- Ability of a clinical test to be
ANTIGEN – ANTIBODY REACTION
TWO WAYS that antigen and antibody react
together;
• IN VITRO – outside, observable
outside = involves serological testing
• IN VIVO – inside = usually related to
our immune response
DIFFERENT TYPES
❖ IgM
not require specific antigens.
❖ IgG
❖ IgE – requires specific antigen
❖ IgA – does not require specific
antigen, however this only occurs into
serum and/or involving in our body
fluids.
❖ IgD – does not interact with antigens.
Stimulates B-cells.
- Usually is the second type of
immunoglobulin that is present in
our B-cells
BASIS ON IN VITRO METHOD
- Usually there are only (2) two
immunoglobulins that associated
IgM and IgG.
✓ IgM in serological testing – reacts
best at room temperature or cold
temperature.
✓ IgG – reacts best at body
temperature.
IgM – largest immunoglobulin
IgG – smallest immunoglobulin
SENSITIVITY
positive in the presence of its
homologous antigen
- The probability that an individual
with the disease will have a
negative result
Sensitivity – in a small amount this will react.
SPECIFICITY
- Ability of a clinical test to be
negative in the absence of its
homogenous antigen
- The probability that an individual
without disease will have a
negative result
Specificity – specific
CROSS REACTION
- Reaction of an antibody with an
antigen (heterologous) other than
the one that induced its formation;
reaction may be weaker that with
the inducing (homologous) antigen
Heterologous – not same
- Same structure different specie or
similar structure but different
specie
- Ex. Cowpox interact to Smallpox
AFFINITY
- The binding strength of a single
antibody combining site for a
ligand (PER)
- is the initial force of attraction that
exists between a single Fab site
on an antibody molecule and a
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 single epitope or determinant site
on the corresponding antigen.
(Stevens)
Affinity – only one will react.
These are the different bonds; though they
are in single form but they do react still, or we
can still observe reactions.
I. lonic bond
VAN DER WAALS
II. Hydrogen bond
III. Hydrophobic bond
IV. Van der Waals force
IONIC BOND
- It occurs between oppositely
charged particles;
- Are interactions between positive
and negatively charged groups
ions.
- "Electrostatic Interactions" –
because this require electricity to
form an interaction between
positive and negative.
- (+) & (-) – a chance of
compatibility
- If in hemolytic transfusion
reaction it will lead to death
(nakakamatay)
HYDROGEN BOND
- It involves an attraction between
polar molecules that have a slight
charge separation and in which
the positive charge resides on a
hydrogen atom
- This require HYDROGEN, with the
help of hydrogen this allow the
antigen interact with antibody.
HYDROPHOBIC BOND
- It occurs between nonpolar
molecules that associate with one
another and exclude molecules of
water as they do so.
- Remove water (sagabal), to let
the antigen and antibody interact
to each other
- "Electrostatic Interactions"
- Forces occur because of the
interaction between the electron
clouds of oscillating dipoles.
- It requires electricity
- Difference to ionic bond is that it
needs an interaction of the
electron clouds of oscillating
dipoles.
- With the help of the hydrogen this
allowed the antigen and antibody
interact to each other, for the
electrostatic forces which is an
example of ionic bond, opposite
attracts. Van der Waals forces
require which is similar with the
ionic bond this require also an
electrostatic force. Hydrophobic
bond this require water, water
should be removed so that there
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 would be an interaction of antigen
and antibody.
AVIDITY
- The binding strength of an
antibody for an antigen (the sum
of the binding affinities of all the
individual Combining sites on the
antibody) - PER
- It represents the sum of all the
attractive forces between an
antigen and an antibody. This
involves the strength with which a
multivalent antibody binds a
multivalent antigen, and it is a
measure of the overall stability of
an antigen-antibody complex -
Stevens
Avidity – multivalent, all immunoglobulin
binds to each other.
✓ More than single.
✓ Sum of all antigen and antibodies.
- A measure of the overall strength
of interaction between the
antigen and antibody must take
into consideration all the binding
sites (Stanley)
- Overall strength - mas dumadami
IMMUNOLOGIC REACTIONS:
PRECIPITATION
• 1897 by Kraus (Stevens)
• 1938 Marrack (Bryant); Heidelberger
and Kendall & It involves combining
antigen with soluble antibody to
produce insoluble complexes that are
visible) (Stevens)
• It is the term for the aggregation of
soluble test antigens.
• It is the combination of soluble antigen
with soluble antibody to produce a
visible insoluble complex. (Turgeon)
• The soluble antigens used in the
precipitin reaction are solution of
molecule with are usually CHON or
carbohydrate in nature.
Most of the time in serological testing terms
that is used are the ff.
1. Precipitation
2. Agglutination
3. Color reaction
4. Inhibition
o Antigen is perfectly fit/ interact
with antibody
o If soluble reaction, walang
makikita visible na insoluble na or
aggregation. Ang magkikita lang
is ang changes in color which its
either transparent or no color or
with color at all.
o In precipitation both antigen and
antibody bind together. However,
the end protect is INSOLUBLE
COMPLEXES.
PRECIPITIN
❖ Soluble antibody combines with
soluble antigen in two steps
i. (Rapid invisible formation of antigen-
antibody aggregates
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 - hindi nakikita if how they interact or • involves antigen-antibody
bind in the immunoglobulin combination
• Initial binding
ii. Slowly forming visible complex lattice
of interlocking aggregates
- nagkakaroon ng precipitation or
reaction
❖ Simplest method for detection of
antigen-antibody reactions
❖ Relative concentrations of antigen and 2nd LATTICE FORMATION
antibody at site of reaction critical to
amount to form precipitate form • representing the sum of interactions
- PRECIPITIN - end product of between antibody and multiple
precipitation antigenic determinants on a particle
- Not all antigen and antibody ay • formation of large aggregates
nag kakaroon ng precipitation. ✓ Affinity - no visible reaction
✓ Avidity- visible reaction
AGGLUTINATION
❖ Gruber and Durham, Widal and Sicar
❖ Agglutination is the process by which
particulate antigens such as cells
aggregate to form larger complexes
when a specific antibody is present.
❖ Agglutination is the visible
aggregation of particles caused by
AGGLUTININ
combination with specific antibody.
(Stevens) - end product of agglutination
❖ It is the process whereby specific
antigens (e.g. red blood cells) • Soluble antibody forms a lattice with
aggregate to form larger visible an insoluble particulate or cellular)
clumps when the corresponding surface antigen
specific antibody is present in the • Agglutination tests are inly semi-
serum. (Turgeon) quantitative, but highly sensitive
✓ AGG: Insoluble antigen + soluble
STAGES UNDER AGGLUTINATION antibody
✓ PREP: soluble both antigen and
BORDET (AGGLUTINATION) antibody but insoluble complexes
1st SENSITIZATION

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FACTORS AFFECTING ANTIGEN- region may prohibit multivalent
ANTIBODY REACTION binding.
- IgM antibodies, on the other hand,
I. Zone of Equivalence - equal form have a diameter of about 35m, so
- Optimum precipitation occurs in they are strong agglutinins.
the zone of equivalence, in which ✓ flexible, big type of
the number of multivalent sites of immunoglobulin
antigen and antibody are ✓ they are pentamer, multivalent
approximately equal. binding
✓ ANTIGEN: ANTIBODY = 1:1 V. Potentiator - allow the antigen -
✓ PROZONE: there is an ANTIBODY antibody reacts better, enhance the
excess reaction.
✓ POST ZONE: there is ANTIGEN a. Low ionic strength saline (LISS)
excess - common in the blood bank
✓ A false-negative reaction may b. Albumin
take place in the c. Dextran
prozone/postzone due to high d. Polyethylene glycol (PEG)
antibody/antigen concentration e. Enzymes
f. Bromelin, Papain, Trypsin, Ficin
VI. Agitation and Centrifugation - if
centrifuge, specific gravitational force
that require para ang antigen -
antibody natin ay perfectly bind to
each other. Usually 5 minutes,
II. Temperature depends on the rpm.
- IgG agglutinate best a 30°C to VII. Antiglobulin reagent - use this to
37°C - Body temp detect the presences of IgG (since
- IgM react best at temperatures small). Common in blood bank
between 4°C and 27°C - Room specifically in cross matching.
temperature
III. pH TYPES OF IMMUNOLOGIC REACTION
- Most reactions produce optimal 1. PRIMARY
antigen-antibody combination
• Specific recognition and combination
when the pH is between 6.5 and
of antigen with the binding site of its
7.5
corresponding antibody.
IV. Type of Antibodies
• A technique to QUANTITATE the
- IgG class often cannot bridge the
antigen or antibody with the use of
distance between particles,
radioisotope (radioimmunoassay),
because their small size and
enzyme (ELISA), fluorescent label
restricted flexibility at the hinge

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 assay or a method to separate the ▪ Chemotaxis
antigen-antibody complex from free ▪ Immune adherence
antibody or antigen in solution. ▪ Cellular degradation
✓ no value, non-quantitative, no visible
QUANTITATIVE TEST: NUMERICAL reaction so it is less sensitive
VALUE ✓ not masyado specific at sensitive
• Immunofluorescence: Fluorescent label
• Radioimmunoassay: Radioisotope
• Immunoenzymatic Assay: Enzyme or
ELISA
• More sensitive; Non-Visible reaction
✓ no precipitation, no
agglutination, only numbers
2. SECONDARY
• Conformation of the amino acid chain
resulting from interchain hydrogen
bonding.
• Demonstrate antigen antibody
reaction.
- Less sensitive; Visible
Example:
▪ Precipitation in gel or solution
▪ Direct
Agglutination/Hemagglutinati
on
▪ Complement Fixation
✓ no number, no value but with reaction
✓ not sensitive
3. TERTIARY
• It involves the folding of polypeptide
chains through hydro phobic and
hydrogen bonds
• Immunologically in vivo; biologic
reaction is detectable.
- LESS SENSITIVE
Example:
▪ Phagocytosis
▪ Opsonization

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