Sera Of Different Species Research Articles

Successful Development of Nonclinical Anti-Drug Antibody Assays to Support Zinpentraxin Alfa Reproductive Toxicology Studies.

Immunogenicity assessment is an essential part of biotherapeutic drug development. While the immune response in animals is not always representative of the human immune response, immunogenicity data obtained in animal models is still informative for the evaluation of drug exposure and safety. The most common assay format used for the detection of anti-drug antibodies (ADAs) in preclinical and clinical studies is the bridging format. The advantage of this method is that it can detect all antibody isotypes generated against the therapeutic. However, the method development can be time-consuming and labor-intensive, due to the need for labeling of the drug which is used both as capture and detection. Various generic ADA assays have been successfully implemented to overcome these disadvantages and to enable faster assay development timelines to support nonclinical toxicology studies. Here, we describe the challenges in the development of an assay to detect antibodies to zinpentraxin alfa, a recombinant human pentraxin-2, in rabbit and rat toxicology studies. Our initial efforts to develop a bridging assay failed, prompting us to develop a method adapted from generic assay formats to detect anti-zinpentraxin alfa antibodies in the serum of different species with minimal optimization. However, while the general assay format remained similar, assay reagents were adapted between the different species, resulting in the development of two distinct assays for the detection of ADAs in rat and rabbit. Here, we share the final development/validation data and the immunogenicity study results. Our work highlights the need for the evaluation of alternate assay formats when evaluating novel drug modalities.

Sero-Epidemiological Survey of Orthopoxvirus in Stray Cats and in Different Domestic, Wild and Exotic Animal Species of Central Italy.

Orthpoxvirus infection can spread more easily in a population with a waning immunity with the subsequent emergence/re-emergence of the viruses pertaining to this genus. In the last two decades, several cases of Orthopoxvirus, and in particular Cowpoxvirus infections in humans were reported in different parts of the world, possibly due to the suspension of smallpox vaccinations. To date, in Italy, few investigations were conducted on the presence of these infections, and because of this a serosurvey was carried out to evaluate Cowpoxvirus infection in feline colonies situated in the province of Rome, since these are also susceptible to other zoonotic viruses belonging to Orthopoxvirus, and from which humans may contract the infection. The sample design was set at an expected minimum seroprevalence of 7.5%, a 5% standard error and 95% confidence level. In parallel, a serological investigation was conducted using convenience sampling in domestic, exotic and wild susceptible animals of the Latium and Tuscany Regions, which are areas in the jurisdiction of the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, coordinating this study. The serological methods employed were indirect immunofluorescence for 36 sera of nonhuman primate and virus neutralization for 1198 sera of different species. All the 1234 sera examined were negative for the presence of antibodies against Cowpoxvirus, indicating its limited circulation in the areas of investigation. The methodology applied for the serosurveillance could be adopted in the case of outbreaks of this infection and for the evaluation of the spread of this infection in the area of interest, to obtain essential information crucial for animal and public health policies according to the One Health concept.

Seroprevalence of small ruminant lentivirus (SRLV) infection in wild cervids in Poland

Small ruminant lentiviruses (SRLV) are widespread amongst domesticated sheep and goats worldwide. Infection of wild ruminants in close contact with affected domesticated small ruminants has been proposed as an actor in SRLV epidemiology, but studies are limited. The aim of this study was to estimate the apparent (AP) and estimated prevalence (EP) of exposure to SRLV infection in wild ruminants from Poland. Samples originating from 198 free-living cervids comprising 142 European red deer and 56 roe deer were serologically tested using a multi-epitope recombinant antigen ELISA representing subtypes A1, A13, B1, and B2 of SRLV and a commercial ELISA test. The estimated prevalence of SRLV infection was estimated using the Bayesian approach with models that adjusted for the misclassification of animals because of a small population and lack of sampling method, the imperfect performance of the ELISAs and because sera of different species were tested. The calculated estimated prevalence ranged from 5.3 % (95 % CI 0.3, 12.5) to 24.6 % (95 % CI 3.3, 38.5) for the ELISA with multi-epitope antigens while estimated prevalence using the commercial ELISA was 2.5 % (95 % CI 0.2, 6.6). These results may suggest the existence of a new SRLV reservoir in Poland and highlight the importance of surveilling and controlling SRLV infection in domestic and wild ruminants sharing pasture areas.

First investigations on serum resistance and sensitivity of Borrelia turcica

Borrelia turcica is a reptile-associated Borrelia species that is vectored by the hard tick Hyalomma aegyptium. Tortoises of the genus Testudo represent the principal host of adult H. aegyptium, while immature stages are less host-specific and can be found on various vertebrates and even on humans. Borrelia turcica isolates were already successfully obtained from exotic tortoises suggesting that they are putative hosts. To the best of our knowledge, no further investigations on additional host association of B. turcica were conducted. Since many but not all adult Hyalomma ticks collected from tortoises are infected, questions arise about the direction of transmission between tick and tortoises for this Borrelia species. In addition, there is no information on the potential pathogenicity of B. turcica for humans. For other Borrelia species it has been shown that resistance or sensitivity to complement-active serum can be indicative of host species association(s). In this study, we explored for the first time the in vitro survival of B. turcica isolates from Turkey (IST7) and Greece (171601G) in the presence of 50% complement-active serum of different species (tortoise, turtle, human and bird). Both isolates showed resistance to tortoise serum, partial resistance to turtle serum but did not survive human and bird serum. These data suggest that indeed tortoises are reservoir host species for B. turcica while birds or humans are not. By implication these data suggest that B. turcica is not human pathogenic. Whether or not other reptile species, such as lizards, are also potential hosts, requires further investigation. However, as the life cycle of Borrelia is closely linked to that of their hosts and vectors, in vitro studies can only give clues about the actual in vivo behavior.

A sensitive antibody-free 2D-LC-MS/MS assay for the quantitation of myostatin in the serum of different species.

Aim: Myostatin (MSTN) is an attractive therapeutic target for the treatment of muscle degeneration-related diseases and is being evaluated as a target engagement biomarker. Methods: A sensitive 2D-LC-MS/MS assay was developed to quantify MSTN in different animal species. Sample preparation involved SDS denaturation of serum proteins followed by tryptic digestion and peptide enrichment by SPE. Results: The assay was validated with LLOQ of 2.5ng/ml in rat and monkey serum. The precision was within 13.7%, and the bias was within±12.6% for all quality control samples in authentic matrices. Conclusion: This new assay was successfully applied to measure MSTN in mouse, rat, monkey and human serum. The total MSTN in rat and monkey serum was elevated following administration of an MSTN inhibitor.

Irisin Is Elevated in Skeletal Muscle and Serum of Mice Immediately after Acute Exercise

Recent findings regarding the response of fibronectin type III domain-containing protein 5 (Fndc5) and irisin to exercise are partly controversial. While the 25 kDa form of Fndc5 can be observed in muscle and serum of different species, the ~12 kDa irisin band was not detectable up to now. The present study aimed to clarify whether irisin exists in its theoretical size of ~12 kDa in mice and if it is affected by exercise. Male mice were randomly assigned to a sedentary control group (CO), a group with free access to running wheels (RW), and a treadmill group (TM). Blood and leg muscles were collected to investigate the regulatory cascade including peroxisome proliferator-activated receptor gamma co-activator 1-alpha (Ppargc1a) and Fndc5. In western blot analysis, antibodies were used capable of differentiation between full-length Fndc5 and irisin. This enabled us to demonstrate that irisin exists in muscle and serum of mice independent of exercise and that it is increased immediately after acute exercise. Different transcripts of Ppargc1a mRNA, but not Fndc5 mRNA, were up-regulated in the TM group. Furthermore, neither Fndc5 (25 kDa) nor Ppargc1a protein was elevated in muscle tissue. The Ppargc1a-Fndc5/irisin pathway did not clearly respond to mild exercise in the RW group. Our results provide evidence for the existence of irisin and for its immediate response to acute exercise in mice.

A polymorphism in the 3′-flanking region of insulin-like growth factor binding protein 2 gene associated with abdominal fat in chickens

Insulin-like growth factor binding protein 2 (IGFBP2) is the predominant insulin-like growth factor binding protein in serum of different species. It binds insulin-like growth factors and plays an important role in growth and fat metabolism. The current study was designed to investigate the associations of IGFBP2 gene 3'-flanking region polymorphisms with chicken fatness traits. The random populations (5 distinct populations: Arbor Acres broiler, Hyline Brown layer, Shiqiza, BeijingYou, and Baier), the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF), and the the Northeast Agricultural University F(2) (NEAU F(2)) resource population were used in the current study. Body weight and body composition traits were measured in NEAUHLF and NEAU F(2) populations. A SNP of 1196C > A in the 3'-flanking region of the IGFBP2 gene was detected, and the PCR-single strand conformation polymorphism method was then used to genotype all of the individuals derived from the above populations. There was a highly significant difference (P < 0.01) in the allele frequency among the 5 distinct populations. The IG-FBP2 polymorphism was significantly associated with abdominal fat weight and percentage of abdominal fat (P < 0.05) in NEAUHLF and NEAU F(2) populations. The research suggests that the IGFBP2 gene could be a candidate locus or linked to a major gene that affects fatness traits in chickens.

Factors Affecting Mitogenic Response of Turkey Lymphocytes

Loa C. C ., T. L . Lin, C . C. W u: Factors Affecting Mitogenic Response of Turkey Lymphocytes. Acta Vet. Brno 2001, 70: 433-442. The objective of the present study was to determine the parameters and conditions for measuring mitogenic response of turkey whole blood and spleen lymphocytes that may be useful in studying cellular immunity of turkeys. Heparinized whole blood or spleen was collected from normal turkeys. The mitogenic assay of whole blood lymphocytes was examined by different combinations of blood dilution (1:10 to 1:80), concanavalin A (Con A) concentration (0 to 320 g/ml), and serum of different species added. The mitogenic assay of spleen lymphocytes was examined by different combinations of spleen cell concentration (1 × 10 6 to 1 × 10 7 cells / ml), Con A concentration (0 to 25 µg / ml), and serum of different species added. The effect of age on the mitogenic response of turkey whole blood cells and spleen cells to Con A was also examined. Both turkey serum and fetal bovine serum enhanced the counts per minute (CPM) readings of the whole blood assay. The presence of fetal bovine serum produced higher stimulation index (SI) than the presence of turkey serum. In the presence of fetal bovine serum, the highest CPM and SI for the whole blood assay were observed with blood dilution at 1:40 and Con A concentration of 40 µg/ml. The highest CPM and SI for the spleen cell assay were observed using turkey serum and Con A concentration of 12.5 µg/ml. Spleen cell concentrations for the highest CPM were 5 × 10 6 and 1 × 10 7 cells/ml whereas those for the highest SI were 2.5 × 10 6 and 5 × 10 6 cells/ml. The mitogenic responses of both whole blood assay and spleen cell assay varied among turkeys of different ages. The results indicated that addition of fetal bovine serum is better for the mitogenic response of turkey whole blood lymphocytes and addition of turkey serum is better for the mitogenic response of turkey spleen lymphocytes. In the presence of appropriate serum, the greatest mitogenic response of turkey lymphocyte to Con A could be obtained by checkerboard tests of whole blood dilutions, spleen cell concentration, or Con A concentration.

Inhibition of a type B monoamine oxidase inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (MDL-72974A), on semicarbazide-sensitive amine oxidases isolated from vascular tissues and sera of different species

(E)-2-(4-Fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A) has been discovered recently to be a very potent and highly selective type B monoamine oxidase inhibitor. We have found that this inhibitor is also capable of inhibiting semicarbazide-sensitive amine oxidases (SSAOs) obtained from vascular tissues and sera of different species. The inhibition of SSAO by MDL-72974A was irreversible and time dependent. It was competitive without preincubation of the enzyme with the inhibitor and demonstrated a mixed-type of inhibition when the enzyme was preincubated with the inhibitor. The ic 50 values were estimated to be 2 × 10 −9 M, 5 × 10 −9 M, 8 × 10 −8 M and 2 × 10 −9 M for SSAO from dog aorta, rat aorta, bovine aorta and human umbilical artery, respectively. SSAO obtained from bovine serum was relatively insensitive to MDL-72974A ( ic 50 = 3 × 10 −7 M . Following intraperitoneal administration of MDL-72974A, rat brain MAO-B was inhibited with the ed 50 value being about 0.2 mg/kg. Rat aorta SSAO was also inhibited and to a similar extent by the same dose. MDL-72974A is the most potent SSAO inhibitor that has been described thus far.

High-performance liquid chromatographic assay for domoic acid in serum of different species

High-performance liquid chromatographic assay for domoic acid in serum of different species

Development and Validation of a Radioimmunoassay for Serum Melatonin

A radioimmunoassay using N-[3-(4-hydroxy-3-[4-hydroxy-3- [125I]iodophenylpropionyl)]-5-methoxytryptamine as tracer for determination of melatonin in the serum of different species is described. Melatonin antisera were raised in rabbits by immunization with a bovine serum albumin conjugate of N-[3-(2-aminoethyl)-5-methoxy indole] hemisuccinamide. A single high affinity, specific antiserum was obtained. In contrast to previous studies, the tracer was synthesised in one step in the absence of water, giving an excellent yield of highly pure product. No chromatographic purification step was needed. Polyethylene glycol in combination with goat antirabbit immunoglobulins was used to separate bound and unbound tracer. Sera were delipidized with Lipoclean prior the extraction of melatonin with diethyl ether. This sample preparation allows the determination of melatonin in the presence of widely varying amounts of lipids in human, rat and hamster serum. Using this extraction procedure, the sensitivity of the radioimmunoassay was approximately 1 ng/l of serum. Dilutions of sera and of synthetic melatonin gave the same parallel response in the radioimmunoassay. High performance liquid chromatography analysis of a serum extract showed only one immunoreactive peak co-eluting with synthetic melatonin. Characteristic diurnal rhythms of melatonin were observed in all species. All assay components including standards and serum controls are stable for at least 1 year at 4 degrees C, thus facilitating the determination of melatonin in a routine laboratory.

Quantitative determination of transferrins in serum of different species

Quantitative determination of transferrins in serum of different species

Quantitative determination of transferrins in serum of different species

Quantitative determination of transferrins in serum of different species

Isolation, Characterization and Implications of Anti-TF (Thomsen-Friedenreich) Agglutinins from Different Sources

Anti-TF agglutinins from peanut ( Arachis hypogaea) and from vertebrate sera of different species have been successfully isolated by affinity chromatography on acid-activated Sepharose 4 B. The proteins were characterized by immunoelectrophoresis, polyacrylamide gel electrophoresis in the presence of SDS and with respect to their carbohydrate binding specificities. Anti-TF substances from sera showed one precipitin arc in immunoelectrophoresis, but quantitative immunoprecipitation revealed our human anti-TF to be a mixture of the three Igclasses IgG, IgA and IgM. This finding was confirmed on SDS gel electrophoresis, where high molecular weight aggregates were found before reduction. Hemagglutination inhibition revealed that all isolated anti-TF compounds exhibit an exceptionally high affinity for the immunodominant group of the TF-antigen, namely the βD-galactosyl-(1→3)-N-acetyl-D-galactosamine disaccharide. On examination of formalinfixed and neuraminidase treated tissue sections (kidney, mammary gland), fluorescein-labelled anti-TF from horse serum showed a virtually identical pattern when compared with fluorescein labelled peanut lectin. Likewise isolated IgA-class myeloma J 539, which shows specificity against β-(1→6)-galacoys, only bound to the appropriate Gal-β-(1→6)-Gal structures, such as those found on bovine lung or the albumin gland of Helix pomatia. Rabbit anti-VCN ( Vibrio cholerae neuraminidase) activity could be selectively abolished by β-galactosyl-containing inhibitors, whereas papain F (ab) fragments from rabbit anti-VCN immunoglobulin did not compete with anti-TF for binding sites on VCN-treated human red cells. Anti-TF, on the other hand, did not compete with anti-VCN for active VCN.

Serum concentration of inorganic sulfate in mammals: Species differences and circadian rhythm

1.1. The concentration of inorganic sulphate in serum of different species ranges between 0.3 (man) and 2.5 mM (goat, rooster).2.2. In the rat a circadian rhythm in serum sulphate is observed. The concentration reaches its maximum in the afternoon, and when the lights are switched off it decreases rapidly, staying relatively low during the night.3.3. Urinary sulphate excretion was significantly lower during daytime than at night.4.4. Sulphate was excreted for 20% in the conjugated form, while 80% was free inorganic sulphate.5.5. There is no relation between serum sulphate concentration and the extent of sulphation of xenobiotics in various mammalian species.

Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.

In vitro exchanges of esterified cholesterol between serum lipoprotein fractions: Studies of humans and rabbits

Lipoproteins labeled with 3H in the free- and esterified-cholesterol moieties were isolated from the serum of rabbits that had been injected 14 hr before with 3H-mevalonic acid and from human serum that had been incubated with 3H-cholesterol for 6 hr at 37°C. Each of the labeled lipoprotein fractions of each species was subsequently incubated in vitro at 37°C with unlabeled lipoprotein fractions isolated from the same species in the presence of an inhibitor of lecithin:cholesterol acyltransferase (LCAT) activity. Incubations contained either the lipoprotein-free (the dialysed 1.21 g/ml infranate) fraction of serum or an equivalent volume of buffer. In incubations of both rabbit and human lipoproteins there was a significant bidirectional transfer of esterified 3H-cholesterol between each pair of lipoproteins so long as the 1.21 g/ml infranate was present, but in its absence there were no transfers. A minimum estimate of the rate at which esterified cholesterol exchanged between human lipoproteins was obtained; expressed as μmole/liter serum/hr, it was 30–166 between very low density lipoproteins (VLDL) and low density lipoproteins (LDL) 31–156 between VLDL and high density lipoproteins (HDL) and 97–140 between LDL and HDL. In subsequent experiments, labeled human VLDL was incubated with unlabeled human LDL in the presence of the 1.21 g/ml infranate isolated from the serum of different species. In terms of promoting a transfer of esterified 3H-cholesterol into the LDL, the rabbit 1.21 g/ml infranate proved to be more effective than that of the human, but the 1.21 g/ml infranate of rat serum was totally ineffective. It has been concluded that a process of esterified-cholesterol exchange between human serum lipoproteins may be rapid enough to have the potential of major physiologic importance.

Basophil chemotaxis under agarose: Variability among guinea pig donors and response to sera of different species

Guinea pig basophils were tested against chemotactic factor(s) generated from homologous and heterologous sera. An “under agarose” method of quantitating leukocyte chemotaxis was adapted to measure serum-derived basophil chemotactic factor(s) (BCF). The method was found to be simple, reproducible, and sparing of time and reagents and it provided simultaneous controls. Because of the economy of reagents compared with the Boyden chamber technique, basophils from individual guinea pigs could be studied and were found to be variable in responsiveness. A definite difference in the response of guinea pig basophils to zymosan-activated sera of various other species was demonstrated. Guinea pig basophils responded to zymosan-activated serum from guinea pig and rat but failed to respond to zymosan-activated serum from human, hamster, rabbit, or cow. Optimal conditions were determined for cell numbers, serum activation and dilution, and migration times for studies of guinea pig BCF. The under agarose technique provides a practical alternative to chamber methods for the study of basophil chemotaxis. Although guinea pig basophils do not respond to human serum-derived BCF, the economy of reagents with this technique gives promise toward adaptation of the method to studies of human basophil chemotaxis.

Application of an improved system of electrophoresis in acrylamide gel to studies on the sera of different species.

Polyacrylamide gels constitute a generally better matrix for routine electrophoresis of serum proteins than other media commonly employed, but the immunoglobulin fractions of the largest size may not migrate into gels in which the acrylamide concentration exceeds 5%. To facilitate adequate separation, slabs containing a semi-solid layer were prepared from discontinuous gels consisting of 2 to 8% acrylamide. Serum samples were subjected to electrophoresis, under carefully controlled conditions at pH 9.0, by means of a pulsed constant power supply. The method provided a rapid, reproducible, and relatively simple technique for the study of human serum proteins, either by electrophoresis or by immunoelectrophoresis, and for differentiation of serum samples from various animal species.

Degradation of radioiodinated porcine and human synthetic calcitonins by sera of different species

The in vitro degradation of porcine and human synthetic calcitonin was measured in the presence of sera of different species (rat, pig, human) using a chromatoelectrophoresis method. Degradation is greater when heterologous sera are used. The degradation was not prevented by enzyme inhibition. Damage was greatest at acid pH and was not removed by heating of the serum. Ammonium sulfate precipitations and euglobulin separation of serum proteins did not result in the isolation of the factor responsible for the degradation of calcitonin. Mercaptoethanol prevented the damage from appearing which suggested that this damage was in part due to breakage of the disulfide bond.