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 Anatomic barriers Inflammation (complement) Phagocytic cells ADAPTIVE/ACQUIRED/SPECIFIC IMMUNITY: Lymphocytes HUMORAL: B lymphocytes Plasma cells Antibody CELLULLAR: T lymphocytescells Lymphokines

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

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

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

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

RBC/Haemagglutination 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 LIMITED SENSITIVITY & SPECIFICITY RAPID EASY TO PERFORM LIMITED SENSITIVITY & SPECIFICITY

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

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

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