Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2.

An assay for the autoubiquitination activity of the E3 ligase HDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used to measure the activity of other E3s and may be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display.

Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. "Phage display" enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.

Conjugates of COL-1 monoclonal antibody and beta-D-galactosidase can specifically kill tumor cells by generation of 5-fluorouridine from the prodrug beta-D-galactosyl-5-fluorouridine.

5'-O-beta-D-galactosyl-5-fluorouridine is a prodrug that can be converted by the enzyme beta-D-galactosidase to the potent antineoplastic drug 5-fluorouridine. The prodrug is more than 100x less toxic than the drug to bone marrow cells in Balb/c mice. The ratio of the IC50 of the prodrug to that of the drug determined on a variety of tumor cell lines in vitro ranged from 500:1-1000:1. An antibody-enzyme conjugate (AEC) was synthesized and purified. Maleimide-substituted COL-1 anti-CEA monoclonal antibody was linked to free thiol groups of beta-D-galactosidase. The conjugate was purified by size exclusion and ion exchange chromatography. It retained full immunoreactivity and enzyme activity. After binding to antigen-positive tumor cells, the conjugate was able to activate the prodrug and specifically kill the cells. We are continuing to investigate this model for its potential use in antibody-directed enzyme prodrug therapy (ADEPT).

Isoabzymes: structurally and mechanistically similar catalytic antibodies from the same immunization.

Mechanistic and structural comparisons of five catalytic monoclonal antibodies generated from the same hybridoma fusion indicated that all five hydrolyze phenyl acetate by subtle variations of the same mechanism. All of the antibodies showed a pre-steady-state multi-turnover burst in which kcat and Km declined but kcat/Km did not change. The burst of one of the antibodies, 20G9, has previously been found to result from inhibition by the product, phenol. Although all of the antibodies showed the burst, their individual values for kcat, Km, and hapten Ki differed substantially. Three of the antibodies that were investigated for the effect of pH on kcat showed an acid limb pK of 9.5-9.6. Substrate inhibition was seen in four of the five antibodies. Variable region nucleotide sequencing of the heavy and light chains confirmed that all five antibodies were structurally similar and also revealed several potentially critical tyrosines. Despite their structural similarities, analysis of their sequences suggested that the antibodies are products of distinct, independent rearrangements of immunoglobulin gene segments that took place in different progenitor B cells. A plot of Ki for hapten inhibition vs Km/kcat for substrate hydrolysis for the mechanistically related antibodies ("isoabzymes") gave a linear relationship suggesting a catalytic role for transition-state complementarity. Taken together with previous work [Martin et al. (1991) Biochemistry 30, 9757-9761], the data conform to a mechanism in which the antibodies exploit both transition-state complementarity and an acyl-tyrosyl intermediate during phenyl acetate hydrolysis.

Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products, using 3'-labelled oligonucleotide probes.

Described are rapid assays for the analysis of PCR products in a one step, non-separation assay based on the use of electrochemiluminescence generated from a tris-bipyridine ruthenium (II) label. The assay uses PCR incorporation of a biotinylated oligonucleotide as a primer, with the inclusion of a labelled oligonucleotide. Oligonucleotides were labelled with an N-hydroxy succinimide ester of tris-bipyridine ruthenium (II) dihexafluorophosphate (Origen-label) by modifying the 3' and 3' 5' ends of the oligonucleotide probes. The assay makes use of the inherent thermal stability and absence of polymerase activity on such probes to allow the PCR and probe hybridization to be completed automatically on the thermocycler. The assay is concluded by the addition of PCR samples to streptavidin beads on an electrochemiluminescence analyser for binding and analysis. Target genes evaluated were the HIV-1 gag gene, and cystic fibrosis delta F-508 deletion mutation. The results obtained from these assays demonstrated the detection of 10 copies of the HIV-1 gag gene, and cystic fibrosis delta F-508 mutation in 1 ng of human DNA within 15 min. This assay format allows a rapid and simple determination of specific amplified DNA sequences, reducing the contamination risks due to washes and multiple pipetting.

Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products.

We describe the characterization and utility of a new electrochemiluminescent (ECL) label for oligonucleotides, utilizing phosphoramidite chemistry. This phosphoramidite of the tris(2,2-bipyridine)ruthenium(II) complex, bis(2,2-bipyridine)(4-[4-(2-cyanoethoxy-N,N-diisopropyl-amino) phosphinoxybutyl]4'-methyl)2,2-bipyridine ruthenium(II) dihexafluorophosphate or Origen phosphoramidite, enables the direct incorporation of the label during automated DNA synthesis. Efficiency of this automated synthesis allows the direct utilization of probes without further purification. Introduction of this labeling group is reproducible, and the ECL signal recovered is not influenced by hybridization. Furthermore, neither hybridization kinetics nor hybrid stability was affected by our label. We also demonstrate the utility of these labels for the development of rapid assays with oligonucleotides direct from automated synthesis. The clinical utility of these labeled oligonucleotides is shown with assays of total nucleic acid, extracted from peripheral blood lymphocytes of patients with acquired immunodeficiency syndrome (AIDS), to detect the human immunodeficiency virus (HIV-1). The results demonstrate the ability of the assay to quantify 30-2000 copies of HIV1 gag genes and to rapidly detect (less than 45 min) HIV-1 gag genes in a nonseparation assay. The application of this assay to clinical samples demonstrates the utility of these assays for rapid and quantitative analysis.

Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics.

Electrochemiluminescence (ECL) has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. This new technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (approximately 1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the chemiluminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds. We illustrate ECL in nonseparation immunoassays for digoxin and thyrotropin and in separation immunoassays for carcinoembryonic antigen and alpha-fetoprotein. The application of ECL for detection of polymerase chain reaction products is described and exemplified by quantifying the HIV1 gag gene.

Rapid electrochemiluminescence assays of polymerase chain reaction products.

We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.

A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene. This chimeric antibody has allowed the development of a sensitive luminescent immunoassay. Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in Escherichia coli. The gene was expressed as apoaequorin and, by using luciferin isolated from Renilla reniformis, its activity was found essentially identical to native aequorin. The aequorin gene was subcloned into a mammalian expression vector to produce a fusion protein directing secretion of apoaequorin; the aequorin gene was fused to the 3' terminus of an immunoglobulin heavy-chain gene that directed expression of an anti-4-hydroxy-3-nitrophenacetyl antibody. The gene fusion contained the variable region, the constant region domain 1, and part of domain 2 for the IgG2b mouse immunoglobulin, followed by the aequorin gene. Transfection of the chimeric gene into a cell line expressing the complementary lambda 1 light chain, J558L, allowed recovery of a chimeric antibody with binding specificity for the 4-hydroxy-3-nitrophenacetyl group and the related 4-hydroxy-3-iodo-5-nitrophenacetyl hapten. The Ca2(+)-dependent bioluminescent activity of aequorin was also recovered.

Characterization of a chimeric aequorin molecule expressed in myeloma cells.

We have constructed a chimeric aequorin consisting of a fragment of the anti-NP immunoglobulin gene fused to the aequorin gene. Expression in a myeloma cell line has produced a Fab'-like molecule that has the ability to bind NIP specifically and generate bioluminescent activity. It takes approximately 8 h at 4 degrees C in the presence of 2-mercaptoethanol and coelenterazine to regenerate luminescent activity. While the flash kinetics of this recombinant molecule are similar to native aequorin, its quantum efficiency is ten times lower. Preliminary studies have been conducted to ascertain its usefulness for immunoassays. We have shown for this chimeric aequorin 7 x 10(-19) moles can be detected in solution, also it can be used in a solid-phase assay and is stably stored at -70 degrees C for at least 2 months.

Preliminary crystallographic data, primary sequence, and binding data for an anti-peptide Fab and its complex with a synthetic peptide from influenza virus hemagglutinin.

X-ray quality crystals which diffract to high resolution (less than or equal to 1.9-2.1 A) have been grown of an anti-peptide Fab and its complex with a 9-residue peptide antigen. Both crystals are monoclinic P2(1), with unit cell dimensions a = 90.3 A, b = 82.9 A, c = 73.4 A, beta = 122.5 degrees for the native Fab and a = 63.9 A, b = 73.0 A, c = 49.1 A, beta = 120.6 degrees for the complex. The peptide sequence corresponds to residues 100-108 of all influenza virus hemagglutinins (HA1) of the H3 subtype (1968-1987). The peptide antigen has been well characterized immunologically (Wilson, I.A., Niman, H.L., Houghton, R.A., Cherenson, A.R., Connolly, M.L., and Lerner, R.A. (1984) Cell 37, 767-778; Wilson, I.A., Bergmann, K.F., and Stura, E.A. (1986) in Vaccines '86 (Channock, R.M., Lerner, R.A., and Brown, F., eds) pp. 33-37, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), structurally, as a free peptide by NMR (Dyson, J.H., Cross, K.J., Houghton, R.A., Wilson, I.A., Wright, P.E., and Lerner, R.A. (1985) Nature 318, 480-483; Dyson, J.H., Lerner, R.A., and Wright, P.E., (1988) Annu. Rev. Biophys. Chem. 17, 305-324), as part of the intact antigen by x-ray crystallography (Wilson, I.A., Skehel, J.J., and Wiley, D. C. (1981) Nature 289, 366-373) and by binding studies to the HA molecule (White, J.M., and Wilson, I.A. (1987) J. Cell Biol. 105, 2887-2896). Knowledge of the three-dimensional structure of the complex will elucidate the details of how anti-peptide antibodies recognize a small peptide antigen and provide insights into the recognition of the same sequence in the intact protein antigen. As both native Fab and the peptide-Fab complex have been crystallized, we can also determine in addition whether changes in the structure of the antibody accompany antigen binding. The nucleotide sequence of the mRNA coding region of the anti-peptide Fab has been determined to provide the amino acid sequence ultimately required for the high resolution three-dimensional structure determination.

A sensitive, nondestructive assay for transfected genes.

We have adapted the fibrin overlay assay for plasminogen activators (Jones et al., 1975) into a gene transfer expression assay which has the advantage of being very sensitive and nondestructive. In this assay plasminogen activators convert plasminogen to plasmin, which then degrades fibrin, resulting in clearings in a fibrin overlay. Furthermore, the assay can be used as a signal indicating the efficiency of gene transfer or the loss of introduced genetic elements in unstable cell lines.

The synthesis and in vivo assembly of functional antibodies in yeast.

The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here the synthesis, processing and secretion of light and heavy chains, the glycosylation of heavy chain, the intracellular localization of these foreign proteins by immunofluorescence, and the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo. Furthermore, only low-level assembly of these chains was found in vitro.

Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised in E. coli.

Genes for a murine mu heavy chain and a lambda light chain immunoglobulin have been inserted into bacterial expression plasmids containing the Escherichia coli trp promoter and ribosome binding site. Induction of transcription from the trp promoter results in accumulation of both light and heavy chain polypeptides in appropriate host strains. Both proteins were found as insoluble products. Following extraction and purification of the immunoglobulin containing fractions, antigen binding activity was recovered. The activity demonstrates essentially the same properties as the antibody from the hybridoma from which the genes were cloned.

Properties of a human immunoglobulin epsilon-chain fragment synthesized in Escherichia coli.

A fragment of the cloned gene for the human myeloma ND epsilon chain, coding for the second, third, and fourth domains of the immunoglobulin, has been coupled to the tryptophan control region of an expression plasmid and subcloned in Escherichia coli. Induction of gene expression results in the synthesis of the expected, antigenically active polypeptide of Mr 40,000, which constitutes 18% of total bacterial protein and yields 55 mg/liter of culture. The immunoglobulin, which is aggregated and packed into large inclusion bodies within the bacterial cell, can be dissolved by denaturing solvents and purified by affinity chromatography using anti-IgE Sepharose. Reduced monomeric chains assemble spontaneously into dimers. On assay to measure the inhibition of binding of 125I-labeled human E myeloma protein to Fc epsilon receptors on cultured human basophils, the cloned gene product exhibited 20% of the activity of the native protein.

Cloning and sequence determination of the gene for the human immunoglobulin epsilon chain expressed in a myeloma cell line.

Messenger RNA has been isolated from cells of the human myeloma line 266BL which synthesizes IgE of the myeloma ND. A fraction enriched in mRNA for the epsilon heavy chain was copied into DNA and the DNA was cloned in Escherichia coli. A chemically synthesized oligonucleotide probe, based on the experimentally determined sequence of the specific message, was used to screen colonies. The largest epsilon chain cDNA cloned, 2.0 kilobases, was characterized by restriction endonuclease mapping and DNA sequence analysis. It appears to encode the complete amino acid sequence of the epsilon chain, including a signal peptide at the NH2 terminus as well as untranslated sequences at the 5' and 3' ends of the mRNA. The missing part of the previously published amino acid sequence of the ND epsilon chain was determined from the DNA sequence.