IMMUNODIAGNOSTIC

Anna Tjandrawati

Clinical Pathology Department

Medical Faculty Padjadjaran University

Dr Hasan Sadikin General Hosptital Bandung

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

THE COMPONENT OF IMMUNITY

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

Diseases involving the immune system

A Healthy immune system is fundamental to overall good health

Immunocompetent Immunocompromised

Acquired:

immunosupression

drugs, microorganism

Inherited:

Chromosomal, gene

Overactive/misdirected

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

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

Testing methods that depend on formation of immune complex

Immunodiffusion

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 testAGGLUTINATION 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 :

• 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

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

Wicking MaterialWicking Material

Colloidal Gold PadColloidal Gold Pad• Flavivirus specific MAb Flavivirus specific MAb conjugated to gold colloidconjugated to gold colloid• Dengue 1-4 Recombinant AntigensDengue 1-4 Recombinant Antigens

Add 10µL of blood or serum

Membrane with Membrane with Immobilised AntibodiesImmobilised Antibodies

• IgM capture lineIgM capture line• IgG capture lineIgG capture line• Control LineControl Line

Blood Separation DeviceBlood Separation Device

Absorbent PadAbsorbent Pad

Backing SheetBacking Sheet

IgM Capture LineIgM Capture Line

IgG Capture LineIgG Capture Line

Control LineControl Line

Add 2 drops of Add 2 drops of running bufferrunning buffer

Assay Principle

Cassette EnclosureRelease of Serum Release of Serum

ComponentsComponents

Release of Assay Reagents

Antibody Complexing

15 min

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 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