Intensely luminescent immunoreactive conjugates of proteins and dipicolinate-based polymeric Tb (III) chelates.

Partial alkylation of polylysine with 4-(iodoacetamido)-2,6-dimethylpyridine dicarboxylate (IADP), followed by exhaustive reaction with succinic anhydride, yielded polymers (PLDS, polymer of lysine, dipicolinate, and succinate) containing large numbers (50-100) of 4-substituted dipicolinic acid moieties per molecule, with the remaining lysyl side chains succinylated. Competition experiments showed that PLDS binds Tb(III) ions with much higher affinity than does EDTA and strongly enhances the visible luminescence they emit when excited with ultraviolet light. Carbodiimide-mediated coupling to proteins, including bovine serum albumin, ovalbumin, and protein A, yielded PLDS-protein conjugates whose Tb(III) chelates displayed intense green luminescence and millisecond excited state lifetimes. These conjugates retained sufficient immunoreactivity to allow their use in sensitive luminescence-based immunodetection schemes for proteins immobilized on nitrocellulose. The presence of 10 ng of ovalbumin could be easily visualized by eye when probed with rabbit anti-ovalbumin and PLDS-protein A-Tb(III). The ease of preparation of PLDS-protein-Tb(III) conjugates, and their favorable luminescence properties, make them promising reagents for use in time-resolved luminescence immunoassays and other ultrasensitive detection schemes for macromolecules.

Low affinity interactions of GDPbetaS and ribose- or phosphoryl-substituted GTP analogues with the heterotrimeric G protein, transducin.

We have examined the effects of three commonly used classes of guanine nucleotide analogues on the retinal G protein, transducin (Gt), and found them to be quite different from those that might be expected from results with other GTP-binding proteins. The most surprising results were with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS); rather than inhibiting activation of Gt, GDPbetaS addition activated Gt as a result of a trace contaminant. Even when the contaminant levels were reduced 5-fold by chromatography, its effects dominated those of GDPbetaS, which binds Gt at least 1500-fold more weakly than guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The affinity of Gt for GDP was found to be at least 300-fold weaker than for GTPgammaS, while the affinities of GTP and GTPgammaS were similar. Ribose-modified GTP analogues, including 2'(3')-O-(N-methylanthraniloyl) GTP (mantGTP), 2'(3')-O-[(2-aminoethyl)carbamyl] GTP (edGTP), and adducts of fluorescein 5-isothiocyanate and rhodamine B-isothiocyanate with edGTP, interacted extremely weakly, if at all, with the GTP binding site of the alpha subunit of Gt. They were neither effective activators of Gt nor effective inhibitors of activation by GTP or GTPgammaS. A gamma-phosphoryl-modified analogue, an adduct of GTPgammaS and (5-(2(iodoacetyl)aminoethyl)amino)naphthalene-1-sulfonic acid (dnsGTP), also activated Gt weakly, if at all, and did not inhibit its activation. The exclusion of these analogues points to the highly restrictive and specific nature of the GTP binding site of Gt, in contrast to those of numerous other GTP-binding proteins which are potently activated or inhibited by these analogues.

Direct detection of nucleic acid hybridization on the surface of a charge coupled device.

A method is described for the detection of DNA hybrids formed on a solid support, based upon the pairing of oligonucleotide chemistry and the technologies of electronic microdevice design. Surface matrices have been created in which oligonucleotide probes are covalently linked to a thin SiO2 film. 32P labeled target nucleic acid is then hybridized to this probe matrix under conditions of high stringency. The salient feature of the method is that to achieve the highest possible collection efficiency, the hybridization matrix is placed directly on the surface of a charge coupled device (CCD), which is used to detect 32P decay from hybridized target molecules (1, Eggers, M.D., Hogan, M.E., Reich, R.K., Lamture, J.B., Beattie, K.L., Hollis, M.A., Ehrilich, D.J., Kosicki, B.B., Shumaker, J.M., Varma, R.S., Burke, B.E., Murphy, A., and Rathman, D.D., (1993), Advances in DNA Sequencing Technology, Proc. SPIE, 1891, 13-26). Two implementations of the technology have been employed. The first involves direct attachment of the matrix to the surface of a CCD. The second involves attachment of the matrix to a disposible SiO2 coated chip, which is then placed face to face upon the CCD surface. As can be predicted from this favorable collection geometry and the known characteristics of a CCD, it is found that as measured by the time required to obtain equivalent signal to noise ratios, 32P detection speed by the direct CCD approach is at least 10 fold greater than can be obtained with a commercial gas phase array detector, and at least 100 fold greater than when X-ray film is used for 32P detection. Thus, it is shown that excellent quality hybridization signals can be obtained from a standard hybridization reaction, after only 1 second of CCD data acquisition.