Antibodies of restricted heterogeneity directed against the cardiac glycoside digoxin.

Intravenous injection of New Zealand White rabbits with type III pneumococcal polysaccharide vaccine conjugated with the cardiac glycoside digoxin resulted in the production of both antidigoxin and anti-type III pneumococcal polysacharide antibodies. Among antisera of 12 rabbits examined during their peak antibody production periods, 1 to 20 mg (mean, 5.4 mg) of antidigoxin antibody could be recovered from 1 ml of serum. Antisera from five of these 12 rabbits contained antidigoxin antibodies of restricted heterogeneity as demonstrated by urea-polyacrylamide disc gel electrophoresis of fully reduced and alkylated antibodies. From the antisera of four of these five rabbits, electrophoretically homogeneous antibodies (1 to 5 mg/ml antiserum) could be isolated by affinity chromatography on ouabain-amine-Sepharose columns. The structural homogeneity of two of these antidigoxin antibodies was confirmed by amino acid sequence analysis of purified light chains through the first hypervariable region. These data suggest that the conjugation of small molecules to bacterial polysaccharide vaccines may provide a general method for synthesis of immunogens that can regularly elicit antihapten antibodies of restricted heterogeneity.     

Simultaneous determination of alpha-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay

A novel simultaneous measurement method for alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT-Eu(3+) and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT-Sm(3+)-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10(-5) M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.     

The effect of heterologous antisera on embryonic development. XIII. Lack of effect of antisera to alpha fetoprotein.

This investigation was carried out to determine whether heterologous antisera to alpha fetoprotein (AFP) are embryotoxic to developing rat embryos. Homogeneous rat AFP was isolated and antisera directed against this glycoprotein were produced in rabbits, horse and goat. The effect of the antisera on embryonic development was examined by injecting the antisera intraperitoneally into pregnant rats on the ninth, eleventh and thirteenth days of gestation. The results demonstrated that there was no evidence of increased incidence of fetal abnormalities in 472 surviving fetuses of 42 injected rats. There was no evidence of increase embryonic death or retardation of intrauterine growth following administration of the antisera on the ninth, eleventh and thirteenth days of gestation. The localization of the injected antisera was examined by the indirect immunofluorescent method. The results showed that the heterologous AFP antibodies localized specifically in the visceral yolk sac placenta. No antibody localization was observed in the embryo proper or the chorioallantoic placenta. It is speculated that the localization of AFP antibodies in the visceral yolk sac does not interfere with the embryotrophic function of the visceral yolk sac placenta.     

Evaluation of the safety and pharmacokinetics of the multi-targeted receptor tyrosine kinase inhibitor sunitinib during embryo–fetal development in rats and rabbits

BACKGROUND : Angiogenesis plays a key role in embryo–fetal development and, based on nonclinical safety data, the majority of vascular endothelial growth factor (VEGF)-targeted antiangiogenic agents used in cancer therapy are not recommended during pregnancy. We investigated the effects of sunitinib (an oral inhibitor of multiple receptor tyrosine kinases [RTKs] including VEGF-receptors) on embryo–fetal development. METHODS : Presumed-pregnant Sprague-Dawley rats and New Zealand White rabbits received repeated daily oral doses of sunitinib (0–30 mg/kg/day), during the major period of organogenesis. Clinical/physical examinations were performed throughout the gestation phase, and blood samples were collected to determine systemic exposure. Necropsy (including uterine examination) was performed on all animals and fetal morphology was examined. RESULTS : The no-observed-adverse-effect level was 1–5 mg/kg/day for maternal toxicity and 3 mg/kg/day for developmental toxicity in rats; 1 and 0.5 mg/kg/day, respectively, in rabbits. Embryo–fetal toxicity included decreases in the number of live fetuses and increases in the numbers of resorptions and post-implantation/complete litter losses; these were observed at doses of ≥5 mg/kg/day in rats and 5 mg/kg/day in rabbits. Malformations included fetal skeletal malformations (generally thoracic/lumbar vertebral alterations) in rats and cleft lip/palate in rabbits. These developmental effects were observed at ∼5.5- (rats) and ∼0.3-times (rabbits) the human systemic exposure at the approved sunitinib dose (50 mg/day). CONCLUSIONS : Similar effects have been reported with the prototype monoclonal antibody bevacizumab. As is typically observed for potent inhibitors of RTKs involved in angiogenesis, sunitinib was associated with embryo–fetal developmental toxicity in rats and rabbits at clinically relevant dose levels. Birth Defects Res (Part B) 33:204–213, 2009. © 2009 Wiley-Liss, Inc.     

Macrophage migration inhibition by T-cells in rats after overcoming natural tolerance to alpha fetoprotein]

Cellular immunity to alpha-fetoprein (AFP) was studied in rats by the macrophage migration inhibition (MMI) test after breakage of tolerance to homologous AFP of rat (RAFP). A single injection of cross-reacting AFP of mouse (MAFP) has induced the capacity of peritoneal exudate cells of rats to react in the MMI test to both MAFP and RAFP. The second injection of MAFP results in reduction of the MMI reaction to both antigens and the appearance of stimulation in the macrophage migration together with further elevation of antibody titer to MAFP and RAFP. The fractionation of rat peritoneal cells has shown the T-cell nature of MMI reaction to both MAFP and RAFP.     

Intravenous tolerability of an acid vehicle in Sprague Dawley rats and beagle dogs after daily slow bolus injection and infusion for one hour during 14 days

To assess the tolerability of an acid vehicle to be used in toxicology studies, a low pH aqueous solution containing 16.4 mg/ml of citric acid, 4.2 mg/ml of disodium phosphate, 25 mg/ml of mannitol, adjusted with phosphoric acid/NaOH 1 M to pH 3 was daily administered intravenously to rats and dogs for 14 consecutive days. The dosing regimen consisted of a slow intravenous bolus injection given over 30 s (0.75 and 0.625 ml/kg, for rats and dogs, respectively) followed by intravenous infusion for one hour (3.75 and 2.75 ml/kg/h, for rats and dogs, respectively). In rats, the dose was administered via the lateral tail vein. In dogs, the intravenous bolus dose was administered via the vena cephalica, vena saphena or vena jugularis, whilst the infusion dose was given into the vena cephalica or vena saphena. In rats, administration of the vehicle was associated with clinical signs (occasional mild vocalization and agitation) which were considered to be due to local irritation during the dosing procedure. Nevertheless, only mild histopathological changes at the injection site were found, while no relevant clinical chemistry changes were found in this species. However, the vehicle caused significant vascular damage with thrombus formation in the dog. It is therefore concluded that this vehicle is suitable for 2-week rat toxicity studies, if carefully applied. The vehicle with its present regimen should not be used in dogs, in view of the prohibitive findings.     

Label-free immunosensing for alpha-fetoprotein in human plasma using surface plasmon resonance

In this study, we attempted to develop a surface plasmon resonance (SPR)-based immunoassay sensor to detect alpha-fetoprotein (AFP) in human plasma at the nanogram level, as is required for clinical diagnosis of hepatocellular tumors. A self-assembled monolayer (SAM) surface of tri(ethylene glycol) (TEG) and carboxyl group-terminated hexa(ethylene glycol) (HEG) was employed to suppress the nonspecific adsorption of plasma components onto the sensor surface. AFP was detected by a sandwich-type immunoassay using two kinds of antibodies, primary and secondary, in this system. The SPR signal shift was further enhanced by applying an antibody (polyclonal) against the second antibody. With this method, the SPR signals were highly intensified, and so nanogram levels (ng/ml) of AFP could be easily detected with a high signal/noise ratio, as is necessary for clinical diagnosis. It is expected that our SPR-based immunoassay method can also be applicable to the detection of several other tumor markers that are present in low concentrations in human blood.     

Immune response in dog. 3. Antibody formation in dogs and rabbits to human serum proteins and keyhole limpet haemocyanin.

Antibody formation in dogs and rabbits to human serum proteins and keyhole limpet haemocyanin (KLH) following primary, secondary and multiple stimulation was analysed. Primary injection of human serum stimulates the formation of precipitin antibodies to beta2 lipoprotein in dogs, beta2 lipoprotein and beta globulin in rabbits. After a secondary dose the rabbits formed precipitins to a whole range of human serum proteins, while the dogs to beta2 lipoprotein, albumin, beta globulin. When the primary dose of antigen was divided over a period of 8 weeks, the dog produced precipitins to beta2 lipoprotein while the rabbit to a wide range of serum proteins. Secondary stimulation of these animals did not increase the number of precipitins formed. Quantitative analysis of the antibody produced show that the best response was with beta2 lipoprotein followed by albumin and beta globulin. As the immunogenicity of the antigen was greater the differences between the two species were narrow. These differences were less pronounced following the primary injection than after the secondary and multiple stimulations. The primary response to KLH (which represents mainly IgM) is better in the dog than in the rabbit, while the secondary response (IgG) was better in the rabbit. The poorer IgG response in the dog compared to the rabbit, observed in all the experiments, is discussed.     

抗冻蛋白应用于水稻悬浮细胞超低温保存的研究

本文将鱼类抗冻蛋白应用于植物细胞的超低温保存。结果表明,在水稻悬浮细胞的两步法保存中,浓度为0.01mg/ml的抗冻蛋白具有特别的负作用,相对高浓度的抗冻蛋白则能减小细胞存活率的波动性。在玻璃化法保存中,浓度为0.2mg/ml的抗冻蛋白能改善保存效果,更高浓度的抗冻蛋白(>5mg/ml)反而会降低保存效果。环境冰晶量、抗冻蛋白浓度、低温保护剂浓度和细胞膜组成等是影响抗冻蛋白使用效果的几大因素。作者在机理分析中认为,一方面,抗冻蛋白能和冰晶作用,抑制重冰晶,防止去玻璃化;另一方面,抗冻蛋白也能和细胞膜作用,诱发膜表面冰晶形成。     

A screening test to detect HGH-antibodies in pituitary dwarfs treated with HGH preparation by the radio-immunoassay with double antibody technique

Sixty-four blood samples, obtained from 25 pituitary dwarfs treated with KABIHGH preparation, were checked for serum HGH concentration by a double antibody radioimmunoassay and for antibodies against HGH by the method of propylethyleneglycol separation. Antibodies to HGH were detected in the samples whose HGH concentration was falsely estimated to be more than 5 ng/ml, but not in those whose HGH concentration was 5 ng/ml or less. The measurement of HGH concentration by a double antibody technique in the serum obtained from pituitary dwarfs 3-4 days after the last injection can be used to screen the presence of antibodies to HGH peparation used.     

抗冻蛋白应用于水稻悬浮细胞超低温保存的研究

AFP from winter flounder was utilized in cryopreservation of plant cells. During cryopreservation of rice suspension cells by two-step method, AFP at 0.01 mg/ml damaged the cells extremely. The data obtained at relatively high concentration, however, decreased the variability of survival rate. During vitrification of rice cells, AFP at 0.2 mg/ml enhanced the viability. However, high concentration AFP (> 5 mg/ml) decreased the recovery rate. Studies indicated that the results of application of AFP in cryopreservation were closely related to the concentration of cryoprotectant. The amount of ice crystal in environment, the concentration of AFP and cryoprotectant, and the composition of plasma membrane were several key factors affecting the results of AFP application. In mechanism analysis, the authors suggested that on one hand AFP can interact with ice crystal, which inhibits ice recrystallization and prevent the cells from devitrification. On the other hand, AFP also can interact with cell membrane, resulting in the ice growth around the plasma membrane.     

A disposable amperometric immunosensor for alpha-1-fetoprotein based on enzyme-labeled antibody/chitosan-membrane-modified screen-printed carbon electrode

A screen-printed three-electrode system is fabricated to prepare a novel disposable screen-printed immunosensor for rapid determination of alpha-1-fetoprotein (AFP) in human serum. The immunosensor is prepared by entrapping horseradish peroxidase (HRP)-labeled AFP antibody in chitosan membrane to modify the screen-printed carbon electrode. The membrane is characterized with scanning electron microscope and electrochemical methods. After the immunosensor is incubated with AFP at 30 degrees C for 35 min, the access of the active center of HRP catalyzing the oxidation reaction of thionine by H(2)O(2) is partly inhibited. In presence of 1.2 mM thionine and 6 mM H(2)O(2), the electrocatalytic current decreases linearly in two concentration ranges of AFP from 0 to 20 and from 20 to 150 ng/mL with a detection limit of 0.74 ng/mL. The immunosensor shows an acceptable accuracy compared with those obtained from immunoradiometric assays. The interassay coefficients of variation are 6.6 and 4.2% at 10 and 100 ng/mL, respectively. The storage stability is acceptable in pH 7.0 phosphate buffer solution at 4 degrees C for more than 10 days. The proposed method can detect the AFP through one-step immunoassay and would be valuable for clinical immunoassay.     

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.     

Radioimmunoassay of 3 alpha-hydroxy-5 alpha-pregnan-20-one in rat and human plasma

A radioimmunoassay for measuring 3 alpha-hydroxy-5 alpha-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3 alpha-hydroxy-20-oxo-5 alpha-pregnan-11 alpha-yl carboxymethyl ether coupled to bovine serum albumin. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was purified from either extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was detected in plasma from female rats on the day of estrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.     

Capillary electrophoretic immunoassay for alpha-fetoprotein with chemiluminescence detection

A capillary electrophoretic immunoassay with chemiluminescence detection (CEIA-CL) using a non-competitive format for analyzing tumor marker alpha-fetoprotein (AFP) has been developed. In this method, antigen (Ag) AFP reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ab*-Ag complex produced in the solution are separated by CE in a separation capillary. Then they catalyze the reaction of enzyme substrate luminol and H(2)O(2) in a reaction capillary following the separation capillary. Parameters affecting the CE separation and CL detection were investigated. Under the optimal conditions, the free Ab* and the Ab*-Ag complex were well separated within 4 min, the linear range and the detection limit (S/N=3) for AFP were 5-500 ng/ml and 0.85 ng/ml (1.2 x 10(-11)M), respectively. The proposed method has been applied satisfactorily in the analysis of human sera samples.     

Monoclonal antibodies as a tool for phylogenetic studies of major histocompatibility antigens and β 2-microglobulin

The cross-reactivity of several monoclonal antibodies recognizing monomorphic determinants of human HLA-A, B, C, and DR antigens and human 2-microglobulin (2m) has been studied on peripheral blood leukocytes in 24 different species. An monoclonal HLA-A-, B-, and C-specific antibody and four monoclonal HLA-DR-specific antibodies cross-reacted with cells from all the primate species tested. Furthermore, antibodies HLA-DR-specific were positive with peripheral blood leukocytes (PBL) from cows, goats, sheep, horses, and dogs. Two monoclonal 2m-specific antibodies, which were positive with PBL from certain primates, also reacted with cells from cows, goats, sheep, horses, and dogs. Two other #2m-specific antibodies reacted only with PBL from chimpanzees. No reaction could be detected with all our reagents in other classes tested (birds, reptiles, amphibians, and Teleostei).     

A comparative characteristic of effector innervation of cerebral arteries in mammals and humans.

The neural fibres of cerebral arteries in humans, rats, guinea-pigs, rabbits, cats, dogs, swine, cows and horses have been studied using the methods of Flack-Hillarp and Koelle. The large arteries of the carotid and vertebral systems bear dense cholinergic and adrenergic plexuses formed by transversal and longitudial neural fibres, which are located in the superficial and deep adventitial layers. The highest density of cholinergic and adrenergic fibres was observed on the arteries of humans and large mammals. Depending on the density of cholinergic and adrenergic fibres in the said arteries, the animals may be listed in the following order: rats, guinea-pigs, rabbits, cats, dogs, swine, cows and horses. Basic differences in the structure of neural plexuses of humans and animals were not observed.     

Suppression of collagen arthritis with antibodies to an arthritogenic, oligoclonal T cell line.

Rats immunized with type II collagen (CII) develop an immunologically mediated polyarthritis. T cells have been implicated in the pathogenesis of this model since they can adoptively transfer the disease. A CII-specific T cell line (VA), consisting of three distinct clones by Southern blot analysis, has been shown to be arthritogenic. Antibodies specific for this line were generated by immunizing rabbits. In an attempt to prevent collagen-induced arthritis (CIA), Louvain rats were injected with 1 ml of anti-VA ip on Days -1, +1, +3 and 0.5 ml on Day +5 (early treatment). To evaluate its effect on existing disease, rats received anti-VA on the day of arthritis onset and subsequently on 4 successive alternate days using the same dosage protocol (late treatment). Control rats received no therapeutic injections or were administered normal rabbit serum. All rats were immunized with CII on Day 0 to induce CIA. Rats administered antibodies using the early anti-VA treatment protocol had a significantly diminished incidence of arthritis compared to controls. Established arthritis was significantly diminished compared to controls in rats given the late anti-VA treatment. In both protocols, radiographic evidence of joint destruction was significantly reduced compared to controls. T cell phenotyping using flow cytometry analysis demonstrated that the anti-VA antibody therapy selectively eliminated a small subset of T cells since there was little difference in total T cell counts in the experimental versus control groups. Delayed type hypersensitivity and IgG antibody titers to CII were minimally decreased in the experimental versus control group. These results suggest that antibodies raised to an oligoclonal arthritogenic T cell line can suppress collagen arthritis. This may have implications with respect to 1) the size of the T cell receptor repertoire involved in the pathogenesis of collagen arthritis and 2) immunospecific protocols for CIA and other autoimmune diseases.     

Development of an inexpensive and rapid two site microenzyme linked immunosorbent assay kit for human alpha fetoprotein

Laboratory scale development of a two site micro enzyme linked immuno assay kit is described. The kit comprises rabbit anti human alphafetoprotein (AFP), anti human AFP IgG peroxidase conjugate and standard AFP. All the above reagents were prepared in the laboratory. The kit is eminently suitable for early screening of blood sample of pregnant women for neural tube defects of their fetuses and for the quantitation of AFP as a tumor marker. The assay kit was used to determine AFP in 76 sera from women at different stages of pregnancy. During 1st trimester AFP level was 18 to 119 ng/ml, during 2nd trimester the concentration varied from 85 to 302 ng/ml and during 3rd from 103 to 580 ng/ml. No evidence for maternal antibody to AFP was found. The above data agree with AFP level in pregnant women reported by earlier workers, using RIA or ELISA. The present ELISA kit would hopefully be much cheaper than internationally available ELISA kits for human AFP.     

Effect of caffeic acid phenethyl ester on treatment of experimentally induced methicillin-resistant Staphylococcus epidermidis endophthalmitis in a rabbit model

This study investigated the anti-inflammatory effects of caffeic acid phenethyl ester (CAPE), a natural bee-produced compound, and compared it with corticosteroids in the treatment of experimentally induced methicillin-resistant Staphylococcus epidermidis (MRSE) endophthalmitis in addition to intravitreal antibiotics. An experimental endophthalmitis model was produced in 24 New Zealand albino rabbits by unilateral intravitreal injection of 0.1 ml of 4.7 x 10(4) colony-forming units (CFU) methicillin-resistant S. epidermidis. The animals were then divided randomly into three treatment groups and a control group, group 1 (six rabbits), received only intravitreal vancomycin (1.0 mg/0.1 ml); group 2 (six rabbits), received both intravitreal vancomycin (1.0 mg/0.1 ml) and intravitreal dexamethasone (400 microg/0.1 ml) and group 3 (six rabbits), received both intravitreal vancomycin (1.0 mg/0.1 ml) and subtenon CAPE (10 mg/0.3 ml) after 24 h post-infection. No treatment was given to the control group. Treatment efficacy was assessed by clinical examination, vitreous culture and histopathology. There were no statististically significant differences between clinical scores of all groups in examinations at 24 and 48 h post-infection (p = 0.915 and p = 0.067 respectively), but in examinations at 72 h post-infection and after 7 days post-infection, although the clinical scores of treatment groups were not significantly different from each other, they were significantly lower than the control group (p < 0.05). The culture results of all groups were sterile. As a result, CAPE was found to be as effective as dexamethasone in reducing inflammation in the treatment of experimental MRSE endophthalmitis when used with antibiotics. More studies are needed to determine the optimal administration route and effective dosage of this compound.