Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

The longue durée of contraceptive methods

Medical history has so far paid hardly any attention to the longue durée, a history in which, according to Braudel, 'all change is slow'. It is therefore one of the challenges of the emerging field of the social history of medicine to work audaciously across time as well as across space. An interesting subject, for example, is the history of contraception. Throughout history, there has been almost nothing people have worried about more than having sex without fear of consequence. At first sight, the story of contraception seems to be one of progress, from the Ancient Egyptian crocodile-dung pessary to the modern-day condom and contraceptive pill. A closer look, however, shows a pattern of remarkable continuities in the reproductive behaviour of men and women over the last two thousand years. In this paper, I shall attempt to show why traditional male-dominated contraceptive methods still linger on despite the sexual revolution brought about by the invention of the 'pill' in the late 1950s.     

Which cervical sampler? A comparison of four methods

Four cytology sampling methods were compared in 1063 patients referred for colposcopy with a recent abnormal smear. A dyskaryotic smear of any grade was considered a positive result, though comparisons were limited to cases with a subsequent biopsy confirming CINII or III. There were no differences between the abilities of any of the four methods to detect higher grades of CIN (χ²₃=4.603, P >0.20). the presence or absence of endocervical cells in a smear was not significantly associated with any variation in success rate (χ²₁=0.959, P >0.30). the joint analysis of the four methods and the presence/absence of endocervical cells also showed no significant effects (χ²₇=12.768, 0.1 > P >0.05). In the latter analysis the trend towards a conventional level of significance was accounted for by the Aylesbury spatula giving a relatively high success rate when endocervical cells were present. the suggestion of advantage for the Aylesbury spatula merits further investigation.     

Precision nutrition — review of methods for point-of-care assessment of nutritional status

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Simple methods for the 3′ biotinylation of RNA

Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3′ ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3′-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3′ ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of ϕ29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme''s proofreading activity, can cope with more extended 3′ heterogeneities.     

Condensed tannins in the diets of primates: a matter of methods?

To understand the ways in which condensed tannins (CT) affect primate diet selection and nutritional status, correct measurements are essential. In the majority of studies of the CT contents of primate foods, a tannin source such as "quebracho" is used to standardize CT assays, but the CT in quebracho tannin may not be similar to those in the plants of interest. We investigated how the choice of standard to calibrate CT assays affects the estimation of CT in the diets of mountain gorillas (Gorilla beringei). We purified the CT from gorilla foods and compared the actual amounts of CT in the foods with estimates produced by using the quebracho tannin. When quebracho was used, the estimates of CT contents of gorilla foods were, on average, 3.6 times the actual content of CT so that the amounts in frequently eaten gorilla foods were substantially overestimated. The overestimation for a given plant could not be predicted reliably and the ranking of plants by tannin content differed according to the standard used. Our results demonstrate that accurate measurements of CT necessitate the use of tannins purified from the plant species of interest. A reevaluation of primatology studies using interspecific comparisons of tannin content will provide new insights into primate food selection and nutritional ecology.     

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.     

Computational methods for evaluation of cell-based data assessment—Bioconductor

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Highlights► Bioconductor proposes more than 20 R packages for FCM analysis. ► Data infrastructure proposed by flowCore has become the standard for many packages. ► Automated gating is improving but remains an intensive field of research.     

Far‐eastern strains of bean common mosaic virus and methods of their immunodiagnostics

The paper presents data of investigation on the physico‐chemical and antigenic properties of capsid proteins of the Bean common mosaic virus isolated from Phaseolus plants in the Russian Far East (BCMV‐R) and from China (BCMV‐C). A method for isolation of the virus preparation was selected. The purified preparations of two isolates BCMV have been obtained. The presence of one polypeptide in structural proteins of virions was established and their molecular masses determined (BCMV‐R ‐ 31,6 kD; BCMV‐C ‐ 32,1 kD). Polyclonal antiserum was obtained with titre 1:12800 and the indirect and “sandwich"‐variants of ELISA were developed to detect this virus. The allied relationships were established with the bean yellow mosaic virus and with the type representative of the genus Potyvirus ‐ PVY. Based on the data of physico‐chemical and antigenic properties it was concluded that isolates BCMV‐R and BCMV‐C are two independent strains of this virus. The presence of strain‐, virus‐ and genusspecific epitopes of capsid proteine was revealed as a result of comparison of antigenic characteristics of the Russian Far Eastern and Chinese strains of BCMV. A high antigenic activity of capsid protein of the Russian Far Eastern strain was observed.     

Power of counter movement jumps with external load – coherence of three assessment methods

Background

The purpose of this study was to evaluate the coherence between three different methods assessing the power driven from a counter movement jump (CMJ); the Powertimer 300-series contact mat (C-mat), the MuscleLab 4010 infrared mat (IR-mat) and the MuscleLab 4010 linear encoder (M-encoder), and to evaluate the test-retest reliability of the M-encoder.

Methods

Twenty-two males and 29 female, elite athletes performed two test sessions with three days in between. Each test session included counter movement jumps (CMJ) performed on a Smith-machine with external loads of 40 kg. Jump height and flight time were assessed with C-mat and IR-mat, and power was additionally assessed with C-mat. Variables analyzed from the M-encoder were average power (AP), average force (AV), average velocity (AV), and distance (D).

Results

The results from the C-mat were systematically higher than the ones obtained from the M-encoder and IR-mat. The correlation between the C-mat, M-encoder and the IR-mat was strong (r_p?=?0.95-0.98). The results showed a high test-retest reliability for all indices assessed with the M-encoder, AP (r_p?=?0.97, p?<?0.001; TE%?=?3.9%), AF (r_p?=?0.99, p?<?0.001; TE%?=?1.4%). Furthermore, the AV had high values (r_p?=?0.94, p?<?0.001; TE%?=?2.9%) as well as D (r_p?=?0.87, p?<?0.001; TE%?=?5.4%).

Conclusion

It is important to use the same equipment in both pre- and post-testing, since all three methods were reliable, coherent but not interchangeable to each other.

     

Analysis of informativeness of immunohistochemical and flow cytometric methods for estrogen receptor α assessment

Informative capacity analysis of immunohistochemistry (IHC) and flow cytometry (FCM) in the assessment of estrogen receptor α (ERα) expression in breast cancer tissue was performed. Similar frequencies of expression were shown by both methods: 27% of ERα-negative and 73% ERα-positive cases. However, IHC evaluation detected low levels in only 20% of ERα-positive cases, whereas low levels of ERα detected by FCM were 2 times more often (48%). Moreover, FCM revealed positive expression (23–60%) in 33% of IHC ERα-negative cases. Among IHC ER-positive cases, zero ERα expression was detected by FCM in 12.5%. The approaches to minimize errors in routine clinical determination of the estrogen receptor status were proposed.     

Study on the production of 6-pentyl-α-pyrone using two methods of fermentation

 The lactone 6-pentyl-α-pyrone has a characteristic coconut aroma and is produced by Trichoderma species. A study on the fermentative production of 6-pentyl-α-pyrone in both surface and submerged conditions by Trichoderma harzianum was carried out. Maximum concentrations of 455 mg/l and 167 mg/l after 96 h and 48 h of fermentation in surface and submerged conditions, respectively, were obtained without using any additional recovery operations. The resultant yields are higher than those previously reported in the literature, which may be attributable to strain characteristics in combination with the choice of fermentation conditions employed in the present study. Enough scope exists for further improvement in the yields by optimizing the cultural and nutritional parameters. Received: 13 July 1999 / Received revision: 19 November 1999 / Accepted: 19 November 1999     

Standardisation of data from real-time quantitative PCR methods – evaluation of outliers and comparison of calibration curves

Background  

As real-time quantitative PCR (RT-QPCR) is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR technique, through investigation of the experimental design and operating procedure. However, measurement uncertainty contributions made during the data analysis procedure have not been studied in detail. This paper presents two additional approaches for standardising data analysis through the novel application of statistical methods to RT-QPCR, in order to minimise potential uncertainty in results.     

Tag–probe labeling methods for live-cell imaging of membrane proteins

Instead of using reconstituted proteoliposomes, in situ investigations of membrane proteins in living cell membranes are important because the heterogeneous and dynamic nature of biomembranes significantly affects their behavior. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence measurements, particularly fluorescence microscopy. However, conventional genetic fusion with fluorescent proteins has several shortcomings. Post-translational labeling methods using a genetically encodable tag and synthetic probes targeting to the tag can overcome these limitations. This review summarizes emerging tag–probe techniques for labeling specific membrane proteins and their applications, including endocytotic internalization, partitioning to specific membrane domains, interprotein interactions, and conformational changes.     

Mild disintegration methods of microalgae–bacteria flocs from wastewater treatment

The use of microalgae in biological wastewater treatment has been widely studied. However, there is a dearth of information about estimating the microalgae and bacteria concentrations. In order to maintain a stable algal-bacterial system, it is necessary to quantify both the algal and bacterial biomasses. Typically, microalgae and bacteria from flocs in activated sludge contribute to better biomass settleability. However, flocs cause problems when it comes to estimating the individual biomass concentrations of microalgae and bacteria in a symbiotic algae-bacteria aggregate. This study aimed to find the best disintegration treatment with low influence on the viability of the microalgal cell determined by its photosynthetic activity. In the present work, biological (enzyme solution), chemical (formaldehyde), mechanical (glass bead-beating), and physical (sonication) treatments were performed on microalgae–bacteria flocs (ALBA flocs) to disintegrate the community as a pre-treatment step in order to develop a method for estimating the algal and bacterial concentration and to quantify the degree of disintegration. The effectiveness of the methods to disintegrate ALBA flocs in descending order are the following: sonication, bead-beating, formaldehyde and enzyme application. Sonication treatment (40 W, 6 min) showed the best disintegration performance of the microalgal-bacterial flocs, up to 90 % with 17 % loss of the algal photosynthetic activity. Bead-beating (3 mm diameter, 80 s) achieved 80 % of disintegration with only 6 % loss of its photosynthetic activity. These results demonstrate the possibility of mild disintegration of compact ALBA flocs without having any adverse impact on the microalgae cell. After these treatments, it becomes possible to estimate the individual biomass concentrations of algae and bacteria manually such as with a cell-counting chamber.     

New methods to approach immunotherapy of cancer – and strategies of tumours to avoid elimination

Owing to the intense effort of numerous investigators, the number of tumour antigens potentially of use for clinical immunotherapy continues to increase. At the same time, further strategies employed by tumour cells to avoid destruction by the immune system are being uncovered. A combined onslaught to target tumour cells and prevent their “escape” will be required for successful immunotherapy. Progress in this area was the subject of a meeting supported by the European Cancer Research Consortium “EUCAPS”, which was held in London in February 2000. This conference was the second of a series, the first of which was summarised previously in this journal [Pawelec G et al. (1999) Cancer Immunol Immunother 48: 214]. Received: 14 March 2000 / Accepted: 30 March 2000