A general strategy to control antibody specificity against targets showing molecular and biological similarity: Salmonella case study.

The control of antibody specificity plays pivotal roles in key technological fields such as diagnostics and therapeutics. During the development of immunoassays (IAs) for the biosensing of pathogens in food matrices, we have found a way to rationalize and control the specificity of polyclonal antibodies (sera) for a complex analytical target (the Salmonella genus), in terms of number of analytes (Salmonella species) and potential cross-reactivity with similar analytes (other bacteria strains). Indeed, the biosensing of Salmonella required the development of sera and serum mixtures displaying homogeneous specificity for a large set of strains showing broad biochemical variety (54 Salmonella serovars tested in this study), which partially overlaps with the molecular features of other class of bacteria (like specific serogroups of E. coli). To achieve a trade-off between specificity harmonisation and maximization, we have developed a strategy based on the conversion of the specificity profiles of individual sera in to numerical descriptors, which allow predicting the capacity of serum mixtures to detect multiple bacteria strains. This approach does not imply laborious purification steps and results advantageous for process scaling-up, and may help in the customization of the specificity profiles of antibodies needed for diagnostic and therapeutic applications such as multi-analyte detection and recombinant antibody engineering, respectively.

Detection of wheat allergens using 2D Western blot and mass spectrometry.

BACKGROUND: Wheat allergy is relatively common and the associated clinical manifestations depend on the involved molecular allergens as well as on the way of exposure. Different symptoms have been described: wheat-dependent exercise-induced anaphylaxis (WDEIA), atopic dermatitis (AD) and pollen rhinitis (PR). Traditional diagnostic methods do not allow accurate molecular identification of the allergens that are essential for risk assessment and for the choice of the most adapted treatment. METHODS: Standardized total protein extracts obtained from wheat seeds were separated by 2D electrophoresis. Twenty-one sera with high wheat-specific immunoglobulin E (sIgE) levels were classified into three patients groups based on their clinical profile. These sera were tested by Western blot on 2D separated standardized wheat protein extract and their sIgE sensitization profiles were compared. RESULTS: Specific sensitization profiles were identified for each phenotype group. For WDEIA, protein spots around 37 kDa (pH 6-9) and 37-50 kDa (pH 5-6) were identified. For AD, spots were observed around 50 kDa (pH 9), 10 kDa (pH 9) and 20 to 75 kDa (pH3). For PR, specific spots were located around 90 kDa (pH 9). The mass spectrometry (UHPLC-MS/MS) analysis of these identified spots pointed out several potential interesting allergens: Tri a 26, Tri a bA, Tri a 34, Tri a tritin. CONCLUSIONS: The present study allowed the identification of different protein areas specific to these studied groups. The protein spots of interest were identified by UHPLC-MS/MS. It has been possible to establish a link between a specific symptomatology and the newly identified responsible allergens.

Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory.

Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.

Selection of egg peptide biomarkers in processed food products by high resolution mass spectrometry.

Food allergy is a growing health problem worldwide; thus, there is an urgent need for robust, specific, and sensitive analytical methods for detecting allergens. Mass spectrometry is an alternative to the existing methods, and it can overcome their limitations. One of the first steps in the development of any analytical method is the identification of the analytes to be further studied. In the case of allergen detection by mass spectrometry, the analytes are peptides. In this study, a strategy was developed for identifying potential peptide biomarkers in processed food products. This strategy was applied to processed egg matrices, and 16 potential peptide biomarkers were identified for the further detection and quantification of egg by means of mass spectrometry. With an empirical approach based on dedicated sample preparation, including tandem Lys-C/trypsin enzymatic digestion and high-resolution mass spectrometry analysis, hundreds of peptides from egg proteins were identified. This list of peptides was further refined with a series of criteria, obtained from empirical evidence, to identify the ideal biomarkers for the development of a quantitative method. These criteria include the resistance to food processing and the specificity of the peptides for eggs but also the effects of amino acid modifications and enzymatic digestion efficiency.

A mass spectrometry method for sensitive, specific and simultaneous detection of bovine blood meal, blood products and milk products in compound feed.

Feed sustainability is one of the biggest challenges for the next few years. Solutions have to be found that take feed quality and safety into account. Animal by-products are one valuable source of proteins. However, since the bovine spongiform encephalopathy (BSE) crisis, their use has been strictly regulated. The objective of this study was to propose a routine, sensitive and specific method using ultra-high performance liquid chromatography coupled to tandem mass spectrometry for the detection of blood-derived products and milk powder in feed. Contaminated aquafeeds were analysed in order to evaluate the sensitivity and specificity of the method. This new method meets both selectivity and sensitivity (0.1% (w/w)) requirements imposed by the European Commission for animal proteins detection methods. It offers an innovative and complementary solution for the simultaneously identification of authorised and unauthorised animal by-products such as processed animal proteins (PAPs).

Liquid chromatography coupled to tandem mass spectrometry for detecting ten allergens in complex and incurred foodstuffs.

Food allergy is a considerable heath problem, as undesirable contaminations by allergens during food production are still widespread and may be dangerous for human health. To protect the population, laboratories need to develop reliable analytical methods in order to detect allergens in various food products. Currently, a large majority of allergen-related food recalls concern bakery products. It is therefore essential to detect allergens in unprocessed and processed foodstuffs. In this study, we developed a method for detecting ten allergens in complex (chocolate, ice cream) and processed (cookie, sauce) foodstuffs, based on ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Using a single protocol and considering a signal-to-noise ratio higher than 10 for the most abundant multiple reaction monitoring (MRM) transition, we were able to detect target allergens at 0.5mg/kg for milk proteins, 2.5mg/kg for peanut, hazelnut, pistachio, and cashew proteins, 3mg/kg for egg proteins, and 5mg/kg for soy, almond, walnut, and pecan proteins. The ability of the method to detect 10 allergens with a single protocol in complex and incurred food products makes it an attractive alternative to the ELISA method for routine laboratories.

Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs.

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity.

Preliminary study on the presence of prednisolone in porcine urine and liver - How to distinguish endogenous from therapeutically administered prednisolone.

In animal breeding in Europe, synthetic corticosteroids are not allowed as growth-promoting agents. However, prednisolone residues have recently been found in porcine urine samples collected at slaughterhouses. The aim of this work was therefore to look for prednisolone in porcine urine and liver, to determine if detected residues might be of endogenous origin, and to check the possible relation with stress. An analytical method developed in-house was validated, combining immunoaffinity-based purification and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). This method was applied to urine and liver samples collected from sows experimentally treated either with prednisolone or tetracosactide hexaacetate (synthetic analogue of ACTH). Thanks to the performance of the analytical method, both cortisol and prednisolone were detected in all pig urine samples collected before or after administration of prednisolone or tetracosactide hexaacetate. High levels of prednisolone were found in porcine urine just after prednisolone administration, decreasing quickly to within the range detected in non-treated animals. In urine, the cortisol level varied depending on the time lapse between administration and sampling. On the other hand, prednisolone was detected also in liver samples of treated pigs. In this matrix, the cortisol level remained constant and prednisolone/cortisol level could be used to detect prednisolone administration at least 4 days after injection. In conclusion, the best indicator for detecting illicit prednisolone administration to pigs seems to be the prednisolone/cortisol ratio in liver samples. This preliminary work must be confirmed by a larger-scale study and metabolites should also be included.

Rapid multi-residue and multi-class qualitative screening for veterinary drugs in foods of animal origin by UHPLC-MS/MS.

Multi-class UHPLC-MS/MS was developed for the analysis of more than 160 regulated or banned compounds of various classes: anthelmintics including benzimidazoles, avermectins and others; antibiotics including amphenicols, beta-lactams, macrolides, pyrimidines, quinolones, sulphonamides and tetracyclines; beta-agonists; corticosteroids; ionophores; nitroimidazoles; non-steroidal anti-inflammatory agents; steroids; and tranquillisers. Samples were extracted with acetonitrile, without any additional purification step, and analysed by using UHPLC-MS/MS. Validation was done in accordance with the guidelines laid down by European Commission Decision 2002/657/EC for qualitative screening methods. This simple method proved applicable to routine screening for residues in egg, honey, milk and muscle samples at half the maximum concentration permitted by the European Union for each drug. In most cases, the target value was set at 5 µg kg(-1) for unauthorised compounds.

Multi-residue method for detecting coccidiostats at carry-over level in feed by HPLC-MS/MS.

A multi-residue HPLC-ESI-MS/MS method has been developed for the simultaneous extraction, detection and confirmation of the 11 coccidiostats referenced by Regulation 2009/8/EC (lasalocid sodium, narasin, salinomycin sodium, monensin sodium, semduramicin sodium, maduramicin ammonium alpha, robenidine hydrochloride, decoquinate, halofuginone hydrobromide, nicarbazin, and diclazuril) in feedstuffs at carry-over level. The sensitivity of the method allows quantification and confirmation for all coccidiostats below target concentration. The method was in-house validated and meets all criteria of European legislation (2002/657/EC). The precision of the method was determined under repeatability and within-laboratory reproducibility conditions; RSD(r) and RSD(R) were below the maximum permitted values for every tested concentration. The specificity was checked by analysing representative blank samples and blank samples fortified with potentially interfering substances (benzimidazoles, corticosteroides, triphenylmethane dyes, quinolones, nitrofurans, nitroimidazoles, phenicols); no interference were found. Concerning quantification, a quadratic regression model was fitted to every calibration curve with a regression coefficient r2 above 0.99 on each data set. Finally, the expanded uncertainty U was calculated with data obtained within the laboratory while applying the method during validation and in routine tests.

gamma-Aminobutyric acid type A receptors in the rat brain can contain both gamma 2 and gamma 3 subunits, but gamma 1 does not exist in combination with another gamma subunit.

Antibodies specific for the gamma 1, gamma 2, and gamma 3 subunits of the gamma-aminobutyric acid (GABA)A receptor have been used to probe the composition of naturally occurring GABAA receptors in the rat brain. Most GABAA receptors contain at least one of these three subunits. The percentage of each, determined by immunoprecipitation of [3H]muscimol binding, was 11 +/- 1%, 59 +/- 3%, and 14 +/- 2% for gamma 1, gamma 2, and gamma 3 subunits, respectively. Receptors containing gamma 2 or gamma 3 subunits were labeled by benzodiazepine site ligands with high affinity, whereas gamma 1-containing receptors could be labeled only by [3H]muscimol. Receptors immunoprecipitated by anti-gamma 2 or anti-gamma 3 antibodies were labeled with [3H]Ro 15-1788 with similar affinities (Kd for anti-gamma 2-immunoprecipitated receptors, 1.9 nM; Kd for anti-gamma 3-immunoprecipitated receptors, 1.7 nM). Immunoprecipitation or Western blot analysis of GABAA receptors solubilized from rat cerebellar or whole-brain preparations indicated that gamma 1 was not present coassembled with any other gamma subunit. Western blot analysis of receptors purified on alpha-specific immunoaffinity resins showed that gamma 1 was predominantly assembled with the alpha 2 subunit. Some GABAA receptors may contain more than one type of gamma subunit. Quantitative immunoprecipitation and Western blot analysis both indicated that gamma 2 and gamma 3 subunits can exist in the same receptor complex. A large proportion of GABAA receptors immunopurified on a gamma 3 affinity resin also appeared to contain a gamma 2 subunit. In contrast, when receptors were purified on a gamma 2 affinity resin a small proportion also appeared to contain a gamma 3 subunit. We conclude that most gamma 1-containing receptors have no other gamma subunit in the same receptor complex but some GABAA receptors contain both gamma 2 and gamma 3 subunits.

Model of subunit composition of gamma-aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their alpha and gamma/delta subunits.

Antibodies specific for subunits of the gamma-aminobutyric acid A (GABAA) receptor have been used to immunoprecipitate [3H]muscimol, [3H]Ro 15-4513, and [3H]Ro 15-1788 binding sites from deoxycholate-solubilized preparations of rat cerebellum. Of the antisera raised against alpha subunits, those specific for alpha 6 immunoprecipitated the largest proportion of receptors. Two populations of alpha 6-containing GABAA receptors were identified. The first was labeled with [3H]Ro 15-4513 and exhibited a pharmacological profile consistent with that observed for alpha 6 beta 2 gamma 2 in transfected cells (Lüddens, H., Pritchett, D. B., Kohler, M., Killisch, I., Keinanen, K., Monyer, H., Sprengel, R., and Seeberg, P. H. (1990) Nature 346, 648-651). The second population was labeled only with [3H]muscimol and was deduced, from quantitative immunoprecipitation studies using combinations of antibodies, to contain both alpha 6 and delta subunits. The alpha 6 subunit was not observed to be present in combination with other alpha subunits or the gamma 1 subunit. Each of the other alpha subunits was found to be present in only one population of receptors in the cerebellum. Some subunits (alpha 4, alpha 5, and gamma 3) were not detectable. By combining information from quantitative immunoprecipitation experiments and Western blot analysis, a model describing the composition of all GABAA receptors in the cerebellum was constructed that defined the following alpha and gamma/delta combinations (percentage of cerebellar GABAA receptors): alpha 6 gamma 2 (36%), alpha 6 delta (23%), alpha 1 gamma 2 (28%), alpha 2 gamma 1 (8%), and alpha 3 gamma 2 (5%).

GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties.

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.

Differential expression of GABAA receptor alpha-subunits in rat brain during development.

Unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor have been expressed in E. coli and used to generate polyclonal antisera specific for these subunits. The antibodies identify proteins by SDS-polyacrylamide gel electrophoresis and western blotting of molecular size 51 kDa, 53 kDa, 59 kDa and 55 kDa, respectively, which show differential patterns of expression during development. Whereas the alpha 2 and alpha 3 subunits are present at early stages, the expression of alpha 1 and alpha 3 subunits is low at birth and increases with age. This differential expression could be correlated with previous studies examining the developmental expression of BZ1 and BZ2 benzodiazepine binding sites.

[125I]iodoclonazepam, a specific high affinity radioligand for the identification of BZ1 and BZ2 sites in rat brain.

Binding of the radioligand [125I]iodoclonazepam to three different areas of rat brain (cerebellum, hippocampus and striatum) has been characterised. In all three regions binding is rapid, saturable and of high affinity (cerebellum Bmax = 1.49 +/- 0.3 pmol/mg of protein, Kd = 0.39 +/- 0.06 nM; hippocampus Bmax = 1.5 +/- 0.14 pmol/mg of protein, Kd = 0.38 +/- 0.6 nM and striatum Bmax = 0.53 +/- 0.1 pmol/mg of protein, Kd = 0.34 +/- 0.03 nm, n = 3). In all regions only one population of sites was apparent. However, competition for [125I]iodoclonazepam sites by a series of benzodiazepine agonists and antagonists showed some regional differences. The BZ1 selective compounds zolpidem and CL218,872 showed a 4.3-fold and 5.2-fold selectivity for cerebellar binding sites respectively. We conclude that [125I]iodoclonazepam is a novel, high affinity ligand which recognises both BZ1 and BZ2 classes of receptor and should be a useful addition to the panel of benzodiazepine ligands currently available.

Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells.

The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN'-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [( 3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells.

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.