IMMUNOLOGY & SEROLOGY REVIEW NOTES

George Vincent Gellena, RMT, MLS (ASCPi)

Immunology - Study concerned with the processes by which all living organisms defend themselves against infection
Immune System - Integrated system of cells, substances & organs responsible for destruction of foreign substances & keeps the body safe
from injury & infectious agents
Antigens - Refers to substances which are considered foreign to the host

HISTORICAL PERSPECTIVE
YEAR SCIENTIST/S CONTRIBUTION
1798 Edward Jenner Demonstrated Cross Immunity
1885 Louis Pasteur Developed Live, attenuated Vaccine, “Father of Immunology”
1880-1900 Ellie Metchnikoff Demonstrated Phagocytosis, Cellular Theory of Immunology
1891 Robert Koch Demonstration of delayed hypersensitivity reaction
1901 Bordet & Gengou Complement Fixation
1949 Salk & Sabin Polio Vaccine both Injected (Salk) & Oral (Sabin)
1972 Gerald Edelman & Rodney Porter Antibody Structure
1975 Kohler & Milstein Hybridoma technology
1978-1987 Susumo Tonegawa Antibody Diversity
1980 George Snell, Jean Dausset & Baruj Benaceraf Major Histocompatibility Complex
1984 ----------------------------------------------------------- Discovery of the T-Cell Receptor Gene

NATURAL/INNATE IMMUNITY
ANATOMICAL BARRIERS RESIDENT FLORA
- Skin - Mucus - Earwax - Skin – S. epidermidis - Intestines – G(-) anaerobes

- Cilia - Secretions - Oral cavity – Viridans - Vagina – Lactobacillus acidophilus

HEREDITARY & GENETIC INFLUENCE TOLL – LIKE RECEPTORS
CELLULAR DEFENSE MECHANISM - Highest Conc in: Monocytes, Macrophages, Neutrophils
- Neutrophils - Monocytes - Basophils - Examples: TLR2 – G(+), TLR4 – G(-)
- Dendritic cells - Eosinophils - Natural Killer Cells
PHAGOCYTOSIS
1. INITIATION 3. RECOGNITION
- Increased Surface receptors that allow adherence a. Direct Recognition via PPRR
2. CHEMOTAXIS - Primitive Pattern Recognition Receptors
- Process by which cells tend to move in a certain direction b. Indirect Recogniton via Opsonization
under stimulation of chemical substances - Opsonins include: C3b, IgG, CRP
- W/O Chemotaxis, cell motion is Random 4. INGESTION/ENGULFMENT
- Chemotactic agents: C5a & C3a a. Pseudopod Formation
- Process of movement of WBCs from blood, WBCs through b. Phagosome Formation
the blood vessel walls into tissues: Diapedesis c. Phagolysosome Formation
- Impaired Chemotaxis: Jobs Syndrome 5. DIGESTION
- Impaired Random Movement & Chemotaxis - Lysosomal Enzymes (Lactoferrin, Lysozymes, Defensins)
Lazy Leukocyte Syndrome - Defect: Chediak Higashi
- Reactive Oxygen Intermediates
(Superoxide Anion, H2O2, Hydroxyl radicals)
- Defect: Chronic Granulomatous Disease
HUMORAL FACTORS INFLAMMATION
- Soluble substances present in bodily fluids - Involves cellular & vascular responses by the tissues of the body
- Acute Phase Reactants in response to injury
- Complement Components - CARDINAL SIGNS
- Tumor Necrosis Factor a. CALOR: Heat Production
- Against G(-) organisms; from LPS activated phagocyte b. TUMOR: Swelling
- Beta Lysins c. RUBOR: Redness
- Interferon (INF) d. DOLOR: Pain
- Interferon – Alpha: From Null lymphocytes, activates NK e. FUNCTIO LAESA: Loss of Function
cells, aka Leukocyte Interferon - Primary Response: LOCALIZED VASODILATION
- Interferon – Beta: From Fibroblasts: aka Epithelial INF - Acute Phase Reactants
- Interferon – Gamma: From T Cells: aka Immunointerferon - Most Potent: CRP
- Interleukin - Negative Acute Phase Reactant: ALBUMIN
- Small Polypeptides produced by WBCs that act as - Final stages of inflammatory process include resolution &
chemical messages repair
- Ex: IL - 1 - induces Fever; proinflammatory

ADAPTIVE/SPECIFIC IMMUNITY
- Characteristics: Specificity & Memory - Components: B & T cells, Cytokines & Antibodies
FORMS ACTIVE IMMUNITY PASSIVE IMMUNITY

NATURAL Natural Disease Process Transplacental Transfer (IgG), Colostrum

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 Hepa B Immuneglobulin (HBIg), Anti-tetanus,
ARTIFICIAL Vaccination, Toxoids
Rhogam
LYMPHOCYTES
- T CELLS - B CELLS
- Thymus – dependent - Thymus – independent
- 60-80 % of total Lymphocytes - 20-30% of total Lymphocytes
- CELL MEDIATED Immunity - HUMORAL MEDIATED Immunity
- Secrete: Lymphokines - Secrete: Antibodies
- Identified by Erythrocyte-Rosette Assay - Identified by Surface Immunoglobulin
- T Cell Maturation - B Cell Maturation
- Double Negative Thymocyte: No CD4 & CD8 - Pro-B Cell: CD19 & CD45
- Double Positive Thymocyte: Contains CD4 & CD8 - Pre-B Cell: With mu chains in cytoplasm
- Mature T Cell: Either CD4 or CD8 Positive - Immature B Cell: CD21, IgM on Surface
- CD4: Helper T Cells (2/3 of T Cells) - Mature B Cells: IgM & IgD on Surface
- CD8: Cytotoxic/Suppressor T Cells (1/3 of T Cells) - Activated B Cell: added CD25 marker
- Activated T Cell: CD25 positive - PLASMA CELL: produce Antibodies
- Located in the Paracortex of the Lymph nodes - Located in the Cortex of the Lymph nodes
- Periarteriolar regions of the spleen - Primary Follicles & Red pulp of the spleen
- Mitogens of Lymphocytes - Obtaining Lymphocytes from the blood specimen:
- Activate B Cells: Lipopolysaccharide (LPS) - Density Gradient Centrifugation with Ficoll-Hypaque
- Activate T Cells: Concanavaline A & Polyhemagglutinin
- Activate both T & B Cells: Pokeweed Mitogen
- Surface Markers on T Cells & B Cells (CD = Cluster of Differentiation)
CD CELL(S) ASSOCIATED COMMENT(S)
CD2 T Cells SHEEP RED BLOOD CELL RECEPTOR
CD3 T Cells Part of the T Cell Antigen Receptor
CD4 Helper T Cells Coreceptor for MHC Class II; Receptor for HIV
CD8 Cytotoxic/Suppressor T Cells Coreceptor for MHC Class I
CD16 & CD56 NATURAL KILLER CELLS Marker for NATURAL KILLER CELLS
CD21 B Cells Receptor for C3d & EPSTEIN BARR VIRUS
CD34 STEM CELL Marker for HEMATOPOIETIC STEM CELL MARKER

LYMPHOID TISSUES
PRIMARY LYMPHOID TISSUES FUNCTIONS
- BONE MARROW - Tissues or organs in which immune cells undergo maturation
- THYMUS &/or differentiation, & proliferation
SECONDARY LYMPHOID TISSUES - Act as Antigen-trapping sites – initial response depends on how
- Spleen - Peyer’s patches antigens enter the body
- Tonsils - Appendix
- Lymph nodes - Mucosa-assoc lymphoid tissue (MALT)

ANTIGENS (Ag)
- “Antibody Generators”; Antigenic determinants - EPITOPE
FORMS OF ANTIGENS
- IMMUNOGENS: Antigens capable of illiciting an - HAPTEN: Small molecules that are not
immune reponse immunogenic themselves but can be immunogenic when
- AUTOLOGOUS ANTIGEN: The host’s own antigens coupled with a Carrier
- ALLOANTIGEN: Ag derived from different individuals - HETEROANTIGEN/XENOGENEIC: Ag from other species
- Homologous Ag: Induces an Ab production & reacts specifically - Heterologous Ag: Reacts with an Ab it did not induce, causing
with it cross reaction
FACTORS AFFECTING IMMUNOGENICITY OF ANTIGENS
1. Foreignness 4. Chemical Composition
- Must be considered as non-self - Proteins > Polysaccharides > Lipids & Nucleic Acids
2. Complexity 5. Route & Dosage
- Molecules with repeating units are Non-complex & are - Intravenous & Intraperitoneal are effective
less immunogenic - Higher dose of exposure = Greater immune response
3. Size 6. Genetic Composition (MHCs & HLA)
- bigger molecules are more immunogenic
ADJUVANTS
- Included in the Antigen preparations of vaccines & enhance immune response to the Antigen

ANTIBODIES/IMMUNOGLOBULINS (Ab)
- Antibody determinant – PARATOPE FUNCTIONS
- Antibodies can be: - Binds Exogenous Antigens; binding would initiate
- Naturally occurring vs Immunogenic phagocytosis by WBCs or Lysis by Complement action
- Warm-Reactive vs Cold Reactive - Neutralize viruses & toxins
STRUCTURE ANTIBODY STRUCTURE:
- Has a pair of Heavy Chains & Light Chains
- Light Chains are divided into two types: κ, λ (2:1)
- Heavy Chains are divided into five types: α, γ, ε, μ, δ
- Antigen Binding Site involves: 1 Light & ½ Heavy Chain
- Hinge Region is between: CH1 & CH2
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 - Complement Binding Site: CH2 (IgG), CH3 (IgM)
- WBC Binding Site: Fc
FRAGMENTATION OF THE MONOMER
- PEPSIN: Digestion hydrolyzes Ab into 2 fragments - PAPAIN: Digestion hydrolyzes Ab into 3 fragments
- Fc’ + F(ab)2 - Fab + Fab + Fc

VARIATIONS IN ANTIBODIES
- ISOTYPES - SUBCLASSES
- Constant regions of heavy & light chains - Differences in Constant region present within an individual
- Subdivided into Subclasses - ALLOTYPES
- IDIOTYPES - Unique, minor variations w/in the constant region of
- Variations in variable regions that give individual Ab gamma & alpha heavy chains
molecules their specificity to an Ag - Differences in the Constant region between individuals
TYPES OF ANTIBODIES
- IgG - IgM
- Smallest, most abundant, longest living Ab - Largest Ab, & most efficient Agglutination & C’ fixation
- Coating Ab & Clinically Significant - SUBCLASSES
- SUBCLASSES - Pentameric: Found in Plasma; contains J Chain
- IgG1:Most Efficient in Crossing the Placenta - Monomeric: Found on surface of naïve B Cells
- IgG2:Cannot Cross the Placenta - Assoc with the Primary response to Ag
- IgG3:Most Efficient in Complement Fixation - Roles: Classic C’ Pathway Activation, ABO hemagglutinin
- IgG4:Cannot activate Complement - IgA
- Assoc with the Secondary/Anamnestic immune response - SUBCLASSES
- Roles: Complement activation, Opsonization, Neutralization - Monomeric: Found in Blood
Mediator of Ab-dependent cell mediated Cytotoxicity - Dimeric: Found in secretions, contains SecretoryComp
- IgE w/c enables IgA to be transported in mucosal surface
- Previously known as Reaginic Antibody - IgD
- Binds to Eosinophils, Mast Cells, & Basophils - Present as surface Ig of naïve B Cells
- For immune response to helminthic infections & allergies
ANTIGEN-ANTIBODY INTERACTIONS
- CROSS REACTIVITY: Phenomenon where some Ab - AFFINITY: Assoc between Ab & a univalent
secreted by one plasma cell can bind a similar but not identical Ag
epitope. - AVIDITY: Measure of overall binding
- Antigen-Antibody reactions are REVERSIBLE between Ag-binding sites & Multivalent Ag
- PRIMARY INTERACTIONS - SECONDARY INTERACTIONS
- Van der Waals, & Ionic/Hydrogen Bonds - Precipitation & Agglutination
IMMUNE RESPONSE
PRIMARY RESPONSE SECONDARY RESPONSE
- Lag
- Log Initial Exposure to Antigen Subsequent Exposure
- Stationary Longer Lag Shorter Lag
- Decline
Low Antibody Titer High Antibody Titer
Predominantly IgM Predominantly IgG

COMPLEMENT (C’)
FUNCTIONS
- Promoting Phagocytosis through Opsonisation - Promotes inflammatory response by Anaphylatoxins
- Promotes directed movement of WBCs as Chemotacting agents - Promotes clearance of immune complex & cell lysis
GENERAL CONCEPTS
- IgM most potent Ab in terms of Complement fixation - IgG3: Most Active IgG in Complement activation
- To activate, complement proteins bind the CH2(IgG) & CH3(IgM) - IgG4: IgG that does not activate the complement
of antibodies
CLASSICAL PATHWAY ALTERNATE PATHWAY
- Trigger: Immune Complex - Triggers: LPS, Fungal Cell Wall, Virally infected cells…
- Recognition Unit: C1 - Factor D: Cleaves Factor B into Bb in the presence of C1 & Mg +
- Activation Unit: C1q, C1r, C1s - Factor P (Properdin): Binds to C3Bb to prevent decay
- Factor B: Bind C3b to form C3 convertase
MANNA-BINDING LECTIN PATHWAY
- Does not require C1
- Activating factor: Mannose groups of CHO in microbial cell
- Effectors: Mannan Binding Protein/Lectin, MASP (MBP Assoc Serine Protease)
CLASSICAL ALTERNATE LECTIN
ACTIVATING SUBSTANCE Immune Complex LPS, IgA Mannose group in microbial cell
RECOGNITION UNIT C1q, C1r, C1s C3, Factor B, Factor D MBP, MASP-1
C3 CONVERTASE C4b2a C3bBb C4b2a
C5 CONVERTASE C4b2a3b C3bBb3bP C4b2a3b
MAC C5b6789
END RESULT CELL LYSIS

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COMPLEMENT REGULATION
- C1 Inhibitor (C1 INH): Dissociates C1r & C1s from C1q
- C4 binding protein: cofactor of Factor I; Inactivate C4b
- S Protein/Vitronectin: prevents attachment of C5b67
complex to the cell membrane
DEFICIENCIES OF COMPLEMENT PROTEINS & ASSOCIATED DISORDERS
- C1 INH: Hereditary Angioneurotic edema
- C5 – C8: Neisseria Infections
- C2: Most common of the human complement deficiencies
- Factor I: Cleaves C3b & C4b
- Factor H: Prevent binding of B to C3b; Cofactor of Factor I
- Decay Accelerating Factor (DAF): capable of dissociating
C3 Convertase
- C2 & C4: Immune complex disorder, SLE-like syndrome
- C3: Severe recurrent infections, glomerulonephritis
- C3: Most severe type of complement deficiency

MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)

- Genes located on the Short Arm of Chromosome 6
- in Humans, it is referred to as Human Leukocyte Antigen (HLA) Complex – encoded by MHC genes
MHC CLASS - 1 MHC CLASS - 2
- Present in ALL NUCLEATED CELLS - Found in B Cells, Marcophages, PMNs, Dendritic Cells (APCs)
- Includes: HLA – ABC - Includes: HLA – DP, DQ, DR
- Presents Ag to: CD8(+) T cells - Presents Ag to: CD4(+) T cells
- Mainly present peptides that have been synthesized w/in the - Mainly bind exogenous peptides; w/c are Ag taken from
cell outside of the cell & then degraded by the cell
MHC CLASS - 3
- Minor MHC Antigens
- Involves Complement components C2, C4, & Factor B
- HLA on RBC: Benett-Goodspeed
HLA PHENOTYPING HLA GENOTYPING
- PRINCIPLE: Complement Dependent Cytotoxicity - Detects specific HLA genes
- Panels of antisera that define the individual are incubated with - Makes use of molecular-based tests such as PCR
Lymphocytes from the individual to be HLA typed HLA ANTIBODY SCREENING & IDENTIFICATION
- Purified T cells are used for HLA class I typing - Antibodies to HLA can be detected in candidates & recipients
- Purified B cells are used for HLA class 2 typing of solid organ transplantation
- After incubation, Complement is added
- Lymphocytes with the HLA Ag will have an Ab bound to it, w/c
reacts with Complement, leading to cell lysis
- Vital dye(Trypan blue) is then added to distinguish live from
dead cell
HLA SIGNIFICANCE
- Involved in Antigen presentation
- Used for Paternity Testing
- Can be used as Anthropological marker
- An important factor in Organ Transplantation
- DISEASE ASSOCIATION
- HLA – B27: Ankylosing Spondylitis
- HLA – DR2, D3: Systemic Lupus Erythematosus
- HLA – DR4: Rheumatoid Arthritis
- HLA – DR3, DR4: Type 1 Diabetes Mellitus

SEROLOGY
- SEROLOGICAL Tests: uses Ag & Ab interactions as tool for - Analytes: substances to be measured
diagnosis
- Serological reactions can be measured & expressed using Titers - Titer is the RECIPROCAL of the highest dilution of the sample
that still results in a positive result
- Mathematics of Serology – Dilution - Serological Tests can be grouped into
- Formula: - Agglutination, Precipitation, Complement Fixation,
- Diluent: NSS/0.85% NaCl Solution Neutralization, Labelled Immunoassay, Nucleic acid tests
- ANTIBODY-ANTIGEN RATIO
- ZONE OF EQUIVALENCE - point at which the most Ab is - PROZONE: Excess Antibody
precipitated by the least amount of Ag - POSTZONE: Excess Antigen
- Optimal Ratio of serum to cell: 2:1

PRECIPITATION
- Involve combination of Soluble Ag & Soluble Ab
- Measured by either Turbidimetry or Nephelometry
PASSIVE IMMUNODIFFUSION
I. Single Immunodiffusion
1. Single Linear Immunodiffusion 2. Single Radial Immunodiffusion
- Ab (serum) is in agar tube; Ag is overlain on top a. MANCINI
- Ag moves through the gel to form Precipitin band - Endpoint method
- Example: Oudin - Measurement at 24 hrs (IgG) & 50-72 hrs (IgM)
- diameter2 = Concentration
b. FAHEY-MCKELVEY
- Kinetic method
- Measurement after 18 hrs

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 - diameter = long of concentration
II. Double Immunodiffusion (Ouchterlony)
- Ag is placed in outer wells, Ab are placed in the inner well
- PATTERNS
1. IDENTITY: Presence of a smooth arc
2. NON-IDENTITY: Presence of crossed lines
3. PARTIAL IDENTITY: Presence of a single spur
ELECTROPHORESIS
I. One Stage Electrophoresis II. Two-Stage Electrophoresis
A. Rocket Electrophoresis A. Classic Immunoelectrophoresis
- aka Laurell electrophoresis - Electrophoresis + Immunodiffusion
- Ab is incorporated in the gel, only the Ag diffuses w/ - Ab is placed in trough
the aid of electric current - (+): Precipitin arc
- (+): Precipitin rockets (bullet-shaped) B. Immunofixation electrophoresis
B. Counterimmunoelectrophoresis - Electrophoresis + Immunodiffusion
- Aka Counter current immunoelectrophoresis - Ab is overlain in surface in gel
- Ag moves toward the ANODE - (+): Precipitin bands
- Ab moves toward the CATHODE C. Crossed (2 dimensional) immunoelectrophoresis
- (+): Precipitin line - Electrophoresis + Electrophoresis
- Ab is incorporated in the 2nd gel
- (+): Precipitin Rockets

AGGLUTINATION
- Involves Particulate Ag interaction with an Ab
- Stages of Agglutination: Sensitization/Coating  Lattice Formation
DIRECT AGGLUTINATION INDIRECT AGGLUTINATION
- Ag is Naturally attached to the carrier molecule - Ag is Artificially attached to the carrier molecule
- Carrier can be RBCs or Bacteria - Carriers include: Latex, Bentonite, Beads, or Charcoal
- Ex: Febrile Agg’n Test & ABO Forward typing - Used to detect Ab
- Ex: ASO Latex Agg’n test & TPPA
REVERSE PASSIVE AGGLUTINATION AGGLUTINATION INHIBITION
- Ab is Artificially attached to the carrier molecule - RESULTS
- Attachment is through Fc region not Fab region - POSITIVE: NO AGGLUTINATION
- Used to detect Ag - NEGATIVE: AGGLUTINATION
- Ex: CRP Latex Agg’n Test - Two Stages: Neutralization Phase & Indicator Phase
- Ex: HCG for Pregnancy Testing
ANTIGLOBULIN TEST (COOMB’S)
- Anti-Human Globulin (AHG) serves as a bridge to connect two non-agglutinating antibodies
I. Direct Antiglobulin Test (DAT) II. Indirect Antiglobulin Test (IAT)
- Detects for IN-VIVO sensitization or coating - Detects for IN-VITRO sensitization or coating
- Specimen: RBCs - Specimen: Serum
- Conditions Associated: - Application:
A. Hemolytic Transfusion Reaction (HTR) A. Crossmatching/Compatibility Testing
B. Hemolytic Disease of the Newborn (HDN) B. Antibody Screening & Panel
C. Autoimmune Hemolytic Anemia (AIHA) C. Antibody Titration
- SOURCES OF ERROR
FALSE POSITIVE FALSE NEGATIVE
Overcentrifugation Undercentrifugation COMPLEMENT FIXATION
Contaminated glassware Inadequate washing - (+) No Hemolysis; (-) Hemolysis
Autoagglutination Reagents not active - STEPS:
Presence of cross-reactivity Delays in testing procedure a. Add reagent Ag to Serum
Presence of rheumatoid factor Incorrect/Insufficient b. Add Complement
Delay in reading a slide test incubation time c. Add sensitized RBC
Drying Prozone & Postzone d. Observe for hemolysis/absence of hemolysis
Failure to add AHG reagent

LABELED IMMUNOASSAY
- Involves Ag & Ab interaction, w/ either reactants labelled w/ a - Separation Step
marker so that the amount of binding may be monitored - Provides a simple way to separate bound & free reactants
- Labels: Act as marker to detect Ag-Ab reaction - Use of Physical Means
- Enzyme Immunoassay uses enzymes - Includes: Decanting, Centrifugation, or Filtration
- Fluorescence Immunoassay uses fluorochrome dyes - Followed by a washing step
- Radioimmunoassay uses radioisotopes - Use of Solid Phase Vehicle
- Labeled Species: may be an Ag or Ab to w/c the label is attached - Detection Step
- Solid Phase: Media such as the well or microplate, to w/c Ag or - Radioimmunoassays: GAMMA (SCINTILLATION) COUNTER
Ab is attached - Enzyme Immunoassays: SPECTROPHOTOMETRY
- Heterogenous: Requires a step to physically separate any - Fluorescence Immunoassays: FLUOROMETERS
unbound analyte
- Homogenous: No separation step is necessary
FORMS OF LABELED IMMUNOASSAY
I. Non-Competitive
A. Direct
- Only Two Layers - NO Solid Phase - Labeled species: Ab that targets Ag
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 - Detects: Antigen - Concentration: Direct
B. Indirect
- Detects: Antibodies - Labeled species: Anti-immunoglobulin
- Solid Phase: Antigen - Concentration: Direct
C. Capture/Sandwich
- Detects: Antigen - Labeled species: Ab that targets Ag
- Solid Phase: Antibody - Concentration: Direct
II. Competitive
- Detects: Antigen - Labeled species: Antigen
- Solid Phase: Antibody - Concentration: Inverse
ENZYME IMMUNOASSAY FLUORESCENCE IMMUNOASSAY
- Advantages: Cheap & readily available; have a long shelf life - Fluorochromes used: FITC (Fluorescein thiocyanate), europrim,
easily adapted to automation; no disposal problems; no tetramethylrhodamine, phyocoerythrin, Lucifer yellow, Texas
health hazards red
- Alkaline phosphatase & Horseradish peroxidase are routinely - FORMS
used a. Direct FIA
- Additonal Method: Immunoenzymetric - Ex: Direct Fluorescence for T. pallidum
- Detects: Antigen - Labeled species: Ab - Specimen: Fluid from Chancre
- Solid Phase: Unattached Ag - Concentration: Direct
RADIOIMMUNOASSAY
- First type of Immunoassay developed by Yalow & Berson
- Radioisotopes used: 131 I, 123 I, & 3H
- Disadvantages: Health hazard; difficult & expensive; disposal b. Indirect FIA
problems; short shelf life - Ex: FTA - Abs
- Forms: Non-competitive, Competitive, & Immunoradiometric - Specimen: Serum (Ab to T. pallidum)
- Applications:
a. Radioimmunosorbent Test (RIST): TOTAL IgE
b. Radioallergosorbent Test (RAST): ALLERGE SPECIFIC IgE

NUCLEIC ACID TESTS

POLYMERASE CHAIN REACTION MOLECULAR BLOTTING TECHNIQUES
- Amplifies tiny quantities of nucleic acid up to detectable levels - Southern Blot
- STEPS: - Northern Blot
1. Denaturation
2. Annealing
3. Extension

DISEASES OF THE IMMUNE SYSTEM

AUTOIMMUNITY
- The body’s immune system mounts an immune response against the host’s Ag
- Autoimmune Diseases
- Systemic Lupus Erythematosus: Anti-dsDNA, Anti-Sm - Wegener’s Granulomatis: Anti-Neutrophil Cytoplasmic Ab
- Rheumatoid Arthritis: Rhematoid factor - Goodpasture’s Syndrome: Anti-glomerular/alveolar
- Diabetes Mellitus Type 1: Anti-Beta cells, Anti-insulin basement membrane
- Pernicious Anemia: Anti-Parietal cells - Addison’s disease: Ab against adrenal glands
- Autoimmune Hemolytic Anemia: Cold/Warm AutoAb - Multiple Sclerosis: Anti-myelin sheath
- Hashimoto’s thyroiditis: Anti-microsomal/TPO/ - Myasthenia gravis: Anti-acetylcholine receptor
Thyroglobulin - Primary Biliary Cirrhosis: Anti-mitochondria
- Grave’s Disease: Anti-TSH receptor - Chronic Active Hepatitis: Anti-Smooth Muscle
- CREST: Anti-centromere - ITP: Ab against Platelets (after viral infection)
- Tests related to Autoimmune disorders
- Rheumatoid Arthritis - Systemic Lupus Erythematosus
- Charac. by: Rf – IgM that targets Fc of IgG - Charac. by: LE Cell, Lupus inhibitor, Butterfly rash
- Latex tests are more sensitive but sheep cell Agg’n test - Substrate to detect anti-dsDNA: Crithidia lucilae
is more specific - Test: ANA by immunofluorescence
- Rf Latex slide test - Specimen: Serum
- Reagent: Latex coated with human gamma - PATTERNS
globulin a. Homogenous: anti-DNA (SLE), anti-DNP
- Specimen: Serum dilutions b. Peripheral: anti-dsDNA (SLE)
- (+): Agglutination c. Speckled: anti-Smith (SLE), anti-RNP
- Rf Latex tube test d. Nucleolar: anti-RNA precursors
- the same as the slide test although uses tube
HYPERSENSITIVITY
- Types of Hypersensitivity
I – Allergic II – Cytotoxicity III – Immune Complex IV – Delayed
Immune Mediator BASOPHILS, IgE IgM, IgG IgM, IgG T CELLS

Antigen Involved NO YES YES NO

Complement Involvement ALLERGEN
Release of Inflammatory
Mechanism Mediators from Basophils &
Mast Cells
Examples Anaphylaxis, Hay Fever,
CELL BOUND ANTIGEN SOLUBLE ANTIGEN SENSITIZED ANTIGEN
Cell Lysis due to Antibody & Deposition of Antigen- Release of Cytokines
Complement Antibody Complex
HTRs, AIHA, HDN Serum sickness, Arthus Contact Dermatitis,

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 Allergies, Asthma
IMMUNODEFICIENCY
- B Cell Disorders
- X linked agammaglobulinemia: lack of Ig in all classes
- Selective IgA deficiency: Most Common immunodeficiency
- Combined T & B Cell Disorders
- Severe Combined Immunodeficiency
- Wiskott-Aldrich syndrome: Immunodeficiency, eczema
& thrombocytopenia
- Ataxia-Telangiectasia: Charac. by Cerebrallar ataxia
& telangiectasias on the earlobes & conjunctiva
ORGAN TRANSPLANTATION
TYPES OF GRAFT
HOST-vs-GRAFT DISEASE (ORGAN REJECTION)
- Autograft: from the same individual
- Isograft/Syngraft: from identical twins
- Allograft: from different individual, same species
- Xenograft: from different species
IMMUNOGENICITY OF ORGANS
- Most Immunogenic: Bone Marrow
- Least Immunogenic: Cornea
- Other organs: Skin, Heart, Kidney, Liver, & Bone
SYPHILIS
NON-TREPONEMAL/NON-SPECIFIC TESTS
- used to detect REAGIN (anti-cardiolipin Ag)
- Non-specific; Often used as Screening Procedure
a. VDRL (Venereal Disease Research Laboratory)
- Principle: Quali or Quanti Slide Flocculation test
- Rotator: Serum – 180 rpm for 4 minutes
CSF – 180 rpm for 8 minutes
- Reagent: CARDIOLIPIN = Responsible for reactivity
CHOLESTEROL = Increases size of Ag
LECITHIN = Standard Reactivity
- Requires Serum inactivation:
- Inactivation is done by heating serum for 56C for 30 mins
- Inactivated serum must be used w/in 4 hrs
- If >4 hrs, reinactivate by heating it at 56C for 10 mins
b. RPR (Rapid Plasma Reagin)
- Principle: Flocculation
- Reagent: Cardiolipin, Lecithin, Cholesterol, Charcoal,
Choline chloride (C’ inactivator), & thimerosal
- Rotator: 100 rpm for 8 minutes
TREPONEMAL TESTS
- Detects Ab directed towards T. pallidum
- More Specific than NTT; used as Confirmatory test
a. TPI (T. pallidum Immobilization Test)
- Treponemal test of Reference
- Specimen: Serum
- Principle: Ab produced against T. pallidum plus C’ can
immobilize the live treponemes
- Reagent: Live actively motile T. pallidum organism
- (+): >50% immobilized treponemes
c. MHA-TP (Microhemagglutination T. pallidum test)
- Principle: Hemagglutination
- Reagent: Tanned formalin Sheep RBC with treponemal Ag
GROUP A STREPTOCOCCAL INFECTION
- S. pyogenes causes suppurative conditions (impetigo, pharyngitis), toxigenic conditions (scarlet fever), non-suppurative conditions
(acute rheumatic fevr, acute glomerulonephritis)
- Sequela of S. pyogenes: Acute Rheumatic Fever, Post-streptococcal Acute Glomerulonephritis
ASO (ANTI-STREPTOLYSIN O) TEST
- Latex Agglutination Test
- Based on Indirect Agglutination
- Tube test is based on Neutralization (ASO Titration)
- Dilutions of Patient serum is incubated with SLO reagent
- RBCs are added & observed for presence or absence of
hemolysis
- Inhibition of hemolysis is related to the presence of ASO
in Patient serum
- Report of Titer
- Reported using Todd units
IMMUNO-SERO NOTES
reaction, SLE Tuberculin test
- T Cell Disorders
- DiGeorge’s Anomaly: Defect in thymus development
- PNP deficiency: defect in T cells due to purine accumulation
- Phagocytic Disorders
- Chronic Granulomatous Disease: Defect in the respiratory
or oxidative burst
- Chediak Higashi Syndrome: Lysosomal granules defect
- Job Syndrome: defect in Chemotaxis
- Lazy Leukocyte: defect in chemotaxis & random movement
TYPE TIME OF TISSUE DAMAGE
Hyperacute Within minutes
Accelerated 2-5 days
Acute 7-21 days
Chronic Later than 3 months
- Gauge numbers for delivering the Ag suspension
- Quali test: 18g = 60+2 drops of Ag susp/mL
- Quanti test: 19g = 75+2 drops of Saline/mL
- Reporting
- Reactive: Medium to large clumps
- Weakly Reactive: Small clumps
- Non-reactive: No clumping
- Positive VDRL test on Spinal fluid is diagnostic of
Neurosyphilis
- Results are read MICROSCOPICALLY
- Advantages: No heat inactivation req’d
Charcoal: Make reaction more visible
Results are read MACROSCOPICALLY
b. FTA-Abs (Fluorescent T. pallidum Ab-Absorbed Test)
- Principle: Indirect Fluorescent Immunoassay
- Reagent Antigen: Nichol’s strain dried & fixed on slide
- Sorbent: Reiter treponemes
- Label: FITC - AHG
d. HATTS (Hemagglutination Treponemal test for Syphilis)
- Principle: Hemagglutination
- Reagent: Turkey RBC coated with treponemal Ag
ANTI-DNAse B
- Detects Ab that targets DNAse
- Principle: Neutralization
- Reagent: DNA-methyl green substrate
- If anti-DNAse is present, they will neutralize the reagent DNAse
- Presence of DNAse is measured by its effect on a DNA methyl
green is reduced & become colorless
GVGGELLENA,RMT,MLS(ASCPi)
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 - Controls used: SLO control: Hemolysis
RBC control: No Hemolysis
FEBRILE AGGLUTININS
SALMONELLA RICKETTSIA
- Test: Widal Test - Test: WEIL-FELIX
- Detects Ab against Salmonella - Detects Rickettsial Antibodies
- Principle: Direct Agglutination - Antigens used: OX-19 & OX-2 (P. vulgaris)
- Antigens used: Somatic – O (Thermostable) OX-K (P. mirabilis)
Capsular – K (Thermolabile - Scrub Typhus: OX-K
Flagellar – H (Thermolabile) - RMSF, Epidemic, & Murine Typhus: OX-19 & OX-2
- Typhidot: Detects IgG & IgM to S. typhi - Rickettsial Pox & Q Fever: Q Fever

MYCOPLASMA PNEUMONIAE MALARIA

- Causes: Primary Atypical Pneumonia
- Associated with: Anti-I
- Laboratory testing involved testing for Cold Agglutinins
- Cold agglutinins are AutoAb that agglutinate RBCs at
temperatures below 37C
- Definitive diagnosis: Thick & Thin Smears
- Immunochromatographic Assays
a. OptiMal Assay: Detects Parasitic LDH
b. MalaQuick: Detects P. falciparum’s Histidine Rich Protein
HRP - 2

HUMAN IMMUNODEFICIENCY VIRUS (HIV) HEPATITIS VIRUSES

- Aka HTLV-III, LAV, & ARV HEPATITS A
- First Retrovirus isolated from man: HTLV-I - Infectious Hepatitis (Self-limiting)
- Genes related to HIV: - Agent: HAV (Picornaviridae)
a. Env: gp 120 c. Pol - MOT: Fecal-oral route
b. Gag: p15, p17, p24 - MARKERS: a. Early shedding of virus in stool
- First marker to be Positive: p24 antigen & viral RNA b. Appearance of IgM anti-HAV
- Screening Test: ELISA c. Development of anti-HAV IgG & immunity
- A positive initial screening test must be repeated HEPATITIS B
- Confirmatory Test: WESTERN BLOT - Serum Hepatitis
- Bands: p24, gp41, & gp 120/160 - Agent: HBV (Hepadnaviridae), Dane Particle
- At least 2 out of 3 bands must be present - MOT: Parenteral, Vertical, Sexual
- Ratio of CD4:CD8 in HIV: 1:2 - Markers
- Indicative of AIDS if CD4 count is: < 200/Ml a. HBsAg: First marker to appear;current HBV infection
b. HBeAg: Active Hepatitis B with high degree of
INFECTIOUS MONONUCLEOSIS (IM) infectivity
- Agent: Epstein-Barr Virus c. HBcAg: Not part of Hepatitis B profile test
- Target Cells: B Lymphocytes d. IgM anti-HBc: First Ab to appear; detecting infection
- Charac. by the presence of: Downey cells (Atypical T cells) in the window period
- Assoc with Burkitt’s lymphoma & Autoanti-i e. anti-HBc Total: Predominantly IgG; life long marker
- Heterophile Antibodies: Ab that body produces as part of an of Hepatitis B
immune response to an infection but that are not related f. Anti-HBe: Sign of recovery
to the causative agent g. Anti-HBs/HBsAb: Marker of Immunity
SEROLOGICAL TESTS FOR IM - Tests for HBV
a. Paul Bunnel Test a. First Generation: Ouchterlony
- Screening Test b. Second Generation: Counterelectrophoresis,
- Principle: Hemagglutination Complement Fixation, Rheophoresis
- Reagent: 2% suspension of Sheep RBCs c. Third Generation: RIA, RPHA, ELISA, RPA
- (+): Agglutination HEPATITIS C
b. Davidson Differential Test - Post Transfusion Hepatitis
- Confirmatory Test - Agent: HCV (NANB)
- Principle: Absorption-Hemagglutination - MOT: Parenteral (Blood Transfusions)
- Antigen: Guinea pig kidney cells & Beef RBCs - Tests for HCV
- Indicator Cells: Sheep RBCs a. Surrogate Test: ALT & anti-HBc
ABSORPTION PATTERN b. Serologic Test for anti-HCV: ELISA & RIA
Guinea Pig Kidney c. Quantitative Test for HCV: Measuring HCV viral load
Heterophil Beef RBCs HEPATITIS D
Cells
Forssman NO YES - Delta Hepatitis
IM YES NO - Agent: Hepatitis D Virus (HDV)
Serum Sickness YES YES - A defective virus; requires HBV for replication
- MOT: Parenteral & Sexual
AGGLUTINATION PATTERN
- Co-infection & Super infection with HBV
After Absorption
After Absorption - Laboratory Diagnosis
Heterophil with Guinea Pig
with Beef Cells - Indirect ELISA:
Kidney Cells
a. Anti-HDB
Forssman High Titer Low Titer b. Anti-HBc IgM differentiates coinfection (+) from
IM Low Titer High Titer super infections (-)
Serum Sickness Low Titer Low Titer - PCR

c. Monospot (Slide Method) SOURCES:

- Principle: Absorption-Hemagglutination 1. Henry’s Clinical Diagnosis & Mngt by Lab Methods
- Indicator cells: Horse RBC 2. Clinical Immunology & Serology Internat’l by Stevens
3. Immunology & Serology in Lab Medicine by Turgeon
IMMUNO-SERO NOTES GVGGELLENA,RMT,MLS(ASCPi)
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4. Essentials of Immunology & Serology by Stanley
5. Lec Handouts of Prof Judea Policarpio & Prof Jude Trinidad

IMMUNO-SERO NOTES GVGGELLENA,RMT,MLS(ASCPi)

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