Radioimmunoassay for prednisone

A radioimmunoassay for the measurement of prednisone in serum has been developed. The method is accurate, precise, specific, and very sensitive. Using a primary antibody elicited against prednisone 21-hemisuccinate-bovine serum albumin and a chemical precipitation step, the assay is capable of detecting 6.25 picograms of prednisone in 0.1 ml of unextracted diluted serum or plasma.The specificity of the assay is influenced by the carbonyl groups at position 11 and 20, and the double bond at the 1-position of the steroid nucleus. Physiological levels of endogenous steroid interfered only slightly with the primary antibody.Measurement of serum concentrations of prednisone in man and dog were accomplished following the administration of prednisone (Deltasone^®). This assay, used in conjunction with a published radioimmunoassay for prednisolone (1), can be used to determine the interconversion of these two drug products following prednisone or prednisolone administration.     

The use of immobilized estradiol antiserum in the study of receptors and other estradiol-binding proteins.

Antiestradiol antibody immobilized on a polymer film is set in competition for tritium-labeled estradiol with estradiol receptor or antiestradiol antibody in solution. Measurement of equilibrium quantity of radiolabel in test solutions in the presence and in the absence of dissolved antibody or receptor gives a data base for evaluating the association constant and the quantity of the estradiol-complexing species in solution. The immobilized antibody method avoids the problems and uncertainties arising in the step of separating the bound from the free hormone which is the pivotal step in all currently used procedures for the determination and characterization of steroid receptors. With the exception of equilibrium dialysis, it is the only procedure presently available to study steroid antibodies and receptors in solution at unperturbed equilibrium. Using the immobilized antibody method at 4°C, 0.86 ± 0.12 × 10¹¹m^?1 was the association constant found for estradiol receptor in rat uterine cytosol, and 2.6 ± 0.5 × 10¹¹m^?1 was the association constant found for antiestradiol antibody raised in a rabbit to estradiol linked to bovine serum albumin via a C-6 carboxymethyloxime.     

Cytotoxicity testing of burn wound dressings: first results

Topical antimicrobial therapy represents an essential part of burn wound care. In order to prevent and treat burn wound infection dressings with antimicrobial properties are applied directly on the wound surface. Not only the infection control but also promotion of healing is very important in burn wound management. It is well known, that a dressing in bactericidal concentration might also delay wound healing. This study was aimed to evaluate the potential toxic effect of topical antimicrobial agents on murine and human dermal cells. For toxicity testing the method by Vittekova et al. was used to evaluate potential toxic effects of 16 agents and 6 control samples on two in vitro cultured cell systems [3T3 cells and dermal fibroblasts] during the first 24 h. Following the 24 h cell culture with the tested agents the live cell counts were evaluated. According to results obtained on both cell systems, the tested samples were divided into three groups—nontoxic, semi-toxic and toxic. Nontoxic samples included Acetic acid 1%, Acticoat^®, Dermacyn^®, Framykoin^®, Silverlon^®, gauze, acellular human allodermis and acellular porcine xenodermis. Semi-toxic group included Algivon^®Plus, Aquacel^®Ag, Betadine^®, Nitrofurazone, Octenisept^®, Suprasorb^® A and a porcine dermal scaffold Xeno-Impl. Finally, the toxic group included Algivon^®, Dermazin^®, Ialugen^®Plus, Prontoderm^®, Suprasorb^® A Ag and 20% SDS. As the preliminary results of this study have shown, our findings may serve as a potential guide to selection of the most appropriate topical antimicrobial dressings for treatmet of burns. However before they can be translated into clinical practice recommendations, more research on antimicrobial dressings cytotoxicity testing will be necessary.     

Aggregation of biopharmaceuticals in human plasma and human serum

Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin^® (trastuzumab) or Avastin^® (bevacizumab) but not Remicade^® (infliximab). The aggregates in the plasma-Herceptin^®-5% dextrose solution were globular, size range 0.5–9 μm, with a mean diameter of 4 μm. The aggregates in the plasma-Avastin^®-5% dextrose samples had a mean size of 2 μm. No aggregation was observed when 0.9% NaCl solutions of Herceptin^®, Avastin^® and Remicade^® were mixed with human plasma. 90° light-scattering measurements showed that aggregates were still present 2.5 h after mixing Herceptin^® or Avastin^® with 5% dextrose-plasma solution. A SPR method was utilized to qualitatively describe the extent of interactions of surface-bound antibodies with undiluted human serum. Increased binding was observed in the case of Erbitux^® (cetuximab), whereas no binding was measured for Humira^® (adalimumab). The binding of sera components to 13 monoclonal antibodies was measured and correlated with known serum binding properties of the antibodies. The data presented in this paper provide analytical methods to study the intrinsic and buffer-dependent aggregation tendencies of therapeutic proteins when mixed with human plasma and serum.     

Radioimmunoassay for fluoxymesterone (halotestin®

A specific, sensitive, precise and accurate radioimmunoassay has been developed for fluoxymesterone, 9α-fluoro-11β, 17β-dihydroxy-17-methyl-4-androsten-3-one (Halotestin^®). The method is capable of detecting 25 picograms of drug in 0. 1 ml of unextracted serum. The primary antibody was prepared against fluoxymesterone 3-[0-(carboxymethoxime)] (CMO) bovine serum albumin. The specificity of the assay is greatly influenced by the hydroxyl group at position 11 and the methyl group at position 17. Physiological levels of endogenous steroids did not cross-react significantly with the primary antibody. Blood levels of fluoxymesterone were determined in both human subjects and male beagle dogs after oral administration of Halotestin.     

Radioimmunoassay for methylprednisolone (Medrol)

A precise, accurate, sensitive and specific double antibody radio-immunoassay for methylprednisolone (Medrol^®) has been developed. The assay is capable of detecting femtogram (10^?9 μgrams) quantities of the drug in unextracted serum. The primary antibody was prepared against methylprednisolone-3-(O-Carboxymethyl)oxime-bovine serum albumin.     

Kepone serum half-life in humans.

Blood samples were obtained from twelve Allied Chemical Company plant workers in Baltimore, Maryland who were exposed to unknown amounts of Kepone^®. The post-exposure serum concentrations of the insecticide were determined by gas chromatography. Serum Kepone concentrations ranging from 120 to 2,109 ng/ml wereobserved on the last day of exposure. Six to seven months later, the insecticide's concentration dropped to 37–486 ng/ml. The half-life of elimination of Kepone was variable and ranged from a low of 63 to a high 148 days. No correletion was observed between the half-life and the serum Kepone concentrations on the last day of exposure.     

Comparison of SurePath® and ThinPrep® liquid‐based cervical cytology using positive predictive value,atypical predictive value and total predictive value as performance indicators

P. K. Wright, J. Marshall and M. Desai Comparison of SurePath ^® and ThinPrep ^® liquid‐based cervical cytology using positive predictive value, atypical predictive value and total predictive value as performance indicators Objective: Two liquid‐based cytology (LBC) systems are in widespread use in the UK: ThinPrep^® and SurePath^®. A number of studies have now compared LBC with conventional cytology in cervical screening. However, to date, we are aware of no studies that have compared ThinPrep^® with SurePath^® LBC. As the selection and use of specific diagnostic systems in a laboratory has significant clinical and economic implications, there is a clear need to compare directly existing LBC technology. The objective of this study was to compare ThinPrep^® with SurePath^® LBC in a single cytology laboratory using performance indicators. Methods: Data were collected for all cervical cytology samples processed at Manchester Cytology Centre over a 1‐year period. ThinPrep^® LBC was compared with SurePath^® LBC using positive predictive value (PPV), atypical predictive value (APV) and total predictive value (TPV), reflecting outcome of cervical intraepithelial neoplasia (CIN) grade 2 or worse for high‐grade dyskaryosis (PPV), low‐grade dyskaryosis or borderline (atypical) cytology (APV) and all (total) abnormal cytology (TPV). Results: 2287 (out of 56 467) (ThinPrep^®) and 586 (out of 22 824) (SurePath^®) samples showed borderline or worse cytology after exclusion criteria. PPV, APV and TPV were within acceptable ranges for both ThinPrep^® and SurePath^®. Conclusions: ThinPrep^® and SurePath^® were equivalent based on three performance indicators. We suggest that APV and TPV should be used as an adjunct to PPV and other methods of quality assurance for cervical screening.     

Biomechanical properties of bovine tendon xenografts treated with a modern processing method

Xenograft tendons have been used in few human studies, with variable results. With the advent of novel tissue processing techniques, which may mitigate against an immune-mediated rejection response without adversely affecting mechanical properties, there may now be a clinical role for xenograft tendons, particularly in knee ligament reconstruction. We hypothesize that ‘BioCleanse®’ processed bovine extensor digitorum medialis (EDM) tendons exhibit favorable time-zero pre-implantation biomechanical characteristics when compared to both unprocessed bovine EDM tendons and BioCleanse® processed human cadaveric allograft tibialis anterior tendons.In this in vitro case controlled laboratory study, three groups of tendons underwent a 5-stage static loading test protocol: 15 BioCleanse^® bovine (BCB), 15 fresh frozen unprocessed bovine (FFB), and 12 BioCleanse^® human allograft (BCA) tendons. Cross-sectional area of the grafts was measured using an alginate molding technique, and tendons were mounted within an Instron^® 5565 Materials Testing System using cryogenic clamps.BCB tendons displayed a higher ultimate tensile stress (p<0.05), with equivalent ultimate failure load, creep, and modulus of elasticity when compared to the FFB tendons (p>0.05). BCB tendons had an equivalent cross-sectional area to the BCA tendons (p>0.05) whilst exhibiting a greater failure load, ultimate tensile stress, less creep and a higher modulus of elasticity (p<0.05).The BioCleanse^® process did not adversely affect the time-zero biomechanical properties of bovine xenograft EDM tendons. BioCleanse^® processed bovine xenograft EDM tendons exhibited superior biomechanical characteristics when compared with BioCleanse^® processed allograft tibialis anterior tendons. These findings support further investigation of xenograft tendons in orthopedic soft tissue reconstructive surgery.     

Development of an immunochromatographic strip test for the rapid detection of okadaic acid in shellfish sample

A rapid detection technology for okadaic acid (OA) in shellfish with one-step immunochromatographic assay using colloidal gold-labeled monoclonal antibody (Mab) probe was developed. OA is one of the diarrhetic shellfish toxins. Firstly, OA was conjugated to bovine serum albumin, and the conjugations as immunogen were injected into mice to raise the polyclonal antibody against OA. Hybridoma cells fused between spleen cells from immunized mouse and myeloma cells (Sp2/0) were prepared and injected into mice intraperitoneally at 1?×?10⁶?cells to produce monoclonal antibody in the ascitic fluid. With the monoclonal antibody against OA, the idc-ELISA assay was established to detect OA. The calibration curve for OA was linear over the concentration range of 0.31–50 ng mL^?1, and the detection limit for OA was 0.45 ng mL^?1. On that basis, paper test strips for detecting OA were prepared, and a fast detection method for okadaic acid using gold-labeled immunological assay was established. With the paper test strips, the detection limit was 6.25 ng mL^?1, and whole detection process for OA in shellfish samples needed only about 40 min.     

Detection of periodontopathogenic bacteria in pregnant women by traditional anaerobic culture method and by a commercial molecular genetic method

To culture facultative and strict anaerobic bacteria is a well-established method for analyzing subgingival plaque samples. Micro-IDent^® and micro-IDent^® Plus (HAIN Lifescience GmbH, Nehren, Germany) tests are two commercially available rapid PCR-based methods for the identification and quantification of putative periodontopathogen bacteria. In this study, we compared these commercial PCR-based hybridization methods with conventional anaerobic culture technique. A total of 36 subgingival plaque samples were collected from periodontal pockets of pregnant women with chronic localized periodontitis. Aliquots of these samples were evaluated with species-specific probes provided by micro-IDent^® and micro-IDent^® Plus tests simultaneously, and from the same samples anaerobic and capnophylic bacteria were cultured on selective media. The overall agreement between both methods was excellent for Eubacterium nodatum, Tannerella forsythia and Porphyromonas gingivalis (97–92%), fair for Capnocytophaga sp, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Prevotella intermedia (91–89%) and poor for Fusobacterium nucleatum, Parvimonas micra (Micromonas micros), and Campylobacter rectus (86–78%). Discrepancies in the results may be explained by inability of culture method to distinguish between closely related taxa (e.i P. intermedia/Prevotella. nigrescens), and problems of keeping periodontopathogen bacteria viable, which is required for successful detection by standard culture method. Nucleic acid-based methods may replace cultivation method as frequently used methods in microbiological diagnosis of progressive periodontitis, thus micro-IDent^® andmicro-IDent^® Plus tests can be recommended where culture of periodontopathogenic bacteria is not performed in routine microbiology laboratories to analyze subgingival plaque samples.     

An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes

Leptospira spp. serovars Hardjo and Wollfi from Sejroe serogroup have been detected in livestock in Brazil, where the main control procedures rely on vaccination. The potency of two commercial vaccines available in this country was monitored by microagglutination test-MAT and in vitro growth inhibition test-GIT in serum samples from 33 female buffaloes divided into: G1-unvaccinated control; G2-vaccinated with Leptobac-6^® containing serovars Hardjo and Wolffi and G3-vaccinated with Triangle-9^® containing serovar Hardjo. G2 and G3 animals were vaccinated on day zero, and received a booster and two revaccinations on days 30, 210 and 390 and G1 animals received phosphate buffered saline. Serum samples were collected at 15-day intervals between days 0 and 60; and at 30-day intervals between days 60 and 540 and were tested by MAT and GIT with serovars Hardjo and Wolffi. G1 remained negative throughout the experiment. Both vaccines were able to induce agglutinating and growth inhibition antibodies. Six months after the last revaccination, all animals tested negative by MAT, but still were positive by GIT until the end of experimental period. GIT could be a good tool to evaluate the potency and to monitor antibodies responses of vaccines of Sejroe group serovars.     

Validation of an automated method for compounding monoclonal antibody patient doses

Automation robots have recently come to the market as an alternative for manual compounding of drugs for intravenous administration. Our aim was to assess whether robotic compounding can be performed with monoclonal antibodies (mAbs) without influencing the aggregation state of the proteins. Three frequently used mAbs were studied: infliximab (Remicade^®, Janssen Biotech) and trastuzumab (Herceptin^®, Roche) in lyophilised form, and bevacizumab (Avastin^®, Roche) as a liquid formulation stored at 2°C to 8°C. The effects of different procedures to prepare the patient doses on antibody aggregation were evaluated. Remicade^® and Herceptin^® were reconstituted both manually and by a robotic arm (i.v.STATION^®, Health Robotics). Additionally, the influence of vigorous shaking during reconstitution was investigated. The effects of rapid aspiration and dispensing on antibody aggregation were investigated for all three mAbs. Aggregation state was assessed by UV-Vis absorbance, 90° light scatter, fluorescence spectroscopy, Nile red fluorescence microscopy, and field flow fractionation without cross and focus flow. Robotic reconstituted samples showed similar findings compared with manual reconstitution if performed exactly according to the summary of product characteristics (SPC). Vials that were vigorously shaken showed a significant increase in aggregates. Similarly, rapid aspiration/dispense cycles resulted in a strong increase in the number and sizes of aggregates for all three mAbs; this result was observed after just one rapid aspiration/dispense cycle. Our study showed that robotic compounding of mAbs is feasible if the robot is exactly programmed according to the SPC, indicating that robotic compounding can be used to achieve reproducible high-quality compounding for delicate formulations.     

Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine

The aim of this study is to develop an orally disintegrating film (ODF) containing a microparticulate measles vaccine formulation for buccal delivery. The measles vaccine microparticles were made with biocompatible and biodegradable bovine serum albumin (BSA) and processed by spray drying. These vaccine microparticles were incorporated in the ODF, consisting of Lycoat RS720®, Neosorb P60W® and Tween 80. The yield of the microparticles was approximately 85–95%, w/w. The mean size of the vaccine microparticles was 3.65?±?1.89 μm and had a slightly negative surface charge of 32.65?±?2.4 mV. The vaccine particles were nontoxic to normal cells at high concentrations (500 μg/2.5?×?10⁵ cells) of vaccine particles. There was a significant induction of innate immune response by vaccine microparticles which was observed in vitro when compared to blank microparticles (P?<?0.05). The vaccine microparticles also significantly increased the antigen presentation and co-stimulatory molecules expression on antigen presenting cells, which is a prerequisite for Th1 and Th2 immune responses. When the ODF vaccine formulation was dosed in juvenile pigs, significantly higher antibody titers were observed after week 2, with a significant increase at week 4 and plateauing through week 6 comparative to naïve predose titers. The results suggest that the ODF measles vaccine formulation is a viable dosage form alternative to noninvasive immunization that may increase patient compliance and commercial distribution.     

Raman spectroscopy characterization of antibody phases in serum

When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separation of mAbs in serum could guide lead optimization. However, serum contains numerous components, making it difficult to resolve mAb/serum mixtures at a scale amenable to analysis in a discovery setting. To address these challenges, a miniaturized assay was developed that combined confocal microscopy with Raman spectroscopy. The method was examined using CNTO607, a poorly-soluble anti-interleukin-13 human mAb, and CNTO3930, a soluble anti-respiratory syncytial virus humanized mAb. When CNTO607 was diluted into serum above 4.5 mg/mL, phase separation occurred, resulting in droplet formation. Raman spectra of droplet phases in mixtures included bands at 1240 and 1670 cm^?1, which are typical of mAb β-sheets, and lacked bands at 1270 and 1655 cm^?1, which are typical of α-helices. The continuous phases included bands at 1270 and 1655 cm^?1 and lacked those at 1240 and 1670 cm^?1. Therefore, CNTO607 appeared to be sequestered within the droplets, while albumin and other α-helix-forming serum proteins remained within the continuous phases. In contrast, CNTO3930 formed only one phase, and its Raman spectra contained bands at 1240, 1670, 1270 and 1655 cm,^?1 demonstrating homogeneous distribution of components. Our results indicate that this plate-based method utilizing confocal Raman spectroscopy to probe liquid-liquid phases in mAb/serum mixtures can provide a screen for phase separation of mAb candidates in a discovery setting.     

Intra-laboratory validation of the Ridascreen® SET Total kit for detecting staphylococcal enterotoxins SEA to SEE in cheese

Aim: To determine the performance of the Ridascreen^® SET Total kit, after sample extraction and concentration by dialysis, with regard to its use in official controls for staphylococcal enterotoxins under European Regulation (EC) No. 2073/2005 modified. This study was conducted on naturally contaminated cheese samples and compared with the results of the previously validated Vidas^® SET2 kit. Methods and Results: The effectiveness of the Ridascreen^® SET Total kit on naturally contaminated cheeses was compared to that of the Vidas^® SET2 kit by applying the EN ISO 16140 standard. Sensitivity and specificity were also compared using spiked buffer solutions and cheese samples with SEA to SEE toxins. Conclusions: This study showed that the Ridascreen^® SET Total kit is as effective as the Vidas^® SET2 kit. Significance and Impact of the Study: The Ridascreen^® SET Total kit was found to specifically detect SEA to SEE in cheeses. The Ridascreen^® SET Total can therefore be used to check the staphylococcal enterotoxin content and ensure consumer protection.     

Japanese encephalitis virus infection induces changes of mRNA profile of mouse spleen and brain

Background

Rotaviruses are the single most important cause of severe diarrhea in young children worldwide. The current study was conducted to assess whether colostrum containing rotavirus-specific antibodies (Gastrogard-R^®) could protect against rotavirus infection. In addition, this illness model was used to study modulatory effects of intervention on several immune parameters after re-infection.

Methods

BALB/c mice were treated by gavage once daily with Gastrogard-R^® from the age of 4 to 10 days, and were inoculated with rhesus rotavirus (RRV) at 7 days of age. A secondary inoculation with epizootic-diarrhea infant-mouse (EDIM) virus was administered at 17 days of age. Disease symptoms were scored daily and viral shedding was measured in fecal samples during the post-inoculation periods. Rotavirus-specific IgM, IgG and IgG subclasses in serum, T cell proliferation and rotavirus-specific delayed-type hypersensitivity (DTH) responses were also measured.

Results

Primary inoculation with RRV induced a mild but consistent level of diarrhea during 3-4 days post-inoculation. All mice receiving Gastrogard-R^® were 100% protected against rotavirus-induced diarrhea. Mice receiving both RRV and EDIM inoculation had a lower faecal-viral load following EDIM inoculation then mice receiving EDIM alone or Gastrogard-R^®. Mice receiving Gastrogard-R^® however displayed an enhanced rotavirus-specific T-cell proliferation whereas rotavirus-specific antibody subtypes were not affected.

Conclusions

Preventing RRV-induced diarrhea by Gastrogard-R^® early in life showed a diminished protection against EDIM re-infection, but a rotavirus-specific immune response was developed including both B cell and T cell responses. In general, this intervention model can be used for studying clinical symptoms as well as the immune responses required for protection against viral re-infection.     

Hsp90 inhibition and co‐incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T‐DM1

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody‐drug conjugate trastuzumab emtansine (T‐DM1; Kadcyla^®) is recommended as a first‐line treatment for patients with HER2‐positive metastatic breast cancer. T‐DM1 combines the antibody‐induced effects of the anti‐HER2 antibody trastuzumab (Herceptin^®) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T‐DM1‐HER2 complex has to be internalized and the trastuzumab part of T‐DM1 has to be degraded. HER2 is, however, considered endocytosis‐resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat‐shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti‐HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T‐DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti‐HER2 antibody pertuzumab (Perjeta^®).     

Modulation of hybridoma cell growth and antibody production by coating cell culture material with extracellular matrix proteins

The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC^® and COSTAR^® cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis.It was shown that coatings with fibronectin (0.2 μg/ml) lead to a substantial improvement of cell growth by 50–70% and an increase of monoclonal antibody production by 100–120%.Collagen I coatings showed an improvement in cell growth by 30–70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the α₂-chain of the α₂β₁-integrin, which is responsible for binding to collagen and laminin.However, the presence of β₁-integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells.