Search

Background and Objectives: The detection of treponemal antibodies, which are used to make a diagnosis of syphilis, is important both for diagnostic purposes and as a mandatory blood donor test in most countries. We evaluated the feasibility of using Kode Technology to make syphilis peptide red cell kodecytes for use in column agglutination serologic platforms. Materials and Methods: Candidate Kode Technology function‐spacer‐lipid (FSL) constructs were made for the Treponema pallidum lipoprotein (TmpA) of T. pallidum, using the peptide and FSL selection algorithms, and then used to make kodecytes. Developmental kodecytes were evaluated against a large range of syphilis antibody reactive and non‐reactive samples in column agglutination platforms and compared against established methodologies. Overall, 150 reactive and 2072 non‐reactive Syphicheck assay (a modified T. pallidum particle agglutination) blood donor samples were used to evaluate the agreement rate of the developed kodecyte assay. Results: From three FSL‐peptide candidate constructs, one was found to be the most suitable for diagnostics. Of 150 Syphicheck assay reactive samples, 146 were TmpA–kodecyte reactive (97.3% agreement), compared with 58.0% with the rapid plasmin reagin (RPR) assay for the same samples. Against the 2072 expected syphilis non‐reactive samples the agreement rate for TmpA–kodecytes was 98.8%. Conclusion: TmpA–kodecytes are viable for use as cost‐effective serologic reagent red cells for the detection of treponemal antibodies to diagnose syphilis with a high level of specificity in blood centres. This kodecyte methodology also potentially allows for introduction of the reverse‐algorithm testing into low‐volume laboratories, by utilizing existing transfusion laboratory infrastructure. [ABSTRACT FROM AUTHOR]

Carbohydrate‐specific antibodies are significant mediators of xenograft rejection. This study analyzed the carbohydrate specificity of antibodies in baboons before and after xenotransplantation of organs or injection of porcine red blood cells from hDAF transgenic pigs, using a glycan array with structurally defined glycans. Antibodies against hyaluronic acid disaccharide (HA2) showed the highest reactivity at baseline and rose after xenogeneic exposure. We also investigated in the serum of baboons that underwent xenotransplantation with either hDAF or hDAF/hMCP transgenic pig organs and Lewis rats after hamster‐skin xenotransplantation the specificity of anti‐HA antibodies on a glycan microarray representing HA oligosaccharides containing from two to 40 saccharides. Notably, the HA oligosaccharides ranging from 32 to 40 saccharides exhibited the highest antibody binding intensities at baseline in baboon and rat sera. After xenotransplantation, antibodies against HA38 and HA40 in baboons, and HA32, HA34, and HA36 in rats showed the highest titer increases. The changes of anti‐HA IgM and IgG antibodies in rats after skin xenotransplantation was also confirmed by an ELISA specific for HA2, HA24, and HA85 antibodies. Thus, xenotransplantation is associated with increased antibodies against HA‐oligosaccharides, which may represent a new target for intervention. [ABSTRACT FROM AUTHOR]

A possible mechanism of the immune tolerance in pregnancy is production of blocking antibodies which reside in placenta and protect foetal allogeneic cells from the mother's immune system. Their epitope specificity, as well as the nature of the biomolecules masked by them, is unknown. For better understanding of this phenomenon, we attempted to characterize the specificity of antibodies isolated from placentas of women with healthy pregnancy and pre‐eclampsia (PE). It was found that: (1) the repertoire of placental antibodies is significantly less variable and qualitatively different from the peripheral blood; (2) with PE, the repertoire of placental antibodies is narrower than in healthy pregnancy; (3) some antibodies are found almost exclusively in the placenta, and some – only in the placenta of healthy women. [ABSTRACT FROM AUTHOR]

Background: The Coronavirus disease 2019 (COVID-19) pandemic is having a major global impact, and the resultant response in the development of new diagnostics is unprecedented. The detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a role in managing the pandemic. We evaluated the feasibility of using SARS-CoV-2 peptide Kode Technology-modified red cells (C19-kodecytes) to develop an assay compatible with existing routine serologic platforms.Study Design and Methods: A panel of eight unique red cells modified using Kode Technology function-spacer-lipid constructs and bearing short SARS-CoV-2 peptides was developed (C19-kodecyte assay). Kodecytes were tested against undiluted expected antibody-negative and -positive plasma samples in manual tube and three column agglutination technology (CAT) platforms. Parallel analysis with the same peptides in solid phase by enzyme immunoassays was performed. Evaluation samples included >120 expected negative blood donor samples and >140 COVID-19 convalescent plasma samples, with independent serologic analysis from two centers.Results: Specificity (negative reaction rate against expected negative samples) in three different CAT platforms against novel C19-kodecytes was >91%, which correlated with published literature. Sensitivity (positive reaction rate against expected positive convalescent, PCR-confirmed samples) ranged from 82% to 97% compared to 77% with the Abbott Architect SARS-CoV-2 IgG assay. Manual tube serology was less sensitive than CAT. Enzyme immunoassay results with some Kode Technology constructs also had high sensitivity.Conclusions: C19-kodecytes are viable for use as serologic reagent red cells for the detection of SARS-CoV-2 antibody with routine blood antibody screening equipment. [ABSTRACT FROM AUTHOR]

Colorectal cancer ( CRC) is the third most common malignancy in industrialized countries. Despite the advances in diagnostics and development of new drugs, the 5-year survival remains only 60-65%. Our approach to early diagnostics of CRC is based on the determination of serological signatures with an array of hemispherical hydrogel cells containing immobilized proteins and oligosaccharides (glycochip). The compounds immobilized on the glycochip include tumor-associated glycans (SiaTn, Tn, TF, LeC, LeY, SiaLeA, and Man β1-4Glc NAc β) and antibodies against human immunoglobulins IgG, IgA, and IgM. The glycochip detects antibodies against tumor-associated glycans in patients' sera. The simultaneous measurement of the levels of immunoglobulins enhances the diagnostic impact of the signatures. In this work, we found previously unreported increase in antibodies against oligosaccharide Man β1-4Glc NAc β in patients with CRC. In parallel with these experiments, we determined the levels of oncomarkers carcinoembryonic antigen ( CEA), cancer antigen ( CA) 19-9, CA 125, CA 15-3, human chorionic gonadotropin ( HCG), and alpha-fetoprotein ( AFP) using another gel-based biochip with immobilized antibodies (oncochip) developed earlier in our laboratory. In total, 69 samples from healthy donors, 33 from patients with colorectal carcinoma, and 27 from patients with inflammatory bowel diseases were studied. The use of combined signatures of antiglycan antibodies and oncomarkers provides much better predictive value than the conventional measurement of oncomarkers CEA and CA 19-9. Positive predictive value of CRC diagnoses using together glycochip and oncochip reached 95% with the sensitivity and specificity 88% and 98%, respectively. Thus, the combination of antibody profiling with detection of conventional oncomarkers proved to be a promising tool in diagnostics of CRC. [ABSTRACT FROM AUTHOR]

Highlights: [•] Background: The human chitotriosidase contributes to defense against chitin-containing pathogens. [•] Results: Chitotriosidase hydrolyzes model substrates for glycolipids and glycoproteins motifs. [•] Conclusion: The GalNAcβ1-4GlcNAc (LacdiNAc) motif was a hitherto unrecognized substrate. [•] Significance: The chitotriosidase may target LacdiNAc motifs found in mammalian and invertebrate cells. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer has the highest mortality rate among gynecological cancers. Altered glycosylation is associated with oncogenic transformation producing tumor-associated carbohydrate antigens. We investigated the potential of natural occurring antiglycan antibodies in the diagnosis of ovarian cancer by using printed glycan array. Antiglycan antibodies bound to 203 chemically synthesized printed glycans were detected via biotin-streptavidin fluorescence system in serum of women with normal operative findings (healthy controls; n = 24) and nonmucinous borderline or ovarian cancer of various FIGO stages (n = 33). Data were validated measuring blood group associated di-, tri and tetrasaccharide antigens on known ABO blood groups. Antiglycan antibodies demonstrated high reproducibility (rc > 0.9). Cluster analysis identified repetitive patterns of specific core carbohydrate structures: 11 N-linked glycans, 3 O-linked glycans and 2 glycosphingolipids. Biomarker detection revealed 24 glycans including P1 (Galα1-4Galβ1-4GlcNAcβ; p < 0.001) significantly discriminating between (low-) malignant tumors and healthy controls. Comparable sensitivity and specificity with tumor marker CA125 was achieved by a panel of multivariate selected and linear combined antiglycan antibody signals (79.2 and 84.8%, respectively). Our findings demonstrate the potential of glycan arrays in the development of a new generation of biomarkers for ovarian cancer. [ABSTRACT FROM AUTHOR]

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/cm2) 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 m2 surface area for an average body blood volume of 5 L). © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010. [ABSTRACT FROM AUTHOR]

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‐PAA1000‐biotin) enhanced antibody binding capacity by two to three fold compared with the low molecular weight conjugate (Atri‐PAA30‐biotin), whereas varying the carbohydrate content in Atri‐PAA1000‐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. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [ABSTRACT FROM AUTHOR]

Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. Methods: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. Results: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 ± 12 min (PBS control) to 74 ± 23 and 77 ± 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. Conclusions: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ‘‘endothelial protective’’ substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection. [ABSTRACT FROM AUTHOR]

Abstract: Background: Activation of endothelial cells (EC) in xenotransplantation is mostly induced through binding of antibodies (Ab) and activation of the complement system. Activated EC lose their heparan sulfate proteoglycan (HSPG) layer and exhibit a procoagulant and pro-inflammatory cell surface. We have recently shown that the semi-synthetic proteoglycan analog dextran sulfate (DXS, MW 5000) blocks activation of the complement cascade and acts as an EC-protectant both in vitro and in vivo. However, DXS is a strong anticoagulant and systemic use of this substance in a clinical setting might therefore be compromised. It was the aim of this study to investigate a novel, fully synthetic EC-protectant with reduced inhibition of the coagulation system. Method: By screening with standard complement (CH50) and coagulation assays (activated partial thromboplastin time, aPTT), a conjugate of tyrosine sulfate to a polymer-backbone (sTyr-PAA) was identified as a candidate EC-protectant. The pathway-specificity of complement inhibition by sTyr-PAA was tested in hemolytic assays. To further characterize the substance, the effects of sTyr-PAA and DXS on complement deposition on pig cells were compared by flow cytometry and cytotoxicity assays. Using fluorescein-labeled sTyr-PAA (sTyr-PAA-Fluo), the binding of sTyr-PAA to cell surfaces was also investigated. Results: Of all tested compounds, sTyr-PAA was the most effective substance in inhibiting all three pathways of complement activation. Its capacity to inhibit the coagulation cascade was significantly reduced as compared with DXS. sTyr-PAA also dose-dependently inhibited deposition of human complement on pig cells and this inhibition correlated with the binding of sTyr-PAA to the cells. Moreover, we were able to demonstrate that sTyr-PAA binds preferentially and dose-dependently to damaged EC. Conclusions: We could show that sTyr-PAA acts as an EC-protectant by binding to the cells and protecting them from complement-mediated damage. It has less effect on the coagulation system than DXS and may therefore have potential for in vivo application. [ABSTRACT FROM AUTHOR]

Abstract: Extracorporeal immunoabsorption for removal of anti-Galα1–3Gal (anti-Gal) antibodies in putative pig-to-human xenotransplantation is considered a major prophylactic measure to avoid hyperacute and acute vascular rejections. However, the efficacy of the procedure does depend on choosing the appropriate oligosaccharide epitopes for the binding of human anti-Gal. The synthetic oligosaccharides Galα1–3Gal (B-disaccharide, Bdi) and Galα1–3Galβ1–4Glc (‘type 6’ trisaccharide, Tri6), covalently coupled to Sepharose via polyacrylamide (Sorbents Bdi and –Tri6, respectively), as well as a mixture thereof (Sorbent Mix), were examined for their efficacy to absorb anti-Gal from 20 human serum samples. Sorbent Bdi removed 81% of anti-Gal IgM and 85% of –IgG when assessed on Bdi by ELISA, but only 49% of IgG and 75% of IgM when assessed on Tri6. Sorbent Tri6 and –Mix showed significantly better absorption capacities in so far as Sorbent Tri6 removed 65% of anti-Gal IgM and 80% of –IgG as assessed on Bdi and 85% of IgM/87% of IgG when tested on Tri6, and Sorbent Mix absorbed > 90% anti-Gal of both isotypes of either specificity. Direct hemagglutination of rabbit erythrocytes (ER) was reduced by 75% (median value, range 0–94%) and the median cytotoxicity to PK15 target cells by > 94% after absorption on Sorbent Mix. Neither the decrease in ER agglutination titers nor the reduction of PK15 cytotoxicity revealed significant differences between the three immunoabsorbents tested. The large variation ranges of absorption efficacies within the 20 tested sera suggest that “tailor-made” immunoabsorption treatments may be needed for putative xenotransplant recipients. [ABSTRACT FROM AUTHOR]

Abstract: Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of pre-formed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galα1-3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galα1-3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and functional properties of IgG and IgM XAb. Both classes were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galα1-3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and for IgM, pointing to Galα1-3Gal as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement- dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity- purified IgG anti-Galα1-3Gal antibodies were 22 times less potent than IgM in induction of cytotoxicity. These results indicate a quantitative, but not a qualitative, difference between IgM and IgG anti-pig antibodies concerning their complement-activating properties. Therefore, both classes of XAb are of importance in the pathogenesis of hyperacute rejection, and the relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum. [ABSTRACT FROM AUTHOR]

To study conformational parameters of ligands before and after complex formation with the galactoside-binding agglutinin of Viscum album L. (VAA) in solution, combined computer-assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two-dimensional rotating-frame nuclear Overhauser effect (ROE) and two-dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galβ1-3GlcNAcβ1-R and Galβ1-3GalNAcβ1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Galβ1-2Galβ1-R′, were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between KY combinations of (40+/60+) and (40+/-60+). Calculations of the de-N-acetylated sugars revealed that presence of this group did not markedly influence the distribution of low-energy conformers in the KY, E plot. Occupation of side minima at KY (180+/0+) or (0+/180+) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free-state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free-state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galβ1-3GlcNAcβ1-R and Galβ1-2Galβ1-R′. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One- and two-dimensional transferred nuclear Overhauser enhancement (TRNOE)... [ABSTRACT FROM AUTHOR]