A generic particle-based nonradioactive homogeneous multiplex method for high-throughput screening using microvolume fluorimetry.

We have developed a novel fluorescence-based homogeneous binding assay for high-throughput screening of chemical compounds. In this assay, a Cy5- or Cy5.5-labeled ligand binds to receptor immobilized on a particle, either a bead or a cell. The resulting localized signal can be detected by a modified microvolume fluorimeter (MVF). When a molecule which competes with the labeled ligand is present, the localized fluorescence on cells or beads is reduced. Image processing software enumerates events and analyzes fluorescence intensity. We describe MVF assays for the IL-1 and IL-5 receptors. Using synthetic peptides with a range of affinities for the IL-1 receptor, we obtained IC(50) data consistent with those determined by radioligand binding assays. Because the image processing software can discriminate among events with different diameters, we were able to develop a multiplex assay, in which the IL-1R and IL-5R assays were carried out in the same well with each receptor immobilized on a different size of bead. IC(50) values generated in the multiplex assay for ligands specific to each receptor were comparable to those determined independently. Finally, similar IC(50) values were obtained in a 16-microl volume in an 864-well plate. This homogeneous, nonradioactive, miniaturizable, and multiplex-capable assay holds much promise for screening of combinatorial libraries and compound collections.

A generic method for expression and use of "tagged" soluble versions of cell surface receptors.

A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.

The use of light-directed combinatorial peptide synthesis in epitope mapping.

The application of light-directed combinatorial peptide synthesis to epitope mapping is described. Photolithography and solid phase peptide synthesis were combined in an automated fashion to assemble arrays containing 1024 peptide sequences on a glass support in ten steps with the precise location of each peptide known. The simultaneous synthesis of two slides containing three arrays of peptides each allowed for the independent screening of both a monoclonal antibody (mAb) and its Fab fragment at two different concentrations. A binary synthesis strategy was used to assemble the arrays, resulting in all deletions and truncations possible within the FLRRQFKVVT sequence being present and available for screening. The relative binding interactions of each peptide was determined by incubating the arrays with either mAb D32.39 and goat antimouse immunoglobulin G-FITC or mAb D32.39 Fab-FITC conjugate, followed by scanning the surface for fluorescence with an epifluorescence microscope. The fragment RQFKVVT was found to bind tightly to both the mAb and Fab fragment while tethered to the surface, and was measured to have 0.49 nM affinity in solution. The frame-shifted RRQFKVV sequence was found to have lower affinity both in solution (1.3 mM) and on the surface. The fragment RQFKVV was determined to be responsible for antibody recognition and was found to bind tightly when tethered to the surface, yet exhibited no binding in solution as the free acid, suggesting the requirement of an amidated C-terminus or an additional flanking residue. A deletion analysis revealed that the novel RQFKVT sequence exhibited higher affinity than the RQFKVV sequence while tethered to the surface.

Urinary hormone analysis as a diagnostic tool to evaluate the ovarian function of female gorillas (Gorilla gorilla).

Daily urine samples were collected from 4 adult female gorillas over 7 menstrual cycles. Urinary oestrone conjugate and pregnanediol-3-glucuronide (PDG) were measured by radioimmunoassay; LH was measured by enzyme immunoassay and each hormone was indexed by creatinine. The quantity of urinary LH during the ovulatory surge was positively correlated with the quantity of PDG excreted during the luteal phase (r = 0.87, P = 0.0013). The observations indicate a relationship between the quality of the LH surge and the levels of PDG in the luteal phase and suggest that both the LH surge and subsequent luteal phase function may be predictable from the oestrogen excretion profile during the follicular phase.