Ramon A. Evangelista

Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays

Enzyme-amplified lanthanide luminescence (EALL) is a new method which has been developed for enzymatically amplified signal detection in ultrasensitive bioanalytical assays where an enzyme is used as label or is itself the analyte of interest. Signal generation is performed by enzymatically transforming a substrate into a product which forms a luminescent lanthanide chelate; the product chelate can then be detected using time-resolved or normal fluorescence methods. Alkaline phosphatase substrates have been developed and demonstrated in a model immunoassay in microwell format. The method has also been demonstrated for detection of a variety of other hydrolytic and oxidative enzymes. Thus the EALL method shows promise for use in a wide variety of bioanalytical applications.

Analysis of structural specificity in antibody—antigen reactions by capillary electrophoresis with laser-induced fluorescence detection

A rapid and simple procedure for screening antibodies for binding to an antigen is proposed. A fluorescent hapten-dye conjugate was prepared by labeling the amino moiety of the hapten with a commercially available reactive cyanine dye, Cy5 (excitation maximum: 650 nm, emission maximum: 670 nm). A fixed amount of the Cy5-hapten was titrated with serial dilution of the antibody. Each of the titration mixture was analyzed by capillary electrophoresis (25 cm x 20 microns column) monitored by laser-induced fluorescence (laser: 10 mW helium-neon, 632.8 nm). Free and antibody-bound Cy5-hapten were analyzed simultaneously on the electropherogram. Competitive immunoassay of hapten was demonstrated with low-end sensitivity of 5.10(-8) M, about 10x more sensitive than the present drug screening methods. Using morphine as an example, the screening of various antibodies (from different vendors) and cross-reactivity of morphine analogues using the present procedure will be discussed.

Alkyl- and aryl-substituted salicyl phosphates as detection reagents in enzyme-amplified fluorescence DNA hybridization assays on solid support

Nine salicyl phosphate esters with hydrophobic substituents (5-phenyl, 5-(2,4-difluorophenyl), 5-tert-octyl, 5-cumyl, 5-(4-tert-butylphenyl, 5-(1-adamantyl), 5-(n-dodecyl), 5-(1,1-diphenylethyl, and 5-trityl) were synthesized and found to be good substrates for calf intestinal alkaline phosphatase. The enzymatic hydrolysis produced the corresponding salicylates, which were strongly fluorescent when excited by ultraviolet light around 300 nm with maximum emission at 420-435 nm. The salicylates were less soluble and/or more adhesive than the nonfluorescent salicyl phosphate substrates, resulting in localization of fluorescence signal, which is a requirement for membrane-based assays. The salicyl phosphates bearing 8-14 carbon substitutents were found to be suitable detection reagents for dot-blot DNA hybridization assays on nylon membrane using a biotinylated probe, allowing the detection of 125 pg of target pBR322 plasmid DNA using a simple apparatus consisting of a transilluminator, a camera. and a 455-nm cutoff optical filter.

Detection of amplified Y chromosome-specific sequence by capillary electrophoresis with laser-induced fluorescence**Supported by Advanced Technology Center, Beckman Instruments Inc., Fullerton, California.

OBJECTIVEnTo develop a sensitive method for genetic diagnosis using capillary electrophoresis with laser-induced fluorescence.nnnDESIGNnUsing male DNA diluted with female DNA as an example, ZFY gene from Y chromosome was amplified specifically by polymerase chain reaction (PCR) with fluorescence-labeled primers and detected by capillary electrophoresis with laser-induced fluorescence.nnnSETTINGnLaser operating laboratory.nnnPARTICIPANTSnHuman male and female DNA were extracted from healthy human male and female subjects.nnnINTERVENTIONSnNone.nnnMAIN OUTCOME MEASURESnThe concentration of human DNA was determined by using a spectrophotometer at an absorbance of 260 nm.nnnRESULTnDeoxyribonucleic acid fragments amplified from a single copy of ZFY gene were detected by capillary electrophoresis with laser-induced fluorescence after 35 cycles of PCR amplification.nnnCONCLUSIONnThis method is potentially applicable for rapid and sensitive detection of fetal Y chromosome DNA sequence in maternal circulation and of single-cell DNA diagnosis.