Characterizing specific phage-protein interactions by fluorescence correlation spectroscopy

The interactions of several affinity reagent displayed T7 and M13 phage particles with their corresponding target molecules were examined using Fluorescence Correlation Spectroscopy (FCS). Diffusion times, relative fractions of each component in the recognition reactions at the equilibrium state, and ultimately the dissociation constants were deduced from analyzing the fluorescence autocorrelation curves. Although the sample preparation and FCS characterization of icosahedral T7-related systems were relatively straight forward, procedures with filamentous M13-related systems were complicated by the physical size of M13 and its aggregate formation. Methods that accommodate the FCS measurement of the M13 phage via changing confocal optics, fitting procedures, and aggregate discrimination are presented and discussed.     

Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles

Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.     

Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display

The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. On the basis of phage display technology, we recently reported the first humanization of a rabbit monoclonal antibody. The allotypic diversity of rabbit immunoglobulins prompted us to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, we evaluated the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes. Most rabbit light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intrachain disulfide bridges shared with mouse and human kappa light chains. Here we evaluate the impact of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries. We demonstrate that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones with b9 allotype is a rabbit/human Fab that binds with a dissociation constant of 1nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer. Examination of 228 new rabbit antibody sequences allowed for a comprehensive comparison of the LCDR3 and HCDR3 length diversity in rabbits. This study revealed that rabbits exhibit an HCDR3 length distribution more closely related to human antibodies than mouse antibodies.     

Development of human monoclonal antibodies: A review

This article describes the current status in the development of human monoclonal antibodies. Over the last ten years a lot of information about the human immune system has emerged. Combining these with the many new (bio-)technologies it is plausible that the long awaited breakthrough of this technology is close. This paper focuses on the classical cell-biological methods of achieving stable, antibody-producing human cell lines via cell fusion methods or virus derived transformations of human B-lymphocytes, as well as genetic engineering methods e.g. DNA libraries or phage display technology. The available in vitro immunization methods are critically reviewed and their impact on this topic is discussed. Therapeutic applications for cancer treatment or passive immunization against infectious diseases with antibodies derived by both ways are also reviewed.     

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

TEM-1 beta-lactamase as a scaffold for protein recognition and assay

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.     

Construction of a combinatorial immune library of human single-chain antibodies to orthopoxviruses and selection of antibodies to recombinant prA30L of the variola virus

A combinatorial immune library of human single-chain antibodies (scAbs) was constructed using the genes coding for the variable domains of the heavy and light chains of human immunoglobulins. The genes were cloned from lymphocytes of four subjects vaccinated with the vaccinia virus (VACV). The library included 3 · 10⁷ independent clones. After enrichment with clones producing scAbs against a recombinant analog of the variola virus envelope protein prA30L, the library was used to select a panel of scAbs binding both prA30L and VACV. All scAbs selected were tested for virus-neutralizing activity, and two scAbs proved to suppress VACV plaque formation in monolayers of Vero E6 cells. The specificity of antigen binding was verified by ELISA and Western blotting. The amino acid sequences of the virus-neutralizing scAbs were determined by sequencing their genes.     

A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75NTR

The neurotrophin receptor p75^NTR is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75^NTR antibody or phage scFv library pre-panned against p75^NTR are internalized by neurons expressing p75^NTR; (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75^NTR antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75^NTR expression is upregulated in motor neurons in response to injury and in disease, the p75^NTR antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.     

Fluorescence correlation spectroscopy as a new method for the investigation of aptamer/target interactions

Fluorescence correlation spectroscopy is an attractive tool for monitoring molecular interactions in solution. We report here a new and highly sensitive method for studying the interaction of aptamers with their targets using this technique. In vitro selection technology is a combinatorial method for the generation of nucleic acid receptors (aptamers) that are capable of binding to various target molecules. Using the in vitro selection approach we isolated RNAs which bind to the antibiotic moenomycin with high affinity. The formation of RNA-moenomycin complexes was studied by fluorescence correlation spectroscopy with a tetramethylrhodamine-labeled derivative of moenomycin.     

Generation and characterization of human monoclonal antibodies to G5, a linear neutralization epitope on glycoprotein of rabies virus, by phage display technology

The aim of the present study was to discover distinct human MAbs to RV with high neutralizing potency and a broad neutralization spectrum. A phage display technology was used to produce human scFv to G5, a conserved linear neutralization epitope on Gp of RV. A phage display scFv library with 6 x 10(7) members was constructed and the phage-scFv with 'antigen-binding' activities were selected with synthetic peptide G5-24. The obtained scFv genes were cloned into pET22b(+)/BL21(DE3) and from this we prepared purified scFv fragments. The assay of the specificity characteristics and neutralization capacity showed that two distinct clones with new human immunoglobulin V genes can recognize G5 specifically as well as neutralize different RV strains. They have potential for inclusion in an antibodies combination aimed for use in rabies PEP.     

Application of photon correlation spectroscopy as a technique for detecting culture contamination

The application of photon correlation spectroscopy (PCS) to detect culture contamination in chemostats was studied. It was found that the presence of a given particle size in a population of particles of a different size could be detected, but this ability was strongly dependent on particles of a different size could be detected, but this ability was strongly dependent on particle size difference and was most sensitive when contaminants are larger than the host. The inherent polydisparity of actively growing and dividing microbial cells negates any advantage in the use of multi-angle PCS to detect contaminants.     

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.     

Isolation and characterization of a novel scFv antibody fragments specific for Hsp70 as a tumor biomarker

Many studies have shown that more than 50% of tumors express heat shock protein 70 kDa (Hsp70) at the plasma membrane surface while not seen in normal cells, therefore it is a promising therapeutic target in human cancers. Hence, we used phage display technology to produce a single-chain fragment variable (scFv) antibody against human Hsp70. For this, a target peptide from human Hsp70 was designed using bioinformatics studies and was chemically synthesized. Then, the selection was performed using four rounds of biopanning with a stepwise decreased amount of the target peptide. Fourteen positive scFv clones were selected using monoclonal phage enzyme-linked immunosorbent assay screening, which was further characterized by means of the polymerase chain reaction and DNA sequencing. Among them, the G6 clone was selected to express scFv into the Escherichia coli. Expression and purification of the scFv shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blot analysis. In silico analysis confirmed specific binding of the scFv to Hsp70 in CDR regions. The specificity of the scFv measured by surface plasmon resonance and immunofluorescence of the A549 human lung carcinoma cell line confirmed the in vitro function of the scFv. Based upon these findings, we propose a novel anti-human Hsp70 scFv as potential immunotherapy agents that may be translated into preclinical/clinical applications.     

The use of fluorescence correlation spectroscopy (FCS) as an alternative biomarker detection technique: a preliminary study

Biomarkers are essential part of daily medical practice. Currently, biomarkers are being used both for diagnostic and prognostic purposes. There are many approaches e.g. ELISA by which biomarker levels are detected from patient samples. However, all these approaches are laborious, time consuming and expensive. There is therefore a general need for exploring new technique which can overcome these drawbacks. Here, we present a preliminary study for detection of serum biomarkers by fluorescence correlation spectroscopy (FCS) based diagnostic technique. FCS is a technique basically used for spatial and temporal analysis of molecular interactions of extremely low-concentration biomolecules in solution. FCS is able to measure diffusion time of the fluorescent molecules passing through the open detection volume and it can also measure the average number of fluorescent molecules passing through the detection volume. Because diffusion speed is correlated with shape and molecular mass of the fluorescent molecule, this property makes it possible to study the complex formation between a small fluorescently labelled and a large unlabelled molecule. In this preliminary study, we utilize this FCS property for detection of serum biomarker. Further studies on various pathological serum samples are warranted to explore further aspects of this technique.     

Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations

Homotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies. Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics. In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen (2008; this issue, pp. 597–609 ). In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces.     

Fluorescence spectroscopy as a tool for monitoring solubility and aggregation behavior of beta-lactoglobulin after heat treatment

Denaturation and aggregation of whey proteins are of interest to the food and pharmaceutical industry due to the importance of final structure in functionality, impact on food texture, and the chemical stability of the final product. In this study, we demonstrate the potential of fluorescence spectrometry combined with multivariate chemometric methods for quantifying solubility and aggregation behavior of beta-lactoglobulin (beta-LG); a major whey protein and a frequent food ingredient. Heat-induced aggregation of beta-LG was studied under different conditions including pH, temperature and heating durations. Results showed very good agreement between the fluorescence-based predictions and measurements obtained by HPLC and gravimetric analysis regardless of the conditions. Standard normal variate (SNV), a signal preprocessing and filtering tool, was found to enhance the predictive accuracy and robustness of the fluorescence-based model.     

Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model

The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.     

The oligomeric state and stability of the mannitol transporter, EnzymeII(mtl), from Escherichia coli: a fluorescence correlation spectroscopy study

Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeII(mtl), is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EII(mtl) functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EII(mtl) using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EII(mtl) is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect.     

Fluorescence resonance energy transfer between two quantum dots with immunocomplexes of antigen and antibody as a bridge.

In this study, 573 nm quantum dots (QDs)-rabbit IgG-goat anti-rabbit IgG-638 nm QDs immunocomplexes were prepared, utilizing antigen-antibody interaction. 573 nm-emitting QDs were conjugated to antigen (rabbit IgG) and 638 nm-emitting QDs were conjugated to antibody (goat anti-rabbit IgG) via electrostatic/hydrophilic self-assembly, respectively. The mutual affinity of the antigen and antibody brought two kinds of QDs close enough to result in fluorescence resonance energy transfer (FRET) between them; the luminescence emission of 573 nm QDs was quenched, while that of 638 nm QDs was enhanced. The luminescence emission of 573 nm QDs could be recovered when the immunocomplexes were exposed to the unlabelled rabbit IgG antigen. The FRET efficiency (E) and the distance between the donor and the acceptor were calculated.     

Fluorescence spectroscopic investigation of competitive interactions between ochratoxin A and 13 drug molecules for binding to human serum albumin

Ochratoxin A (OTA) is a highly toxic mycotoxin found worldwide in cereals, foods, animal feeds and different drinks. Based on previous studies, OTA is one of the major causes of the chronic tubulointerstitial nephropathy known as Balkan endemic nephropathy (BEN) and exerts several other adverse effects shown by cell and/or animal models. It is a well‐known fact that OTA binds to various albumins with very high affinity. Recently, a few studies suggested that reducing the bound fraction of OTA might reduce its toxicity. Hypothetically, certain drugs can be effective competitors displacing OTA from its albumin complex. Therefore, we examined 13 different drug molecules to determine their competing abilities to displace OTA from human serum albumin (HSA). Competitors and ineffective chemicals were identified with a steady‐state fluorescence polarization‐based method. After characterization the competitive abilities of individual drugs, drug pairs were formed and their displacing activity were tested in OTA‐HSA system. Indometacin, phenylbutazone, warfarin and furosemide showed the highest competing capacity but ibuprofen, glipizide and simvastatin represented detectable interaction too. Investigations of drug pairs raised the possibility of the presence of diverse binding sites of competing drugs. Apart from the chemical information obtained in our model, this explorative research might initiate future designs for epidemiologic studies to gain further in vivo evidence of long‐term (potentially protective) effects of competing drugs administered to human patients. Copyright © 2012 John Wiley & Sons, Ltd.