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Introduction toserology - immunology
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What do we mean by immunology ?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 ?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 immunoglobulinStructure 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 & FCFab & FC
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Different immunoglobulins: IgGDifferent immunoglobulins: IgG
IgG is the major Ig in extra vascular spaces placental transfer - IgG is the only class of Ig that crosses the placenta.
Monomeer major Ig: 75% of total Ig’s 4 subclasses IgG1 to IgG4)
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Different immunoglobulins: IgMDifferent immunoglobulins: IgM
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.
pentamer (can also exist as monomeer)third most common Ig
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Different immunoglobulins: IgADifferent immunoglobulins: IgA
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.
monomeer but in secretions a dimersecond most common Ig
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Other immunoglobulinsOther immunoglobulins
IgEIgEmonomer with extra domain in the constant regionleast common Iginvolved in allergic reactionsIgE also plays a role in parasitic helminth diseases does not fix complement
IgDIgDmonomerlow levels in serumuncertain roledoes not fix complement
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ImmunoreactionImmunoreactionprimary 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
- IgG shoots up rapidly and earlier than in primary infection
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Criteria for diagnosing primary InfectionCriteria for diagnosing primary Infection
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
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 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 Criteria for diagnosing re-infection/re-activationre-infection/re-activation
In general:
- sharp rise of IgG
- low or absent IgM
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/re-activation from primary infection
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Recovery primary infection or old infection ?Recovery primary infection or old infection ?
How to differentiate ? - Low avidity IgG recovery primary infection & recent infection - High avidity IgG old infection
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
Screening population: - IgG present at acute & recovery phase and will remain detectable for mainy years, even for life time
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Avidity & Affinity Avidity & Affinity The difference ?The difference ?
Avidity: - avidity is a measure of the overall strength of binding of an antigen (Ag) with many antigenic determinants and multivalent Ab’s … 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
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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IgG Avidity
Avidity increases with the maturation of the immune response
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Polyclonal and monoclonal Ab’sPolyclonal and monoclonal Ab’s
Monoclonal Ab’s: - derived from a single plasma cell and specific for one epitope- useful for in vitro & in vivo diagnostic products
Polyclonal Ab’s: - each B-cell clone produces Ab specific for one particular Ag epitope- but serum Ab’s are produced as a response to complex Ag, therefore contains a mixture of Ab’s raised against different Ag epitopes- each cell clone produce Ab’s 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
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Cross-reactivityCross-reactivitywith diagnostic productswith diagnostic products
« Non artificial » cross-reactivity:
- a polyclonal reaction after infection with other Ag
- Auto-immune diseases
- Other non viral, bacteriological or parasitological agentse.g. rheumatoid factor, anti-caseine, other proteins, …
« Artificial » cross-reactivity:
- caused by non specific monoclonals/polyclonals used in test kitcross-reactivity with related Ag’s, e.g. the herpesviridea
giving mainly false positive results
- appearance of the sample testede.g. haemolytic, icteric & lipemic samples
giving false positive or negative results
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AntigensAntigensViruses – Bacteria - ParasitesViruses – Bacteria - Parasites
Bacteria:procariotic organisms
single-celled organisms able to reproduce by duplicating themselves
responsive to antibiotics typical bacterium: rigid cell wall, thin 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
some have flagella
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envelope: outermost spiky layer
protein coat
core of genetic material: DNA or RNA
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)
AntigensAntigensViruses – Bacteria - ParasitesViruses – Bacteria - Parasites
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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:
Cryptosporidium parvumoocytes
Giardia intestinalis
AntigensAntigensViruses – Bacteria - ParasitesViruses – Bacteria - Parasites
Toxoplasma gondii
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Infectious diseases Infectious diseases Chorus Elisa lineChorus Elisa line
Kits currently available for the detection of Ab’s against:
TORCH panel:Toxoplasma gondiiRubella virusCytomegalovirus (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: Viruses: different families different families (1)(1)
HERPESVIRIDEA (DNA viruses)8 human herpes types
Alpha group 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
HV-7 T cells (CD4+)
HV-8 Sarcoma, lymphoma
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Viruses: Viruses: different families (2)different families (2)
PARAMYXOVIRIDEA (RNA viruses)
Subfamily Genus Human Species
Paramyxoviridea Paramyxovirus Human parainfluenza viruses types 1 & 3
Rubulavirus Human parainfluenza viruses types 2, 4a and 4b,
Mumps virus
Morbillivirus Measles virus
Pneumovirnea Pneumovirus Human respiratory syncytial virus
Viruses: Viruses: different families different families (2)(2)
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Viruses: Viruses: different families (3)different families (3)
TOGAVIRIDEA (RNA viruses)
*: rubella (German measles)
Genus Species
Alphavirus Sindbis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Ross River virus, O’nyong’nyong virus
Rubivirus Rubella virus*
Viruses: Viruses: different families different families (3)(3)
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BacteriaBacteriaHelicobacter 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 ulcer.
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
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ParasitesParasitesToxoplasma 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 diseasesChildhood 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 diseasesOther 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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VaccinationsVaccinations
1. Aim is to prevent epidemics which caused many deaths in the past.
2. Vaccines help the body produce antibodies protection.3. 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 ……….
4. Pipeline: hepatitis C, RSV, HIV, CMV, EBV, …..5. Far future: HSV 1&2, adenovirus, toxoplasmose
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HSV-1HSV-1
• oral-facial lesions
• most common HSV
• usually acquired in childhood
• fever blisters inside mouth
• transmitted by infected saliva
• 90% of adults have Ab’s
HSV-1HSV-1
• oral-facial lesions
• most common HSV
• usually acquired in childhood
• fever blisters inside mouth
• transmitted by infected saliva
• 90% of adults have Ab’s
Herpes Simplex 1 & 2 Herpes Simplex 1 & 2 (1)(1)
both belonging to the Herpesviridea slight antigenic difference between HSV-1 and HSV-2
HSV-2HSV-2
• genital lesions (ulcers or sores)
• possible complications: meningoencephalitis or infection of the eye (acquired at burth, passing through infected mother’s birth canal)
• can also be asymptomatic
• sexually transmitted
• 25% of 25-45 years old of US population
HSV-2HSV-2
• genital lesions (ulcers or sores)
• possible complications: meningoencephalitis or infection of the eye (acquired at burth, passing through infected mother’s birth canal)
• can also be asymptomatic
• sexually transmitted
• 25% of 25-45 years old of US population
Cross-infection of type 1& 2 viruses may occur from oral-genital contact.
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Herpes Simplex 1 & 2 Herpes Simplex 1 & 2 (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 you’ve 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 Herpes Simplex 1 & 2 (3)(3)
Recommended testing:
Seronegative
NEGATIVE
Acute/Recentin fection
IgMPO SITIVE
Old infection(Ig G p ositive)
IgMNEG ATIVE
Herpes Sim plex 1+2 IgM
POSITIVEIgG or IgM ?
HSV 1 Screenand
HSV 2 Screen(IgM + IgG)
Chorus line:- HSV-1 Screen (IgM & IgG)- HSV-2 Screen (IgM & IgG)- Herpes Simplex 1+2 IgM
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Helicobacter pylori Helicobacter pylori (1)(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 Helicobacter pylori (2)(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 Ab’s does not correlate with disease severity but
succesful treatment can be monitored by decrease of Ab’s 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 Helicobacter pylori (3)(3)
Recommended testing: IgA and IgG
Chorus line:- H. pylori IgA- H. pylori IgG
Seronegative
H. Pylori IgGNEG AT IVE
H. Pylori IgANEG AT IVE
Case 1
Start prim ar infection
H. Pylori IgGNEG AT IVE
H. Pylori IgAPO SIT IVE
Case 2
Follow -up sam pleIncrease IgG = acute inf.
Decrease IgG = im pact drug or recoveryStable = past infection
Acute infectionor
past infection ?Drug m onitoring
H. Pylori IgGPO SIT IVE
H. Pylori IgAPO SIT IVE
Case 3
Treated infection ?F o llo w -up sam p le
H. Pylori IgGPO SIT IVE
H. Pylori IgANEG AT IVE
Case 4