Serology Review: Tom Kairo T. Pacquing

SEROLOGY REVIEW
Tom Kairo T. Pacquing

TYPES OF IMMUNOLOGIC
REACTIONS
I. PRIMARY REACTIONS
Specific recognition & combination of antigen w/ binding site of
its corresponding antibody
“Labeled immunoassays (LIA)”
Radioimmunoassay (RIA)
_____________________________________
Enzyme immunoassay (EIA)
_____________________________________
Immunofluorescent assay (IFA)
_____________________________________
Fluorescence polarization immunoassay (FPIA)
_____________________________________
Chemiluminescent immunoassay
_____________________________________
REACTIONS
II. SECONDARY REACTIONS
 Conformation of amino acid chain resulting from interchain hydrogen
bonding
 “Nonlabelled immunoassays”
Precipitation
 Light scattering techniques
Nephelometry
 _________________
 _________________
Turbidimetry
REACTIONS
 Without electrical involvement – “passive immunodiffusion”
Oudin technique
 Single diffusion-single dimension – _____________________________________
Radial immunodiffusion
 Single diffusion-double dimension – _____________________________________
Oakley and Fulthorpe technique
 Double diffusion-single dimension – _____________________________________
Ouchterlony double diffusion
 Double diffusion-double dimension – _____________________________________
 With electrical involvement – “electrophoretic techniques”
Rocket immunoelectrophoresis (Laurell)
 1 reactant moving in 1 dimension – _____________________________________
Ressler’s method
 1 reactant moving in 2 dimensions – _____________________________________
Countercurrent immunoelectrophoresis
 2 reactants moving in 1 dimension – _____________________________________
Immunoelectrophoresis
 2 reactants moving in 2 dimensions – _____________________________________
Immunofixation electrophoresis
 _____________________________________
REACTIONS
 Agglutination
Direct agglutination
 _____________________________________
Passive/indirect agglutination
 _____________________________________
Reverse passive agglutination
 _____________________________________
Coagglutination/conglutination
 _____________________________________
Agglutination inhibition
 _____________________________________
Antiglobulin-mediated agglutination
 _____________________________________
REACTIONS
I. TERTIARY REACTIONS
 Folding of polypeptide chains through hydrophobic and hydrogen bonds
 Phagocytosis
 Opsonization
 Chemotaxis
 Immune adherence
 Cellular degradation
ANTIGEN-ANTIBODY BINDING
Affinity
PROPERTIES
 ___________
 Initial force of attraction that exists between a single Fab site of an antibody
molecule & a single epitope on the corresponding antigen
 Several types of weak noncovalent bonds hold them together:
Ionic bonds
 ___________________
 Occur between oppositely charged particles
Hydrogen bonds
 ___________________
 Attraction between polar molecules that have a slight charge separation & in
which positive charge resides on a hydrogen atom
 ___________________
Hydrophobic bonds nonpolar molecules that associate with one another and
 Occur between
exclude molecules of water as they do so
ANTIGEN-ANTIBODY BINDING
Van der Waals forces
PROPERTIES
 ___________________
 Occur because of the interaction between the electron clouds of oscillating
dipoles
 Antibody binds with an antigen which is structurally similar to the original antigen
that induces the antibody formation: ________________
cross reactivity
 ____________
Avidity
 Represents the sum of all the attractive force between an antigen and an antibody
 Measure of the overall stability of an antigen–antibody complex
 High avidity can compensate for low affinity
01
PRECIPI
TATION
PRECIPITATION
 Antigen-antibody reaction wherein a soluble antigen binds with a soluble antibody,
forming an insoluble complex
 Requires that both precipitin and precipitinogen should be at least _____________
bivalent
 Precipitins:
 ______________
IgG>IgM>IgA
 Nonprecipitating antibody: ______
IgE
ZONING PHENOMENON
Zone of equivalence
 ____________________
 The number of multivalent sites of antigen and antibody are approximately equal
 All of the reactants are involved in the formation of precipitation lattice
 Formulated by Marrack
 Neither free antigen nor free antibody can be detected in the solution
 Optimal precipitation
 Should be seen in every precipitation test performed
 ____________________
 Antibody in excess
 Too many free antibodies are found in the solution
Prozone
 No lattice formed
 Resolution: _____________________________________
Dilute antibody in the patient serum and retest

ZONING PHENOMENON
Postzone
 ____________________
 Antigen in excess
 Too many free antigens are found in the solution
 No lattice formed
Retest additional patient serum at least a week later
 Resolution: _____________________________________
 Notes:
false negative result
 Both prozone and postzone lead to __________________
 Zoning phenomenon is a major deterrent to the use of fluid precipitation tests
in the clinical setting
 Agglutinating systems are LESS SUSCEPTIBLE to prozone, but they can
still occur (e.g., VDRL)
PRECIPITATION BY LIGHT
SCATTERING
I. TURBIDIMETRY
 Measurement of turbidity/cloudiness of a solution
reduction in light intensity
 Measures the _______________________________ caused by reflection, absorption,
or scatter
 Amount of scatter is proportional to the size, shape, and concentration of
molecules in the solution
PRECIPITATION BY LIGHT
SCATTERING
II. NEPHELOMETRY
Heidelberger and _______________
 Described by _____________ Kendall
 Measures the light that is scattered at a particular angle from the incident beam as it
passes through a suspension
 Can be used for either antigen or antibody detection
 Usually, the reagent is antibody
 Note: Quantification of IgG, IgA, IgM, and IgE, and kappa and lambda chains are done
almost exclusively in nephelometry
 Other methods are more labor-intensive
More sensitive than turbidimetry
PASSIVE IMMUNODIFFUSION TECHNIQUES
 Movement of either soluble antigen, antibody or both in a semisolid media
(gels) to form precipitation reactions WITHOUT the involvement of
electrical current
 Allowing precipitinogens and precipitins to react in semisolid media has
neither of these disadvantages:
 Prozone or postzone phenomena
 Ambiguity in the number of antigen-antibody systems in heterogeneous
mixtures
 General factors that affect the rate of diffusion:
Size of the molecule
 ______________________
 Inversely proportional
 Larger = slower diffusion & vice versa
Molecular weight
 ______________________
 Inversely proportional
 Higher MW = slower diffusion & vice versa
 Gelling substances used as support medium:
Agarose
 _____________ – most widely used gel
Agar
 _____________
Polyacrylamide
 _____________
Cellulose acetate
 _____________
Gelatin
 _____________
Starch
 _____________
I. SINGLE DIFFUSION-SINGLE DIMENSION (OUDIN)
Simple immunodiffusion
 Aka ____________________________
 Earliest of the gel techniques
 Mobile phase: antigen
 Stationary phase: antibody
 Incorporated into agarose in a test tube
 Positive result: single band of precipitate
 Note: if two bands of precipitate are present, it is inferred that at least 2 antigens are
present
 Negative result: no precipitation
II. SINGLE DIFFUSION-DOUBLE DIMENSION (RADIAL
IMMUNODIFFUSION)
 Modification of Oudin technique
 Application: serum protein quantification (IgG, IgA, complement)
 Stationary phase: Antibody
 Incorporated in the gel medium
 Mobile phase: Antigen
 Applied to a well which is cut into the gel
IMMUNODIFFUSION)
 Techniques for the measurement of RID:
 Kinetic/Timed method
Fahey/Fahey & McKelvey method
 Aka _____________________________________
 Diameter of precipitin ring is measured at 18 hrs.
 Logarithm of the concentration of standards is proportional to diameter of the precipitin ring
 X – axis → diameter of the ring
 Y – axis → Ag concentration
 End-point method
 Aka _____________________________________
Mancini
 Equivalence method
occurs between 24 and 72 hrs.
 IgG → 24 hrs.
 IgM → 50-72 hrs.
 Square of diameter is proportional to Antigen concentration
 X – axis → Ag concentration
 Y – axis → diameter of the ring
IMMUNODIFFUSION)
 Sources of errors:
 Over- or underfilling of the wells
 Spilling of patient serum on the gel
 Nicking the side of the well
 Improper incubation time and temperature
 Specimen contamination
III. DOUBLE DIFFUSION-SINGLE DIMENSION (OAKLEY &
FULTHORPE)
 Modified Oudin technique by overlaying Ab with neutral agar, allowing it to gel, and
then overlaying the gel with Ag
 Ag & Ab diffuses toward the center of the medium, forming precipitin lines at the
plain agar column
 Has similar application with Oudin technique
IV. DOUBLE DIFFUSION-DOUBLE DIMENSION (OUCHTERLONY)
 First method used in establishing the relationship of HBsAg to type B hepatitis
 Application:
 Detection of fungal antigens
 Detection of extractable nuclear antigens (ENAs) in autoimmune diseases
 Ag and a mixture of Ab diffuse independently through a semisolid medium
 A suitable pattern of wells is cut in an agarose plate (petri dish or microscope slide),
loaded with reactants
 Covered to prevent evaporation
 Incubated at room temp or ref temp until line of precipitate have fully developed
(between 12 and 48 hrs.)
 Pattern: _____________________________________
 Antibacterial: _____________________
 Two known reagents (known
imaginary antibody
equilateral and known antigen) and the unknown patient
triangle
sample are utilized insodium
the setup
azide
 Interpretation:
 Serological identity
smooth curve (arc) or solid chevron
 Line of precipitation: ________________________
 The unknown Ag shares a common epitope & have similar complexity with known Ag
 Note: They may not need to be exactly identical
 Serological non-identity
 Line of precipitation: crossed lines
 The unknown Ag is totally distinct from the known Ag
 Serological partial identity
 Line of precipitation: _______________________
 The unknown Ag shares spur formation
a common epitope with the known Ag, but different in complexity (one
complex Ag & one simple Ag)
 Some Ab are not captured by the simple Ag, and so continues to form precipitin lines with the
more complex Ag
 The spur always points to the simpler Ag
ELECTROPHORETIC TECHNIQUES
 Immunodiffusion coupled with electrical current
 Electrophoresis separates molecules according to their differences in electric
charge when placed in an electric field
faster and more accurate results
 Main advantage: _____________________________________
 Can be applied to single diffusion, or double diffusion methods
I. SINGLE REACTANT MOVING IN ONE DIRECTION
 Aka _____________________________________
Rocket technique of Laurell
 Differs from Oudin in that:
 It is performed on plates rather than tubes
 Quantitative rather than qualitative
 Employs electrophoresis rather than simple diffusion
I. SINGLE REACTANT MOVING IN ONE DIRECTION
 pH of medium (particularly agar medium): 8.6 (isoelectric point of immunoglobulins)
 Stationary phase: antibody
 Mobile phase: antigen (negatively charged at pH 8.6)
 Ag is placed on the well which is cut into the gel
 As Ag diffuses out towards the anode with the help of electrophoresis, precipitin
lines are formed upon binding with the stationary Ab
 Precipitin lines are formed in a conical shape, like a rocket
 Interpretation: length of the rocket is proportional to the log of the Ag
concentration
II. SINGLE REACTANT MOVING IN TWO DIMENSIONS
Ressler’s method of two-dimensional electrophoresis
 Aka _________________________________________________
 Research technique for fractionating serologically active components of a
complex mixture of proteins such as whole serum
III. TWO REACTANTS MOVING IN ONE DIRECTION
Countercurrent immunoelectrophoresis (counter-immunoelectrophoresis or CIEP)
 Aka ________________________________________________________
 Two columns of well are cut (Ag in one well, Ab in the other)
 When electrophoresed, Ag will diffuse towards the anode, and the Ab will diffuse towards the
cathode, and will form precipitin lines as they meet at the center of the medium
 Applications:
 Autoantibodies
 Antibodies to infectious agents and microbial antigens
 Sources of error:
 Reversal of the wells such that the current is applied at the wrong direction
 Improper pH of the buffer
 Insufficient electrophoresis time
 Prozone or postzone
 Wells that are not parallel
IV. TWO REACTANTS MOVING IN TWO DIMENSIONS
immunoelectrophoresis (IEP)
 Aka _____________________________________
 Introduced by Grabar and Williams
 Applications:
 Identification of monoclonal proteins (free κ and λ chains)
 Screening for the detection of Ab classes
 Troughs and wells are cut, wherein Ag is placed in the well, and the Ab is placed in the
trough
 Gel is electrophoresed, and the Ab and Ag would form precipitin arcs
 Considered as semiquantitative procedure (size of arc = amount of Ag present)
 Sources of error:
 Prolonged diffusion results in artifacts
 Marked Ab excess may result in multiple concentric arcs
V. IMMUNOFIXATION ELECTROPHORESIS (IFE)
 Aka immunofixation
 First described by Alper and Johnson
 Replaced IEP in the evaluation of monoclonal gammopathies because of its
rapidity and ease of interpretation
 Primary use: Characterization of monoclonal proteins
 Similar to IEP except that after electrophoresis takes place, Antiserum
(containing the Ab) is applied directly on the surface of the gel rather than
placed in a trough
02
AGGLUTI
NATION
AGGLUTINATION
 Combination of an agglutinating antibody (agglutinin) with a particulate antigen (agglutinogen) in the
correct proportion that results in visible clumping of the antigen
 Agglutinins: _____________
IgM>IgG
 Particulate antigens: _____________________________________
 Particles: antigen is carried by a particle
 _______________
RBCs
 _______________
Bacteria/Yeast
 Inert particles:
o ____________________
Latex (polystyrene beads)
o ____________________
Charcoal
o ____________________
Gelatin
o ____________________
Plastic beads
o ____________________
 Note: in order forSilicates
agglutination to occur, both the agglutinin and agglutinogen should be multivalent (for
crosslinking)
AGGLUTINATION
 Advantages of agglutination over precipitation:
 Can be used either for antigen or antibody detection
 Simple
 Uncomplicated instruments
 Availability of kits (minimum reagent preparation)
 Macroscopic endpoints
 Less zoning
serial dilution
 Qualitative (semiquantitative: _________________)
AGGLUTINATION
 Stages:
a) Sensitization
 Initial binding
 Ag-Ab combination through single antigenic determinants on the particle
surface
 Rapid & reversible
 Factors affecting sensitization:
 Ab nature (affinity, avidity, etc.)
 Nature of Ag-bearing surface
o Number of epitopes
o Epitopes obscured by other surface molecules
AGGLUTINATION
b) Lattice formation
 Crosslinking – visible aggregates
 Stabilization
 Factors affecting lattice formation:
 Environmental conditions
o Ionic strength
o pH
o Temperature
 Concentration of Ag and Ab
 Nature of Ab
AGGLUTINATION
 Enhancement of the lattice formation
 Chemical potentiators
______________________ – substances that facilitate Ag-Ab interactions in-vitro
 LISS (Low Ionic Strength Solution/ Low Ionic Strength Saline/ Low Ionic Salt Solution)
 Made of NaCl, glycine, and albumin
 Mechanism of potentiation:
o Lowers the zeta potential
o ___________________________________
Provides
 Bovine Albumin Ab uptake by the RBCs
(5-30%)
 Most common concentration: 22%
 High molecular weight protein
o Reduces zeta potential by dispersing some of the cations surrounding each negatively
charged RBC
o ________________________________ (measure of ability to dissipate a charge)
Increases the dielectric constant
AGGLUTINATION
 Dextran or polyethylene glycol (PEG)
o Removes water
__________________ from the test system, thereby concentrating any antibody present
 Can cause nonspecific cellular aggregation
o Note: tests using PEG CANNOT be centrifuged and evaluated following a 37˚C
incubation
 Testing should proceed immediately to the wash phase with a minimum of 4 washes
performed
 Proteolytic enzymes (Bromelin, papain, ficin, trypsin)
o Reduce the surface charge on RBCs through cleaving of chemical bonds and decreasing
hydration
 Ficin cleaves sialoglycoproteins from RBCs
o This may change the external configuration of the RBC membrane to expose further its
antigen for antibody binding
AGGLUTINATION
 Physical/Mechanical Potentiators
 Centrifugation
 Physically binds the Ag to Ab closer to each other which results to a more
likely interaction
 Agitation
 Increases kinetic energy of the reactants (antigen and antibody) which
increases the likelihood of collision
 Other factors affecting agglutination reactions
 Temperature
 IgG (Warm-reacting antibody)
 IgM (Cold-reacting antibody)
PHASES OF AGGLUTINATION
Immediate Spin Phase Incubation Phase Antihuman globulin
(Room Temp. Phase) (37˚C phase) Phase (AHG Phase)

Antibody detected
IgM IgG IgG

Procedure
1. Ag + Ab 1. Ag + Ab + 1. Ag + Ab +
2. Centrifuge chemical potentiator incubate
3. Grade 2. Incubate at 37˚C 2. Wash
agglutination 3. Centrifuge 3. AHG
4. Grade 4. Incubate
agglutination 5. Centrifuge
6. Grade
agglutination
PHASES OF AGGLUTINATION
Description
Grading
Cells Supernatant
One, large solid agglutinate, no free cells Clear
4+
Several large agglutinates, few free cells Clear

3+
Many medium-sized agglutinates, Moderate number of free Clear

cells
2+
Many small agglutinates, many free cells Turbid

1+
Many tiny agglutinates, many free cells, microscopically visible Dark, turbid
W+
No agglutinates Dark, turbid, homogeneous

0
TYPES OF AGGLUTINATION REACTIONS
 Based on:
 Particle used
 Ab/Ag on particle
I. DIRECT AGGLUTINATION
 Occurs when antigens are found naturally on a particle
 Patient serum is diluted (which may or may not contain antibody to the reagent antigen)
and reacted with antigen specific for the suspected disease
 Widal test
 Principle: Direct bacterial agglutination
 Rapid screening test for Typhoid fever
 Antigens:
 _____________________________________
Salmonella somatic (O) antigen
 _____________________________________
Salmonella
 Paired flagellar (H)
sera: four-fold (or antigen
greater) increase in Ab titer indicates infection
 Direct hemagglutination
 Principle of ABO blood typing
 Anti-A dyes:
Bromphenol blue
 ____________________
Thymol blue
 ____________________
Patent blue
 ____________________
Alcian blue
 ____________________
Methylene blue
 ____________________
 Anti-B dyes
Acriflavin
 ____________________
Tartrazine yellow
 ____________________
 Preservative/antibacterial substance present in the antisera: 0.1% sodium azide
 Minimum titer: ________
>256
GRADING & SCORING OF HEMAGGLUTINATION
Grade Appearance Score
H
Hemolysis – presence of free Hgb in serum 10
4+ 1 solid aggregate, clear supernatant 10
3+ supernatant
Several medium to large aggregates, clear 8
2+ Many small to medium aggregates, clear supernatant 5
1+
Many small aggregates, turbid background 3
+ or w
Few small aggregates with many unagglutinated cells 2
+m or +m
Aggregates visible only under microscopic magnification 1
0 cells unagglutinated
Negative – absence of aggregates with all 0
mf minor population of agglutinated cells
Mixed-field agglutination – presence of NA
superimposed on a negative background
R Rouleaux – nonspecific aggregation appearing like a stack of coins; disappears NA

with addition of saline
Clinical applications involving hemagglutination reactions
involve detection of Ab to:
 HAV
 HBV
 HCV
 HIV-I and HIV-II
II. PASSIVE AGGLUTINATION
indirect agglutination
 Aka ________________________
 Uses particles coated with Ag not normally found on their surfaces
 Coating is performed through adsorption
 Particles used:
 RBCs
 Polysaccharide antigens adsorb readily
 Con: possibility of cross-reactivity especially with heterophile antibody
 Latex
 Initially used by Singer & Plotz
 IgG naturally absorbed to the surface of polystyrene latex
 Inexpensive, relatively stable, not subject to cross-reactivity with other antibodies
 Synthetic beads
II. PASSIVE AGGLUTINATION
 Clinical applications – used for the detection of:
 Rheumatoid factor
 Antinuclear Ab in SLE
 Ab to group A Streptococcus antigens
 Ab to Trichinella spiralis
 Ab to Treponema pallidum
 Ab to viruses (CMV, rubella, VZV, HIV-1, HIV-2)
 Cons:
 Risk of nonspecific agglutination reactions caused by presence of other IgM
antibodies
 Remedy: always run positive and negative controls
 Note: usually used as a screening test only
III. REVERSE PASSIVE AGGLUTINATION
 Antibody, rather than the antigen is adsorbed to the carrier particle
 Joined in a manner that the antigen-binding sites are faced outwards (Fc portion attached to the
surface of particle)
 Clinical application: used for the detection of microbial antigens
 Rapid identification of:
 Group A & Group B Streptococci
 Staphylococcus aureus
 Neisseria meningitidis
 Haemophilus influenzae
 Rotavirus
 Cryptococcus neoformans – very high sensitivity
 Vibrio cholerae
 Leptospira
 Identification of soluble antigens in the urine, CSF, or serum
 Cons:
rheumatoid factor will cause false-positive as it reacts
 In all these reactions, ___________________
with any IgG antibody
 Remedy:
Pretreatment with heat (boiling) for 5 mins.
 _____________________________________
Use of EDTA
 _____________________________________
Proteolytic enzymes (2-Mercaptoethanol, pronase)
 _____________________________________
 Latex agglutination
 Immunologic assays performed by latex particle agglutination includes:
 _______________________________
C-reactive protein (CRP)
 _______________________________
IgM rheumatoid factor
 _______________________________
IgG rheumatoid factor
 _______________________________
Rubella antibody (neonates)
 Variations:
 _____________________________________
Coagglutination
 _____________________________________
Liposome-enhanced latex agglutination
IV. AGGLUTINATION INHIBITION
 Based on the competition between particulate and soluble antigens for limited
antibody-combining sites
Lack of agglutination
 Positive result: _________________________
 Clinical applications:
 Highly sensitive assay capable of detecting small quantities of antigen
 Detection of illicit drugs (cocaine or heroin)
 Detection of β-HCG in serum/urine (pregnancy testing)
 Most common principle for detection of β-HCG: ______________
 False-positive results: ELISA/EIA
 hCG injection (Pregyl) 10 days previously
 ____________________
 Chorioepithelioma
____________________
 Hydatidiform mole
____________________
 Excessive ingestion of aspirin
____________________
Testicular tumor (males)
 Hemagglutination inhibition
 Similar principle as with agglutination inhibition, with the use of RBCs as indicator
particles
 Used to detect Abs to certain viruses (rubella, mumps, measles, influenza,
parainfluenza, HBV, herpesvirus, RSV, adenovirus)
 Serologic principle of secretor status determination
 Controls are necessary because there may be a factor in the serum that causes
agglutination, or the virus may have lost its ability to agglutinate
V. COAGGLUTINATION
 Agglutination principle that utilizes bacteria as inert particles to which the antibody is attached
 Pros: Exhibit greater stability than latex particles and are more refractory to changes in ionic
strength
 Cons: reactions are often difficult to read because bacteria are not colored
 Most frequently used bacteria: _______________________
 _________: naturally binds to the FcStaphylococcus
portion of IgGaureus
antibodies except _______
Protein
 Clinical A IgG3
applications:
 Identification of:
 Streptococci
 Neisseria meningitidis
 Neisseria gonorrhoeae
 Vibrio cholerae 0139
 Haemophilus influenzae
VI. ANTIGLOBULIN-MEDIATED AGGLUTINATION TESTS (AGT)
Coomb’s test
 Aka _______________
 Makes use of reagent antibodies that detect antibodies on RBCs
 Antibodies against human globulin (Antihuman globulin or AHG)
 Green solution (blue dye + yellow dye)
 Preparation of reagent antibodies:
 Purified human globulin (IgG or C3b/C3d) is injected into rabbits/mouse
 The rabbit/mouse produces Abs against the human globulin (Anti-IgG or Anti-C3b/C3d)
 Anti-IgG – detects IgG on the surface of RBCs
 Anti-C3b/C3d – detects C3b/C3d on the surface of RBCs (evidence of complement activation
due to the presence of IgM activity)
 Types of AHG reagents according to their specificity
 Monospecific – contains either anti-IgG or anti-C3b/C3d
 Polyspecific – contains both anti-IgG and anti-C3b/C3d
 Two types of AGT
a) Direct antiglobulin test (DAT)
 Also known as:
 Direct Coomb’s test
__________________
 1-step AGT
__________________
 Detects in-vivo sensitization with IgG or complement components
 Clinical application – DAT is used for the investigation of:
 _____________________________________
Hemolytic transfusion reactions (HTRs)
 _____________________________________
Hemolytic Disease of the Fetus and the Newborn (HDFN)
 _____________________________________
Autoimmune Hemolytic Anemia (AIHA)
 _____________________________________
Drug-induced Hemolytic Anemia (DIHA)
b) Indirect antiglobulin test (IAT)
 Also known as:
Indirect Coomb’s test
 _____________________
2-step AGT
 _____________________
 Detects in-vitro sensitization of RBCs by IgG or complement components
 Clinical application – IAT is the serological principle of the ff.
immunohematology tests:
 _____________________________________
Crossmatching (Major & Minor)
 _____________________________________
Antibody screening/detection
 _____________________________________
Antibody identification
 _____________________________________
RBC phenotyping
 _____________________________________
Antibody titration
 Note:
 When performing AGTs and a negative result occurs (tube method), Coomb’s
control cells (check cells) must be added, and a positive result SHOULD
OCCUR
 Check cells will prove that:
AHG was added
 _____________________________________
AHG added was working properly
 _____________________________________
Washing was adequate
 _____________________________________
 If result is still negative, then AGT is invalidated and should be repeated
 Most common error made when performing AGTs:
___________________________
inadequate washing
03
LABELED
IMMUNOA
SSAYS
LABELED IMMUNOASSAYS
 Analyte
 Unlabeled analyte
 Labeled analyte
 Competitive vs. Noncompetitive assays

 Homogeneous vs. Heterogeneous assays

 Property of the reagent antibody:
 High specificity
 High affinity
 Stable throughout the reaction
 Property should not change upon labelling/tagging
I. RADIOIMMUNOASSAY (RIA)
Yalow & Berson
 Discovered by __________________
 Originally for determination of insulin-anti-insulin complex in DM
 Uses scintillation counter
liquid scintillation counter
 Beta radiation uses _____________________________________
crystal scintillation counter
 Gamma radiation uses _____________________________________
 Labels: radioactive elements
125
I
 _____
 Most common
 Half-life: 60 days
 Emits gamma radiation
 131I
 Half-life: 8.1 days
 Emits beta and gamma radiation
 3H (Tritium)
 Half-life: 12.3 years
 Emits beta radiation
 14C
 Half-life: 5760 years
 32P
 Half-life: 14.3 days
 Uses competitive binding
 Unlabeled analyte vs. Radiolabeled analyte for a limited number of binding sites on a high affinity antibody
 If patient antigen is present = some binding sites will be filled with unlabeled analyte, decreasing amount of bound
radioactive label
 Interpretation:
_____________________________________________________________________________________
amount of radiolabel in bound phase is inversely proportional to the patient analyte
 Applications:
 Primarily for determination of analytes with small size
 Measurement of TSH
 Measurement of IgE
 Practical use in type I hypersensitivity reactions
 _____________________________________ – quantitation of total IgE
RIST (Radioimmunosorbent test)
 _____________________________________ – quantitation of allergen-specific IgE
 Disadvantages:RAST (Radioallergosorbent test)
 Health hazards and disposing problems (i.e., radioactive wastes) – primary problem
 Regulations
 Short shelf-life
 Need for expensive instrument
II. ENZYME IMMUNOASSAY (EIA)
 E + S → E + P (chromogenic, fluorogenic, or luminescent)
 Enzymes as labels can be proven to be advantageous through the ff.:
 Cheap and readily available
 Long shelf-life
 Easily adapted to automation; products are measured using inexpensive instruments
 Non-hazardous
 Only little amount of reagent required
 Can be used qualitatively or quantitatively
ENZYME SOURCE/S
Horseradish peroxidase Horseradish
Alkaline Phosphatase Escherichia coli

β-D-galactosidase Escherichia coli
Glucose-6-phosphate dehydrogenase Leuconostoc mesenteroides
Acetylcholinesterase
Electrophorus electricus
Glucose oxidase
Aspergillus niger
Lysozyme
Egg white
Malate dehydrogenase
Pig heart
Solid phase supports:
Microtiter plates – most common
Nitrocellulose membranes (used for rapid EIA if
combined with nylon. i.e., pregnancy kit)
Magnetic latex beads
 Classification:
a) Heterogeneous EIA: COMPETITIVE
Direct ELISA
 Aka ________________
 Principle: enzyme-labeled Ag and unlabeled Ag compete for a limited
binding site in antibodies bound to a solid phase
 These two antigens are simultaneously added in the test system
o Patient antigen is being detected
 Attached to the solid phase: Reagent antibody
 Washing is involved
 Interpretation:
Enzyme activity is inversely proportional to the concentration of the patient
____________________________________________________________________
antigen
_________________
 If patient antigen is high, less enzyme-labeled Ag could bind to the antibody,
leading to a low enzyme activity
 If patient antigen is low, more enzyme-labeled Ag can bind to the antibody,
leading to a high enzyme activity
 Sensitivity: nanograms (10-9 g/mL)
 Application: primarily used for the measurement of small antigens (e.g., insulin &
estrogen)
b) Heterogeneous EIA: NONCOMPETITIVE
Indirect ELISA
 Aka ______________
 Difference from competitive: enzyme-labeled reagent does not
participate in the initial Ag-Ab reaction
 Attached to the solid phase: Reagent antigen
 Patient antibody is being detected
 Interpretation:
Enzyme activity is directly proportional to the concentration of the patient antibody
_________________________________________________________________________________
 If patient antibody is present, it will attach to the bound antigen, and a secondary enzyme-
labeled antiglobulin will bind to the patient antibody, resulting to increase in enzyme activity
after addition of substrate
 If patient antibody is absent, no antibodies will attach to the bound antigen, leading to enzyme-
labeled antiglobulin not binding and negative result ensues
 Clinical applications:
 Primarily used for the detection of microorganisms that are difficult to isolate
 Remains as the primary screening test for the detection of antibodies to the following
organisms:
o _______________
o HIV
_______________
o Hepatitis A& C
_______________
EBV (IM)
c) Heterogeneous EIA: CAPTURE ASSAYS
Sandwich immunoassay
 Aka _____________________________________
 Attached to solid phase: Reagent antibody
 Known antibody should have high affinity and specificity
 Patient antigen is detected
 Patient antigen should have multiple epitopes
 Interpretation:
Enzyme activity is directly proportional to the concentration of patient’s analyte
_____________________________________________________________________________________
 Clinical applications:
 Primarily used for the detection of antigens with multiple epitopes like:
o Antibodies
 Quantitative IgM determination
 Quantitative IgE determination
 Quantitation of allergen-specific IgE
o Polypeptide hormones
o Proteins
o Tumor markers
o Microorganisms (viruses)
 The epitope must be unique to the organism being tested and must be present in all strains of that
organism
→Rotavirus in stool
→Respiratory syncytial virus in respiratory tract specimens
→Giardia and Cryptosporidium in stool
→Aspergillus, Candida, and Cryptococcus
 Disadvantage:
Hook effect
 ______________
o Unexpected fall in the amount of measured analyte when an extremely high
concentration is present
serum dilution
o Solution: _________________
 There may be problems with nonspecific protein binding on the presence of
antibodies to various components in the testing systems
d) Homogeneous EIA
Washing step is omitted
 Difference from heterogeneous EIAs: ________________________
 Based on the change (decrease) in enzyme activity as a result of Ag-Ab interactions
 Note: Enzyme is inactivated when the labeled antigen reacts with bound antibody
 This is primarily competitive assay
o Increased unlabeled Ag = increased enzyme activity
 Attached to solid phase: Reagent antibody
 Unlabeled antigen competes with enzyme-labeled antigen at the limited binding sites available
from the bound antibody
 Interpretation: Enzyme activity is directly proportional to the concentration of patient analyte
 If patient antigen is high, less enzyme-labeled Ag could bind to the antibody, leading to a high
enzyme activity
 If patient antigen is low, more enzyme-labeled Ag can bind to the antibody, leading to a low
enzyme activity
 Sensitivity: microgram (μg/mL) (less sensitive than heterogeneous)
 Factors affecting sensitivity:
o Detectability of enzyme activity
o Change in activity when antibody binds with antigen
o Strength of antibody binding
o Susceptibility of the assay to interference from endogenous enzyme activity, cross-reacting
antigens, or enzyme inhibitors
 Clinical applications: primarily used for the detection of low-molecular weight analytes
 Hormones
 Therapeutic drugs
 Drugs of abuse
 Note: EMIT (enzyme multiple immunoassay technique) employs Homogeneous EIA
 Comments on EIA:
 Indirect ELISA is more sensitive than direct ELISA
 All patient Ag has chance to participate in the reaction
 Indirect ELISA requires more manipulation than direct ELISA
 Two incubations and two washings
 Heterogeneous EIA has equal sensitivity with RIA
III. FLUORESCENT IMMUNOASSAY
 Discovered by Albert Coons
Fluorophores/fluorochromes
 Label: _____________________________________
 Ringed organic molecules with optimum absorption range
 Absorb incident light then convert this into a light of longer wavelength (lower energy)
 Measured in nanoseconds
 Examples:
Fluorescein isothiocyanate (FITC)
 _____________________________________
• Absorbance: 490-495 nm
• Emission: 517-520 nm
o Color: green
Rhodamine isothiocyanate (RITC)
 _____________________________________
• Aka Tetramethyl rhodamine
• Absorbance: 550 nm
• Emission: 585 nm
o Color: red
Phycoerythrin
 _____________________________________
Europium (β-naphthyl trifluoroacetone)
 _____________________________________
Lucifer yellow VS
 _____________________________________
 Initially used for histochemistry (Ag localization in tissues)
 Clinical applications:
 Rapid identification of:
 Microorganism in cell culture or infected tissue
 Tumor specific antigen on neoplastic tissue
 CD markers on T and B cells
 Transplantation antigens
 Grading of fluorescence:
 Negative – no apple green fluorescence
 1+ – faint apple green fluorescence
 2+ – apple green fluorescence
 3+ – bright apple green fluorescence
 4+ – brilliant apple green fluorescence
 Classifications:
a) Direct immunofluorescence assay (DFA)
 Attached to a solid phase (i.e., fixed to a slide): Unknown
antigen (patient antigen)
 Fluorescent-labeled Ab is added
 Washing is involved
 Positive result: Bright apple-green fluorescence or orange-
yellow objects against dark background
 Interpretation:
degree of fluorescence is directly proportional to the amount of analyte
____________________________________________________________________
 Clinical application:
 Ag detection in tissues and body fluids (e.g., Legionella pneumophila,
Pneumocystis, Chlamydia trachomatis, RSV)
b) Indirect immunofluorescence assay (IFA)
 Attached to solid phase: Known antigen
 Patient Ab binds to the known antigen
 Secondary Ab labeled with fluorophore is added, binding to the
patient Ab
 Interpretation:
Degree of fluorescence is directly proportional to the patient antibody
____________________________________________________________________
 Clinical application – used for Ab identification in:
 Syphilis
 Antinuclear antibodies (FANA)
 Chlamydia and Toxoplasma
 HSV, EBV, CMV
c) Inhibition immunofluorescence assay
 Blocking test in which Ag is first exposed to unlabeled Ab, then to fluorescent-
labeled Ab
 Positive result: NO FLUORESCENCE
o Unlabeled antibody is homologous with the labeled antibody
 Notes in immunofluorescence:
Subjectivity in reading the result
 Cons: _____________________________________
Fluorescence Polarization Immunoassay (FPIA)
 Quantitative Fluorescence immunoassay: _____________________________________
 Based on polarization of fluorescent light emitted from a labeled molecule when it is bound by Ab
 Incident light directed at the specimen is polarized with a lens or prism
 If molecule is small enough and rotates quickly enough, the emitted light is unpolarized
 If a molecule is large (as in the case of antibody, binding to an antigen resulting to a larger
complex), it is unable to tumble as rapidly, and it emits polarized light
 Competitive assay
 Attached to solid phase: reagent antibody
 Labeled antigens compete with unlabeled antigens over a limited number of Ab binding sites
 Interpretation:
______________________________________________________________________
The degree of fluorescence polarization is inversely proportional to the concentration of the unlabeled analyte
 If patient Ag is present, smaller number of fluorescent-labeled antigens will react to

bound antibody, emitting less amount of polarized light
 If patient Ag is absent, more fluorescent-labeled antigens will react to bound
antibody, emitting more amount of polarized light
 Clinical application – this technique is limited to low molecular weight analytes such
as:
 Therapeutic drugs
 Hormones
IV. CHEMILUMINESCENT IMMUNOASSAY
 Emission of light caused by chemical reaction (oxidation)
 Excited molecule that decays back to its original state
 Label: chemiluminescent substances
Luminol
 ______________________
Dioxetane
 ______________________
Acridinium ester
 ______________________
Ruthenium derivatives
 ______________________
Nitrophenyl oxalates (peroxyoxalates)
 ______________________
 These substances are oxidized primarily by hydrogen peroxide coupled with enzyme catalyst
 Once oxidized, they become excited
 As they return to their ground state, they release light (rapid flash or continuous glow)
 Classification:
 Heterogeneous
 Competitive – therapeutic drugs and steroid hormones
 Sandwich – protein hormones
 Homogeneous
 Advantages:
 Sensitivity equals that of RIA & EIA
 Stable, non-toxic reagents
 Little reagent requirement (inexpensive)
 Relatively high speed of detection resulting to faster turnaround time
 Disadvantages:
 Lack of precision in hydrogen peroxide injection
 Quenching by other biological materials
 Fluorescence of an analyte is reduced due to the excited molecule losing some of its energy by interacting with
other substances in the solution
 Detection is through photomultiplier (PM) tubes
In the end, you shall succeed. And no
one can stop you

Serology Review: Tom Kairo T. Pacquing

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SEROLOGY REVIEW

Tom Kairo T. Pacquing

Dilute antibody in the patient serum and retest

Several large agglutinates, few free cells Clear

Many medium-sized agglutinates, Moderate number of free Clear

Many small agglutinates, many free cells Turbid

No agglutinates Dark, turbid, homogeneous

R Rouleaux – nonspecific aggregation appearing like a stack of coins; disappears NA

Alkaline Phosphatase Escherichia coli

 If patient Ag is present, smaller number of fluorescent-labeled antigens will react to

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