Quantitative immunoassay of biotoxins on hydrogel-based protein microchips

Three-dimensional gel-based microchips with immobilized proteins were used for quantitative immunoassay of a series of plant (ricin and viscumin) and bacterial (staphylococcal enterotoxin B, tetanus and diphtheria toxins, and lethal factor of anthrax) toxins. It was shown that different types of immunoassays (direct, competitive, and sandwich type) could be carried out on gel microchips. As shown by confocal microscope studies, antigen-antibody interactions involving the formation of tertiary antibody-antigen-antibody complex occur in the whole volume of microchip gel elements. Sandwich assay on microchips with immobilized antibodies provided the highest sensitivity of detection (0.1 ng/ml for ricin). Antibodies labeled with fluorescent dyes, horseradish peroxidase conjugates, or biotinylated antibodies with subsequent treatment with labeled avidin were used as developing antibodies. The results of immunoassays were recorded using fluorescence, chemiluminescence, or matrix-assisted laser desorption ionization mass spectrometry directly from microchip gel elements. Gel microchips with immobilized capture antibodies were used to analyze the sample simultaneously for the presence of all six biotoxins with the same sensitivity as that for any single toxin.     

Biological microchips with hydrogel-immobilized nucleic acids,proteins, and other compounds: properties and applications in genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain the gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes proceeding on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, the protein microchips have been created, containing the immobilized antibodies, antigens, enzymes, and many other substances, as well as the microchips with the gel-immobilized live cells.     

Biological Microchips with Hydrogel-Immobilized Nucleic Acids,Proteins, and Other Compounds: Properties and Applications in Genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single-nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, protein microchips have been created, containing immobilized antibodies, antigens, enzymes, and many other substances, as well as microchips with gel-immobilized live cells.     

Comparison of surface and hydrogel-based protein microchips

Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.     

Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips.

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.     

Gel-Based Microchips: History and Prospects

The review describes the history of formation and development of the microchip technology and its role in the human genome project in Russia. The main accent was done on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous models. The microchips are manufactured by photoinitiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical properties and stability; this allowed one to work with the DNA fragments of up to 500 nt in length, as well as with quite large protein molecules. At present, the gel-based microchips are widely applied to solve different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA–protein interactions. The oligonucleotide microchips are a cheap and reliable diagnostic tool designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; of orthopoxviruses, including the smallpox virus; of the anthrax pathogen; and chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteo-mics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which opens a wide potential for creating biosensors on the basis of microchips.     

Microchips based on three dimensional gel cells: history and perspective

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.     

Hydrogel-based protein microchips: manufacturing,properties, and applications

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.     

Analysis of protein interaction and function with a 3-dimensional MALDI-MS protein array

Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.     

Microfabricated polymer chip for capillary gel electrophoresis

A polymer (PDMS: poly(dimethylsiloxane)) microchip for capillary gel electrophoresis that can separate different sizes of DNA molecules in a small experimental scale is presented. This microchip can be easily produced by a simple PDMS molding method against a microfabricated master without the use of elaborate bonding processes. This PDMS microchip could be used as a single use device unlike conventional microchips made of glass, quartz or silicon. The capillary channel on the chip was partially filled with agarose gel that can enhance separation resolution of different sizes of DNA molecules and can shorten the channel length required for the separation of the sample compared to capillary electrophoresis in free-flow or polymer solution format. We discuss the optimal conditions for the gel preparation that could be used in the microchannel. DNA molecules were successfully driven by an electric field and separated to form bands in the range of 100 bp to 1 kbp in a 2.0% agarose-filled microchannel with 8 mm of effective separation length.     

An RNA microchip containing immobilized oligoribonucleotides with protective groups at 2'-O-positions

To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.     

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T_m) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T_m. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.     

Hydrogel glycan microarrays

The technology of hydrogel microchips manufacturing, which was developed previously for covalent immobilization of DNA and proteins, was applied for the preparation of glycochips and combined glyco/protein chips. Microchips consist of hydrogel drops separated with hydrophobic surface. Spacered amino-saccharides and polyacrylamide glycoconjugates were used for immobilization. Gel elements were approximately 1 nl in volume (150 microm in diameter and 25 microm in height), and the amount of covalently immobilized saccharide in the glycoarray was 0.4-1.7 pmol per gel element. Hydrogel glycan microchips were used for quantitative assay of antibodies against blood group antigens and assay of lectins with fluorescent detection. In all cases, only specific interaction with chip-immobilized saccharides was observed, whereas the background signal was very low. The detection limit of on-chip assays was comparable to that of the standard 96-well plate assays. Mixing of reaction solution allowed us to decrease the duration of the assays significantly: 2-3 h for incubation and development steps and 10 min for washing. A method for determination of association constants for binding of compounds with chip-immobilized ligands from the kinetics of their binding is proposed. Combined microchips containing different types of biomolecules can be designed and used for simultaneous detection of different compounds.     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

Effect of mixing on reaction-diffusion kinetics for protein hydrogel-based microchips

Protein hydrogel-based microchips are being developed for high-throughput evaluation of the concentrations and activities of various proteins. To shorten the time of analysis, the reaction-diffusion kinetics on gel microchips should be accelerated. Here we present the results of the experimental and theoretical analysis of the reaction-diffusion kinetics enforced by mixing with peristaltic pump. The experiments were carried out on gel-based protein microchips with immobilized antibodies under the conditions utilized for on-chip immunoassay. The dependence of fluorescence signals at saturation and corresponding saturation times on the concentrations of immobilized antibodies and antigen in solution proved to be in good agreement with theoretical predictions. It is shown that the enhancement of transport with peristaltic pump results in more than five-fold acceleration of binding kinetics. Our results suggest useful criteria for the optimal conditions for assays on gel microchips to balance high sensitivity and rapid fluorescence saturation kinetics.     

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.     

Immune interactions with cells infected with herpes simplex virus: antibodies to radioiodinated surface antigens.

Lactoperoxidase-catalyzed radioiodination was used to study reactions between surface antigens and antibodies on BHK-21 cells infected with HSV-1 and HSV-2. Isolation of iodinated surface antigens was achieved by indirect immune precipitation of Triton X-100 disrupted cells with antisera to HSV and IgG. Analysis of immune precipitates by polyacrylamide gel electrophoresis (PAGE) revealed at least 10 antigens, ranging in m.w. from 35 x 103 to 160 x 103 daltons. Antigens were detectable on cell surfaces as early as 2 hr post-infection. Electrophoretic patterns of surface antigens induced by HSV-1 were similar to those induced by HSV-2. In both cases the major portion of activity was associated with glycoprotein(s) in the range of 115 x 103 to 130 x 103 daltons. A reduced amount of radioactivity was obtained if cells were reacted with anti-HSV sera before disruption with Triton X-100, suggesting that less surface antigen was accessible to HSV antibody applied directly to intact cells.     

IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.