CCD-blocking solution
A convenient feature in the AllergyScreen®/AlleisaScreen® test kit
CCD-blocking solution is used to inhibit anti-CCD IgE in human serum/plasma samples, which are tested for sensitization of a patient to specific allergens using in vitro allergy tests but is not a therapy-accompanying in vitro diagnostic tool.
Many allergens are glycoproteins, which consist of a protein part and one or more glycan chains. Glycan chains are composed of different sugars linked together and are bound via an amino-group (N-glycan) or a hydroxyl-group (O-glycan) to the protein part. N-glycans are particularly immunogenic and can induce the production of IgE, specific for N-glycans, which are usually pathologically irrelevant.
These IgE antibodies are highly cross-reactive for glycoproteins of plants, insects and molluscs. Therefore, glycan chains are named “cross-reactive carbohydrate determinants” (CCDs). CCDs have been found in allergen extracts of plant origin like tree, weed and grass pollen, vegetables, fruits, seeds, nuts and latex but also in insect venoms, snails and parasites.
More than 20 % of all allergic patients produce anti-CCD IgE antibodies. The sensitization of patients against CCDs probably results from contact with pollen or from insect stings. While usually the occurrence of CCDs in allergen extracts induce no positive results in skin tests, they can lead to positive results or enhanced positive results in in vitro test systems without any pathologically relevance. For these reasons the possible occurrence of CCD-specific IgE antibodies must be considered if in vitro test systems are used.
Benefit by the advantages of the CCD-blocking solution
- Improvement of the specificity of the allergy diagnostic according to the recommendations of the German Society for Allergology and Clinical Immunology (Deutsche Gesellschaft für Allergologie und klinische Immunologie)
- Inhibition of glycan-specific IgE antibodies - only peptide-specific IgE antibodies are detected
- Just a short preincubation step of 30 min before running the test
- No change in the required serum/plasma amount of 250 µl (AllergyScreen®) or 300 µl (AlleisaScreen®) — the serum/plasma is mixed with 10 % of the CCD-blocking solution. This serum-CCD-mix can be directly used in the test
- Further test procedure for the AllergyScreen®/AlleisaScreen® test stays unaffected
CCD reaction scheme
Recognition of antigens in an in vitro test system
The same allergen with a glycan epitope and a peptidyl epitope can lead to a positive result in an in vitro test in three different cases:
- The tested serum contains only gylcan-specific antibodies (anti-CCD-IgE antibodies)
> positive result because of CCDs (cross-reactive carbohydrate determinants) - The tested serum contains only peptide-specific antibodies
> positive result due to a peptid-based sensitisation - The tested serum contains both peptide- and glycan-specific antibodies
> positive result due to a peptid-based sensitisation with an enhanced signal because of anti-CCD-IgE antibodies
Positive or enhanced positive signals due to glycan-specific antibodies (1 and 3) can be prevented by blocking the binding of the anti-CCD-IgE antibodies to the allergen.
How the CCD-blocking solution works
The CCD-blocking solution consists of a mixture of different CCD-containing glycoproteins. Before starting the AllergyScreen®/AlleisaScreen® procedure the serum is mixed with the CCD-blocking solution. Thereby, the glycan-specific anti-CCD-IgE antibodies are captured and do not react with the allergen. Positive results that caused by binding of anti-CCD IgE antibodies to the allergen can be reduced or even completely inhibited with this method.
- The glycan-specific antibodies (anti-CCD-IgE antibodies) bind to the glycoproteins of the CCD-blocking solution and not to the glycan epitope of the allergen
> positive result due to a peptide-based sensitization - The peptide-specific antibodies stay unaffected by the glycoproteins of the CCD-blocking solution and bind to the peptidyl epitope of the allergen
> positive result due to a peptide-based sensitization - The peptid-specific antibodies bind to the peptidyl epitope of the allergen, only the glycan-specific antibodies bind to the glycoproteins of the CCD-blocking solution
> positive result due to a peptide-based sensitization without amplification caused by anti-CCD-IgE antibodies
Positive or enhanced positive signals due to glycan-specific antibodies (1 and 3) can be prevented by blocking the binding of the anti-CCD-IgE antibodies to the allergen.
Examples of use
1. Only glycan-specific IgE antibodies
Panel 30 Food A (30 different food allergen lines) with positive control at the top
Left side:
Test result without CCD-blocking solution
Right side:
Preincubation of the serum with CCD-blocking solution, the same serum and procedure as for the left side
The CCD-inhibition shows that the serum only contains anti-CCD IgE antibodies and no peptide-specific antibodies against the tested allergens.
2. Mixture of glycan- and peptide-specific IgE antibodies
Panel 30 Food A (30 different food allergen lines) with positive control at the top
Left side:
Test result without CCD-blocking solution
Right side:
Preincubation of the serum with CCD-blocking solution, the same serum and procedure as for the left side
The CCD-inhibition shows that the serum contains anti-CCD IgE antibodies but also peptide-specific antibodies against the tested allergens.
3. Only peptide-specific IgE antibodies
Panel 30 Food A (30 different food allergen lines) with positive control at the top
Left side:
Test result without CCD-blocking solution
Right side:
Preincubation of the serum with CCD-blocking solution, the same serum and procedure as for the left side
The CCD-inhibition shows that the serum contains only peptide-specific antibodies against the tested allergens.
Explanation
The tested sera in the examples show several positive results with varying intensities for a selection of allergens (membranes each on the left side). After the preincubation with the CCD-blocking solution no (example 3, right membrane), some (example 2, right membrane) or all (example 1, right membrane) of the signals except the control line disappear.
The reduction (example 2) or the complete disappearance (example 1) of signals after a preincubation with the CCD-blocking solution indicates that the positive responses are partly or completely due to a reaction of anti-CCD-IgE antibodies with the glycan epitopes (CCDs) of the allergens.
Since a sensitisation to CCDs is usually not linked to allergies the CCD-blocking solution suppresses positive signals for usually pathologically irrelevant sensitisations. The visible signals after the preincubation with the CCD-blocking solution in example 2 and 3 indicate a pathologically relevant sensitization against the tested allergens. These peptid-based signals are not blocked by the CCD-blocking solution.
The use of the CCD-blocking solution makes it possible to distinguish between a usually pathologically irrelevant glycan-based sensitisation against CCDs and a protein-based sensitisation.