Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria). Ib. Gel antibodies against proteins (hemoglobins)

Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.     

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria): Ia. gel antibodies against proteins (transferrins)

Artificial antibodies in the form of gel granules were prepared by the molecular imprinting technique from the monomers acrylamide and N,N'-methylene-bis-acrylamide. Gel granules, freed from the selectively adsorbed protein (the antigen), are neutral and, accordingly, do not migrate in an electrical field. However, upon selective interaction with the antigen at a pH different from its pI, the granules become charged. The selectivity of the gel antibodies was studied by free zone electrophoresis in a tube with inside diameter larger than the size of the granules. Such electrophoretic analyses showed that gel antibodies against iron-free transferrin had a high selectivity for this protein, although some crossreaction took place with iron-saturated transferrin, indicating that these artificial antibodies can easily distinguish the minute differences in the 3-D structure of the transferrins. Analogously, gel antibodies against iron-saturated transferrin were highly selective for this protein with some crossreaction with iron-free transferrin. The mobilities of iron-free and iron-saturated transferrin are very similar, and, therefore, capillary free zone electrophoresis cannot distinguish between these structurally related proteins. However, significant differences in the mobilities of the selective gel granules can be observed depending on their interaction with iron-free or iron-saturated transferrin, i.e., the artificial gel antibodies may become powerful analytical tools.     

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria). III: gel antibodies against cells (bacteria)

Artificial antibodies in the form of gel granules were synthesized from the monomers acrylamide and N,N'-methylenebisacrylamide by the imprinting method in the presence of Echerichia coli bacteria as template. The electrophoretic migration velocities of the gel antibodies (i) saturated with the antigen (Escherichia coli MRE-600), (ii) freed of the antigen, and (iii) resaturated with bacteria, were determinated by electrophoresis in a rotating narrow-bore tube of 245 mm length and the 2.5 and 9.6 mm inner and outer diameters, respectively. Removal of bacteria from the gel antibodies was made by treatment with enzymes, followed by washing with SDS and buffer. Gel granules becoming charged by adsorption of bacteria move in an electrical field. We obtained a significant selectivity of gel antibodies for E. coli MRE-600, since the granules did not interact with Lactococcus lactis; and when E. coli BL21 bacteria were added to the gels selective for E. coli MRE-600, a significant difference in the migration rate of the complexes formed with the two strains was observed indicating the ability of differentiation between the two strains. The gel antibodies can be used repeatedly. The new imprinting method for the synthesis of artificial gel antibodies against bioparticles described herein, and the classical electrophoretic analysis technique employed, thus represent - when combined - a new approach to distinguish between different types and strains of bacteria. The application area can certainly be extended to cover other classes of cells.     

Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples. I. Spectrophotometric approach to design the calibration curve for the quantification

High selectivity of a biomarker is a basic requirement when it is used for diagnosis, prognosis and treatment of a disease. The artificial gel antibodies, which we synthesise by a molecular imprinting method, have this property not only for proteins, but also for bioparticles, such as viruses and bacteria. However, diagnosis of a disease requires not only that the biomarker can be “fished out” from a body fluid with high selectivity, but also that its concentration in the sample can rapidly be determined and preferably by a simple technique. This paper deals primarily with the development of a spectrophotometric method, which is so simple and fast that it can be used with advantage in a Doctor's Office. The development of this method was not straight‐forward. However, by modifications of the performance of these measurements we can now design standard curves in the form of a straight line, when we plot the true (not the recorded “apparent” absorption) against known protein concentrations. In an additional publication (see the following paper in this issue of JSS) we show an application of such a plot: determination of the concentration of albumin in serum and cerebrospinal fluid from patients with neurological disorders to investigate whether albumin is a biomarker for these diseases.     

Monolithic beds of artificial gel antibodies

The introductions of the continuous beds, now often called monoliths [S. Hjertén, J.-L. Liao, R. Zhang, J. Chromatogr. 473 (1989), 273-275] and the artificial, highly selective gel antibodies against antigens as large as proteins, viruses and cells [J.-L. Liao, Y. Wang, S. Hjertén, Chromatographia 42 (1996), 259-262] were breakthroughs in the design of chromatographic beds. This paper deals with a combination of these two methods, i.e., the artificial gel antibodies have been synthesized in the monolithic mode. As antigen we have used human hemoglobin. A comparison of the ion-exchange chromatograms of the eluates from the monolithic columns shows that the monolith prepared in the presence of hemoglobin adsorbed this protein, but not the other proteins in the sample (ribonuclease A and cytochrome c), i.e., this monolith was selective for hemoglobin, whereas the blank column (prepared in the absence of hemoglobin) had no selective properties, since none of the applied proteins were adsorbed. The diameter of the column was 6mm, but the same approach to synthesize a monolithic selective bed can very likely also be used for capillaries and microchips.     

Immunosensor based on fluorescence quenching matrix of the conducting polymer polypyrrole

In this study, the combination of autofluorescent proteins and fluorescence quenching polymers was shown to be a design which can increase the selectivity and sensitivity of immunosensors. With this objective, the conducting polymer polypyrrole (Ppy) was used as a matrix for immobilization of proteins, which enables biological recognition of the analyte, and as a fluorescence quencher, which increases the selectivity of fluorescence-based detection. In this study, bovine leukemia virus proteins gp51 were immobilized within the Ppy matrix and formed a polymeric layer with affinity for antibodies against protein gp51 (anti-gp51). The anti-gp51 antibodies are present at high levels in the blood serum of cattle infected by bovine leukemia virus. Secondary antibodies labeled with horseradish peroxidase (HRP) were used as specific fluorescent probes for detection of a particular target, because the fluorescence of HRP was readily detectable at the required sensitivity. The Ppy was used as fluorescent background, because its fluorescence was almost undetectable when excited by near UV light at 325 nm. Moreover the Ppy quenched the fluorescence of some fluorescent agents including fluorescein-5(6)-isothiocyanate (fluorescein), rhodamine B, and HRP by almost 100% when these fluorescent agents were adsorbed on the surface of Ppy. It is predicted that Ppy-induced fluorescence quenching could be used in the design of immunosensors to increase selectivity and sensitivity.     

Poly(methylene green) employed as molecularly imprinted polymer matrix for electrochemical sensing

This paper describes the development of a molecularly imprinted polymer (MIP) for theophylline that can be used for electrochemical sensing. Theophylline is a commonly used medication for the treatment of asthma. Due to its very narrow therapeutic index, it may have toxic and potentially fatal effects on the individual. Electrochemical detection of theophylline is difficult, because its molecular structure and standard reduction potential are very similar to that of caffeine. A new method for fabricating molecularly imprinted polymers is proposed utilizing methylene green. Poly(methylene green)(PMG), prepared by electropolymerization of an azine, methylene green, was imprinted for theophylline. PMG-based MIP-coated electrodes showed sensitivity towards the presence of the imprint molecule in solutions, as well as selectivity for the imprint over the interferent molecule caffeine. The PMG-based MIP-coated electrode described in this paper had an improved selectivity factor and reproducibility compared to other theophylline-imprinted MIP-coated electrodes in literature.     

Selection of phage-displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices

A method development aimed for high-throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets, and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Here, we present a phage-selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high-affinity phage-displayed antibodies against virus glycoproteins, including human immunodeficiency virus-1 glycoprotein 120 or the Ebola virus glycoprotein (EBOV-GP). Phages were differentially and laterally swept depending on their antigen affinity; the high-affinity phages were recovered at channels proximal to the application site, whereas low-affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage-selection is rapid, sensitive, and effective. Therefore, this is an efficient and cost-effective method that allowed highly controlled assay conditions for isolating and sorting high-affinity ligands displayed in phages.     

Antibody-scanning and epitope-tagging methods; molecular mapping of proteins using antibodies

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.     

Gels mimicking antibodies in their selective recognition of proteins

Summary In a previous paper we presented preliminary experiments aimed at the preparation of gel particles with the property to recognize selectively some particular protein (hemoglobin, cytochrome C, transferrin) [1]. Using the same method we show in this article that human growth hormone, ribonuclease and myoglobin from horse can also be adsorbed specifically, indicating that the method may be universal or at least applicable to a great number of proteins. A gel with specific adsorption of three model proteins was synthesized in order to demonstrate that the beds can be employed to remove (traces of) several proteins contaminating a sample (“negative purification”). The degree of selective recognition is high, to judge from the fact that myoglobin from horse, but not that from whale, was adsorbed onto a column designed to bind specifically the former protein. This selectivity is noteworthy, since these two proteins have similar amino acid sequences and 3-D structures. The method for the synthesis of the specific gels involves polymerization of appropriate monomers (for instance acrylamide and its derivatives) in the presence of the protein to be adsorbed specifically, granulation of the gel formed, packing a column with the gel particles, washing the column to remove the protein and finally application of the sample for selective adsorption of the protein. The approach resembles that used for entrapment (immobilization) of proteins for affinity chromatography and that for molecular imprinting, with the distinct difference that the monomer composition is quite different and thereby the binding mechanism. This mechanism is discussed, for instance, in terms of (1) a new classification system for chromatographic beds based on the number of bonds between the solute and the matrix and the strength of each bond and (2) “non-specific bonds” (these bonds are often harmful in conventional chromatography, but we have used them to advantage). In this classification system the selective recognition is characterized by a large number of weak bonds. Therefore, so-called functional monomers are not used for the preparation of the gels because they often are charged and, accordingly, give rise to strong electrostatic interactions, i.e. the beds behave to some extent as ion-exchangers. In most experiments we have used a polyacrylamide gel with large pores to facilitate diffusion of proteins into and out of the gel granules. When used in chromatography these soft gels (which can be used repeatedly) allow only rather low flow rates. This problem can be overcome by a new approach to prepare the granules. Potential applications of the selective beds are discussed, as well as future improvements. YW's visit to the Department was sponsored by the Swedish Institute, Stockholm, Sweden.     

Identification of phage antibodies toward the Werner protein by selection on Western blots

A procedure was established for selecting phage antibodies (phage-abs) from phage-displayed antibody repertoires by panning against proteins, separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Western blots). This immobilization strategy is applicable for secondary rounds of panning in selections against semipurified proteins, and directs the selection toward antibodies suitable as immunochemical reagents in Western blots. In model experiments, enrichment factors as high as 1.9x10(5) were obtained in a single round of panning. Furthermore, we demonstrate the application of this approach by selection of phage-abs recognizing the human Werner protein, which is defective in a premature aging syndrome.     

A simple direct enzyme immunoassay of antibodies based on the differences in diffusion rates in a gel of a synthetic antigen and of its complex with antibodies

A simple direct enzyme immunoassay for semiquantitative detec tion of antibodies is suggested. It is based on the difference in diffusion rates in a gel for a synthetic low-mol-wt antigen and of its complexes with antibodies to be detected. Sensitivity and specificity of the devel oped assay are equal to an ELISA method. The assay has been tested with antibodies against HIV protein gp41 in rabbit serum. Possible applications and limitations of the method are discussed.     

Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection

Recently we described the design of a hybrid microdevice for micro(nano)electrophoresis and electrochromatography, discussed its advantages and disadvantages compared to conventional microdevices and presented a few applications with low-molecular-weight samples. In this paper, we demonstrate the broad application range of this device using UV-based analyses of (i) peptides by free-zone electrophoresis and electrophoresis in a recently introduced gel (polyacrylamide cross-linked with allyl-beta-cyclodextrin), (ii) proteins by electrophoretic molecular-sieving in a polymer solution supplemented with SDS, (iii) DNA fragments by electrophoresis in the above gel, (iv) virus particles in this gel, as well as in free buffer and (v) bacteria in free buffer. To illustrate the advantages of the hybrid microdevice we can mention that electrophoresis of proteins in a polymer-containing buffer, supplemented with sodium dodecyl sulfate (SDS), in a 4.30 (2.75) cm long channel gave a resolution similar to that in conventional capillary electrophoresis in a 23.5 (18.6) cm long capillary and analysis times which were 15-fold shorter.     

Detection of Staphylococcus aureus cell walls by enzyme-linked immunoassay using antibodies prepared from a semi-synthetic peptidoglycan precursor

The peptidoglycan layer of Staphylococcus aureus contains a (Gly)(5) cross-link which is not found in other bacteria, and which could be used to develop a specific immunoassay for detection of S. aureus in MRSA infections. A semi-synthetic route was used to prepare the S. aureus peptidoglycan precursor UDPMurNAc-L-Ala-γ-D-Glu-L-Lys(Gly)(5)-D-Ala-D-Ala, which was covalently attached to carrier protein bovine serum albumin via the UDP nucleotide. Serum raised using this antigen showed specificity for chemically immobilised peptidoglycan monomer containing (Gly)(5), using an ELISA immunoassay. ELISA assays using 0.1 or 1.0 μg samples of cell walls prepared from two MRSA strains and one penicillin-sensitive S. aureus strain, and from three other bacteria, showed the highest response against cell walls containing (Gly)(5), with a particularly high response against cell walls from one MRSA strain. Competition assays to investigate antibody selectivity demonstrated that the antibody response could be most effectively antagonised using ligands containing (Gly)(5). These data demonstrate that it is possible to generate antibodies with high affinity and selectivity for the (Gly)(5) containing monomer in S. aureus peptidoglycan, that could be used to develop an immunoassay for S. aureus.     

Development of a new parallelized,optical biosensor platform for label-free detection of autoimmunity-related antibodies

Autoimmune diseases are characterized by the presence of autoantibodies in serum of affected patients. The heterogeneity of autoimmune relevant antigens creates a variety of different antibodies, which requires a simultaneous detection mode. For this reason, we developed a tool for parallelized, label-free, optical detection that accomplishes the characterization of multiple antigen–antibody interactions within a single measurement on a timescale of minutes. Using 11-aminoundecyltrimethoxysilane, we were able to immobilize proteinogenic antigens as well as an amino-functionalized cardiolipin on a glass surface. Assay conditions were optimized for serum measurements with a single spot antigen chip on a single spot 1-λ detection system. Minimized background signal allows a differentiation between patients and healthy controls with a good sensitivity and specificity. Applying polarized imaging reflectometric interference spectroscopy, we evaluated samples from three APS patients and three control subjects for this proof-of-principle and already obtained good results for β2-glycoprotein I and cardiolipin.     

Development of superior antibodies against the S-protein of SARS-Cov-2 using macrocyclic epitopes

One of the proven methods to prevent and inhibit viral infections is to use antibodies to block the initial Receptor Binding Domain (RBD) of SARS-CoV-2 S protein and avoid its binding with the host cells. Thus, developing these RBD-targeting antibodies would be a promising approach for treating the SARS-CoV-2 infectious disease and stop virus replication. Macrocyclic epitopes constitute closer mimics of the receptor's actual topology and, as such, are expected to be superior epitopes for antibody generation. This work demonstrated the vital effect of the three-dimensional shape of epitopes on the developed antibodies' activity against RBD protein of SARS-CoV-2. The molecular dynamics studies showed the greater stability of the cyclic epitopes in comparison with the linear counterpart, which was reflected in the activity of their produced antibodies. Indeed, the antibodies we developed using macrocyclic epitopes showed superiority with respect to binding to RBD proteins compared to antibodies formed from a linear peptide. The results of the present work constitute a roadmap for developing superior antibodies that could be used to inhibit the activity of the SARS-CoV-2 and prevent its reproduction.     

Recombinant single chain antibodies in bioelectrochemical sensors

Recombinant antibodies provide an emerging strategy in the development of new immunosensors. In particular, single chain antibodies (scFvs) can be isolated and expressed in bacterial systems that also allow their in vitro manipulation at the gene level. In this work, we present for the first time results of single-chain phage displayed antibodies combined with amperometric detection and its application as an immunosensor. The scFv is immobilized on a carbon electrode and used to capture and quantify its specific target antigen. We describe the detection of the sugar milk lactose, the bacteria Listeria monocytogenes, and the enzyme MtKatG, which is expressed by Mycobacteriumtuberculosis.     

Direct antigen capture by soluble scFv antibodies

For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag.     

Metal–Organic Gels Derived from Iron(III) and Pyridine Ligands: Morphology,Self‐Healing and Catalysis for Ethylene Selective Dimerization

Metal‐organic gels showing potential application in catalysis have received much concern. In this work, we designed and synthesized two metal‐organic gels based on coordination between Fe^III and pyridine ligands at room temperature. The gels were characterized by X‐ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to reveal their assembly structures and morphologies, and it was found the metal‐organic gel derived from di‐topic ligand was composed of three‐dimensional network of nanofibers, while the gel derived from tri‐topic ligand was constituted of sponge‐like structure with amorphous phase. Rheological analysis showed the gel consisting of nanofiber networks displayed self‐healing property. The gels were used as catalysts for selective ethylene dimerization, and the optimum catalysis results of the gel with nanofibers reached the maximal catalytic activity of 1.48×10⁵ g/(mol Fe?h) with C₄ yield more than 90 %, whereas the sponge‐like gel only gave 38 % C₄ products at the same condition. The higher dimerization selectivity of the former Fe^III gel was attributed to its regular assembly structure and lower steric hindrance of the surface metal sites. Due to its catalytic activity, high selectivity and preparation simplicity, the Fe^III gel might be potentially applicable for the preparation of C₄ α‐olefins.     

Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples. II. Albumin in body fluids of patients with neurological disorders

We have previously used the molecular‐imprinting method for the synthesis of artificial gel antibodies, highly selective for various proteins. In the present work, we have synthesized artificial gel antibodies against human albumin with the aim to develop a simple and rapid procedure to measure the concentration of this protein in samples of clinical interest. The procedure, based on the design of a standard curve (see the preceding paper), was applied on a quantitative analysis of albumin in human plasma and cerebrospinal fluid (CSF). We found that our technique permitted detection of albumin in these body fluids with high precision and that the concentration of this protein was significantly enhanced in CSF from patients with amyotrophic lateral sclerosis (ALS), compared to control samples. This finding is in agreement with results from earlier studies, which confirms the validity of our analysis technique and suggests that the barrier permeability may be affected in ALS, perhaps also for other proteins. No enhancement in plasma levels of albumin was seen in patients with ALS, but rather a decrease. The results further indicate that our approach might also apply well to other biomarkers for the actual neurological disease and other disorders.