IMMUNOLOGICAL DIAGNOSIS OF PARASITIC INFECTIONS

INTRODUCTION

Antigenic nature of parasites immune reactions 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 most pronounced 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 available lysis. (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.
Rocket electrophoresis Laurell Used to quantitate Ag Also for Ab (single radial electro-Immunodiffusion: electrically driven) One dimensional pH chosen Ab imobile Ag, with negative charge Quant: Height of rocket proportional to ag conc.

7.

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 probes

Specific tests Schisto: Circumoval precipitin Test (COPT) of Olivier Gonsalesvery 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 immunological techniques using antibodies (monoclonal) to detect malarial antigens, through antibody-antigen interactions. For malaria diagnosis, 2 important soluble antigens secreted by erythrocytic forms of the parasite in the blood are targeted:
Histidine rich Protein-2 (HRP-2),

Test kits include: Paracheck, ICT, Parasight-Ftest,

Parasite Lactate dehydrogenase (pLDH),

Optimal test

Optimal Rapid test

A qualitative, sandwich immunoassay for the detection and differentiation of P. falciparum and P. vivax in whole blood samples It is based on the detection of an abundant intracellular metabolic enzyme, produced by malarial parasites in the blood. The enzyme, Lactate Dehydrogenase (pLDH), is released from parasitized red blood cells and is rapidly detected by a series of monoclonal antibodies. Differentiation between malarial species is based on antigenic differences between pLDH isoforms.

pLDH detection kitsOptiMAL

Test principle
-Add sample to sample pad
Control window
Test window

-Antigen (pLDH binds antibody + gold particle -Ag-Ab- Gold particle complx migrate through test zone for Pf and Pv

Sample application well Buffer application trough

-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 be amplified a billion fold provided that part of its nucleotide sequence is already known. Known part of sequence used to design two synthetic DNA Oligonucleotides, one complementary to each strand of the DNA double helix and lying on opposite side of region to be amplified.

If DNA present, then parasite present