Immunology & SerologyFebruary 2006

The Immune System• The human immune system responds from attacks

from outside the body.• Consists of skin, tears, saliva, mucous• Thymus • Spleen • Lymph system • Bone marrow • White blood cells • Antibodies • Complement system • Hormones

AntibodiesAntibodies, also called immunoglobulins

The antibodies inactivate antigens by• Attaching to antigen and causing them to lyse• neutralization (binding to specific sites to prevent

attachment—this is the same as taking their parking space)

• (c) agglutination (clumping)• (d) precipitation (forcing insolubility and settling out of

solution), and other more arcane methods.

The Immune Response1. Recognition of antigens

2. Removal of antigen

• Lymphocytes - WBCs produced by bone marrow & spleen. Lymphocytes enter the blood and the lymph nodes.

• T-cells - lymphocytes that pass through thymus gland• B-cells - lymphocytes that do not pass through thymus

gland; divide to form clones and product Abs.• Macrophages - WBC also involved in immunity.

The Immune Response cont…..• Both B-cells and T-cells have antibodies on their

surface. When bacteria enter the body, macrophages change the bacteria slightly and present them to the lymphocytes. The lymphocytes have antibodies that match the bacterial antigens. The Ags and Abs join. Once the B-cell is linked to an antigen, the B-cell becomes activated and begins to enlarge. This large B-cell divides to form a clone of cells called plasma cells which produces only one type of antibody. These antibodies are poured into the blood and other tissues and are free to attach to the bacterial antigen.

The Immune Response cont…..• T-cells help stimulate B-cell growth and antibody

production. T-cells also have Abs on their surface. Entire T-cells can attach to antigens.

• Ag-Ab combination can lead to:

1. The Abs make viruses/bacteria more susceptible to attack by macrophages.

2. Macrophages engulf the microbes by phagocytosis• Digested microbes enter the lymph and are destroyed.• Ab can cause microbes to clump making them

inactive.

Immune System

Antigen

Activates

B-lymphocytes

T-lymphocyteCellular Immunity

Humoral ImmunityImmunoglobulinsspecific antibodies

Cellular ImmunityCell lysisWhite blood cell productionLymphokines

Antibodies• The part of the immune response that is mounted

when a germ (antigen) invades the body.• Bind to the antigen (Ag) enabling the immune cells to

recognise the foreign invaders and therefore remove & destroy them.

• There are 5 classes of antibodies produced by the body- IgG, IgA, IgM, IgE and IgD.– IgA, IgM and IgG are the main antibodies formed in

response to viral or bacterial infection and are the antibodies tested for by Panbio kits.

IgA Antibodies

• Predominant Ab in seromucous secretions (saliva, tracheobronchial secretions).

• Critical first line defence system that protects against invasion by microorganisms.

• Indicative of acute infection.

• Important marker for mucosal infections such as Pertussis and Mycoplasma.

IgA Dimer

IgM Antibodies

• First antibodies to appear after primary antigenic stimulus.

• Marker of acute phase of an infection.

• Disappear usually within 1-3 months after infection.

IgM Pentamer

IgG Antibodies

• Major antibody of secondary (anamnestic) responses.

• Provide life long protection.

• Indicator of past exposure or infection and immune status.

• Marker of active infection in paired sera.

Antibody Response Curve

Immune Response to Dengue Infection

An

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od

y L

ev

el

P rim a ry In fe c tio n

S e c o n d a ryIn fe c tio n

Ig G

Ig M Ig M

O n s e t o f S y m p to m s

O n s e t o fS y m p to m s

V iru s V iru s Ig MC u to ff

Ig G C u to ff

H A I 1 :2 5 6 0

Immunological Tests & Techniques

There are a number of tests that diagnose disease based on the detection of antibodies. Some common techniques are listed below.

Immunofluorescence (IFA) Neutralisation Haemagglutination Inhibition (HAI) Complement Fixation (CFT) Enzyme-linked Immunosorbent assay (ELISA)

Immunofluoresence Assay (IFA)

Method for identification of Ags in tissue sections & on cells, or for identifying Abs to them.

Direct- Fluorescent Ab is incubated with cells or tissue section. The Ab binding to Ag is visualised by UV light.

Indirect (IFA)- Cells or tissue are incubated with test serum Ab, which is then visualised by the addition of a second layer fluorescent anti-antibody.

Disadvantages- subjective, time consuming, inconvenient for large scale screening.

Neutralisation• Virus is mixed with patient’s serum and indicator cells.

If serum contains specific Ab, it will bind to the virus. If sufficient Ab binds to the virus it will prevent the virus from infecting the indicator cells. If virus infects the indicator cells it destroys them.

• Used as a confirmatory test

• Disadvantages: time consuming, labour intensive, takes 5-7 days for result.

Complement fixation Detects antibody (or antigen).

Test Ab is mixed with Ag and complement. Ag-Ab complexes form if specific Ab is present and fix the complement. If specific Abs are not present active complement remains. Active complement is detected by adding Ab sensitised red cells which lyse if complement is present.

Disadvantages- results can differ between laboratories, time consuming, labour intensive, takes 24hrs for a result.

Haemagglutination Inhibition (HAI)• Based on the principle that some viruses will

agglutinate red blood cells providing the appropriate species of RBC is used.

• Antibody reacts with the haemagglutinin molecule on the virus, thus preventing the virus from agglutinating the RBCs.

• Disadvantages- results can differ between laboratories, time consuming, labour intensive.

ELISA• Used to detect antibody • Panbio kits are of two types:-

– Standard Indirect ELISA

• majority of Panbio kits

• Ag coated onto plate binds specific Ab in patient’s serum. The bound Ab is detected using a labelled Ab. The reaction is visualised by a colour change.

– Capture ELISA

• Anti-human IgG or IgM coated onto plate captures patient’s Ab. Specific Ag bound to the plate also or added individually then binds to specific patient Ab. The bound Ag is then detected by a labelled monoclonal Ab. The reaction is visualised by a colour change.

Panbio Dengue Duo IgM & IgG Capture ELISA

Panbio Indirect ELISA

Advantages of Panbio ELISAs• Consistent results• Standardised format• Fast results (assay time 1hr 30min)• Ease of use

– colour coded reagents

– all liquid reagents ready to use (in indirect kits)

• IgM & IgA ELISAs contain Absorbent (RF & IgG removal agent)

• Convenient for large scale screening

IgM / IgA ELISAs & Absorbent• Panbio IgM & IgA ELISA kits contain Absorbent (goat

anti-human IgG).• Absorbent has two functions:-

1. Remove competing IgG that can cause false negative results.

2. Remove Rheumatoid Factor (RF) that can cause false positive results.

Competing IgG & False negatives

False positives & Rheumatoid factor