Detection of Antibodies against Endemic and SARS-CoV-2 Coronaviruses with Short Peptide Epitopes.

(1) Background: Coronavirus proteins are quite conserved amongst endemic strains (eCoV) and SARS-CoV-2. We aimed to evaluate whether peptide epitopes might serve as useful diagnostic biomarkers to stratify previous infections and COVID-19. (2) Methods: Peptide epitopes were identified at an amino acid resolution that applied a novel statistical approach to generate data sets of potential antibody binding peptides. (3) Results: Data sets from more than 120 COVID-19 or eCoV-infected patients, as well as vaccinated persons, have been used to generate data sets that have been used to search in silico for potential epitopes in proteins of SARS-CoV-2 and eCoV. Peptide epitopes were validated with >300 serum samples in synthetic peptide micro arrays and epitopes specific for different viruses, in addition to the identified cross reactive epitopes. (4) Conclusions: Most patients develop antibodies against non-structural proteins, which are useful general markers for recent infections. However, there are differences in the epitope patterns of COVID-19, and eCoV, and the S-protein vaccine, which can only be explained by a high degree of cross-reactivity between the viruses, a pre-existing immune response against some epitopes, and even an alternate processing of the vaccine proteins.

Glycosylation of bacterial antigens changes epitope patterns.

Introduction: Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines. Methods: Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level. Results: The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination. Discussion: This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.

Development and characterization of a disposable plastic microarray printhead.

During the last decade microarrays have become a powerful analytical tool. Commonly microarrays are produced in a non-contact manner using silicone printheads. However, silicone printheads are expensive and not able to be used as a disposable. Here, we show the development and functional characterization of 8-channel plastic microarray printheads that overcome both disadvantages of their conventional silicone counterparts. A combination of injection-molding and laser processing allows us to produce a high quantity of cheap, customizable and disposable microarray printheads. The use of plastics (e.g., polystyrene) minimizes the need for surface modifications required previously for proper printing results. Time-consuming regeneration processes, cleaning procedures and contaminations caused by residual samples are avoided. The utilization of plastic printheads for viscous liquids, such as cell suspensions or whole blood, is possible. Furthermore, functional parts within the plastic printhead (e.g., particle filters) can be included. Our printhead is compatible with commercially available TopSpot devices but provides additional economic and technical benefits as compared to conventional TopSpot printheads, while fulfilling all requirements demanded on the latter. All in all, this work describes how the field of traditional microarray spotting can be extended significantly by low cost plastic printheads.

Label-free serodiagnosis on a grating coupler.

The unique feature of the label-free measurement techniques for screening specific binding molecules against a certain ligand is that knowledge about the analyte is not required. Due to the direct monitoring of the binding event, no further detection step, e.g., by a fluorescently labeled antibody, is necessary. This technique enables not only the analysis of binding properties, but also applications in serodiagnosis and in primary screening in drug discovery. Especially when complex biological solutions such as blood serum are used as sample fluids, the minimization of unspecific attachment is the crucial point of the assay. In this chapter, the basic handling of the grating coupler as an example of a label-free transducer is described, together with a simple protocol to minimize unspecific attachment when measuring undiluted blood serum.

Detection of angiotensin II type 1 receptor ligands by a cell-based assay.

This work describes a cell-based assay that does not depend on radioactivity or laboratory animals for the detection of ligands of angiotensin II type 1 receptor (AT1R). The assay makes use of stable transfected Chinese hamster ovary cells (CHO-AT1R) expressing the AT1R. A sequential saturation assay principle was used in which receptor binding sites of the CHO-AT1R cells are blocked by the analyte in a concentration-dependent manner. Afterwards, TAMRA-angiotensin II, a fluorescence-labeled ligand, was added to bind to the remaining free binding sites of the receptor. In consequence, the fluorescence signal determined is inversely proportional to the concentration of the analyte.

Biosensor-based on-site explosives detection using aptamers as recognition elements.

Reliable observation, detection and characterisation of polluted soil are of major concern in regions with military activities in order to prepare efficient decontamination. Flexible on-site analysis may be facilitated by biosensor devices. With use of fibre-optic evanescent field techniques, it has been shown that immunoaffinity reactions can be used to determine explosives sensitively. Besides antibodies as molecular recognition elements, high-affinity nucleic acids (aptamers) can be employed. Aptamers are synthetically generated and highly efficient binding molecules that can be derived for any ligand, including small organic molecules like drugs, explosives or derivatives thereof. In this paper we describe the development of specific aptamers detecting the explosives molecule TNT. The aptamers are used as a sensitive capture molecule in a fibre-optic biosensor. In addition, through the biosensor measurements the aptamers could be characterised. The advantages of the aptamer biosensor include its robustness, its ability to discriminate between different explosives molecules while being insensitive to other chemical entities in natural soil and its potential to be incorporated into a portable device. Results can be obtained within minutes. The measurement is equally useful for soil that has been contaminated for a long time and for urgent hazardous spills.

Development of peptide microarrays for epitope mapping of antibodies against the human TSH receptor.

Accurate characterization of the antigen binding region of antibodies is of great value in many fields of research, assay development and clinical diagnostics. Up to now, there is an unmet clinical need to use antibodies as diagnostic markers for the prediction of both prognosis and therapeutic response. To this end, comprehensive but differentiated immunoassays need to be generated. We have developed a peptide microarray for the diagnosis and epitope mapping of anti-thyrotropin receptor antibodies. The primary sequence of the human thyrotropin receptor (hTSHR) was represented by a library of 251 synthetic peptides. The peptides were site-specifically immobilized in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde coated glass surface. The technology was used to map the epitopes of seven commercially available murine monoclonal antibodies specific for the human TSH receptor (mTSHRAb). A previously unknown epitope recognized by mTSHRAb 4C1 was identified at amino acids (AA) 379 through 384 and the epitope recognized by mTSHRAb A9 was also localized (AA 214-222). Previously identified epitopes recognized by mTSHRAbs 2C11 (AA 349-360), 28 (AA 34-39), 49 (AA 289-297), A7 (AA 406-411) and A10 (AA 34-39) were confirmed. The peptide microarray exhibited excellent performance in single and multiplex antibody analysis and high specificity. This technology may have potential as a multi-determinate in vitro diagnostic assay for the differential analysis of a heterogeneity of antibodies involved in the pathogenesis of autoimmune diseases.

Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics.

Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.

Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding.

Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.

Chemically synthesized zinc finger molecules as nano-addressable probes for double-stranded DNAs.

Our experiments describe an alternative method of dsDNA recognition using zinc finger (ZF) molecules which bind DNA specifically and with high affinity. Our aim was to develop zinc finger probes which are able to bind to dsDNA molecules at predetermined sites. In our basic approach we used pairs of complementary oligonucleotides to form dsDNAs, containing one of the three SP1-transcription factor motifs as a zinc finger recognition site. Two zinc finger probes of the SP1 motif were chemically synthesized and modified with a Dy-633 fluorophore. The SP1 peptides were folded into functional zinc fingers using zinc chloride. The addressable dsDNAs were immobilized on optical fibres, and the kinetics and binding rates of the artificial zinc finger probes were measured by a fluorescence detecting device (photomultiplying tube). The two zinc fingers and their corresponding DNA recognition sites served as specific probes and controls for the matching site and vice versa. Our experiments showed that a variety of dsDNA-binding probes may be created by modification of the amino acid sequence of natural zinc finger proteins. Our findings offer an alternative approach of addressing dsDNA molecules, for example for use in a nanoarray device.

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer.

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol), because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm(-2). The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.

Detection of progesterone in whole blood samples.

The progesterone concentration in blood samples can be utilised as a marker for the diagnosis of early pregnancy, endocrinopathy and virilism. Here, we describe a method for progesterone detection and measurement in whole blood samples by a surface sensitive biosensor used in conjunction with an integrated optical grating coupler. This device determines refractive index changes near the biosensor's surface. Hence, biological species bound to a surface layer can be measured in real-time without any label. For the measurements, we have modified the indirect competitive immunoassay principle. The concentration of the progesterone antibody was kept at 1 microg/ml. Progesterone concentration was determined in buffer solution and whole blood in a range between 0.005 and 10 ng/ml. The detection limit was determined to be 3 pM. The relative standard deviation was calculated to be 3.5%.

Cohort analysis of a single nucleotide polymorphism on DNA chips.

A method has been developed to determine SNPs on DNA chips by applying a flow-through bioscanner. As a practical application we demonstrated the fast and simple SNP analysis of 24 genotypes in an array of 96 spots with a single hybridisation and dissociation experiment. The main advantage of this methodical concept is the parallel and fast analysis without any need of enzymatic digestion. Additionally, the DNA chip format used is appropriate for parallel analysis up to 400 spots. The polymorphism in the gene of the human phenol sulfotransferase SULT1A1 was studied as a model SNP. Biotinylated PCR products containing the SNP (The SNP summary web site: ) (mutant) and those containing no mutation (wild-type) were brought onto the chips coated with NeutrAvidin using non-contact spotting. This was followed by an analysis which was carried out in a flow-through biochip scanner while constantly rinsing with buffer. After removing the non-biotinylated strand a fluorescent probe was hybridised, which is complementary to the wild-type sequence. If this probe binds to a mutant sequence, then one single base is not fully matching. Thereby, the mismatched hybrid (mutant) is less stable than the full-matched hybrid (wild-type). The final step after hybridisation on the chip involves rinsing with a buffer to start dissociation of the fluorescent probe from the immobilised DNA strand. The online measurement of the fluorescence intensity by the biochip scanner provides the possibility to follow the kinetics of the hybridisation and dissociation processes. According to the different stability of the full-match and the mismatch, either visual discrimination or kinetic analysis is possible to distinguish SNP-containing sequence from the wild-type sequence.

Integrated planar optical waveguide interferometer biosensors: a comparative review.

Integrated planar optical waveguide interferometer biosensors are advantageous combinations of evanescent field sensing and optical phase difference measurement methods. By probing the near surface region of a sensor area with the evanescent field, any change of the refractive index of the probed volume induces a phase shift of the guided mode compared to a reference field typically of a mode propagating through the reference arm of the same waveguide structure. The interfering fields of these modes produce an interference signal detected at the sensor׳s output, whose alteration is proportional to the refractive index change. This signal can be recorded, processed and related to e.g. the concentration of an analyte in the solution of interest. Although this sensing principle is relatively simple, studies about integrated planar optical waveguide interferometer biosensors can mostly be found in the literature covering the past twenty years. During these two decades, several members of this sensor family have been introduced, which have remarkably advantageous properties. These entail label-free and non-destructive detection, outstandingly good sensitivity and detection limit, cost-effective and simple production, ability of multiplexing and miniaturization. Furthermore, these properties lead to low reagent consumption, short analysis time and open prospects for point-of-care applications. The present review collects the most relevant developments of the past twenty years categorizing them into two main groups, such as common- and double path waveguide interferometers. In addition, it tries to maintain the historical order as it is possible and it compares the diverse sensor designs in order to reveal not only the development of this field in time, but to contrast the advantages and disadvantages of the different approaches and sensor families, as well.

Platform technologies for molecular diagnostics near the patient's bedside.

It is believed Lab-on-Chip systems will become a mainstream technology within the next centuries. Especially because of new findings in molecular medicine and global trends such as the changing global population in third world countries and an ageing population in industrial countries, the need for fast and reliable diagnostics is rising tremendously. Hence, diagnostics have to become more frequently and more easily available. In this regard, technologies have to be found that enable the cost-effective production and hence an affordable price. In a joint-project between seven Fraunhofer institutes a Lab-on-Chip system was developed which consists of a credit-card-sized cartridge and a base station. The cartridges contain besides the reagents necessary for a specific assay also functionalities such as pumping or heating enabling a self-contained system without any fluidic interfaces, which tend to be error-prone. Because of the modularity of the system it is possible to integrate an optical sensor as well an electrochemical sensor into the cartridge. Hence, the system can be customized to serve the needs of the particular assays. This chapter will describe the system including generic design rules for such Lab-on-Chip systems, the development of these rules into a modular Lab-on-Chip system, the integration of biomedical assays, and the production possibility of this system.

Miniaturization for Point-of-Care Analysis: Platform Technology for Almost Every Biomedical Assay.

Platform technologies for the changing need of diagnostics are one of the main challenges in medical device technology. From one point-of-view the demand for new and more versatile diagnostic is increasing due to a deeper knowledge of biomarkers and their combination with diseases. From another point-of-view a decentralization of diagnostics will occur since decisions can be made faster resulting in higher success of therapy. Hence, new types of technologies have to be established which enables a multiparameter analysis at the point-of-care. Within this review-like article a system called Fraunhofer ivD-platform is introduced. It consists of a credit-card sized cartridge with integrated reagents, sensors and pumps and a read-out/processing unit. Within the cartridge the assay runs fully automated within 15-20 minutes. Due to the open design of the platform different analyses such as antibody, serological or DNA-assays can be performed. Specific examples of these three different assay types are given to show the broad applicability of the system.

Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.

Highly-integrated lab-on-chip system for point-of-care multiparameter analysis.

A novel innovative approach towards a marketable lab-on-chip system for point-of-care in vitro diagnostics is reported. In a consortium of seven Fraunhofer Institutes a lab-on-chip system called "Fraunhofer ivD-platform" has been established which opens up the possibility for an on-site analysis at low costs. The system features a high degree of modularity and integration. Modularity allows the adaption of common and established assay types of various formats. Integration lets the system move from the laboratory to the point-of-need. By making use of the microarray format the lab-on-chip system also addresses new trends in biomedicine. Research topics such as personalized medicine or companion diagnostics show that multiparameter analyses are an added value for diagnostics, therapy as well as therapy control. These goals are addressed with a low-cost and self-contained cartridge, since reagents, microfluidic actuators and various sensors are integrated within the cartridge. In combination with a fully automated instrumentation (read-out and processing unit) a diagnostic assay can be performed in about 15 min. Via a user-friendly interface the read-out unit itself performs the assay protocol, data acquisition and data analysis. So far, example assays for nucleic acids (detection of different pathogens) and protein markers (such as CRP and PSA) have been established using an electrochemical read-out based on redoxcycling or an optical read-out based on total internal reflectance fluorescence (TIRF). It could be shown that the assay performance within the cartridge is similar to that found for the same assay in a microtiter plate. Furthermore, recent developments are the integration of sample preparation and polymerase chain reaction (PCR) on-chip. Hence, the instrument is capable of providing heating-and-cooling cycles necessary for DNA-amplification. In addition to scientific aspects also the production of such a lab-on-chip system was part of the development since this heavily affects the success of a later market launch. In summary, the Fraunhofer ivD-platform covers the whole value chain ranging from microfluidics, material and polymer sciences, assay and sensor development to the production and assembly design. In this consortium the gap between diagnostic needs and available technologies can be closed.

DNA microarrays.

Microarray technology provides new analytical devices that allow the parallel and simultaneous detection of several thousands of probes within one sample. Microarrays, sometimes called DNA chips, are widely used in gene-expression analysis, genotyping of individuals, analysis of point mutations and single nucleotide polymorphisms (SNP) as well as other genomic or transcriptomic variations. In this chapter we give a survey of common microarray manufacturing, the selection of support material, immobilisation and hybridisation and the detection with labelled complementary strands. However, DNA arrays may also serve as the basis for more complex analysis based on the action of enzymes on the immobilized templates. This property gives DNA microarrays the potential for being the template for whole PCR and transcription experiments with high parallelism, as will be discussed in the last section of this chapter.

DNA microarray for detection of antibiotic resistance determinants in Bacillus anthracis and closely related Bacillus cereus.

We developed a multiplex PCR for amplification of ten genes involved in resistance to ciprofloxacin, doxycycline, rifampin, and vancomycin in Bacillus anthracis and closely related Bacillus cereus. Enzymatic labelling of PCR products followed by hybridization to oligonucleotide probes on a DNA microarray enabled simultaneous detection of resistance genes tetK, tetL, tetM, tetO, vanA, and vanB and resistance-mediating point mutations in genes gyrA, gyrB, parC, and rpoB. The presented assay allows detection of clinically relevant antibiotic resistance determinants within 4h and can be used as a time-saving tool supporting conventional culture-based diagnostics.