Using FAM labeled DNA oligos to do RNA electrophoretic mobility shift assay

The electrophoretic mobility shift assay is a useful tool to identify proteins and nucleic acids interactions. Traditionally, the nucleic acids fragments are end-labeled with ³²P. We present here the use of fluorescent methodologies for studies of RNA in place of radioactivity. The method is highly sensitive and quantitative with the use of an infrared fluorescence imaging system. Fluorescently labeled primers can be used to monitor protein–RNA interactions by fluorescent mobility shift assays. The simplicity and validity of this approach may have more advantages than that of previous methods that traditionally used hazardous radioisotopes. This method was successfully tested in the study of the interactions between MS2 Coat Protein and its RNA target.     

High sensitivity immunoassays using particulate fluorescent labels.

The use of polystyrene fluorescent microspheres as sensitive labels in direct-detection (not enzymatically amplified) heterogeneous equilibrium "sandwich" immunoassays in 96-well plates is described. With mouse IgG as a model antigen, a fluorescent particulate label is more sensitive than a corresponding soluble reporter. The limit of detection of mouse IgG in the multiparametrically optimized assay was 0.2 ng/ml (7.6 x 10(8) antigens/ml) for the particulate reporter and 50 ng/ml (1.9 x 10(11) antigens/ml) for the soluble reporter. The sensitivities of assays using the particulate label were dependent on the surface densities of the capture and reporter antibodies and the concentration of reporter beads. Sensitivity was improved by adding the preformed reporter antibody/fluorescent microsphere complex to trapped antigen on the well surfaces instead of sequentially adding the reporter antibody and then the fluorescent microspheres. Maximal (equilibrium) binding of the particulate reporter to captured antigen occurred after 20 h with a concentration of 1.4 x 10(9) reporter beads/ml. Thus, particulate fluorescent labels provide high sensitivity in direct-detection immunoassays.     

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.Phosphorylation of proteins is an integral part of the signal transduction of eukaryotic cells as it modulates the activity of complex protein networks. Although Western blot- and immunoprecipitation-based MS approaches (1, 2) can lead to detailed insights into these processes, most of the integrated approaches only allow a static view of protein phosphorylation because they are not suitable for the screening of hundreds of samples. Either planar or bead array-based sandwich immunoassays can be used to analyze the quantity and activation state of signaling molecules in multiplex, enabling the systematic profiling of protein abundance and post-translational modifications (3–6) in hundreds of samples. However, multiplex immunoassays are only suitable for the simultaneous analysis of a limited number of proteins. The detection of comprehensive phosphorylation patterns is difficult as this involves assay systems that are incompatible with multiplexing.In principle, two sandwich immunoassay setups are possible for probing the phosphorylation state of a protein. The first setup applies a capture antibody specific for a non-modified part of the protein and uses a phosphorylation state-specific detection antibody. When applied to an array-based format, however, this setup does not allow for the simultaneous measurement of the abundance and the degree of phosphorylation (3, 4). A mixture of detection antibodies, one specific for the phosphorylation site and one specific for the non-modified site of the protein, would bind simultaneously to the two different epitopes, and assay signals could not be further deconvoluted by the spatial or color code of the array. The second sandwich immunoassay setup for the analysis of protein phosphorylation applies a phosphorylation state-specific capture antibody and a protein-specific detection antibody. In such a setup, an anti-phosphotyrosine antibody (e.g. mAb 4G10) cannot be applied as a capture antibody because a huge variety of tyrosine phosphorylated proteins would be captured, and specific signals could rarely be deconvoluted. Using capture antibodies that bind to phosphorylated epitopes in the context of their flanking amino acids is not a problem until a multiplex readout is desired. If one antibody specific for the phosphosite and one antibody specific for the abundance of a protein are used together in a multiplex assay panel they might compete for their analyte. The situation becomes even more complex if the protein of interest contains various phosphorylation sites such as e.g. the epidermal growth factor receptor. Several capture antibodies target different epitopes of the same protein and therefore compete for the overall amount of targeted protein in the sample, thus making a valid simultaneous measurement problematic.Although different ways of tackling the problem of assay multiplexing are in use, we demonstrated the feasibility to sequentially perform such incompatible assays from the same sample using a magnetic particle handler that moves particles through the samples and reagents (Fig. 1). Using a model assay, we confirmed that suspension bead array-based immunoassays work under ambient analyte conditions. As described by Roger Ekins (7), decreasing of the amount of capture antibody in a sandwich immunoassay setup from a macrospot (e.g. a microtiter plate assay) to a microspot generates a scenario where only a tiny fraction of the present target analytes is captured on the microspot. Therefore, the overall concentration of the analyte molecules in the sample does not change significantly even in the case of low target concentrations and high affinity binding reactions. Furthermore, as the initial concentration of the analyte is not significantly changed when performing a miniaturized sandwich immunoassay, multiple post-translational modifications within the same protein can be measured either in sequence or in parallel in the same multiplex panel.Open in a separate windowFig. 1.Sequential multiplex analyte capturing. Magnetic suspension bead array assays can be performed sequentially, reusing the same sample material (indicated by the blue arrow). The use of a magnetic particle handler enables the quantitative transfer (black arrow) of the magnetic beads from the sample well into the wells containing washing solutions or other assay reagents. Magnetic beads from the first bead array panel are incubated with the samples to capture their respective analyte. Then the magnetic beads are subjected to washing and detection steps and are finally transferred into the readout plate (first row). After retracting the magnetic suspension bead array of the first assay panel from the sample, a bead array from the second assay panel is added and processed as described above but using different detection antibodies (second row). A third bead array assay panel can be applied after removing the second panel (third row) and so on.By probing tumor cell lines for the abundance of seven different receptor tyrosine kinases and their generic tyrosine phosphorylation, we generated complex phosphorylation patterns and thereby demonstrated the potential of this approach. More importantly, demonstrating ambient analyte conditions allowed the parallel detection of phosphorylation at different sites of the EGFR1 using phosphorylation site-specific antibodies as capture molecules with one assay panel. Phosphorylation of eight different sites and the abundance of the EGFR could be quantified relative to one another without any interference of the different immunoassays during multiplexing because competition for the analyte can be prevented by running the assays under ambient analyte conditions.     

Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface

It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology.     

VERÄNDERUNGEN DER AMINOSÄUREKONZENTRATIONEN IM RATTENHIRN BEI VERRINGERUNG DER K+-ZUFUHR IN VIVO

Abstract—

• 1 Resonium A, a cation exchange resin, administered orally caused no decrease of the potassium content in the CNS of the rat, but it provoked a potassium depletion in the liver tissue. However a slight increase could be detected in the ‘cortex’ and ‘striatum’.
• 2 A rise of the concentration of the free amino acids was found in ‘cortex’, ‘striatum’, ‘thalamus’ and cerebellum. Glutamic acid showed an increase of 70–80 per cent. GABA and glycine showed a remarkable increase of 280–330 per cent.
• 3 Restitution of K⁺ by feeding a potassium-rich diet brought the amino acid concentrations in the ‘cortex’ and cerebellum within a normal range. In ‘striatum’ and ‘thalamus’ an overshoot could be observed.
• 4 The experimental procedure for the estimation of free amino acids in brain tissue is discussed.

     

Scintillation proximity assay: A sensitive and continuous isotopic method for monitoring ligand/receptor and antigen/antibody interactions

Scintillation proximity assay (SPA) makes it possible to use radioisotopes for monitoring binding reactions continuously without the need to separate free from bound components. As a result SPA can be carried out more rapidly than most other methods used to monitor binding reactions. The methodology also lends itself to automation. The sensitivities already achieved with SPA procedures are comparable to the sensitivities of other procedures in use today. Another feature of SPA is that the key reagents (beads, ¹²⁵I labeling) are relatively inexpensive. The principles of SPA, utilizing ¹²⁵I-labeled molecules, are discussed and some applications to immunology, receptor binding, and measurement of potential across membranes are presented. SPA should also be applicable to monitoring interactions involving nucleic acids, lipids, and carbohydrates. Characteristics of some radionuclides, other than tritium and ¹²⁵I, that may be used in SPA are presented.     

Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA

We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein-RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.     

The immunoassay of gibberellins

The development of sensitive and specific solid-phase enzyme immunoassays for gibberellic acid and gibberellins A₄ and A₇ is reported. The use of antisera of high apparent affinity (K_a over 10¹⁰ l mol^-1) in conjunction with alkaline phosphatase-labeled gibberellins allows, with minimum procedural effort, the quantitative determination of sub-picogram amounts of these gibberellins. The assays reported here are applicable to most gibberellins and can be set up with 1–1.5 mg of starting material. They represent the most sensitive methods for gibberellin determination known.Abbreviations GA gibberellin - GA₃ gibberellic acid - TLC thin-layer-chromatography     

MIP-ligand binding assays (pseudo-immunoassays)

Molecular imprint sorbent assays (MIAs) have been applied to an increasing number of analytes of medical and environmental interest: the sensitivities and selectivities of these assays are comparable to immunoassays employing biological antibodies. In a number of cases complete analytical procedures starting from raw samples (blood, plasma and urine) have been demonstrated. There have been significant advances in applying MIPs in new formats and in the use of non-radioisotope labels. Progress in the field is reviewed, with particular emphasis on the technical aspects and new innovations. It is demonstrated that many of the perceived drawbacks of molecular imprinted polymers (MIPs) do not hinder their application in competitive binding assays: Many MIAs have been applied in aqueous systems and a heterogenous distribution of binding sites is not problematic, provided the recognition sites which bind the probe most strongly are selective.     

Favorably orienting recombinant proteins to develop amperometric biosensors to diagnose Chagas’ disease

Clinical immunoassays often display suitable sensitivity but some lack of specificity or vice versa. As a trade-off between specificity improvement and sensitivity loss, biosensors were designed to perform indirect immunoassays with amperometric detection using tailor-made chimeric receptors to react with the analyte, specific anti-Trypanosoma cruzi immunoglobulin G (IgG). Recombinant chimeras were designed to favor their oriented covalent attachment. This allows the chimeras to properly expose their epitopes, to efficiently capture the analyte, and to withstand severe chemical treatment to reuse the biosensors. By further binding the secondary antibody, horseradish peroxidase-labeled anti-human IgG, in the presence of the soluble mediator and the enzyme substrate, a current that increased with the analyte concentration was measured. Biosensors using the chimeric constructions showed 100% specificity with samples that had revealed false-positive results when using other bioreceptors. A protein bearing a poly-Lys chain and thioredoxin as directing elements displayed the highest signal-to-noise ratio (P < 0.05). The limit of detection was 62 ng ml⁻¹, which is eight times lower than that obtained with a currently used commercial Chagas enzyme-linked immunosorbent assay (ELISA) kit. Reusability of the biosensor was assessed. The signal was approximately 80% of the original one after performing 10 consecutive determinations.     

Time-resolved fluoroimmunoassay of steroid hormones

The use of europium chelates as labels in immunoassays and their sensitive quantitation based on time-resolved fluorescence is reviewed. The technique is applied on competitive solid-phase immunoassays for direct determination of progesterone and estradiol in serum samples. Both antigen- and antibody-labelled competitive assays are described. The nonisotopic label technology, which provides a very high specific activity, as well as the antibody-labelled competitive assays, present several advantages in the assay of haptens as e.g. steroids. As the optimal sensitivity of competitive methods is not limited by the specific activity of the label the steroid assays which employ europium chelates as labels do not show any marked increase in sensitivity as compared to that achieved by using 125I. The potential sensitivity provided by the high specific activity of the label is optimally utilized in noncompetitive immunometric assays.     

Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics

A significant challenge for all biosensor systems is to achieve high assay sensitivity and specificity while minimizing sample preparation requirements, operational complexity, and sample-to-answer time. We have achieved multiplexed, unamplified, femtomolar detection of both DNA and proteins in complex matrices (including whole blood, serum, plasma, and milk) in minutes using as few as two reagents by labeling conventional assay schemes with micrometer-scale magnetic beads, and applying fluidic force discrimination (FFD). In FFD assays, analytes captured onto a microarray surface are labeled with microbeads, and a controlled laminar flow is then used to apply microfluidic forces sufficient to preferentially remove only nonspecifically bound bead labels. The density of beads that remain bound is proportional to the analyte concentration and can be determined with either optical counting or magnetoelectronic detection of the magnetic labels. Combining FFD assays with chip-based magnetoelectronic detection enables a simple, potentially handheld, platform capable of both nucleic acid hybridization assays and immunoassays, including orthogonal detection and identification of bacterial and viral pathogens, and therefore suitable for a wide range of biosensing applications.     

High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction.

A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and beta-Gal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specific monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate co-amplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays by approximately three orders of magnitude. Detection limits for hTSH, beta-Gal and hCG were respectively 1 x 10(-19), 1 x 10(-17) and 1 x 10(-17) mol. Given the enormous amplification afforded by PCR and the existing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays.     

Biochemiluminescence and biomedical applications

Although used for analytical purposes for more than 40 years it is only recently that biochemiluminescence (BCL) has found widespread acceptance. Methods employing BCL reactions now play an important role in biomedical research and laboratory medicine. The main attractions for the assay technology include exquisite sensitivity (attomole-zeptomole), high selectivity, speed and simplicity. In biomedical research, the most important applications of BCL are: (1) to estimate microbial numbers and to assess cellular states (e.g., after exposure to antibiotic or cytotoxic agents) and in reporter gene studies (firefly luciferase gene); (2) NAD(P)H involved in redox/dehydrogenase studies usingVibrio luciferase complex; (3) BCL labels and CL detection of enzyme labels in immunoassays are the most widespread routine application for this technology. BCL enzyme immunoassays represent the most active area of development, e.g., enhanced BCL method for peroxidase and BCL assays for alkaline phosphatase labels using adamantyl 1,2-dioxetane.Abbreviations BCL biochemiluminescence - CL chemiluminescence - RLU relative light unit - ROS reactive oxygen species     

An investigation on the catalytic mechanism of enhanced chemiluminescence: Immunochemical applications of this reaction

The mechanism of peroxidase-catalysed oxidation of luminol by H₂O₂ was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Novel homogenous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfuly tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10^?9M for IgG and 10^?11M for T4, the overall time of the assay being 5–15 min.     

T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay

The use of xanthine oxidase in immunoanalysis has never been reported. We describe here a procedure in which the xanthine oxidase dependent luminescence of luminol is enhanced in the presence of Fe–EDTA complex, providing an highly sensitive assay (3 amol of enzyme) and a long-term signal. This specific amplification has been applied to T₄ and ultrasensitive TSH solid phase immunoassays, with T₄–XO and anti-TSH monoclonal antibody-XO conjugates as tracers. The performances of these assays are at least equivalent to those obtained with iodinated tracers, using the same solid phases and the same calibrators. The major advantages of these immunoassays are: (1) the long-term signal which can be repeatedly recorded over several days, (2) the high detection sensitivity, (3) the long-term stability of the luminescence reagent and (4) the stability of the conjugates.     

Development of Ss-NIE-1 Recombinant Antigen Based Assays for Immunodiagnosis of Strongyloidiasis

Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG₄ specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG₄. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG₄ ELISA were 95% and 93%, respectively. For the IgG₄ Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG₄ ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.