ABSTRACT
Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).
Subject(s)
Coproporphyrins/chemistry , Deoxyuracil Nucleotides/chemistry , Metalloporphyrins/chemistry , Microscopy, Fluorescence/methods , Acyltransferases/analysis , Animals , Deoxyuracil Nucleotides/chemical synthesis , Glucagon/analysis , Glutathione Transferase/analysis , Humans , Immunohistochemistry/methods , In Situ Hybridization/methods , Liver/chemistry , Male , Pancreas/chemistry , Plants/chemistry , Prostate/chemistry , Prostate-Specific Antigen/analysis , RNA, Ribosomal, 28S/analysis , Rats , Receptors, Androgen/analysis , Streptavidin , Time , Tumor Suppressor Protein p53/analysisABSTRACT
We investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized antibody and avidin metalloporphyrin conjugates. The optimal F/P ratio with respect to quantum yield, decay time, and retention of biological activity of these immunoreagents was determined. The reagents were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of hapten-labeled DNA probes, membrane antigens (CD type), and 28S rRNA. All stained samples exhibited bright phosphorescence that could be selectively detected using time-resolved microscopy, especially when glucose/glucose oxidase was added to the embedding medium to deplete oxygen. Applications of time-resolved detection of phosphorescent porphyrins in strongly autofluorescent material (histological sections) are discussed.
