1 Detection methods of IgE : a critical view Allergy School 21st-24th September 2007 Laurence Guilloux

1

Detection methods of IgE : a critical view

Allergy School21st-24th September 2007

Laurence Guilloux

2

Detection method of IgE

The diagnostic algorithm for human allergic disorders begins with • A thorough clinical history• And a physical examination

Temporal associations between allergic symptoms and allergen exposures lead to a high degree of suspicion that the patient has an allergic disorder.

Confirmatory testing for IgE antibody (AB) is often performed to strengthen the probability that the working diagnosis is correct.

• This involve in vivo methods (skin, provocation tests)• Laboratory-based in vitro analysis

Cell-based technology Serum-based technology

3

Serum-based technologyHistory

1960, identification of human « reagin » as IgE Preparation of IgE Fc fragment and production of polyclonal AB

specific for the heavy chain 1967, first total IgE and allergen-specific IgE RAST were developed

(Wide, Lancet) 1974, first generation semi-quantitative IgE AB assay (birch

specific IgE calibration curve and results expressed in classes) was first made commercially available by Pharmacia Laboratories.

Over the past decade there have been improvements in all aspects of technology with

• Second-generation quantitative IgE assays • Third-generation automated IgE assays with a detection limit of

0.1 kU/L (Li CCA 2005)

• Biochip technology, microarrays (Wiltshire, Clin Chem 2000; Kim, Exp Mol Med 2002)

4

The two most representative classes of immunoassays for the measurement of specific IgE are based on the use of Solid phase allergens Liquid phase allergens (the fluid phase allergen is bound to the solid

phase only after the allergen has complexed with fluid phase IgE AB).

Today

The assay of specific IgE AB is considerably more complicated than most other immunoassays

Firstly, the concentration of the IgE in the blood is extremely low, (0.004 mg/100ml; IgG: 1200 mg/100ml)

Secondly, each main allergen, contains a large number of different allergenic components,

The assay must be sensitive enough to capture IgE AB to all relevant components but still with a very low level of non-specific binding.

5

« Architecture » of specific IgE immunoassays

1. Allergen-containing reagent and binding chemistry 2. Anti IgE 3. Calibration system4. And also the tracer and the signal detection system influence the sensitivity

and quality of final results

The analytical performances of immunoassays depend on the whole « architecture » of the method and particularly on some fundamental variables.

6

1. Allergen-containing reagent and binding chemistry

The solid-phase (allergosorbent) or liquid-phase labelled allergen reagent is the principal component of the assay that confers specificity on the IgE AB assay.

The most complex and highly variable component partly due to:

1-1) the heterogeneity of most allergen extracts

1-2) the different supports

1-3) and the different chemical methods used to insolubilise or label the allergenic material

7

1-1) Heterogeneity of « allergens » Whole extract

Heterogeneity has many possible causes, even when the same manufacturer uses; • the same extract source (variable stability, inherent biological variation of the raw material,

contamination…) • the same chemical and manufacturing procedures (extraction process, variable binding of

allergen, variable stability….)

Purified native and recombinant allergenic moleculesThe advent of these allergenic molecules offers new perspectives:• rAllergens produced in almost unlimited quantities will avoid the variability of natural

allergenic extracts and allow standardisation (but different of the native form).• Allergenic molecules are essential for the development of innovative techniques using

microchip technologies and to improve our understanding of problems related to cross-reactivity of IgE AB to allergens.

• Spiking of allergen extract with purified native or recombinant allergenic molecules (known to be clinically relevant), can improve the sensitivity of the diagnostic tests.

8

1-2) Different supports

Solid phase allergens (allergosorbent) Insoluble dextran Cellulose

• filter paper disc (RAST)• Minicolumns (CAP)• Thread (CLA)

Mycrocrystalline particles Nitocellulose (dot blot) Polystyrene tube (FAST) Microtiter well activated silica

chip (ISAC)

Liquid phase allergens Biotin-labelled allergens

(Advia Centaur) The ligand (biotin) is

covalently attached to a backbone (soluble polymer and amino acid copolymer) to wich the allergen molecules are also attached (Immulite 2000)

9

1-3) Different chemical methods Characteristics

CNBr CNBr bounds only NH2 bearing molecules, iso urea N-substituted liaisons are not stable, iso urea group is protonated at neutral to

alkaline pH, which implies a positively charged matrix-CNBr-allergen which can bind in a non specific manner negatively charged sIgE.

Weak binding capacity (large amount of bound allergen that may increase NSB and IgG interference),

Slow kineticsBethell J Chromat 1981

CDI, BDE Several coupling agents that allow allergen

coupling by different groups (NH2, SH, OH) Neutral matrix-allergen binding Better allergen representation, no masked

epitopes, conformation close to their native states.

10

Highly specific for unique determinants on heavy chains Used as both capture and detection reagents

Subjected to chemical modification (radio-labelled, enzyme-labelled, chemical and physical immobilisation on solid-phase matrices

Initially polyclonal reagents Monoclonal reagents are specific and lack batch-to-batch variations, however as they have low affinity

and avidity, mixtures of mono and polyclonal antisera are often used

2. Anti - IgE

3. Calibration system (NCCLS 1997)

Homologous interpolation methodsThis is a term that defines a calibration scheme in which the standard or reference (calibration) dose-response curve is constructed using reagents that have the same specificity from those being used to measure the analyte of interest.

Heterologous interpolation methodsThis is a term that defines a calibration scheme in which the standard or reference (calibration) dose-response curve is constructed using reagents that have a different specificity from those being used to measure the analyte of interest. In the contexte of specific IgE assays, this mean that a total serum IgE calibration curve in IU/ml of IgE, which is calibrated back to an IgE primary standard, is used to interpolate unknown allergen-specific IgE response data.

11

Classification of specific IgE antibody immunoassays

Qualitative assays (no calibration curve)Positive/negative based on pre-assigned positive threshold

Semi-quantitative assaysThe variations in the positive signal detected are commonly presented • in terms of a series of increasing grades (e.g., I to IV; low to high) or classes• in arbitrary units (U/ml) determined relative to a supplier-specific, heterologous dose-

response curve • or an end-point dilution to which the signal becomes negative (e.g., titer),• or in comparison to a qualitative grading scheme (e.g., colour chart)

Quantitative assay (multipoint calibration curve)A quantitative assay provides an accurate and reproducible estimate of the concentration of IgE and fulfills the analytical criteria for quantification including:

• Parallelism, recovery, precision and• Linearity across the whole assay’s working range

Is dependent on the degree with which the assay result accurately reflects the quantity of sIgE AB in the test specimen, and the assay’s precision requirements (according to NCCLS approved guidelines)

The most diffuse and typical illustration is the multiallergen screening test in which different allergen specificities are present in a single reagent

The results produced are not traceable to any comon reference material.

12 SQA, semi quantitative assay; QA, quantitative assaySP, solid phase; LP, liquid phase; IM, immunometric assay

Past and current serologic assays for allergen-specific IgE

WithdrawnSQAPhosphatase alcaline

SP-IMCNBr-activatedcellulose

Matrix (Abbott)

CE labelSQAEnzyme (fluorescense)

SP-IMGlass slideISAC (VBC-Genomics)

Enzyme

Phosphatase alcaline




Bêta galactosidase

Acridiniumester-labelledStreptavidin

I 125

Anti E Labelled

CE labelSQASP-IMCNBr-activatedcellulose threads

CLA (Hitachi)

CE labelQA (WHO 75/502)

SP-IMTipsVidas (Biomérieux)


LP-IMBiotin-labelledAllergen

Immulite 2000 (SMS Diag.)


SP-IMCNBr-activatedpaper discs

Hycor(J2L Elitech)

WithdrawnSQASP-IMcupuleFAST (International CIS)


SP-IMCNBr-activatedSponge

CAP (Phadia)

CE labelQA (Bet v 1 standardized/WHO 75/502)

LP-IMBiotin-labeledAllergen

Advia Centaur(SMS Diag.)

WithdrawnSQA (anti birch IgE)


PhadebasRAST (Pharmacia)

StatusCalibrationChemistryAllergosorbentAllergosorbentAssayAssay methodmethod

WithdrawnSQAPhosphatase alcaline

SP-IMCNBr-activatedcellulose

Matrix (Abbott)

CE labelSQAEnzyme (fluorescense)

SP-IMGlass slideISAC (VBC-Genomics)

Enzyme





Bêta galactosidase

Acridiniumester-labelledStreptavidin

I 125

Anti E Labelled

CE labelSQASP-IMCNBr-activatedcellulose threads

CLA (Hitachi)


SP-IMTipsVidas (Biomérieux)


LP-IMBiotin-labelledAllergen

Immulite 2000 (SMS Diag.)



Hycor(J2L Elitech)

WithdrawnSQASP-IMcupuleFAST (International CIS)


SP-IMCNBr-activatedSponge

CAP (Phadia)

CE labelQA (Bet v 1 standardized/WHO 75/502)

LP-IMBiotin-labeledAllergen

Advia Centaur(SMS Diag.)

WithdrawnSQA (anti birch IgE)


PhadebasRAST (Pharmacia)

StatusCalibrationChemistryAllergosorbentAllergosorbentAssayAssay methodmethod

13

Conclusion Excellent analytical performances are generally achieved by third generation

laboratory systems for sIgE as CAP System, IMMULITE 2000 3g and ADVIA Centaur in term of quantitative results.

• On account of differences in term of kU/L results obtained with the different systems and without the avaibality of the « true » value of sIgE we should keep in mind that the different technics are not interchangeable (thresholds using ROC curves, probability curves are not identical). It’s a general rule for immunoassays (ex. tumor markers).

Third generation laboratory systems for sIgE demonstrate acceptably high concordance with objective clinical tests (skin, provocation tests).

• Discrepant results have to be carefully analysed in term of sensitivity and specificity.

Immunoassays for the sIgE AB have undergone significant improvements with, the use of more purified extacts, the capacity to quantitate IgE AB in mass units, and total automation of assay system. However, laboratory information should be totally integrated in rational clinical pathways.

Documents

1 Detection methods of IgE : a critical view Allergy School 21st-24th September 2007 Laurence Guilloux