Limiting dilution analysis

In production of the anti-RANTES monoclonal antibodies, after generation of the hybridoma fusions, plate ELISA was performed on 480 hybridomas. After repeated subcloning using limiting dilution analysis, four reproducibly positive hybridomas were characterized. 

Lohse AW, Dinkelmann M, Kimmig M, Herkel J, Meyer zum Buschenfelde KH (1996) Estimation of the frequency of self-reactive T cells in health and inflammatory diseases by limiting dilution analysis and single cell cloning. J Autoimmun 9 667-675. 

Rossert J, Pelletier L, Pasquier R, Druet P Autoreactive T cells in mercury-induced autoimmunity. Demonstration by limiting dilution analysis. Eur. J. Immunol. 1988 18 1761-1766. 

The accuracy of real-time PCR quantification depends not only on the method chosen to analyze the curves, but also on the quality of calibrators used. Purified PCR products quantified by spectrophotometry are easily obtained. When serially diluted, these calibrators can accurately quantify the amount of target in human genomic DNA. Alternatively, purified plasmids or genomic DNA can be used as calibrators. Limiting dilution analysis to determine the amount of ampMable DNA is seldom necessary. The precision of quantitative real-time PCR depends on the copy number. When the initial target concentration is low, imprecision is high. Part of the variance comes from stochastic limitations as defined by the Poisson distribution as described earlier. In addition, the PCR efficiency may be more variable at low copy numbers. 

Limiting dilution analysis is an all-or-none type of assay, conducted within a range of lymphoid cell dose where there is a measurable probability that a sample will not contain a species necessary for a response (Groves et al., I969). 

The best precision is obtained for isotope ratios near unity (unless the element to be determined is near the detection limit, when the ratio of spike isotope to natural isotope should be between 3 and 10) so that noise contributes only to the uncertainty of natural isotope measurement. Errors also become large when the isotope ratio in the spiked sample approaches the ratio of the isotopes in the spike (overspiking), or the ratio of the isotopes in the sample (underspiking), the two situations being illustrated in Fig. 5.11. The accuracy and precision of the isotope dilution analysis ultimately depend on the accuracy and precision of the isotope ratio measurement, so all the precautions that apply to isotope ratio analysis also apply in this case. 

Accurate determination of the biological important element, selenium, in blood serum by isotope dilution analysis using ICP-QMS with octopole collision cell (Agilent 7500ce, Tokio, Japan) is described by Schaumloffel and coworkers.55 A recovery of selenium from human serum reference material was only 78 % when hydrogen was applied as collision gas but 96.7 % using xenon as a non-reactive collision gas to eliminate isobaric interferences. A detection limit of 3.3p,gl-1 was achieved.55... 

Sector-based ICP-MS instruments have recently been used for seawater analysis. These instruments can provide high resolution to minimize spectral overlaps or high sensitivity when used in a low-resolution mode. The sample can be diluted to reduce sample-dependent variations in sensitivity while maintaining sufficient detection limits. Diluted seawater was introduced into the ICP without further sample preparation in one recent report [324]. Nonspectral matrix effects due to seawater for quadrupole and sector-based ICP-MS instruments have been compared [325]. 

M. Monperrus, E. Krupp, D. Amoiroux, O. F. X. Donard, R. C. Martin-Doimeadios, Potential and limits of speciated isotope-dilution analysis for metrology and assessing environmental reactivity, Trends Anal. Chem., 23 (2004), 261D272. 

For clcmcnt-speciPc detection in GC, a number of dedicated spectrometric detection techniques can be used, for example, quartz furnace AAS or atomic Bu-orescence spectrometry (AFS) for Hg, or microwave-induced plasma atomic emission spectrometry (MIP-AES) for Pb or Sn. However, ICP-MS is virtually the only technique capable of coping, in the on-line mode, with the trace element concentrations in liquid chromatography (LC) and capillary electrophoresis (CE) efBuents. The femtogram level absolute LoDs may still turn out to be insufficient if an element present at the nanogram per milliliter level splits into a number of species, or when the actual amount of sample analyzed is limited to some nanoliters as in the case of CE or nanoBow HPLC. The isotope spcciPcity of ICP-MS offers a still underexploited potential for tracer studies and for improved accuracy via isotope dilution analysis. 

Sanz-Medel and co-workers reported on selenium determination in biological samples by isotope dilution analysis in ICP-QMS with an octopole collision cell." The argon based isobaric interference arising during measurements of °Se was eliminated by using a hydrogen flow of 4 ml min in the octopole cell, so that it is possible to determine Se with a detection limit of 14pgg. ... 

Gas chromatography-olfactometry (GCO) has been used extensively for the identification of characteristic aroma conq)onents of foods (9,10). Aroma extract dilution analysis (AEDA) is a GCO technique in which serial dilutions (e.g. 1 3) of an aroma extract are evaluated by GCO. In AEDA, the highest dilution at which an odorant is last detected during GCO, so-called flavor dilution (FD) factor, is used as a measure of its odor potency (P). One potential drawback to AEDA is that the technique is limited to the analysis of components of intermediate and low volatility. To overcome this limitation, AEDA results have been con5>lemented by results of GCO of decreasing dynamic headspace (DHS) and decreasing static headspace (GCO-H) san5)les (70,77)... 

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