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serology & panbio product overviewmay 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
© 2001 Panbio Limited. All Rights Reserved.
Dengue immune response
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 polystyrenesurface 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 reactionwith acid turns the TMB yellow
Colour development indicates the presencespecific antibodies in the test sample
Panbio Capture ELISA
Serum antibodies combine with the anti-human 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 reactionwith 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 sampleMean absorbance of cut-off
ExampleSample 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
IgG
Antigen
IgM
Without Absorbent
Anti-human IgG
Immobilised IgG
IgM
Antigen
With Absorbent
False positives and rheumatoid factor
Antigen
IgG
RF (IgM or IgA)
Anti-human IgM HRP
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)
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