Immunology Review

12/8/21 1
 Immunology Terms
Acute phase Protein that increases due to infection, injury, or
reactant trauma (e.g., C-reactive protein, alpha-1 antirypsin,
haptoglobin, fibrinogen, ceruloplasmin, alpha-1 acid
glycoprotein, complement)
Alloantibody Antibody formed in response to antigens from
individuals of the same species
Antigen A foreign substance that stimulates antibody
production. Large, complex molecules
(MW>10,000), usually protein or polysaccharide.
Antibody Immunoglobulin produced by plasma cells in
response to an antigen

12/8/21 2
 Autoantibody Antibody against self
Avidity Strength of bond between antigen and antibody
Chemotaxin Chemical messenger that causes migration of cells in
a particular direction
Clusters of Antigenic of leukocytes
differentiation
(CD)
Cytokine Chemical messenger produced by stimulated cells that
affect the function of the other cells
Epitope Determinant site on an antigen
Hapten A low molecular weight substance that can bind to an
antibody once it is formed, but that is incapable of
stimulating antibody production unless it is bound to a
larger carrier molecule

12/8/21 3
 Histamine A vasoactive amine that is released from mast cells and
basophils during an allergic reaction
Hypersensitivity A heightened state of immune responsiveness that causes
tissue damage in the host
Immunity Resistance to infection
Immunogen Any substance that is capable of inducing an immune
response
Immunoglobulin Antibody
Inflammation Cellular and humoral mechanisms involved in the reaction
of the body to injury or infection
Interferons Cytokines produced by T cells and other cells that inhibit
viral synthesis or act as immune regulators
Interleukins Cytokines produced by T cells or macrophages that
stimulate a number of cell types

12/8/21 4
 Ligand A molecule that binds to another molecule of a complementary
configuration. The substance being measured in an immunoassay.
Lymphokines Polypeptides given off by antigen-stimulated T cells, which
regulate the function of other cells
Lysozyme Enzyme found in tears and saliva that attacks cell walls of
microorganisms
MHC Major histocompatoibility complex. Genes that control the
expression of proteins found on all nucleated cells.
Monoclonal An antibody derived from a single B-cell clone
antibody
Opsonin Serum proteins that attach to a foreign substance and enhance
phagocytosis
Phagocytosis The engulfment of cells or particulate matter by neutrophils and
macrophages

12/8/21 5
 Plasma cells Transformed B cell that secretes antibody
Polyclonal antibody Antibody produced by many B-cell clones
Postzone False-negative reactions in a serological test due to antigen excess
Prozone False-negative reactions in a serological test due to antibody
excess
Seroconversion The change of a serological test from negative to positive due to
development of detectable antibody
Serum sickness A type III hypersensitivity reaction that results from the buildup of
antibodies to animal serum used in some passive immunizations
Thymus A small, flat bilobed organ found in the thorax. Site of T-
lymphocyte development.
Titer A means of expressing the concentrations of an antibody. The
reciprocal of the highest dilution in which a positive reaction
occurs.
Vaccination Injection of immunogenic material in order to induced immunity

12/8/21 6
 Branches of the Immune System

Branch Definition Defense Against Cells Involved Examples

Cellular Cell mediated Viruses, fungi, T lymphocytes Graft rejection,

mycobacteria, other hypersensitivity
intracellular pathogens, reaction, elimination of
tumor cells tumor cells

Humoral Antibody Bacteria (extracellular) B lymphocytes, Antibody production

mediated plasma cells

12/8/21 7
 Types of Immunity

Type Explanation Components Memory

Natural or innate Non-specific defenses Intact skin, mucous membranes, No

with which one is born stomach acidity, cilia, mucus,
skin secretions, lysozyme, IgA,
phagocytes, complement.
Interferon, inflammation
Acquired or Specific responses to T cells, B cells, plasma cells, Yes
adaptive eliminate a antibodies, cytokines
microorganism

12/8/21 8
 Adaptive Immunity
Type Explanation Example Specific? Immediate? Long-
term?
Naturally An individual Clinical or Yes No Yes
acquires infected with a subclinical
active microorganism infection
immunity produces an
antibody.
Artificially An individual DPT, MMR, Yes No Yes
acquired exposed to an polio,
active antigen through a tetanus,
immunity vaccine develops Haemophilus
immunity influenzae
without having type b
the infection. vaccine

12/8/21 9
 Adaptive Immunity Continued
Type Explanation Example Specific? Immediate? Long-
term?

Naturally An individual is Babies are Yes Yes No

acquired protected by antibodies protected by
passive produced in another maternal
immunity individual. antibodies that
cross the
placenta and
present in breast
milk

Artificially An individual receives Rh immune Yes Yes No

acquired an immune globulin globulin, HBIG,
passive containing antibodies anti-toxins
immunity
12/8/21 produced in another 10
individual.
 Immunoglobulins
IgG IgM IgA IgD IgE
Form Monomer Pentamer Monomer Monomer Monomer
and dimer
monomer
Molecular 150000 900000 160000 or 180000 190000
weight 400000
(daltons)
Heavy chain Gamma (γ) Mu (μ) Alpha (α) Delta (δ) Epsilon (ε)
Light chain Kappa or κ or λ κ or λ κ or λ κ or λ
lambda (κ or
λ)
% of total 75-80% 10% 15-20% <1% 0.004%
immunoglob
ulin

12/8/21 11
 IgG IgM IgA IgD IgE

Serum 800-1600 120-150 70-350 1-3 0.005mg/dL

concentration mg/dL mg/dL mg/dL mg/dL

Antigen- 2 10 2 or 4 2 2
binding sites

Complement Yes Yes No No No

fixation

Crosses Yes No No No No
placenta?

12/8/21 12
 IgG IgM IgA IgD IgE
Other Defense First Ig produced In tears, Function Role in allergic
against in an immune sweat, unknown reactions.
bacteria and response. Only saliva, Binds to mast
viruses. Ig at initiating respiratory cells and
Neutralizes complement and GI triggers
toxins. cascade. More mucosa. degranulation
Opsonin. efficient at First line and release of
More efficient agglutination of defense. histamine and
at precipitation than IgG. heparin.
than Destroyed by
agglutination. sulfhydryl
compounds.

12/8/21 13
 Cells of the Immune System
Cell Function Comments

T lymphocytes Cell-mediated immunity Derived from cells in bone marrow.

Develop T-cell specific surface antigens
in thymus (e.g., CD2, CD3, CD4, cD8).
60-80% of peripheral lymphocytes.

Helper/ inducer T cells Orchestrates cell-mediated CD4+ (T4). .55-70% of peripheral T

immunity. Activates B cells, cells. Normal CD4 = 1,000/μL. In
cytotoxic cells, and natural AIDS, less than 0.5:1).
killer cells

B lymphocytes Synthesize and secrete Develop in bone marrow. Surface

immunoglobulins after immunoglobulin and CD19, CD20,
antigenic challenge CD21. Less than 15% of lymphs.

12/8/21 14
 Cells of the Immune System Continued
Cell Function Comments

Suppressor/cytotoxic T Suppressor cells inhibit helper T CD8+ (T8). 25-40% of

cells cells. Cytotoxic cells kill other cells. peripheral T cells. (Normal
CD4:CD8 ratio is 2:1. In
AIDS, less than 0.5:1).
Lymphokineactivated Use IL-2 to help lyse tumor cells Non-T, non-B
killer cells (LAK)

Eosinophils Suppress inflammatory reaction. Some phagocytic ability

Kill some parasites

Basophils Granules contain histamine and Some phagocytic ability.

heparin. Role in hypersensitivity Precursor of mast cell.
reactions.

12/8/21 15
 Cells of the Immune System Continued

Cell Function Comments

Neutrophils Principal leukocyte associated with

phagocytosis and localized inflammatory
response. Granules contain bactericidal
enzymes. Respond to chemotaxins.

Monocytes Phagocytes. Respond to chemotaxins. Precursor of macrophages

Process antigens and present to
lymphocytes. Produced interleukin-1 and
other cytokines.

12/8/21 16
 Lymphoid Organs

Primary Lymphoid Organs Secondary Lymphoid Organs

Bone marrow Sleen

Thymus Lymph nodes

Tonsils
Appendix
Peyer’s patches in the intestines
Other mucosal-associated lymphoid tissue
(MALT)

12/8/21 17
 Laboratory Identification of
To Obtain Lymphocytes Lymphocytes
Density gradient centrifugation with Layers from top to bottom: plasma, mononuclear cells,
Ficoll-Hypaque Ficoll-Hypaque, RBCs and granulocytes

To Identify Lymphocytes Use of labeled monoclonal antibodies against specific

Direct or indirect immunofluorescence surface antigens. Slides are read with a fluorescent
microscope.

Flow cytometry (FACS: fluorescence Use of labeled monoclonal antibodies against specific
activated cell sorter) surface antigens. Light scattering is measured as cells
flow through a laser beam.

Surface immunoglobulins Immunoenzymatic method to demonstrate

immunoglobulis on B cells

Rosette technique T lymphocytes form rosettes with sheep RBCs. Old

method.

12/8/21 18
 Common Lymphocyte Markers

T cells B cells

CD2 CD19
CD3 CD20

CD4 CD21

CD8 Surface immunoglobulins

12/8/21 19
 Complement

Definition A series of more than 25 serum proteins that interact to enhance

host defense reactions. Most are inactive enzyme precursors that
are converted to active enzymes in a precise order (cascade).

Functions Opsonization, chemotaxis, cell lysis

Classical Triggered by antigen-antibody reactions. IgM is most efficient

pathway activator. A single molecule attached to two adjacent antigenic
determinants can initiate the cascade. IgG1, 2, and 3 can activate
complement but at least two molecules are required. Recognition
unit: C1 (first to bind).
Activation unit: C4, C2, C3.
Membrane attack complex: C5, C6, C7, C8, C9 (drills holes in cell
membrane).

12/8/21 20
 Immunoglobulin Structure

Basic structure A four-chain polypeptide unit that consists of two heavy chains
and two light chains held together by disulfide bonds

Heavy chains γ, α, μ, δ, and є. The two heavy chains in the immunoglobulin

molecule are always of the same type. They determine the
immunoglobulin class (IgG, IgA, IgM, IgD, and IgE).

Light chains κ or λ. Both κ and λ are found in all classes of immunoglobulins,

but only one type is present in a given molecule.

Fab fragment Fragment antigen-binding. Consists of one light chain and one-
half of a heavy chain. The intact immunoglobulin has two Fab
fragments, each representing one antigen-binding site.

12/8/21 21
 Immunoglobulin Structure Continued
Fc fragment Fragment crystalline. The carboxy-terminal halves of the two
heavy chains. This portion of the molecule has no antigen binding
ability.
Constant region The amino-terminal end of the immunoglobulin molecule, where
the amino acid sequence is the same for all chains of that type.
Variable region The amino-terminal end of the immunoglobulin molecule, where
the amino acid sequence varies. This part of the molecule is
responsible for the specificity of a particular immunoglobulin.
Also known as the antigen-recognition unit.
Hypervariable Regions within the variable region that actually form the antigen-
region binding site. Through changes in the hypervariable region, an
immense diversity of the antigen-binding sites can be created.

12/8/21 22
 Immunoglobulin Structure Continued

Hinge region The flexible portion of the heavy chain, located between the first
and second constant regions. This allows the molecule to bend to
let the two antigen-binding sites operate independently.
Joining chain A glycoprotein that serves to link immunoglobulin monomers
together. Only found in IgM and secretory IgA molecules.

12/8/21 23
 Complement Continued

Definition A series of more than 25 serum proteins that interact to enhance

host defense reactions. Most are inactive enzyme precursors that
are converted to active enzymes in a precise order (cascade).

Functions Opsonization, chemotaxis, cell lysis

Classical Triggered by antigen-antibody reactions. IgM is most efficient

pathway activator. A single molecule attached to two adjacent antigenic
determinants can initiate the cascade. IgG1, 2, and 3 can activate
complement but at least two molecules are required. Recognition
unit: C1 (first to bind).
Activation unit: C4, C2, C3.
Membrane attack complex: C5, C6, C7, C8, C9 (drills holes in cell
membrane).

12/8/21 24
 Immune Phenomena
Explanation Application
Primary Combination of Not easily detectable. Can be measured
phenomena antibody with antigen indirectly by radioimmunoassay (RIA),
enzyme immunoassay (EIA),
immunofluorescence.
Secondary Precipitation, Measured more readily. Basis for many
phenomena agglutination, serological tests.
complement fixation
Tertiary In vivo reactions; e.g., Some are useful diagnostically, e.g., skin
phenomena inflammation, testing.
phagocytosis,
desposition of immune
complexes, immune
adherence, chemotaxis

12/8/21 25
 Hypersensitivity Reactions
Type I: TypeII: Type III: Type IV: Cell
Anaphylactic Cytotoxic Immune Mediated
Complex

Key reactant(s) IgE IgG, IgM, IgG, IgM, T cells and

complement, complement macrophages
antigens found
on cell
surfaces

Result Release of Cytolysis due Deposits of Release of

mediators to antibody antigen lymphokines
from mast and antibody from antigen-
cells basophils compliment complexes stimulated T
cells

12/8/21 26
 Hypersensitivity Reactions Continued
Type I: TypeII: Cytotoxic Type III: Type IV: Cell
Anaphylactic Immune Complex Mediated

Symptoms Immediate Immediate Immediate Delayed

Example Anaphylaxis, Transfusion Serum sickness, Contact

hay fever, reactions, systematic lupus dermatitis,
asthma, food hemolytic disease erythematosus tuberculin
allergies of the newborn, (SLE), test
autoimmune rheumatoid
hemolytic arthritis (RA)
anemia,
idiopathic
thrombocytopenic
purpura (ITP),
Good pasture’s
syndrome

12/8/21 27
 Agglutination Methods
Method Principle Examples

Direct The process by which particulate Febril agglutinins,

agglutination antigens, such as cells, aggregate Salmonella and Shigella
to form large complexes whan serotyping
specific antibody is present

Hemagglutination An antigen-antibody reaction that ABO slide typing

results in the clumping of RBCs

Passive A reaction in which soluble Rhe

agglutination antigens are bound to latex beads,
bentonite, or charcoal. The
particles are agglutinated by the
corresponding antibody.

12/8/21 28
 Agglutination Methods Continued
Method Principle Examples

Passive A reaction in which soluble antigens Cold agglutinins

hemagglutination are absorbed onto RBCs. The RBCs
are agglutinated by the
corresponding antibody.

Reverse passive A reaction in which carrier particles Rapid tests for

agglutination coated with antibody clump together identification of bacteria
due to combination with antigen

Agglutination An agglutination reaction based on Slide tests for human

inhibition competition between particulate chorionic gonadotrophin
antigen (reagent) and soluble antigen (HCG)
(specimen) for limited sites on a
reagent antibody. Lack of
agglutination is a positive result.

12/8/21 29
 Agglutination Methods Continued
Method Principle Examples

Hemagglutination A test for detecting antibodies to Rubella antibody

Inhibition certain viruses that agglutinate
RBCs. In the presence of
antibody, the virus is neutralized
and hemagglutination does not
occur.
Coagglutination An agglutination reaction in Rapid tests for
which bacteria are used as the identification of bacteria
carrier for the antibody. [Protein
A on the surface of S. aureus
aabsorbs the Fc potion of the
antibody molecule.]

• Rheumatoid factor can cause false-positive reactions in agglutination tests

because it reacts with any IgG. Heterophile antibodies can cause false-
positive reactions in hemagglutination tests.
•
12/8/21 30
 Precipitation Methods
Method Principle Application
Precipitation Soluble antigen combines with soluble See following
antibody to produce visible insoluble examples.
complexes. Precipitation methods are
less sensitive than agglutination
methods because more antibody is
needed to produce a visible reaction.
Flocculation Soluble antigens react with specific VDRL, rapid plasma
antibody to form a precipitate of fine reagin (RPR)
particles.
Ouchterlony Antigens and antibodies diffuse out Fungal antigens,
technique from wells cut in gel and form precipitin extractable nuclear
lines where they meet. Double antigens
immunodiffusion.

12/8/21 31
 Precipitation Methods Continued
Counter Antigens and antibodies are placed in Bacterial antigens
immunoelectrophoresis wells that are directly opposite one
(CIE) another in a gel. An electrophoretic
charge is applied to drive the reactants
toward each other. A precipitin band
forms where they meet.
Immunoelectrophoresis Proteins are separated by Identification of
(IEP) electrophoresis, then subjected to immunoglobulins in
double diffusion with reagent monoclonal
antibodies placed in a trough cut in the gammophaties
agar. The shape, intensity, and location
of the precipitin arcs that develop are
compared with those of a normal
control.
12/8/21 32
 Precipitation Methods Continued
Method Principle Application
Radial Antigen is measured based on the Quantitation of
immunodiffusion diameter of a precipitin ring that forms immunoglobulins and
(RID) when it diffuses out of a well nin a gel- complement
containing antibody.
Rocket An electrical charge is applied to an Immunoglobulins,
electrophoresis RID assay. The height of the rocket- complement,
shaped precipitin band obtained is
proportional to the concentration of the
antigen.
Nephelometry Light scattering by immune complexes Immunoglobulins,
is measured. complement, C-
reactive protein

12/8/21 33
  
Method Other
Principle Serological Methods
Comments Example
Complement Patient serum is Patient serum must be Viral, fungal,
fixation incubated with antigen inactivated. Best for rickettsial
and complement. If the demonstration of IgM antibodies
corresponding antibody antibodies. More
is present in the serum, it sensitive than
forms a complex with agglutination and
the antigen and precipitation, but less
complement. When sensitive than labeled
sensitized RBCs are immunoassays. Not
added, there is no free widely used.
complement to lyse
them. No hemolysis is a
positive reaction.

12/8/21 34
 Other Serological Method Continued
Direct The specimen is placed on
fluorescent a glass and overlaid with
antibody fluorescein-labeled
antibody. If the
corresponding antigen is
present, the labeled-antiody
binds and fluorescence will
be seen with a fluorescent
microscope.
12/8/21
Detects antigens. Bacterial
Fluorescent compounds antigens
absorb energy from light
source and convert it
into light of a longer
wavelength (lower
energy) than that
absorbed. Fluorescent
labels: fluorescein
isothiocyanate or
rhodamine B
isothiocyanate.
35
 Other Serological Method Continued
Method Principle Comments Example
Indirect Reagent antigen on a glss “Sandwich Fluorescent
fluorescent slide is overlaid with technique.” Detects antinuclear antibody
antibody patient serum. If the antibodies in serum. (FANA), fluorecent
corresponding antibody is treponemal
present in the serum, it antibody (FTA)
attaches to the antigen.
When fluorescein-labeled
antihuman globulin is
added, it attaches to the
antibody. Fluorescence
will be seen with a
fluorescent microscope.

12/8/21 36
 Labeled Immunoassay Terminology

Ligand The substance being measured in an immunoassay

Isotopic An immunoassay that uses a radioisotope as the label
Nonisotopic An immunoassay that uses something other than a radioisotope as the
label; e.g., enzyme, fluorochrome, chemiluminescent molecule
Competitive An immunoassay in which the patient ligand and the labeled reagent
ligand compete for a limited number of binding sites on a reagent
antibody
Noncompetitive An immunoassay in which the reaction does not involve competition
for binding sites. Noncompetitive assays are more sensitive than
competitive assays.

12/8/21 37
 Labeled Immunoassay Terminology Continued

Heterogeneous An immunoassay in which separation step is required to removed free

reactant from bound reactant. Heterogeneous assays are more
sensitive than homogenous assays.

Homogenous An immunoassay in which a separation step is not required.

Homogenous assays are easier to automate.

12/8/21 38
 Comparison of Radioimmunoassay and
Enzyme Immunoassay
RIA EIA
Label Radioisotope, usually 125I Enzyme
Measurement Counts per minute in Color development after
scintillation counter addition of substrate
Advantages Sensitivity Sensitivity
Specificity Specificity
Long shelf-life of reagents
No radiation hazard
No special disposal
requirements
No need for scintillation
counter

12/8/21 39
 Comparison of Radioimmunoassay and
Enzyme Immunoassay Continued
RIA EIA
Disadvantages Short shelf-life of reagents
Radiation hazard
Regulations governing disposal
of radioisotopes

12/8/21 40
 Immunoassay Labels
Radioisotopes Enzymes Fluorochromes* Chemiluminescent Molecules↑
125 I Horseradish Fluorescein Luminol
peroxidase
3H β-D- Rhodamine Acridium esters
galactosidase
14C Alkaline Dioxetane phospate
phosphatase

• *Fluorochromes absorb energy from light source and convert itinto light of a longer
wavelength and lower energy.
• ↑Chemiluminescent molecules produce light energy from a chemical reaction.
•

12/8/21 41
 Principles of Labeled Immunoassays

Method Type of Assay Principle

RIA Competitive Isotopic Radiolabeled ligand and unlabeled ligand in
Heterogeneous the specimen compete for binding sites on
reagent antibody. The more unlabeled ligand
in the specimen, the less labeled ligand binds.
Free labeled ligand is separated from bound
labeled ligand. Solid-phase attachment is the
most frequent separation is achieved by
aspiration or decantation. The amount of
labeled ligand bound is determined by the
counts per minute (CPM) on a scintillation
counter. CPM are inversely proportional to
the concentration of the ligand in the
specimen.
12/8/21 42
 Principles of Labeled Immunoassays Continued

Method Type of Assay Principle

Immunoradiometric Non-competitive The specimen is incubated with a labeled
assay (IRMA) Isotopic antibody. Solid-phase ligand is added to
Heterogeneous remove unbound labeled antibody. After
centrifugation, the radioactivity in the
supernatant is determined. CPM are
directly proportional to the concentration
of ligand in the specimen.

12/8/21 43
 Principles of Labeled Immunoassays Continued

Method Type of Assay Principle

Enzyme-linked Competitive Enzyme-labeled ligand and unlabeled
immunosorbent Nonisotopic patient ligand compete for binding sites
assay (ELISA) Heterogeneous on antibody molecules attached to a
solid phase. Free labeled ligand is
rem,oved by washing and substrate is
added. The enzyme activity is
proportional to the concentration of the
ligand in the specimen.

12/8/21 44
 Principles of Labeled Immunoassays
Continued
Method Type of Assay Principle
Indirect or no- Noncompetitive Antibody in the specimen attaches to a
competitive ELISA Nonisotopic solid-phase antigen. After incubation and
Heterogeneous washing to remove unbound antibody, an
enzyme-labeled antiglobulin is added. This
second antibody reacts with the Fc portion
of the patient antibody bound to the solid
phase. Following another wash, substrate is
added. The amount of enzyme label
detected is directly proportional to the
amount of antibody in the serum. Indirect
ELISAs are more sensitive than direct
ELISAs.

12/8/21 45
 Principles of Labeled Immunoassays
Continued

Method Type of Assay Principle

Immunoenzymometric Noncompetitive The specimen is incubated with an
assay (IEMA) Nonisotopic enzyme-labeled antibody. A solid-
Heteroogeneous phase ligand (usually on glass beads)
is added and the tubes are centrifuged
to remove unbound labeled antibody.
The amount of labeled antibody in the
supernatant is directly proportional to
the concentration of the ligand in the
specimen. This method is more
sensitive than indirect ELISAs.

12/8/21 46
 Principles of Labeled Immunoassays
Method Type of Assay
Continued
Principle
Sandwich Noncompetitive The antigen in the specimen is sandwiched
enzyme- Nonisotopic between antibody attached to a solid phase and
multiplied Heterogeneous enzyme-labeled antibody. Enzymatic activity is
immunoassay directly proportional to the amount of antigen in
the test sample.
Enzyme- Competitive The antigen in the specimen and an enzyme-
multiplied Nonisotopic labeled antigen compete for binding sites on
immunoassay Homogeneous reagent antibody. When enzyme-labeled antigen
technique binds to antibody, enzyme activity is inhibited.
(EMIT) Enzyme activity is directly proportional to the
concentration of the antigen in the specimen.
This is an automated method that is frequently
used for drug assays.

12/8/21 47
 Principles of Labeled Immunoassays Continued

Method Type of Assay Principle

Substrate-labeled Competitive Unlabeled ligand in the specimen and
fluorescent Nonisotopic fluorogenic ligand compete for sites on
immunoassay Homogeneous reagent antibody. Free labeled ligand
(SLFIA) produces fluorescence. Bound labeled
ligand does not produce fluorescence.
Fluorescence is directly proportional to
the concentration of the ligand in the
specimen. This is an automated method.

12/8/21 48
 Principles of Labeled Immunoassays Continued

Method Type of Assay Principle

Fluorescence Competitive Unlabeled ligand in the specimen and
polarization Nonisotopic fluorogenic ligand compete for sites on reagent
immunoassay Homogeneous antibody. Free labeled ligand rotates rapidly
(FPIA) and emits little polarized fluorescence. Bound
labeled ligand rotates more solely and emits
more polarized fluorescence. The higher the
concentration of bound labeld ligand, the more
polarized fluorescence. The amount of
polarized fluorescence is inversely propotional
to the concentration of the ligand in the
specimen. This is an automated method.

12/8/21 49
 Labeled Immunoassays

Isotopic Non-Isotopic
Competitive Radioimmunoassay (RIA) ELISA
Heterogeneous
Homogeneous EMIT
SLFIS
FPIA
Noncompetitive Immunoradiometric assay Indirect ELISA
Heterogeneous (IRMA) IEMA
Sandwich enzyme-multiplied
immunoassay

12/8/21 50
 Nontreponemal Tests for Syphilis
VDRL RPR
Method Flocculation Flocculation
Detects Reagin Reagin
Reagent(s) Cardiolipin Cardiolipin with charcoal
added
Positive reaction Microscopic clumps Microscopic agglutination
Specimen(s) Inactivated serum, CSF Serum (inactivation not
required), plasma
Reactivity during May be negative in primary stage. Same as VDRL
disease Titers usually peak during
secondary or early late stages.
Titers decline in late stage, eve3n
when untreated. More rapid decline
with treatment. Becomes
nonreactive following successful
treatment.
12/8/21 51
 Nontreponemal Tests for Syphilis
Continued
VDRL RPR
False- 10-30% may be biological false positive; Same as VDRL
positives e.g., infectious mononucleosis (IM),
infectious hepatitis, malaria, leprosy, lupus
erythematosus, rheumatoid arthritis,
advanced age, pregnancy. Positive in other
treponemal infections such as yaws and
pinta.
Other Replaced by RPR for serum. Still Good for screening and
performed on CSF.. treatment monitoring

12/8/21 52
 Treponemal Tests for Syphilis

FTA-ABS Microhemagglutination Assay

Method Indirect fluorescent antibody Hemagglutination

Detects Antibody to T. pallidum Antibody to T. pallidum
Reagent(s) Sorbent (nonpathogenic Sheep RBCs coated with antigens
treponemes-Reiter strain), from Nichols strain T. pallidum,
slides with Nichols strain of T. sorbent
pallidum, fluorescein-labeled
antihuman globulin

Positive reaction Fluorescence Rough or jagged pattern of cells

min microtiter wells

12/8/21 53
 Treponemal Tests for Syphilis Continued
FTA-ABS
Specimen(s) Serum, CSF
Reactivity during Usually positive before regain
disease tests. Some false-negatives in
primary syphilis. Usually
positive for life.
False-positives Fewer than nontreponemalk
tests. Reactivity is seen jwith
other treponemal diseases,
notably yaws and pinta
12/8/21
Microhemagglutination Assay
Serum
Not as sensitive in primary
syphilis as FTA. Sensitivity
close to 100% in secondary
syphilis. Usually positive in
late stages.
Fewer than nontreponemal
tests.
54
 Treponemal Tests for Syphilis Continued

FTA-ABS Microhemagglutination Assay

Other Sorbent removes Sorbent removes nonspecific

nonspecific antibodies. antibodies. Used to confirm
Used to confirm reactive reactive nontreponemal test. Not
nontreponemal tesr. Not good for treatment monitoring.
good for treatment
monitoring.

12/8/21 55
 Test for the Diagnosis of Infectious
Mononucleosis (IM)
Antibodies Interpretation
Heterophile antibodies*
IM antibodies: Agglutinate sheep, beef, Agglutination of sheep or horse cells
ox, and horse RBCs. Absorbed by following adsorption with GPK but not with
beef RBCs but not by guinea pig kidne beef RBCs = IM.
cells (GPK). No agglutination of sheep or horse cells
Serum sickness antibodies: Agglutinate following adsorption with GPK or beef
sheep, beef, and horse RBCs. RBCs = serum sickness.
Adsorbed by beef RBCs and guinea pig
kidney cells. Aggllutination of sheep or horse cells
Forssman antibodies: Agglutinate sheep following adsorption with beef RBCs but
and horse RBCs. Adsorbed by guinea pig not with GPK = Forssman.
kidney cells but not by beef RBCs.

12/8/21 56
 Test for the Diagnosis of Infectious
Mononucleosis (IM) Continued
Antibodies Interpretation
Specific viral antibodies
IgM-VCA (viral capsid antigen): Peaks 3-4 IgM anti-VCA, anti-EA, or IgA anti-
weeks after infection. Undetectable after 12 VCA without EBNA = recent or current
weeks. infection.
IgG-VCA: Appears in late acute phase and Anti-EBNA or IgG anti-VCA without
persists for life. IgM anti-VCA = past infection.
Anti-EA (early antigen): Appoears early and
persists for years.
Anti-EBNA (EB nuclear antigen): Appears 2-
3 months after infection and persists
indefinitely.

*Nonspecific antibodies that cross-react with antigens other than those originally
responsible for their production.
•
12/8/21 57
 Weil-Felix Reaction
Disease Organism Transmission Weil-Felix Reaction
OX-2 OX-19 OX-K
Rocky Rickettsia Dog or wood tick + ++++ 0
Mountain rickettssi
spotted fever*
Epidemic R. prowazekii Body or head louse + ++++ 0
typhus
Endemic R. typhi Rat flea + ++++ 0
typhus
Scrub typhus R. tsutsugamushi Mite 0 0 ++++
Q fever Coxiella burnetii Inhalation of 0 0 0
barnyard dust or
ingestion of infected
meat or milk
• Most common rickettsial disease in U.S.

12/8/21 58
 Antinuclear Antibodies
Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti-ds- Double- Specific for Found in Peripheral or Considered
DNA stranded DNA SLE only 50- homogeneous diagnostic
80% of for SLE,
patients especially
with SLE when C3 is
low.
Anti-Sm Extractable Specific for Only found Speckled
nuclear SLE. Not in 30% of
antigen found in other patients
associated disorders. with SLE
with RNA
12/8/21 59
 Antinuclear Antibodies Continued
Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti-DNP Deoxyribonuc- Present in Present in Peripheral or LE factor
leoprotein SLE, and 70-90% of homogeneous
drug-induced patients
SLE with SLE
Anti-SS- Extractable Present in Present in Speckled Most often in
A/Ro nuclear SLE, 25-40% of patients with
antigen. RNA scleroderma SLE cutaneous
protein and patients manifestations
conjugate. Sjogren’s (photosensiti-
syndrome vity
dermatitis)

12/8/21 60
 Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti-SS- Ribonucl- Present in Present in 10- Speckled
B/La eoprotein SLE, 15% of SLE
scleroderma patients
and
Sjogren’s
syndrome
Anti-RNP Proteins Present in Speckled
complexed SLE, mixed
with connective
nuclear tissue disease
RNA and other
autoimmune
12/8/21 diseases 61
 Antinuclear Antibodies Continued
Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti- Histone Present in Present in Homogeneous Diagnostic
histone (nucleoprot SLE, RA, almost all drug-induced
ein that is a primary patients with lupus. High
major biliary drug-induced levels
constituent cirrhosis lupus associated with
of more active and
chromatin) severe forms of
SLE.

– Multiple ANAs can be present in a specimen. More than on epattern will be observed.
– Fluorescence of nucleoli is due to antibody to RnA. Seen in scleroderma and polymyositis.
•
12/8/21 62
 Hepatitis Tests
Test Indication
Hepatitis A
Anti-HAV (total) Past infection and immunity
Anti-HAV (IgM) Acute hepatitis A
Hepatitis B
HbsAg Acute or chronic infection HBV (carrier), infectivity

HbeAg Acute or chronic HBV (carrier), high degree of infectivity

Anti-HBc (total) Current or previous infection or carrier
Anti-HBc (IgM) Recent acute infection
Anti-Hbe Disease resolution and reduced infectivity
Anri-0HBs Recovery and immunity

12/8/21 63
 Hepatitis Tests Continued

Test Indication
Hepatitis C Acute, chronic, or previous hepatitis C infection
Anti-HCV
Hepatitis D Past or current hepatitis D infection
Anti-HD
Hepatitis E
None

12/8/21 64
 Hepatitis Profiles

Acute Recovery from Acute Recovery from Chronic

Hepatitis A Hepatitis A Hepatitis B Hepatitis B Hepatitis B
Carrier
Anti-HAV Anti-HAV HbsAg + HbsAg – HbsAg +
(IgM) + (total) + HbeAg + HbeAg – HbeAg +
Anti-HBc (IgM) Anti-HBs + Anti-HBc
+ Anti-HBc (total) +
Anti-Hbe + (total) + Anti-HBs –
Anti-Hbe + Anti-Hbe –

12/8/21 65
 HIV Testing

Test Detects Type of Test Principle Comments

ELISA Antibody Screening Viral antigen is Most widely used
to HIV coated on a solid screening procedure.
support. Patient Positives must be
serum added. After confirmed. Anti-HIV-
incubation and 1 and anti-HIV-2 (or
washing, enzyme- combination test)
labeled antihuman required on blood
globulin (AHG) is donors.
added followed by
substrate.

12/8/21 66
 HIV Testing Continued

Test Detects Type of Principle Comments

Test
Slide Antibody Screening HIV antigen is Used when
agglutination to HIV adsorbed onto instrumentation is not
tests carrier particles available
agglutinate in
the presence of
antibody.

12/8/21 67
 HIV Testing Continued

Test Detects Type of Test Principle Comments

Western Antibody to Confirmatory A lysate of HIV Most commonly used
blot HIV antigen is separated confirmatory test.
into components by HIV infection is
electrophoresis and indicated by
transferred to reactivity in two of
nitrocellulose paper. the following bands:
The resulting blot is p24, gp41, or
cut into strips and gp120/160.
reacted with serum.
Labeled antihuman
globulin is added.

12/8/21 68
 HIV Testing Continued

Test Detects Type of Test Principle Comments

Indirect Virus or Confirmatory Antibody test: Serum is Sensitivity and
immunofluo- antibody incubated with virally specificity comparable
rescence infected cells on a glass to western blot.
assays slide. Fluorescein- Simple and rapid to
labeled antihuman perform.
globulin is added.
Antigen test: Patient
cells are fixed to a slide
and incubated with HIV-
specific antiserum.

12/8/21 69
 HIV Testing Continued

Test Detects Type of Principle Comments

Test
P24 antigen test HIV Screening Anti-HIV bound to a P24 antigen
(HIV-1-Ag) antigen solid support is precedes antibody
incubated with serum. by several weeks.
After washing, Positives must be
enzyme-labeled anti- confirmed by a
HIV-1 is added neutralization assay.
followed by substrate Required on blood
donors.

12/8/21 70
 Viral Testing

Test Detects Type of Test Principle Comments

Polymerase Viral Adjunct to Viral DNA from Extremely sensitive
chain reaction genome standard infected cells is technique. Will detect
(PCR) testing amplified then infections during
identified using “window period”. Not
labeled probes. suitable for routine
screening.
Viral culture Virus Adjunct to Virus grown on Expensive, time-
standard cell culture consuming, and
testing hazardous. Not
routinely performed.

12/8/21 71
 Other Serological Tests

Test Common Reagents Interpretation Comments

Method
Rheumatoid Agglutination IgG Positive in 70- Rheumatoid factor is
arthritis (RA) attached to 80% of an autoantibody
latex or patients with (usually IgM) against
RBCs RA IgG. Not specific for
RA. Also found in
SLE, scleroderma,
Sjogren’s syndrome,
B-cell
lymphoproliferative
disorders.

12/8/21 72
 Other Serological Tests Continued

Test Common Method Reagents Interpretation Comments

Cold Hemagglutination Patient or Positive in PAP is caused by
agglutinins Group O primary Mycoplasma pneumoniae.
RBCs atypical Increased titer of Anti-I.
pneumonia Test is incubated in
(PAP) refrigirator. Specimen
must be kept warm prior to
testing to prvent false-
negatives or decreased
titers. Positives must be
confirmed by reversal of
agglutination at 37°C.

12/8/21 73
 Test Common Reagents Interpretation Comments
Method

Anti- Neutralization Streptolysin Their highest Streptolysin O lyses

streptolysin O O, group O dilution showing RBCs. Anti-streptolysin
(ASO) RBCs no hemolysis. High O in patient’s serum
titers with strep neutralizes streptolysin
infections, O reagent so RBCs are
rheumatic fev er, not lysed. Streptolysin
glomerulonephritis. O is oxygen labile. RBC
control (RBCs + buffer)
should show no
hemolysis. Streptolysin
control (streptolysin O +
buffer + RBCs) should
show hemolysis.
Reported in Todd units
(reciprocal of highest
dilution showing no
hemolysis). Normal is
12/8/21 less than74
166.
 Other Serological Tests Continued

Test Common Reagents Interpretation Comments

Method
C-reactive Latex Latex Agglutination = Sensitive, but
protein agglutination particles inflammation nonspecific, indicator of
coated with inflammation.
anti-CRP Originally named
because it was thought
to be an antibody to the
polysaccharide of S.
pneumoniae.

12/8/21 75
 Other Serological Tests Continued

Test Common Reagents Interpretation Comments

Method
Rubella Hemagglutin Viral Titer is highest Rubella = German
ation preparation, dilution showing no measles = 3-day
Inhibition RBCs agglutination. A titer measles. In utero
of 8 or higher = infections cause fetal
immunity to rubella. death or birth defects.
Greatest arisk in first
month of pregnancy.
IgM antibodies in a
neonate are diagnostic
of congenital rubella.

12/8/21 76
 Test Common Reagents Interpretation Comments
Method
Febrile Bacterial Salmonella O Positive reaction Screening test for fever of
agglutinins agglutination and H, is agglutination. unknown origin. Weil-
Brucella, Felix reaction: rickettsial
abortus, antibodies cross react
Proteus OX with OX-2, OX-19, and
19. OX-K strains of Proteus.
Widal’s test: detects
antibodies in typhoid
fever, tularemia,
brucellosis. Antibodies to
O = recent infection.
Antibodies to H = past
infection. Best indicator
of active infection is four
fold increase in titer.
Non-specific test.
12/8/21 77
Infrequently ordered
 Interpretation of Serological Tests

A single titer is not usually meaningful because it can’t differentiate current

infection from past infection or immunity due to immunization.
The acute specimen should be frozen and run with the convalescent
specimen to eliminate differences in titer due to technical variations.
The convalescent specimen should be drawn 10-14 days after the acute
specimen.
A fourfold increase in titer from acute to convalescent specimen is
diagnostic.
IgM antibody is a sign of recent infection.

12/8/21 78
 Serology Calculations

How would you prepare a 5% suspension of human group O red blood cells?
5% = mL per mL.
5 mL of packed RBCs + 95 mL of buffer.
How would you prepare 5 ml of a 1:10 dilution of serum?
1 =x
10 5
10x = 5
x = 0.5 mL
Mix 0.5 mL of serum with 4.5 mL of buffer.
How would you prepare 10 mL of 1:100 dilution from a 1:10 dilution?
A 1:10 dilution of a 1:10 dilution yields a 1:100 dilution (1/10 x 1/10 = 1/100).
Mix 1 mL of the 1:10 dilution with 9 mL of buffer. (1 mL + 9 mL = 1:10)
How would you prepare 10 mL of a 1:500 dilution from a 1:100 dilution?
A 1:5 dilution of a 1:100 dilution yields a 1:500 dilution (1/5 x 1/100 = 1/500).
Mix 2 mL of the 1:100 dilution with 8 mL of buffer. (2 mL + 8 mL = 2:10 = 1:5.)
What is the dilution in tube 4 of a twofold serial dilution, if is undiluted?
1 x ½ x ½ x ½ = 1/8

12/8/21 79
 The End

12/8/21 80