Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain.

Processed milk and milk products produced from bovine milk, commonly contain ß-casein A1 (ßCA1) and ß-casein A2 (ßCA2). Since the presence of ßCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains ßCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between ßCA2 and ßCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discriminate between ßCA2 and ßCA1, this newly developed method was able to detect the addition of common bovine A1A2 milk to A2A2 milk, as low as 1%. Besides the detection of A2A2 milk purity, the developed assay can also be implemented as a rapid phenotyping method at dairy farms to replace the more invasive DNA-based screening. Additionally, the developed method was capable of detecting the addition of common bovine milk up to 1% in sheep, goat, buffalo, horse and donkey milk, which conforms to EU recommendations. In conclusion, a newly developed multiplex method capable of reliably detecting the dilution of A2A2 milk of multiple species, with common bovine milk up to 1%, is presented.

Detection of methionine- and alanine-recombinant bovine somatotropins and their induced antibodies in serum and milk of cows suggests blood-milk barrier specificity for these compounds.

The elimination of recombinant bovine somatotropin (rbST) and its induced antibodies through milk of 2 formulations is studied to propose a control strategy for its use or abuse. Two dairy cows were treated with alanine-rbST (Ala-rbST), which is identical to endogenous bovine somatotropin, and ten dairy cows were treated with methionine-rbST (Met-rbST), which differs by 1 amino acid from endogenous bovine somatotropin. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method able to measure rbST at a decision limit (CCα) of 0.8 ng/mL and 2.3 ng/mL for serum and milk, respectively. The results show that the administered Ala-rbST is transferred from blood to milk but that this is not the case for Met-rbST. This suggests a blood-milk barrier-related specificity for these compounds. In addition, rbST-induced antibodies were formed in animals treated with Ala-rbST and those treated with Met-rbST. In both treatments, the rbST-induced antibodies were transferred from blood to milk, showing no blood-milk barrier specificity for these antibodies. These elimination patterns show that, for enforcement purposes, the detection of rbST-induced antibodies in tank milk can serve to screen for rbST administration, and subsequent confirmatory serum analysis by LC-MS/MS is needed to identify whether Ala-rbST or Met-rbST has been used.

Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST.

Multiplex flow cytometric immunoassay for serum biomarker profiling of recombinant bovine somatotropin.

Recombinant bovine somatotropin (rbST) is licensed for enhancing milk production in dairy cows in some countries, for instance the United States, but is banned in Europe. Serum biomarker profiling can be an adequate approach to discriminate between treated and untreated groups. In this study a multiplex screening tool of a small set of biomarkers for pinpointing recombinant bovine somatotropin (rbST) (ab)use was developed and evaluated: insulin-like growth factor 1 (IGF-1), IGF binding protein 2 (IGFBP2) and rbST-induced antibodies were selected as rbST dependent markers and combined in one parallel assay format. For this, the color-encoded microspheres were used in a suspension array, with a dedicated flow cytometer. Serum samples obtained from an animal experiment with rbST-treated and untreated dairy cows were measured with the developed triplex immunoassay and biomarker responses on rbST treatment were evaluated. This resulted in characteristic treatment-dependent responses for all three individual biomarkers. Combining these results with the statistical prediction model k-nearest neighbours (kNN), resulted in good discrimination of treated and untreated animals: an overall sensitivity (true positive rate) of 89.1% and an overall specificity (true negative rate) of 97.7% were reached. Therefore, this is the first multiplex method which can be applied with high confidence for screening of unknown herds of cattle pinpointing at rbST (ab)use.

Simultaneous quantification of six major allergens in commercial foods for children using a multiplex array on a digital versatile disc.

The hypothesis of this study is centered around the logic that an enhanced analysis of potential allergens during the food production can lead to increased accuracy and reliability of food labeling. The development of a cost-effective and straightforward optoelectrical microanalytical system for the simultaneous quantification of the six most common food allergens (peanut, hazelnut, almond, milk, wheat, and soybean) is presented. The system uses a regular versatile disc (DVD) functionalized with highly selective antibodies in a microarray format and a DVD drive as the optical detector. The multiplexed assay reliably (RSD < 20 %) determines the level of the allergenic proteins ranging from 0.1 to 143.4 ng mL-1. The analysis of food consumables (biscuits, seafood substitutes, and probiotic foods) revealed a 100 % accuracy in identifying the allergens ingredients declared on the label. The method offers potential for application as a high throughput biosensing tool for screening multiple allergens in commercial foods.

Comparison of commercial allergen ELISA kits for egg detection in food matrices.

Consumption of low levels of egg already can evoke harmful physiological responses in humans in those allergic to eggs. By detection of egg in food products, using Egg ELISA kits to determine its unintended presence, food producers can respond to avoid potential safety or quality risks of their products. Selection of an ELISA kit fit for the issue at hand is challenging due to, amongst others, lack of information on assay performances with specified matrices. In this study, performances of seven commercial egg ELISA kits are compared for nine different relevant matrices: cookie, chocolate, pasta, dressing, stock cube, wine, vegetable drink and milk, ice cream and meat/meat replacers. The presence of egg was unified for all ELISA kits to mg total egg protein kg-1 food product. In every matrix, kit performances for recovery, intra- and interassay were compared, and also processing is accounted for by determination of egg in incurred samples. All seven kits were able to detect egg qualitatively at the VITAL3 ED01 level of 0.2 mg total egg protein and the corresponding relevant portion size for each matrix. For quantitative results, each ELISA kit showed an increase in detected egg concentration with increased egg levels and performed within the set criteria for recovery for the cookie, chocolate, stock cube and wine. For pasta, vegetable drink and milk, ice cream, and salad dressing, recovery of egg was within the set criteria for at least 4 ELISA kits. Most challenging matrices were meat/meat replacers, showing high matrix effects which could not be explained by the possible egg presence in the cognate blank. Only one ELISA kit was able to recover egg within the set criteria for the meat/meat replacer matrix. Results enable food industry to choose for ELISA kits suitable for egg detection in the matrix of interest.

Microsphere Peptide-Based Immunoassay for the Detection of Recombinant Bovine Somatotropin in Injection Preparations.

The use of peptides in immunoassays can be favored over the use of the full protein when more cost effective or less toxic approaches are needed, or when access to the full protein is lacking. Due to restricted access to recombinant bovine somatotropin (rbST), a protein enhancing growth and lactating performances of livestock, which use has been banned in the EU, Canada and Australia (amongst others), we developed a peptide-based biorecognition assay on an imaging planar array analyzer. For this, we identified the rbST epitope that is responsible for binding to the rbST-targeting monoclonal antibody 4H12 (MAb 4H12) to be 115DLEEGILALMR125. This linear peptide was synthesized and coupled to microspheres, after which it was tested in a biorecognition competitive inhibition assay format. We observed IC50 values of approximately 0.11 µg mL-1, which are lower than observed for the full rbST protein (IC50 = 0.20 µg mL-1). Importantly, there was no binding with the scrambled peptide. Preliminary results of directly coupled peptides in a microsphere biorecognition assay for detection of rbST are presented. Real-life applicability for detection of somatotropins (STs) in injection preparations of bovine-, porcine- and equine ST are shown. This newly developed immunoassay strongly supports future developments of peptide-based immunoassays to circumvent the limited access to the full protein.

Multiplex ready flow cytometric immunoassay for total insulin like growth factor 1 in serum of cattle.

The European Union has banned the use of recombinant bovine somatotropins (rbST, growth hormones) to increase milk yield in dairy cattle. As direct detection of rbST in serum is problematic, methods based on the detection of changes in multiple rbST-dependent biomarkers have high potential for monitoring rbST abuse. In this study immunoassays were developed for total insulin-like growth factor 1 (IGF-1) in cow sera. Ultimately aiming at combination with other rbST-dependent biomarker assays two multiplex formats were studied and compared critically, a multi-channel surface plasmon resonance (SPR)-based biosensor and flow cytometry combined with color encoded microbeads. Moreover, a new dedicated sample pretreatment was developed for the dissociation of complexed IGF-1 in serum, while keeping other biomarkers in solution. Compared to the SPR biosensor immunoassay, the flow cytometric immunoassay (FCIA) was more sensitive, less antibody-consuming and less vulnerable to necessary but interfering reagents from the sample treatment. In an initial in-house validation study the developed FCIA showed to be fast, specific, robust, and a high repeatability and reliability, and generated realistic IGF-1 values for bovine serum, without compromising the potential for simultaneous detection of other biomarkers. Due to the xMAP technology, in which 100 different bead sets can be measured simultaneously, the total IGF-1 assay can easily be extended with other immunoassays for candidate biomarkers. Preliminary results about a FCIA for IGF-1 multiplexing with insulin-like growth factor binding protein 2 (IGFBP2) are presented which strongly supported both the FCIA multiplex format as well as the generic nature of the developed sample pretreatment.

Biosensor immunoassay for traces of hazelnut protein in olive oil.

The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct biosensor immunoassay, based on a highly specific monoclonal antibody, was developed to detect the presence of hazelnut proteins in olive oils. The sample preparation was easy (extraction with buffer); the assay time was fast (4.5 min only) and the limit of detection was low (0.08 microg/g of hazelnut proteins in olive oil). Recoveries obtained with an olive oil mixed with different amounts of a hazelnut protein containing hazelnut oil varied between 93% and 109%.

A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products.

A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further study.

Potential of treatment-specific protein biomarker profiles for detection of hormone abuse in cattle.

Targeted protein biomarker profiling is suggested as a fast screening approach for detection of illegal hormone treatment in meat production. The advantage of using biomarkers is that they mark the biological response and, thus, are responsive to a panel of substances with similar effects. In a preliminary feasibility study, a 4-plex protein biomarker flow cytometric immunoassay (FCIA) previously developed for the detection of recombinant bovine somatotropin (rbST) was applied to cattle treated with steroids, such as estradiol, dexamethasone, and prednisolone. Each treatment resulted in a specific plasma biomarker profile for insulin-like growth factor-1 (IGF-1), IGF binding protein 2, osteocalcin, and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. In summary, the 4-plex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and therefore clearly shows the potential of biomarker profiling as a screening method in veterinary control. It is proposed to perform additional validation studies covering high numbers of treated and untreated animals to support inclusion or adaptation of protein biomarker approaches in future monitoring regulations.

Development of a flow cytometric immunoassay for recombinant bovine somatotropin-induced antibodies in serum of dairy cows.

Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4 weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle.

Multiple protein biomarker assessment for recombinant bovine somatotropin (rbST) abuse in cattle.

Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin - anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries.

Comparison of non-linear temperature-dependent development rate models applied to in vitro growth of entomopathogenic fungi.

Five non-linear models with three to five parameters, built to quantify the effect of temperature on insect development and microbial growth, were tested to describe the influence of temperature on in vitro-measured growth rates of entomopathogenic hyphomycetes. Data from two isolates of each of the four fungal species, Paecilomyces fumosoroseus, Beauveria bassiana, Metarhizium anisopliae, Metarhizium flavoviride, were used to assess the features of each model. Criteria for model evaluation included the statistical quality of parameters estimates, the goodness of fit to data, as well as the ability to provide estimates of several key parameters: the upper and lower development thresholds, the thermal optimum and the maximal growth rate at thermal optimum. The second model proposed by Brière et al. (1999) was found to be the best, and Ratkowsky's model (1983) also exhibited good features.

Effect of liquid culture media on morphology, growth, propagule production, and pathogenic activity of the Hyphomycete, Metarhizium flavoviride.

Two isolates of Metarhizium spp. were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M. flavoviride (or M. anisopliae var. acridum) strain IMI 330189, and Mf324 = M. flavoviride strain ARSEF324). Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects. Shake-flask experiments were carried out at 28 degress C in the dark. Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity. Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules. Submerged propagule yields were higher with Mf189 than with Mf324 in all seven media. While high concentrations of propagules (1.4 to 2.4 x 10(8) propagules ml(-1) for MF189 and 1.4 to 8.3 x 10(7) propagules ml(-1) for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins-Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media). Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules. In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides. In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia. Infection potential of submerged propagules was assayed on Schistocerca gregaria. Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 10(7) propagules ml(-1). All seven media produced submerged propagules that were highly infectious for S. gregaria larvae. Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins-Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control.

Fast biosensor immunoassays for the detection of cows' milk in the milk of ewes and goats.

Two monoclonal antibodies (MAb) raised against bovine kappa-casein were developed and applied in an automated optical biosensor (Biacore 3000) to create easy and fast direct and inhibition biosensor immunoassays (BIA) for the detection of cows' milk in the milk of ewes and goats. With both assay formats, low limits of detection (<01%) and fast run times (around 5 min) were obtained. For sample preparation, milk was diluted in buffer (direct assay) or in an antibody-containing buffer (inhibition assay) only. For quantitative analysis, calibrants of cows' milk in ewes' or goats' milk were used. Advantages of the direct BIA are: the single reagent format (biosensor chip immobilized antibodies only); the use of small amounts of antibodies (2 microg for >350 tests); and the wide measurement range (0.1 to 10% cows' milk). Despite these advantages, the inhibition BIA (using kappa-casein immobilized on the chip) was preferred because of the possible application of non-purified Mab, the higher responses, the higher sensitivity at relevant low percentages of cows' milk and its robustness (>800 cycles per chip).