professor leke feb 2009
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IMMUNOLOGICAL DIAGNOSIS OF
PARASITIC INFECTIONS
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INTRODUCTION
Antigenic nature of parasites immunereactions
Location in body T, B, M, Inflammation Abs: G,A, M, E., Cytokines
If parasites persist:
Immune Response Modulated:
If Re-infection enhanced, turned off,hypersensitivity.
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INTRODUCTION
Tissue parasites Produce mostpronounced IR.
Ectoparasites, Intestinal worms less
likely to evoke IR. IR: Dynamic event: can determine
levels/types of Abs.
Initial Ab Response: IgM later: IgG, ISgA,
IgE. Helminths more likely than Protozoa to
evoke IgE.
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Biological diagnosis of parasitic infections,
3 methods currently used:
○ Diagnosis with certitude or direct diagnosis.
○ Immunological diagnosis.
○ Presumptive diagnosis or indirect diagnosis.
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Why perform Immunological Tests for Diagnosis?
○ To complement direct methods
○ Early diagnosis
○ Low infestation
○ Follow evolution of disease
○
Confirm recovery
○ To diagnose illnesses where direct methods are difficult to use.
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LIMITATIONS OF SEROLOGICAL TESTS
Complex antigenic composition of parasites -cross reaction ags
Lack of well standardized reagents and test
procedures makes interpretation difficult.
What does presence of Ab mean?
○ Abs present during active infection
○ Abs stay after treatment○ Levels persist after infection has ceased false
positives
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Types Of Assays Used In Parasitic Infection
1. Direct Agglutination:
○ T- Cruzi epimastigotes.
○ CATT = Card Agg - Test – Tryps
2. Indirect Agglutination= Hemagglutination or latex agglutination.
○ Fix ER or latex with tannic acid,gluteraldehyde or formaldehyde
○ Coat with Ag.
○ Dilute antiserum
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3. Hem - Inhibition: to detect small quantities of
soluble ag - (samples)
Principle:
1. Ab + Ag (low conc Ab)
2. Add Ag - coated RBC - agglutinated by
uncombined free Ab
4. Complement fixation
• Add fixed Amt Ag to Test Serum (if ab+)Ag Ab
complex• Add C (known)Ag – Ab – C
• EA indicator cells if C availablelysis.
(RBC + sub agglutinating amt of Ab)
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5. Immunodifussion in gels
• Ouchterlony/Double diffusion
= Ag & Ab moving PPt
•
Single Radial Immunodifusion / Mancini= Ig quantitative
• Immunoelectrophoresis
= Ags separated using electrical charge
PH Chosen = + proteins Neg Proteins move
to +
Cut Trough – Fill with Ab-
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6. Electro-immunodiffussion (EID) :
Electrically drive both ag and ab: – counter –
current electrophoresis:Ab is + charged ; Ag is negative charged.
7. Rocket electrophoresis – Laurell
Used to quantitate Ag – Also for Ab (single radialelectro-Immunodiffusion: electrically driven) Onedimensional
pH chosenAb imobile
Ag, with negative chargeQuant: Height of rocket proportional to ag – conc.
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8. Immunofluorescence:
Direct: Label (Histochemical) Ab – directly
Indirect: serum - labelled Ab – can identify abs to
different Ags in a single test
9. Enzyme Immunoassays
• Enzyme – linked immunoabsorbent assay = ELISA
Principle analogous to IFAT – Couple enzyme to ab or ag
Enzymes: Horseradish peroxidase., Alkaline phosphatase
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Enzyme – Linked immuno-electrodiffusion Assay
(ELIEDA)
- Enhance reaction of EID
- Define class of Ab –
1. IC formed
2. Fix IC with anti – Ig confugated to enzyme
3. Reveal with subsctrate
10. Radioimmunoassay (RIA)
Like ELISA: Instead of using enzyme, use radiolabelled 125I ligand
11. Use DNA probesSpecific tests
• Schisto: Circumoval precipitin Test (COPT) of Olivier Gonsales-
very sensitive
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If antigen is present, infection is there
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1. ANTIGEN DETECTION.
ANTIGEN CAPTURE.
Use monoclonal / polyclonal Abs. Can use recombinant DNA Techniques
to
produce proteins, polypeptides
(antigens). Then make abs.
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RAPID TESTS: Ag DETECTION
e.g. MALARIA
Involves the application of immunologicaltechniques using antibodies (monoclonal) todetect malarial antigens, through antibody-antigeninteractions.
For malaria diagnosis, 2 important solubleantigens secreted by erythrocytic forms of theparasite in the blood are targeted:
○ Histidine rich Protein-2 (HRP-2),
Test kits include: Paracheck, ICT, Parasight-Ftest,
○ Parasite Lactate dehydrogenase (pLDH),
Optimal test
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1- Optimal Rapid test
A qualitative, sandwich immunoassay for thedetection and differentiation of P. falcip arum and P.
vivax in whole blood samples
It is based on the detection of an abundantintracellular metabolic enzyme, produced by
malarial parasites in the blood.
The enzyme, Lactate Dehydrogenase (pLDH), isreleased from parasitized red blood cells and israpidly detected by a series of monoclonal
antibodies.
Differentiation between malarial species is basedon antigenic differences between pLDH isoforms.
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pLDH detection kits – OptiMAL®
Sample application well
Test window
Control window
Buffer application
trough
Test principle
-Add sample to sample pad
-Antigen (pLDH binds
antibody + gold particle
-Ag-Ab- Gold particle
complx migrate through test
zone for Pf and Pv
-Cplx binds Ab to the AG
-Unbound cplx is trapped in
control window
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5. PCR: Polymerase Chain Rection.
• DNA from a selected region of a genome to beamplified a billion fold provided that part of itsnucleotide sequence is already known.
• Known part of sequence used to design twosynthetic DNA Oligonucleotides, onecomplementary to each strand of the DNA
double helix and lying on opposite side of regionto be amplified.
• If DNA present, then parasite present