Analysis of saliva for drugs , On-site testing
A number of on-site test systems for drugs in oral fluid have been developed. These tests are immunochromatographic screening tests. They generally employ lateral diffusion of the oral fluid sample mixed with labelled antibodies in buffer across a linear array of immobilised drugs. When drugs are present in the oral fluid, they bind to the anti-drug antibodies, so the antibodies pass by and do not bind to the corresponding test line that contains the immobilised drug conjugate. Visualisation of the antibody label (colloidal gold, phosphor or other indicator) reveals a lack of response from the array location that corresponds to the drug(s) present. The first on-site saliva test that utilised an electronic reading device was the Cozart Rapiscan oral fluid drug testing system, developed in 1998, which uses a lateral transfer immunoassay with colloidal gold-labelled anti-drug antibodies (Spiehler 2001). The procedure is typical of those used for on-site oral fluid testing. The saliva specimen is collected from the mouth using a collection pad and placed in a test tube that contains 2 mL run buffer. When placed in the mouth, the collection pad absorbs 1 mL of saliva, which is indicated by development of a blue colour in the indicator section of the handle (see Fig. 1). The pad is placed in the tube, where it is diluted with 2 mL of run buffer fluid. The cellulose pad is separated from the plastic handle along a perforated edge. After removing the cap and plastic collector handle, the cotton pad is compressed with a dispenser filter to dispense six drops of the saliva–buffer mixture onto the cassette by directing the tip of the dispense filter tube into the cassette well and gently squeezing the tube.
A fresh disposable cassette and collection kit are used for each test. The cassette or cartridge is inserted into the hand-held instrument for incu bation, reading and reporting. The saliva and run fluid rehydrate gold-labelled anti-drug anti bodies contained within the cartridge. This mixture travels by capillary action across an array of immobilised drug sites (3–5 minutes for single- and two-panel tests; 12 minutes for a five panel test). The absence of colour development at an immobilised drug position indicates the presence of drug. The quality-control position contains anti-mouse IgG to ascertain that complete lateral transfer of the specimen has been achieved. The cassette result (binding of gold-labelled antibody to immobilised drug in the absence of drug in sample) is monitored by the portable, battery-powered reader and reported on the display screen. The results can be printed out on an optional battery-powered printer to provide a permanent record. Results are sent to the printer via the multifunctional port, which also serves to charge the instrument’s batteries and to up load new versions of the instrument software, new drug combinations, etc., via an internet interface module. If the saliva screening test is positive, the remainder of the sample (2.8 mL fluid) may be capped, with tamper-proof tape placed across the cap, and the samples sent to the designated laboratory for confirmation. Alternatively, a urine or blood sample may be collected and sent with the remainder of the positive saliva to the laboratory, depending on the preference of the contracting laboratory (De Giovanni et al. 2002). Other on-site immunochromatographic oral fluid drug tests that use colloidal gold antibody labels are the ORAL Screen System (Avitar, Canton, MA, USA; Barrett et al. 2001) and Drug Wipe and Drug Read (Securitec GmbH, Ottobrunbei Munchen, Germany), which employ the Frontline urine dipstick (Boehringer-Manheim GMbH, Manheim, Germany). A recent on-site immunochromatographic assay uses an up-converting phosphor based on lanthanide particles that absorb infrared light and emit visible light (up-conversion) as the antibody label (Fig. 1; Up-Link Rapid Detection system, OraSure Technologies, Inc., Bethlehem, PA, USA). Biological matrices do not up-convert, which eliminates the test background from the autofluorescence. As in the immunochroma to graphic procedure described above, oral fluid specimens are collected in a device that indicates sample adequacy and retains the oral fluid sample for confirmation testing if required. Specimens are mixed with buffer and introduced to a test cassette. Antibodies labelled with up converting phosphor microparticles contained on a lateral flow membrane in the cassette are mobilised when the liquid sample flows across the pad. The presence of increasing amounts of drug in the sample decreases the amount of antibody-bound label to the corresponding test line that contains the immobilised drug conjugate. A 10-minute incubation is required. The test simultaneously detects amfetamine, methamfetamine, phencyclidine (PCP) and opiates in oral fluid with 40% or better displace ment at 10 lg/L drug (Niedbala et al. 2001a). A reader utilising an infrared excitation laser (980 nm) and photomultiplier tube with a filter to determine visible light visualises the location of the bound phosphor-labelled antibody, which indicates whether drugs are present in the oral fluid. Up-converting phosphors that emit visible light at 475, 505, 550 and 720 nm are available. Different phosphors can be used as labels for different anti-drug antibodies, which allows close spacing of the drug conjugate lines on the immunochromatography strip.
Figure 1 The Up-Link collection device.
