Theodore K. Christopoulos

Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA

OBJECTIVE To develop a quantitative reverse transcriptase-polymerase chain reaction assay for monitoring the prostate-specific antigen (PSA) mRNA. METHODS PSA mRNA is amplified, in parallel, with the mRNA of beta-actin, a housekeeping gene. The ratio of the amplification products obtained reflects the relative amount of PSA mRNA with respect to actin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplified sequences. The PCR products are analyzed separately by time-resolved immunofluorometric hybridization assays, using specific probes immobilized in microtiter wells. The hybrids are reacted with alkaline phosphatase-labeled anti-digoxigenin antibody. The phosphate ester of fluorosalicylate is used as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb(3+)-EDTA which is measured by time-resolved fluorometry. RESULTS The hybridization assays for both PSA and actin amplification products show linearity in the range of 1.4-110 pmol/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mixtures containing various numbers of LNCaP cells in one million cells that do not express PSA and used them as samples in the proposed assay. The ratio of the fluorescence values obtained after analysis of PSA and actin amplification products is linearly related to the number of LNCaP cells in the range of 20 to 3000 cells. Reproducibility studies demonstrate %CVs for the fluorescence ratios of 14.7, 11.8, and 12.2 when samples containing 150, 300 and 1600 LNCaP cells were analyzed (n = 4). CONCLUSIONS A quantitative analytical methodology is provided for monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.

3 – THEORY OF IMMUNOASSAYS

This chapter deals with the theory of immunoassays. The Scatchard model focuses on the individual binding sites of the binder and applies the law of mass action for each site s defining the association constant K and assuming that the affinity of each particular site for the ligand is not influenced by the extent of occupancy of the other sites. Study of the kinetics of an immunoassay system allows the derivation of equations that predict the concentrations of reactants and products at any time, even if the system has not reached equilibrium. The rate of the reaction between a ligand and a homogeneous population of identical binding sites corresponds to the difference between the rates of immunocomplex formation and dissociation. A typical two-site (sandwich) immunoassay consists of several steps. Model based on the mass-action law is theoretically sound and very useful in gaining insights into the behavior of the assay system.

1 – PAST, PRESENT, AND FUTURE OF IMMUNOASSAYS

This chapter deals with the past, present, and future of immunoassays. Immunoassays employ antibodies as analytical reagents. The assays are based on the observation that in a system containing the analyte and a specific antibody, the distribution of the analyte between the bound and free forms is quantitatively related to the total analyte concentration. Immunoassays were first introduced in the 1960s by Berson and Yalow for insulin and by Ekins for thyroxine. In the first immunoassays, the distribution of the analyte between the bound and free forms was monitored by adding a fixed known concentration of radioisotopically labeled analyte followed by separation of the bound and free analyte and measurement of the radioactivity the bound fraction. Immunoassay applications expanded to areas such as therapeutic drug monitoring, measurement of enzymes, tumor markers, lipoproteins, vitamins, and many other metabolites, and the detection and quantification of antibodies and antigens associated with infectious agents. Immunoassays with labeled antibodies were introduced and shortly afterward the first “two-site” immunoassays were described.

Novel Hybridization Assay Configurations Based on In Vitro Expression of DNA Reporter Molecules

OBJECTIVES To develop and study novel microtiter well based DNA hybridization assays in which the DNA serves as a reporter molecule. METHODS Two hybridization assay configurations are proposed. In configuration A the target DNA is end-labeled with biotin and captured to streptavidin-coated wells. The one strand is removed by NaOH treatment and the other is hybridized with a dATP-tailed oligonucleotide probe. Configuration B involves simultaneous hybridization of heat-denatured target DNA with a biotinylated capture probe (immobilized on streptavidin-coated wells) and a dATP-tailed detection probe. In both configurations the hybrids are reacted with dTTP-tailed luciferase-coding DNA fragment followed by in vitro expression of the DNA on the solid phase. This is accomplished either by a coupled transcription/translation or by sequential transcription and translation reactions optimized separately. RESULTS The signal-to-background ratios for configuration A at 0.93 fmoles target DNA were 2.6 and 16.7 with the coupled and the separated transcription/translation protocols, respectively. As low as 0.1 fmoles target DNA can be detected with the separate transcription/translation protocol with a signal-to-background ratio of 2.7. The signal-to-background ratios obtained for 0.1 fmoles target DNA with configuration B using the coupled and the separate expression protocols were 2.2 and 4.6, respectively. The average CV was 10%. CONCLUSION The expression yield is significantly improved with the separate transcription/translation protocol. Both assay configurations offer high sensitivity and are easily automatable.

14 – FLUORESCENCE IMMUNOASSAYS

This chapter discusses applications and limitations of fluorescence immunoassay. Fluorometry is superior to spectrophotometry in terms of sensitivity and specificity. In general, the sensitivity of fluorescence is 10–1000-fold higher in comparison to absorbance measurements. Fluorometry was introduced in immunological assays to improve immunoassay sensitivity. An indication of the potential sensitivity of fluorometry is that the search for single-molecule detection has been based almost exclusively on the use of fluorescent compounds. In addition, fluorometric determination combines several parameters, such as excitation and emission wavelength, lifetime, and polarization, simultaneously. Because these parameters are affected by changes in the microenvironment of the fluorescent compound, fluorescence spectroscopy frequently allows the direct study of molecular processes, such as antigen–antibody interaction, without prior separation of the bound from the free fraction. This forms the basis for the development of homogeneous fluorescence immunoassays.