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Monitoring the levels of therapeutic antibodies in individual patients would allow patient-specific dose optimization, with the potential for major therapeutic and financial benefits. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection directly in blood plasma. In this study, we targeted four clinically important therapeutic antibodies, the Her2-receptor targeting trastuzumab, the anti-CD20 antibodies rituximab and obinutuzumab, and the EGFR-blocking cetuximab. A strong correlation was found between the affinity of the antibody binding peptide and sensor performance. LUMABS sensors with physiologically relevant affinities and decent sensor responses were obtained for trastuzumab and cetuximab using mimotope and meditope peptides, respectively, with affinities in the 10-7 M range. The lower affinity of the CD20-derived cyclic peptide employed in the anti-CD20 LUMABS sensor ( Kd = 10-5 M), translated in a LUMABS sensor with a strongly attenuated sensor response. The trastuzumab and cetuximab sensors were further characterized with respect to binding kinetics and their performance in undiluted blood plasma. For both antibodies, LUMABS-based detection directly in plasma compared well to the analytical performance of commercial ELISA kits. Besides identifying important design parameters for the development of new LUMABS sensors, this work demonstrates the potential of the LUMABS platform for point-of-care detection of therapeutic antibodies.

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Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

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The androgen receptor (AR) signaling is critical for prostate cancer (PCa) progression to the castration-resistant stage with poor clinical outcome. Altered function of AR-interacting factors may contribute to castration-resistant PCa (CRPCa). Inhibitor of growth 1 (ING1) is a tumor suppressor that regulates various cellular processes including cell proliferation. Interestingly, ING1 expression is upregulated in senescent primary human prostate cells; however, its role in AR signaling in PCa was unknown. Using a proteomic approach by surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS) combined with immunological techniques, we provide here evidence that ING1b interacts in vivo with the AR. The interaction was confirmed by co-immunoprecipitation, in vitro GST-pull-down, and quantitative intracellular colocalization analyses. Functionally, ING1b inhibits AR-responsive promoters and endogenous key AR target genes in the human PCa LNCaP cells. Conversely, ING1b knockout (KO) mouse embryonic fibroblasts (MEFs) exhibit enhanced AR activity, suggesting that the interaction with ING1b represses the AR-mediated transcription. Also, data suggest that ING1b expression is downregulated in CRPCa cells compared with androgen-dependent LNCaP cells. Interestingly, its ectopic expression induces cellular senescence and reduces cell migration in both androgen-dependent and CRPCa cells. Intriguingly, ING1b can also inhibit androgen-induced growth in LNCaP cells in a similar manner as AR antagonists. Moreover, ING1b upregulates different cell cycle inhibitors including p27(KIP1), which is a novel target for ING1b. Taken together, our findings reveal a novel corepressor function of ING1b on various AR functions, thereby inhibiting PCa cell growth.

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Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this 'protein microarray on a smartphone'-concept for on-site testing, e.g., in food safety, environment and health monitoring.

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Current contaminant and residue monitoring throughout the food chain is based on sampling, transport, administration, and analysis in specialized control laboratories. This is a highly inefficient and costly process since typically more than 99% of the samples are found to be compliant. On-site simplified prescreening may provide a scenario in which only samples that are suspect are transported and further processed. Such a prescreening can be performed using a small attachment on a cellphone. To this end, a cellphone-based imaging platform for a microsphere fluorescence immunoassay that detects the presence of anti-recombinant bovine somatotropin (rbST) antibodies in milk extracts was developed. RbST administration to cows increases their milk production, but is illegal in the EU and a public health concern in the USA. The cellphone monitors the presence of anti-rbST antibodies (rbST biomarker), which are endogenously produced upon administration of rbST and excreted in milk. The rbST biomarker present in milk extracts was captured by rbST covalently coupled to paramagnetic microspheres and labeled by quantum dot (QD)-coupled detection antibodies. The emitted fluorescence light from these captured QDs was then imaged using the cellphone camera. Additionally, a dark-field image was taken in which all microspheres present were visible. The fluorescence and dark-field microimages were analyzed using a custom-developed Android application running on the same cellphone. With this setup, the microsphere fluorescence immunoassay and cellphone-based detection were successfully applied to milk sample extracts from rbST-treated and untreated cows. An 80% true-positive rate and 95% true-negative rate were achieved using this setup. Next, the cellphone-based detection platform was benchmarked against a newly developed planar imaging array alternative and found to be equally performing versus the much more sophisticated alternative. Using cellphone-based on-site analysis in future residue monitoring can limit the number of samples for laboratory analysis already at an early stage. Therewith, the entire monitoring process can become much more efficient and economical.

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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.

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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.

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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.

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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.

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The Inhibitor of Growth (ING) proteins represent a type II tumor suppressor family comprising five conserved genes, ING1 to ING5. While ING1, ING2 and ING3 proteins are stable components of the mSIN3a-HDAC complexes, the association of ING1, ING4 and ING5 with HAT protein complexes was also reported. Among these the ING1 and ING2 have been analyzed more deeply. Similar to other tumor suppressor factors the ING proteins are also involved in many cellular pathways linked to cancer and cell proliferation such as cell cycle regulation, cellular senescence, DNA repair, apoptosis, inhibition of angiogenesis and modulation of chromatin.A common structural feature of ING factors is the conserved plant homeodomain (PHD), which can bind directly to the histone mark trimethylated lysine of histone H3 (H3K4me3). PHD mutants lose the ability to undergo cellular senescence linking chromatin mark recognition with cellular senescence. ING1 and ING2 are localized in the cell nucleus and associated with chromatin modifying enzymes, linking tumor suppression directly to chromatin regulation. In line with this, the expression of ING1 in tumors is aberrant or identified point mutations are mostly localized in the PHD finger and affect histone binding. Interestingly, ING1 protein levels increase in replicative senescent cells, latter representing an efficient pathway to inhibit cancer proliferation. In association with this, suppression of p33ING1 expression prolongs replicative life span and is also sufficient to bypass oncogene-induced senescence. Recent analyses of ING1- and ING2-deficient mice confirm a tumor suppressive role of ING1 and ING2 and also indicate an essential role of ING2 in meiosis.Here we summarize the activity of ING1 and ING2 as tumor suppressors, chromatin factors and in development.