Heterologous-coating antigen enhancing the sensitivity of enzyme-linked immunosorbent assay for detection of mebendazole residues.

The heterologous strategy could improve the sensitivity of competitive enzyme-linked immunosorbent assay (ELISA) for detection of chemical contaminants in food samples. In this study, the heterologous coating antigen ELISA was developed to evaluate its sensitivity for mebendazole (MBZ). Results showed that the heterologous ELISA had a linear range of (IC20-IC80) 0.34-10.54 ng/mL, an IC50 value of 1.83 ng/mL, and a limit of detection (LOD) of 0.13 ng/mL, in which the sensitivity of ELISA improved 1.7- and 2-fold (IC50 value dropping from 7.41 and 3.65 ng/mL to 4.27 and 1.83 ng/mL) than that of rabbit IgG- and chicken IgY-based homologous ELISA for MBZ, respectively. The heterologous coating antigen ELISA showed negligible cross reactivity (<0.2%) with its structural analogues, including hydroxy-MBZ, albendazole, oxfendazole, fenbendazole, and flubendazole, except the value of 72.6% for amino-MBZ. The average recoveries of MBZ spiked in pork and chicken muscle samples by the assay ranged from 83.7% to 109.8% and agreed well with those of high-performance liquid chromatography. The results suggested that using heterologous coating antigen could distinctly improve the sensitivity of ELISA for routine screening of MBZ residues in food samples.

Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N- and O-glycomes of porcine, bovine and equine heart tissues.

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by ß-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Using hapten cross-reactivity to screen heterologous competitive antigens for improving the sensitivity of ELISA.

In this study, heterologous competitive antigens (HCAs) suitable for improving the sensitivity of ELISA were successfully screened based on their cross-reactivities (CRs) with 19 quinolone analogues; each containing the norfloxacin amino derivative (NOR0) coupled with bovine serum albumin (BSA) as a coating antigen. HCAs prepared with hapten analogues (CRs of 0.77%-49.92%) remarkably enhanced the sensitivity of the subsequent ELISA. ELISA sensitivity for NOR detection improved 26-fold when moxifloxacin-BSA was used as a heterologous coating antigen relative to when NOR0-BSA was used as a homologous coating antigen. This work, therefore, represents a detailed screening method to select suitable heterologous competitive antigens that improve ELISA sensitivity. Secondly, we present new theoretical tools to estimate hapten structures for use in the method, which may also be applied to improve the sensitivity of other immunoassays.

Using molecular descriptors for assisted screening of heterologous competitive antigens to improve the sensitivity of ELISA for detection of enrofloxacin in raw milk.

The use of the heterologous competitive strategy has become a vital method to improve the sensitivity of ELISA. In this work, we prepared an anti-enrofloxacin (ENR) mAb with ENR-bovine serum albumin (BSA) as immunogen. The molecular descriptors of quinolones were then used to screen heterologous coating antigens for the detection of ENR based on an ensemble learning method to improve the sensitivity of the ELISA. Results indicated that 6 of the 7 selected heterologous competitive antigens could enhance the sensitivity of ELISA. The ELISA sensitivity for the detection of ENR with sarafloxacin-BSA as heterologous coating antigen was improved 10-fold (in PBS) and 6-fold (in milk) compared with that with ENR-BSA as homologous antigen. The strategy can effectively screen suitable heterologous competitive antigens to improve the sensitivity of ELISA, followed by preparation of mAb with no additional modification to the corresponding immunogen.

Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts.

Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.

Detection of Hanganutziu-Deicher antigens in O-glycans from pig heart tissues by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: In the α1,3-galactosyltransferase knockout (α-GalT KO) pig era, identification of the non-Gal epitopes is necessary for successful pig-to-human xenotransplantation. Recently, we successfully detected α-Gal epitopes as well as Hanganutziu-Deicher (H-D) antigens from the N-glycans in the pig heart tissues, which have been considered as promising non-Gal antigens. However, the profiling of O-glycan from pig heart tissues had not been performed owing to the difficulty of O-glycan preparation. METHODS: In this study, we established the simple and sensitive method to profile O-glycans from pig heart aortic valve, aortic wall, pulmonary valve, pulmonary wall, and cardiac muscle tissues. To liberate O-glycans from the pig heart tissues, we used non-reductive ß-elimination reagent and subsequently purified the glycans. After permethylation, the glycans were qualitatively analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The comprehensive O-glycan analysis method was successfully validated using model glycoproteins such as bovine serum fetuin (BSF) and bovine submaxillary gland mucin (BSM) glycoproteins, and their O-glycan profiles were in accordance with the data of previous studies. Next, we applied the method for O-glycan release and characterization to analysis of various pig heart tissues. As a result, total 39, 33, 24, 36, and 25 of O-glycans were detected from aortic valve, aortic wall, pulmonary valve, pulmonary wall, and cardiac muscle, respectively. Furthermore, four in aortic valve, one in aortic wall, one in pulmonary valve, one in pulmonary wall, and one in cardiac muscle were particularly determined as terminally N-glycolylneuraminic acid-linked O-glycans, which is considered to be the H-D antigens. CONCLUSIONS: Here, we initially described the O-glycan structures of various pig heart tissues, and additionally, the existence of H-D antigen type O-glycans was firstly identified. These results will be fundamental information for overcoming the xenoantigenic carbohydrate-related immunological rejection in pig-to-human heart tissue xenotransplantation.

Alpha-Gal detectors in xenotransplantation research: a word of caution.

Xenogeneic tissues are currently employed in clinical practice to create biological substitutes (bioprosthetic heart valves) and in the repair of various damaged tissues (pericardium, gastric-mucosa, nerves, cartilage). Many studies have shown that xenogeneic tissues express superficial epitopes as alpha-Gal, capable of triggering hyperacute and acute vascular rejection phenomena. Currently, no tissue treatment has proven able to completely mask or inactivate such epitopes. In fact, neither glutaraldehyde fixation nor decellularisation procedures ensure a definitive solution because of the persistence of reactive xenoantigen residues. The ability to ascertain alpha-Gal epitope removal from a xenogeneic tissue is closely related to the possibility of its quantitative determination. In the past, detection of the alpha-Gal epitope relied on the use of alpha-Gal reactive isolectin molecules and was limited to isolated cells. Recently, the quantitative evaluation of this antigen has been carried out in whole connective tissue through the use of the monoclonal antibody M86. This article provides an overview of the implications of the alpha-Gal epitope in the current clinical scenario and a definitive comparison between the reliability and specificity of isolectines vs. M86 in alpha-Gal determination.

The expression and distribution of xenogeneic targeted antigens on porcine bone tissue.

OBJECTIVE: The objective of this study was to investigate the distribution of α-galactosyl (α-Gal), major histocompatibility complex (MHC)-I, and MHC-II antigens on adult porcine bone tissue. METHODS: Distribution of α-Gal, MHC-I, and MHC-II antigens on porcine bone tissue were observed using immunohistochemistry. RESULTS: α-Gal, MHC-I xenogeneic antigens were extensively observed on the surface of bone marrow cells, osteocytes, osteoblasts, and Harversian canals; MHC-II antigens were mainly expressed on bone marrow cells. CONCLUSION: α-Gal, MHC-I, and MHC-II are the main xenogeneic antigens that must be deleted to avoid xenogeneic immune reactions against bone xenografts.

Significance of screening tests in diagnosis of infectious mononucleosis.

The investigation included 91 patients in who an acute or previous EBV infection was established by ELISA test. All patients were also subjected to the Paul-Bunnell-Davidsohn test, while 20 patients were tested by the rapid screening test Clearview IM. The diagnosis of acute infective mononucleosis was in 61 patients (67%) confirmed by the Elisa test, and in 12 patients (19.67%) by the Paul-Bunnell-Davidsohn test, while the rapid screening test Clearview IM demonstrated too low a detection of heterophile antibodies. The rapid screening test was not reliable. In 25% cases, the test was invalid, at early infection stages the rapid test failed to diagnose any case of the EBV virus infection. Paul-Bunell-Davidsohn was often negative, especially with young children. Therefore, priority should be given to virology tests based on the detection of specific antibodies to EBV antigen.

Deviation of xenogeneic immune response and bystander suppression in rats fed porcine blood mononuclear cells.

Reducing or deviating xenogeneic immune response prior to xenotransplantation may enhance the efficacy of conventional immunosuppressive therapies in prolonging xenograft survival. The potential to suppress or steer immune responses by oral administration of xenoantigens was evaluated. Based on knowledge of oral tolerance, hypotheses tested were that feeding xenoantigens would inhibit cell-mediated immune response (CMIR) and production of antibodies associated with graft rejection and induce bystander suppression. DA and LEW rats, high and low responders to xenoantigens, respectively, were fed dead porcine blood mononuclear cells (PBMC) and subsequently received live PBMC and hen egg-white lysozyme (HEWL, a third-party antigen) by subcutaneous injection. Delayed-type hypersensitivity (DTH) to PBMC was an indicator of CMIR. Quantification of T(H)1 (IgG(2b)) and T(H)2 (IgG(1))-associated antibodies and their ratio measured magnitude and bias of the antibody-mediated response to PBMC and HEWL. Feeding PBMC reduced IgG(2b) antibody production by 90% (DA) and 71% (LEW) and increased IgG(1) antibodies by 116% in DA but not LEW rats (p

[Antigen measurement and biomechanical characteristics of the inbred-line Banna mini-pig acellular bone matrix].

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.

Assessment of plasma profile of pregnancy-associated glycoprotein (PAG) in sheep with a heterologous (anti-caPAG55+59) RIA and its potential for diagnosing pregnancy.

The purpose of the present investigation was to generate pregnancy associated glycoprotein (PAG)-profiles throughout pregnancy in a heterogenous sample of sheep using a radioimmunoassay with a heterologous antibody (anti-caPAG(55+59), #708) and utilize them for the purpose of pregnancy detection. From 2 weeks after the introduction of males into the breeding herd until 4 weeks after parturition, weekly blood samples were collected from 66 pregnant and 25 non-pregnant ewes of various breeds. Between 3 and 5 weeks after conception, plasma PAG levels increased, remained almost stable until week 17, then continued to increase, culminating in a drastic surge during the last 2 weeks of pregnancy. By 4 weeks of gestation, the plasma PAG level exceeded the level typical for non-pregnant ewes by five standard deviations, permitting a reliable pregnancy diagnosis. Plasma PAG levels were higher in twin-bearing ewes than in ewes carrying a single lamb, differences getting more evident as pregnancy proceeded. Neither breed and parity of the mother nor sex and weight of lambs borne exerted a significant effect. The heterologous assay system utilizing a caprine antibody proved to deliver results that are more consistent and less depending on various variables than those used in other studies. It may be concluded that, at the present state of development, the assay provides a reliable means of diagnosing pregnancy in sheep from the 4th week after they have been bred onward.

Toward xeno-free culture of human embryonic stem cells.

The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.

Properties of novel anti-digoxin antisera in radioimmunoassay using homologous and site heterologous tritium-labeled antigens involving a [3H]-leucine moiety.

The specificities of antisera against digoxin C-3' or C-3'' hemisuccinate-bovine serum albumin (BSA) conjugate were assessed by cross-reactivity studies with digoxin metabolites by radioimmunoassay (RIA) using the homologous and the site heterologous tritium-labeled antigens. One of the tracers used was digoxin 3'-hemisuccinyl-[3H]-leucine; the other was digoxin 3''-hemisuccinyl-[3H]-leucine, which had been prepared from digoxin 3''-hemisuccinate. When the tracer with [3H]-leucine at the C-3' position was used, antisera (I-1, I-3) elicited by digoxin 3'-hemisuccinate-BSA conjugate showed the following cross-reactivity: digoxigenin bisdigitoxoside (0.34%, 76%), digoxigenin monodigitoxoside (0.11%, 65%), digoxigenin (0.02%, 26%) and dihydrodigoxin (9.4%, 1.2%). However, when using the homologous antigen, antiserum (I-1) was highly specific against the digitoxose chain. When the site heterologous antigen, digoxin 3''-hemisuccinyl-[3H]-leucine was combined, this antiserum showed high cross-reactivity to digoxin degradation products. This digoxin RIA using antiserum (I-1) with the homologous antigen measures unmetabolized digoxin. On the other hand, the RIA system using antiserum (I-3) with the homologous antigen had cross-reactivity with the metabolites in accordance with their relative cardio-activities, so this system would be useful in therapeutic drug monitoring of digoxin.

Prevention of the Xenogenic Infection Risk and the Spanish and German Constitutions

Si el problema de una infección xenogénica o de una posterior xenozoonosis es probablemente el obstáculo más importante en el camino de un xenotrasplante terapéuticamente aceptable en su salto a su aplicación clínica, es por lo mismo fundamental evaluar Ias posibilidades que ofrece el Derecho para atajar tal riesgo. Este artículo tratará de ofrecer un marco de Ias soluciones preventivas - es decir, tras la operación y antes de la aparición de pruebas inequívocas de infección xenogénica - que sean posibles desde los ámbitos constitucionales de los sistemas jurídicos alemán y español. Principalmente en cuanto a sus dos propuestas más representativas: la posibilidad de experimentar y mantener a los animales fuente de los injertos en condiciones especial y específicamente higiénicas, así como la eventualidad de obligar al paciente (y/o a sus contactos íntimos o próximos) a observar ciertas medidas profilácticas, en tal fase temprana de riesgo, con el fin de prevenir la expansión de una posible xenoenfermedad a terceros (AU)

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Release of pig leukocytes during pig kidney perfusion and characterization of pig lymphocyte carbohydrate xenoantigens.

The Galalpha1-3Gal (alphaGal) antigen is considered the main xenoantigen in the pig to human species combination but other porcine antigens have to be considered such as the swine lymphocyte antigen (SLA), the blood group A/O and the Hanganutziu-Deicher (H-D) antigens. The H-D antigens are N-glycolyl-neuraminic acid (NeuGc) terminated gangliosides that are widely distributed in mammalian species but absent in humans. Upon exposure to a vascularized pig organ, the human recipient can be immunized by direct interaction with the pig tissue or/and by transfer of tissue/cells from the organ into the recipient. In the present work, we describe the release of cells from porcine kidneys upon perfusion and the expression of glycolipid based alphaGal, blood group A/O and H-D antigens in pig lymphocytes. Pig kidneys were flushed with 20 ml of NaCl or Lidocain containing 5000 U heparin, and thereafter perfused with 3000-ml perfusion solution and the cells released were counted and examined microscopically. Neutral glycolipid and ganglioside fractions were extracted from purified pig lymphocytes. The extracted components were characterized by thin layer chromatography, degradation and mass spectrometry. The expression of alphaGal and H-D epitopes on cells released from pig kidneys and purified pig lymphocytes were studied by immune electron microscopy. A total amount of about 300 x 106 leukocytes, mainly lymphocytes were released in the perfusate from the kidneys, of which about 100 x 106 cells were eluated in the 600 to 2400 ml perfusate fraction. Immunelectron microscopical analysis with Griffonia simplicifolia isolectin B4 showed staining of pig leukocytes and other cells, morphologically similar to endothelial cells, released in the perfusate. The purified porcine lymphocytes contained 930 microg neutral glycolipid (4.2 microg/mg cell protein) of which 95% was glycolipids with one to four sugar residues. Immunostaining of the neutral glycolipid fractions revealed alphaGal terminated compounds migrating in the five and 10 to 12 sugar regions and blood group A compounds in the six and eight sugar regions. Two major gangliosides NeuGc-GM3 and NeuGc-GD3 were found in the pig lymphocytes. In a patient extracorporeally xenoperfused with a pig kidney, an increased staining of both alphaGal terminated structures as well as the H-D reactive gangliosides were found in the post-perfusion serum samples. In summary, leukocytes, mainly lymphocytes are released from pig kidneys during perfusion which may contribute to immunization of human xenograft recipients.

Salmonella vaccines for use in humans: present and future perspectives.

In recent years there has been significant progress in the development of attenuated Salmonella enterica serovar Typhi strains as candidate typhoid fever vaccines. In clinical trials these vaccines have been shown to be well tolerated and immunogenic. For example, the attenuated S. enterica var. Typhi strains CVD 908-htrA (aroC aroD htrA), Ty800 (phoP phoQ) and chi4073 (cya crp cdt) are all promising candidate typhoid vaccines. In addition, clinical trials have demonstrated that S. enterica var. Typhi vaccines expressing heterologous antigens, such as the tetanus toxin fragment C, can induce immunity to the expressed antigens in human volunteers. In many cases, the problems associated with expression of antigens in Salmonella have been successfully addressed and the future of Salmonella vaccine development is very promising.