Carbohydrate Epitopes Research Articles

Antigenic cross-reactivity between Schistosoma mansoni and pollen allergens from the birch tree (Betula verrucosa) and Timothy grass (Phleum pratense): involvement of shared glycan epitopes and implications for the hygiene hypothesis

Previous studies have shown that schistosome infection can protect against allergic symptoms, but the underlying mechanisms are still not fully understood. Here we have shown that rabbit IgG antibodies raised against Schistosoma mansoni soluble egg antigens (SmSEA) are cross-reactive with a wide array of molecules in Timothy grass pollen (TGP) and birch tree pollen (BTP). Five of the cross-reactive pollen molecules (two from TGP and three from BTP) were selected randomly and identified by tandem mass spectrometric (TMS) analysis to be, respectively, the TGP allergens Phl p 1 and Phl p 5b, and BTP glutathione S-transferase (GST), and the BTP allergens Bet v 1 and Bet v 6.0102. Rabbit anti-SmSEA IgG antibodies that cross-reacted with each of the five allergens were found to be reactive with three major S. mansoni egg antigens, IPSE/alpha-1, omega-1 and kappa-5. Pairwise alignment of the amino acid sequences of each of the five TMS-identified pollen allergens with each of the three egg antigens revealed a low level of amino acid sequence identity. Further experiments indicated that the schistosome antigen/allergen cross-reactivity was mostly due to similar glycans present in helminths and plants, but not in mammals: so called cross-reactive carbohydrate determinants (CCDs). Previously, CCDs have been implicated in the cross-reactivity between many plants and invertebrates. Furthermore, pollen-induced anti-CCD IgGs have been found in sera of patients undergoing allergen-specific immunotherapy (SIT) and implicated in the treatment of the allergy. Thus, our finding provides not only possible explanations for the allergy-protective effect of helminth/schistosome infections as explained by the hygiene hypothesis, but also a potential starting point for improved SIT.

Theranostic Applications of Antibody-Based Systems in Human Diseases.

Since antibodies are one of the most successful classes of biopharmaceuticals for cancer treatment, it is very important to discuss the functionality and biological activity of endogenous antibodies in the diagnosis and treatment of the diseases. Antibodies act on tumor cells in diverse ways, including engaging in antibody-dependent effector mechanisms, internalizing to deliver toxins, and displaying direct effects on cells. In this review, we outline three kinds of antibody-based therapeutic systems, namely Antibody-based Drug Conjugates (Ab-DCs), Antibody-based Recruiting Small Molecules (Ab-RSMs) and Carbohydrate Epitopes Recruiting Natural Antibodies (CERNA) for human disease treatment and imaging. These antibody-based therapeutic systems are able to strengthen the binding ability of antibodies with disease-relevant cells or viruses, leading to their immuno-mediated therapeutic responses and clearance. Research in these fields shows exciting potential at the junction of organic chemistry and immune-biology. Since the applications of antibodies are of great importance, it is necessary to functionalize antibodies or develop other antibody-based synthetic strategies to modulate the human immune system, and ultimately achieve the treatment of diseases.

A preliminary study of serum IgE against cross-reactive carbohydrate determinants (CCD) in client-owned atopic dogs.

Cross-reactive carbohydrate determinants (CCD) are defined carbohydrate portions of glycoprotein cell surface molecules common to many plant and insect species. Mammalian species recognize CCD as foreign antigens and can mount humoral immune responses against them. Approximately 20-37% of grass and venom allergic people possess circulating IgE against CCD; these antibodies are generally considered clinically irrelevant. Anti-CCD IgE is, however, recognized as a cause of false positive, clinically incongruent serum allergen test results in people; this phenomenon has not been investigated in animals. To determine if anti-CCD IgE could be detected in sera of client-owned atopic dogs and how frequently it is found. Sera from 38 dogs with a clinical diagnosis of atopic dermatitis and prior serological evidence of IgE antibodies, defined as a positive result to at least one mite and pollen (of any type). Sera were analysed for IgE against CCD and environmental allergens with a commercially available multiplex enzyme-labelled allergen-specific IgE assay. Anti-CCD IgE was detected in nine of 38 (24%) of atopic dog sera. As with their human counterparts, all dogs with anti-CCD IgE had strong serological reactivity to grass pollens. Anti-CCD IgE can confound serological allergen testing in people; the same might be true in dogs. Further studies are warranted to investigate the clinical implications of anti-CCD IgE in dogs, including the potential for these antibodies to affect serum allergen-specific IgE assays used for clinical diagnosis, and whether they are relevant to clinical disease.

Antitumor activity and carrier properties of novel hemocyanins coupled to a mimotope of GD2 ganglioside

Conjugation to carrier proteins is a way to improve the immunogenicity of peptides. Such is the case for peptides mimicking carbohydrate tumor-associated antigens in cancer vaccine development. The most used protein for this purpose is the keyhole limpet hemocyanin (KLH) from Megathura crenulata. Its limited bioavailability has prompted interest in finding new candidates; nevertheless, it is not known whether other hemocyanins might be equally efficient as carrier of carbohydrate peptide mimotopes to promotes anti-tumor responses. Here, we evaluated the carrier and antitumor activity of novel hemocyanins with documented immunogenicity obtained from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH), coupled through sulfo-SMCC to P10, a mimetic peptide of GD2, the major ganglioside constituent of neuroectodermal tumors, and incorporating AddaVax as an adjuvant. The humoral immune responses of mice showed that CCH-P10 and FLH-P10 conjugates elicited specific IgM and IgG antibodies against P10 mimotope, similar to those obtained with KLH-P10, which was used as a positive control. The CCH-P10 and FLH-P10 antisera, exhibited cross-reactivity with murine and human melanoma cells, like anti-CCH and anti-FLH sera suggesting a cross-reaction of CCH and FLH glycosylations with carbohydrate epitopes on the tumor cell surfaces, similar to the KLH antisera. When mice were primed with each hemocyanin-P10 and challenged with melanoma cells, better antitumor effects were observed for FLH-P10 than for CCH-P10 and, as for KLH-P10, irrespective of conjugation. These data demonstrate that CCH and FLH are useful carriers of carbohydrate mimotopes; however, the best antitumor activity of FLH preparations, indicate that is a suitable candidate for further cancer vaccines research.

Component-resolved diagnostics to direct in venom immunotherapy: Important steps towards precision medicine.

Stings of Hymenoptera can induce IgE-mediated systemic and even fatal allergic reactions. Venom-specific immunotherapy (VIT) is the only disease-modifying and curative treatment of venom allergy. However, choosing the correct venom for VIT represents a necessary prerequisite for efficient protection against further anaphylactic sting reactions after VIT. In the past, therapeutic decisions based on the measurement of specific IgE (sIgE) levels to whole venom extracts were not always straightforward, especially when the patient was not able to identify the culprit insect. In the last years, the increasing knowledge about the molecular structure and relevance of important venom allergens and their availability as recombinant allergens, devoid of cross-reactive carbohydrate determinants, resulted in the development of an advanced component-resolved diagnostics (CRD) approach in venom allergy. Already to date, CRD has increased the sensitivity of sIgE detection and enabled the discrimination between primary sensitization and cross-reactivity, particularly in patients with sensitization to both honeybee and vespid venom. Hence, CRD in many patients improves the selection of the appropriate immunotherapeutic intervention. Moreover, the detailed knowledge about sensitization profiles on a molecular level might open new options to identify patients who are at increased risk of side-effects or not to respond to immunotherapy. Therefore, increasing potential of CRD becomes evident, to direct therapeutic decisions in a personalized and patient-tailored manner. Reviewed here are the state of the art options, recent developments and future perspectives of CRD of Hymenoptera venom allergy.

Venoms of Neotropical wasps lack cross-reactive carbohydrate determinants enabling reliable protein-based specific IgE determination

Venoms of Neotropical wasps lack cross-reactive carbohydrate determinants enabling reliable protein-based specific IgE determination

Component-resolved diagnosis in selecting patients for yellowjacket venom immunotherapy

BackgroundVenom immunotherapy is effective in preventing systemic allergic reactions (SARs), but the diagnosis of venom allergy is problematic. ObjectiveTo compare the performance of component-resolved diagnosis and conventional tests in patients referred for venom immunotherapy. MethodsWe measured serum-specific immunoglobulin E to yellowjacket and honeybee venoms (Ves v 1 and Ves v 5 and Api m 1), cross-reactive carbohydrate determinants, serum basal tryptase (ImmunoCAP, ThermoFisher Scientific, Uppsala, Sweden), and skin prick test reactions in 84 patients referred to receive venom immunotherapy. History of SAR and its severity were evaluated. ResultsOf the 78 patients with suspected yellowjacket venom (YJV) allergy, a history of SAR was confirmed in 47 (60%) and 31 (40%) had a non-SAR reaction. The most accurate tests to confirm venom allergy after a SAR were serum-specific immunoglobulin E to yellowjacket whole-venom extract spiked with Ves v 5 (area under the curve 0.87, 95% confidence interval 0.77–0.97, P < .001) and Ves v 5 (area under the curve 0.86, 95% confidence interval 0.76–0.96, P < .001). Sensitization to Ves v 1 was infrequent and its area under the curve was low (0.62, 95% confidence interval 0.47–0.76, P = .106). Sensitivity of the YJV skin prick test was 86%, but its specificity was low at 54%. Double sensitization to yellowjacket and honeybee occurred frequently in skin prick tests. Of the patients without a SAR, 26% showed a positive reaction to YJV in any serum test and 46% showed a positive reaction in skin tests. ConclusionSpecific immunoglobulin E to the YJV spiked with Ves v 5 confirmed the allergy after a SAR. A history of SAR should be confirmed before testing, because venom sensitization is frequent in other types of reactions.

Single Site Fluorination of the GM4 Ganglioside Epitope Upregulates Oligodendrocyte Differentiation.

Relapsing multiple sclerosis is synonymous with demyelination, and thus, suppressing and or reversing this process is of paramount clinical significance. While insulating myelin sheath has a large lipid composition (ca. 70-80%), it also has a characteristically large composition of the sialosylgalactosylceramide gangliosde GM4 present. In this study, the effect of the carbohydrate epitope on oligodendrocyte differentiation is determined. While the native epitope had no impact on oligodendroglial cell viability, a single site OH → F substitution is the structural basis of a significant increase in ATP production that is optimal at 50 μg/mL. From a translational perspective, this subtle change increases the amount of MBP+ oligodendrocytes compared to the control studies and may open up novel therapeutic remyelination strategies.

Вплив цилостазолу на перебіг репаративних процесів у пацієнтів з діабетичними трофічними виразками ніг

Мета роботи: аналіз перебігу репаративних процесів у пацієнтів із діабетичними виразками ніг при застосуванні стандартної схеми лікування та при доповненні її цилостазолом.Матеріали і методи. Для дослідження відібрано 60 хворих із діабетичними трофічними виразками і розділено їх на дві групи, пацієнти однієї з яких приймали цилостазол у дозі 100 мг 2 р./добу. Виконано лектиногістохімічні дослідження, для проведення яких здійснювали забір тканин із ран до та після двох і чотирьох тижнів лікування. Набір лектинів включав: Con A – специфічний до DMan, HPA – до NAcDGal, PNA – до βDGal, LABA – до αLFuc, SBA – до NAcDGal, WGA – до NAcDGlc.Результати досліджень та їх обговорення. Активацію регенераційних процесів у групі із застосуванням цилостазолу відстежено вже на 14-ту добу лікування. Це підтверджує поява вуглеводних детермінант у ендотелії новоутворених судин, тоді як у більшості препаратів пацієнтів із контрольної групи таких виражених проявів регенерації не було. Після 28 днів лікування у тканинах із ран пацієнтів ІІ групи спостерігали наростання експресії вуглеводних детермінант у гермінативному шарі епідермісу, локальне посилення рівня експонування специфічних мембранних глікопротеїнів на колагенових волокнах, що ми розцінюємо як свідчення відновлення пучків колагенових волокон.

Construction of Multivalent Homo- and Heterofunctional ABO Blood Group Glycoconjugates Using a Trifunctional Linker Strategy.

The design and synthesis of multivalent ligands displaying complex oligosaccharides is necessary for the development of therapeutics, diagnostics, and research tools. Here, we report an efficient conjugation strategy to prepare complex glycoconjugates with 4 copies of 1 or 2 separate glycan epitopes, providing 4-8 carbohydrate residues on a tetravalent poly(ethylene glycol) scaffold. This strategy provides complex glycoconjugates that approach the size of glycoproteins (15-18 kDa) while remaining well-defined. The synthetic strategy makes use of three orthogonal functional groups, including a reactive N-hydroxysuccinimide (NHS)-ester moiety on the linker to install the first carbohydrate epitope via reaction with an amine. A masked amine functionality on the linker is revealed after the removal of a fluorenylmethyloxycarbonyl (Fmoc)-protecting group, allowing the attachment to the NHS-activated poly(ethylene glycol) (PEG) scaffold. An azide group in the linker was then used to incorporate the second carbohydrate epitope via catalyzed alkyne-azide cycloaddition. Using a known tetravalent PEG scaffold (PDI, 1.025), we prepared homofunctional glycoconjugates that display four copies of lactose and the A-type II or the B-type II human blood group antigens. Using our trifunctional linker, we expanded this strategy to produce heterofunctional conjugates with four copies of two separate glycan epitopes. These heterofunctional conjugates included Neu5Ac, 3'-sialyllactose, or 6'-sialyllactose as a second antigen. Using an alternative strategy, we generated heterofunctional conjugates with three copies of the glycan epitope and one fluorescent group (on average) using a sequential dual-amine coupling strategy. These conjugation strategies should be easily generalized for conjugation of other complex glycans. We demonstrate that the glycan epitopes of heterofunctional conjugates engage and cluster target B-cell receptors and CD22 receptors on B cells, supporting the application of these reagents for investigating cellular response to carbohydrate antigens of the ABO blood group system.

Determination of Glycolipid Ligands of Carbohydrate-Binding Proteins Directly on Thin Layer Chromatograms.

Determination of a glycolipid ligand can quickly reveal the carbohydrate epitope recognized by carbohydrate-binding proteins (CBP)which may include antibodies, lectins, toxins, and virulence factors. Thin layer chromatography (TLC) is a simple, well-known, and heavily used method to separate glycolipids based on their migration through a thin layer of chromatography media, usually silica gel, coated on the surface of a rigid plate. Reacting carbohydrate-binding proteins directly on a TLC plate of separated glycolipids is a powerful technique to quickly determine functional carbohydrate epitopes present in an entire glycolipid extract of a target cell type or tissue.

Використання методів лектинової гістохімії для дослідження впливу дисбалансу тироїдних гормонів на орґанізм

A literature survey concerning application of lectin histochemistry methods for investigating the effect of hypo- and hyperthyroidism on the organ systems. It was established that redistribution of lectin receptor sites on the background of thyroid hormones imbalance showed signs of species and organ specificity. It was demonstrated that hypo- and hyperthyroidism cause a violation of the regulatory influence of the hypothalamic-pituitary axis on the function of peripheral endocrine organs; change the intensity of metabolism and the rate of cell proliferation; and stimulate destructive changes and apoptosis phenomena in target organs. Specifically, in the salivary glands, the thyroid imbalance caused the accumulation of terminal carbohydrate residues DGal and DGalNAc in combination with reduction of DMan, DGlcNAc, NeuNAc and LFuc determinants; on this basis, was formulated the hypothesis of incompleteness of the final glycosilation steps in conjunction with lysosomal glycosidase synthesis disorders, both these processes causing the accumulation of abnormal glycoconjugates in tissues. Storage of secretory products in the glandular cells may be associated with blocking of exocytosis mechanisms. Hypothyroidism caused a decreased reduction rate of DGal and DGalNAc embryospecific carbohydrate determinants (lectin PNA, SBA, HPA receptor sites), which apparently indicate a delay in organ morphogenesis. On the background of maternal hypothyroidism in the testes of offspring rats there was documented an increased proliferation of spermatogenic epithelium, Sertoli and Leydig cells contents, in combination with the increased apoptosis of spermatogenic epithelium. A hypothesis on possible role of HPA lectin receptors as markers of human sperm cells apoptosis on the background of thyroid gland dysfunction was formulated. Hyperthyroidism induced the enhanced collagen synthesis by fibroblasts in the endometrium and ovarian interstitium. New data on the possibilities of lectins application for selective histochemical labeling of distinct cell types and their subpopulations were obtained. In addition, it has been shown that lectins SBA, PNA, SNA can be used for monitoring acrosomal maturation; lectins WGA and HPA - for monitoring the process of gastric glands secretion; WGA, CNFA, SNA, VAA - for monitoring secretion of lingual salivary glands. Since the available literature contains no information concerning the modification of &nbsp;carbohydrate determinants in the adrenal glands of progeny developing under the conditions of maternal hypo- and hyperthyroidism, a conclusion is made on prospective use of lectin histochemistry methods for the experimental investigation of inflences of maternal hypo- and hyperthyroidism on morphogenesis, glycocode and hystophysiology of the progeny adrenal glands. We suppose these data can be of particular interest for basic and applied endocrinology, giving new insight into thyroid-adrenal interactions.&nbsp

Glycoreplica Peptides.

"Glycoreplica peptides" are prepared using a phage display peptide library and monoclonal antibodies that recognize the carbohydrate epitopes of glycoconjugate antigens. The peptides obtained not only mimic the shapes of original glycoconjugate antigens but also have some of their functions. We herein describe how to identify the amino acid alignments of glycoreplica peptides using phage display selection against carbohydrate-binding proteins. Target-specific peptides and proteins may be selected from the large repertory of a peptide/protein library using phage display technology. Glycoreplica peptides have the potential to become alternatives to carbohydrate ligands such as mimotopes for vaccinations and carbohydrate-derived drugs for carbohydrate-related diseases.

Phospholipase A1-based cross-reactivity among venoms of clinically relevant Hymenoptera from Neotropical and temperate regions

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

CILOSTAZOL FOR TREATMENT OF PATIENTS WITH DIABETIC LEG ULCERS

Aim. The research was conducted for the purpose of improving treatment outcomes, reducing time on treatment, declining in disability, improving life quality of patient with diabetic ulcers. Methods. In this research two groups of patients with diabetic leg ulcers were used: I group (51 patients) – treated with a standard procedure (control group) and second group (52 patients) – whether treatment was supplemented cytostazol (100 mg twice a day). For the evaluation the life quality of patients a questionnaire «SF - 36 Health Status Survey» was used during the treatment, the rate of the wound healing was calculated by the formula and 120 histological preparations were analyzed (20 patients of each group). Results. After analysing the questionnaire results, significant differences were observed between groups of patients, manifested by de-emphasized role of physical problems, improved viability and psycho-emotional state with less manifestation of the differences in the control group. The reliable increase in the rate of wound healing was found in patients who took cilostazol. It was observed appearance of carbohydrate determinants in the endothelium of newly formed blood vessels after two weeks of treatment while in the most of the patients’ drugs of the control group there were no such obvious manifestations of regeneration. Discussion. Regarding the results of the research, cilostazol is worth to be examined as perspective means for treatment of patients with diabetic ulcers. In turn, the results of lectinohistochemicals researches can be one of the objective tests of violation of balance of restorative-reparatory and destructively-necrotizing processes for patients with leg ulcers.

Chemical remodeling cell surface glycans for immunotargeting of tumor cells

Recruitment of human endogenous antibodies to target and eliminate tumor cells is a promising therapeutic strategy in the biomedical field. Current antibody-recruiting molecules are typically bi-functional agents that utilize cell-surface receptor binding property for targeting. This approach has intrinsic limitations due to the heterogeneity of tumor cells and the limited number of receptors on the cell surface. Here we report a targeting strategy based on remodeling of cell surface glycans through metabolic engineering and bioorthogonal chemical ligation. In vitro cultured tumor cells and in vivo xenograft tumors were actively remodeled with rhamnose carbohydrate epitopes, which were capable of recruiting endogenous anti-rhamnose antibodies and activating complement-mediated cell cytotoxicity. This study highlights the therapeutic potential for modulating endogenous immune response through cell-surface glycan engineering.

Custom Glycosylation of Cells and Proteins Using Cyclic Carbamate-Derivatized Oligosaccharides

The structural complexity of glycosylation restrains the functional characterization of glycans. We present a versatile carbohydrate ligation technique based on the reaction of cyclic carbamates with primary amines. Cyclic-carbamate-derivatized carbohydrates can be added to primary amine-containing molecules in aqueous solution to yield glycoconjugates. This method enabled the presentation of carbohydrate epitopes on live animal cells, as shown by the acquisition of E-selectin binding sites on mouse MC-38 cells decorated with 3-fucosyllactose or 3-fucosyl-3-sialyllactose. Ligation of 3- and 6-sialyllactose to Escherichia coli demonstrated theimportance of sialic acid linkages in regulating complement factor H binding. Proteins were modified with oligosaccharides to study their role in stimulating cytokine secretion by dendritic cells, thus pointing to interactions between glycoproteins and phosphoinositide 3-kinase signaling in controlling interleukin-12, tumor necrosis factor alpha and interleukin-1β release. Overall, cyclic-carbamate-mediated ligation is useful to study the biology ofcarbohydrate epitopes on proteins and on cell membranes.

Investigation of allergen proteins in five tomato cultivars

Investigation of putative allergens from tomato berries is challenging as differences between human serum IgE specificity and reactivity as well as the non-specific binding of the primary and secondary antibodies often cause difficulties. In this study five tomato cultivars were investigated to evaluate their potential allergenicity in Hungarian tomato sensitive patients. The major allergens proved to be low molecular weight proteins, but several previously described allergens could be identified as well using IgE-Western blotting. IgE binding to cross-reactive carbohydrate determinants (CCDs) was ruled out through the use of CCD inhibitor, but non-specific binding of the secondary antibody remained an issue. IgE binding activity of a purified, immunoblot positive protein (band at 40 kDa), and non-specific binding of the secondary antibody to the same protein, was demonstrated with an Optical Waveguide Lightmode Spectroscopy (OWLS) based immunosensor. LC-ESI-MS/MS analysis showed this protein is an as-yet undescribed vicilin-type putative allergen.

Lateral flow assay for carbohydrate antigen 19-9 in whole blood by using magnetized carbon nanotubes.

The authors describe a rapid, low-cost and sensitive approach for the determination of carbohydrate antigen 19-9 (CA 19-9) in whole blood by using magnetized carbon nanotube (MCNT) and lateral flow strip biosensor (LFSB). MCNTs were synthesized by depositing magnetite (Fe3O4) nanoparticles on multiwalled carbon nanotube (CNT) via co-precipitation of ferric and ferrous ions within a dispersion of shortened multiwalled CNTs. Antibody against CA 19-9 (Ab1) was covalently immobilized on the MCNTs and were used to capture CA 19-9 in blood. After magnetic separation, the formed MCNT-Ab1-CA 19-9 complexes are applied to the LFSB, in which a capture antibody (Ab2) and a secondary antibody (Ab3) are immobilized on the test zone and control zone of the LFSB, respectively. The captured MCNTs on the test zone and control zone are producing characteristic brown bands, and this enables CA 19-9 to be visually detected. Quantitation is accomplished by reading the intensities of the bands with a portable strip reader. Under optimized conditions, the assay has a detection limit as low as 30 U⋅mL-1 of CA19-9 in blood. This is below the cutoff value (37 U mL-1) of CA 19-9. The assay duration for blood samples is 35 min. In our perception, the assay represents a rapid and low-cost tool for rapid determination of CA19-9 in blood that holds promise for clinical applications, particularly in limited resource settings.

Human monoclonal antibody GNX-8 directed to extended type 1 chain: Specific binding to human colorectal cancer.

We observed previously that two carbohydrate epitopes, extended type 1 chain Le(a)-Le(a) and Le(b)-Le(a), are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of "KM mice" with colonic cancer cell line Colo205, and with purified Le(b)-Le(a) glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le(b)-Le(a), bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le(b)-Le(a), whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-alpha-GalNAc, indicating that these epitopes are O-linked glycans.Immunohistological reactivity of GNX-8 at 1 mug/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 mug/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le(b) antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le(b)-Le(a) antigen, provides a novel direction of immunotherapy for human colorectal cancer. (c) 2009 UICC.