Transcript West Nile virus

serology & panbio product
overview
may 2005
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 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.
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
Dengue immune response
© 2001 Panbio Limited. All Rights Reserved.
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
Hemagglutination Inhibition (HAI)
Complement Fixation (CFT)
Enzyme-linked Immunosorbent assay (ELISA)
Rapid immunochromatographic assays
Immunofluorescence (IFA)
• Method for identification of antigens in tissue sections
& on cells, or for identifying antibodies to them.
– Direct - Fluorescent Ab is incubated with cells or tissue
section. The Ab binding to Ag is visualised by UV light.
– Indirect - Cells or tissue are incubated with test serum Ab,
which is then visualised by the addition of a second layer
fluorescent anti-antibody.
• Still used as a reference method for diagnosis of many
diseases
• Panbio offers many IFA kits – based on the indirect IFA
procedure
Panbio IFA kits
Diluted patient serum samples are applied to cultured cells
containing inactivated antigens provided on paint delineated
wells on glass microscope slides. During incubation, specific
antibody forms a complex with the antigens in the cells.
Washing removes nonspecific antibody and other unreacted
serum proteins
Fluorescein-conjugated goat anti-human IgG, IgM or IgA is
applied to the wells of the glass slide. The conjugate
combines with the specific human antibodies, if present, during
the incubation period.
The slides are viewed by fluorescence microscopy. A positive
antibody reaction is denoted by bright green fluorescence at
the antigen sites.
Panbio IFAs
• Procedure will vary slightly depending on the kit
ELISA
• Enzyme-linked immunosorbent assay
• 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 Indirect ELISA
Specific serum antibodies combine with
antigens attached to the polystyrene
surface of the microwells
Washing removes residual serum
Peroxidase-conjugated anti-human specific
immunoglobulin is added
The colourless substrate,
tetramethylbenzidine/hydrogen peroxide (TMB /
H2O2) is hydrolysed to a blue chromogen
Stopping the hydrolytic reaction
with acid turns the TMB yellow
Colour development indicates the presence
specific antibodies in the test sample
Panbio Capture ELISA
Serum antibodies combine with the antihuman IgM or IgG coated on the plate.
Simultaneously the peroxidase
conjugated MAb and antigen supplied
form complexes when incubated
together.
Washing removes residual serum
The antigen-Mab complexes bind if
antigen-specific antibodies have been
captured.
The colourless substrate,
tetramethylbenzidine/hydrogen peroxide
(TMB/H2O2) is hydrolysed to a blue
chromogen
Stopping the hydrolytic reaction
with acid turns the TMB yellow
Colour development indicates the
presence of specific antibodies in the
test sample
Calculation of results
•
On completion of reading the assay, the Panbio Units for
each sample must be calculated. This is done as follows:
1. Calculate the average absorbance of the triplicates of
the cut-off calibrator. This is the cut-off value. In some
Panbio kits a calibrator is supplied rather than a cutoff
sample. In this instance the average absorbance of the
triplicates of the calibrator should be calculated and this
figure multiplied by the calibration factor supplied on the
specification sheet.
2. Panbio units can be calculated by dividing the sample
absorbance by the cut-off value (calculated in step (1)
above) and multiplying by 10.
Panbio Units = 10 X
Absorbance of sample
Mean absorbance of cut-off
Example
Sample A Absorbance = 0.949
Sample B Absorbance = 0.070
Mean absorbance of cut-off = 0.302
Sample A
0.949 / 0.302 X 10 = 31.4 Panbio Units
Sample B
0.070 / 0.302 X 10 = 2.3 Panbio Units
Interpretation: IgM ELISA*
Panbio Units
Result
Interpretation
<9
Negative
No evidence of recent infection
(see Note 1)
9 - 11
Equivocal
Suggest samples be retested
(see Note 2)
>11
Positive
Suggestive of a recent infection
Note 1: If specific IgM antibodies are not detected and a recent infection is
suspected, this can be confirmed by testing a further specimen 7-14 days
later.
Note 2: If specimen remains equivocal following repeat testing then the
specimen may be tested by an alternate method or another patient
specimen obtained and tested.
*General interpretation supplied. May vary depending on disease (i.e. Dengue)
Interpretation: IgG ELISA*
Panbio Units
Result
Interpretation
<9
Negative
No evidence of IgG antibodies
(See Note 1)
9 - 11
Equivocal
Suggest samples be retested
(see Note 2)
>11
Positive
Specific IgG antibodies present.
Suggestive of recent or past
exposure.
Note 1: If specific IgM and IgG antibodies are not detected and a recent infection is
suspected, this can be confirmed by testing a further specimen 7-14 days later.
Note 2: If specimen remains equivocal following repeat testing then the specimen
may be tested by an alternate method or another patient specimen obtained and
tested.
*General interpretation supplied. May vary depending on disease (i.e. Dengue)
Features/benefits: Panbio ELISAs
• Breakapart wells
– Minimise wastage
• Consistent procedures
– Decreases chances of error
– Easier for programming automated instruments
• Short assay times (1 hr 10 min Indirect; 2 hr 10 min Capture)
– Provide results faster
• Ready-to-use colour coded reagents
– Decreases chances of error preparing reagents
– Leads to shorter overall assay time – results generated
faster
• Compatible with standard microplate technology
– Suitable for use on a wide range of automated instruments
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 and false negs
Anti-human IgG
IgM
Immobilised IgG
IgM
IgG
Antigen
Antigen
Without Absorbent
With Absorbent
False positives and rheumatoid
factor
Anti-human IgM HRP
RF (IgM or IgA)
IgG
Antigen
Panbio Rapid
Immunochromatographic tests
• Dengue Duo Cassette
• Dengue Duo IgM & IgG Rapid Strip
Procedure: Dengue Duo
Cassette
Procedure: Dengue Rapid Strip
Advantages of rapid tests
• Fast (results in less than 30 minutes)
• Simple (3 step procedure)