Immunoassay as an analytical tool in agricultural biotechnology

Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.     

Using DNA-binding proteins as an analytical tool

We propose that DNA-binding proteins can be used as highly efficient and versatile tools in analyses of DNA, RNA, and proteins. This work reports assays applying specific affinity probes: hybridization probes for analyses of DNA and RNA, and aptamer probes for analyses of proteins. Both types of probes are single-stranded DNA. In affinity analyses, in general, the probe (P) binds to a target molecule (T), and the amounts of the probe-target complex (P.T) and unbound P are determined. Distinguishing between P and P.T can be achieved by electrophoretic separation. If the electrophoretic mobilities of P and P.T are close in gel-free media, which is always the case for hybridization analyses, separation typically requires the use of a sieving matrix. Here we utilized a single-stranded DNA binding protein (SSB) to facilitate highly efficient gel-free separation of P and P.T in capillary electrophoresis (CE) for three types of targets: DNA, RNA, and proteins. When present in the CE run buffer, SSB binds differently to P and P.T. Due to this selective binding, SSB induces difference in electrophoretic mobilities of P and P.T in an SSB concentration-dependent fashion. The difference in the electrophoretic mobilities allows for affinity analyses of DNA, RNA, and proteins in gel-free CE. The large number of well-characterized DNA- and RNA-binding proteins and the diversity of their properties will allow researchers to design a comprehensive tool set for quantitative analyses of DNA, RNA, and proteins. Such analyses will facilitate identification of genomic DNA in ultra-small samples without error-prone and time-consuming PCR. They can also be used for monitoring gene expression at both mRNA and protein levels.     

The nuclear microprobe as an analytical tool

The nuclear microprobe is a spatially sensitive analytical instrument which exploits measurement of the particles or other radiations emitted when specimens are irradiated with beams of energetic charged-particles, most often having energies lying the range 0.5–4 MeV. Factors affecting the analytical and technical performance are discussed and examples given of application of the instrument to the fields of nuclear science and metallurgy, biology and geology.     

Electrification of gas bubbles as an analytical tool

When a gas passes through a liquid, a charge is produced and this is carried on in the droplets formed when the bubble bursts at the surface of the liquid. A theory to account for the relationship between the bubble charge and concentration of electrolyte, viz. q proportional, variant C(-1 2 ), is derived and applied to a number of uni-univalent and multivalent electrolytes. The general agreement between theory and experiment and the reasonable reproducibility of the technique show that it could have analytical application. Such use is exemplified in laboratory simulations of flowing systems for continuous analysis and in bubble-charge indication of titration end-points.     

Mutagenicity testing as an analytical tool in environmental pollution control

Screening of the environment for mutagens and carcinogens is a task which presents particular problems. Carcinogenicity tests involving mammals are prohibitively expensive for routine purposes and current procedures using the Salmonella reverse mutation test are insufficient.     

Genetic programming as an analytical tool for non-linear dielectric spectroscopy.

By modelling the non-linear effects of membranous enzymes on an applied oscillating electromagnetic field using supervised multivariate analysis methods, Non-Linear Dielectric Spectroscopy (NLDS) has previously been shown to produce quantitative information that is indicative of the metabolic state of various organisms. The use of Genetic Programming (GP) for the multivariate analysis of NLDS data recorded from yeast fermentations is discussed, and GPs are compared with previous results using Partial Least Squares (PLS) and Artificial Neural Nets (NN). GP considerably outperforms these methods, both in terms of the precision of the predictions and their interpretability.     

EPR as an analytical tool in assessing the mineral nutrients and irradiated food products-vegetables

EPR spectral investigations of some commonly available vegetables in south India, which are of global importance like Daucus carota (carrot), Cyamopsis tetragonoloba (cluster beans), Coccinia indica (little gourd) and Beta vulgaris (beet root) have been carried out. In all the vegetable samples a free radical corresponding to cellulose radical is observed. Almost all the samples under investigation exhibit Mn ions in different oxidation states. The temperature variation EPR studies are done and are discussed in view of the paramagnetic oxidation states. The radiation-induced defects have also been assessed by using the EPR spectra of such irradiated food products.     

Differential scanning calorimetry as an analytical tool in the study of drug-cyclodextrin interactions

The interactions of trimethoprim, sulphadiazine and sulphamethoxazole with natural (a- b-, g- ) and amorphous (RAMEB) or crystalline (DIMEB) methylated b-cyclodextrins were investigated both in aqueous solution (using phase-solubility analysis) and in the solid state (using DSC supported by X-ray analysis). In particular, DSC studies enabled determination of the relative degree of crystallinity of each drug in its physical and ground mixtures with the different cyclodextrins on the basis of the variation of its heat of fusion in comparison with that of the pure drug. In all cases, the host cavity size was a prevalent factor for the inclusion complexation in liquid state. On the contrary, it had a negligible effect on solid-state interactions in terms of drug amorphization. DIMEB and RAMEB exhibited similar performances in aqueous solution, showing that the presence of methyl-groups improved the complexing and solubilizing properties of b-cyclodextrin. However, DSC studies revealed that RAMEB was clearly more active in performing solid-state interactions, i.e. drug amorphization, and as stabilizing agent for the amorphous state brought forth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Investigation of high sensitivity GC-FTIR as an analytical tool for structural identification

As part of a detailed investigation into the application of GC-FTIR in industrial and environmental analysis, representative sets of samples have been analyzed in parallel using commercial high-sensitivity instruments. Two of the instruments utilize low temperature storage of the GC eluate to extend the time available for FTIR analysis, yielding greater sensitivity than that possible by conventional ‘light-pipe’ GC-FTIR. In certain circumstances, instruments using both types of sample storage give rise to spectra exhibiting features characteristic of the interface used. Chromatographic resolution was found not to be significantly degraded by use of either sample storage interface. Particular advantages were found in having parallel flame ionization detection and mass spectrometry; this enabled the location of smaller components and gave greater certainty of identification.     

Flow microcalorimetry and thermokinetics of liquid mixtures

The identification of the calorimetric curves corresponding to liquid mixtures for different injection flows, given by a flow microcalorimeter, permits to classify in a kinetic way the studied mixtures. For this purpose, it is determined the establishment time constant of the mixture (τ_mix) that allows us to estimate the length occupied by the dissipation (through the parameter λ_mix) and thus to justify the sensitivity variation obtained in different chemical calibrations.     

Two-dimensional electrophoresis protein profiling as an analytical tool for human acute leukemia classification

Two-dimensional electrophoresis (2-DE) was used to profile the proteins of leukemic cells from 61 cases of akute leukemia (AL) characterized by the French-American-British (FAB) classification. The differentially expressed protein spots were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). The distinct protein profiles (DPPs) of AL FAB subtypes were explored successfully, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5), and acute lymphoid leukemia (ALL), which were homogeneous within different samples of the same subgroup but clearly differed from all other subgroups. We also found a group of proteins differentially expressed between AL cells and normal white blood cells. Among the DPPs of AL subtypes, some proteins have been reported, but most of them were first reported here to mark AML differentiation and to discriminate AML from ALL. These data show that 2-DE protein profiling could be used as an analytical tool for facilitating molecular definition of human AL classification and understanding the mechanism of leukemogensis, and the extension of the present analysis to the currently less well-defined AL will identify additional subgroups and may promote the identification of new targets for specific treatment approaches.     

Silica gel with adsorbed Adogen 464 as an analytical sampling tool for anions

A solid extractant made with the liquid anion-exchanger Adogen 464 supported on silica gel has been prepared and its potential as a resin-like exchange material has been evaluated. In acid media it furnishes a ready available, inexpensive tool for recovery of anionic metal complexes as well as simple anions and for elimination of complex matrices. Copper and cobalt have been recovered (with a concentration factor of 20) from sea-water, natural water, metal alloys and industrial electroplating baths and measured by atomic-absorption spectrophotometry. The detection limits for copper and cobalt are 0.2 and 0.4 ng ml respectively and interferences are minimal. Chromium(VI) has been separated from chromium(III), and a concentration factor of 40 and a detection limit of 0.2 ng ml have been achieved.     

Flow field-flow fractionation as an analytical technique to rapidly quantitate membrane fouling

The initial fouling behavior of a clean membrane surface was studied using flow field-flow fractionation (flow FFF), an analytical technique typically used to separate and characterize macromolecules and particulates. This work represents the first time flow FFF has been used to quantitatively evaluate membrane performance. Flow FFF is an ideal tool for expeditiously studying sample–membrane interactions for the following reasons: membranes can be quickly installed into the flow FFF channel, each analysis requires only microgram amounts of sample, and sample–membrane interactions can be rapidly quantitated for different flowrates and solution compositions.Suwannee River humic acids were used as a probe to investigate the initial fouling of an XLE reverse osmosis membrane and an NF-200 nanofiltration membrane. Flow FFF was successfully used to quantitate the fouling of each membrane and to demonstrate that the majority of sample loss was due to irreversible adsorption. The fouling on both membranes was enhanced by increasing the flowrate perpendicular to the membrane surface and by adding calcium ions to the solution. The NF-200 membrane was more resistant than the XLE membrane to fouling in the presence of calcium ions, whereas, the fouling resistance of both membranes improved to similar levels with the addition of EDTA to a solution containing calcium ions.     

EDXRF as an analytical tool in art: Case studies from pigment identification and treatment assessment

Energy dispersive X-ray fluorescence (EDXRF) was employed for the identification of pigments decorating Hellenistic figurines, and the assessment of the efficiency of a treatment with barium hydroxide applied to stone. Elements present in the colored areas of the figurines, as well as the treated stone was identified by EDXRF. These data together with complementary information obtained by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction analysis (XRD) led to the identification of several precious pigments. As far as the treatment efficiency is concerned, EDXRF analysis revealed that barium is unevenly distributed on the treated surface and reaches a maximum depth of 2.5 mm.     

EDXRF as an analytical tool in art: Case studies from pigment identification and treatment assessment

Energy dispersive X-ray fluorescence (EDXRF) was employed for the identification of pigments decorating Hellenistic figurines, and the assessment of the efficiency of a treatment with barium hydroxide applied to stone. Elements present in the colored areas of the figurines, as well as the treated stone was identified by EDXRF. These data together with complementary information obtained by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction analysis (XRD) led to the identification of several precious pigments. As far as the treatment efficiency is concerned, EDXRF analysis revealed that barium is unevenly distributed on the treated surface and reaches a maximum depth of 2.5 mm.     

Hydrogen exchange mass spectrometry as an analytical tool for the analysis of amyloid fibrillogenesis

In this study, we have used glucagon as a model system for analyzing amyloid fibrillogenesis by hydrogen exchange MALDI mass spectrometry (HXMS). The hydrogen exchange mass spectrometry data correlated well with the traditional method based on Thioflavin T fluorescence and provided quantitative information by measuring the fibrillating molecules directly. The hydrogen exchange mass spectrometry data collected during fibrillogenesis revealed that glucagon fibrillation was a two component system showing an on/off type of interaction where only monomeric and fibrils were present without any substantial amount of intermediate species. This was evident by the extensive deuteration of the monomer and protection of the entire 29 residue glucagon peptide upon fibrillation.. The method complements the traditional procedures and has the potential to provide new information with respect to the nature of transient species, the structure of the growing fibrils and the mechanism of formation.     

Polymerase chain reaction technology as analytical tool in agricultural biotechnology

The agricultural biotechnology industry applies polymerase chain reaction (PCR) technology at numerous points in product development. Commodity and food companies as well as third-party diagnostic testing companies also rely on PCR technology for a number of purposes. The primary use of the technology is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.