Immunology- Mic 451

Lab -Notes

1. Agglutination

• is the aggregation of particulate matter caused by the combination with

specific antibody

• 1896: First observed by Gruber and Durham when serum antibody was

found to react with bacterial cells.

• Agglutination occurs if an antigen is mixed with its corresponding antibody

called isoagglutinin. This term is commonly used in blood grouping.

This occurs in biology in three main examples:

• The clumping of cells such as bacteria or red blood cells in the presence of

an antibody or complement. The antibody or other molecule binds multiple

particles and joins them, creating a large complex. This increases the

efficacy of microbial elimination by phagocytosis as large clumps of

bacteria can be eliminated in one pass, versus the elimination of single

microbial antigens. Another example occurs when people are given blood

transfusions of the wrong blood group.

• Types of particles that participate in such reactions:

• Erythrocytes

• Bacterial cells

• Inert carriers such as latex particles

• Primary phenomenon (SENSITIZATION)

First reaction involving Ag-Ab combination

Initial reaction: rapid and reversible

Cross link formation visible aggregates (stabilization)

Secondary phenomenon: (LATTICE FORMATION)

stable network (visible reaction)

Governed by physiochemical factors:

Ionic strength of milieu

pH

temperature

2. HEMAGGLUTINATION

Hemagglutination is the process by which red blood cells agglutinate,

meaning clump or clot. The agglutin involved in hemagglutination is

called hemagglutinin.

In cross-matching, donor red blood cells and recipient's serum or plasma are

incubated together. If agglutination occurs, this indicates that the donor and

recipient blood types are incompatible

DIRECT AGGLUTINATION

Test patient serum against large, cellular antigens to screen for the presence of

antibodies.

• Antigen is naturally present on the surface of the cells.

• In this case, the Ag-Ab reaction forms an agglutination, which is directly

visible.

PASSIVE AGGLUTINATION

• An agglutination reaction that employs particles that are coated with

antigens not normally found in the cell surfaces

• Particle carriers include:

▫ Red blood cells

▫ Polystyrene latex

▫ Bentonite

▫ Charcoal

The latex particles are coated with IgG and mixed with the patient's serum

REVERSE PASSIVE AGGLUTINATION

Antibody rather than antigen is attached to a carrier particle

For the detection of microbial antigens such as:

Group A and B streptococcus

Staphylococcus aureus

Neisseria meningitidis

Haemophilus influenzae

PRINCIPLE: latex particles coated with antibody are reacted with a patient sample

containing suspected antigen.

Latex Agglutination

• Antibody molecules can be bound to each latex beads

• It will increase the potential number of exposed antigen-binding sites.

• When an antigen is present in test specimen, it may bind to the latex bead

thus forming visible cross-linked aggregates.

• Latex particles can be coated with antigen (pregnancy testing, rubella

antibody testing)

3. Blotting Techniques

What is blotting?

Blots are techniques for transferring DNA , RNA and proteins onto a carrier

so they can be separated, and often follows the use of a gel electrophoresis.

The Southern blot is used for transferring DNA, the Northern blot for RNA

and the western blot for PROTEIN.

TYPES OF BLOTTING TECHNIQUES

Blotting technique

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA.

Western blot

It is used to detect protein.

This method Involves separation, transfer and hybridization.

It is a method routinely used in molecular biology for detection of a specific

DNA sequence in DNA samples. The DNA detected can be a single gene, or

it can be part of a larger piece of DNA such as a viral genome.

Southern blotting combines agarose gel electrophoresis for size separation of

DNA with methods to transfer the size separated DNA to a filter membrane

for probe hybridization.

The key to this method is Hybridization.

Hybridization - Process of forming a double-stranded DNA molecule

between a single-stranded DNA probe and a single-stranded target patient

DNA.

Western blotting

Western blotting (1981) is an Immunoblotting technique which rely on the

specificity of binding between a protein of interest and a probe (antibody

raised against that particular protein) to allow detection of the protein of

interest in a mixture of many other similar molecules.

The SDS PAGE technique is a prerequisite for Western blotting .

Dot blot

A dot blot (or slot blot) is a technique in molecular biology used to

detect biomolecules, and for detecting, analyzing, and identifying proteins .

It represents a simplification of the northern blot, southern blot, or western

blot methods. In a dot blot the biomolecules to be detected are not first

separated by chromatography.

Instead, a mixture containing the molecule to be detected is applied directly

on a membrane as a dot, and then is spotted through circular templates

directly onto the membrane or paper substrate.

This differs from the western blot because protein samples are not separated

electrophoretically. This is then followed by detection by

either nucleotide probes (for a northern blot and southern blot)

or antibodies (for a western blot).

4. What Is An ELISA?

It uses a 96-well plate to measure a protein or substance based on an

antigen/antibody

Analyte

The sample being analyzed and in immunoassays the analyte is either

Antibody or Antigen

ELISA technique

Is a biochemical technique used mainly in immunology to detect the

presence of an antibody or an antigen in a sample.

An ELISA test uses components of the immune system and chemicals to

detect immune responses in the body (for example, to infectious microbes).

The technique is divided into

1- Competitive ELISA

2- Sandwich ELISA (also called direct ELISA)

3- Indirect ELISA

1- Competitive ELISA

The labelled antigen competes for primary antibody binding sites with the

sample antigen (unlabeled). The more antigen in the sample, the less labelled

antigen is retained in the well and the weaker the signal).

2- Sandwich ELISA (also called direct ELISA)

Method

) Plate is coated with a capture antibody;

(2) sample is added, and any antigen present binds to capture antibody;

(3) detecting antibody is added, and binds to antigen;

(4) enzyme-linked secondary antibody is added, and binds to detecting

antibody;

(5) substrate is added, and is converted by enzyme to detectable form.

3- Indirect ELISA

Method

The protein antigen to be tested for is added to each well of ELISA plate,

where it is given time to adhere to the plastic through charge interactions.

A solution of non-reacting protein is added to block any plastic surface in

the well

Then the serum is added, which contains a mixture of the serum antibodies,

of unknown concentration, some of which may bind specifically to the test

antigen that is coating the well.

Afterwards, a secondary antibody is added, which will bind to the antibody

bound to the test antigen in the well. This secondary antibody often has an

enzyme attached to it

A substrate for this enzyme is then added.

This substrate changes colour upon reaction with the enzyme.

The colour change shows that secondary antibody has bound to primary

antibody,

which strongly implies that the donor has had an immune reaction to the test

antigen.

The higher the concentration of the primary antibody that was present in the

serum, the stronger the colour change.

spectrometer is used to give quantitative values for colour strength

Indirect ELISA

5. SEROLOGICAL DIAGNOSIS IN IMMUNOLOGY

METHODS OF DETECTION OF ANTIBODIES

1. Immuno-precipitation Assays

= detect antibodies in solution

= qualitative indication of the presence of antibodies

= end-point is visual flocculation of the antigen and antibody in suspension

2. Complement Fixation

= based on the activation or fixation of complement following binding of

complement factors to Ag-Ab immune complexes

3. Neutralization

= the ineffectivity of an organism or the activity of toxin is neutralized by

specific antibody

= rarely used for diagnostic purposes

= mainly used to detect antibody formation after vaccination

4. Particle Agglutination

= relatively simple and fast

= capable of detecting lower concentration of antibodies

= designed to detect antibodies to viruses, subsequent to interaction or vaccination

= utilize Ag coated latex particles, coal particles, bentonite particles or erythrocytes

5. Immunofluorescence

= requires use of microscope equipped to provide ultraviolet illumination or an

instrument capable of irradiating the assay with UV light and detecting the

resultant fluorescence with a fluorometer

6. Enzyme Immunoassay

= the most sensitive

= usually indirect assay that depends on the use of an antihuman IgG or IgM

antibody conjugate

7. Radioimmunoassay

= high sensitivity

6. What is an Epitope ?

An epitope is the small site on the antigen which is recognized by the antibody.

Usually between one and six sugars or amino acids on the surface of the antigen.

Monoclonal Antibodies

• Much more complicated to produce than polyclonal antibodies.

• Process begins by immunizing an animal (most commonly a mouse) with an

antigen.

• The animal’s spleen is removed.

• Monoclonal antibodies recognize one epitope only.

Polyclonal Antibodies

• Produced by immunizing an animal with the appropriate antigen.

• The immunized animal’s serum is collected.

• Antibodies can then be purified from the serum.

• Since one antigen induces the production of many antibodies the result is a

‘polyclonal’ mixture of antibodies.

Fluorophore

• Fluorophore is the region of the molecule capable of exhibiting fluorescence.

Fluorochrome is a dye or molecule capable of exhibiting fluorescence

How Does Fluorescence Microscopy Work ?

• Excitiation: Energy in the form of a photon of light is absorbed by a

fluorophore.

• While in the excited state, the fluorophore undergoes conformational

change.

• Emission: a photon of energy is emitted and the fluorophore returns to its

original state.

• A single fluorophore can be repeatedly excited and detected.

7. THIN LAYER IMMUNOASSAY

Background In formation

Many antigens adsorbs firmly to the hydrophobic surface of polystyrene. The

exposed antigenic determinants on the monomolecular film are fully reactive with

antibody. Antibody bound to the antigen may be detected by fluorescence in

ultraviolet light after reaction with fluorescein labeled anti-immunoglobulin.

Thin-layer immunoassay (TIA) is a technically simple and accurate method

for the assay of antigen-antibody reaction. The technique basically uses the

principle that macromolecules may be absorbed as a thin layer to a polystyrene

surface. These macromolecules most often preserve their serological reactivity and

hence capable of binding antibodies. The presence of antibodies on the antigen

surface is visualized as a distinct hydrophilic condensation pattern when surface is

exposed to water vapor. TIA has been used for the determination of antibodies

against purified proteins, polysaccharides, and lipopolisaccharides also proved

suitable for screening of serum samples.

8. PHAGOCYTOSIS

Background In formation

Inflammation is one of the ways our body reacts to an infectious agents or

injury. During the inflammatory response, white blood cells with multilobed

nucleus, PMNs (Polymorphonuclear neutrophils) migrate out of capillaries and

into the infected tissue. PMN attracted to the site of infection through a process

known as chemotaxis. In Chemotaxis, phagocyctic cells move migrate towards the

infected cite where phagocytosis begin. The phagocytic cells are PMNs,

monocytes, and macrophages. Monocytes are white blood cells with one large

nucleus. Monocytes circulate in our pheripheral blood for only about 1 day. They

may eventually move into tissue where they mature into a cell called a

macrophages. Macrophage may life for several month. PMNs and macrophage are

the major phagocytic cells of the immune system.

Phagocytosis is the process when phagocytes ingest, kill, and digest

infectious organisms and unwanted cellular debris such as old cells or particulate

matter. The particulate susceptible to the phagocytosis can be intracellular, such as

Mycobacterium tuberculosis, or extracellular such as Streptococcus pneumonia.

The intracellular pathogen are ingested by macrophage when they are released

from a dying cell.

9. PRECITPITIN: Ring Test

Background Information

The ring test is one of the oldest methods for detecting soluble antigen and

antibody. It is simple, quick, and reasonably sensitive for the detection of trace

quantities of antigen, as used forensically to detect adulteration of food meats with

our meat proteins. It is less sensitive for the detection of antibody. A good

antiserum usually gives a ring test visible in minutes at a dilution factor 1:5.

The ring test is based on the propensity of antibodies to form complexes with their

corresponding antigens. When antibodies attach to antigen molecules in solution,

the molecules become part of an insoluble antibody-antigen complex, and a visible

precipitate forms.

Ring Test

10. IMMUNODIFFUSION : Radial Immunodifussion

Background Information

In Immunodiffusion tests, antibodies and or soluble antigens are loaded

into separate wells of a gel and are allowed to difuse, each reagent moving radially

into the gell. An immobile precipitate, visible as a band (precipitin line) in the gel,

develops if specific antibody- antigen binding takes place, and if antibody-antigen

components are present at optimal proportions.

In radial immunodifussion, the antibodies are equally distribute, loaded into

a well, diffuses. When antigens molecules move through the gel, they bind to and

carry antibodies, until the ratio of antibodies are optimal for complex formation.

Procedure (Immunodiffusion)

1. Obtain 5 µl of human serum, and pipette it into designated wells of the RID

assay gel.

2. Reserve the three vails for standard concentrations. High standards, low

standard, and a serum control (Provided in the radial immune diffusion kit)

3. Place the gel into moist box, and incubate for 24 hours at room temperature to

allow diffusion on and banding (The gel can be stored in refrigerator for several

weeks, if the box is keep moist)

4. Read the result of your assay: Measure the diameter of the circle of precipitate

(in centimeters)

11. Antigen & Antibody Reactions

Affinity vs. Avidity

• Affinity

– Measure of the binding strength between an antigenic determinant

(epitope) and an antibody combining site.

– It is the sum of the attractive and repulsive forces operating between

the antigenic determinant and the combining site .

• Avidity

– The cumulative binding strength of all antibody-epitope pairs which

results from multivalent antigen and antibody.

Precipitation Serological Tests

• One of the easiest of serological tests

• Soluble AG & Ab interact and form a lattice that develops into a visible

precipitate.

• Occur best when antigen and antibody are present in optimal proportions

(Equivelance).

• Antibodies that aggregate soluble antigens are called precipitins.

• Polyclonal antibodies can form lattices, or large aggregates.

• However, monoclonal antibody can link only two molecules of antigen and

no precipitate is formed.

Diagrammatic representation of radial & double immunodiffusion.(Precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess.)

The region of equivalence

Simple Immunodiffusion Reactions

• In electro-immunodiffusion, diffusion is combined with electrophoresis

• Electrophoresis separates antigen molecules according to differences in

their electrical charges and molecular weight then specific antibodies

diffuse and react with separated antigen forming precipitin bands.

III- Electro-Immnodiffusion

a) Immunoelectrophoresis

• Method

– Ags are separated by electrophoresis

Ag

-+

Ag

Ab

Ag

Ab

– Ab is placed in trough cut in the agar

• Interpretation- Precipitin arc represent individual antigens

• Qualitative - Relative concentration

c) Rocket Electroimmunodiffusion

• Antigen is electrophoresed into gel containing antibody.

• The precipitin reaction results in a rocket-shaped precipitin formation.

• The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

IV-Measurement of Precipitation by Light

• Principle: Antigen-antibody complexes, when formed, will precipitate in a

solution resulting in a turbid or cloudy appearance that can be measured by:

Turbidimetry

Passing light through a cloudy solution. (Net decrease in light

intensity)

Nephelometry

Measuring light scattered at a particular angle after being passed

through a solution i.e. indirect measure.

Amount of light scattered correlates to the concentration of the

solution

• Evaluation: Ag concentration is proportional to the

turbidity of complexes (end point)