1. IMMUNODIAGNOSTIC Anna Tjandrawati Clinical Pathology Department
Medical Faculty Padjadjaran University
Dr Hasan Sadikin General Hosptital Bandung

2. THE COMPONENT OF IMMUNITY
INNATE/NATURAL/NON SPECIFIC IMMUNITY:
Normal flora
Anatomic barriers
Inflammation (complement)
Phagocytic cells
ADAPTIVE/ACQUIRED/SPECIFIC IMMUNITY: Lymphocytes
HUMORAL:
B lymphocytes
Plasma cells
Antibody
CELLULLAR:
T lymphocytescells
Lymphokines

3. ANTIBODIES/IMMUNOGLOBULINS CLASSES OF IMMUNOGLOBULINS
Proteins produced by plasma cells and secreted into body fluids in response to antigen exposure
CLASSES OF IMMUNOGLOBULINS
IgG:Long lasting immunity, crosses the placenta
IgM:First response antibody
IgA:Present in secretions
IgD:Functions unknown
IgE: Allergic reactions

4. IMMUNE RESPONSE Primary Antibody response:
First exposure to ag
IgM appears 3-5 days,
increased and then drops
over a few weeks-months
IgG detectable 1-2 weeks ,
increased and decreases
over a period of time
Secondary Antibody response/ Anamnestic response:
After reexposure to ag
Antibody production increases rapidly
IgG increase in 2 – 3 days and
increases higher levels than primary
response and remains detectable for
months or years

5. PRIMARY AND SECONDARY Ab IMMUNE RESPONSE

6. A Healthy immune system is fundamental to overall good health
Diseases involving the immune system
Immunocompetent
Immunocompromised
Overactive/misdirected
Acquired:
immunosupression
drugs, microorganism
Inherited:
Chromosomal, gene
Hypersensitivities
Autoimmune disease

7. TYPES OF IMMUNOLOGICAL TESTS
Tests of immune function:
CD4, CD8
Quantitation of Ig subgroup
Tests of leucocyte function
Allergy tests
Tests based on Ag-ab reaction:
The presence of an ab to a defined ag depends on the immune response of the patients
Ab detection (qualitative/quantitative) is used to evaluate N or AbN immune responses

8. Clinical Laboratory Methods
IMMUNODIAGNOSTIC
Clinical Laboratory Methods
for Detection of Antigens(Ag) and Antibodies(Ab)
Antigen-antibody interactions underlie many immunological technique, in which the high specificity of the ab is used to identify,isolate or quantify a particular ag
The technique can identify ag or ab

9. THE PRECIPITIN REACTION
The classical illustration of ag-ab reaction in-vitro is
THE PRECIPITIN REACTION

10. Antibody excess zone:
The amount of Ag is insufficient to react with and precipitate all the ab present, thus free ab can be detected in the supernatant (PROZONE EFFECT)
Equivalence zone:
The added ag is sufficient to combine with and precipitate all the ab present and neither free ag nor ab can be detected in the supernatant
Antigen excess zone:
The amount of ag exceeds that required to bind all the ab and this leads to a reduction in the amount of ab precipitated. This falls is due to the formation of soluble ag-ab complex by the excess ag

11. Testing methods that depend on formation of immune complex
Immunodiffusion
Immunodiffusion (ID) is the simple technique by which ag and abs are placed in separate wells within a semisolid medium (agar) then allowed to mix through the medium by diffusion
When a zone of equivalence is reached, a line of precipitation occurs
Eg. Total Ig G, total IgM and total IgA

12. Precipitin reaction in gels: immuno-double diffusion-Antibody binding

13. Single radial immunodiffusion

14. Agglutination
Agglutination assay that test for the presence of an ab depend on the availability of a particle that is coated with the appropriate ag.
The particle can be an RBC (hemaglutination), synthetic particle (latex agglutination) and can be seen in the tube, microtitres well or simple glass slide.

15. AGGLUTINATION test Y + 
The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen.

16. RBC/Haemagglutination
Latex agglutination
RBC/Haemagglutination
E.g :
RA factor
Pregnancy test
CRP
ASLO
E.g:
* ABO Blood typing
* TPHA

20. Latex Agglutination for Rheumatoid factor
RA/ are autoantibodies, usually of the IgM class, directed agaist human IgG
Principle the test:
Latex particle are coated with specially treated human IgG. When serum containing RF is mixed with the IgG coated latex particle, the RF bind to the IgG and cause agglutination of the particle

21. ELISA(Enzyme-linked immunoassay)
The ab or ag is fixed to a surface, such as a well or
microtiter plate or plastic bead.
The test sample is applied and bound material is
detected by secondary, enzymatically labeled antibody
enzym + substrates  a colored product 
Spectrophotometer
E.g: Anti CMV IgM, Anti Rubella IgG,Anti HAV, Anti HCV
Tumor marker, Hormon

22. Ab/Ag + secondary enzymatically labeled antibody (conjugate) + substrate product (chromogen).

25. IMMUNOCHROMATOGRAPHY LIMITED SENSITIVITY & SPECIFICITY
RAPID
EASY TO PERFORM
LIMITED SENSITIVITY & SPECIFICITY

26. Add 2 drops of running buffer Membrane with Immobilised Antibodies
Assay Principle
Colloidal Gold Pad
Flavivirus specific MAb
conjugated to gold colloid
Dengue 1-4 Recombinant Antigens
Add 2 drops of running buffer
Add 10µL of blood or serum
Membrane with Immobilised Antibodies
IgM capture line
IgG capture line
Control Line
Blood Separation Device
Wicking Material
15 min
Release of Assay Reagents
Cassette Enclosure
Backing Sheet
Release of Serum Components
Absorbent Pad
Antibody Complexing
IgM Capture Line
IgG Capture Line
Control Line

27. PROCEDURE

28. Immunohistochemical methods
INDIRECT Immunofluorescence Assay (IFA)
The specific ag conjugated to the cells or tissues and the patient serum incubated with the cells. Unbound ab removed by washing, and the specifically bound ab are visualized with fluorescently labeled anti-Ig antisera.
When observed in the fluorescence microscope againts a dark background, fluorescent labeled anti-Ig antisera bound specifically to agab complex can be visualized by their bright color
E.g: ANA, anti DS-DNA

31. RESULT OF ANA TEST
NEG. ANA POS. CONTROL NEG. CONTROL
Homogenous

32. RESULT OF ANA TEST POS. ANA POS. CONTROL NEG. CONTROL
Homogenous Homogenous

33. RESULT OF ANA TEST POS. ANA POS. CONTROL NEG. CONTROL
Nucleolar Nucleolar

34. Thank you