Streptavidin-coated Plates Research Articles

Quantification of streptavidin adsorption in microtitration wells

Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037 Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm 2 (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 μl liquid, coated area 1.54 cm 2) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350–380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.

Quantification of streptavidin adsorption in microtitration wells

Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037 Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm2 (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 μl liquid, coated area 1.54 cm2) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350–380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.

An interference-free fluorescent assay of telomerase for the high-throughput analysis of inhibitors

We have developed a telomerase assay that can quickly and accurately rank the ability of molecules to inhibit telomerase activity. It is based on the method of Orlando and co-workers which utilizes PicoGreen to detect dsDNA formed during the polymerase chain reaction (PCR) amplification of telomerase products. PCR cycles were optimized to give as linear a signal as possible relative to telomerase products; 96-well streptavidin-coated PCR plates were used to isolate the preamplification telomerase products and to wash inhibitors away before the amplification step. The inhibitor removal step is critical to prevent false positives potentially caused by inhibition of Taq polymerase during amplification. Use of the streptavidin-coated PCR plate allows this step to be done much more rapidly than use of the liquid/liquid extraction adopted by others. We have demonstrated that this assay can correctly order the ability of four inhibitors to inhibit telomerase and reproduce within a factor of two the absolute IC 50 values determined by the more time-consuming direct assay. We have shown that the difference in IC 50 values determined in this assay versus the direct assay can be corrected for by using the standard curve appropriately. Using this method 96 compounds can be assessed in 3–5 h.

Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices

Ribozymes are RNA or modified RNA polymers capable of catalyzing cleavage reactions in target strands RNA, and are under development as human therapeutics. Previous methods used for quantitation of nucleic acid polymers in serum or plasma required extraction of the polymer followed by capillary electrophoresis, HPLC, or gel electrophoresis. These methods are time consuming and lack sensitivity. A bioanalytical method has been developed that does not require extraction of the ribozyme analyte from serum. This technique relies on hybridization of the ribozyme molecule to two complementary biotin and digoxigenin labeled oligonucleotide probes. Serum containing the ribozyme is mixed with the labeled probes, and the mixture is heated at 75 °C for 5 min to disrupt the ribozyme secondary structure. Samples are then cooled to permit probe annealing and are added to a streptavidin-coated 96-well plate. The bound complex is detected with an anti-digoxigenin alkaline phosphatase (AP) conjugate using PNPP ( p-nitrophenyl phosphate) as a substrate. The amount of colored product is measured on a microtiter plate reader at a wavelength of 405 nm. Concentrations of unknown ribozyme samples are estimated based on a standard curve (0.37–270 ng/ml) prepared in serum. The validated lower and upper limits of quantification are 5.0 and 120 ng/ml, respectively. The assay can be completed in approximately 5 h and does not require extraction procedures or electrophoretic/chromatographic separation. It is therefore a simple, sensitive and rapid technique. This assay has been validated and has been used for quantitation of serum levels of the HEPTAZYME™ ribozyme in mouse, monkey, and human pharmacokinetic studies.

Two-layer antibody capture of enzymes on the surface of microtiter plates: application to the study of the regulation of phospholipase C-γ1 catalytic activity

In vitro quantification of the catalytic activity of an enzyme isoform requires the availability of selective agents that allow for either measurements in the presence of the other enzyme isoforms or purification of the isoform and subsequent performance of these measurements on the purified enzyme. Isozyme-specific antibodies are useful tools for these types of analyses. In the present report, we detail a method for the measurement of phospholipase C-γ1 enzyme activity employing native enzyme that is immobilized on microtiter plates. The method uses biotinylated antiglobulin bound to streptavidin-coated microtiter plates to immobilize antiphospholipase C-γ1 antibody and subsequently capture phospholipase C-γ1 from brain tissue lysates. This method avoids biotinylation of the primary (antiphospholipase C-γ1) antibody, making it less labor intensive than previously described methods for using streptavidin-coated plates; in addition, it is highly reproducible and sensitive and allows for quantification of enzyme activity. We employ the technique to show that one or more tyrosine kinases copurify with rat brain phospholipase C-γ1. The method is applicable to the study of any enzyme isoform for which antibodies that capture the native form of the enzyme are available and could easily be employed in high-throughput procedures.

Effect of low molecular weight heparin and different heparin molecular weight fractions on the activity of the matrix degrading enzyme aggrecanase: structure-function relationship

The matrix-degrading enzyme aggrecanase has been identified in cartilage and is largely responsible for cartilage breakdown. The present study determined the efficacy of different heparin molecular weight fractions (HMWFs) and low molecular weight heparins (LMWHs) on aggrecanase activity. Aggrecanase activity was determined using biotinylated peptide substrate, which was immobilized onto streptavidin-coated 96-well plates; aggrecanase enzyme was then added. Proteolysis of the substrate at the specific amide bond was detected using specific antibody for the neoepitope generated. HMWFs ranging from 1,700 to 12,000 Da demonstrated a concentration-dependent inhibitory efficacy of aggrecanase activity, with a Ki ranging from 5,000 nM down to 1 nM as a function of the molecular weight. The higher the molecular weight distribution, the greater the inhibitory efficacy of the heparin fragments toward aggrecanase activity. The absence or presence of antithrombin did not alter the affinity of heparin in inhibiting aggrecanase. Additionally, tissue factor pathway inhibitor at various levels did not alter the activity of aggrecanase. LMWHs demonstrated different levels of potency in inhibiting aggrecanase activity as a function of their average molecular weight distribution. Tinzaparin (average molecular weight = 6,500 Da) and enoxaparin (average molecular weight = 4,500 Da) demonstrated a Ki of 20 and 80 nM, respectively. The aggrecanase inhibitory effect of LMWH might contribute to blocking inflammation and tumor invasion by inhibiting aggrecanase activity and maintaining an intact extracellular matrix barrier.

Macroscopic-scale surface properties of streptavidin and their influence on aspecific interactions between streptavidin and dissolved biopolymers

To avoid aspecific attractions between carrier surfaces for either ligand or receptor molecules in, e.g., immunoassays, or kinetic rate constant measurements, it has long been established that a background consisting of an aspecific, very hydrophilic carrier surface is generally quite effective. However, it is not often realized that one achieves such a non-reactive background, even with electrostatically neutral materials, at the price of creating a strong (polar) hydrophilic repulsion between dissolved biopolymer (e.g., protein) molecules and the non-adsorbing carrier surface. To investigate the quantitative effects of this type of repulsion in systems involving streptavidin, the surface properties of a streptavidin-coated glass plate were determined by contact angle measurements, from which the aspecific, macroscopic-scale free energies of repulsion between a streptavidin-coated surface and dissolved proteins such as immunoglobulin-G (IgG), and human serum albumin (HSA), could be derived. Streptavidin, even at neutral pH (at which it has virtually no electric surface charge as determined by electrophoresis) is very hydrophilic and strongly repels both IgG and HSA molecules. At neutral pH, molecules such as IgG and HSA, in aqueous solution, cannot approach a streptavidin layer more closely than to approximately 3.0 nm, which suffices to prevent IgG or HSA from any aspecific adherence to the streptavidin layer (as determined by extended DLVO analysis). This aspecific repulsion however also has the (usually unsuspected) effect of causing a decreased specific attachment between ligand and receptor molecules. In addition, it decreases the measured kinetic on-rate constants, often by about two decimal orders of magnitude. However, once the surface-thermodynamic properties of all the aspecific (macroscopic-scale) and specific (microscopic-scale) entities, as well as the specific equilibrium binding constant are known, the real kinetic on-rate constant between just the ligand and the receptor determinants can be determined, yielding the value it would have if the measurement of that constant were unhindered by the repulsive interactions exerted by the background of hydrophilic carrier molecules.

Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.

A microplate assay specific for the enzyme aggrecanase

We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.

Application of Random Peptide Phage Display to the Study of Nuclear Hormone Receptors

This chapter focuses on the application of random peptide phage display to the study of nuclear hormone receptors. The chapter is organized into four sections: (1) the construction of a peptide phage display library, (2) a step-by-step protocol for biopanning (the affinity selection process), (3) the discussion of a mammalian cell-based screening technology to validate and complement the in vitro phage display approach, and (4) the use of phage display selected peptides as antagonists of nuclear receptor function. The selection of peptides that bind to the target proteins of interest is achieved by incubating phage libraries containing small random peptide inserts with the target proteins that are immobilized on a solid support. Phage display has been widely used as a method to map protein–protein interactions, identify peptide ligands for cell surface receptors, and map antibody–antigen epitopes. By using phage display, random peptide libraries can be generated and specific target-interacting peptides can be identified. The random peptide libraries are created by inserting short random oligonucleotides within the coding sequence of the M13 bacteriophage capsid protein, with the subsequent display of random peptides on the outer surface of the phage. In addition to using phage display to study receptor conformation and SERM pharmacology, this powerful technique has been used to develop highly specific peptide antagonists that block receptor function by interfering with required protein–protein interactions. A very good alternative to immobilizing steroid–nuclear hormone receptors and other DNA-binding transcription factors is by using biotinylated oligonucleotides (corresponding to their cognate response element) pre-captured on streptavidin-coated plates.

1P-0029 Angiotensin-converting enzyme inhibitor promotes coronary collateral circulation in patients with coronary heart disease

The use of antisense oligodeoxynucleotides (AS-ODNs) in therapeutic applications requires the development of appropriate analysis and delivery systems. Here, we report a quantitation method and a carrier-mediated AS-ODN delivery system. AS-ODN levels were quantitated using an enzyme-linked immunosorbent assay (ELISA) in which biotinylated AS-ODNs bound to streptavidin-coated plates were detected by binding of a complementary, dinitrophenol-labeled detector ODN. The ELISA-based assay could detect AS-ODNs at the femtomole level. AS-ODN delivery systems based on opsinized erythrocyte ghosts (EGs) were developed using various combinations of hypotonic solution and resealing buffer to optimize AS-ODN encapsulation efficiencies. AS-ODN and polyethyleneimine (PEI) complex formation did not affect encapsulation into EGs. The ELISA-based assay showed that the pharmacokinetics of AS-ODNs differed significantly among the various delivery methods. Opsonized EG-encapsulated AS-ODNs exhibited a mean residence time (MRT) significantly shorter than AS-ODN encapsulated in EGs. The biodistribution of EG-loaded AS-ODNs depended on opsonization, with opsonized EG carriers producing 4.5-fold higher levels of AS-ODN in the liver compared with unopsonized EGs. These results indicate that opsonized EGs can be used for liver-targeted delivery of AS-ODN and suggest that an ELISA-based method may be useful for studying the in vivo fate of AS-ODNs.

A high-capacity streptavidin-coated microtitration plate.

A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). The maximum immobilization capacity of the modified plate was up to 2.5 times higher than that of the control plate. The higher binding capacity was especially emphasized with small-size molecules. The modified high capacity plate increased the linear ranges of the immunoassays and thus delayed the high-dose hook effect. At high antigen concentrations the signal increased up to 59%, and at the conventional linear ranges of the assays, the increase was up to 29%. We conclude that the modified coating method will be valuable for the future miniaturized systems, where high immobilization capacity is needed at limited areas.

Rinderpest virus lineage differentiation using RT-PCR and SNAP-ELISA

An RT-PCR/ELISA system has been developed that detects and differentiates Rinderpest virus (RPV) from the other closely related morbillivirus of ruminants, Peste des petits Ruminants virus (PPRV). In addition, using lineage specific probes, it is possible to determine whether the virus sample is wild-type or vaccine, and the likely origin of the outbreak if it is wild-type. It involves carrying out a RT-PCR with one digoxygenin (Dig)-labelled primer followed by a hybridisation step with a virus-specific, biotin-labelled, probe. The hybridisation step is carried out in an ELISA format on a streptavidin-coated plate. The DIG-labelled products are detected using a specific anti-DIG monoclonal antibody and an anti-mouse horseradish peroxidase conjugate. The hybridisation step replaces nucleotide sequencing or nested PCR for confirmation of the identity of DNA product. The assay is fast and easy to carry out and can give semi-quantitative estimates of the virus content of samples.

Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays

We have achieved sensitive, rapid and reproducible detection of three biological threat agents in a variety of biological and environmental matrices using the DELFIA time-resolved fluorometry (TRF) assay system (Perkin-Elmer Life Sciences, Akron, OH). Existing ELISA assays for the detection of Francisella tularensis, Clostridium botulinum A/B neurotoxin (BotNT A/B), and Staphylococcus aureus enterotoxin B (SEB) were converted to TRF assays. They use 100 μl of positive control or unknown per test well and require just over 2 h to run. Fluorescent signal read time is a fraction of a second per well. The assay format consists of a capture ELISA utilizing a biotinylated capture antibody, prebound to a streptavidin-coated 96-well plate and a lanthanide (Europium, Eu 3+)-labeled detector antibody. The bound Eu-labeled detector antibody produces a fluorescent signal upon the addition of an enhancement solution. The signal results from the dissociation of the Europium from the antibody, creating a micelle, thus amplifying the signal nearly one million-fold. Sensitivities achieved by these assays were between 4 and 20 pg/ml in buffer. Additionally, we have tested this system in different matrices such as serum, urine, dirt, and sewage. Concentration curves generated from standard solutions produced a wide linear range making serial dilutions of unknown samples unnecessary. DELFIA TRF assays are significantly better in terms of sensitivity, linear range, and run time than standard capture ELISAs and should facilitate early detection of potential biological warfare agents in clinical and environmental samples.

In Vitro High-Throughput Screening Assay for Modulators of Transcription

We developed a 96-well microtiter-plate high-throughput screening (HTS) assay for the detection of modulators of transcription. This HTS assay consists of three steps: (1) the in vitro transcription reaction; (2) modification and hybridization of RNA products; and (3) washing and quantification. During the first step, a DNA template containing the promoter of interest upstream of a cassette lacking guanosine residues in one of its strands (G-less cassette) is incubated with nuclear extract and the necessary cofactors/activators and substrates. During the second step, the in vitro synthesized transcripts are digested with RNase T1 and hybridized to two DNA oligonucleotides. One oligonucleotide is biotinylated for trapping of the RNA products to a streptavidin-coated plate, and the other is europium-labeled for detection by time-resolved fluorescence. We show that this assay is highly reproducible and robust, yielding results comparable to those obtained by standard methodologies employing radioactive nucleotide incorporation and gel electrophoresis while offering a very significant advantage in terms of throughput (>2000 assay points per operator per day). We demonstrate the usefulness of the assay for the discovery of small molecule inhibitors of transcription, and applications of this approach for the high-throughput discovery of transcriptional modulators are discussed.

Microbial identification by immunohybridization assay of artificial RNA labels

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA–RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA–RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

Engineering the prion protein using chemical synthesis.

In recent years, the technology of solid-phase peptide synthesis (SPPS) has improved to the extent that chemical synthesis of small proteins may be a viable complementary strategy to recombinant expression. We have prepared several modified and wild-type prion protein (PrP) polypeptides, of up to 112 residues, that demonstrate the flexibility of a chemical approach to protein synthesis. The principal event in prion disease is the conformational change of the normal, alpha-helical cellular protein (PrPc) into a beta-sheet-rich pathogenic isoform (PrP(Sc)). The ability to form PrP(Sc) in transgenic mice is retained by a 106 residue 'mini-prion' (PrP106), with the deletions 23-88 and 141-176. Synthetic PrP106 (sPrP106) and a His-tagged analog (sPrP106HT) have been prepared successfully using a highly optimized Fmoc chemical methodology involving DCC/HOBt activation and an efficient capping procedure with N-(2-chlorobenzyloxycarbonyloxy) succinimide. A single reversed-phase purification step gave homogeneous protein, in excellent yield. With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP106 was indistinguishable from its recombinant analog (rPrP106). Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine (Bo) PrP(90-200), were successfully prepared using a combination of the highly activated coupling reagent HATU and t-Boc chemistry. To mimic the glycosylphosphatidyl inositol (GPI) anchor and target sPrP to cholesterol-rich domains on the cell surface, where the conversion of PrPc is believed to occur, a lipophilic group or biotin, was added to an orthogonally side-chain-protected Lys residue at the C-terminus of sPrP sequences. These groups enabled sPrP to be immobilized on either the cell surface or a streptavidin-coated ELISA plate, respectively, in an orientation analogous to that of membrane-bound, GPI-anchored PrPc. The chemical manipulation of such biologically relevant forms of PrP by the introduction of point mutations or groups that mimic post-translational modifications should enhance our understanding of the processes that cause prion diseases and may lead to the chemical synthesis of an infectious agent.

Quantitative reverse transcription polymerase chain reaction of a molluscan metallothionein mRNA

A quantitative assay based on competitive reverse transcription polymerase chain reaction (RT-PCR) was developed for metallothionein (MT) mRNA of the mollusc Crassostrea virginica and applied to analysis of MT mRNA of hemocytes. The assay was based on titration of a competitive external standard cRNA derived from the coding region of the oyster MT mRNA. Serial dilutions of the cRNA standard were coamplified with a constant amount of total RNA using biotinylated primers common to both target and standard sequences. Amplified products were bound to streptavidin-coated plates and hybridized to sequence-specific fluorescein-labeled probes. Detection was based on single photon counting of chemiluminescence generated by an alkaline phosphatase-conjugated antifluorescein antibody. For quantification, the target chemilumincescence was normalized to that of the standard, and the amount of target MT mRNA in the sample was derived from the titration. Cadmium-induced MT mRNA equivalent to that in 180 hemocytes was easily detected, and, for routine quantitative analysis, was sufficiently sensitive to quantify basal and induced MT mRNA. Basal hemocyte MT mRNA of 133±8 (1 S.E.) amol per microgram total RNA was induced 5-fold to 573±14 amol per microgram total RNA by in vitro exposure to 15 μM CdCl 2 for 20 h.

Development of an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay for detection of equine and swine IgM antibodies to vesicular stomatitis virus.

An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competitive ELISA (C-ELISA) and the standard microtiter serum neutralization (MTSN) assay by testing serum samples from horses and pigs experimentally infected with VSV-NJ or VSV-IN. The MC-ELISA detected specific homologous IgM antibodies from equine and swine sera as early as 5 and 4 days postinfection (DPI), respectively, and as late as 35 DPI. The MTSN test also detected antibodies as early as 5 DPI and as late as 160 DPI. In a similar fashion, the C-ELISA detected antibodies from 6 to 7 DPI and as late as 160 DPI. These results demonstrated that the MC-ELISA is a useful test for serodiagnosis of primary VSV infection in horses and pigs.

Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300.

The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20O whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300-p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities.