Biotin-labeled Lectins Research Articles

Differential cellular localization of lectins in the testes of dromedary camel (Camelus dromedarius) during active and inactive breeding seasons

The reproductive activity of the male dromedary camel (Camelus dromedarius) as a seasonal breeder is affected by various seasonal changes that reflect on the reproductive performance. In the current study, we explored a differential cellular localization of lectins in eight dromedary camel testes utilizing lectin histochemistry (LHC). The glycoconjugates’ localizations were detected within the testicular tissue utilizing 13 biotin-labeled lectins (PNA, ConA, LCA, RCA120, GS IB4, WGA, BPL, DBA, ECA, PHA-E4, UEA-1, PTL-II, and SBA) distributed into six sets. The cellular structures revealed diverse lectins distribution that may reflect various glycoproteins’ structures and their compositional modifications during spermatogenesis. Some of the investigated lectins were restricted to acrosomes of spermatids that will help study different stages during the spermatogenic cycle of dromedary camel, particularly PNA, and ECA. The statistical analysis showed a marked positive correlation between the response intensity of various lectins and the breeding season (P < 0.05). We can conclude that lectins have a fundamental role during camel spermatogenesis and are associated with the reproductive activity of dromedary camel.

Comparative Cellular Localization of Sugar Residues in Bull (Bos taurus) and Donkey (Equus asinus) Testes Using Lectin Histochemistry.

Lectins are glycoproteins of a non-immune origin often used as histochemical reagents to study the distribution of glycoconjugates in different types of tissues. In this study, we performed a comparative cellular localization of sugar residues in bull and donkey testes using immunofluorescent lectin histochemistry. We inspected the cellular localization of the glycoconjugates within the testes using 11 biotin-labeled lectins (LCA, ConA, PNA, WGA, DBA, SBA, ECA, BPL, PTL-II, UEA-1, and PHA-E4) classified under six groups. Although the basic testicular structure in both species was similar, the cellular components showed different lectin localization patterns. The statistical analysis revealed no significant association between the intensity of labeling and different variables, including group and type of lectin and type of cell examined, at p < 0.05. However, a stronger response tended to occur in the donkey than in the bull testes (odds ratio: 1.3). These findings may be associated with the different cellular compositions of the glycoproteins and modification changes during spermatogenesis. Moreover, glycoconjugate profiling through lectin histochemistry can characterize some cell-type selective markers that will be helpful in studying bull and donkey spermatogenesis.

P456 Ig glycosylation in ulcerative colitis: it’s time for new biomarkers

Abstract Background Ulcerative colitis (UC) is a chronic relapsing disease, which needs a continue monitoring, especially during biological therapies. An increasing number of patients is treated with anti-Tumor Necrosis factor (TNF) drugs, and current research is focalized to identify biomarkers able to monitor the disease and to predict therapeutic outcome. Methods We enrolled consecutive UC patients treated with anti-TNF, naïve to biologic drugs. Therapeutic outcome was evaluated after 54 weeks of treatment in terms of clinical remission (Partial Mayo Score -PMS- &amp;lt;2) and mucosal healing (Mayo Endoscopic Score &amp;lt;2). On serum samples collected at baseline and after 54 weeks of treatment, a Lectin-based ELISA assay was performed, and specific glycosylation patterns were evaluated by biotin-labelled lectins. We have also collected 21 healthy controls (NHS) samples, age and sex-matched. Results Out of 44 UC patients enrolled, 22 achieved clinical remission and mucosal healing after 54 weeks. At baseline, when Protein A was used as coating, UC patients non-responders showed a reduced reactivity to Jacalin (JAC) in comparison with NHS (p=0,04). After one year of treatment, a decrease in JAC binding was seen only in responders, in comparison with baseline (p=0,04). When JAC binding was tested selecting IgG by means of Fab anti-IgG Fab, UC patients displayed an increased reactivity after anti-TNF therapy (p&amp;lt;0,0001 vs controls). At baseline, PMS inversely correlates with JAC binding when Fab anti-IgG Fab was used in solid phase (r2= 0,2211; p=0,0033). Patients with higher PMS at baseline (PMS ≥5) presented lower binding capacity for JAC in comparison with NHS and with lower PMS patients (p= 0,0135 and p=0,0089 , respectively). Conclusion Ig glycosylation was correlated with clinical and endoscopic activity in patients with UC. JAC protein A-selected Ig showed a possible role in predicting therapeutic effectiveness. If these data would be confirmed, Ig glycosylation could be used as biomarker in UC.

Ig Glycosylation in Ulcerative Colitis: It's Time for New Biomarkers.

Background: Ulcerative colitis (UC) is a chronic relapsing disease, which needs a continue monitoring, especially during biological therapies. An increasing number of patients is treated with anti-Tumor Necrosis factor (TNF) drugs, and current research is focalized to identify biomarkers able to monitor the disease and to predict therapeutic outcome. Methods: We enrolled consecutive UC patients treated with anti-TNF, naïve to biologic drugs. Therapeutic outcome was evaluated after 54 weeks of treatment in terms of clinical remission (Partial Mayo Score -PMS- <2) and mucosal healing (Mayo Endoscopic Score <2). On serum samples collected at baseline and after 54 weeks of treatment, a Lectin-based ELISA assay was performed, and specific glycosylation patterns were evaluated by biotin-labelled lectins. We have also collected 21 healthy controls (NHS) samples, age and sex-matched. Results: Out of 44 UC patients enrolled, 22 achieved clinical remission and mucosal healing after 54 weeks. At baseline, when Protein A was used as coating, UC patients non-responders showed a reduced reactivity to Jacalin (JAC) in comparison with NHS (p = 0.04). After one year of treatment, a decrease in JAC binding was seen only in responders, in comparison with baseline (p = 0.04). When JAC binding was tested selecting IgG by means of Fab anti-IgG Fab, UC patients displayed an increased reactivity after anti-TNF therapy (p < 0,0001 vs controls). At baseline, PMS inversely correlates with JAC binding when Fab anti-IgG Fab was used in solid phase (r 2 = 0,2211; p = 0,0033). Patients with higher PMS at baseline (PMS ≥5) presented lower binding capacity for JAC in comparison with NHS and with lower PMS patients (p = 0,0135 and p = 0,0089, respectively). Conclusion: Ig glycosylation was correlated with clinical and endoscopic activity in patients with UC. JAC protein A-selected Ig showed a possible role in predicting therapeutic effectiveness. If these data would be confirmed, Ig glycosylation could be used as biomarker in UC.

Differences between the Glycosylation Patterns of Haptoglobin Isolated from Skin Scales and Plasma of Psoriatic Patients

Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt), a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.

Photoimmobilization of unmodified carbohydrates on activated surface

Herein, we demonstrate a simple, versatile and efficient method for immobilization of unmodified carbohydrates onto a solid surface. The method employs a photoreactive-cellulose membrane which is prepared by the introduction of a photoreactive group to a cellulose membrane through 1-fluoro-2-nitro-4-azidobenzene (FNAB). Upon exposure to UV light photo-reactive azido group of the activated cellulose membrane transforms itself into highly reactive nitrene which covalently binds to underivatized carbohydrates through an insertion reaction. Maximum immobilization of carbohydrate is achieved at the UV exposure time of 60min and carbohydrate concentration of 100microg/disk. The immobilized carbohydrate is assayed by affinity binding of biotin-labeled lectins. The amount of Con A required for detecting immobilized carbohydrate ranges from 4-10microg/ml. The binding affinities of the lectin to the immobilized carbohydrates are analyzed by calculating their IC(50) values. Overall, the present work demonstrates an efficient immobilization of underivatized carbohydrate onto cellulose surface and has the potential to be applied to other surfaces.

Polyester Nanoparticles Presenting Mannose Residues: Toward the Development of New Vaccine Delivery Systems Combining Biodegradability and Targeting Properties

We report the synthesis of fully biodegradable polymeric nanoparticles presenting mannose residues at their surface and their interaction with lectins. A simple and versatile method was used to reach the surface functionalization of poly(D,L-lactic acid) (PLA) nanoparticles by mannose moieties: It consists in using an amphiphilic mannosylated poly(ethylene oxide)-b-poly(E-caprolactone) (PEO-b-PCL) diblock copolymer as a bioresorbable surface modifier in a simple nanoprecipitation-evaporation procedure. The size and zeta potential of the nanoparticles were found to depend on the molar copolymer/PLA ratio, demonstrating the influence of the copolymer on the formation of the nanoparticles. The bioavailability of the mannose residues as specific recognition sites on the nanoparticle surface could be demonstrated by a modified enzyme-linked lectin assay (ELLA) using biotin-labeled lectins which interact specifically with alpha-D-mannopyrannoside derivatives. Besides specific interaction by lectin-mannose complex formation, nonspecific adsorption of the proteins on the nanoparticle surface was observed. These results were fully supported by isothermal titration calorimetry experiments which suggested that the balance between specific and nonspecific interactions can be controlled by the amount of glycosylated polymer used for the preparation of the nanoparticles. Such nanoparticles are expected to be specifically recognized by mannose receptors, which are highly expressed in cells of the immune system. The targeting properties of these carrier systems combined with their potential adjuvant effects due to their size in the range of 200-300 nm make them attractive candidates as vaccine delivery systems.

Isolation of the receptor for the Amaranthus leucocarpus lectin from human T lymphocytes

Amaranthus leucocarpus lectin (ALL) is specific for GalNAc, and recognizes human T cells. The receptor for ALL was purified from T cells using biotin-labeled lectin and avidin-agarose as affinity matrix. It is a 70-kDa glycoprotein, constituted mainly by serine, glycine, and glutamic acid; its glycosidic portion contains mainly GalNAc; galactose, sialic acid, mannose, and GlcNAc were identified at a lower proportion. By ionic strength chromatography, as well as double dimension electrophoresis, we identified four isoforms of the ALL-receptor. N-terminal amino acid was blocked both in the ALL-receptor and its isoforms, therefore, tryptic peptides of ALL-receptor, analyzed through MALDI-TOF, were compared with the relative values obtained from the NCBInr (ProFound 2004/06/01) database. Our results indicated that the tryptic peptides obtained showed 54% homology with a DnaK-core molecular chaperone, 47% with human KIAA protein, and 44% with heat shock protein 8. The most frequent phenotype of the CD4 or CD8 ALL+ T cells was CD45RA+ CD27+; 26% of ALL+ T cells were CD25+ and 13% were CD69+, indicating that the glycoprotein recognized by ALL is present mainly on naive or quiescent T cells.

Synthesis of cytoadhesive poly(methylmethacrylate) for applications in targeted drug delivery.

The objective of this work was to incorporate cytoadhesive properties into poly(methylmethacrylate) (PMMA) for potential applications in highly localized tissue-specific drug delivery. First, the PMMA was chemically modified by aminolysis to yield amine-terminated surfaces. X-ray photoelectron microscopy confirmed the presence of surface nitrogen entities, and the distribution of amine groups was found to be relatively uniform, as characterized by atomic force microscopy. The availability of these groups for attachment of biologically active molecules was characterized by fluorescence microscopy after immobilization of avidin-FITC. To render the PMMA cytoadhesive, avidin molecules were conjugated to the amine-terminated surfaces with a hydroxy-succinimide-catalyzed carbodiimide reagent and biotin-labeled lectins (tomato, which binds selectively to Caco-2 cells, and peanut, an unrelated lectin) subsequently were attached utilizing avidin-biotin chemistry. Cytoadhesive activity was evaluated by characterizing the interactions between microfabricated PMMA particles and Caco-2 monolayers. After 15-, 30-, 60-, and 120-min incubation periods, the tomato lectin-conjugated PMMA showed a two to sixfold increase in Caco-2 cell recognition over control particles. Furthermore, the stability of the cytoadhesive PMMA interactions appeared to be three to seven times greater than that of the control surfaces. These findings demonstrate that cytoadhesive properties of modified PMMA, making this novel bioactive polymer very promising for applications in targeted drug delivery.

Lectin histochemistry of microvascular endothelium in chick and quail musculature.

The lectin binding pattern of muscular microvessels in chick, quail and chick/quail chimeras was analysed. Paraffin wax sections of muscles from embryonic and adult animals were used. The biotin-labelled lectins were detected by avidin-alkaline phosphatase complex. The following lectins bound to muscular microvessels including arterioles, capillaries and venules of both species: SNA-I (Sambucus nigra agglutinin), MAA (Maackia amurensis agglutinin), AIA (Artocarpus integrifolia agglutinin), VAA-I, VAA-II and VAA-III (Viscum album agglutinin I-III), WGA (wheat germ agglutinin), LEA (Lycopersicon esculentum agglutinin). Endomysium and basement membranes of muscle fibres were also stained to a variable extent and intensity. Only SNA-I stained almost exclusively the endothelium of blood vessels. WFA (Wisteria floribunda agglutinin) bound to the quail endothelium only. MPA (Maclura pomifera agglutinin) marked vessels in adult muscles of chick and quail, but embryonic vessels were stained in quail only. Our results show that lectin histochemistry is a useful tool for visualisation of microvasculature in avian species. In particular, WFA and MPA can be used to determine the origin of endothelia in chick/quail chimeras.

Mast cell distribution and density in the normal uterus — metachromatic staining using lectins

This study investigates the number and the distribution of mast cells in the normal human uterus. Reliability of results was ensured by prompt tissue fixation and the use of biotin-labelled lectins in conjunction with the avidin–biotin peroxidase complex (ABC) method. This design revealed that mast cells are, indeed, normal constituents of the human uterus. They occur in large numbers in the myometrium, but are only scanty in the endometrium where they tend to be confined to the stratum basalis. The mean mast cell counts per high power field (MC/HPF), after staining with Canavalia ensiformis agglutinin (Con A), were 17.9 MC/HPF in the inner half of the myometrium, and 8.3 MC/HPF in the outer half of the myometrium; 2.7 MC/HPF in the basalis, and 0.3 MC/HPF in the functionalis ( P<0.05). There are no apparent differences in the number of mast cells between the normal proliferative and secretory phase endometrium, however, endometrial mast cells are considerably reduced and, apparently, depleted of metachromatic granules during the immediate pre-menstrual phase of the menstrual cycle. It is presumed that this, almost exclusive, presence of mast cells in the basal layer of the endometrial matrix, combined with the discharge of their cytoplasmic granules towards the end of the cycle, may be related with the contracting process preceding menstruation. On the other hand, the relative paucity of mast cells in the functional layer may contribute to the immune tolerance of the gestational endometrium to the implantation of the blastocyst.

Lymph node metastasis-related carbohydrate epitopes of gastric cancer with submucosal invasion.

This study was conducted to examine the lymph node metastasis-related carbohydrate epitopes of cancer cells in primary lesions of gastric cancer with submucosal invasion (sm gastric cancer). A total of 118 formalin-fixed and paraffin-embedded surgical specimens were studied. Carbohydrate epitopes were detected histochemically using 17 kinds of biotin-labeled lectins and three kinds of monoclonal antibodies (mAbs) against Thomsen-Friedenreich antigen (HB-T1), Tn (HB-Tn1), and sialyl Tn antigens (HB-STn1). The node-positive group showed significantly lower reactivities with Ricinus communis I (RCA-I), Lotus tetragonolobus (Lotus), Triticum vulgaris (WGA), and Concanavalia ensiformis (ConA), and higher reactivities with HB-STn1. When sm gastric cancer was classified into differentiated and undifferentiated types, lower reactivities with RCA-I and ConA were observed in the differentiated type cancers, whereas lower reactivities with Lotus, WGA, and higher reactivity with HB-STn1 were observed in the undifferentiated type cancers. Nodal status was able to be predicted with high precision by a combination of these carbohydrates and histological subtypes. Since neuraminidase treatment rarely changed the reactivity of cancer cells with these lectins, we conclude that the loss of certain kinds of carbohydrates that are restricted by histological subtypes is related to lymph node metastasis in sm gastric cancer.

Isolation of the receptor for Amaranthus leucocarpus lectin from murine naive thymocytes

From murine medullary thymocytes we purified the receptor for the Amaranthus leucocarpus lectin (ALL) using a complex with the biotin-labeled lectin and avidin-agarose as the affinity matrix. Most ALL(+)thymocytes (83%) are naive cells with the CD4(+)CD8(-)CD45RB(+)phenotype. The receptor for this lectin is a 70 kDa glycoprotein that contains 20% of sugar by mass. It is constituted mainly by aspartic and glutamic acids, serine, proline, and glycine; its glycosidic portion contains mainly O-glycosidically linked glycans with Gal, GalNAc and NeuAc residues as well as one N-glycosidically linked glycan per molecule. Ionic strength chromatography revealed that the ALL-thymocyte receptor (ALLTr) is made up by three isoforms, which possess similar amino acid composition but show slight differences in their sugar composition. The N-terminal amino acid residues are blocked both in the receptor and its purified isoforms. Analyses of the receptor's peptides, obtained by trypsin digestion with MALDI-TOF (matrix assisted laser desorption ionization-time of flight), were compared with the relative values obtained from the NCBInr (Swiss-Prot 10/01/99) database. Our results indicate that the peptides of ALLTr show low homology (<17%) with the human KIIA protein, the Fas-associated death domain protein, and the transforming growth factor-beta type II receptor. Our results suggest that the ALL thymocyte receptor could be considered a novel phenotypic marker specific for naive T cells.

ULTRASTRUCTURAL AND LECTIN-HISTOCHEMICAL DIFFERENCES BETWEEN THE SCOLEX/STROBILA AND BLADDER TEGUMENTS OF THETAENIA TAENIAEFORMISSTROBILOCERCUS

The strobilocercus stage of the cat tapeworm Taenia taeniaeformis is surrounded by a single syncytial sheet of cytoplasm called the tegument. The outer membrane of the tegument covers both the scolex/strobila (S/S) and the bladder portions of the strobilocercus, but only the S/S region is resistant to intestinal digestion. It has been suggested that the glycocalyx, the surface-exposed glycoconjugates of the outer membrane, may serve to insulate underlying surface membrane components from digestion. In this study, we used lectin binding to test the hypothesis that the glycocalyx of the S/S is different from that of the bladder and that this may serve as the resistance mechanism of the S/S to digestion. Biotin-labeled lectins and an avidin-glucose oxidase detection system were applied to whole strobilocerci and to 1-microm epon-araldite plastic-embedded sections. Lectins bound to either both regions of the strobilocerci, to the S/S regions only, or did not bind at all. The restriction of some glycoconjugates to the glycocalyx of the S/S region only is consistent with our hypothesis.

Changing surface antigen and carbohydrate patterns during the development of Oesophagostomum dentatum.

Living and fixed specimen of Oesophagostomum dentatum were labelled in situ with serum antibodies or a panel of biotin-labelled lectins. Specific binding of antibodies was observed in all parasitic stages--freshly exsheathed 3rd-stage larvae (L3), 3rd- and 4th-stage (L4) larvae cultured in vitro and L3 and L4 and adults isolated from pig intestines. The shedding of the stained layer by motile larvae was inhibited by levamisole-induced paralysis. Larvae cultured in vitro exposed serum-derived proteins on their surface which could be labelled with secondary antibody directed against the respective serum donor species. While freshly exsheathed larvae were recognized by O. dentatum-positive serum only, older larvae and adults cross-reacted with serum from pigs infected with O. quadrispinulatum, a closely related species. Lectin binding varied considerably between stages. While binding was not observed in pre-parasitic stages, Concanavalin A, Soybean Agglutinin, Wheat Germ Agglutinin, Ricinus communis Agglutinin and Peanut Agglutinin bound to developing larvae in varying degrees. Dolichos biflorus Agglutinin only bound to advanced (luminal) larval stages, while adults generally displayed only weak or partial lectin binding (except with Concanavalin A and Wheat Germ Agglutinin). Ulex europaeus Agglutinin only labelled larvae derived from cultures containing 10% pig serum. Cleavage of the carbohydrate residues by sodium periodate treatment resulted in reduction of antibody binding to cultured larvae, but not to freshly exsheathed L3. Concanavalin A, Soybean Agglutinin, and Peanut Agglutinin binding was also reduced by periodate treatment, while binding of Wheat Germ Agglutinin and Ricinus communis Agglutinin was inhibited only in early L3, but not in older stages. The different lectin labelling patterns are related to the different stages of the nematode--infective, invasive, histotropic, and luminal--and may serve as a mode of adaptation for the parasite against the host's immune attack by surface glycoprotein variation, together with antigen shedding (as demonstrated by labelling of motile larvae) and a possible acquisition of host molecules at the parasite's surface. Furthermore, a possible role of this developmental variation in surface carbohydrates in parasite-parasite interactions is discussed.

O-linked glycosylation occurs on basic parotid salivary proline-rich proteins.

Interactions between salivary glycoproteins and many oral bacteria have been shown to depend on O-linked glycans on salivary glycoproteins. Basic proline-rich proteins form the largest group of proteins within human parotid saliva. In the present study human parotid salivary glycoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or two-dimensional electrophoresis, electroblotted onto nitrocellulose and probed with two biotin-labelled lectins from Maclura pomifera (MPA) and Arachis hypogaea (PNA) which are specific for O-linked (galactose beta 1,3 N-Acetylgalactosamine) glycans. Lectin binding was detected with avidin-biotin complex and enhanced chemiluminescence. Two-dimensional electrophoresis in combination with lectin binding indicated that only basic parotid salivary glycoproteins bind the lectin MPA. Following removal of terminal sialic acid residues by sialidase digestion the same glycoproteins were detected by the lectin PNA. Glycosidase digestion with endo-alpha-N-acetylgalactosaminidase (O-glycanase) in conjunction with sialidase eliminated MPA binding. Taken together these results indicate that many basic parotid salivary glycoproteins contain O-glycans, all of which are sialylated.

Lectin ELISA for the c-erb-B2 Tumor Marker Protein p185 in Patients with Breast Cancer and Controls

The search for diagnostic and prognostic factors in breast cancer has included several oncogenes, particularly c- erb -B2, which encodes a 185-kDa transmembrane glycoprotein receptor, denoted p185, with tyrosine kinase activity (1)(2). p185 is frequently investigated by immunohistochemical techniques. The extracellular domain is shed from tumors in vitro (3) and is detectable in serum by immunoassay (4); however, the considerable overlap between patient and control values renders its estimation of little value in individuals. However, many glycoproteins exhibit carbohydrate changes in cancer (5). Thus a test combining the concentration with changes in glycosylation might be more specific than either alone. We therefore investigated potential glycosylation changes in p185 in the serum of breast cancer patients and controls collected sequentially from a patient series immediately before surgery, using the convenient Lectin ELISA format (6). The protein was specifically captured immunochemically and quantified with one of two biotin-labeled lectins binding particular sugars on the protein: wheat germ agglutinin (WGA), with affinity for sialic acid and N -acetylglucosamine, and Concanavalin A (Con A), which binds preferentially to mannose. In preliminary experiments, we investigated these assays in lysates of SKBR3 breast cancer cells, which express p185, and normal lymphocytes. Monoclonal antibody raised against a synthetic peptide sequence of p185 extracellular domain (OM-11-954) was obtained from Cambridge Research Biochemicals. In immunoblotting studies, the antibody detects an ∼170-kDa protein in SKBR3 cell membranes blocked by preincubation with synthetic peptide. In immunocytochemical studies using frozen and paraffin-embedded material, staining was blocked by preincubation with the synthetic peptide. The antibody does not recognize the epidermal growth factor receptor. Human breast carcinoma cells, SKBR3, were a gift from Professor B.R.Westley, Pathology Department, University of Newcastle, Newcastle upon Tyne, United Kingdom. The tetramethylbenzidine peroxidase substrate (K-blue) was from Bionostics; all other reagents were from Sigma. Absorbances were measured …

An antibody-lectin sandwich assay for quantifying protein glycoforms.

We describe an antibody-lectin sandwich assay for quantitation of glycoforms of proteins. The assay uses deglycosylated IgG antibody immobilized on a microtiter plate to capture the protein of interest from the sample. The particular glycoform is then identified by reaction with biotin-labeled lectin, which is measured using streptavidin/alkaline phosphatase. The assay can be adapted to quantitate any protein's glycoforms by simply substituting the antibody and lectin with specific alternatives.

Compositional changes in glycoconjugates recognized histochemically with lectins in Purkinje cells in suckling and adult rats.

Cerebella from suckling and adult rats were examined histochemically with 19 different biotin-labeled lectins. Purkinje cells from postnatal rats had a marked ability to combine with many lectins, but minimal ability was found in adult rats except for Con-A, LEL, and MAL lectins. The cell body of Purkinje cells on postnatal day 7 was strongly labeled with 6 lectins (Con-A, LTL, MAL, SJA, UEA-I, and VVA). Only moderate staining was observed with these lectins on postnatal day 5. The dendritic tree of the cells showed a moderate labeling ability with LTL and UEA-I on postnatal days 15 and 20. The dendritic tree was strongly labeled with MAL on postnatal days 10, 15, 20 and adult. Positive reactions were observed in the cells when cerebellar sections from rats on postnatal day 7 were incubated with 3 other lectins (AAL, LEL, and SBA). The cells on postnatal day 7 were rarely labeled with BSL-II, DBA, DSL, LCA, PNA, PSA, RCA120, SSA, STL, and WGA. Purkinje cells on postnatal day 7 may be rich in N-linked oligosaccharides, with terminal sugar structures that resemble blood-group-related antigens (type H) and/or tumor-related antigens. These glycoconjugates may be present at low levels in the Purkinje cells of adult rats. Dendrites of Purkinje cells of adult rats were strongly labeled by Con-A, LEL, and MAL. The dendrites of Purkinje cells may be rich in highly branched oligosaccharides.

Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG

IgA1 proteins from sera of patients with IgA nephropathy (IgAN) are galactosylated to a lesser degree than those from healthy controls. The increased reactivity of intact or de-sialylated serum IgA1 with N-acetylgalactosamine (GalNAc)-specific lectins, Helix aspersa (HAA) and Caragana arborescens (CAA) and de-sialylated IgA1 with Helix pomatia (HPA) and Bauhinia purpurea (BPA) indicated that the Gal deficiency is in glycans located in the hinge region of IgA1 molecules. De-sialylated IgA from sera of 81 IgAN patients bound biotin-labeled lectin HAA more effectively than did de-sialylated IgA from 56 healthy controls (P < 0.0001). Similar results were observed for 67 IgAN patients and 52 controls with second lectin, CAA (P < 0.001). The binding patterns for 9 patients with mesangial proliferative glomerulonephritis of non-IgA origin were similar to those for controls. Incompletely galactosylated IgA1 capable of binding GalNAc-specific lectins was detected in complexes with IgG as demonstrated by ELISA, size-exclusion chromatography and sucrose gradient ultracentrifugation. The formation of IgA1-IgG complexes may affect the serum level of IgA1 by reducing the rate of its elimination and catabolic degradation by the liver.