Detection of opiate use in a methadone maintenance treatment population with the CEDIA 6-acetylmorphine and CEDIA DAU opiate assays.

Heroin, with a plasma half-life of approximately 5 min, is rapidly metabolized to 6-acetylmorphine (6-AM). 6-AM, a specific marker for heroin use, which also has a short half-life of only 0.6 h, is detected in urine for only a few hours after heroin exposure. Ingestion of poppy seeds and/or licit opiate analgesics can produce positive urine opiate tests. This has complicated the interpretation of positive opiate results and contributed to the decision to raise opiate cutoff concentrations and to require 6-AM confirmation in federally mandated workplace drug-testing programs. Microgenics Corp. has developed the CEDIA 6-AM assay, a homogeneous enzyme immunoassay for semiquantitative determination of 6-AM in human urine, in addition to its CEDIA DAU opiate assay. Urine specimens were collected 3 times per week from 27 participants enrolled in a clinical research trial evaluating a contingency management treatment program for heroin and cocaine abuse. Of the 1377 urine specimens screened, 261 (18.9%) were positive for opiates at > or = 300 ng/mL, 153 (11.1%) were positive for opiates at > or = 2000 ng/mL, and 55 (4.0%) were positive for 6-AM at > or = 10 ng/mL. For opiate-positive screens > or = 300 and > or = 2000 ng/mL, 91.3% and 80.8% confirmed positive for morphine or codeine at the respective gas chromatography-mass spectrometry (GC-MS) cutoffs. All specimens screening positive for 6-AM also confirmed positive by GC-MS at > or = 10 ng/mL. Increasing the opiate screening and confirmation cutoffs for the federal workplace drug-testing program resulted in 8% fewer opiate-positive tests; however, recent heroin use was not affected by this change.

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.