Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg⁻¹ almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg⁻¹.We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman^® probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg⁻¹ almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg⁻¹. Further, between 100 and 100,000 mg kg⁻¹ spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman^® real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals.     

Tracking growth hormone abuse in sport: A comparison of distinct isoform-based assays

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22 kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20 kDa hGH isoforms and also a ratio 22/20 kDa is derived.     

Comparison Between Two Assays for Plasma Cortisol

Abstract

We have compared the results obtained for plasma cortisol when two methods were used to measure this steroid. The first method, a Competitive protein binding (CPB) assay, used transcortin as binding protein for cortisol. The second method is a radioimmunoassay (RIA) utilizing as binding reagent, a specific antibody against cortisol. Nineteen samples were tested covering a wide range of values. The CPB assay consistently overestimated the levels of plasma cortisol compared to the RIA method.     

Detection of Aflatoxins in Capsicum Spice Using an Electrochemical Immunosensor

Abstract

Electrochemical immunosensor based on immobilized aflatoxin B1 (AFB)–albumin conjugate and polyclonal antibody against AFB1 was performed for a competitive assay of AFB1 in capsicum spice. Spiked samples were used for construction of calibration curve. The proved limit of detection was 2.4 ppb. Immunosensor long-term stability was also estimated. Decrease of immunosensor sensitivity was less than 10% when stored in a refrigerator and approximately 22% for the immunosensor preserved at laboratory temperature for two weeks. Consequent performance of immunosensors for assay of real capsicum spice samples with proven aflatoxins presence produced good correlation with the data from valid method (high-performance liquid chromatography with fluorescence detector).     

The Determination of Methenamine and its Salts in Tablet Dosage Forms Using Ion-Pair Extraction

Abstract

This paper reports a simple, accurate, reproducible, stability-indicating procedure which may be used to quantitatively determine methenamine base, methenamine mandelate or methenamine hippurate in tablet dosage forms. The procedure initially involves the separation of methenamine from the dosage form and from formaldehyde, the decomposition product, by a cartridge ion-pair extraction process. The methenamine may then be assayed by a number of methods. In this paper the methenamine was hydrolyzed to formaldehyde and the formaldehyde determined by the Nash procedure. The method compares favorably with the USP XXI procedure for methenamine tablets. Advantages of the procedure include the ability to use a single method for the base and salt forms of the drug and the potential application to stability and quality control studies of methenamine dosage forms.     

A Sensitive Gas Chromatographic Method for the Determination of Metoprolol in Human Plasma

Abstract

A sensitive method for the determination of metoprolol in plasma has been developed. The procedure is based on gas chromatographic measurements of derivatized metoprolol, using 9-bromophenanthrene as internal standard. Metoprolol is derivatized with pentafluoropropionic anhydride. The resulting derivative gives a four-fold increase in sensitivity compared to the published methods where trifluoroacetic anhydride was used for derivatization.     

A Novel Protein Assay Method Using Tetraphenylporphin tetrasulfonate (TPPS4)

Abstract

A sensitive protein assay method which involves the reaction of TPPS₄ with protein is described. When protein is added to TPPS₄ solution, an absorption band with the maximum at 488 nm appears and the absorbance is proportional to the concentration of protein. Just Like the Soret absorption of the porphyrin, the new band is very narrow and there is no overlap at all between them, which means the free dyes would not give any background for the detection of the protein-TPPS₄ complexes. A new spectrophotometric method for determination of protein has been constructed and applied to the determination of human plasma protein and urinary protein; The assay using microtiter plates has also been studied.     

Pre-Incubation and Low Temperatures in Quantitative Radioreceptor Assays

Abstract

The detection limits of drugs in quantitative RRA are primarily determined by their affinities towards the receptor. Yet, the concentration of radiolabeled ligand, necessary for quantification of receptor-bound drug, increases the theoretical detection limit. Therefore the influences of low temperatures and pre-incubation on the detection limit was studied.     

Determination of Phenols Using Various Peroxidases

Abstract

Peroxidases of different origin — horseradish peroxidase isozyme C, alfalfa and peanut cationic peroxidases, tobacco leaves and novel fungal anionic peroxidases – were used to determine phenol and its analogues. Phenol and resorcinol were shown to be the inhibitors of the peroxidase activity towards o—dianisidine for all the enzymes tested, whereas pyrogallol and hydroquinone caused an appearance of a lag—period on a kinetic curve. The duration of a lag—period was proportional to the effector concentration and could be used to determine it. The novel fungal peroxidase from Phellinus igniarius exhibited the highest sensitivity towards phenols and they could be determined at the 10–6 – 10–7 M concentration levels.     

A Direct Spectrophotometric Assay for Pyruvic Oxime Hydrolase

Abstract

A sensitive spectrophotometric method, which directly measures the formation of pyruvic oxime from pyruvate and NH₂OH in the UV, is described for the detection of pyruvic oxime hydrolase activity in biochemical systems. The method was used in an attempt to detect pyruvic oxime hydrolase activity in cell-free extracts from an Alcaligenes sp. which can grow on pyruvic oxime. Although substantial pyruvic oxime + nitrite oxidative activity was detected in cells and cell-free extract of this bacterium, catalysis of pyruvic oxime formation was not observed within the error of the method (～20% of the uncatalyzed rate constant of 1.9x10^?3 s^?1 at pH 7, 22°C). The rate of pyruvic oxime oxidation by cells and cell-free extract was at least 10⁵?10⁶ times greater than the rate of its hydrolysis, thus implying that oxidation of pyruvic oxime need not require prior hydrolysis. The method would appear to be applicable to hydrolases directed toward a variety of oximes.