Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
  • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/24—Immunology or allergic disorders

Definitions

• the present invention relates to a method for removing cross-reactive carbohydrate determinant (CCD) specific antibodies from a sample.
• CCD carbohydrate determinant
• Carbohydrate cross-reactive determinants are carbohydrate moieties of glycoproteins that induce the production of highly cross-reactive IgE.
• Many allergens are glycoproteins containing carbohydrate moieties (N-glycans or O-glycans depending to their site of attachment to the protein) . Till today N- glycans containing ⁇ l,2-xylose and ⁇ l,3-fucose have been found in many glycoproteins. Despite the great diversity of carbohydrate structures, some common patterns are present in glycoproteins of invertebrate animals and plants.
• Anti-CCD IgE has also been observed in response to several vegetables, fruits and seeds, like tomato, apple, peanut, hazelnut .
• anti-CCD IgE has no clinical relevance predominates because the presence of anti-CCD IgE continues to be associated with negative reactions to skin prick tests and lack of clinical symptoms. Because many glycoproteins carry only one IgE-binding glycan, they would be unable to cross-link IgE bound to the receptors of the mast cells and basophils for reasons related to epitope accessibility and antibody affinity; thus clinical symptoms might not appear in patients whose IgE response is restricted to CCDs. Nevertheless, it has been reported that anti-CCD IgE can be detected as such by in vitro tests. CCDs should be considered a potential cause of interference in these tests. Data on the prevalence and distribution of anti-CCD IgE as well as anti-CCD IgG prevalence in a large cohort of subjects with allergy symptoms are limited.
• the potential interference by CCD can have several effects. If the patient is sensitised to said allergen but the assay detects IgE directed against both the protein and the carbohydrate structures of said allergen, positive test results for this allergen will be exaggerated. If the patient is not sensitised to said allergen but the assay detects anti-CCD IgE, test results for this allergen will result in a false positive diagnosis. Such misdiagnosis could result in unnecessary avoidance and/or inappropriate immunotherapy, which may even pose a risk of inducing a new allergy.
• glycoproteins as an anti-CCD IgE "marker" can indicate the presence in serum of IgE directed against carbohydrate epitopes.
• the glycoprotein most widely used to identify IgE reactivity to carbohydrate determinants is the pineapple protease bromelain. It is known to be a good marker because its carbohydrate chain is very similar to those of many other plant proteins and because a true allergy to bromelain is very rare. Bromelain can be used as an inhibitor in quantitative or qualitative competitive inhibition measurements. Inhibition procedures are well-standardised procedures to distinguish between cross-reactivity and true double sensitisation . An elevated result for a bromelain-specific IgE test suggests normally the presence of anti-CCD IgE in the sample.
• Jappe U. et al . (Allergy 61 (2006) : 1220-1229) relates to a method for determining the allergen sensitivity of individuals against insect venoms of honeybee and yellow jacket.
• CCD-specific antibodies were determined. The authors come to the conclusion that antibodies from sera of individuals which have been contacted with the insect venom are able to bind to CCD.
• a further object of the present invention is providing a method for removing CCD specific antibodies from a sample.
• the present invention relates to a method for removing cross-reactive carbohydrate determinant (CCD) specific antibodies from a sample comprising the steps of:
• the method of the present invention allows to remove efficiently CCD specific antibodies from a sample. This removal is achieved by contacting the sample with a solid support having immobilised on its surface at least one cross-reactive carbohydrate determinant.
• the removal of CCD specific antibodies from a sample is particularly advantageous when the sensitivity of a patient to certain allergen is analysed. Allergens having N- or O-glycans bound to them are able to induce in an individual the formation of antibodies directed to these glycan structures. Since the glycan structures are present in many different aller- gens, the presence of CCD specific antibodies in a sample obtained from an individual may lead to false positive results and consequently to a false diagnosis (CCD' s mask allergy diagnosis) . Therefore it is advantageous to remove prior such test assays CCD specific antibodies from the sample to be analysed, so that preferably only protein/polypeptide specific antibodies remain in the sample.
• the CCD may be immobilised on the surface of the solid support via at least one amino acid residue, which is preferably asparagine, which represents the natural amino acid residue to which CCDs usually are bound.
• amino acid residue which is preferably asparagine, which represents the natural amino acid residue to which CCDs usually are bound.
• linker it is of course possible to use another molecule or amino acid residue as linker, provided that said molecule or amino acid residue does not change the conformation or structural properties of the CCD or its linking sugar residue.
• Such a change can be determined by performing binding assays of CCD specific antibodies to said altered linking. If CCD specific antibodies still bind to the CCD bound to another molecule than asparagine said molecule may be used as an alternative linker.
• said molecule or asparagine, acting as a linker may be part of a peptide, polypeptide or even protein, provided that said peptide, polypeptide or protein does not comprise allergen (T-cell or B-cell epitopes) .
• allergen T-cell or B-cell epitopes
• antibodies may be removed from the sample which are specific for the proteinaceous portion of an allergen. This will lead to a false negative result, if the sample obtained by the method of the present invention is used to determine the presence of antibodies directed to an allergen.
• the linker or the CCD is preferably bound only to one amino acid residue (preferably asparagine) , which itself is bound to a maximum of one, preferably a maximum of two, three, four, five, ten, 15, 20, 30, 50, amino acid residues provided that these amino acid residues do not comprise an allergen-specific T-cell or B-cell epitope.
• one amino acid residue preferably asparagine
• the linker or the CCD is preferably bound only to one amino acid residue (preferably asparagine) , which itself is bound to a maximum of one, preferably a maximum of two, three, four, five, ten, 15, 20, 30, 50, amino acid residues provided that these amino acid residues do not comprise an allergen-specific T-cell or B-cell epitope.
• CCD carbohydrate cross-reactive determinant
• glycan structures of proteins in particular allergens derived from plants and insects.
• CCDs are able to provoke a specific immune response in a mammal.
• CCDs are usually asparagine-linked oligosaccharides (N-glycan) and contain core ⁇ l,3-linked fucose and/or ⁇ l,2-xylose.
• N-glycan oligosaccharides
• fucose and/or ⁇ l,2-xylose ⁇ l,3-linked fucose and/or ⁇ l,2-xylose.
• mammalian glycopro- teins may contain a mammalian CCD comprised of the sugar sequence Gal ⁇ l-3Gal ⁇ l-4GlcNAc ⁇ l- that is immunogenic in humans (Galili U.
• the sample comprises allergen specific antibodies.
• the sample from which the CCD specific antibodies are removed comprises preferably allergen specific antibodies.
• the allergen specific antibodies are of the immunoglobulin isotype IgE, IgA and/or IgG.
• the specificity and/or amount of the allergen specific antibodies present in the sample is preferably determined. Due to said removal cross reactivity between antibodies and various allergens can be avoided and only antibodies which are specific for the proteinaceous moiety of the allergen are detected,
• Another aspect of the present invention relates to a method for diagnosing an allergy in an individual comprising the steps of:
• CCD carbohydrate determinant
• CCD specific antibodies The removal of CCD specific antibodies from a sample allows, if the sample is obtained from an individual, to determine the presence of IgE molecules specifically binding to the proteinaceous moiety of an allergen.
• Methods for the qualitative and quantitative detection of allergen specific IgE 's are well known in the art (e.g. ImmunoCAP®, radioallergosorbent test (RAST), multiple radioallergosorbent test (MAST) , fluorescent aller- gosorbent test (FAST) , paper radioimmunosorbent test (PRIST) , radioimmunosorbent test (RIST) , enzyme-linked immunosorbent assay (ELISA) ) .
• the sample is of human or animal origin and preferably selected from the group consisting of whole blood, serum and plasma.
• the method of the present invention may be performed with any kind of sample, provided that said sample comprises or is suspected to comprise CCD specific antibodies.
• a sample suspected to comprise CCD specific antibodies is a sample from which is not exactly known whether said sample comprises such antibodies. Therefore the sample is preferably obtained from a patient who is suspected to be allergic against a certain allergen or which is known to be sensitive for an allergen.
• the CCD is selected from the group consisting of ⁇ l,2-xylose and ⁇ l,3-fucose and is preferably bound to a further sugar moiety comprising or containing Manp ( ⁇ l, 4) -GlcpNAc ( ⁇ l, 4) -GlcpNAc ( ⁇ l, N) resulting in a glycan selected from the group consisting of (Man ⁇ l-3)Man ⁇ l-6 (Xyl ⁇ l-2) Man ⁇ l-4GlcNAc ⁇ l-4GlcNAc (MMX), (Man ⁇ l- 3)Man ⁇ l-6 (Xyl ⁇ l-2) Man ⁇ l-4GlcNAc ⁇ l-4 (Fuc ⁇ l-3) GIcNAc (MMXF) , Man ⁇ l-6 (Xyl ⁇ l-2) Man ⁇ l-4GlcNAc ⁇ l-4 (Fuc ⁇ l-3) GIcNAc (MUXF), Man ⁇ l- 6 (Man ⁇ l-3)Man ⁇
• the glycan or CCD which is immobilised on a solid carrier is preferably isolated from a plant or mammalian glycoprotein. Due to the natural occurrence of CCD structures it is particularly advantageous to obtain the sugar moieties to be immobilised from a natural source. Therefore, fragments of the glycoprotein comprising the glycan or CCD or entire glycoprotein may be used for immobilisation. However, it should be noted that the protein- aceous portion of the isolated plant or mammalian glycoprotein does not show any, or shows only a very reduced cross reactivity with the allergens analysed.
• the CCD may also be coupled to a proteinaceous or non-proteinaceous carrier molecule via an in vitro reaction known in the state of the art and described exem- plarily in the example section under "preparation of glycan allergens" .
• the glycan or CCD may further be synthesized chemically with methods known in the art. Furthermore, it is of course also possible to synthesize then in vitro with corresponding enzymes which catalyse the formation of glycan structures.
• the plant glycoprotein is obtained from bromelain, patatin, horseradish peroxidase, or other plant glycoproteins, which are not significant allergens based on their protein-moiety (such as phospholipase A), and which have a defined glycan structure.
• the mammalian glycoprotein is obtained from fibrin and/or other vertebrate glycoproteins containing a substantial amount of diantennary N-glycans (e.g. serotransferrin, protease- inhibitors, immunoglobulins or egg yolk glycoproteins) is preferably modified with glycosidases or glycosyl transferases.
• diantennary N-glycans e.g. serotransferrin, protease- inhibitors, immunoglobulins or egg yolk glycoproteins
• glycoprotein used according to the present invention does not comprise all structural features of the CCD 's the glycan structure of such proteins may be modified accordingly by using glycosidases and/or glycosyl transferases.
• the solid support is a conventional affinity matrix consisting of a matrix such as porous beads, monolithic chromatography bed or a functionalised permeable membrane, functionalised or activated cellulose, agarose, dextran, poly-methacrylate, polystyrol, polyacrylamide or silica.
• CCDs may be Gal ⁇ l-3Gal ⁇ l-4GlcNAc ⁇ l-.
• ⁇ -Gal CCDs occur on many glycoproteins of mammals but not of man. They may be isolated from natural glycoproteins similar as plant/insect CCDs, e.g. from calf intestine phosphatase (Bublitz et al . , J. Mass Spectrom.
• ⁇ l, 3-galcto- syl-transferase on desialylated mammalian serum glycoproteins e.g. asialo-forms of fetuin, ⁇ l-acid glycoprotein, Tamm-Horse- fall glycoprotein, serotransferrin, protease-inhibitor, immunoglobulins, egg yolk glycoproteins or other glycoproteins with preferably known N-glycans structures lacking interfering determinants
• the determinant may also be fully or partially synthesised chemically.
• a solid support carrying this CCD would help to eliminate antibodies against this mammalian CCD, which in some allergic patients may lead to false positive results with animal allergens.
• Solid supports having on their surface immobilised CCD may be suitably employed in allergy diagnosis.
• allergen specific antibodies Prior to diagnose an allergy sensitivity in an individual CCD specific antibodies are removed and allergen specific antibodies are determined in the sample which is preferably blood, plasma, serum or nasal fluid. The determination of allergen specific antibodies may be performed with methods known in the art.
• kits for diagnosing an allergen sensitivity comprising:
• solid support comprising at least one cross-reactive carbohydrate determinant, immobilised on its surface, wherein the solid support is contained in a cartridge, filter or pipette tip or packed in a column, preferably chromatographic column,
• the means for determining the presence of allergen specific antibodies comprise an allergen and means for detecting and optionally quantifying the binding of the allergen specific antibodies to the allergen.
• ImmunoCAP® radioallergosorbent test (RAST) , multiple radioallergosorbent test (MAST) , fluorescent allergosorbent test (FAST) , paper radioimmunosorbent test (PRIST), radioimmunosorbent test (RIST), enzyme-linked immunosorbent assay (ELISA) .
• RAST radioallergosorbent test
• MAST multiple radioallergosorbent test
• FAST fluorescent allergosorbent test
• PRIST paper radioimmunosorbent test
• RIST radioimmunosorbent test
• ELISA enzyme-linked immunosorbent assay
• Fig. 1 shows a plot of levels of CCD-IgE versus that of CCD- IgG in sera of allergic patients. Comparison of ELISA readings of antibodies binding to a plant glycoprotein (HRP) serving as a CCD-allergen . Panel A shows the results obtained with the 170 sera from which those used for affinity measurement were selected. Panel B shows another group of 110 sera analyzed in the course of a recent study. The values are unstandardized absorb- ance readings of single measurements. Blank-correction leads to negative values in some cases.
• HRP plant glycoprotein
• Fig. 2 shows binding of human IgG and IgE to various glycan structures. Equal amounts of various glycoforms of human transferrin were subjected to SDS-PAGE and either stained with Coomassie blue (lane A) or blotted onto nitrocellulose. Lane B was incubated with rabbit anti-HRP antiserum. Lane C shows the binding of IgG from the serum pool to virtually all glycoforms. Lane D demonstrates the strong preference of IgE from the pool for fucosylated glycoforms.
• Fig. 3 shows the role of xylose for human IgG and IgE. Dilutions of the serum pool were incubated in ELISA plates with a plant-glycoprotein (HRP) containing xylose and fucose or with the snail glycoprotein hemocyanin containing xylose. The wells were stained either for bound IgG (panel A) or IgE (panel B) . Values obtained for HRP are shown as open squares, for hemocyanin as black dots.
• HRP plant-glycoprotein
• Fig. 4 shows the purification of specific IgE and IgG.
• the scheme depicts the steps of the antibody purification as described in the methods section.
• the flags symbolize serum or antibody preparations, the boxes chromatography columns.
• the CCD agarose contained a mixture of three BSA-glycopeptides conjugates.
• the glycopeptides were prepared from fibrin and were gly- comodified to obtain MMX, MMF and MMXF-glycans .
• Fig. 5 shows the binding specificity of purified human anti- MMXF IgG and IgE.
• ELISA plate wells were coated with different glycomodified transferrins.
• the black bars indicate the binding of human IgG to different glycoforms; the grey bars that of human IgE. Blank values, which were quite high in the case of IgG, were substracted.
• Fig. 6 shows the glycan molecules comprising the CCDs of the present invention.
• Fig. 7 shows the efficiency of anti-CCD IgE removal from serum of a wasp-allergic patient.
• N-glycans of plant and insect glycoproteins are the most widely distributed group of pan-epitopes of relevance in allergy, but the clinical impact of these cross-reactive carbohydrate determinants (CCDs) is currently considered as negligable and therefore the binding of IgE to CCDs in in vitro diagnostic tests causes false-positive results.
• CCDs carbohydrate determinants
• the key elements of CCDs are the core ⁇ l,3-fucose occuring in insect glycoproteins, or this fucose plus a ⁇ l,2-xylose in plant glycoproteins as reviewed recently.
• Anti-CCD IgE was found in 15 to 30% of sera of allergic patients. The frequent occurence of sensitization against CCDs stands opposite to their apparent inability to cause clinical manifestations.
• Truly polyvalent food glycoproteins might be tomato invertase (Lye e 2, Q8RVW4), a tomato peroxidase (P15003) or zucchini ascorbate oxidase (P37064).
• Such "paucivalent" glycoproteins may represent borderline cases with an only low ability to elicit physiological reactions.
• the only example of a convincingly polyvalent plant glycoprotein is HRP with its seven N-glycans all having CCD structures.
• the inability of HRP to provoke positive skin tests in about 80% of CCD-reactive patients is a clear indication for the clinical insignificance even of polyvalent glycoproteins.
• a second explanation assumes low binding affinity of anti- CCD IgE.
• Low affinity was in fact observed for the interaction between glycoproteins and glycan-binding protein such as C-type lectin, siglecs, and galectins.
• the affinities of these lectins for their glycan ligands are typically low (K 0 of micromolar to millimolar) and therefore it requires multivalent interactions to achieve a biological effect.
• K 0 of micromolar to millimolar
• This does not necessarily apply to human anti-carbohydrate antibodies.
• the notorious ⁇ -Gal epitope can cause hyperacute rejection in pig-to-primate xenotransplantation.
• the IC 50 value of 4 x 10 "8 M for the binding of ⁇ -Gal-polyacrylamide to anti- ⁇ -Gal antibody or the K 0 of 10 "9 M for an anti-LPS antibody demonstrates the principal possibility of high binding affinity between glycan determinants and antibodies .
• Patients and sera Sera were collected from patients undergoing routine allergy testing for inhalant or insect venom allergy.
• Patients with inhalant allergy were skin-prick tested with common inhalant allergens, including pollens (hazel, alder, birch, grass, rye, ash, plantain, nettle, mugwort, ragweed, oilseed rape, plane tree) , house dust mites, animal danders (cat, dog, horse, guinea pig) , molds (Cladosporium, Alternaria, Penicillum) , and rubber latex (all Soluprick, ALK, Demark) .
• pollens hazel, alder, birch, grass, rye, ash, plantain, nettle, mugwort, ragweed, oilseed rape, plane tree
• house dust mites animal danders (cat, dog, horse, guinea pig) , molds (Cladosporium, Alternaria
• Glyco-modified human transferrins with N-glycans with or without core ⁇ l, 3-fucose, xylose or terminal GIcNAc (GnGn-, MM-, GnGnX-, GnGnF-, GnGnXF-, MMX-, MMF-, and MMXF-Tf) were prepared from human apo-transferrin (Sigma-Aldrich) as described (Bencurova M et al . , (Glycobiology 14 (2004): 457-466); Jin C et al., (Glycobiology 16 (2006): 349-357).
• BSA conjugates of glycopeptides with bovine serum albumin were prepared using 2, 5-dinitro-l, 4-difluoro benzene as linker and fibrin-gly- copeptides previously modified by glycosidases and glycosyltransferases similarily as the transferrin-derivates .
• the BSA conjugates (MMX-, MMF-, and MMXF-BSA) were subjected to amino sugar analysis (Altmann, Anal. Biochem. 204 (1992) : 215- 219) and found to contain 2-3 mol glycopeptides per mol of BSA. No glycosyltransferases or glycosidases of plant origin were used for these preparations to exclude contamination of the substances with plant glycoproteins.
• human anti-CCD IgG For determination of human anti-CCD IgG, sera at a dilution of 1:1,000 and alkaline phosphatase (AKP) conjugated goat anti- human IgG (H+L) (Jackson ImmunoResearch, PA, USA) at a dilution of 1:2,000 were used.
• human anti-CCD IgE measurement human sera at a dilution of 1:10 and alkaline phosphatase-conjugated mouse anti-human IgE (mAb G7-26, BD Pharmingen, Heidelberg, Germany) at dilution of 1:500 were used. Purified antibodies, both IgG and IgE were diluted 1:100.
• anti-CCD IgE and IgG was quantitated with the CAP-FEIA system (Phadia , Uppsala, Sweden) with bromelain serving as glyco-allergen .
• Total IgE was likewise determined with the CAP-FEIA system.
• Total IgG was quantitated by the Im- munoCAP system (Phadia) with some modifications.
• the calibration curve used in this assay was based on six different IgG calibrators (International reference preparation 67/86 from World Health Organization) with various concentrations of 0.02, 0.04, 0.1, 0.3, 1.0 and 2.0 mg/L, respectively.
• the calibrators were incubated with ImmunoCAP IgG/IgA Calibrator which coated with monoclonal mouse antibodies against IgG to capture IgG.
• ImmunoCAP IgG/IgA Calibrator instead of antigen-specific ImmunoCAP, and the assay otherwise performed as for specific IgG measurement according to the manufacture's instructions.
• Serum samples from 19 CCD-IgE positive patients were mixed to give an anti-CCD serum pool of about 20 mL .
• Human IgG unex- pectedly also bound to human transferrin. Therefore, the first purification step consisted of an affinity column with human transferrin conjugated to Affi-Gel 15 (BioRad, Richmond, CA) according to the manufacturer's instructions.
• the serum pool was diluted with the same volume of TBS buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN 3 , 1:1) and applied to 1 mL of trans- ferrin-gel. The non-binding antibody fraction was collected.
• an affinity column with immobilized BSA-conjugates was employed.
• an equimolar mixture of MMX-, MMF-, and MMXF-BSA was coupled to Affi-Gel 15.
• the bound human anti-CCD antibodies were eluted with 0.2 M glycine-HCl, pH 2.2.
• the eluate was immediately neutralized by 100 ⁇ L 1 M Tris-HCl, pH 7.5 already present in the collection vials.
• Fractions containing anti-CCD antibodies were detected by ELISA.
• the pooled sample was concentrated and brought into TBS buffer by centrifuge filter devices with 10 kDa cut-off (Milli- pore, Vienna, Austria) .
• human anti-CCD IgG was separated from anti-CCD IgE with the help of protein G plus agarose (Calbiochem, Darmstadt, Germany) .
• the breakthrough of this column contained the CCD-spe- cific IgE.
• the bound anti-CCD IgG was eluted and neutralized as describe above.
• the breakthrough (mainly IgE) and eluate (IgG) were concentrated by centrifugal ultrafiltration, and finally brought into TBS buffer containing 10% glycerol.
• the antibodies were kept at -20 0 C until use.
• the specificities of human anti-CCD IgG and IgE were determined by ELISA and Western blot using different transferrin gly- coforms (unmodified Tf, NaNa-, GnGn-, MM-, GnGnX-, GnGnF-, GnGnXF-, MMX-, MMF-, and MMXF-Tf) , the natural, xylose-containing glycoprotein hemocyanin from Helix pomatia (Sigma-Aldrich) or HRP. All glycoproteins were coated to plates at a concentration of 5 ⁇ g/mL. Otherwise ⁇ the procedure described above was followed.
• the affinity constants of human anti-MMXF IgG and IgE were determined by surface plasmon resonance (SPR) .
• MMX-, MMF-, or MMXF-Tf were immoblized to CM5 sensor chips to yield a response of 4,000 to 8,000 RU.
• Purified human IgG and IgE in HBS-P buffer ran over the surface for 7 min, followed by another 7 min washing with protein-free buffer at a flow rate of 5 ⁇ L/min.
• Rabbit anti-HRP polyclonal serum Sigma-Aldrich
• the affinity constants were determined by fitting to a single binding model by using BIAevaluation 3. Otherwise the procedures described in the previous studies have been followed. (Hantusch et al . , 2005; Jin et al . , 2006)
• the ratio of IgGl+3 versus IgG4 in total and specific IgG was accomplished by mass-spectrometric procedure.
• SDS-PAGE band of IgG was excised, digested with trypsin and analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) as described previously.
• the ratio of IgG4 in relation to the IgGl (plus IgG3) was determined from the signal height of the peaks of the homologous peptides peptides ALPAPIEK (IgGl & IgG3), GLPAPIEK (IgG2) and GLPSSIEK (IgG4); as well as SLSLSPG (+/-K) (IgGl, IgG2 & IgG3) and SLSLSLG (+/-K) (IgG4) .
• ALPAPIEK IgGl & IgG3
• GLPAPIEK IgG2
• GLPSSIEK GLPSSIEK
• SLSLSPG (+/-K) IgGl, IgG2 & IgG3
• SLSLSLG (+/-K) IgG4
• the specificity of the purified IgE resembled that from the serum pool whereas the purified IgG no longer bound to MM-glyco- forms (Fig. 5) .
• a noteworthy observation is the rather strong binding to glycoforms terminating with GlcNAc-residues (GnGnF, GnGnXF) .
• GnGnF, GnGnXF GlcNAc-residues
• This hitherto untested trait of human IgE contrasts earlier findings on commercial rabbit anti-HRP antibodies, where capping with GIcNAc drastically reduced antibody binding.
• rabbits can generate antibodies that are not kept off by terminal GIcNAc and apparently humans also have this ability.
• the antibodies have been purified over affinity columns with glycans devoid of terminal GIcNAc indicating that the same antibody population binds to both MMXF and GnGnXF glycans.
• the binding affinity between CCD-reactive antibodies and their ligands was assessed by SPR using sensor chips with immobilized glycoforms of human transferrin. Using evaluation, the purified anti-MMXF IgG and IgE were applied to SPR analysis. The reference chip was coupled with unmodified transferrin. Various amounts of human anti-MMXF IgG and IgE were injected in triplicates onto the sensor chip. The "on-rate" k a (M -1 S "1 ) was determined from the association phase of the binding curve, whereas k d (s "1 ) was deduced from the dissociation phase. The results are summarized in Table 1.
• Anti-CCD IgE antibodies appear incapable of provoking clinical symptoms despite their frequent presence in serum. This empirical evidence implies that positive IgE tests must be rated as false-positive when they are solely based on CCD-reactivity .
• Anti-CCD-IgE can be detected by using glycoproteins, which are unlikely to contain peptideepitopes for human IgE such as pineapple stem bromelain or HRP. An even better choice might be artificial glyco-allergens . Inhibition experiments with such glyco-proteins can help to discriminate allergen binding via peptide or glycan epitopes. The more difficult question is on the reason for the biological insignificance.
• CCD-specific IgE concentration of CCD-specific IgE in the serum pool was 3,7 kU/L (1.54 ⁇ g/mL) , which is only RAST class II, but single sera with much higher titers of CCD-specific IgE are often found.
• the affinity of CCD-specific IgG was determined to be about 100 times lower than that of IgE. This agrees at first sight with the results obtained for prominent allergens such as Bet v 1 and PhI p 5 (Table 2), or - measured by a method other than SPR - Amb a 5 . However, a closer look suggests the affinity of CCD-IgG to be comparably higher than that of anti-peptide IgG.
• IgE/IgG affinity ratio is about 2 orders of magnitude in the case of the CCD system, it is about 3 orders for Bet v 1 and PhI p 5 and even a bit higher for Amb a 5.
• the picture is considerable complicated by a report where the affinities of IgE, IgGl and IgG4 have been determined in individual patients having or having not received birch pollen SIT. These authors rather found individual differences of affinity than systematic differences between IgE and the IgG subclasses.
• Table 2 Comparison of Binding affinities for antibodies to CCDs, PhI p 5 and Bet v 1.
• a supporting or maybe even predominant mechanism might be antagonistic signalling via the tyrosine inhibition motif-containing immunore- ceptor Fc ⁇ RIIb on mast cells and basophils upon binding of IgG allergen complexes. Other, cellular mechanism may also take place .
• the substantially higher affinity for structures with xylose in addition to the indispensable core ⁇ l,3-fucose may mean that plant glycoproteins play a vital role for induction and/or maturation of the IgE response.
• the initial trigger for the anti- CCD immune reaction may be an insect sting. Short phase of allergic sensitization is soon followed by natural de- sensitization via incidential glyco-specific immunotherapy. The transient occurence of symptomatic allergic reactions towards CCD appears possible. Later, blocking antibodies would protect from symptoms and much later, s-IgE would gradually disappear - as in SLIT. It does not escape our attention that the mechanism proposed here for carbohydrate determinants could also apply to "mimickers of allergy" of protein nature.
• IgE against protein panallergens such as profilins is likewise known to be of clinical irrelevance in many cases (even though they may provoke skin reactions in SPT) .
• These examples make a point for allergen resolved allergy diagnosis. More importantly for this example, they share with CCDs that patients are frequently and repeatedly exposed to them. Presumably, ubiquitous glyco-epitopes and panallergens probably loose their allergenic potency by the same mechanism.
• T Reg cells have been described as the key element in allergen tolerance development.
• T Reg cells and their products are thought to suppress activation of effector cells in an allergen-dependent manner.
• This model for allergen tolerance suggests that a tolerated allergen, say a glycoprotein with CCDs, would attenuate the reaction against a simultaneously present perilous allergen.
• a tolerated allergen say a glycoprotein with CCDs
• Such an anti-allergic effect of CCDs has, however, not been described so far.
• Lane 1 of Fig. 7 shows that the patient's serum binds to all three allergens.
• Lane 2 of Fig. 7 was incubated with serum in the presence of an arteficial inhibitory glycoprotein (bromelain-glycopeptide coupled to bovine serum albumin) (Altmann Int. Arch. Allergy Immunol. 142 (2007): 99-115).
• an arteficial inhibitory glycoprotein bromelain-glycopeptide coupled to bovine serum albumin
• the absence of a stained band at the position of hyaluronidase indicates that the serum only reacted with the carbohydrate moiety of hyaluronidase but not with the polypeptide as was the case for the phospholipase and antigen 5.
• Lane 3 of Fig. 7 shows the immunoblot result with the same patient's serum after removal of the anti-CCD IgE using an affinity matrix according to the present invention.

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