Analyte detection with DNA-labeled antibodies and polymerase chain reaction.

We demonstrate immuno-polymerase chain reaction (PCR) assays for two clinical analytes--human thyroid-stimulating hormone and chorionic gonadotropin (hTSH, hCG)--using DNA-labeled antibodies and PCR for amplification of assay response. DNA-antibody conjugates were synthesized by using heterobifunctional cross-linker chemistries to covalently attach single- or double-stranded DNA labels through amine or sulfhydryl groups on the analyte antibodies. These approaches yielded molecular chimeras possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demonstrated for immuno-PCR assays of both analytes in a microtiter plate-based, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exceeded those of conventional enzyme immunoassays by 2-3 orders of magnitude. We also evaluated various DNA design factors influencing label amplification and assay performance, such as primer sequence, strand number, and DNA length. Our findings, in concert with previous reports, suggest that this hybrid technology could provide the basis for a new generation of ultra-sensitive immunoassays offering multianalyte capabilities.

High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction.

A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and beta-Gal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specific monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate co-amplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays by approximately three orders of magnitude. Detection limits for hTSH, beta-Gal and hCG were respectively 1 x 10(-19), 1 x 10(-17) and 1 x 10(-17) mol. Given the enormous amplification afforded by PCR and the existing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays.

Separation and isolation of viable bacteria by capillary zone electrophoresis.

Mixtures of bacteria, Enterococcus faecalis (ATCC 29212), Streptococcus pyogenes (ATCC 19615), Streptococcus agalactiae (ATCC 13813), Streptococcus pneumoniae (ATCC 6303), and Staphylococcus aureus (ATCC 29213), were resolved into discrete electrophoretic bands using capillary zone electrophoresis (CZE). Bacteria remained viable (> 90%) during the electrophoretic process. Analysis of peak separation indicates organisms were quantitatively resolved and recovered at greater than 98% purity. E. faecalis was resolved into two discrete fractions with different chain assemblages that may reflect different developmental stages. These findings suggest that CZE can afford the microbiologist a new tool for studying the composition and distribution of microorganisms in mixed populations.

Piezoelectric cell growth sensor.

We have developed a reusable piezoelectric sensor that enables rapid characterization of cell viability and response to cell-affecting agents. This is accomplished via a novel polymer transduction principle that involves reaction of a pH-sensitive amphoteric polymer with metabolically generated acid. Subsequent adhesion of the protonated polymer to the transducer surface causes a decrease in the sensor resonant frequency corresponding to the cell metabolic rate. This disclosure provides the first example of a piezoelectric sensor capable of detecting metabolic responses of viable cells. The sensor provides real-time measurement of cell metabolism and division rates, and antibiotic sensitivity. This technology provides the basis for an advanced piezoelectric sensor that does not require immobilized biological receptors and can be miniaturized without compromising signal-to-noise factors.

Effect of halothane anesthesia on muscle, liver, thyroid, and adrenal-function tests in man.

Seven healthy men volunteers received 6.6 +/- 1.3 (SD) percent-hours of halothane oxygen anesthesia without surgery. Serum bilirubin, alanine aminotransferase, and aspartate aminotransferase significantly increased after anesthesia, which may indicate subclinical liver-cell damage. Creatine kinase of skeletal muscle origin increased above 90 U/liter in six subjects, indicating subclinical muscle-cell damage. Cortisol, triiodothyronine uptake, thyroxine, and free thyroxine index increased significantly immediately after anesthesia. Serum bromide concentrations had increased by fivefold on the second day after anesthesia, and on the ninth day was still elevated fourfold. Oral temperatures increased 0.7 degrees C 6 h post-anesthesia, possibly because of increased thyroxine activity. Lactate dehydrogenase, hydroxybutyrate dehydrogenase and gamma-glutamyltransferase activities did not change significantly. No drugs administered during the course of this study chemically interfered with any of the test methods used.

Increased serum bromide concentration after halothane anesthesia in man.

Seven healthy male volunteers received 6.6 plus or minus 0.5 (SE) per cent-hours of halothane-oxygen anesthesia without surgery. Serum bromide concentrations increased from 0.6 plus or minus 0.1 before anesthesia to 2.9 plus or minus 0.2 mEq/l on the second day after anesthesia. On the ninth day serum bromide was still elevated to 2.5 plus or minus 0.1 mEq/l. These bromide concentrations represent psychoactive levels and may account for previously recognized prolonged changes in mood and intellectual function after halothane. Plasma and urinary fluoride concentrations did not increase significantly.