Dual-Color Bioluminescent Sensor Proteins for Therapeutic Drug Monitoring of Antitumor Antibodies.

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.

Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.

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.

Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone.

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.

Cellphone-based detection platform for rbST biomarker analysis in milk extracts using a microsphere fluorescence immunoassay.

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.

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.