Introduction to

serology - immunology

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What do we mean by immunology ?

The production of immunoglobulins (antibodies) as a

reaction of the human body when infected with an
agent.

Immunoglobulins: glycoprotein molecules that are

produced by plasmacells in response to an immunogen
and which function as antibodies.
Each immunoglobulin actually binds to a specific
antigenic determinant. Antigen binding by antibodies
is the primary function of antibodies and can result
in protection of the host.

All lab tests where Ag is used to detect Ab or Ab to

capture the Ag, present in human specimen (blood,
faeces, urine, CSF, etc. )
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What do we mean by serology ?

All immunology tests performed on human serum

Immuno-serology:
all tests designed for the detection of human
antibodies in human fluids
all tests using monoclonal or polyclonal antibodies
for capturing antigens

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Structure of an immunoglobulin

four chain structure as

basic unit
two identical light
chains and two identical
heavy chains
Variable and Constant
regions
Inter- & intra-disulfide
bindings
Hinge region
Oligosaccharides
(Carbohydrates)

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Fab & FC

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Different immunoglobulins: IgG

Monomeer
major Ig: 75% of total Igs
4 subclasses IgG1 to IgG4)

IgG is the major Ig in extra vascular spaces

placental transfer - IgG is the only class of Ig that

crosses the placenta.

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Different immunoglobulins: IgM

pentamer (can also exist as monomeer)
third most common Ig

IgM is the first Ig to be made by the fetus and the first Ig to be made

by virgin B cells when stimulated by antigen.

pentameric structure => IgM is a good complement fixing Antibody
IgM antibodies are very good in clumping microorganisms for
eventual elimination from the body.

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Different immunoglobulins: IgA

monomeer but in secretions a dimer
second most common Ig

IgA is major class Ig in secretions - tears, saliva,

colostrum, mucus. Therefore important in local (mucosal)
immunity.
Normally IgA does not fix complement, unless aggregated.

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Other immunoglobulins
IgD
monomer
low levels in serum
uncertain role
does not fix complement
IgE
monomer with extra domain in the constant region
least common Ig
involved in allergic reactions
IgE also plays a role in parasitic helminth diseases
does not fix complement

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Immunoreaction
primary humoral immune response to an antigen:
- following exposure, IgM is the first antibody to appear
- followed by a much higher titre of IgG

Reinfection:
- IgM remains the same or rises slightly

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- IgG shoots up rapidly and earlier than in primary infection

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Criteria for diagnosing primary Infection

Seroconversion: changing from a previously antibody (Ab)
negative status (seronegative) to a positive status (seropositive).
Negative IgM & IgG do not exclude exposure to antigen (incubation
time).

Presence of IgM:
- offers rapid diagnosis
- might be negative at convalescent phase
- possible interference with RF, polyclonal stimulation (other antigen)
- also with re-infection and sometimes unexplained persistence of
IgM years after primary infection

Presence of IgG:
- significant rise between acute and convalescent (recovery) phase
- titre of IgG depending on individual
- single high titre of IgG is a very unreliable means of serological
diagnosis

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Criteria for diagnosing

re-infection/re-activation
Importance of diagnosing:
- diseases like e.g. Rubella where re-infection cause no
fetal damage in pregnant women, whereas a primary
infection in the first trimester does
- often very difficult to differentiate re-infection/reactivation from primary infection
In general:
- sharp rise of IgG
- low or absent IgM

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Recovery primary infection or old infection ?

Screening population:
- IgG present at acute & recovery phase and will remain
detectable for mainy years, even for life time
Importance of differentiation:
- primary infection of TORCH (Toxo, Rubella, CMV)
during first trimester of pregnant women causes fetal
damage
- checking if vaccination has been succesfull
How to differentiate ?
- Low avidity IgG recovery primary infection & recent
infection
- High avidity IgG old infection

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Avidity & Affinity

The difference ?
Affinity:
- Ab affinity is the strength of the reaction between a
single antigenic determinant and a single combining site
on the antibody . in other words: the degree of match
between Ag & Ab

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Avidity:
- avidity is a measure of the overall strength of binding of
an antigen (Ag) with many antigenic determinants and
multivalent Abs with other words: the eagerness of
the Ab to match with an Ag
- avidity is more than the sum of the individual affinities
- the older the infection, the higher the avidity
- the higher the avidity, the stronger the Ag-Ab binding
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IgG Avidity
Avidity increases
with the maturation
of the immune
response

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Polyclonal and monoclonal Abs

Polyclonal Abs:
- each B-cell clone produces Ab specific for one particular Ag
epitope
- but serum Abs are produced as a response to complex Ag,
therefore contains a mixture of Abs raised against different Ag
epitopes
- each cell clone produce Abs specific for one of the various
epitopes of the Ag
this polyclonal response is advantageous for the immune
system because it helps localization, phagocytosis, and
complement mediated lysis of the Ag

Monoclonal Abs:
- derived from a single plasma cell and specific for one epitope
- useful for in vitro & in vivo diagnostic products

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Cross-reactivity
with diagnostic products
Artificial cross-reactivity:
- caused by non specific monoclonals/polyclonals used in test kit
cross-reactivity with related Ags, e.g. the herpesviridea
giving mainly false positive results
- appearance of the sample tested
e.g. haemolytic, icteric & lipemic samples
giving false positive or negative results

Non artificial cross-reactivity:

- a polyclonal reaction after infection with other Ag
- Auto-immune diseases
- Other non viral, bacteriological or parasitological agents
e.g. rheumatoid factor, anti-caseine, other proteins,

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Antigens

Viruses Bacteria - Parasites

Bacteria:procariotic organisms
single-celled organisms able to reproduce by duplicating
themselves
typical bacterium: rigid cell wall, thin
responsive to antibiotics
rubbery cell membrane surrounding the
cytoplasma, various shapes (coccus, rod,
vibrion etc)
contains all genetic information to copy
itself DNA in the chromosome
plasmids: extra circular bits of DNA
ribosomes: necessary for copying DNA

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some have flagella

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Antigens

Viruses Bacteria - Parasites

The biggest viruses are only as large as the
tiniest bacteria.
Virus:
lives within a cell (intracellular) to survive and needs host cell for
multiplication
not responsive to antibiotics
protection through vaccination (not for all viruses)
envelope: outermost spiky layer
protein coat
core of genetic material: DNA or RNA

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Antigens

Viruses Bacteria - Parasites

Parasite: eukaryotic organisms
lives in or on the living tissue of a host organism
lives at the expense of the host
parasitism = biological interaction between host and parasite

Some parasites:

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Cryptosporidium parvum
oocytes

Toxoplasma gondii
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Giardia intestinalis

Infectious diseases
Chorus Elisa line
Kits currently available for the detection of Abs against:
TORCH panel:
Toxoplasma gondii
Rubella virus
Cytomegalovirus (CMV)
Epstein-Barr virus (EBV)
Measles
Mumps
Herpes Simplex virus type 1 & 2
Varicella zoster virus (VZV)
Helicobacter pylori
Treponema pallidum (Syphilis)

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Viruses:
different families (1)
HERPESVIRIDEA (DNA viruses)
8 human herpes types
HV-1

Herpes simplex 1

Fever blisters, encephalitis

HV-2

Herpes simplex 2

Genital, meningitis/enecephalitis
(newborns)

HV-3

Varicella zoster

Chicken pox, shingles

Beta group

HV-4

Cytomegalovirus

Infectious mononucleosis,
neonatal fatality

Gamma group

HV-5

Epstein-Barr virus

Infectious mononucleosis, cancer

(Lymphoma)

Other

HV-6

HBLV
(human B-lymphotropic virus)

B cells

Alpha group

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HV-7

T cells (CD4+)

HV-8

Sarcoma, lymphoma

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Viruses:
Viruses:
differentfamilies
families(2)
(2)
different
PARAMYXOVIRIDEA (RNA viruses)
Subfamily

Genus

Human Species

Paramyxoviridea

Paramyxovirus

Human parainfluenza viruses types 1 & 3

Rubulavirus

Human parainfluenza viruses types 2, 4a and 4b,

Morbillivirus

Measles virus

Pneumovirus

Human respiratory syncytial virus

Pneumovirnea

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Mumps virus

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Viruses:
Viruses:
differentfamilies
families(3)
(3)
different
TOGAVIRIDEA (RNA viruses)
Genus

Species

Alphavirus

Sindbis virus, Eastern equine encephalitis virus, Western

equine encephalitis virus, Ross River virus, Onyongnyong
virus

Rubivirus

Rubella virus*

*: rubella (German measles)

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Bacteria
Helicobacter pylori

slow-growing Gram spiral shaped

bacterium. It lives in the stomach and
duodenum.
H. pylori is very common. About 2/3 of
the world's adult population are infected
with it. Almost everyone who has a
duodenal ulcer has H. pylori, but not
everyone that has H. pylori develops an

Treponema pallidum

It is a spirochete, a helical to sinusoidal

bacterium
It is the causative agent of Syphilis
Despite the availability of effective
therapy (penicillin), syphilis remains a
commonly sexually transmitted disease
worldwide

ulcer.

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Parasites
Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite

Toxoplasma gondii has very low host specificity, will probably infect
almost any mammal.
2 life cycles: the intestinal and extraintestinal phases
intestinal phase occurs in cats only produces oocysts
extratestinal phase in all infected animals (including cats)
produces tachyzoites and eventually bradyzoites & zoitocysts

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Infectious Diseases

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Childhood diseases
The most common childhood diseases are:

Viral infections
Measles
Mumps
Rubella*
Chicken pox
Respiratory syncytial virus (RSV)
Rotavirus
The fifth disease (Parvovirus)
Bacterial infections

Wooping Cough (pertussis)

Meningitis
Scarlet fever

* Risk for pregnant women

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Other infectious diseases

Toxoplasmose (Toxoplasma gondii)*

Cytomegalovirus (CMV)*
Infectious Mononucleosis with Epstein-Barr virus (EBV)
Herpes Simplex virus, type 1 (oral-facial lesions)
Herpes Simplex virus, type 2 (genital lesions)
Influenza A & B
Hepatitis A & B
Helicobacter Pylori
Syphilis

* Risk for pregnant women

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For Internal Use Only

Vaccinations
1.
2.
3.

4.
5.

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Aim is to prevent epidemics which caused many deaths in the

past.
Vaccines help the body produce antibodies protection.
Most common vaccines:

MMR: measles, mumps & rubella

IPV: inactivated poliovirus

DTaP: diphetheria, tetanus & pertussis

Varicella vaccine

HBV & HAV: hepatitis A & B

Influenza

PCV: pneumococcal conjugate vaccine

.
Pipeline: hepatitis C, RSV, HIV, CMV, EBV, ..
Far future: HSV 1&2, adenovirus, toxoplasmose
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Herpes Simplex 1 & 2 (1)

both belonging to the Herpesviridea

slight antigenic difference between HSV-1 and HSV-2

HSV-1
HSV-1

HSV-2
HSV-2

oral-facial
oral-faciallesions
lesions

genital
genitallesions
lesions(ulcers
(ulcersororsores)
sores)

most
mostcommon
commonHSV
HSV

possible
possiblecomplications:
complications:

usually
usuallyacquired
acquiredininchildhood
childhood
fever
feverblisters
blistersinside
insidemouth
mouth
transmitted
transmittedby
byinfected
infectedsaliva
saliva
90%
90%of
ofadults
adultshave
haveAbs
Abs

meningoencephalitis
meningoencephalitisororinfection
infectionofof
the
theeye
eye(acquired
(acquiredatatburth,
burth,passing
passing
through
infected
mothers
birth
through infected mothers birthcanal)
canal)

can
canalso
alsobe
beasymptomatic
asymptomatic
sexually
sexuallytransmitted
transmitted
25%
25%ofof25-45
25-45years
yearsold
oldofofUS
US
population
population

Cross-infection of type 1& 2 viruses may occur from oral-genital contact.

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Herpes Simplex 1 & 2 (2)

Human response to infection:

primary infection often subclinal and rarely diagnosed

primary infection ? first IgM followed by IgG
virus stays in your system once youve been infected
reactivation after latency period, may or may not rise lesions

Why testing ?
Infection contracted during birth is of particular interest
important cause of morbidity and mortality
!! Important to determine the immunitary state of women during
pregnancy in order to detect serum conversion !!

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Herpes Simplex 1 & 2 (3)

Recommended testing:
H S V 1 S c re e n
and

H S V 2 S c re e n
(Ig M + Ig G )

N E G A T IV E

P O S IT IV E
Ig G o r Ig M ?

S e r o n e g a tiv e

H e r p e s S im p le x 1 + 2 Ig M

Chorus line:
- HSV-1 Screen (IgM & IgG)
- HSV-2 Screen (IgM & IgG)
- Herpes Simplex 1+2 IgM

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Ig M

Ig M

P O S IT IV E

N E G A T IV E

A c u t e /R e c e n t

O ld in fe c tio n

in fe c tio n

(Ig G p o s itiv e )

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Helicobacter pylori (1)

a bacteria responsible for most ulcers and many cases of

chronic gastritis

weakens protective coating of stomach & duodendum

digestive juices will iritate sensitive lining of these
organs
H. pylori infection is linked to stomach cancer
having H. pylori in gastrointestinal tract is not always
leading to ulcer or gastritis other factors increase risk

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Helicobacter pylori (2)

Human response to infection:

most patients exhibit a strong immune response

locally mostly IgA antibodies

circulating antibodies are primarily IgG

IgGs remain constantly high until infection is eliminated

(antibiotics)
Treatment of H. pylori:

concentration of Abs does not correlate with disease severity but

succesful treatment can be monitored by decrease of Abs

Efficacy of treatment decrease in IgG level over time

No treatment:
Spontaneous clearing of H. pylori by IgA & IgG is rare
elevated IgA & IgG levels, indicates current infection

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Helicobacter pylori (3)

Recommended testing: IgA and IgG
Case 1

Case 2

H . P y lo ri Ig G

H . P y lo ri Ig G

N E G A T IV E

N E G A T IV E

H . P y lo r i I g A

H . P y lo r i I g A

N E G A T IV E

P O S IT IV E

S e r o n e g a tiv e

Case 4

H . P y lo ri Ig G

H . P y lo ri Ig G

P O S IT IV E

P O S IT IV E

H . P y lo r i Ig A

H . P y lo r i I g A

P O S IT IV E

S ta r t p r im a r in fe c tio n

Chorus line:
- H. pylori IgA
- H. pylori IgG

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Case 3

A c u t e in f e c t i o n
or
p a s t in fe c tio n ?
D r u g m o n ito r in g
F o l l o w - u p s a m p le
I n c r e a s e Ig G = a c u te i n f.
D e c re a s e Ig G = im p a c t d r u g o r r e c o v e ry
S t a b le = p a s t in f e c tio n

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N E G A T IV E

T r e a t e d in f e c t i o n ?
F o l l o w - u p s a m p le