Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor.

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFbeta isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFbeta1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

High-level expression of Camelid nanobodies in Nicotiana benthamiana.

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative 'universal' nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.

The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.

Cytokines, chemokines and soluble adhesion molecules interact in a complex network within the immune system. Fingerprinting of these proteins may allow the use of these proteins as biomarkers for identification of disease, disease subtyping and monitoring therapeutic interventions. We developed a multiplex immunoassay (MIA) for the detection of 30 proteins in a variety of human body fluids such as plasma and synovial fluid (SF). The measurement of these proteins is hampered by the presence of human (auto-) antibodies, which can cause non-specific binding. We have validated a novel approach for the removal of interfering immunoglobulins using pre-absorption with protein-L. Interfering (auto-) antibodies, such as rheumatoid factor (RF), were removed using three methods; polyethylene glycol (PEG) precipitation, pre-absorption with human gamma-globulin or pre-absorption with protein-L. A significant decrease of RF was observed after a 2 h incubation with protein-L. RF IgM levels were reduced by 89% whereas total IgM, IgG and IgA levels were reduced by 60%. Residual immunoglobulins were blocked with rodent serum and did not interfere with the multiplex immunoassay. Comparing the MIA with a conventional enzyme-linked immunosorbent assay (ELISA) using a panel of spiked plasma samples resulted in correlation coefficients for all mediators between R2 = 0.88 and R2 = 0.99. Intra-assay variance was less than 10% whereas inter-assay variance ranged between 6% and 16%. Pathological samples with heterophilic antibodies hamper immunoassays such as ELISA and MIA. We show that pre-absorption with protein-L is a powerful tool for removal of interfering immunoglobulins from human bodily fluids to be used in immunoassays for studying changes in protein patterns.

Selection of phage-displayed anti-guinea pig C5 or C5a antibodies and their application in xenotransplantation.

Xenogeneic liver transplantation in the discordant guinea pig (gp) to rat model results in hyperacute rejection within a few minutes, which is due to activation of the complement system. Currently no antibodies against gp complement factors are available, which allow activation of the gp complement system in serum or complement deposition in tissue to be detected. To close this gap, we started developing single chain Fvs (scFvs) against gpC5 and gpC5a. We generated a combinatorial library of scFv antibodies comprising the variable heavy and light chain repertoire from mice immunized with gpC5. Out of this library we selected several antibodies against gpC5 and C5a after four and six rounds of biopanning, respectively. Selected gpC5-specific scFvs were purified by metal affinity chromatography followed by size exclusion chromatography or by affinity chromatography using Protein L. Purified scFvs were able to inhibit gp complement system in a hemolytic assay and to detect gpC5 deposition in tissue. A surface plasmon resonance based assay on BIAcore was established, with which the C5 concentration in gp serum was determined to 240 microg/ml. As at least 0.04% of the normal gpC5 concentration can be detected, the test provides a powerful tool to investigate the development and the consequence of a hybrid complement system after liver xenotransplantation from gp to rat.

A specific enzyme-linked immunosorbent assay (ELISA) procedure for detection of heterophile Hanganutziu and Deicher (HD) antibodies.

A specific, relatively sensitive, quantitative and standardized enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of heterophile Hanganutziu and Deicher (HD) antibodies which are occasionally elevated in pathologic human sera. The HD antigen-active molecule used for the assay was a ganglioside (N-glycolylneuraminyllactosylceramide, abbreviated as NeuGc-LacCer) previously purified from horse erythrocyte membranes. The test used antigen-coated plastic microtiter plates and anti-human immunoglobulin G (IgG, Fab fragment) conjugated with alkaline phosphatase. Fifty-four normal human sera gave ELISA values ranging from -2 to 2%. Random sera from hospitalized patients were first screened by the horse erythrocyte hemagglutination (HA) test, whereby 5.7% (76 cases) gave abnormal HA titers of 128-4096 compared to titers in normal sera equal to or less than 64. Ninety-seven % of the patients' sera gave abnormal ELISA values (3-200%). They were classified into 3 groups: cancer (42 cases), infection (10 cases), and others (24 cases). The potential value of this ELISA method is discussed.

In vivo depletion of xenoreactive natural antibodies with an anti-mu monoclonal antibody.

Hyperacute rejection of vascularized discordant xenografts, such as pig-to-primate kidney or heart xenotransplants, is thought to be mediated by xenoreactive natural antibodies (XNA) of the IgM isotype and the activation of the classic pathway of complement. Using the guinea pig-to-rat discordant xenograft model, we have developed a potential therapeutic protocol leading to long-term depletion of circulating IgM in adult animals. This protocol consists of the injection into adult LOU/C rats of an antirat IgM MAb (MARM-7) after splenectomy, plasma exchange, and the administration of an anti-B cell immunosuppressant, mycophenylate mofetil (RS61443). Splectomized plasma exchanged adult rats receiving RS61443 showed strongly decreased IgG and IgM serum concentrations for a relatively short period during which these isotypes remained nevertheless detectable by a sensitive ELISA technique. In contrast to IgM, IgG in serum returned, shortly after the end of this treatment, to normal concentrations. Splenectomy alone was able to significantly decrease, for a long period (more than 70 days), IgM but not IgG serum concentrations in these rats. During this treatment, IgM XNA concentration mirrored total IgM. The injection of MARM-7 MAb to adult LOU/C rats was able to deplete circulating IgM and IgM XNA for a period of several weeks during which IgM was undetectable by a sensitive ELISA technique. Depletion time was dose-dependent--the higher the dose of injected MARM-7, the longer the period for which IgM and IgM XNA remained undetectable. Moreover depletion of circulating IgM was correlated with the detection in the serum of these rats of noncomplexed, free MARM-7. Finally, MARM-7 administration was significantly more efficacious in rats that had decreased levels of circulating IgM after splenectomy, plasma exchange, and administration of RS61443. These experiments suggest that the anti-mu approach may allow depletion of IgM XNA for a sufficiently long period to test the hypothesis of "accommodation" in other xenograft models such as the pig-to-primate xenograft or even in ABO-incompatible allografts.

Application of immunoapheresis for delaying hyperacute rejection during isolated xenogeneic pig liver perfusion.

BACKGROUND: Extracorporeal liver perfusion in hepatic coma, used to eliminate toxic metabolites causing hepatic encephalopathy, is limited by the antibody (Ab) and complement-mediated hyperacute rejection of discordant xenografts. Thus, the efficacy of highly selective immunoadsorption columns to deplete xenoreactive human anti-porcine antibodies before ex vivo liver perfusion was examined in this study. METHODS: Eighteen domestic pigs were hepatectomized according to standard techniques. The livers were ex vivo perfused for 4 hr. The perfusion protocol closely followed the physiological conditions of a human liver. Parameters of liver function and damage were analyzed. Three groups were formed differing in the treatment of the perfusate. As basic control, livers were perfused with heparinized human blood (group 1; n=6). Immunoapheresis was applied in group 3 (n=6). Immunoapheresis was performed using Ig Therasorb columns consisting of sheep-anti-human IgG Abs covalently coupled to Sepharose CL-4B. Additionally, the effect of pure Sepharose CL-4B without immobilized Ab was tested in group 2 (n=6). RESULTS: The use of Ig Therasorb 100 columns in group 3 resulted in a reduction of IgG, IgM, and IgA in the order of 95.0%, 72.3%, and 81.5%, respectively. In group 2, IgG, IgM, and IgA were lowered by 30% to 39%. Determination of liver-specific enzymes and tolerance tests revealed a significant reduction of cellular damage and functional restrictions in group 3 compared with the control groups. CONCLUSIONS: Immunoapheresis conducted according to this protocol appears to be an effective approach for delaying antibody-mediated hyperacute xenogeneic rejection.

The role of anti-Galalpha1-3Gal antibodies in acute vascular rejection and accommodation of xenografts.

BACKGROUND: A major impediment to the transplanting of porcine organs into humans is the susceptibility of porcine organs to acute vascular rejection, which can destroy a vascularized xenograft over a period of hours to days. Acute vascular rejection of porcine-to-primate xenografts is thought to be triggered by binding of xenoreactive antibodies to the graft. We tested whether antibodies, binding to Galalpha1-3Gal epitopes in porcine tissue, initiate this phenomenon. METHODS AND RESULTS: Specific depletion of anti-Galalpha1-3Gal antibodies from the blood of baboons, using extracorporeal perfusion of separated plasma through columns of Sepharose beads covalently linked to the antigenic trisaccharide, Galalpha1-3Galbeta1-4GlcAc, averted the development of acute vascular rejection in porcine organs transgenic for human decay-accelerating factor and CD59. More importantly, after immunodepletion was stopped and Gala1-3Gal antibodies were allowed to return, these same organs continued to function and remained pathologically normal and thus seemed to achieve a state of accommodation. CONCLUSION: These results demonstrate that anti-Galalpha1-3Gal antibodies cause acute vascular rejection and suggest that depletion of these antibodies leads to accommodation of the donor cardiac xenograft and could supply an important model for additional study.

Heterophile antibodies to rabbit erythrocytes in human sera and identification of the antigen as a glycolipid.

Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer.

Antibody depletion prolongs xenograft survival.

BACKGROUND: The lack of human organ donors has prompted a renewed interest in xenotransplantation. Xenoantibody is believed to be an initiator of a complex cascade of events ultimately ending in rapid xenograft destruction. METHODS: Cell-free plasma was obtained via plasmapheresis of the recipient canine. This plasma was then perfused in an ex vivo fashion through either the donor (pig) spleen or liver, allowing for specific antidonor antibody deposition in the "screening" organ, and is then returned to the animal. A porcine kidney is then transplanted to the dog, and the outcome is observed. RESULTS: We have used specific antibody depletion to prolong xenotransplant survival. In the untreated pig-to-dog combination, the transplanted pig kidney is destroyed by the dog in 13 minutes (mean). Adsorption using either the donor spleen or liver resulted in an increase of 3.4 hours and 7.8 hours of graft survival, respectively. The histologic picture of rejected kidneys after adsorption shows a modified form of rejection. CONCLUSIONS: In this pig-to-dog xenograft combination, xenoantibody adsorption allows for prolonged graft survival as compared with control animals. Subsequent xenograft destruction occurs but in a modified manner.

Removal of primate xenoreactive natural antibodies by extracorporeal perfusion of pig kidneys and livers.

Organ perfusion is one of the possible strategies to attenuate rejection of discordant xenografts by reducing the levels of the recipient's xenoreactive natural antibodies (XNA). Its efficacy in terms of XNA removal was studied in models of primate blood or plasma perfusion through porcine kidneys or livers, with special attention to haematological consequences and potential side-effects. We first perfused the blood of rhesus monkeys through pig kidneys and livers, and demonstrated that the perfusion of a pig liver resulted in higher XNA adsorption (72 +/- 13%) than the perfusion of a pig kidney (51 +/- 25%). However, when we normalized for the weight of the perfused organs and for levels of natural antibodies in individual monkeys, livers adsorbed less antibody (1.4 +/- 0.9 U antibody/g) than kidneys (7.2 +/- 7 U antibody/g). Histological signs of rejection were observed in perfused kidneys, but not in perfused livers. A major drawback of the perfusion of blood through livers was a considerable decrease in the primates' haemoglobin and platelet levels. To avoid this, we developed a plasma liver perfusion device. This method allowed a significant improvement in the haemodynamic state of primates and was particularly effective in preventing anaemia. Moreover, plasma liver perfusion was as effective as blood liver perfusion to remove natural antibodies and, resulted in a marked decrease in their functional activity as assessed by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The level of other plasma proteins was not significantly affected, apart from a dilution effect. After xenoperfusion a strong antibody response was evidenced by ELISA, CDC and ADCC between days 7 and 14 and then decreased progressively. We conclude that the separation of blood to allow the perfusion of plasma through a pig organ is safer than the perfusion of unseparated blood and is associated with efficient natural antibody removal. However, organ perfusion is limited by a rebound in antibody levels after a few days, and thus will have to be associated with anti-B cell immunosuppressive therapy for long-term or repeated applications.

Nonspecific reactions with the intradermal test for hydatidosis in persons with other helminth infections.

A partially purified Echinococcus antigen solution, prepared by boiling hydatid cyst fluid, was used in the intradermal test for hydatid disease in a Peruvian population. An unexpectedly high rate of positive reactions and poor agreement with serological tests suggested the presence of some agent(s) which produced cross-reactions with Echinococcus granulosus antigens in the intradermal test. Testing of hospital patients infected with a variety of helminths demonstrated nonspecific reactions in persons with Hymenolepis nana, Taenia spp., and Fasciola hepatica infections, mixed parasitisms, and several non-helminth pathological conditions. The findings contraindicated the use of the intradermal test for epidemiological surveys of hydatid disease. It is pointed out that intradermal test positivity rates cannot be used as synonymous with infection prevalence and regional differences do not necessarily reflect differences in the prevalence of E. granulosus infection. The greater specificity of some serological tests favor their use for epidemiological purposes.

Fallacies in the interpretation of Paul-Bunnel Davidsohn differential test.

Serological findings in five cases where Paul-Bunnel Davidsohn (PBD) test results were misleading, are presented. Three patients' with Chronic renal disorder and positive PBD test had specific serology results, signifying Cyto Megalo Virus infection. A fourth patient with Hepatitis B Virus infection also had positive PBD test. Forssman type of antibody response was demonstrable in a boy with recent Epstein-Barr virus infection and high Cyto Megalo Virus antibody titres.

[A morphological study of the keratin cytoskeleton of the oocyte from the clawed toad using heterologous monoclonal antibodies]. / Izuchenie morfologii keratinovogo tsitoskeleta ootsita shportsevoi liagushki s pomoshch'iu geterologicheskikh monoklonal'nykh antitel.

Heterologous monoclonal antibodies E2 (against rat cytokeratin 8) and OSC-1 (against cytoskeletal preparations of mouse oocytes) were used to study the presence and distribution of cytokeratins in Xenopus oocytes during their maturation and growth. To improve visualization of cytokeratins, more adequate methods of oocyte fixation and processing were developed. The results on distribution of cytokeratins obtained with the above mentioned antibodies were compared with one another and with published data. It was found that data obtained by different authors, who used different types of antibodies and different methods for fixation and visualization of Xenopus oocyte cytokeratins, are often contradictory and inconsistent. Along with that, common features of cytokeratin distribution are revealed that have been noticed by every author: animal-vegetal asymmetry of cytokeratin distribution and the existence of two keratin domains, cortical and ooplasmic ones. Specific for the cortical domain of the animal hemisphere is the arrangement of filaments in parallels to the surface, whereas filaments of the ooplasmic domain are oriented radially. The vegetal hemisphere is characterized by the presence of a network formed by filaments with different orientation. We also describe certain peculiarities of cytokeratin distribution in previtellogenic oocytes, as well as disintegration of the keratin system by the end of oocyte maturation.

[Study of human secretory immunoglobulin A. I. Obtaining monospecific antiserum to human secretory immunoglobulin A]. / Izuchenie sekretornogo immunoglobulina A cheloveka. Soobshchenie I. Poluchenie monospetsificheskoi antisyvorotki k sekretornomu immunoglobulinu A cheloveka

A method of obtaining monospecific antiserum to the human secretory IgA is described. Immunochemically pure secretory IgA (isolated from human colostrum by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200) was used for immunization of rabbits or sheep. Heterologous antibodies were removed by adsorption with commercial gamma globulin, normal serum, the serum of a patient suffering from A-myeloma with the IgA polymere and purified lactoferrin. Monospecific antiserum to the secretory IgA gave a reaction of complete immunological identity with the secretory IgA and a free secretory component.

[Monoclonal antibodies with anti-N specificity]. / Monoklonal'nye antitela s anti-N-spetsifichnost'iu.

In this article serologic characteristics of monoclonal antibodies with anti-N specificity is given. Antibodies are directed to homologous sequence of N-form of A glycophorine (group-specific N-antigen) and B glycophorine, expressed both on N- and on M-erythrocytes. Higher titre and avidity of monoclonal antibodies with anti-N specificity in relation to erythrocytes with N-phenotype make it possible to detect N-antigen in material subjected to expert evaluation.

Heterophilic antibody interference in immunometric assays.

Immunometric assays are inherently vulnerable to interference from heterophilic antibodies, endogenous antibodies that bind assay antibodies. The consequences of such interference can be devastating. In this review, we discuss strategies that reduce the damage caused by heterophilic antibodies. Clinicians should only order blood tests that are indicated for the patient and clinical setting at hand, and have the confidence to question laboratory results discordant with the clinical picture. Laboratorians should familiarize themselves with the vulnerability of the assays they offer, and be able to perform and interpret adequate confirmatory measures correctly. When designing immunoassays, the immunoassay industry should invest the necessary resources in specific protective measures against heterophilic antibody interference. Examples include using antibody fragments and the addition of effective blockers to assay reagents. The increasing use of modified monoclonal mouse antibodies both in therapy and diagnostics could present a particular challenge in the future.

Interference from anti-streptavidin antibody.

Immunoassays are commonly used for clinical diagnosis, although interferences have been well documented. The streptavidin-biotin interaction provides an efficient and convenient method to manipulate assay components and is currently used in several immunoassay platforms. To date, there has been no report in the literature of interference from endogenous anti-streptavidin antibodies; however, such antibodies would potentially affect multiple diagnostic platforms. We report results from a patient being treated for thyroid dysfunction who demonstrated a T-uptake result of less than 0.2 and a nonlinear thyroid stimulating hormone dilution that suggested an immunoassay interference. Protein-A sepharose pretreatment corrected the nonlinear dilution and revealed an interference trend of falsely decreased results, as measured by sandwich assay, and falsely elevated results, as measured by competitive assay. The results of streptavidin-agarose adsorption were comparable to adsorption with protein-A sepharose. To our knowledge, this is the first published description of an endogenous anti-streptavidin antibody interfering with clinical laboratory assays.