CELL-MEDIATED IMMUNITY

LYMPHOCYTES
T cells B cells NK cells
Population % 60-70% 10-20% 10-15%
Immunity Cell-mediated Humoral-mediated Antibody-Dependent Cellular Cytotoxicity (ADCC)
Products Cytotoxins (Perforins, Granzymes) Antibodies Cytotoxins (Perforins, Granzymes)
Defense against Viral, Tumor, Fungal, Graft rejections Bacterial Viral, Tumor
CD: 2, 3, 4, 5/7, 8 CD: 19, 20, 21, 22, 10 CD: 16, 56
CD2 E-rosette receptor (Affinity to Sheep RBC) CD21 C3d and EBV receptor CD16 Receptor for IgG
CD3 Part of T-cell Ag Receptor (TCR) CD19 Part of B cell co-receptor CD56 Unknown function
Surface markers
CD5/7 Early stages of T-cells CD10 Early stage of B cells/CALLA
(Flow cytometry)
CD4 Helper T-cell/ T-regulatory cell CD20 Found on all stages of B cells
Subpopulation: Th1, Th2, Th3, Th9 CD22 Found mostly on mature B cells
CD8 Cytotoxic T-cell
• Pokeweed: Blood • Pokeweed: Blood • None
Mitogens
• Concanavalin A: Jack beans • Staphylococcal Protein A (LPS)
(Enhance mitosis)
• Phytohemagglutinin: Red beans
• NK cells + IL-2 = Lymphokine Activated Killer (LAK) cells:
Both cannot be identified morphologically
o LAK cells: Kill fresh autologous and allogeneic
Remarks human tumor cells in vitro
Granzymes: Induce apoptosis Cytotoxic T cell: Specific
• Produces perforins: Lyses infected cells → apoptosis
Perforins: Membrane-disrupting protein/ Pore-forming NK cell: Nonspecific
• Transitional cell: Bridges innate and humoral immunity

LYMPHOCYTE: MATURATION STAGES

T CELLS B CELLS
Primary Lymphoid Organ: Thymus Primary Lymphoid Organ: Bone Marrow
1. Double Negative 2. Double Positive 1. Pro-B cells/Progenitor B cells 2. Pre-B cells/Precursor B cells 3. Immature B cells
√ CD2, CD5, CD7 × CD4, CD8 √ CD3, CD4, CD8 Heavy & light chains Mu heavy & surrogate light chains Complete surface IgM
• Rearrangement of genes coding for • Positive Selection: MHC restriction • Heavy chain: Chromosome 14 • Mu chain: 1st heavy chain • IgM: Earliest Ab that appears
TCR o Selection of thymocytes that will • Light chain: synthesized in B cell’s surface
• Thymocytes in this stage proliferate only interact with the MHC Ag on o Chromosome 2: kappa
d/t IL-7 host cells o Chromosome 22: lambda
o Very high/low affinity to self Ag →
Apoptosis
• Negative Selection: Clonal deletion
o Elimination of clones of T cells
capable of an autoimmune
response
o Strong reactions w/ self-peptides
other than MHC Ag → Apoptosis
97%: Apoptosis 3%: Mature 90%: Apoptosis 10%: Mature
Secondary Lymphoid Organs: Spleen & Lymph Node Secondary Lymphoid Organ: Spleen & Lymph Node
3. Mature T Cells/Single Positive Stage 4. Activated T Cells 4. Mature B cells 5. Activated B cells 6. Plasma cells
√ Either CD4 or CD8 √ CD25: Receptor for IL-2 Surface IgM & IgD (Ag recognition) CD25: Receptor for IL-2
• Normal CD4:CD8 ratio → 2:1 • IL-2: Causes proliferation of cells • Marginal: Remains in the • IL-2: Causes proliferation of • Size: 8-20 um
o ↓ in AIDS spleen cells • Shape: Oval
• Follicular: Migrates to the • Nucleus: Eccentric
lymph nodes and other 2o LO • Nuclear chromatin: Cartwheel
• IgD: Prolongs life span of • Perinuclear Hof = Golgi body
Mature B cells near nucleus

SUBPOPULATION OF CD4 T CELLS

Th1 cells (Cell-mediated) Th2 cells (Humoral-mediated) Th3 cells (T-regulatory cell) Th9 cells
• Protects against intracellular pathogens by: • Protects against extracellular pathogens by: • Suppresses the immune response to self- • Protects against fungi and bacteria
o Activating cytotoxic lymphocytes & o Helping B cells produce Ab antigens • Produces IL-9; Has a proinflammatory effect
macrophages o Regulating B cell activity • Inhibits proliferation of other T cell • Stimulate mast cell production
populations by secreting inhibitory cytokines
 CLASSES OF ANTIBODIES
IgG IgM IgA1 IgA2 IgD IgE
Structure Monomer Pentamer Monomer Dimer Monomer Monomer
Serum Concentration 70-75% 10% 10-15% 0.0001% 0.0005%
Molecular weight 150, 000 da 900, 000 da 160, 000 - 350, 000 da 180, 000 da 190, 000 da
Sedimentation rate 7s 19s 7s 7s 8s
Half-life 23 days 6 days 5 days 1-3 days 2-3 days
Fixes complement Yes Yes Alternative No
Crosses placenta Yes No
Valence 2 10 2 4 2 2

IgG IgM IgA1 IgA2 IgD IgE

• Smallest immunoglobulin • Largest immunoglobulin • Serum IgA • Secretory IgA • Shortest lifespan • Reaginic Ab: High affinity to
• Most efficient in • Most efficient in • Major Ab in secretions • Regulates B cell mast cells and basophils
precipitation agglutination and differentiation • Heat-labile Ab: 56 deg C for
• Major antibody in complement fixation • More susceptible to 30 mins
Anamnestic response • Most primitive proteolysis
• Major Ab in serum • For immunoregulation

Classical (1st pathway discovered: 1900) Alternative/ Properdin Lectin

Requirement for activation Ag-Ab complex (1 IgM & 2 IgG) Bacterial polysaccharide / IgA binding Mannose/Mannan sugar in cell wall
Order of activation 1, 4, 2, 3, MAC 3, MAC MBL, 4, 2, 3, MAC
C3 convertase C4b2a C3bBb C4b2a
C5 convertase C4b2a3b C3bBb3b C4b2a3b
Notes • C1 cleaves C4 → C4a + C4b • Bacterial cell wall exposed cleaves C3 → C3a • Mannose-Binding Lectin Serine-Associated Protease
• C1 cleaves C2 → C2a + C2b + C3b (MASP) 1, 2, 3, complex attaches to
o C4b + C2a = C4b2a/ C3 convertase • Factor B + C3b → C3Bb Mannose/Mannan will act on C4 and C2
• C3 convertase cleaves C3 → C3a + C3b • Factor D cleaves Factor B in C3Bb → Ba and Bb • C3 convertase cleaves C3 → C3a + C3b
o C3a (smaller): Anaphylatoxin o Ba → Plasma o C3a (smaller): Anaphylatoxin
o C3b (bigger): Opsonin o Resulting to: C3bBb (C3 convertase) o C3b (bigger): Opsonin
• C3b + C4b2a (C3 convertase) = C4b2a3b/C5 • C3 convertase cleaves other C3 → C3a + C3b • C3b + C4b2a (C3 convertase) = C4b2a3b/C5
convertase • C3 convertase + C3b = C3bBb3b (C5 convertase
• C5 convertase cleaves C5 → C5a + C5b convertase) • C5 convertase cleaves C5 → C5a + C5b
• C6, C7, C8, C9 attaches to C5b = MAC • C5 convertase cleaves C5 → C5a + C5b • C6, C7, C8, C9 attaches to C5b = MAC
o C8: Start of lysis • C6, C7, C8, C9 attaches to C5b = MAC o C8: Start of lysis
o C9: Complete lysis o C8: Start of lysis o C9: Complete lysis
o C9: Complete lysis
 HYPERSENSITIVITY
• Heightened state of immune responsiveness
• An exaggerated response to a harmless antigen that results in injury to the tissue, disease, or even death
CLASSIFICATION BY PHILLIP GELL & ROBIN COOMBS
Type I Type II Type III Type IV
Other term Immediate/ Anaphylactic Antibody mediated/ Cytotoxic Immune-complex Cell-mediated/ Delayed
Immune Mediator IgE IgG/ IgM IgG/ IgM Sensitized T cells (Th1)
Immune Mechanism • Release of mediators like histamine from IgE- • Cell lysis due to antibody & • Deposition of immune • Release of lymphokines from sensitized T cells
sensitized basophils/mast cells complement complexes to host tissues • May also cause formation granuloma
• Granules of mast cells (found in tissue)/ basophils o multinucleated fused macrophages
(found in peripheral blood) • Release of IL-1 & IL-6 (Proinflammatory
o Histamine cytokines) secreted by monocytes &
o Heparin macrophages
o Prostaglandins
o Leukotrienes
• Release of IL-1 & IL-6 (Proinflammatory cytokines)
secreted by monocytes & macrophages
End result - Cell lysis Tissue damage Inflammation d/t cytokines
Complement No Yes Yes No
Examples • Anaphylaxis • Transfusion reaction • Serum Sickness • Contact Dermatitis
• Urticaria • AIHA • Arthus reaction • Pneumonitis
• Hay fever • HDN • SLE • Granuloma Formation
• Allergic rhinitis • Goodpasture Syndrome • RA • Poison Ivy
• Food allergies • Graves’ Disease • Post-streptococcal • Tuberculin & Allergy Skin Test
• Bee sting • Myasthenia Gravis glomerulonephritis
• Bronchial asthma • Drug reaction* • Drug reaction*
• Wheal and Flare reaction
Tests • Skin allergy testing (Penicillin drug) DAT (+): • SLE: Detect ANA • Tuberculin/ Purified Protein Derivative Test
• Measurement of IgE levels • HTR • RA: Detect RF (Mantoux)
o Radio Immunosorbent Assay (RIST): Total IgE • HDN • Von Pirquet Test
o Radio Allergosorbent Assay (RAST): Allergen- • AIHA
specific IgE • DIHA
Remarks • Serum Sickness: Contact Dermatitis:
• Systemic inflammation d/t • Caused by low MW compounds/ haptens
• Injection of animal serum that have contact w/ skin
to humans • Nickel, rubber, poison ivy, cosmetics
Arthus Reaction: Tuberculin/ Purified Protein Derivative (Mantoux)
• Localized inflammation d/t • Based on the principle that soluble Ags from
• Injection of Ag to M. tuberculosis induce a reaction in people
subcutaneous tissue who have past or present infection of
tuberculosis
• PPD is injected under the skin and the
reaction is read at 48 to 72 hours (erythema /
induration – local swelling / raised bump)
• The diameter is measured
• (+) Result >15 mm
 Precipitation (Kraus, 1897) Agglutination (Gruber & Durham, 1896)
Soluble antibody + soluble antigen (small particles) = insoluble complexes Antibody + particulate antigen (large particles; e.g. cells, latex particles) = cellular aggregates

PRECIPITATION
Antigen-Antibody Binding
Affinity • Initial force of attraction that exist between a single Fab (paratope) and single epitope of an antigen
• Refers to the single force of binding
Avidity • Represents the overall strength of antigen-antibody binding and is the sum of all the affinities involved
• ↑ avidity = IgM
• ↑ avidity = ↓ chances of dissociation
• Refers to the total antigen-antibody binding
Zone of Equivalence • Where optimum precipitation/agglutination occurs
• Number of multivalent sites of antigen and antibody are approximately equal
Prozone phenomenon Postzone phenomenon
Excess antibodies Excess antigens

PRECIPITATION TECHNIQUES
Precipitation in Fluid Medium Precipitation in Agar Gel (Passive Immunodiffusion) Precipitation by Electroimmunodiffusion Precipitation by Electrophoretic Techniques
1. Turbidimetry 1. Oudin / Single Diffusion in One Dimension 1. Rocket Immunoelectrophoresis / Laurell IEP 1. Immunoelectrophoresis
2. Nephelometry 2. Oakley-Fulthorpe / Double Diffusion in One Dimension 2. Countercurrent IEP 2. Immunofixation electrophoresis
3. Radial Immunodiffusion (RID) / Single Diffusion in Two Dimension
a) Mancini/Endpoint method
b) Fahey & McKelvey/Kinetic method
4. Ouchterlony/Double Diffusion in Two Dimension

PRECIPITATION IN FLUID MEDIUM

Turbidimetry Nephelometry
• Measure the decrease in light intensity caused by absorption in a solution of antibody-antigen • Measures the amount of light scattered in a solution containing antibody-antigen complexes
complexes • Widely used to quantify immunoglobulins
• Measurements are made using a spectrophotometer or automated clinical chemistry analyzer • Amount of scattered light = Concentration (mg/dL or IU/mL)
• ↑ Turbidity = ↑ Concentration (absorbance units)

PRECIPITATION IN AGAR GEL (PASSIVE IMMUNODIFFUSION)

AKA Migration Ab Ag (+) Result
Oudin 1 Diffusion in
Only the Ag moves toward the Ab to form the precipitin line
(James Oudin) 1 Dimension Tube/ layered at the top of agar
Precipitin band
2 Diffusion in column incorporated with gel
Oakley-Fulthorpe Both Ag and Ab moves toward each other to form the precipitin line
1 Dimension the agar gel
Radial 1 Diffusion in Mancini Ag is allowed to diffuse completely
Petri added to pre-cut wells Precipitin ring
Immunodiffusion (RID) 2 Dimension Fahey & McKelvey Ag is not allowed to diffuse completely
dish/
2 Diffusion in Known Ab: placed Known & Unknown Ag: Distinct pattern
Ouchterlony slide Both Ag and Ab diffuse independently in agar gel
2 Dimension on center well placed on surrounding wells of precipitation

Radial Immunodiffusion (RID) → Quantitative

Mancini/Endpoint method Fahey & McKelvey/Kinetic method
The square of the diameter (d2) is directly proportional to the antigen concentration The diameter (d) is proportional to the log of Antigen concentration
d2 = Ag concentration d = log (Ag concentration)
• Antigen is allowed to diffuse completely • Antigen is not allowed to diffuse completely
o Measurements taken after the point of equivalence is reached o Measurements taken before the point of equivalence is reached
• Incubation: • Incubation: 19 hours
o IgG= 24 hours • A graph is plotted using semi-log paper
o IgM = 50-72 hours o X-axis = diameter
o Y-axis = concentration
 Ouchterlony/Double Diffusion in Two Dimension → Qualitative
• 2 antigens have common epitope
Serologic Identity • Smooth curve/Arc

• 2 antigens do not have common epitope

Non-Identity • Crossed line

• 2 antigens have common epitope Antigen 1 xy antigen

o but the other possess an antigenic specificity that is not present in the other antigen Antigen 2 x antigen only
Partial Identity
• Spur formation is then produced towards the simpler antigen Antibody Anti-xy

PRECIPITATION BY ELECTROIMMUNODIFFUSION
Rocket / Laurell Immunoelectrophoresis Countercurrent Immunoelectrophoresis
RID + Electrophoresis Double diffusion + Electrophoresis
• Ab: incorporated with the gel • Ab and Ag are placed on a well directly opposite to
• Ag: placed in wells each other
• Migration: toward anode (+) • Ab → Cathode (-)
• Ag → Anode (+)
• Source of Antigen: Patient’s serum which is
electrophoresed in separate protein fractions
Height/ Apex of rocket = Antigen concentration Any change in shape, location and intensity of “precipitin arc” indicates an abnormality

PRECIPITATION BY ELECTROPHORETIC TECHNIQUES

Immunoelectrophoresis Immunofixation Electrophoresis (IFE)
Grabar & Williams Alper & Johnson
Protein electrophoresis + immunodiffusion Protein electrophoresis + immunoprecipitation

Abnormal contour of precipitin arc = Monoclonal gammopathy (Multiple Myeloma Monoclonal Immunoglobulin: Dark bands after staining
• Ag: placed on well and electrophoresed • Specimen is electrophoresed at 6 positions
• Ab: placed on trough parallel to separated proteins • Monospecific antisera are layered on each position, includes antibody to:
• It has been replaced by the Immunofixation technique o Gamma, alpha, mu (heavy chains)
Serum • Detection of monoclonal gammopathy o Kappa, lambda (light chains)
Immunoelectrophoresis o can speciate which immunoglobulin is the cause of increase • Antigen-Antibody complex form, washed and stain to visualize
Serum Protein • Used to identify heavy and light chains involved in monoclonal gammopathies
• Quantifies the “M component” of the spike but cannot speciate Determines whether the monoclonality is due to Gamma, alpha, mu (heavy chains) or
Electrophoresis o
Urine • Detection of Bence Jones protein Kappa, lambda (light chains)
Immunoelectrophoresis o can detect if Bence Jones protein is a Lambda or Kappa • First lane is considered as the Reference Lane (antisera for all serum proteins is added)
Urine Protein • Hypogammaglobulinemia: Faintly staining bands
• Quantify the number of light chains but cannot speciate • Polyclonal Hypergammaglobulinemia: Darkly staining band in the gamma region
Electrophoresis
• Monoclonal Antibody: Dark narrow band in specific lane

AGGLUTINATION REACTIONS (Particulate/Large Antigens)

DIRECT INDIRECT/PASSIVE REVERSE PASSIVE CO-AGGLUTINATION AGGLUTINATION-INHIBITION
(+) agglutination (+) agglutination (+) agglutination (+) agglutination (+) lack of agglutination
Ag: Found naturally on the surface of Ag: Attached to particulate carrier Ab: Attached to particulate carrier Ab: bacterial protein as inert particles Haptens attached to carrier particle
particles
Detect: Ab (viruses) Detect: Ag (microbial Ag) Detect: Ab to viruses (rubella, influenza,
RSV)
 AGGLUTINATION
Sensitization Lattice Formation
• Initial reaction & involves antigen-antibody complex combination through single antigenic • Represents the stabilization of antigen antibody complexes
determinants • Formation of cross-links that form the visible aggregates
• Coating of cells with the antibodies

AGGLUTINATION INSTRUMENTATION SYSTEM LABELED IMMUNOASSAY

Particle-Enhanced Immunoassay Particle-Counting Immunoassay (PACIA) Heterogenous Immunoassays Homogenous Immunoassays
• Nephelometry has been applied to the • Measuring the # of residual non- • “Hetero” = means many • “Homo” means one
reading of agglutination reactions agglutinating particles in a specimen • Require separation step (washing) • Does not require separation step
• As agglutination occurs, clumps of antigens • Requires secondary reaction (washing)
increase in size; these large clumps are not • More sensitive • Does not require secondary reaction
counted • Less sensitive
• ↑# free (unagglutinated) particles = ↓
Antigen concentration

LABELED IMMUNOASSAY
RADIOIMMUNOASSAY ENZYME IMMUNOASSAY FLUORESCENT IMMUNOASSAY
Rosalyn Yalow & Solomon Berson (1950) - Albert Coons (1941)
• Iodine-125: Gamma counter (Most common) • HRP, ALP, G-6PD, glucose oxidase, beta-galactosidase • Fluorescein Isothiocyanate (FITC): green @ 520 nm
• Iodine-131: Gamma counter o HRP & ALP: highest turnover rate and highest sensitivity • Tetramethyl Rhodamine Isothiocyanate (TRITC):
• Hydrogen-3: Beta counter orange red @ 585 nm
• Carbon-14: Beta counter • Phycocyanin: red
• Reporting: Counts per minute (CPM) • Texas red: red
Examples Applications Examples Applications Examples Applications
1. Competitive RIA: • RIST: Total IgE 1. Heterogeneous EIA • Indirect EIA 1. Direct IF • FPIA
Result: Inversely • RAST: Allergen-specific IgE a. Competitive EIA o HIV, HBV, HCV 2. Indirect IF • CLIA
proportional • Immunoradiometric assay b. Non-competitive/Indirect EIA • Sandwich EIA 3. Inhibition IF/Blocking test
Most sensitive for drug (IRMA) → Non-competitive c. Sandwich/Capture EIA o Tumor markers
assay 2. Homogeneous EIA o Plasma proteins
2. Non-competitive RIA: a. Enzyme multiplied o Infectious agents
Result: Directly immunoassay technique (EMIT) • EMIT: therapeutic drugs
proportional

RADIOIMMUNOASSAY
Rosalyn Yalow & Solomon Berson (1950)
• Iodine-125: Gamma counter (Most common) • Reporting: Counts per minute (CPM)
• Iodine-131: Gamma counter • Scintillation counter
• Hydrogen-3: Beta counter • Philippine Nuclear Research Institute (PNRI): Government agency that monitors, evaluates,
• Carbon-14: Beta counter regulates, and disposes anything radioactive
Examples
Competitive RIA Non-Competitive RIA
Ag (px) + Ab (Beads/Wells) + Radiolabeled Ag Ag (px) + Radiolabeled Ab
↑ Radioactivity = ↓ Ag conc. ↑ Radioactivity = ↑ Ag conc.
1 wash 2 washes

ENZYME IMMUNOASSAY
Enzyme Substrate Product
Ag/Ab Chromogenic Substance Color Formation (Spectrophotometer)
• HRP, ALP, G-6PD, glucose oxidase, beta-galactosidase
o HRP & ALP: highest turnover rate and highest sensitivity
 HETEROGENEOUS EIA HOMOGENEOUS EIA
COMPETITIVE EIA NON-COMPETITIVE/INDIRECT EIA CAPTURE/SANDWICH EIA ENZYME MULTIPLIED IMMUNOASSAY TECHNIQUE (EMIT)
Px Ag + Enzyme-labeled Ag → Solid phase Ab Solid phase Ag + Px Ab → Wash + Enzyme- Solid phase Ab + Px Ag → Incubate +
labeled Ab → Wash + Enzyme substrate Enzyme-labeled Ab + Enzyme substrate
Captured Ag must have multiple epitopes
Inversely proportional Directly proportional Directly proportional Directly proportional
Drugs and hormones Screening for detecting Ab to HIV, Hepa B and C Measurement of immunoglobulins Hormones, therapeutic drugs, and drugs of abuse in
both serum and urine

FLUORESCENT IMMUNOASSAY
Albert Coons (1941)
• Fluorescein Isothiocyanate (FITC): green @ 520 nm • Phycocyanin: red
• Tetramethyl Rhodamine Isothiocyanate (TRITC): orange red @ 585 nm • Texas red: red
FLUORESCENT IMMUNOASSAY (FIA)/ IMMUNOFLUORESCENCE (IF)
FLUORESCENCE POLARIZATION IMMUNOASSAYS
DIRECT IMMUNOFLUORESCENT ASSAY INDIRECT IMMUNOFLUORESCENT ASSAY
• Competitive Assay: Inversely proportional
• Based on the change in polarization of fluorescent light
emitted from a labeled molecule when it is bound by
antibody

Detects Antigen Detects Antibodies ↑ Ag = ↓ Fluorescence-labeled Ag bound = ↓ Polarization

Reagent Known Ab + FITC Reagent Known Ag + FITC + AHG ↓ Ag = ↑ Fluorescence-labeled Ag bound = ↑ Polarization
Applications Legionella, Chlamydia, HSV, CMV, EBV Applications FANA in SLE, FTA-ABS in Syphilis Applications Therapeutic drugs and hormones

MOLECULAR DIAGNOSTIC TECHNIQUES

HYBRIDIZATION TECHNIQUE FLUORESCENCE IN SITU HYBRIDIZATION (FISH) POLYMERASE CHAIN REACTION (PCR)
• Hybridization: Binding of two specific complementary nucleic • In situ hybridization: Additional technique that utilizes nucleic • Amplifies small quantities of nucleic acid up to levels that can
acid strands together acid probes to identify DNA be detected by hybridization reaction using nucleic acid
• Probe technology: Basis of identification of individual genes or o However, target DNA is found in intact cells probes
DNA sequences o Thin sections of solid tissue or deposits of cells are • Thermal cyclers are run 20x to 50x
o Probe binds to target area of nucleic acid and gives of deposited on glass slides Denaturation 94oC Separation of dsDNA into ssDNA
fluorescent signal to identify genome sequence o Cell membranes are permeabilized to allow entry of the Binding of primer to ssDNA; lays
probe Annealing 55 C
o
Southern Northern O Western foundation for NA synthesis
DNA RNA O Protein Probes are applied to the prepared slides of cells where DNA polymerase synthesizes new
they hybridize to their complementary sequence Extension 72oC
complementary strands
 Hepatitis Virus
Hepa Type Family Transmission Serological Markers Incubation Disease
Anti-HAV IgG, Anti-HAV Only hepatitis virus that can be
HAV ssRNA Picornaviridae Fecal-Oral, Blood Transfusion HAV RNA 4 weeks Foodborne/Traveler’s Hepatitis
IgM cultured
HBV DNA
Anti-HBc IgG, Anti-HBc IgM 4-26 Serum/Long-incubation/Hospital 1st to be discovered;
HBV dsDNA Hepadnaviridae Parenteral, Sexual, Perinatal HBsAg,
Anti-HBe, Anti-HBs weeks Personnel Hepatitis Most significant
HBeAg
Non-A Non-B/ Post-transfusion Most common bloodborne
HCV RNA Flaviviridae Parenteral, Sexual, Perinatal HCV RNA Anti-HCV 7 weeks
Hepatitis infection in US
Anti-HDV IgG, Anti-HDV Co-infection (+) and Superinfection (-) Requires HBV for replication
HDV ssRNA None Parenteral, Sexual, Perinatal HDV RNA 4-7 weeks
IgM (Anti-HBc IgM to differentiate) Utilizes HBsAg as envelope
HEV ssRNA Hepeviridae Fecal-Oral, Blood Transfusion HEV RNA Anti-HEV IgG, Anti-HEV IgM 2-9 weeks Water-borne Hepatitis

HAV HCV HDV HEV

HAV RNA RT-PCR HCV RNA PCR, Viral load measurement HDV RNA Used to confirm (+) HDV Ab HEV RNA PCR (blood/stool)
screen
Anti-HAV IgM Current infection Anti-HCV Indirect ELISA, Recombinant Anti-HDV IgM ↑ acute phase; Short period of Anti-HEV IgM Acute infection; ↓ in early recovery
Immunoblot Assay (RIBA) time
Anti-HAV IgG Past infection/ Immunization Anti-HDV IgG Acute/ Chronic/ Past infection Anti-HEV IgG Later stage of infection; Past exposure

HBV
HBsAg/Australian Ag Anti-HBs/HBsAg HBeAg Anti-HBe HBcAg Anti-HBc/ Total Anti-HBc
• Marker of active infection • Marker of recovery/ • Marker of ↑ degree of • Marker of ↓ degree of • Not detected in serum • Best marker for window period
(acute/ chronic) immunity/ vaccination infectivity infectivity • Detected by: biopsy • Only Ab that can detect recent
• 1st detectable marker in • Viral clearance of HBV • Active viral replication • 1st serologic evidence HBV infection
serum • Protective titer: 10 mIU/mL of convalescent phase • Detect previous/ ongoing
infection
• Detected by: EIA
Anti-HBc IgM Anti-HBc IgG
Only marker detected during Predominant Ab during late recovery
window phase phase
Recent acute infection Past infection