horse serum
contant)

=> the antitoxin diffusing

from paper will precipitate
the toxin diffusing from
the toxigenic cultures

-sensitivity: 20-60 mg Ab /mL

 Antigen in the gel

Diameter2

Antigen concentration

Radial immunodiffusion (semiquantitative method, the

increasing concentration of antibody leads to increasing
precipitation diameter

radial immunodiffusion (e.g. to measure IgG, IgM, complement components)

- Immunoelectrophoresis (precipitation in agar with an electric field; e.g. this
permits the serum proteins to be characterized in terms of their presence,
abscence, or unusual pattern)

- Immuno-osmophoresis (counter immune electrophoresis)

- to detect of bacterial and fungal polysaccharide antigens in
cerebrospinal fluid

Immunelectropho-
resis (the antigens
separated in the
electric field react
with the specific
antibody providing
precipitation)
ICT – immunochromatography test
II. Indirect methods

Ag + Ab reaction is not visible but indirectly

detected by means of an indicator system.
1) Methods utilising labelled antibodies or antigens

(a) Immunofluorescent reactions (IF)

 labelling: fluorescein isothiocyanate (FITC, rhodamine)

Fluorescent dyes can be covalently attached to antibody molecules and made
visible by ultraviolet light in the fluorescence microscope.
Such labelled antibody can be used to identify antigens
(eg, on the surfaces of bacteria such
as streptococci or treponemes)
or in cells in histological section
or other specimens.

 sensitivity 100 mg Ab/ml

 epitope

Unknown
antigen
(bacteria)

Antibody of
known antigen

Fluorescent
dye

Direct immunfluorescentia /DIF/(antigen detection with

fluorescent specific antibody)
=> known labelled antibody interacts directly with unknown
antigen
 Solid phase
Indirect immunfluorescencia for antibody detection/IDIF/ (e.g. Chlamydia, EBV
detection)
=> two – stage process is used / it’s more sensitive, because more labelled antibody
adheres per antigenic site/
o the unknown serum antibody matches the antigen, it will remain fixed to it on the
slide
o it can be detected by adding a fluorescent-labelled antiglobulin antibody or other
antibody-specific reagent such as staphylococcus protein A

Furthermore, the labelled antiglobulin becomes a “universal reagent” (independent of

the nature of the antigen used) reactive with all IgG of that species)
 Streptococcus „A” Fluorescent Fluorescent
in the throat sample antibody for streptococci
of the patient Streptococcus A

Direct fluorescent antibody

T. pallidum specific antibody in Antibody- Fluorescent

(laboratory strain) the patient’s blood T.pallidum binding antihuman Fluorescent
serumglobulin spirocheatas
Indirect fluorescent antibody
(b) Radio immune assay (RIA)
 labelling: radioactive isotopes

It is based on the competition for specific

antibody between the labelled (known) and
the unlabeled (unknown) concentration of
material.

The complexes that form between antigen

and antibody can then be separated and
the amount of radioactivity measured.

The concentration of the unknown

(unlabeled) antigen or hapten is
determined by comparing the results with
those obtained using several
concentrations of a predetermined
standard antigen.

RIA is a highly sensitive method applied to

the assay of hormones or drugs in serum.
(e.g. HBcAg, HBsAg)
c) Enzyme linked immunosorbent
assay (ELISA)
 labelling: enzymes (e.g.: peroxidase,
alkaline phosphatase etc.)

EIA which has many variations, depends

on conjugation of an enzyme to either an
antigen or an antibody.
The enzyme is detected by assaying for
enzyme activity with its substrate.

To measure antibody, known antigens are

fixed to a solid phase (e.g., plastic micro
dilution plate), incubated with test serum
dilution, washed, and incubated with an
anti-immunoglobulin labelled with an
enzyme (e.g., horseradish peroxidase).

Enzyme activity, measured by adding the

specific substrate and estimating the
colour reaction, is a direct function of the
amount of antibody bound.
(sensitivity: 0,001 mL Ab/ml)
 To detect antibody; indirect ELISA
-Coat the microtiter plate with purified antigen by
letting an antigen solution sit in the wells for 30-60
minutes.
- Wash away unbound antigen with buffer and cover
any sites that might non-specifically bind antibody with
unrelated protein (such as solution of powdered milk),
again washing away unbound protein.
-Add serum sample to be tested for specific
antibody to plate and allow specific antibody to
bind to the antigen.
- Wash off unbound antibody.
- Add anti-Ig that will bind to Fc region of specific
antibody (for example, anti-human gamma chain that
will bind human IgG). The Fc region of the anti-Ig is
covalently linked with enzyme.
- Wash off unbound antibody-enzyme complex.
- Add chromogenic substrate: colourless substrate
that the enzyme will convert to a coluored product.
Incubate until colour develops; measure colour in a
spectrophotometer.

-The more colour that is detected, the more

specific antibody is present in the unknown
sample.
Negative controls include omit the antigen omit the
test antiserum or substitute with an antibody that will
not bind the antigen.
Positive control substitutes known positive serum for
unknown serum.
ELISA: the yellow colour is the positive reaction
•Coat the microtiter plate with To detect antigen (sandwich ELISA):
purified antibody to the antigen.
Wash away unbound antibody and
cover any sites that might non-
specifically bind with unrelated
protein.

•Add sample to be tested for

antigen to plate and allow antigen
to bind antibody. Wash off unbound
antigen.

•Add enzyme-labeled specific

antibody to a different epitope of the
antigen to make a "sandwich"; wash
away unbound antibody.

•Add chromogenic substrate for

enzyme that will be converted to a
coloured product.

•Negative control omits unknown

antigen; positive control uses
known antigen.
Competitive ELISA :
the weaker the signal,
the higher the original antigen
concentration was.

 
Automatization!
 Additional comments
 The ELISA is probably the most commonly used immunological
assay because of its versatility,
 sensitivity (ability to detect small amounts of antigen or antibody),
 specificity (ability to discriminate between closely related but
antigenically different molecules),
 and ease of automation.

 Although some of the substrates are carcinogenic, they are generally

considered safer than radioisotopes used in RIA (radioimmunoassay). By
binding the initial layer to plastic, washing away unbound reagents becomes
rapid and requires no expensive equipment - the plate can be flodded with
buffer and shaken over a beaker to remove the wash fluid and unbound antigen
or antibody. The process can be easily automated for performance of large
numbers of tests.

 False positives can result from impure reagents: for example,

tissue culture-grown HIV used in the HIV ELISA can react with
antibodies to tissue-culture grown influenza present in someone
who has just had an influenza vaccination.
3) Western blot

- to determine whether a positive result in a screening

immunologic test is a true-positive or a false-positive result

- e.g. verification ELISA positive result for HIV infection or for

Lyme disease
Western Blot Procedure
 ELISA high fals positivity =>
WB verification

IgM: p24, 39, 41 – 2

IgG: p18, 21, 28, 30, 39, 41, 45, 58, 66, 93 - 5
2. Methods utilising the complement system
 Complement fixation (CF)

The Ag + Ab complex activates and consumes the proteins of the complement

system and the lysis of RBC + haemolysin complex (indicator system) is not
seen (e.g. Wassermann-reaction).

- sensitivity: 1 mL Ab/mL
 Negative: red blood cells are lysed

Positive: red blood cells are

in the bottom of the well
Neutralization
- to determine the ability of antibodies to block the effect of
toxins or the infectivity of viruses (in cell culture or in host
animals)

Toxin molecules cell

cell damaged by toxin

Toxin molecules
Antitoxin: antibody neutralized toxin and intact cell
for toxin
Why we use serological reactions?

1. Detection and identification of infectious agent by

means of serological methods

Aim:

(1) to detect Ag of infectious agents

* extracellularly in body fluids or secretions
* intracellularly

(2) to identify and type infectious agent from their cultures

Why we use serological reactions?

2. Testing the immune response of the host

Aim: by using a known specific antigen of the suspected infectious agent,

the presence of specific antibodies is tested

Commonly used methods: agglutination, precipitation, CF, IDIF, ELISA

Note well:
Titre = reciprocal of the highest rate of dilution at which the given reaction
still gives a visible/detectable result
/Immunoassays report results as absorbance values, optical densities, or
international units/

The significance of use of paired sera !  Changes in titres !

Ab-s is produced in response to a primary infection
- specific IgM, found during the first 2 to 3 weeks (7-10 days)
- specific IgG production begins approximately 10 days after exposure

Typically, as the IgM decreases, the IgG level will increase => by the end of the
2. or 3. month, only IgG class antibody is detectable

reinfection, or recurrence later in

life 
secondary=anamnestic=booster
response
- lag phase is shorter (1-3 d)
-IgG-class antibodies are the
primary isotype
- ~ 1 month plateau
- Ab titers may remain high
(for life)
-IgM +/-; IgA+/-
CAP - Chlamydia pneumoniae?
Use of serologic analysis as a measure of recent versus remote
infection
- A single IgM or IgG serum result should not be used to determine the stage of
infection
- Antibody levels are highly host specific (age, genetic disposition, comorbidities…)
- Some antigens are more effecting than others in stimulating the immune system

 IgM class antibodies = acute infection/recent infection

But! May persist beyond the acute stage (for months, years)
/e.g. M. pneumoniae, HAV, T. gondii, Borrelia burgdorferi…/
Negative IgM does not immediately exclude a recent exposure
Time! Reinfection!

 Significant change in antibody titers

the finding of at least a fourfold increase in the antibody titers between serum obtained during
the acute phase of disease and that obtained at least 2 to 3 weeks later, during the
convalescent phase

Seroconversion (conversion from negative to positive)

/=> molecular techniques are the preferred diagnostic methods

for early clinical management/
(IgG) Avidity testing

- Avidity is subsequently estimated

by comparing the levels of IgG in
(urea) treated versus untreated
samples
- One sample!

! pregnant women
T. gondii, rubella, CMV
Typing techniques
 Pulsed-field gel electrophoresis (PFGE)
 DNA will be extracted from
bacteria, cleaved with enzymes
and run in agarose gel.
 The position of the positive and
negative poles will be changed
several times during running
that will allow better band
separation.
 If strains differ in at least two
bands they are considered
distinct. PFGE assay of Pseudomonas aeruginosa
isolates /1-7: nosocomial outbreak/
Advantage: a very sensitive technique; allows the separation of closely related
isolates. Excellent for the investigation of individual outbreaks.
Drawbacks:
- assays done in different laboratories can not always be compared
- too sensitive for the comparison of strains isolated in geographically distant locations
or between considerable time intervals.
Macrorestriction pattern of SHV-type β-
lactamase producing klebsiella isolates
 Typing techniques based on nucleotide sequencing I.
Multi locus sequence typing (MLST)
 Six or seven genes of the bacterium are selected sequenced
and the sequences compared
 All bacteria belonging to the species must carry the selected
genes. (Housekeeping genes are selected.)
 The evolutionary changes in the selected genes should not be
either too fast or too slow.
Advantages:
- gives unequivocal results; results obtained in various laboratories can be
compared
- appropriate for the comparison of strains isolated in distant geographical
locations or between considerable time intervals
- it can usually be performed also directly from sample
Drawback: not sufficiently sensitive for the investigation of individual outbreaks
MLS typing of selected klebsiellae strains
isolated in Hungary
Result of Alleles Seq.
PFGE typing type
rpoB gapA mdh pgi phoE infB tonB

HECIa (n=4) 1 1 1 1 1 1 1 15
HECIb (n=1) 1 1 1 1 1 1 1 15
EC II (n=3) 4 3 6 1 7 4 38 147
EC III (n=2) 1 3 1 1 1 3 4 11
 Typing techniques based on nucleotide sequencing II.
„Spa” typing
 Spa: Staphylococcus protein A
 Principle: sequencing and comparison of a single gene; the protein A
gene of S. aureus (protein A is a major virulence factor on the surface of
S. aureus)
 The sensitivity of spa typing is lower than that of MLST, however, it
allows an appropriate separation of S. aureus strains and is much
cheaper than MLST
 Sequencing of total bacterial genome

 The „final” typing technique

 Allows „total” comparison of bacterial genomes
 Can be done within four hours with up-to-date pyro
sequencing systems
 Apart from typing the strains provides all relevant
information on them (factors of pathogenicity,
presence of antibiotic resistance genes/markers)