Non-isotopic immunoassay drug tests in racing horses: a review of their application to pre- and post-race testing, drug quantitation, and human drug testing.

We have introduced large scale non-isotopic immunoassay testing into pre- and post-race drug testing in racehorses. The technologies utilized are Particle Concentration Fluorescence Immuno Assay (PCFIA) and the one-step Enzyme Linked Immuno Sorbent Assay (ELISA). These technologies are rapid, inexpensive, and highly effective. On introduction into post-race testing in the Western United States, these ELISA tests exposed several previously undetected patterns of drug abuse. The drugs detected were buprenorphine, oxymorphone, mazindol, sufentanil and cocaine. This led to the suspension of a large number of trainers and exposed the high false negative rate of thin layer chromatography (TLC) based testing. More recently, we have introduced both PCFIA and ELISA assays into pre- and post-race testing in Illinois. Within days, our pre-race PCFIA tests detected signs of acepromazine abuse. Directed searches of post-race urines from these horses showed evidence for acepromazine metabolites in the urine of these horses. Examination of frozen samples from associated horses yielded about 70 ELISA "positives" for acepromazine. To date, about 25 of these ELISA "positives" have been confirmed by mass spectrometry. We have also raised antibodies to phenylbutazone and furosemide to enable rapid and inexpensive quantitation of these permitted medications. Furosemide is a particular problem since its use requires a pre-race detention barn. For furosemide, we have developed a regulatory schedule based on our immunoassay test that allows elimination of the detention barn. For phenylbutazone, we have developed a similar immunoassay that allows rapid and inexpensive quantitation of this drug in blood. To enable racing authorities to test jockeys and other racetrack personnel, we have adapted PCFIA technology to human drug testing, and a full range of very sensitive tests for human drugs of abuse is available. These immunoassays are sufficiently sensitive to control abuse of the most potent drugs available to horsemen. In principle, an immunoassay can be raised to any drug within about six months, and made available worldwide at competitive rates. It appears clear that these non-isotopic immunoassays provide racing with the only technological basis that is sufficiently sensitive to detect the most potent abused drugs pre- and post-race, and has the flexibility to be readily adaptable to different drugs. Because of the high false negative rate generated by TLC, credible pre- and post-race testing programs cannot be based on TLC alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoassay detection of drugs in racing horses. III. Detection of morphine in equine blood and urine by a one step ELISA assay.

A one step enzyme-linked immunosorbent assay (ELISA) test for morphine was evaluated as part of a panel of pre- and post-race tests for narcotic analgesics in racing horses. This ELISA test is very sensitive to morphine with an I-50 for morphine of about 400 pg/ml. The test is also rapid, and ten samples, a normal pre-race complement, can be analyzed in about thirty minutes. The test can be read with an inexpensive spectrophotometer, or even by eye. The test readily detects the presence of morphine or its metabolites in equine blood for up to six hours after administration of sub-therapeutic doses. The antibody also cross-reacts with hydromorphone, orymorphone, nalorphine, levorphanol, and codeine, and the test either can detect or is likely to detect these drugs in blood or urine shortly after their administration to horses. As such this test is capable of dramatically improving the speed and efficacy of both pre-race and post-race testing for morphine and its congeners in racing horses. On initial introduction into post-race urine screening this test flagged 18 of 166 samples positive for opiates, and 13 of these samples were confirmed positive for opiates by mass spectrometry.

Immunoassay detection of drugs in horses. I. Particle concentration fluoroimmunoassay detection of fentanyl and its congeners.

We investigated the use of particle concentration fluorescence immunoassay (PCFIA) as a technique for drug detection in racing horses. The test was constructed from an antiserum to a carboxyfentanyl-BSA conjugate and carboxyfentanyl linked to b-Phycoerythrin. Using these reagents and a PCFIA apparatus levels of fentanyl as low as 0.1 ng/ml could be detected by the assay. In addition, cross-reactivity studies on this assay showed that the anti-serum cross-reacted well with carfentanil, sufentanil and the methylated analogs of fentanyl. We therefore evaluated the ability of these agents to produce pharmacological effects in the horse and the ability of this test to detect pharmacologically significant doses of this drug in racing horses. All of these agents produced good locomotor responses in horses at doses of between 0.1 and 10 micrograms/kg. Of these agents, carfentanyl was the most potent followed by 3-methylfentanyl, sufentanyl, alpha-methylfentanyl, and fentanyl. Similarly, when these agents were administered to horses at doses sufficient to produce a pharmacological response, all produced sufficient inhibition of fluorescence in the PCFIA system to enable their detection in post-race urines from these horses. Since PCFIA is a much faster technique than radioimmunoassay, is of approximately similar sensitivity, and requires much less instrumentation we concluded that this technique holds considerable promise as an equine drug testing technique.

Inhibition of T-antigen-binding cells by idiotypic antisera.

Shared idiotypy between B- and T-cell receptors specific for the antigen L-tyrosine-p-azophenyltrimethylammonium [tyr(TMA)] was studied in an antigen-binding assay using idiotypic antisera. These idiotypic reagents were prepared by inoculation of rabbits with purified anti-tyr(TMA) antibody raised in strain 13 guinea pigs. The antisera blocked 78-83% of the antigen-binding T cells (T-ABC) and 50-55% of the antigen-binding B cells (B-ABC) from tyr(TMA)-immune strain 13 and outbred lymph node cells (LNC). An excess of normal guinea pig Ig in the ABC assay did not affect the ability of the idiotypic antisera to block T- and B-ABC. Nylon wool-passed tyr(TMA)-immune LNC were trypsin treated resulting in a 75% loss of T-ABC. The trypsin-treated population was then cultured for 16 h which resulted in a return of T-ABC to 92% of pretrypsin values. 77% of these regenerated T-ABC could be blocked with idiotypic antisera. Specificity of the idiotypic antisera was tested in L-tyrosine-p-azobenzenearsonate-immune guinea pig LNC. Neither T- nor B-ABC were blocked in this heterologous system. Further blocking experiments were performed to characterize the nature of the T-ABC receptor. A variety of anti-Ig reagents, some of which block B-ABC, do not inhibit T-ABC suggesting that variable regions on T cells are not linked to Ig Constant regions.

Antigen-binding lymphocytes in guinea pigs. I.B cell expansion to the monovalent antigen L-tyrosine-p-azophenyl trimethylammonium (tyr(TMA)) in the absence of antibody production.

The monofunctional antigen L-tyrosine-p-azophenyltrimethylammonium chloride, tyr(TMA), and the polyfunctional antigen, TMA-human gamma-globulin (TMA-HGG), were used to investigate the antigen structural requirements necessary for clonal proliferation of B cells. This clonal expansion was characterized with respect to receptor immunoglobulin class and affinity maturation. Antigen-binding analysis revealed that inoculation of tyr(TMA), although only of m.w. 344, triggers clonal expansion of B lymphocytes 9-fold in the absence of any apparent antibody production. There does not appear to be any maturation with respect to antibody class since greater than 90% of the tyr(TMA)-specific B cells bear the micron receptor in the nonimmune and immune state. However, the average avidity of the B cells for this antigen increases with time after immunization. In contrast, immunization with TMA-HGG results in an 18-fold increase in B lymphocytes with significant amounts of anti-TMA antibody production. With time after immunization, both maturation of average avidity and class of Ig receptor (micron leads to gamma shift) occur. These findings indicate that the functionally T cell-specific antigen tyr(TMA) can trigger clonal B cell expansion and affinity maturation at the receptor level in the absence of detectable antibody production.