Download IgM - Akademik Ciamik 2010

IMMUNODIAGNOSTIC
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
ADAPTIVE/ACQUIRED/SPECIFIC
IMMUNITY: Lymphocytes
• Anatomic barriers
• Inflammation (complement)
• Phagocytic cells
HUMORAL:
CELLULLAR:
B lymphocytes
T lymphocytescells
Plasma cells
Lymphokines
Antibody
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:
Inherited:
Hypersensitivities
immunosupression
Chromosomal, gene
Autoimmune disease
drugs, microorganism
TYPES OF IMMUNOLOGICAL TESTS
Tests of immune function:
Tests based on Ag-ab reaction:
• CD4, CD8
The presence of an ab to a defined
ag depends on the immune
response of the patients
• Quantitation of Ig subgroup
• Tests of leucocyte function
• Allergy tests
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.
AGGLUTINATION test
The antibody is mixed with the particulate
antigen and a positive test is indicated by the
agglutination of the particulate antigen.
+
Agglutination
Latex
agglutination
RBC/Haemagglutination
E.g :
E.g:
• RA factor
* ABO Blood typing
• Pregnancy test
* TPHA
• CRP
• ASLO
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
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
Ab/Ag + secondary enzymatically labeled antibody
(conjugate) + substrate
product (chromogen)
.
IMMUNOCHROMATOGRAPHY
•RAPID
•EASY TO PERFORM
• LIMITED SENSITIVITY & SPECIFICITY
Assay Principle
Membrane with
Immobilised Antibodies
Add 10µL
bloodline
or serum
• IgM of
capture
• IgG capture line
• Control Line
Add 2 drops of
running buffer
Blood Separation Device
Wicking Material
15 min
Cassette Enclosure
Absorbent Pad
Colloidal Gold Pad
• Flavivirus specific MAb
conjugated to gold colloid
• Dengue 1-4 Recombinant Antigens
Release of Serum
Components
Release of Assay
Reagents
Backing Sheet
IgG Capture Line
Antibody
Complexing
Control Line
IgM Capture Line
PROCEDURE
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
RESULT OF ANA TEST
NEG. ANA
POS. CONTROL
Homogenous
NEG. CONTROL
RESULT OF ANA TEST
POS. ANA
Homogenous
POS. CONTROL
Homogenous
NEG. CONTROL
RESULT OF ANA TEST
POS. ANA
Nucleolar
POS. CONTROL
Nucleolar
NEG. CONTROL