Download Antibodies

Immunology & Serology
February 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
Antibodies
Antibodies, 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 Response
1. 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
Humoral Immunity
Immunoglobulins
specific antibodies
B-lymphocytes
Activates
T-lymphocyte
Cellular Immunity
Antigen
Cellular Immunity
Cell lysis
White blood cell
production
Lymphokines
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
Ab/Ag titre
IgM
IgG
IgA
Viremia
5
10
15
20
Day
25
30
A n tib o d y L e v e l
Immune Response to Dengue Infection
Ig G
O nset of
S y m p to m s
O nset of
S y m p to m s
Ig G C u to ff
H A I 1 :2 5 6 0
V iru s
P rim a ry
In fe c tio n
Ig M
V iru s
S e c o n d a ry
In fe c tio n
Ig M
Ig M
C u to ff
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