Wolkite University

Department of Medical Laboratory Sciences

Molecular Biology, Serology & Immunology
Course Team
 Topics for Tutorial Class
 Perform immunological assays
 Perform serological assays
 Utilize serologic equipment
 Interpret report and document

By:Dereje Abebe(MSc) 12/23/2023

WKU,MLS,IMMUNOLGY and SEROLOGY ASSAYS
1
 Quote
“If the purpose of learning is to score well
on a tests, we have lost our sight of the
real reason for learning.”

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 overview of Serology and immunology
objectives
 At the end of this session, you should be able to:
 Define the term serology
 Discuss the general immunology overview

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 Introduction
 Serology is the scientific study of blood serum or other body
fluids.
 In practice, the term usually refers to the diagnostic
identification of antibodies in the serum.
 Such antibodies are typically formed in response to:
 an infection (against microorganisms)
 against other foreign proteins ( example, to a mismatched
blood transfusion)
 own proteins (in instances of autoimmune disease).

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Immunology
 The study of immune system or immunity
 the study of all aspects of host defense against infection and of
adverse consequences of immune responses.
 The study of the physiological mechanisms which enable the body
to recognize materials as foreign and to neutralize, metabolize or
eliminate them without injury to the host tissue.
Immunity
 State of protection from infectious diseases.
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Definition cont’d…
Immune system
 A remarkably versatile defense system that has evolved to protect
animals from invading pathogenic microorganisms and cancer.
 It is able to generate an enormous variety of cells and molecules
capable of specifically recognizing and eliminating a variety of
foreign invaders.

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 Overview of immune system

Immune
System

Innate Adaptive
(Nonspecific) (Specific)

Cellular Humoral Cell- Humoral

Components Components Mediated (Ab)

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 Structural configuration of Antibody

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 Antibodies bond to antigenic determinants
Antigenic determinants are portions of the antigen

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 Primary vs Secondary Humoral
Response

IgG

IgM

IgM

IgG

First Second
exposure
exposure
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Antigen-Antibody
Interactions:
Principles & Applications

- A bimolecular association
involving various noncovalent interactions
- Is similar to an enzyme-substrate interactions,
but not lead to an irreversible chemical alteration

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 Nature of Ag/Ab Reactions

- Four types of non-covalent forces operates over a very short distance

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 immunological Assays
Objectives
 Describe the principle of different types of primary,
secondary and tertiary binding tests
 Under standing and defining all aspects of primary
secondary and tertiary binding tests
 Differentiate the polyclonal & monoclonal antibody

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 immunological techniques

Definition
 Immunological techniques: it is the technique that
include both experimental methods to study the
immune system and methods to generate or use
immunological reagents as experimental tools.
 The most common immunological methods relate to
the production and use of antibodies to detect specific
proteins in biological samples.

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 The three immunological techniques
are :
 Primary binding: measure the direct binding of the antibody
to its antigen; or
 allowing Ag and Ab to combine and then measuring or
visualizing the amount of immune complex formed.
 Secondary binding: measure the amount of antibody
present by the change it induces in the physical state of the
antigen.
 measure the results of antigen – antibody interaction in vitro.
 Tertiary binding: measure the consequences of immune
responses in vivo.
E.g. measurement of the protective effects of antibody.

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 [Link] binding tests
 Fluorescent immunoassay
 Radioimmunoassay
 Enzyme immunoassay

 Blotting -western
-Northern
-Southern
 “Chemiluminescent immunoassays”

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 [Link] test
 There are two type of (FAT):
1. Direct
2. Indirect
1. Direct fluorescent antibody tests

 Used to detect and identify unknown antigen in specimens.

E.g. Viral, bacterial, and parasitic antigens
 It is called direct test because the fluorescent dye is attached, or
labeled, directly to the antibody.

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 Direct immunofluorescence tests…cont

 The fluorochrome used is usually fluorescein isothyocynate

(FITC), which gives a yellow-green fluorescence.

 A fluorescent substance is one that, when absorbing light of

one wavelength, emits light of another (longer) wavelength.

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 2. Indirect Fluorescent Antibody Test
 Un-labelled antibody combines with antigen and the antigen
antibody complex is detected by attaching a fluorescent-labeled
anti-species globulin to the antibody.
 The antibody, therefore, is labeled indirectly.

 Fluorescent-labeled antihuman globulin is used if the antibody

is of human origin.

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 IFAT…cont’d
 The indirect FAT is used in two main ways:
 To detect and identify unknown antigen in specimens

 To detect antibodies in a patient's serum using a known

antigen (microorganism).

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 Immunofluorescence

Fluorochromes
-Fluorescein (490→517nm)
-Rhodamine (515→546nm)
-Phycoerythrin
: absorb light of one wavelength & emit
fluorescence at a longer wavelength than
fluorescein.

mIgM-producing B cells indirectly stained with rhodamine-conjugated

secondary Ab under a fluorescence microscope.
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 [Link]
 Radioimmunoassay is widely-used because of its great
sensitivity.
 Using antibodies of high affinity, it is possible to detect a few
picograms (10−12 pg) of antigen in the tube.

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  Conventional RIA
 is a competitive immunologic procedure
 It measures very low concentrations of antigens (or
antibodies) by using radioactively labeled antigens as
competitors.
 Radioactive isotopes such as 3H , 14C, 35S, 30P or 125I
can be used for labeling

 Radioactive isotopes are molecules with unstable nuclei

and therefore emit radiation spontaneously.

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  RIA technique utilizes three components
 Patient antigen
o the specific compound we wish to determine.
 Labeled antigen
o the same compound as above to which is attached a
radioactive label.
 Antibody
o specific for the sample and labeled antigen.

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 Competitive Binding Assays
 Labeled Ag competes with patient
Ag for a limited number of
binding sites on solid-phase Ab.
 Very little patient Ag is present,
making radioactivity of the solid
phase to be high.
 More patient Ag is present, & the
radioactivity of the solid phase is
reduced in proportion to the
amount of patient Ag bound.

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  RIA is highly sensitive method to detect low concentration
of unknown (unlabeled) antigen
 RIA is used to assay:
 Hormones,
 Drugs,
 Enzymes,
 Microbial antigens
 It also used to the detection of antibody

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 [Link]
 Enzyme-linked immunosorbent assay use to detect the level of
an antibody or an antigen in a sample.

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 Enzymes and Substrates
Horse radish peroxidase (HRP) enzyme
 its substrates are
 OPD o-phenylenediamine dihydrochloride & its
maximum absorbance: 492 nm
TMB 3,3',5,5'-tetramethylbenzidine & its maximum
absorbance: 450 nm
ABTS 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic
acid]-diammonium salt & its maximum absorbance: 410 nm

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 Enzymes and Substrates
alkaline phosphatase (AP) enzyme
Its substrates are

PNPP p-Nitrophenyl Phosphate, Disodium Salt & its

maxium absorbance: 405 nm

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 Types of ELISA
1. Direct ELISA (antigen-coated plate; screening Ab)

2. Indirect ELISA(antigen-coated plate; screening Ab)

3. Sandwich ELISA(antibody-coated plate; screening Ag)

4. Competitive ELISA(screening soluble Ag)

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ELISA Strengths Weaknesses
1) High sensitivity due to the fact that multiple
enzyme-conjugated secondary antibodies can bind
to the primary antibody
2) Many different primary antibodies can be 1) High background signal may
recognized by a single enzyme-conjugated occur because the coating of the
Direct and
secondary antibody giving the user the flexibility of antigen of interest to the plate is not
Indirect
using the same enzyme-conjugated secondary specific (i.e., all proteins in the
antibody in many different ELISA (regardless of the sample will coat the plate)
antigen being detected)
3) Best choice when only a single antibody for the
antigen of interest is available
1) The use of antigen-specific capture and detection
1) Optimizing the concentrations of
monoclonal antibody increases the sensitivity and
the capture and detection
specificity of the assay (compared to the indirect
Sandwich monoclonal antibodies can be
ELISA)
difficult (especially for non-
2) Best choice for detecting a large protein with
commercial kits)
multiple epitopes (such as a cytokine)
1) Impure samples can be used
2) Less sensitivity to reagent dilution effects 1) Requires a large amount of highly
Competitive
3) Ideal for detecting small molecules (such as a pure antigen to be used to coat plate
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hapten)
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 [Link] Immunoassays

 Reading assignment

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 [Link] binding tests

Agglutination

Precipitation

Complement fixation

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 Antigen-Antibody interaction
 It is a two-step process:
 Sensitization

 Lattice formation

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 Factors affecting antigen-antibody
interaction
 Each stage of the process is affected by different factors
 Sensitization is affected by:
 Nature of the antibody molecules
 Nature of the antigen-bearing surface
 Lattice Formation dependent on
 Physicochemical factors(like ionic strength, pH,
temperature, duration of incubation, others)
 Relative concentrations of antigen and antibody

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 Fig .show the relationship between Ag-Ab

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 Enhancement of agglutination
 Controlling surface charge
 Increasing Viscosity
 Agitation and centrifugation
 Use of antiglobulins(AHG)
 Use of carrier particles

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Agglutination method

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 Agglutination tests
 Agglutination:
 The clumping of particles that have antigens on their
surface, such as erythrocytes, by antibody molecules
that form bridges between the antigenic determinants.

 Antibodies that produce such reactions are often called

agglutinins.

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 Agglutination tests…cont
 Used for organisms that are difficult to grow in the lab.
 Used for rapid identification of the pathogen to initiate the
treatment early.
 Used in district setting, because they are:
 Simpler to perform
 Require no special equipment
 Usually less expensive

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 Agglutination tests…cont
 Agglutination tests can be performed:
 On slides or tiles
 In tubes
 In micro titration plates
 Most agglutination tests are qualitative, but dilutions can
be made to provide quantitative information.

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Types of agglutination reactions
[Link] Agglutination
occurs when antigens are found naturally on a particle
 Direct Bacterial agglutination
 Can be used to detect antibodies directed against the
pathogens
 Can be used to detect the presence of unknown antibodies in
the patient using a known to bacterial antigen
 e.g. Widal test
 Hemagglutination
 an agglutination reaction involves red blood cells
 e.g. ABO blood group
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 Direct Agg. Cont’d
 The antigen is a natural part of the solid’s surface.
 Often performed at room temperature.
 May use centrifugation to bring antigen and antibody into closer
proximity.
 Can be used to detect antigen or antibody

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 [Link]/Indirect agglutination
 Also known as indirect agglutination
 Employs particles that are coated with Ag not normally found
on their surfaces.
 Artificial carrier particles use to indicate visibly of antibody-
antigen reaction has taken place.
 Examples:
 Latex particles
 Gelatin
 Silicates
 Colloidal charcoal

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Passive agglutination: Antigen is attached to the carrier
particle, and agglutination occurs if patient antibody is
present.

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 Passive agglutination cont…
 The use of particles provides the advantage of consistency,
uniformity, and stability.
 Used to detect:
 Rheumatoid factor
 Antinuclear antibody occurring in autoantibody disease
 Antibodies to group A streptococcus, Treponema pallidum
and viruses such CMV, rubella, and HIV-1/HIV-2
 There is ‘always’ the risk of nonspecific agglutination

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 [Link] passive agglutination
 Antibody is attached to a carrier particle.
 Often used to detect microbial antigens
 E.g. Group B streptococcus, Staphylococcus aureus,
Neisseria meningitidis, streptococcal groups A & B,
Haemophilus influenzae, rotavirus, Cryptococcus
neoformans, Vibrio cholera, and Leptospira.
 Use of monoclonal antibodies has greatly cut down on cross-
reactivity.

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Reverse passive agglutination: Antibody is attached to the carrier
particle, & agglutination occurs if patient antigen is present

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 Precipitation
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 Definition
 Precipitation: is the combination of soluble antigen with
antibody to produce a visible insoluble complex.

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Measurement of precipitation by light scattering
 Turbiditmetry: measures the decrease in the amount of light as it
passes through a particulate solution.
 In turbidity the decrease in transmittance is measured at a zero
degree angle to the light passing through the cuvette.
 Nephelometry: is used to measure the amount of light that is
scattered as it passes through the particulate solution.
 In nephelometry the particles scatter the light at a 90 degree angle
and the rate of light scatter is measured.
 Both methods measure Scattered Light and used to determine the
concentration of the particulate solution.

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 Nephelometry
 When light is passed through the
solution, the precipitates cause
the light to scatter at various
angles. Light
source
 The light that is scattered at a
particular angle is measured.
 The amount of scattered light is
corresponds to the level of
antigen in the sample. Detector

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 Reading assignment
 Other Measurement of precipitation
such as:-
Immunodiffusion Techniques
electrophoretic techniques

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 Complement fixation

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 Complement Fixation
 is an immunological test that can be used to detect the presence
of either specific antibody or specific antigen in a patient's serum.

 It is widely used to diagnose infections, particularly with

microbes that are not easily detected by culture methods, and in
rheumatic diseases.

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 Complement Fixation principle

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 [Link] immunological tests
 It is used to determine the protective value of an anti-serum in
animal.
 Measures the consequences of immune responses in vivo
 These tests are much more complex than primary and secondary
tests but results reflect the practical significance of the immune
response
 E.g. measurement of the protective effects of antibody.

 Reading assignment for more understanding about it.

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Polyclonal and monoclonal
antibody production

 How is antibody and Antigen can be

produced and purified???

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