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

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

ANTIBODIES/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

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

PRIMARY AND SECONDARY Ab IMMUNE
RESPONSE

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

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

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

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

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

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

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

Single radial immunodiffusion

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.

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

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

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

 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

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

IMMUNOCHROMATOGRAPHY
• RAPID
• EASY TO PERFORM
• LIMITED SENSITIVITY & SPECIFICITY

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

PROCEDURE

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

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

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

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