Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

Rapid colorimetric detection of antibody-epitope recognition at a biomimetic membrane interface.

Biomolecular recognition of antigens and epitopes by antibodies is a fundamental event in the initiation of immune response and plays a central role in a variety of biochemical processes. Peptide binding requires, in many cases, presentation of the peptides at interfaces, such as protein surfaces, cellular membranes, and synthetic polymer surfaces. We describe a novel molecular system in which interactions between antibodies and peptide epitopes displayed at a biomimetic membrane interface can be detected through induction of visible, rapid color transitions. The colorimetric assembly consists of a phospholipid/polydiacetylene matrix anchoring a hydrophobic peptide displaying the epitope at its N-terminus. The colorimetric transitions observed in the assembly, corresponding to perturbation of the polydiacetylene framework, are induced only upon recognition of the displayed epitope by its specific antibody present in the aqueous solution. Significantly, the color changes occur after a single mixing step, without further chemical reactions or enzymatic processing. The new molecular system could be utilized for studying antigen-antibody interactions and peptide-protein recognition, epitope mapping, and rapid screening of biological and chemical libraries.     

Surface characterization and efficiency of a matrix-free and flat carboxylated gold sensor chip for surface plasmon resonance (SPR)

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM–MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen–antibody interaction. For both interactions, the well characterized densely functionalized SAM–MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.     

Recognition of peptides by antibodies and investigations of affinity using biosensor technology

The study of peptide-antibody interactions has many applications in biology and medicine. Synthetic peptides corresponding to single protein epitopes are used instead of intact proteins as reagents for the diagnosis of viral and autoimmune diseases. Furthermore, antibodies raised against peptides are useful reagents for isolating and characterizing gene products. In this review, methods for analysing the molecular basis of peptide-antibody interactions are described, such as amino acid replacement studies, X-ray crystallography of peptide-antibody complexes and biosensor technology based on surface plasmon resonance. The importance of peptide conformation in antibody recognition is discussed, and the antigenic reactivity of epitopes in synthetic peptides and in cognate, intact proteins is compared.     

3D-Epitope-Explorer (3DEX): localization of conformational epitopes within three-dimensional structures of proteins

Neutralizing antibodies often recognize conformational, discontinuous epitopes. Linear peptides mimicking such conformational epitopes can be selected from phage display peptide libraries by screening with the respective antibodies. However, it is difficult to localize these "mimotopes" within the three-dimensional (3D) structures of the target proteins. Knowledge of conformational epitopes of neutralizing antibodies would help to design antigens able to elicit protective immune responses. Therefore, we provide here a software that allows to localize linear peptide sequences within 3D structures of proteins. The 3D-Epitope-Explorer (3DEX) software allows to map conformational epitopes in 3D protein structures based on an algorithm that takes into account the physicochemical neighborhood of C(alpha)- or C(beta)-atoms of individual amino acids. A given amino acid of a peptide sequence is localized within the protein and the software searches within predefined distances for the amino acids neighboring that amino acid in the peptide. Surface exposure of the amino acids can also be taken into consideration. The procedure is then repeated for the remaining amino acids of the peptide. The introduction of a joker function allows to map peptide mimotopes, which do not necessarily have 100% sequence homology to the protein. Using this software we were able to localize mimotopes selected from phage displayed peptide libraries with polyclonal antibodies from HIV-positive patient plasma within the 3D structure of gp120, the exterior glycoprotein of HIV-1. We also analyzed two recently published peptide sequences corresponding to known conformational epitopes to further confirm the integrity of 3DEX.     

Design and application of GB virus C (GBV-C) peptide microarrays for diagnosis of GBV-C/HIV-1 co-infection

The main objectives of the design of GB virus C (GBV-C) peptide microarrays are the miniaturisation of antigen–antibody interaction assays, the simultaneous analysis of several peptide sequences and the reduction in the volume of serum required from patients since this always represents a limiting factor in studies to develop new systems for diagnosing human diseases. We herein report the design of a microarray immunoassay based on synthetic peptides derived from the GBV-C E2 protein to evaluate their diagnostic value in detecting anti-E2 antibodies in HIV-1 patients. To this end, peptide microarrays were initially prepared to identify the most relevant epitopes in the GBV-C E2 protein. Thus, 124 peptides composed of 18 amino acids covering the whole E2-protein sequence, with 15 residue overlaps, were spotted in triplicate onto γ-aminopropyl silane-functionalised adsorbent binding slides. The procedure to select the E2 protein epitopes was carried out using serum samples from HIV-1-infected patients. The samples had previously been tested for the presence or absence of GBV-C anti-E2 antibodies by means of the Abbott test. Thus, 11 specific epitopes in the GBV-C E2 protein were identified. Subsequently, peptide antigen microarrays were constructed using the E2 epitopes identified to detect GBV-C anti-E2 antibodies in the serum of HIV-1-infected patients with no known GBV-C co-infection. The 11 peptides selected identified anti-E2 GBV-C antibodies among HIV-1-infected patients, and a reactivity of 47 % was established. The potential antigenic peptides selected could be considered a useful tool for designing a new diagnostic system based on peptide microarrays to determine anti-GBV-C E2 antibodies in the serum of HIV-1-infected patients.     

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87–90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81–95), NESP (81–95), and NESP (86–104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide–OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81–95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84–106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86–91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.     

MIMOTOPES,CONTINUOUS PARATOPES AND HYDROPATHIC COMPLEMENTARITY: NOVEL APPROXIMATIONS IN THE DESCRIPTION OF IMMUNOCHEMICAL SPECIFICITY

Most antigenic sites of proteins, known as discontinuous epitopes, are made up of residues on different loops that are brought together by the folding of the polypeptide chain. The individual loops are sometimes able, on their own, to bind to the antibody and they are then known as continuous epitopes. The binding sites of antibodies, known as paratopes, are built up from residues on six hypervariable loops known as complementarity determining regions (CDRs). Peptides corresponding to individual CDR loops are often able to bind the antigen and such peptides may be viewed as continuous paratopes. Using random combinatorial peptide libraries, it is possible to obtain peptides that bind to an antiprotein antibody without showing any sequence similarity with any part of the protein. Such epitope mimics are called mimotopes provided they are able also to elicit antibodies that react with the original antigen. The binding activity of mimotopes may partly be due to the phenomenon of hydropathic complementarity between epitope and paratope peptides. Although these concepts are vague in their structural connotation, they are useful for describing the immunological activity of linear peptides.     

Development of field-deployable instrumentation based on “antigen–antibody” reactions for detection of hemorrhagic disease in ruminants

Development of field-deployable methodology utilizing antigen–antibody reactions and the surface plasmon resonance (SPR) effect to provide a rapid diagnostic test for recognition of the blue tongue virus (BTV) and epizootic hemorrhage disease virus (EHDV) in wild and domestic ruminants is reported. A Spreeta chip, which utilizes microelectronic technology to implement the SPR effect, is shown to possess sufficient sensitivity and operating speed to detect either BTV and EHVD antigens or antibodies in real time. The biosensor consists of an outer active surface layer comprised of either an antibody or antigen immobilized by covalent bonds through several other organic layers including a self assembled monolayer to a gold surface. Parallel experiments were run on the biosensor surface using either a home-built high resolution SPR instrument or a low resolution solid state Spreeta SPR chip. Both instruments were capable of monitoring the antigen–antibody reaction used to selectively detect the presence of BTV and EHDV viral pathogens. Results for the antibody and antigen reactive layers with antigen or antibody solutions as well as the modeling of these layers are discussed. The characteristics of these biosensors – specificity and time of reaction – were assessed. The antibody surface biosensors exhibited a high degree of specificity, even when using low resolution instrumentation. The time of analysis was under 20 min, which was the arbitrary exposure time.     

CMOS image sensor for detection of interferon gamma protein interaction as a point-of-care approach

Complementary metal oxide semiconductor (CMOS)-based image sensors have received increased attention owing to the possibility of incorporating them into portable diagnostic devices. The present research examined the efficiency and sensitivity of a CMOS image sensor for the detection of antigen–antibody interactions involving interferon gamma protein without the aid of expensive instruments. The highest detection sensitivity of about 1 fg/ml primary antibody was achieved simply by a transmission mechanism. When photons are prevented from hitting the sensor surface, a reduction in digital output occurs in which the number of photons hitting the sensor surface is approximately proportional to the digital number. Nanoscale variation in substrate thickness after protein binding can be detected with high sensitivity by the CMOS image sensor. Therefore, this technique can be easily applied to smartphones or any clinical diagnostic devices for the detection of several biological entities, with high impact on the development of point-of-care applications.     

Comparison between RGD-peptide-modified titanium and borosilicate surfaces

The use of synthetic peptides containing adhesive sequences, such as the Arg-Gly-Asp (RGD) motif, represents a promising strategy to control biological interactions at the cell–material interface. These peptides are known to improve the tissue–material contact owing to highly specific binding to cellular membrane receptors known as integrins, thereby promoting the adhesion, migration and proliferation of cells. The peptides were coupled to borosilicate glass and titanium surfaces using silanisation chemistry. A tryptophan residue was incorporated into the amino acid sequences of selected peptides to facilitate the detection of the covalently bound peptides. Successful peptide immobilisation was proven by fluorimetric measurements. The confocal imaging analysis suggests a homogeneous distribution of the immobilised peptide across the biomaterial surface. In vitro cell proliferation assays were employed to compare the adhesion potentials of the well-known RGD-containing peptides GRGDSP, GRADSP and RGDS to the three peptides designed by our group. The results demonstrate that the RGD sequence is not necessarily required to enhance the adhesion of cells to non-biological surfaces. Moreover, it is shown that the number of adhering cells can be increased by changes in the peptide hydrophobicity. Changes in the cytoskeleton are observed depending on the type of RGD-peptide modification.     

Label-free quantification of cystatin C as an improved marker for renal failure

A label-free biosensor has been developed, allowing quantification of cystatin C in human serum. This was achieved by using reflectometric interference spectroscopy as detection method. Cystatin C is a small serum protein that allows detection of renal failure more reliably than established parameters as creatinine. The protein was immobilized on the surface of a glass transducer, forming the sensitive layer of the sensor chip. Based on a binding-inhibition assay, two different types of monoclonal cystatin C antibodies were compared, by their behavior and their obtained working range in buffer and serum as matrix. Both antibodies allowed quantification of the protein in serum as matrix within the required clinical ranges of 0.53–1.02 mg/L. Detected recovery rates are in a range between 84.8% and 116.1%. The developed sensor shows high inner chip reproducibility and low cross-sensitivity.     

Multiplex localization of sequential peptide epitopes by use of a planar microbead chip

Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.     

Use of polyclonal antibodies to ochratoxin A with a quartz–crystal microbalance for developing real-time mycotoxin piezoelectric immunosensors

A piezoelectric immunosensor was tested for ochratoxin A (OTA) mycotoxin detection through the immobilization of OTA–bovine serum albumin (OTA–BSA) conjugate on gold-coated quartz crystals (AT-cut/5 MHz). Immunoassays were performed in a flow-injection system through frequency decreases in a quartz–crystal microbalance (QCM) because of a mass increasing during immunoreaction with anti-OTA antibodies. Three immobilization procedures for OTA–BSA (direct adsorption and covalent attachment to two alkane thiol self-assembled monolayers) were characterized with QCM in real time. Covalent attachment of the OTA–BSA conjugates through gold nanoparticles was also tested for amplifying the signal. Binding of the excess of antibodies to the immobilized OTA in an indirect competitive analysis decreased linearly the resonant frequency in the range of the OTA concentration from 10 to 128 ng/mL, with a detection limit of 8 ng/mL (signal/noise ratio of 3). A pepsin 2 mg/mL (pH = 2.1) solution was used to release antigen–antibody complexes, regenerating the biorecognition surface.     

Development of a Protein Chip to Measure PKCβ Activity

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.     

Multiple Peptide Synthesis Methods and Their Applications. New Synthetic Methods (87)

Rapid developments in the biotechnology of new proteins, as well as advances in immunology and the development of pharmaceuticals based on inhibitors and antagonists, have led to immense demands for synthetic peptides. Simultaneous preparation of 100–150 completely different peptides, having chain lengths of up to 20 amino acids can nowadays be achieved using multiple synthesis methods. The yields and qualities of the peptides so obtained are high enough to permit reliable in vivo and in vitro screening for biological activities. Moreover, it is possible to optimize synthetic conditions and to carry out comparative studies on the secondary structures and conformational mapping of proteins. Special multiple synthesis methods facilitate the epitope mapping of larger peptides for diagnostic purposes and for the development of vaccines based on a few hundreds of free or rod-bound peptides that are useful for immunoassays. Multiple methods of peptide synthesis also enable the preparation of so-called peptide libraries which could comprise hundreds of thousands of peptides, and by which new perspectives for the screening of lead structures will be opened up. Peptide synthesis using a combination of photolabile protecting groups and photolithographic procedures enables the assembling of peptide libraries on small plates for use in miniature immunoassays. Furthermore, lipopeptide-antigen conjugates allow both the preparation of peptide-specific and monoclonal antibodies as well as a complete screening of epitopes of B-, T-helper and T-killer cells. Applications in the areas of AIDS diagnosis, the development of vaccines, and screening for the hormone analogues, demonstrate just some of the possibilities that have been opened up by multiple peptide synthesis methods.     

Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5–16) and (17–23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.     

Multifunctional nanoparticles as simulants for a gravimetric immunoassay

Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and ¹H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au–tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.     

A piezoelectric immunosensor for <Emphasis Type="Italic">Leishmania chagasi</Emphasis> antibodies in canine serum

The American visceral leishmaniasis is an important cause of morbidity and mortality in Brazil for both humans and dogs. Attempts to make a diagnosis of this disease need to be improved, especially in endemic areas, and in the tracking and screening of asymptomatic dogs, which are their main host in urban areas. A quartz crystal microbalance immunosensor for the diagnosis of the canine visceral leishmaniasis using a recombinant antigen of Leishmania chagasi (rLci2B-NH6) was developed. The rLci2B-NH6 was tightly immobilized on a quartz crystal gold electrode by self-assembled monolayer based on short-chain length thiol. The strategy was the use of the antigen-histidine tail covalently linked to glutaraldehyde performing a Schift base which permits a major exposure of epitopes and a reduced steric hindrance. The immunosensor showed good results regarding sensitivity and reproducibility, being able to distinguish positive and negative canine serum for L. chagasi. Furthermore, the immunosensor can be reused through exposure to sodium dodecyl sulfate solution, which promotes the dissociation of antigen–antibody binding, restoring the sensor surface with immobilized biologically active antigens for further analysis.     

Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision–mass spectrometry

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision–mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP^C) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision–MS, with complete suppression of nonspecific bPrP binding.