Antibody Binding Capacity Research Articles

Flow cytometric study of potential target antigens (CD19, CD20, CD22, CD33) for antibody-based immunotherapy in acute lymphoblastic leukemia: analysis of 552 cases

Monoclonal antibody (MoAb)-based therapies have opened innovative treatment avenues that have impacted on the management of patients with both neoplastic and non-neoplastic hematological diseases. The aim of our study was to evaluate in a large series of cases of acute lymphoblastic leukemia (ALL) the expression of specific antigens, CD19, CD20, CD22, and CD33, for which MoAbs are available for clinical use. For each antigen, evaluation was based on the percentage of positive leukemic cells and the degree of antigen expression by mean fluorescence intensity (MFI) and antibody binding capacity (ABC) that were correlated with age, immunophenotype, and presence/absence of particular molecular markers. We can document that some of the analyzed antigens showed a degree of expression related to the B-cell maturation profile, and that the antigen expression intensity appeared to vary according to the presence of specific genetic markers. These findings suggest that the possible clinical use of a given MoAb in patients with ALL should take into account both the maturation profile of the leukemic cells and the presence of a given molecular transcript. Only clinical studies will conclusively demonstrate whether the differences in antigenic expression truly correlate with the different therapeutic efficacies of the various clinical grade MoAbs.

Immunology in the heart of the High Tatra mountains

Immunology in the heart of the High Tatra mountains

Constitutive activation and accelerated maturation of peripheral blood t cells in healthy adults in burkina faso compared to Germany: The case of malaria?

ObjectiveIt is not exactly known how frequent exposure to Plasmodium falciparum shapes the peripheral blood T-cell population in healthy West Africans.MethodsThe frequency of peripheral blood CD4+ lymphocytes responding to Plasmodium falciparum merozoite surface protein 1 (PfMSP-1) by production of interferon-gamma (IFN-γ), interleukin-2 (IL-2) or tumor necrosis factor-alpha (TNF-α) was determined using a commercially available flow cytometric activation assay (Fastlmmune) in 17 healthy adults in Nouna, Burkina Faso. T-cell activation and maturation in peripheral blood of healthy adults in Burkina Faso (n = 40) and Germany (n = 20) were compared using immunophenotyping and three-colour flow cytometry.ResultsSignificant numbers of PfMSV-1 -specific CD4+ lymphocytes producing IFN-γ, IL-2 and/or TNF-α were detected in 14 healthy adults in Nouna. Cytokine profiles showed predominant production of IFN-γ and TNF-α. Compared to Germans, Burkinabé showed markedly lower proportions of CCR7+ CD45RA+ naïve CD4+ cells and slightly higher frequencies of CD95+ CD4+ T-cells and of CD38+ CD8+ T-cells. The median antibody-binding capacity of CD95dim CD4+ T-cells in Burkinabé was more than twice the value observed in Germans (263 vs. 108 binding sites per cell, p < 0.0001).ConclusionsWe hypothesize that an IFN-γ-induced increase in the expression level of CD95 on CD4+ lymphocytes may lower the activation threshold of resting naïve CD4+ T-cells in healthy adults living in Burkina Faso. Bystander activation of these cells deserves further study as a molecular mechanism linking strong IFN-γ responses against Plasmodium falciparum to decreased susceptibility to parasitemia observed in specific ethnic groups in West Africa.

Recovery of Functional EBV Specific T Cells Is Not Impaired In Patients with Chronic Gvhd.

AbstractAbstract 1286 Introduction:T cell expression of PD-1, a marker of functional exhaustion manifested by inability to produce cytokines upon stimulation, is upregulated in patients with acute GHVD. This is thought to explain at least in part why patients with acute GVHD have frequent infections (Gallez-Hawkins et al, BBMT15:872, 2009). Here we wished to evaluate whether this is true also for chronic GVHD. Patients and Methods:We studied 17 allogeneic HCT recipients for AML who have not developed acute or chronic GVHD by day 84. Their blood was drawn on day 84, 180 and 365. Between day 84 and 365, 7 patients did and 10 patients did not develop chronic GVHD needing systemic immunosuppressive therapy (de novo, ie, without preceding acute GVHD). Onset of the chronic GVHD was on median day 103 (range, 90–147). We studied total CD4 and CD8 T cells as well as Epstein-Barr virus (EBV)-specific CD4 and CD8 T cells, as patients with chronic GVHD are at risk of EBV disease (Landgren et al, Blood,14:4992, 2009). Blood mononuclear cells were stimulated with Epstein-Barr virus (EBV) lysate, EBNA3A+B+C overlapping peptides, no or irrelevant stimulus as negative control, or Staphylococcal enterotoxin B as positive control. After overnight incubation, expression of IFNγ, TNFα, IL2 and PD-1 on CD3+CD4+CD8- or CD3+CD4-CD8+ cells was determined by flow cytometry. Cells expressing IFNγ, TNFα, IL2 or their combinations were enumerated. PD-1 expression was quantified using beads coated with anti-mouse antibody (Quantum Simply Cellular, Bangs Laboratories) and expressed as antibody binding capacity units (ABC) (dynamic range, approximately 300 to 500,000 ABC units per cell). Results:PD-1 expression on total, EBV lysate-specific or EBNA3-specific CD4 or CD8 T cells was not significantly higher among patients who did vs did not develop chronic GVHD. On the contrary, there was a trend toward lower PD-1 expression on EBV lysate-specific CD4 and CD8 T cells and EBNA3-specific CD4 T cells in patients who developed chronic GVHD. This was significant (p<.05, Mann-Whitney test) for EBV lysate-specific CD4 T cells on day 84, EBV lysate-specific CD8 T cells on day 180, EBV lysate-specific CD4 and CD8 T cells on day 365, EBNA3-specific CD4 T cells on day 84 and EBNA3-specific CD4 T cells on day 365. Consistent with that, absolute counts of total, EBV lysate-specific or EBNA3-specific T cells were not significantly lower in patients who did vs did not develop chronic GVHD. On the contrary, there was a trend toward higher EBV lysate-specific and EBNA3-specific CD4 or CD8 T cell counts in patients who developed chronic GVHD. This was significant on day 84 for total EBV lysate-specific CD4 and CD8 cells, EBV lysate-specific CD4+IFNγ+ cells and CD8+IFNγ+ cells, and total EBNA3-specific CD4 cells, EBNA3-specific CD4+IFNγ+ cells, CD4+IL2+ cells, CD4+IFNγ+TNFα+IL2+ cells and CD8+IFNγ+ cells. Conclusion:De novo chronic GvHD and its treatment do not adversely affect the counts of functional EBV specific T cells. Disclosures:No relevant conflicts of interest to declare.

Specific antibody filter (SAF) binding capacity enhancement to remove anti‐A antibodies

Removal of Anti-A/B antibodies prior to ABO-incompatible transplantation can prevent hyperacute organ rejection. We are developing a specific antibody filter (SAF) device to selectively remove ABO blood group antibodies from the whole blood by utilizing immunoaffinity adsorption. The device consists of ultrafiltration hollow fiber membranes with synthetic antigens specific to bind blood group antibodies immobilized on the inner lumenal walls of the fibers. The aim of this study was to evaluate the effect of antigen molecular weight and surface activation process to increase the antibody binding capacity of the fiber membrane surface. A new higher molecular weight antigen Atri-pNSA-1000 compared with Atri-pNPA-30 (A-trisaccharide (Atri) conjugated to activated polymers of Mol. wt. 1000 kDa and 30 kDa, respectively) was employed to improve accessibility of the antigen to bind antibodies. Also, a cyanogen bromide (CNBr) based surface activation method mediated by TEA in neutral pH medium was used to enhance the number of active sites for antigen binding compared to a strong basic medium of NaOH. Using a CNBr/TEA activation method and by immobilizing Atri-pNSA-1000 antigen, an antibody binding capacity (∼0.01 monoclonal anti-A IgM nmol/cm(2)) was achieved on the fiber surface. This binding capacity was sufficient to reduce monoclonal antibody titer from 1:128 to final titer below 1:4 with a surface area to volume ratio that is similar to commercial dialysis device (∼1.1 m(2) surface area for an average body blood volume of 5 L).

A new bioassay for the immunocytokine L19-IL2 for simultaneous analysis of both functional moieties

Currently, cancer directed new biological entities (NBEs) in the pharmaceutical R&D pipelines are derived from monoclonal antibodies in various formats, such as immunocytokines. Generally, immunocytokines are bi-functional molecules that consist of a specific targeting antibody-based portion and a linked cytokine. To confirm the quality of the drug product both moieties have to be characterized using appropriate techniques. Until now, the binding capacity of antibodies is usually examined by ligand binding assays whereas the biological activity of the linked cytokine is determined by cell-based potency assays. However, the simultaneous analysis of both functional moieties in a single assay format has not been described so far. In this paper we present a newly designed bioassay format for the anti-cancer immunocytokine L19-IL2, comprising of the human vascular targeting single-chain Fv L19 and human interleukin 2 (IL2). This new potency assay allows simultaneous analysis of both moieties, thus specific L19 binding capacity and the ability of IL2 to induce the proliferation of the detector cytotoxic T-cell line CTLL-2. Assay development was performed with special focus on application of different fitting models for the sigmoid dose–response curves to evaluate the influence of model optimization on the validity of assay results. For assay validation generally accepted characteristics were determined. Assay specificity was shown by testing L19-IL2 related compounds. All other validation parameters were derived from 25 batch runs using five nominal L19-IL2 concentrations, covering a range from 60% to 140% of the standard's potency. Accuracy ranged from −3.4% to −6.9% relative error (%RE). Interbatch precision ranged from 6.1% to 10.6% coefficient of variation (%CV). For assay linearity a coefficient of determination ( R 2) of 0.9992 was found. Assay robustness was shown with L19-IL2 samples after three freeze–thaw cycles and also with different cell passages of the used cytotoxic T-cell line. Based on the data, we conclude that this assay is valid for potency estimation of the immunocytokine L19-IL2. Moreover, this format represents a major improvement compared to other approaches which only allow potency evaluation of both functional moieties in separate assays. In general the underlying assay principle described seems suitable for potency determination of other immunocytokines.

Transferrin receptor expression on reticulocytes as a marker of iron status in dialyzed patients

Erythropoietin therapy should be accompanied by an adequate iron supply in order to avoid functional iron deficiency (FID) related to enhanced erythropoiesis. Therefore, accurate monitoring of the body's iron homeostasis is needed. This study was conducted to investigate whether transferrin receptor (TfR) expression on reticulocytes can reflect iron status in patients with chronic renal failure (CRF). TfR expression [antibody binding capacity (ABC)] and the proportion of TfR positive reticulocytes (%TfR+ Ret) relative to all reticulocytes were measured by a quantitative flow cytometric method at baseline and at 3 weeks in 34 dialysis patients. Iron status (plasma ferritin and soluble TfR) and hemoglobin (Hb) with advanced cellular indices, such as the percentage of hypochromic reticulocytes (%HYPOr) and cellular Hb in reticulocytes (CHr), were also analyzed. Patients with FID had significantly higher TfR ABC and %TfR+ Ret compared with patients with replete iron status (p=0.034 and p=0.006, respectively). In patients whose Hb concentrations showed a reduction, the mean increase (3 weeks- baseline) in TfR ABC was four-fold higher and %TfR(+)Ret was three-fold higher when compared with patients whose Hb was stable or had increased. The changes in TfR expression correlated significantly with the changes in reticulocyte indices [CHr (negatively), %HYPOr (positively)] and plasma ferritin (negatively). Reticulocyte TfR expression reflected the changes in the Hb level and the iron availability at the cellular level, and therefore it might be useful in the assessment of iron status in patients with CRF.

Quantification of Interleukin-6 in cell culture medium using surface plasmon resonance biosensors

Interleukin (IL)-6, a multifunctional cytokine, is widely used as an index for illnesses such as inflammatory and autoimmune disorders, coronary artery disease, neurological disease, and gestational problems. It is thus very important to be able to precisely quantify the level of IL-6 for disease diagnosis and any subsequent therapy. Surface plasmon resonance (SPR) biosensors are sensitive in detecting the interaction between biomolecules by sensing the changes in the refractive index on the sensor chip. This study investigated the SPR technique to determine the IL-6 secretion of human fibroblast MRC5-CVI cells induced by lipopolysaccharide (LPS). To reduce non-specific binding, a mixed self-assembled monolayer of mercaptoundecanoic acid (MUA) and mercaptohexanol (MCH) was attached to the sensor, and then used for IL-6 determination using a sandwich type immunoassay. In addition, two antibody immobilization methods were applied to the sensor surface—direct immobilization and indirect immobilization via protein G affinity. The results demonstrated that the direct immobilization method had a better antibody binding capacity on the sensor surface. The level of cellular IL-6 secretion detected by the SPR biosensor showed a consistent correlation with the commercial kit of IL-6 enzyme-linked immunosorbent assay.

Influence of different distinct differentiation of trophoblastic cells on binding characteristics of targeted microbubble contrast agents in vitro

Objective In this study, we investigated the in vitro binding capacity of β-HCG antibody targeted SonoVue® microbubbles to trophoblasts with distinct differentiation in order to explore the possibility of utility of targeted SonoVue® microbubbles imaging for early locating diagnosis of malignant trophoblastic cell disease.

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.

Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products

GVAX ® immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-to-lot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis

Background:The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested.Methods:The antibody was modified using EBNA cells cotransfected with β-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes.Results:The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcγRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcγRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcγRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model.Conclusion:The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

A disposable electrochemical immunofiltration test strip for rapid detection of α-fetoprotein

A disposable electrochemical immunofiltration test strip for the rapid detection of α-fetoprotein (AFP) was developed. The test strip was constructed by assembly of screen-printed carbon electrodes, absorption-water pad, nitrocellulose membrane modified by anti-AFP antibody and glass fiber membrane conjugated with ferrocene monocarboxylic acid (FC) labeling AFP. The analytical system utilizes flow-through immunofiltration and competitive immunoassay techniques in combination with an amperometric sensor. The parameters affecting the immunoassay such as selection of filter membrane, membrane pore-size, and antibody binding capacity were investigated and optimized. The immunofiltration system allows us to specifically and directly detect AFP in serum with a low detection limit of 6 ng/mL. The working range is from 6 to 500 ng/mL with an overall analysis time of 5 min for one sample. This electrochemical immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of biomarker including AFP.

The role of insulin antibodies in insulin treatment of type I diabetes.

We investigated equilibrium plasma binding patterns of insulin in 45 juvenile diabetics treated with conventional insulin preparations. Insulin binding parameters were evaluated by Scatchard analysis of the binding data. Stable diabetics had significantly lower equilibrium dissociation constants than labile, thus suggesting an enhanced insulin depot effect due to stronger insulin binding. Correlation of insulin binding data with a glycemic control index yielded a positive relationship between insulin antibody binding and the degree of glycemic control. Insulin neutralization as detected by a relationship between maximum binding capacity of high affinity antibodies and insulin requirement could only be found if patients with poor diabetes control were excluded. Similarly, the well-known promoting influence of residual beta-cell functional capacity (assessed by C-peptide levels) on diabetic stability was observed only after exclusion of patients with higher insulin antibody binding. These data suggest that insulin antibodies are influencing insulin treatment of diabetics in a dual way. They may neutralize therapeutic insulin but at the same time they exert an insulin-sparing action by improvement of diabetes control. Occasionally the latter effect may abolish the correlation between diabetes control and beta-cell functional capacity.

High molecular weight blood group A trisaccharide-polyacrylamide glycoconjugates as synthetic blood group A antigens for anti-A antibody removal devices.

Specific immunoadsorption of blood group antibodies by synthetic antigens immobilized on support matrices in the peri-transplantation period provides a promising solution to hyperacute rejection risk following ABO-incompatible transplantation. In this study, we investigated binding interactions between anti-A antibodies and synthetic blood group A trisaccharide conjugated with polyacrylamide of different molecular weights (30 and 1000 kDa). The glycopolymers were equipped with biotin tags and deposited on streptavidin-coated sensor chips. The affinity and kinetics of anti-A antibodies binding to glycoconjugates were studied using surface plasmon resonance (SPR). The high molecular weight conjugate (Atri-PAA(1000)-biotin) enhanced antibody binding capacity by two to three fold compared with the low molecular weight conjugate (Atri-PAA(30)-biotin), whereas varying the carbohydrate content in Atri-PAA(1000)-biotin (20 mol % or 50 mol %) did not affect antibody binding capacity of the glycoconjugate. The obtained results suggest that immunoadsorption devices, especially hollow fiber-based antibody filters which are limited in available surface area for antigen immobilization, may greatly benefit from the new synthetic high molecular weight polyacrylamide glycoconjugates.

Influence of ligand density on antibody binding capacity of cation-exchange adsorbents

Adsorption properties of a set of polymethacrylate-based cation exchangers designed for purification of monoclonal antibodies were investigated. The materials differed significantly in the density of sulphoisobutyl ligand groups. The ligand density had a pronounced effect on the static adsorption capacity of a polyclonal human immunoglobulin G. An optimal ligand density was observed at any pH and NaCl concentration tested when sharp optima were observed at low pH and ionic strength values. This was caused by effective clogging of pore mouth at high ligand densities. An anomalous effect of ionic strength was observed for the adsorbents with the high ligand density when the adsorption capacity increased with the addition of NaCl at low pH.

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically.

Specific binding of magnetic nanoparticle probes to platelets in whole blood detected by magnetorelaxometry

The binding of monoclonal antibodies labelled with magnetic nanoparticles to CD61 surface proteins expressed by platelets in whole blood samples was measured by magnetorelaxometry. This technique is sensitive to immobilization of the magnetic labels upon binding. Control experiments with previous saturation of the epitopes on the platelet surfaces demonstrated the specificity of the binding. The kinetics of the antibody antigen reaction is accessible with a temporal resolution of 12 s. The minimal detectable platelet concentration is about 2000 μL −1 (sample volume 150 μL). The proportionality of the magnetic relaxation amplitude to the number of bound labels allows a quantification of the antibody binding capacity.

Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, Western blotting and Q-TOF mass spectrometry

Peanut allergy is triggered by several proteins known as allergens. In this study, the complexity the peanut allergome is investigated with proteomic tools. The strength of this investigation resides in combining the high-resolving power and reproducibility of fluorescence two-dimensional differential gel electrophoresis with specific immunological detection as well as polypeptide sequencing by high-resolution mass spectrometry. Matching of the peanut proteins in 2D gels was achieved by differential labelling whereby peanut proteins and purified allergens (Ara h 1, Ara h 2 or Ara h 3/4) were run on the same gel. Ten protein spots on a mass line of ca. 63-68 kDa were likely to correspond to Ara h 1. Two doublets on two different mass lines at ca. 16 and 18 kDa matched with purified allergen Ara h 2. The basic and acidic sub-units of Ara h 3/4 were observed at masses of ca. 25 kDa and 40-45 kDa, respectively. Subsequently the antibody-binding capacity of spots corresponding to peanut allergens was investigated by Western blotting of 2D gels using antibodies (IgY) raised against Ara h 1, Ara h 2 and the recombinant 40 kDa sub-unit of Ara h 3/4. Final confirmation of the identity of the protein spots matched after 2D electrophoresis and identified by Western blotting was obtained by in-gel digestion of protein spots and analysis by quadrupole time-of-flight mass spectrometry. By using the method developed in our work, the location and identification of two different isoforms of the allergen Ara h 1, the allergen Ara h 2 and six isoforms of the allergen Ara h 3/4 in 2D peanut protein maps was established.

Synthesis, characterization and protein binding properties of supported dendrons

Novel benzylether type aldehyde and acetal terminated dendrons were synthesized and attached to a silica gel support; a linear spacer was also introduced as a control material. The supported dendritic compounds were mainly characterized by solid state 13C CPMAS NMR, elemental analysis and X-ray photoelectron spectroscopy (XPS) and the presence of free aldehydes was determined by the purpald test. Bovine serum albumin (BSA) protein was coupled to the dendronized support by imine bond formation, followed by irreversible reduction of the carbon–nitrogen double bond. A significant positive dendritic effect was observed on the antibody binding capacity of immobilised BSA as measured by fluorescence immunoassay (FIA).