HPTLC assay of nicotine and cotinine in biological samples

This study presents the development of a simple high-performance thin layer chromatography (HPTLC) method for the determination of nicotine and its metabolite cotinine in human plasma and urine. The following mobile phases: methanol: ammonia (100:1.5, v:v), chloroform: acetone: ammonia (48.75: 48.75: 2.5, v:v:v), methanol: chloroform: ammonia (48.75: 48.75: 0.5, v:v:v) and glass plates precoated with silicagel 60 F254 (20x20) as a stationary phase were used. Densitometric scanning was performed at 263 nm. Two different extraction procedures have been applied: liquid-liquid extraction using dichloromethane at alkaline pH and solid-phase extraction using C18 cartridges. Preliminary tests in order to establish the system of solvents for development, as well as the range of linearity, were conducted. The best separation of nicotine and cotinine was obtained by using methanol: chloroform: ammonia (48.75: 48.75: 0.5, v:v:v) as the mobile phase. The liquid-liquid extraction technique led to better results than solid phase extraction. The regression curves were linear (with a corresponding correlation coefficient higher than 0.99) in the quantities range of 200 ng–1000 ng/spot for both nicotine and cotinine. The UV spectra confirm the identification of nicotine and cotinine both in the standards and in the extracts after liquid-liquid extraction. The proposed method can be applied for the simultaneous evaluation of nicotine and cotinine in biological samples at toxic/lethal levels. Thus, the method may be applicable in lethal nicotine intoxication cases in forensic toxicological analysis

An analytical basis for assaying buried biological contamination Interim report

Assay techniques for determining biological contamination of spacecraft material

The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure

Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally exposed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis. The main disadvantage of the CBMN assay is related to the variable micronucleus ( MN) background frequency, by which only in vivo exposures in excess of 0.2-0.3 Gy X-rays can be detected. In the last years, several improvements have been achieved, with the ultimate goals (i) of further increasing the sensitivity of the CBMN assay for low-dose detection by combining the assay with a fluorescence in situ hybridisation centromere staining technique, (ii) of increasing the specificity of the test for radiation by scoring nucleoplasmic bridges in binucleated cells and (iii) of making the assay optimally suitable for rapid automated analysis of a large number of samples, viz. in case of a large-scale radiation accident. The development of a combined automated MN-centromere scoring procedure remains a challenge for the future, as it will allow systematic biomonitoring of radiation workers exposed to low-dose radiation

Immunofunctional assay of human growth hormone (hGH) in serum: A possible consensus for quantitative hGH measurement

Confirmation of the diagnosis of GH deficiency in adults and children involves provocative testing for human (h) GH. Different commercially available immunoassays yield largely discrepant results in the measurement of GH levels in human serum. These discrepancies result in doubtful relevance of cut-off levels proposed for GH provocative testing. We have developed an immunofunctional assay method that allows quantitation of only those GH forms in circulation that possess both binding sites of the hormone for its receptor and thus can initiate a biological signal in target cells. An anti-hGH monoclonal antibody recognizing binding site 2 of hGH is immobilized and used to capture hGH from the serum sample. Biotin-labeled recombinant GH-binding protein in a second incubation step forms a complex with those hGH molecular isoforms that have both binding sites for the receptor. The signal is detected after a short third incubation step with labeled streptavidin. The assay is sensitive (detection range, 0.1-100 micrograms/L) and has average inter- and intraassay precisions of 10.3% and 7.3% respectively. Endogenous GH-binding protein does not interfere with the hGH result; placental lactogen slows no detectable cross-reaction in this immunofunctional assay. The degree of immunofunctionally active hGH forms in serum samples, calculated by comparison of immunofunctional assay and RIA results, varied between 52-93%. We propose this immunofunctional assay for GH measurement as a new reference method for hGH quantitation in serum. The immunofunction assay translates only hGH forms into an assay signal that are capable of dimerizing GH receptors and, thus, of initiating a biological effect in target cells

The effect of changing gravity and weightlessness on vasopressin control systems Progress report, 21 Oct. 1969 - 15 Feb. 1970

Changing gravity and weightlessness effects on vasopressin control systems, with immunochemical and biological assay studie

Development and application of a bioassay for follicle-stimulating hormone : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Physiology at Massey University

Follicle-stimulating hormone (FSH) is involved in the regulation and maintenance of vital reproductive processes, such as gametogenesis, follicular development and ovulation. Produced in the anterior pituitary, FSH is a glycoprotein hormone that exists as a family of isohormones. Follicle-stimulating hormone concentrations have traditionally been measured by radioimmunoassay (RIA). However, results generated using RIA are a determination of the immunological activity of FSH. The potential of FSH to generate a biological response cannot be measured by RIA. Therefore, the identification of physiologically significant differences in the activity of these isoforms requires the use of assay systems that can differentiate between the biological activity of the FSH isoforms. Commonly used assays for measuring the biological activity of FSH are based on the measurement of aromatase activity in cultured rat Sertoli cells following stimulation with FSH. However, these assays have an inherently high ethical cost involved due to the use of primary tissue culture. In addition, the variation in these assays associated with differences between animals is difficult to eliminate. Recently a bioassay for human FSH has been described based on FSH stimulation of cyclic AMP production by a Chinese hamster ovary (CHO) cell line stably expressing the human FSH receptor (FSH-R). The purpose of this study was to evaluate the potential usefulness of this CHO FSH-R cell line expressing the human receptor for FSH to be used as a bioassay to measure the biological activity of ovine FSH. The receptor cell line bioassay described in this study is based on the ability of FSH to stimulate cAMP production by cultured CHO FSH-R cells. Optimisation of the culture system to enable the bioactivity of ovine FSH to be measured by bioassay was undertaken. This involved optimising the density of cultured cells, the time in culture and time exposed to FSH and the most suitable dose range for FSH. The influence of matrix effects, such as those exerted by serum was also investigated. The specificity of the assay towards FSH was also determined as was the sensitivity, accuracy and precision of the assay. No stimulation of cAMP production was seen in CHO FSH-R cells following treatment with α-FSH, β-FSH, LH, TSH, GH, prolactin or vasopressin at concentrations up to 10 μg/ml. Although the methodology used differed slightly depending on the presence or absence of serum, all assayed were performed using the following methods and materials. Freshly thawed FSH-R cells were bulked up in culture, and aliquots of 1 x 105 to 5 x 105 cells/well dispensed into 48 well culture dishes and incubated overnight at 37°C. The assay culture media was then replaced with 0.25 ml fresh media (α-MEM + 0.1% BSA + 0.25 mM 3-isobutyl-1-methyl-xanthine) containing varying doses of NIH-FSH-RP2 (RP2) FSH preparations or FSH containing samples, and the cells incubated for 4 hours at 37°C. The assay culture media was then removed and stored frozen at -20°C until assayed for cyclic adenosine monophosphate (cAMP) by RIA. Once optimal assay conditions were determined, the CHO FSH-R cell bioassay was used to measure FSH concentrations in ovine serum, pituitary extracts and medium from cultures of ovine pituitary cells. It was found that the concentrations of FSH in serum from intact sheep was close to the detection limit of the assay. Thus, while FSH concentrations could be measured in serum from some sheep, other animals had concentrations that were too low to be accurately measured by the bioassay in its present form. The assay was, however, well suited to measuring FSH concentrations in serum from sheep that had elevated concentrations of FSH. In one study, FSH concentrations measured by the bioassay were compared to those measured by RIA in sheep that had been ovariectomised and then hypophysectomised. It was found that the profile of FSH concentrations following hypophysectomy was similar whether measured by RIA or by bioassay (R2=0.7513), though absolute concentrations sometimes differed. This suggested that the immunoassay and bioassay were not always measuring the same characteristics of FSH. The assay was also used to measure FSH concentrations in samples of ovine hypophyseal venous blood. However, the results obtained for these samples indicated a poor correlation between FSH concentrations obtained by bioassay and RIA. Levels of bioactive FSH in hypophyseal venous blood fluctuated markedly and were up to 10-fold higher than the associated RIA concentrations. The CHO-cell bioassay was also found to be very suitable for measuring pituitary concentrations of FSH. In one study, pituitary extracts underwent chromatography and the separated isoforms of FSH were analysed by bioassay and RIA. Again, there was excellent correlation (R2=0.9328) between the concentrations of FSH measured both assay types. However, some differences were apparent suggesting a discrepancy in the biological and immunological characteristics of different FSH isoforms. The bioassay was also used to measure FSH concentrations in media from pituitary cells in tissue culture where serially diluted samples displayed good parallelism with the RP2 FSH standard curve. Results of this study demonstrate that the CHO FSH-R cell bioassay is suitable for measuring the biological activity of ovine FSH in a variety of biological fluids. The use of a permanent cell line eliminates the high ethical cost associated with primary tissue culture that other bioassay systems have. The inherent variation associated with culture systems utilising tissue from different sources is also avoided. The sensitivity of the bioassay is suitable for measuring FSH in surgically altered sheep or hypophyseal blood concentrations where FSH levels are generally higher than those in the peripheral circulation. In addition to blood samples, the bioassay is also excellent for monitoring FSH activity in pituitary extracts and in media from tissue culture. However, the sensitivity of the bioassay currently does not always allow measurement of bioactive FSH concentrations in serum samples with low FSH levels. In summary, the CHO FSH-R cell bioassay described in this study offers a useful alternative to RIA and other bioassays for monitoring the biological activity of ovine FSH and its isoforms in various biological fluids. It is concluded that this convenient and robust bioassay may have considerable application in future investigations of ovine FSH bioactivity

A novel NGS library preparation method to characterize native termini of fragmented DNA.

Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report relative abundances of all lengths and types (5' and 3') of single-stranded overhangs, if present, on each DNA fragment with an overall accuracy between 80-90%. In addition, XACTLY retains the sequence of each native DNA molecule after fragmentation and can capture the genomic landscape of cleavage events at single nucleotide resolution. The XACTLY assay can be applied as a novel research and discovery tool for fragmentation analyses and in cell-free DNA

Remote Cell Growth Sensing Using Self-Sustained Bio-Oscillations

A smart sensor system for cell culture real-time supervision is proposed, allowing for a significant reduction in human effort applied to this type of assay. The approach converts the cell culture under test into a suitable “biological” oscillator. The system enables the remote acquisition and management of the “biological” oscillation signals through a secure web interface. The indirectly observed biological properties are cell growth and cell number, which are straightforwardly related to the measured bio-oscillation signal parameters, i.e., frequency and amplitude. The sensor extracts the information without complex circuitry for acquisition and measurement, taking advantage of the microcontroller features. A discrete prototype for sensing and remote monitoring is presented along with the experimental results obtained from the performed measurements, achieving the expected performance and outcomes