Preparative IEF Research Articles

Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing.

Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.

New solution IEF device for micropreparative separation of peptides and proteins

The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel-based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution-based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V-shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of pH gradient and final local pH is visually traced by colored low-molecular pI markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired pH range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones.

Isoelectric focusing of small non‐covalent metal species from plants

IEF is known as a powerful electrophoretic separation technique for amphoteric molecules, in particular for proteins. The objective of the present work is to prove the suitability of IEF also for the separation of small, non-covalent metal species. Investigations are performed with copper-glutathione complexes, with the synthetic ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) and respective metal complexes (Fe, Ga, Al, Ni, Zn), and with the phytosiderophore 2'-deoxymugineic acid (DMA) and its ferric complex. It is shown that ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid and DMA species are stable during preparative scale IEF, whereas copper-glutathione dissociates considerably. It is also shown that preparative scale IEF can be applied successfully to isolate ferric DMA from real plant samples, and that multidimensional separations are possible by combining preparative scale IEF with subsequent HPLC-MS analysis. Focusing of free ligands and respective metal complexes with di- and trivalent metals results in different pIs, but CIEF is usually needed for a reliable estimation of pI values. Limitations of the proposed methods (preparative IEF and CIEF) and consequences of the results with respect to metal speciation in plants are discussed.

Quantitative Proteomic Profiling of Matched Normal and Tumor Breast Tissues

Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but <22% in common with an adenoma patient. In invasive carcinoma patients, several cellular and extracellular proteins that were significantly increased (Periostin, Small breast epithelial mucin) or decreased (Kinectin) have previously been associated with breast cancer, thereby supporting this approach for a larger disease-stage characterization effort.

Analysis of the phosphoproteome of the multicellular bacterium Streptomyces coelicolor A3(2) by protein/peptide fractionation, phosphopeptide enrichment and high‐accuracy mass spectrometry

The serine (Ser)/threonine (Thr)/tyrosine (Tyr) phosphoproteome of exponentially growing Streptomyces coelicolor A3(2) was analysed using the gel-free approaches of preparative IEF for protein fractionation, followed by strong cation exchange peptide fractionation and phosphopeptide enrichment by TiO(2) metal oxide affinity chromatography. Phosphopeptides were identified using LC-ESI-LTQ-Orbitra MS. Forty-six novel phosphorylation sites were identified on 40 proteins involved in gene regulation or signalling, central metabolism, protein biosynthesis, membrane transport and cell division, as well as several of unknown function. In contrast to other studies, Thr phosphorylation appeared to be preferred, with relative levels of Ser, Thr and Tyr phosphorylation of 34, 52 and 14%, respectively. Genes for most of the 40 phosphorylated proteins reside in the central "housekeeping" region of the linear S. coelicolor chromosome, suggesting that in general Ser, Thr and Tyr phosphorylation play a role in regulating essential aspects of metabolism in streptomycetes. A greater number of regulators and putative regulators were also identified compared with other bacterial phosphoproteome studies, potentially reflecting the complex heterotrophic and developmental life style of S. coelicolor. This study is the first analysis of the phosphoproteome of a member of this morphologically complex and industrially important group of microorganisms.

Microfluidic preparative free‐flow isoelectric focusing in a triangular channel: System development and characterization

A preparative scale free-flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular-shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion-permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50 x 70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free-flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule-heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370+/-20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.

Characterization of Antibody Charge Heterogeneity Resolved by Preparative Immobilized pH Gradients

A capillary isoelectric focusing (cIEF) method has been developed as an alternative to cation exchange chromatography to determine charge heterogeneity for a therapeutic antibody. Characterization of the cIEF profile is important to understand the charged isoform distribution. A variety of preparative IEF methods have been developed over the years but have had various limitations including high levels of contaminating ampholytes and complex fractionation and isolation procedures. More recently, an off-line method that uses pI-based separation on immobilized pH gradients was developed to preparatively isolate material with convenient liquid phase recovery. This method uses the Agilent OFFGEL 3100 Fractionator and was optimized to produce fractions of antibody charge isoforms differing by as little as 0.1 pI units. The isolation of highly resolved fractions then allowed for the identification of N- and C-terminal basic charge modifications including noncyclized glutamine, signal peptide extensions, and various levels of C-terminal lysine processing and high mannose structures. These species could then be correlated to specific peaks in the cIEF profile. This work shows that a preparative IEF method using immobilized pH gradients can be optimized to generate highly resolved, pI-based fractions in solution which can be used for successful cIEF profile characterization. Access to preparative amounts of discrete charged species allows for a better understanding of the underlying covalent modifications responsible for the charge differences and facilitates evaluation of the impact of these modifications on stability and potency of therapeutic antibodies.

Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples

Efficient separation method is a crucial part of the process in which components of highly complex biological sample are identified and characterized. Based on the principles of recently newly established electrophoretic method called divergent flow IEF (DF IEF), we have tested the DF IEF instrument which is able to operate without the use of background carrier ampholytes. We have verified that during separation and focusing of sample consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be created by autofocusing of the sample components themselves without any addition of carrier ampholytes. In DF IEF, the proteins are separated, desalted and concentrated in one step. The fractions of yeast lysate sample, collected at the DF IEF output and subjected to gel IEF, contained the zones of proteins gradually covering the pI values from 3.7 to 8.5. In our experimental arrangement, the highest number of proteins has been found in fractions with pI values around 5.3 as detected by polyacrylamide gel IEF with CBB staining. During DF IEF, the selected protein bands have been concentrated up to 16.8-fold.

Proteomics Analysis of Cells in Whole Saliva from Oral Cancer Patients via Value-added Three-dimensional Peptide Fractionation and Tandem Mass Spectrometry

Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for conditions within the oral cavity such as oral cancer. Although many have studied the soluble fraction of whole saliva, few have taken advantage of the diagnostic potential of the cells present in saliva, and none have taken advantage of proteomics capabilities for their study. We report on a novel proteomics method with which we characterized for the first time cells contained in whole saliva from patients diagnosed with oral squamous cell carcinoma. Our method uses three dimensions of peptide fractionation, combining the following steps: preparative IEF using free flow electrophoresis, strong cation exchange step gradient chromatography, and microcapillary reverse-phase liquid chromatography. We determined that the whole saliva samples contained enough cells, mostly exfoliated epithelial cells, providing adequate amounts of total protein for proteomics analysis. From a mixture of four oral cancer patient samples, the analysis resulted in a catalogue of over 1000 human proteins, each identified from at least two peptides, including numerous proteins with a role in oral squamous cell carcinoma signaling and tumorigenesis pathways. Additionally proteins from over 30 different bacteria were identified, some of which putatively contribute to cancer development. The combination of preparative IEF followed by strong cation exchange chromatography effectively fractionated the complex peptide mixtures despite the closely related physiochemical peptide properties of these separations (pI and solution phase charge, respectively). Furthermore compared with our two-step method combining preparative IEF and reverse-phase liquid chromatography, our three-step method identified significantly more cellular proteins while retaining higher confidence protein identification enabled by peptide pI information gained through IEF. Thus, for detecting salivary markers of oral cancer and possibly other conditions of the oral cavity, the results confirm both the potential of analyzing the cells in whole saliva and doing so with our proteomics method.

The Alpher, Bethe, Gamow of isoelectric focusing, the alpha‐Centaury of electrokinetic methodologies. Part I

The birth and evolution of IEF in conventional carrier ampholyte buffers is reviewed here, from a shaky start during World War II, via desperate attempts of Svensson to create pH gradients by stationary electrolysis of salts, to the development of the IEF theory and the solution of the steady-state equation. The remarkable synthetic process of Ampholines, as ingeniously devised by Vesterberg, is additionally recalled, with a thorough description of the fundamental properties of these amphoteric buffers, creating and maintaining the pH gradient under strong electric fields. The review ends with a mention of the major contributions of B. J. Radola to this field, namely analytical and preparative IEF in granulated Sephadex layers and the development of ultrathin IEF, in polyacrylamide gels as thin as 20-100 mum. The latter technique paved the way to DNA sequencing gels and to CZE. The symptoms of decay are here presented through the simulations of Mosher and Thormann, clearly indicating an isotachophoretic mechanism for pH gradient decay with time. The decay of IEF was the birth of IPGs.

Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Three α-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of α-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with p I values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS–PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25–55°C). The three purified isozymes were inhibited at low concentration by paraoxon (10 −10 M), chlorpyrifos (10 −6 M), DEF (10 −6 M), and PMSF (10 −6 M) indicating that they were “B” type serine esterases. Conversely, inhibition was not observed at 10 −4 M eserine, PHMB, or CaCl 2, further supporting the conclusion that the microsomal esterases were of the “B” type. None of the isozymes was inhibited by 10 −4 M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.

A highly specific glucosyltransferase is involved in the synthesis of crocetin glucosylesters in Crocus sativus cultured cells

Saffron (Crocus sativus) stigmata contain rare water-soluble carotenoids and the major one is crocin, the crocetin digentiobiosyl-ester. Previous studies indicated that two glucosyltransferases might be involved in the formation of crocetin glucosyl- and gentiobiosyl-esters (Dufresne et al. 1997). A UDP-Glc: crocetin 8,8′-glucosyltransferase involved in the biosynthesis of crocetin monoglucosyl- and diglucosyl-esters was extracted from saffron cell cultures and purified 300-fold by gel filtration chromatography and preparative IEF electrophoresis, with a recovery of 13 percnt;. The purified enzyme preparation was highly specific for crocetin and formed ester bonds between the glucose moiety of UDP-Glc and the free carboxyl functions of crocetin. The enzyme did not add other glucose units to the glucosyl-esters to form crocetin gentiobiosyl-esters. A crude desalted extract of the same material was less specific and formed glucosyl-esters with several other compounds, including abscisic and retinoic acids. The purified preparation was active between pH 4.4 and 4.6. SDS-PAGE revealed a major band at 26 kDa while the native molecular mass determined by gel filtration was in the range of 49 to 55 kDa. The study provides concrete evidence for the hypothesis that more than one glucosyltransferase is involved in the biosynthesis of crocetin glycosyl-esters in saffron.

Characterisation of xylanases produced in liquid and compost cultures of the cultivated mushroom Agaricus bisporus

The production and properties of Agaricus bisporus endoxylanase and β-xylosidase were studied in compost and liquid cultures. A. bisporus was grown on minimal medium with solka floc BW40, oat spelt xylan, Whatman CC41 microcrystalline cellulose, D-xylose and D-glucose as carbon sources. Endoxylanase activity and β-xylosidase activity were found during growth with complex carbon sources such as cellulose or xylan and no activity was present with sugars such as glucose or xylose. In compost culture endoxylanase activity increased during and after the button stage of fruit body development and decreased after fruit body harvest. The production of β-xylosidase differed in that it was only detectable during the button stage of development. Using activity staining and preparative IEF, three endoxylanase activities were separated from liquid cultures and three principal endoxylanase activities were separated from compost extract. The pH optima and IEF profiles of these activities were similar. Only one β-xylosidase activity was found in liquid culture, whereas two were found in compost culture; the IEF profiles of the compost activities differed markedly from those of the β-xylosidase found in liquid culture.

Isoforms of purified methyltransferase from human blood platelets

A membrane-bound protein with N-methyltransferase activity, associated with phospholipid metabolism, has been isolated from purified human blood platelet plasma membranes. The activity of this enzyme has been detected in crude platelet preparations. However, the nature and properties of this enzyme and its purification from normal human blood platelets have not been investigated, hence, the aim of this study was to purify, characterise the enzyme from human blood platelets and determine its possible role in phospholipid transmethylation. The plasma membranes were purified by velocity and sucrose gradient centrifugation and solubilized in buffer containing 0.5% Triton-X 100. The partially purified extracts were further purified using a combination of ion-exchange chromatographic procedures, preparative IEF and preparative SDS-PAGE. The preparative rotorfor electrofocusing, using pH 3-10 ampholyte at 40EC, yielded activities at both acidic and basic pHs of approximately 3.5 and 8.5, respectively. The pH 8.5 activity yielded predominantly a single 67 KDa band on SDS-PAGE gel and the pH 3.5 activity also gave a similar band and a few lighter bands at 35 KDa and 50 KDa. The acidic and basic fractions catalysed the transfer of methyl groups from S-adenosyl inethionine to PE with a predominant formation of PME (ca 90%). The fraction purified by preparative SDS-PAGE also yielded a major methylating activity that eluted between 60-70 KDa. This latter protein was washed free of SDS and its activity measured; it, too yielded predominaly the monomethylated PE. These data are consistent with the hypothesis that several SAM-dependent isozymes also exist in human blood platelets that convert PE to PC. JOURNAL OF THE GHANA SCIENCE ASSOCIATION Volume 2 No. 3 (2000) pp. 98-102

Polymorphic glutamate dehydrogenase in lilac vitroplants as revealed by combined preparative IEF and native PAGE: Effect of ammonium deprivation, darkness and atmospheric CO2 enrichment upon isomerization

The activity and polymorphism of glutamate dehydrogenase (GDH) were studied in basal callus of lilac (Syringa vulgaris L.) vitroplants. Native PAGE alone revealed seven bands staggered at regular intervals. Preparative liquid‐vein IEF allowed the separation of six to ten GDH fractions with charges ranging between 5.18 and 7.08. Analysis of these GDH fractions in native PAGE indicated that up to seven GDH bands can be detected for each fraction. This suggests the existence of seven isoforms of the enzyme with subunits presenting different isoelectric points. Dark‐ and ammonium‐controlled forms were found to be the more acidic and faster migrating ones in native PAGE. The results support for the first time that atmospheric CO2 enrichment increases GDH activity dramatically and modifies isomerization of the enzyme.

Engineering protein for X-ray crystallography: the murine Major Histocompatibility Complex class II molecule I-Ad.

Class II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I-Ad, with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced in Drosophila melanogaster SC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six-residue linker to the amino terminus of the I-Adbeta chain. This modified I-Ad molecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X-ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I-A and other class II MHC-peptide complexes.

Purification and properties of the soluble midgut trehalase from the gypsy moth, lymantria dispar

The midgut trehalese (THA) from fifth instar Lymantria dispar (gypsy moth) larvae was purified to homogeneity by two separate methods: gel filtration followed by Rotofor preparative IEF, and affinity chromatography on trehalose coupled to Sepharose 6B followed by preparative polyacrylamide gel electrophoresis. Midgut THA from the last stadium L. dispar larvae existed mainly in soluble form and displayed a single band of activity in nondenaturing polyacrylamide gels when stained by a THA-specific staining procedure. Analytical IEF of purified mifgut THA revealed a single protein band with an apparent pI of 4.6. SDS-PAGE and gel permeation studies indicated that the smlallest active form of THA in the late fifth instar larval midgut was a monomeric protein with an approximate size of 60 kDa. A specific activity of 67 units/mg of protein at 30°C and at pH 6.4 was determined for the enzyme purified by affinity chromatography and preparative gel electrophoresis. The midgut enzyme exhibited a very high substrate specificity with a K m of 0.4 mM for trehalose. The enzyme was maximally active at pH 5.4–6.0 and was thermally stable at temperature up to 65°C. The midgut THA was insensitive to inhibition by a high concentration of Tris, sucrose, p- nitrophenyl-β- d- glucoside or phloridzin. Divalent cation, metal ions, hypertrehalosaemic hormone and octomine had no significant effect on the activity of the purified enzyme in vitro. The purified enzyme was inactivated by modification with DEP and was competitively inhibited by castanosperine with an apparent K i of 0.8 × 10 −6 M at pH 6.4.

Characterization of hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera: Notonectidae) reveals high electrophoretic heterogeneity

Backswimmers (Notonectidae) are one of the three families of insects which contain species known to make hemoglobin. In backswimmers the hemoglobin allows the insect to maintain a prolonged neutral buoyancy during its dive. Sephadex G-200 column chromatography, SDS-polyacrylamide gel electrophoresis and HPLC demonstrate a molecular weight for the monomeric hemoglobin og Buenoa margaritacea of 15,000–19,000 Da. u.v.-visible spectra at eight hemoglobin derivatives (Fe 2+:deoxy, O 2, CO, NO; Fe 3+:H 2O, OH −, CN −, imidazole) are given. Native gel electrophoresis of the cyanomet form shows nine bands, while isoelectric focusing shows 12. Bands isolated from preparative IEF gels form multiple bands when rerun, indicative of a change in aggregation status after isolation. Interactions among various hemoglobin components are seen using 2-dimensional “titration” electrophoresis.

Binding site mapping of a photoaffinity-labeled juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) of larval Manduca sexta was labeled by a photoaffinity analog of JH II and purified by preparative IEF and ion-exchange HPLC. The purified [ 3H]EHDA-labeled JHBP was selectively cleaved by CNBr and by endoproteinases Lys-C and Glu-C. The radioactive peptides were separated by tricine SDS-PAGE and sequenced after blotting to a PVDF membrane. The sequence revealed that Ala 184-Asn 226 contained a primary binding site of [ 3H]EHDA. Furthermore, peptide mapping indicated that Asp 1-Glu 34 also contained a second covalent attachment site of [ 3H]EHDA. Labeling of the N-terminal region increased when the photolysis was performed at lower temperature. Since Ala 184-Asn 226 is predicted to be a hydrophobic β-sheet region, it may participate in the recognition of lipophilic backbone of JH. Five out of six cysteines are located in these two regions, consistent with a model in which the two binding regions connected by disulfide bridges provide a two-sided binding pocket for juvenile hormone.

Isolation and characterization of a glycoprotein allergen, Art V II, from pollen of mugwort ( Artemisia vulgaris L.)

A glycoprotein allergen, Art v II, was isolated from pollen of mugwort by two different isolation procedures. Art v II-A was isolated by a combination of ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Con A-Sepharose and ion-exchange chromatography on Mono P. Art v II-B was isolated by a combination of preparative IEF in Ultrodex granulated gel, affinity chromatography on Con A-Sepharose and HPLC size exclusion on Ultropac TSK G2000SW. Art v II-A and Art v II-B were shown to be antigenically identical with the allergen we have formerly denoted Ag7. The MW of Art v II A/B was determined to be 34,000–38,000 by HPLC size exclusion, and 35,000 and 20,000 by SDS-PAGE under non-reducing and reducing conditions, respectively. An v II was found to consist of 6(7) isoforms with pI 4.10, 4.20 (major component), 4.35, 4.45, 4.55, 4.65, (4.80). The glycoprotein allergen had a protein to carbohydrate ratio of 10:1 and the carbohydrate part contained mannose (70.7%), N-acetyl-glucosamine (17.0%), glucose (7.0%) and galactose (5.3%). In R(R)IE the purified allergen bound IgE from 5 (33%) of 15 sera from patients with clinical allergy against mugwort pollen and from 13 (52%) of 25 sera from patients selected only on the basis of a RAST-class 4 against mugwort pollen.