professor leke feb 2009

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IMMUNOLOGICAL DIAGNOSIS OF

PARASITIC INFECTIONS



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.



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.



Biological diagnosis of parasitic infections,

3 methods currently used:

○ Diagnosis with certitude or direct diagnosis.

○ Immunological diagnosis.

○ Presumptive diagnosis or indirect diagnosis.



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.



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



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



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)



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-



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.



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



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



If antigen is present, infection is there



1. ANTIGEN DETECTION.

ANTIGEN CAPTURE.

Use monoclonal / polyclonal Abs. Can use recombinant DNA Techniques

to

produce proteins, polypeptides

(antigens). Then make abs.



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



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.



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



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

professor leke feb 2009

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