Serology as a Diagnostic

Technique

Characteristics of Any Diagnostic

Techniques

Any useful detection strategy must be: Specific: yield a positive response for only the target

organism or molecule.

Sensitive: identify very small amount of the target the target organism or molecule, even in the presence of other potentially interfering organisms or substances

Simple: to be run efficiently, effectively, and inexpensively on a routine basis

Replicable (or Reliability ): to be repeated different times with different technicians under the same experimental conditions and leading for the same results

Diagnostic Methods

Biological assays: Symptomatology

Microscope: Observation

Serological Assays: Antibodies

Molecular Diagnostic Tools: DNA

Serological Assays

Immunology (sometimes called serology ): is the

study of the serum, and the properties and functions

of its components.

Immuno-technology, is an important arm of

biotechnology, constituting the industrial scale

application of immunological procedures to produce

vaccines, for mass immunisation to prevent prevalent

diseases and/or producing immunological therapeutic

agents to cure the afflicted.

When an infecting organism gains entry into the

mammalian system for the first time, the immune

system of the mammal reacts, mainly in response to

the proteins of the invading organism, generally

Serological Assays As A Diagnostic Tool

Antiserum: Blood serum containing antibodies arising out of immunisation or after an infectious disease.

Production of antibodies: Antibodies are produced by the lymphocytes. The process of antibody production and immune response are complex and both the lymphatic and the blood systems are very closely involved: Polyclonal antibodies: antibodies produced by molecules with

several different antigenic determinants (epitopes) and/or several different cell populations.

Monoclonal antibodies: antibodies produced against a single antigenic determinant (epitope) and/or by a single cell population; hence are very specific.

Antigen-binding sitesAntibody A

Antigen

Antibody BAntibody C

Epitopes(antigenicdeterminants)

Antigen Recognition by Lymphocytes An antigen is any foreign molecule that is specifically

recognized by lymphocytes and elicits a response from

them

A lymphocyte actually recognizes and binds to just a small,

accessible portion of the antigen called an epitope.

Antigen Recognition by

Lymphocytes Epitope: a part of a protein

molecule that acts as an

immunogenic/antigenic

determinant [and so

determines specificities]

a macromolecule, such as a

protein, may contain many

different epitopes, each

capable of stimulating the

production of specific

antibodies, each with a

correspondingly specific

binding site.

B Cell Receptors for Antigens B cell receptors

bind to specific,

intact antigens, are

often called

membrane

antibodies or

membrane

immunoglobulins

Antigen-bindingsite

Antigen-binding site

Disulfidebridge

Lightchain

Heavy chains

Cytoplasm of B cell

A B cell receptor consists of two identical heavy chains and two identical light chains linked by several disulfide bridges.

(a)

Variableregions

Constantregions

Transmembraneregion

Plasmamembrane

B cell

C C

Antibodies: (also known as immunoglobulins, abbreviated

Ig) are gamma globulin proteins that are produced by the

immune system of an organism in response to exposure to a

foreign molecule and characterised by its specific binding to a

site, related to an epitope of that molecule; induced response

proteins.

The antibodies, like all proteins, are formed of chains of

amino acids, which undergo very complex packing, giving the

proteins a specific and functionally significant final shape

(tertiary configuration), which determines most of the

characteristics of the protein.

Molecular Structure of Antibodies The conventional model of the

Ig molecules is a ‘Y’ shapedconfiguration, with two heavy chains and two light chains, with two open arms containing the antigen combining sites, which occur on both the light and the heavy chains.

The two heavy chains are bound together by disulphide bonds.

At any point, the molecule has two chain sections, parallel to each other.

Clonal Selection

of Lymphocytes

In a primary immune

response binding of

antigen to a mature

lymphocyte induces the

lymphocyte’s

proliferation and

differentiation, a process

called clonal selection

Clonal selection of B

cells generates a clone of

short-lived activated

effector cells and a clone of

Antigen molecules

Antigenreceptor

B cells thatdiffer inantigen

specificity

Antibodymolecules

Clone of memory cells

Clone of plasma cells

Antigen moleculesbind to the antigenreceptors of only oneof the three B cellsshown.

The selected B cellproliferates, forminga clone of identicalcells bearingreceptors for theselecting antigen.

Some proliferatingcells develop intoshort-lived plasmacells that secreteantibodies specificfor the antigen.

Some proliferating cells develop into

long-lived memory cells that can

respond rapidly uponsubsequent exposureto the same antigen.

Clonal Selection of Lymphocytes

In the secondary immune response memory cells

facilitate a faster, more efficient responseAntibody c

oncentr

ation

(arb

itra

ry u

nits)

104

103

102

101

100

0 7 14 21 28 35 42 49 56

Time (days)

Antibodiesto A

Antibodiesto B

Primaryresponse toantigen Aproduces anti-bodies to A

2Day 1: First exposure toantigen A

1 Day 28: Second exposureto antigen A; firstexposure to antigen B

3 Secondary response to anti-gen A produces antibodiesto A; primary response to anti-gen B produces antibodies to B

4

Antibody

Classes

These are the five

major classes of

antibodies, or

immunoglobulins

differ in their

distributions and

functions within the

body

IgM: primary response

and 10 active site, 5 Y-

shape

IgG: one single Y-

shape 2 active sites,

secondary response

First Ig class produced after initial exposure to antigen; then its concentration in the blood declines

Most abundant Ig class in blood; also present in tissue fluids

Only Ig class that crosses placenta, thus conferring passive immunity on fetus

Promotes opsonization, neutralization, and agglutination of antigens; less effective in complement activation than IgM

Present in secretions such as tears, saliva, mucus, and breast milk

Triggers release from mast cells and basophils of histamine and other chemicals that cause allergic reactions

Present primarily on surface of naive B cells that havenot been exposed to antigens

IgM(pentamer)

IgG(monomer)

IgA(dimer)

IgE(monomer)

J chain

Secretorycomponent

J chain

Transmembraneregion

IgD(monomer)

Promotes neutralization and agglutination of antigens; very effective in complement activation

Provides localized defense of mucous membranes byagglutination and neutralization of antigens

Presence in breast milk confers passive immunity onnursing infant

Acts as antigen receptor in antigen-stimulated proliferation and differentiation of B cells (clonal selection)

Binding of antibodies to antigensinactivates antigens by

Viral neutralization(blocks binding to host)

and opsonization (increasesphagocytosis)

Agglutination ofantigen-bearing particles,

such as microbes

Precipitation ofsoluble antigens

Activation of complement systemand pore formation

Bacterium

Virus Bacteria

Solubleantigens Foreign cell

Complementproteins

MAC

Pore

Enhances

Phagocytosis

Leads to

Cell lysis

Macrophage

Antibody-mediated mechanisms of antigen disposal

Antiserum PreparationObtaining PAb for the diagnostic purposes:

1. Choose of the animal: decide wither polyclonal or

monoclonal

2. Preparation of antigen

3. Immunization: the antigen is emulsified sometimes

with with an equal volume of complete adjuvant

(Freund’s adjuvant- mannitol monoleate mixed

with paraffin oil)

4. Collection of sera: (the supernatant)

5. Titration….

6. Storage: (addition of antiseptic substances (NaN3);

lyophilized; at temperature below -20°C)

Typical Serological Profile After Acute

Infection

Note that during reinfection, IgM may be absent or present at a low level transiently

IgG Purification with Protein A-

spherosis column:1. Column preparation by washing with Eluting Buffer.

[Glycine 0.1 M pH 3] and then with 50 ml PBS 1x.

2. Adding of antiserum [previously preabsorbed with healthy

material] to the column

3. Washing the column with 50 ml PBS 1x.

4. Eluting the IgG with Eluting Buffer and collect fraction

of 1 ml. Then bring pH to 7.3 with Neutralizing Buffer

[Tris 1M pH 9]

5. Titration: Calculate the absorption at the 280 nm for each

fraction. Form fractions of 1 ml with concentration of 1

mg/ml (1.445 OD) and store them at -20ºC

6. Wash the column with 50 ml of PBS 1x and conserve them

at +4ºC.

Monoclonal Antibodies Immunochemical techniques are extremely useful for the

rapid and accurate routine detection of host pathogens

and ultimately the diagnosis of host disease and their

relatedness

The introduction of hybridoma technology has provided

methods for the production of homologous and biochemically

defined immunological reagents of identical specificity

Hybridoma are produced by a single cell line and are directed

against a unique epitop of the immunizing antigen.

Antibodies-

Production

1. Spleen cells are removed washed

minced and gently agitated to

release individual cells, some of

which are

antibody-producing B cells.

2. Myeloma cells (immortal B-cell Line)

are genetically defective for the

enzyme:

“Hypoxanthine-Guanine

PhosphoRibosylTransferase

(HGPRT-)

3. The combined cells are mixed with

35% PEG (to facilitate fusion

between cells) for few minutes to

form hybridoma, then transferred to

a growth medium containing:

Hypoxanthine, Aminopterin, and

Thymidine (HAT medium).

4. The HAT medium allow only the

Explanation of use HAT

medium

The myeloma (HGPRT-) will not be able to synthesize

purines in HAT medium:

The (HGPRT-) myeloma alone or as myeloma-myeloma fusion

cells cannot use the Hypoxanthine as a precursor for

biosynthesis of purines (adenine and guanine), which of

course essential for nucleic acid synthesis.

They may use the other pathway for purine synthesis by the

enzyme dihydrofolate reductase, but the enzyme

Aminopterin which included in the media will inhibit the

enzyme dihydrofolate reductase activity.

Spleen+myeloma fused cells would be the only

survivors in HAT medium as they contain HGPRT

which allow the use of Hypoxanthine for biosynthesis

of purines.

Although Thymidine will overcome the blockage of

dihydrofolate reductase activity by Aminopterin

Monoclonal Antibodies

Production

Serological Techniques In serology Ab acts as a probe for detection of Ag => ppt

Direct Methods: it is possible to visualize the Antigen

Antibody reaction without the help of other reactions

Precipitation or agglutination

Immunoelectron microscopy

Agar precipitation

Indirect Methods:

ELISA

Comparison among the two

Antiserum Preparation

Polyclonal Antibodies (Pabs) has two drawbacks:

The prepared amount of PAbs produced each time

could be varied.

PAbs couldn't be used to distinguish two similar

target protein.

Monoclonal Antibodies (MAbs) are useful for their:

Reproducibility

Specificity

Serological Relationships1. Serological distinguished

2. Serological related:A. Partial identity reaction

B. Non-identity reaction

3. Serological identical

Ag2 Ag

As

Ag2 Ag

As

Ag2 Ag

As

Ag2 Ag

As

A B

Serological Relationships

Gel preparation

0.8 g agarose

0.2 g NaN3 +

100 ml distilled water

Preparation of antiserum:

Mix with saline solution (0.85 g NaCl in

100 ml distilled water)

The first reading is made after 2 hours

and the second reading at 18 hours.

Immunological Diagnosis: ELISA

ELISA Diagnostic Kits – Some

Characteristics

A number of disease detection kits have been developed for use at the site where a disease is suspected.

These kits, which in most cases do not require laboratory equipment, are very useful

Some tests only take five minutes to perform.

The diagnostic kits are based on a method that uses proteins called antibodies to detect disease causal agents called antigen.

One of the technique used is called ELISA (enzyme-linked immunosorbent assay).

ELISA Diagnostic Kits This assay is based on the ability of an antibody to recognize and

bind to a specific antigen, a substance associated with a host pathogen.

The antibodies used in the diagnostic kits are highly purified proteins produced by injecting a warm-blooded animal (like a rabbit) with an antigen associated with one particular host disease.

The animal reacts to the antigen and produces antibodies. The antibodies produced recognize and react only with the proteins associated with the causal agent of that host disease.

Color changes on the unit’s surface indicate a positive (disease present) reaction.

ELISA Diagnostic Kits

ELISA-test

Sample preparations

Generalized ELISA-test protocol1) Coating of the plates

(wells):

with IgG diluted in

coating buffer

2) Adding of the extract

: Coating antibody

: specific Protein

: non specific proteins

Incubation at 37°C

Incubation at 37°C

Generalized ELISA-test

protocol

5) Observe or measure the amount of colored product

3) Adding of conjugated IgG

diluted in conjugating buffer.

: Labeled antibody with Alkaline phosphatase

4) Adding of substrate solution

p-nitrophenyl-phosphate

diluted in substrate buffer.

: Substrate Enzyme

Incubation at 37°C

ELISA-test

Light Filter 405

Healthy O.D * constant (2.5) >

Positive control

Different Types of ELISA-test

Double Antibody

Sandwich ELISA (DAS)

Triple Antibody

Sandwich-Indirect

ELISA (TAS)

Enhanced Double

Antibody Sandwich-

Indirect ELISA (DAS-I)

: Coating antibody

: Antigen

: Labeled antibody

: Substrate Enzyme

: The intermediate antibody

: The labeled anti-mouse IgG

: Biotinylated anti-body

: Streptavidine-enzyme

conjugates

F(ab')2 Antibodies Production

Papain cleavage: (Fc)

Pepsin cleavage [F(ab)2]

F(ab')2 Antibodies Production

• A system has been devised to carry out DAS-I using asingle antiserum

• The antibodies are treated with the enzyme pepsin toremove the Fc portion of the molecule.

• The remaining F(ab')2, fragment still has the antigenbinding sites and will bind to the immunosorbent, but willnot bind to protein A.

• The F(ab')2 fragments are used as trapping “antibody”and the whole antibody is used as the intermediateantibody.

• It is possible to use specific substances that recognize Fcportion for increase the reaction sensitivity.