SEROLOGIC

PROCEDURES
ASSOC . PROF. JOCELYN D. DOMINGO
MR. ALVIN REY FLORES

UST FACULTY OF PHARMACY

• SEROLOGY - medical science that deals with blood sera, their components,
immunologic properties and reactions

• SEROLOGIC TESTS/ PROCEDURES –

• blood tests or any of the several laboratory procedures carried out on sample of
blood serum
• detect the presence of antibodies against microorganism;
• designed to demonstrate Ag-Ab reactions in vitro
RECAP: ANTIGEN AND ANTIBODY

What is an ANTIGEN?
• Foreign substances that stimulates antibody formation but may not always
able to evoke and immune response

• What is an IMMUNOGEN?
• IMMUNOGENS- macromolecules capable of triggering an adaptive
immune response by inducing antibodies or sensitized T cells in an
immunocompetent host.
ANTIGENS AND IMMUNOGENS

• Epitope: portion of the Antigen that is recognized by the Antibody

• AKA: Antigenic Determinant
• Hapten: Non immunogenic small substances that need to be complexed to
larger molecules to be able to stimulate immune response
• Non-immunogenic by themselves
PREREQUISITES FOR IMMUNOGENECITY

HIGHLY IMMUNOGENIC SUBSTANCES ARE BASED ON:

1. MOLECULAR SIZE OR WEIGHT
2. CHEMICAL COMPOSITION AND MOLECULAR COMPLEXITY
1. Proteins
2. Polysaccharides
3. Lipids
4. Nucleic Acids
3. FOREIGNNESS
4. ABILITY TO BE PROCESSED AND PRESENTED WITH MHC
MOLECULES
ANTIBODY

• AKA: IMMUNOGLOBULINS
• Composed of 4 Polypeptide Chains
• Specific proteins that combines with an antigen
• Paratope: Ag binding site
• Antibody Classes: IgG, IgA, IgM, IgD, IgE
 IMMUNOGLOBULIN CONSISTS OF 4
POLYPEPTIDE CHAINS
N terminus
Ag binding site Variable regions Ag binding site

VH
VL
CH1
CL

Disulfide
CH2
bonds
Constant
EM Rabbit Ig
region
CH3 X 2,042,500
Light chain

C terminus Heavy chain

ANTIGEN AND ANTIBODY INTERACTIONS

• Immune Reaction involving formation of Antigen and Antibody Complexes

• Involves the interaction of the Epitope and the Fab Region
• Result of NON-COVALENT, and REVERSIBLE INTERMOLECULAR
INTERACTIONS
• Ag + Ab Ag- Ab
• Four Types of Intermolecular Forces
1. Ionic or Electrostatic
2. Hydrogen Bonds
3. Van der Waals
4. Hydrophobic bonds
WHAT IS AN ANTIGEN –ANTIBODY REACTION?

• Characteristics : invisible and precise ; Engagement

comparable to a “Lock and Key”.
• KINETICS of ANTIGEN- ANTIBODY REACTION –
complies with the “ Law of Mass Action”
AFFINITY VS. AVIDITY
TYPES OF IMMUNOLOGIC REACTIONS

A. Primary Reactions
• Specific recognition and combination of Ag and a corresponding Ab
• Examples: Immunofluorescence, Radioimmunoassay, Enzyme Immunoassays,
Chemiluminescence
B. Secondary Reactions
• Conformation of Amino Acids Chain due to interchain hydrogen bonding
• Examples: Precipitation, Agglutination, Complement Fixation, Neutralization
C. Tertiary Reactions
• Occur as biologic reactions and involves folding of polypeptide chains through
hydrophobic and hydrogen bonds
• Examples: Phagocytosis, Opsonization, Chemotaxis, Immune Adherence, Cellular
Degradation
IMMUNOASSAYS

• are quick and accurate tests that can be used on-site and in the laboratory
to detect specific molecules and /or measure the concentration of a
molecule in a solution through the use of an antibody or immunoglobulin.
• Immunoassays rely on the inherent ability of an antibody to bind to the
specific structure of a molecule referred to as "analyte" and which
chemically in many cases a protein.
 IMMUNOASSAYS

• In an immunoassay, an antibody molecule recognizes and binds to an

antigen.
• Binding is related to:
• Concentration of each reactant
• Specificity of antibody for antigen
• Affinity & avidity for pair
• Environmental conditions
TYPES OF IMMUNOASSAY FORMATS

• HOMOGENEOUS

üConsists only of a liquid phase

ü allowing to make an assay-measurement by a simple mix and read
procedure
üNo need for washing or separation step
• HETEROGENEOUS
üInvolves the use of a solid phase (microwell, bead)
ü requires a the use of a separation method (washing) to discriminate the
free from the bound components
HOMOGENEOUS VS. HETEROGENEOUS
PRIMARY REACTIONS

• Since the Ag-Ab Reaction is NOT VISIBLE, labels are employed in

IMMUNOASSAYS
• LABELED IMMUNOASSAYS
• LABELS improves the analytic sensitivity of the test
• Addition of labels to Ag-Ab interactions allows QUANTITATIVE
measurement of target analyte
PRIMARY REACTIONS

• Indicator Labels for Immunoassay fall into 3 Categories:

1. Radioactive Isotopes – with unstable nuclei and emit radiation
spontaneously
• 125I and 131I are commonly used isotopes in the clinical laboratory
• Application: RADIOIMMUNOASSAYS
2. Fluorochromes – molecules that absorb light and becomes transiently
excited. The excited molecule becomes stable and is emitted as visible light
(FLUORESCENCE)
• Application: Immunofluorescence
3. Enzymes – biological catalysts;
• Acts upon the the substrate added in the reaction leading to a change that
can be measured
• Enzymatic Activity can be a: Colored Product or Chemiluminescence
• Ex. ALP, Horseradish Peroxidase
• Application: ELISA
INDICATOR LABELED IMMUNOASSAYS CAN BE
CLASSIFIED AS
• COMPETITIVE
üPrinciple: Labeled and Unlabeled Analytes (usually Antigens) can
compete for the same binding site on the antibody
ü can be carried out in an antigen-capture or antibody-capture
format,
üthe measured signal is inversely correlated to the concentrations of
analyte in the sample
üEx. RIA, EMIT, FPIA
• NON COMPETITIVE
üDetection of either Ag or Ab in a biological sample
üAg and Ab are permitted to interact without deliberate
introduction of competing molecules
DIRECT, INDIRECT, SANDWICH IMMUNOASSAY

• DIRECT DETECTION
• method uses a labeled primary antibody that reacts directly with the antigen.
• Ag can be immobilized on the assay plate or in a capture assay format
• Application: Immunohistochemistry

• INDIRECT DETECTION
• method is the most popular format for ELISA.
• It uses a labeled secondary antibody for detection. The secondary antibody
has specificity for the primary antibody.
DIRECT, INDIRECT, SANDWICH IMMUNOASSAY

• SANDWICH ASSAY-
• Sensitive, Robust and Most Powerful ELISA Assay Format
• Analyte (to be measured) bound between 2 Primary Antibodies
(Capture and Detection Ab)
• Ab Sources: Mouse IgG and Rabbit IgG
• For sandwich assays, it is beneficial to use secondary antibodies that
have been cross-adsorbed to remove any antibodies that have affinity
for the capture antibody.
COMPONENTS OF LABELED IMMUNOASSAY

1. Labelled and unlabelled analyte

2. Receptor Molecule
3. Standard or Calibrator Solutions
4. A means to separate the free from the bound components
5. A mean to measure the labelled product
 SEPARATION METHODS

• ADSORPTION ONTO PARTICLES

• Uses particles to trap small antigens (labeled or not)
• Mixture of charcoal & cross-linked dextran is most commonly used.
• PRECIPITATION
• Occurs when environment is altered, affecting solubility of protein
• USE OF SOLID PHASE
• Used to immobilize reagent antibody or antigen and separate free from
bound-labeled reactant after washing
 SECONDARY REACTIONS
PRECIPITATION
• Involves the interaction of antigen with antibody resulting in a precipitate.
• 2 phases:
• INITIAL
• FORMATION of LATTICE WORK
• ZONE OF EQUIVALENCE
• Ag involved: SOLUBLE
 PRECIPITATION

• Procedures:
BETA : Ramon procedure
Ab concentration (varying), Ag concentration. (fixed)
ALPHA: Dean Webb procedure
Ag concentration (varying), Ab (fixed)
TYPES OF IMMUNOPRECIPITATION REACTION

Precipitation Using Liquid Medium

• Test tube Method: turbidity
• Slide Method: use microscope
• Capillary method: dense granulation in the interphase
• Ring test: precipitin ring formation
• The antigen is carefully layered over the antiserum, without mixing so that an
interphase is formed. Diffusion of each reagent occurs into the other.
• If the system is homologous, precipitation will occur at the point in the tube
where the proper ratio of antigen to antibody is reached.
• Example: Ascoli’s test for anthrax diagnosis.
 PRECIPITATION
• B. Precipitation in Semi Solid Medium (Agarose Gel)
• B.1. Single Immunodiffusion
• B.2 Double Immunodiffusion
• B.3. Immunodiffusion in Two Dimension
• Single Diffusion
• Double Diffusion
 PRECIPITATION
SINGLE
IMMUNODIFFUSION –
ONE DIMENSION (OUDIN)
Patient sample with soluble antigen

(+) reaction- formation of pptin band

Agar impregnated with known Ab

 PRECIPITATION
Double Diffusion in One Dimension
• AKA: OAKLEY- FULTHORPE TEST
• Antibody (antiserum) is incorporated in agar, poured into a tube and allowed to harden.
• A second layer of agar without antibody is placed above and allowed to solidify.
• Antigen solution is placed above the agar.
• The precipitin band appears in the plain agar column.
OAKLEY- FULTHORPE TEST
 PRECIPITATION

Single Diffusion, Double Dimension (RADIAL IMMUNODIFFUSION)

• uses a plate containing agar with known antibody
• Patient serum is placed on the wells
• Diameter of the pptn ring is directly proportional to the concentration of the
target antigen
RADIAL IMMUNODIFFUSION
 SINGLE DIFFUSION , DOUBLE DIMENSION

• Mancini : end point method (24-72 h)

: the square of the diameter is directly proportional to the conc. of Ag

• Fahey and McKelvey: kinetic method (18 h)

: the diameter is proportional to the log of the concentration of Ag
 IMMUNOPRECIPITATION

Double Diffusion, Double Dimension (OUCHTERLONY TECHNIQUE OR DOUBLE

ANGULAR IMMUNODIFFUSION)
• Diffusion of both Ag and Ab
• Diffusion of 3 reagents placed in wells (2 Ag and 1 Ab)
• Diffusion occurs in all directions
• This test can be used to find out relationship between antigens.
• Complicated Diffusion Patterns
OUCHTERLONY TECHNIQUE OR DOUBLE ANGULAR
IMMUNODIFFUSION)
 IMMUNOPRECIPITATION
3. IMMUNOELECTROPHORESIS
• Double-diffusion technique that incorporates current to enhance results.
• Serum: source of Ag
• Trough: place the antiserum
• Double diffusion occurs at right angles
• Precipitin line/arc develops
 IMMUNOELECTROPHORESIS

a. Ags migrate under an electric current (-) Ag (+)

b. Rgt. Ab Added. Diffusion through gel (-) (+)

An;body

c. (+) rxn - Pptn arcs formed (-) (+)

 IMMUNOPRECIPITATION
4. COUNTER IMMUNOELECTROPHORESIS OR COUNTER CURRENT
IMMUNOELECTROPHORESIS OR VOLTAGE FACILITATED DOUBLE
IMMUNODIFFUSION
• Double immunodiffusion assay with voltage applied w/ pH = 8.6 to the medium
• Apply Ag to one side , Ab to the other (Placed in Parallel Wells)
• End result: precipitin line formation
• Rapid but not sensitive
 IMMUNOPRECIPITATION
5. Rocket Electrophoresis (LAURELL TECHNIQUE)
• antiserum is incorporated into the agar and the unknown antigen is placed
in the well and electrophoresed
• The height of the rocket (conical), measured from the well to the apex, is
directly proportional to the amount of Ag
 IMMUNOPRECIPITATION
6. IMMUNOFIXATION ELECTROPHORESIS

• First described by Alper and Johnson

• 2 Stage Technique
• A. Electrophoresis
• B. Immunoprecipitation
• Similar to immunoelectrophoresis except that antiserum is applied directly on the
gel’s surface rather than placed in a trough.
• Agarose or cellulose acetate can be used
• Eg: Western, Southern, and Northern blotting
 AGGLUTINATION
• Ag is insoluble/particulate
• Formation of agglutinates/aggregates
• 2 mechanisms:
1. Sensitization
2. Lattice formation
 TYPES OF AGGLUTINATION REACTIONS
• DIRECT AGGLUTINATION
• Antigen is found naturally on the cell surface
• Application: Blood Typing, Febrile Agglutination Tests;
 TYPES OF AGGLUTINATION REACTIONS
• PASSIVE AGGLUTINATION
• Ag is attached/coated to a carrier particle Example : ___
• Application: RF Latex Test;
 TYPES OF AGGLUTINATION REACTIONS
• REVERSE PASSIVE AGGLUTINATION
• Antibodies are attached to a carrier particle
• For Detection of Antigens
• Application: Detection of CRP
 TYPES OF AGGLUTINATION REACTIONS

• AGGLUTINATION INHIBITION
• 2 Step Procedure
• Homologous Ag inhibits aggn of Ag coated particle
• 1st step: soluble Ag in px sample + known Ab rgt.
• 2nd step: particulate Ag is added
(+) result : _____________________________
AGGLUTINATION - INHIBITION
 TYPES OF AGGLUTINATION REACTIONS
• ANTIGLOBULIN TECHNIQUES
• COOMB’S TEST
• Primarily employed in Blood banking Procedures
• Coomb’s Reagent is added to bridge the gap between the cells
• to demonstrate incomplete antibodies
• TYPES:
• DIRECT COOMB’s
• INDIRECT COOMB’s
 TYPES OF AGGLUTINATION REACTIONS
• COAGGLUTINATION
• Term given to systems using bacteria as the inert particles to which antibody
is attached.
• Staphylococcus aureus is most frequently used
• With surface protein called protein A which naturally adsorbs the Fc portion
of Ab
• The active sites face outward and are capable of reacting with specific
antigen.
• Exhibits greater stability than latex particles and are more refractory to
changes in ionic strength.
 COMPLEMENT FIXATION
• Basis: Ag combines with an Ab in the presence of complement, the
complement is “FIXED” by the immune complex
• Inability of the complement to react with cells sensitized with other
immune complexes
• Two systems involved for complement fixation:
• 1. Test system/Bacteriolytic system
• 2. Indicator system/Hemolytic system
 COMPLEMENT FIXATION
• Components:
• Known target Ag rgt (ex. beef heart extract, bacterial Ag)
• Complement - Best source : Guinea pig serum
• Hemolysin or Amboreceptor - the Ab used to sensitize indicator cell
• Best source: Rabbit antisera
• Indicator cells - Sensitized Sheep RBCs
PROCEDURE:

+ + +

POSITIVE REACTION:

+ +
+

NO HEMOLYSIS TEST SYSTEM FIXES

COMPLEMENT
 NEUTRALIZATION
• This is the reaction in which the antigenic activity is stopped/nullified
(neutralized) by its specific antibody.
• Target: To detect toxins, viral agents or Abs to the toxin or viral agents
• Types of Neutralization Tests
• 1. Toxin Neutralization (e.g. Schick Test, Dick Test, ASO Titration Test)
• 2. Virus Neutralization
 FLOCCULATION
• AKA : Natural Clumping
• Involves aggregation of colloidal particles
• Observed as fleecy mass when a suspension of Ag and Ab is agitated
• Midway reaction between agglutination and precipitation
• Involves noncellular particulate Ags
• Example: RPR, VDRL slide test
 RAPID IMMUNOASSAYS
• Immunochromatography
• Membrane Based Cassette Assays
• For Point of Care Testing
• Lateral Flow Test
• Based on antigen-antibody interactions employing capillary flow
through a membrane
COMPONENTS :
 Lysing agend Test band Control band
Bound Labled AB. (bound AB) (bound AB)
AB

Free labled
AB

Nitrocellulose strip

• Dye-labelled antibody, specific for target antigen, is present on the lower end of
nitrocellulose strip or in a plastic well provided with the strip

• Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line, and
either antibody specific for the labelled antibody, or antigen, is bound at the control line
 Immunochromatography

Parasitized
Blood
Test band Control band
(bound AB) (bound AB)

Parasite antigen (AG.)

Captured by labled AB.

Blood and labled Ab flushed along the strip

Blood and buffer, which have been placed on strip or in the well,
are mixed with labelled antibody and are drawn up strip across
the lines of bound antibody
Source: http://www.wpro.who.int/rdt
Immunochromatography

Captured Ag-labled Captured labled Ab

Ab-complex

Labled AB-AG- Labled AB-AG-

complex complex
Captured by Captured by
bound AB of bound AB of
test band control band

If antigen is present, some labelled antibody will be trapped on

the test line. Excess-labelled antibody is trapped on the control
line
Source: http://www.wpro.who.int/rdt
RESULTS :
 IMMUNOCHROMATOGRAPHY

• 1-2. components of the reaction port

• 3. site of application of unknown samples
• 4. site where immune factor in the sample cross-link Ag/orAb/ conjugate
• 5. This part serves as the procedural control
• 6. Area where the Unknown analyte bound to the Ag/orAb/ conjugate is captured