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

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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)