© 2016 MEDIWISS Analytic GmbH

CCD-Blocking-Solution

A convenient feature in the
AlleisaScreen® system

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 sensitisation 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.

Profit 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 amount of 300 µl in the AlleisaScreen® test procedure – the serum 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 AlleisaScreen® 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:

  1. The tested serum contains only gylcan-specific antibodies (anti-CCD-IgE antibodies)
    > positive result because of CCDs (cross-reactive carbohydrate determinants)
  2. The tested serum contains only peptide-specific antibodies
    > positive result due to a peptid-based sensitisation
  3. 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 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.

  1. 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
  2. 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
  3. 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 allergens) 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 allergens) 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 site

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 allergens) 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 site

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.

Literature

[1] Aberer et al, Inhibition of cross-reactive carbohydrate determinants (CCDs) enhances the accuracy of in vitro allergy diagnosis, Allergologie, 2014, 45–53

[2] Jappe U, Raulf-Heimsoth M. Kreuzreagierende Kohlenhydratdeterminanten (cross-reactive carbohydrate determinants, CCD). Allergologie 2008; 31: 82–90

[3] Kochuyt AM, Hoeyveld EM van, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immuno globulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005; 35: 441–7

[4] Mari A, Iacovacci P, Afferni C, Barletta B, Tinghino R, Di Felice G, Pini C. Specific IgE to cross-reactive carbohydrate determinants strongly affect the in vitro diagnosis of allergic diseases. J Allergy Clin Immunol 1999; 103: 1005–11

[5] Renz H et al, Leitlinie der Deutschen Gesellschaft für Allergologie und klinische Immunologie (DGAKI) unter Beteiligung des Ärzteverbandes Deutscher Allergologen (ÄDA), der Gesellschaft für Pädiatrische Allergologie und Umweltmedizin (GPA) und der Deutschen Dermatologische Gesellschaft (DDG), Allergo Journal, 2010; 19: 110–28
English version: http://link.springer.com/journal/40629