Notes
Slide Show
Outline
1
ELISA TESTING OF RACING
HORSES:
A REGULATORY REVOLUTION
  • By
  • Thomas Tobin
  • University of Kentucky
  • Maxwell H. Gluck Equine Research Center
  • for the
  • California Association of Toxicologists
  • Tempe, Arizona
  • May 18th, 2002
  • Last Modified: May 30th, 2002 3:00 pm



2
HISTORY OF DRUGS IN HORSES
  • The less than proper medication of racing horses has a long history, with an absolutely impeccable starting point, Greek mythology
3
 
4
 
5
 
6
 
7
 
8
How do Some Horsemen try to Win Races?
  • Any way they can, including but not limited to the creative use of drugs and medications
9
Acute Stimulant Medication
  • Classic stimulants: cocaine, caffeine, amphetamine, methylphenidate, methamphetamine, mazindol
  • Opiates: etorphine, fentanyl, morphine, buprenorphine
  • Tranquilizers: “washy horses”: acepromazine, promazine, fluphenazine, detomidine, etc.
  • Bronchodilators: clenbuterol, terbutaline, atropine, ipratropium bromide
  • Always illegal and usually an “inside job”
10
Narcotic Analgesics
  • Morphine and synthetic analgesics
  • Locomotor stimulant
  • Suppress pain
  • Prolongs endurance
  • Potentiated by stimulants
  • Reliable, classic stimulant medication: widely used pre-ELISA
11
 
12
Bronchodilators
  • Beta-2 agonist bronchodilators: clenbuterol, albuterol, terbutaline, etc.
  • Highly potent agents
  • Presumably improve oxygen delivery to blood and musculature
  • Widely used close to post prior to ELISA
  • Post-ELISA: use cut back dramatically
13
Tranquilizers as Stimulants
  • Used to treat “washy” horses (“running” race in paddock)
  • Allows jockey to “rate” horse in the race
  • Detomidine, reserpine, acepromazine, promazine
  • All legitimate therapeutic agents: rate of improper use?
14
Chronic Stimulant Medication
  • Anabolic steroids, growth hormone, erythropoietin (EPO)
  • Special food supplements
  • Vitamins
  • By definition an “inside job”
  • With the exception of anabolic steroids, not currently a very active area
15
“Stopping” Horses
  • “Nobbling” the English term
  • Horses heavily backed “ante-post”
  • Administer tranquilizer or depressant to slow or stop a horse
  • Oldest form of illegal medication
  • Usually considered an “outside” job
16
 
17
Medication to Lose
  • The oldest form of illegal medication
  • In principle, always possible to medicate a horse so that it will lose
  • Challenge is to treat a horse such that he goes to post but does not win his race
  • More likely where there is heavy ante-post betting
  • Also possible to medicate a horse to lose so that a favored horse will win
  • Always illegal, and usually considered an “outside” job
18
Medication to Restore
“Normal” Performance
  • Thought is that you can medicate a horse to restore “normal” performance
  • No stimulant or depressant actions
  • Non-steroidal anti-inflammatory agents (NSAIDs)
  • Prophylaxis of EIPH: furosemide
  • Corticosteroids?
19
Medication to “Mask” Other Agents
  • Administer agents that will interfere with testing
  • As testing improves, a less likely scenario
  • Furosemide: dilution
  • Polyethylene glycol: smear TLC plates
  • Thiamine: interfere with UV analysis
20
Medication to Dilute Out Other Drugs
  • Administer a drug that dilutes drugs/drug metabolites and makes them more difficult to detect
  • Diuretics generally do not affect blood levels of drugs
  • Dilute out water soluble drugs/drug metabolites in equine urine
  • Effect is generally up to but not greater than the diluting effect
  • Rules on furosemide in the US specify dose and time of administration
21
Can Drugs/Medications Influence Performance?
22
 
23
 
24
 
25
 
26
Equine Drug Testing
  • Equine drug testing has been in place longer (since 1910), and is more rigorous and extensive (250-2,500 agents) than any other ongoing human drug testing endeavor
  •                  WHAT HAPPENS
  • Horsemen use drugs ® Chemists develop tests ® Horsemen switch to new drugs ® Chemists develop new tests; one outcomes is that:
  • Neogen has a large and developing panel of Forensic Drug Tests
                               ad infinitum?
27
 
28
Equine Drug Testing
  • Based on post-race urine samples
  • About 10% of runners sampled/500,000 samples/year worldwide
  • Winners, beaten favorites, selected horses that the stewards nominate
  • Usually a central university-based laboratory, testing 10-40K samples/year
  • Scope of testing very broad: 250-2,500 agents
29
 
30
High Potency Agents
  • Any drug given to a horse < 10 mg/horse


    • Etorphine, Fentanyl, Acepromazine, Mazindol, Terbutaline, Buprenorphine, Cocaine, Oxymorphone, Detomidine

  • Not being reliably detected pre-ELISA
  • Being used at will by some horsemen
31
 
32
The 1980’s: ELISA Testing
  • Immunoassay program starts; Illinois Racing Board Laboratory, IDS, a Biotech Company, and the University of Kentucky
  • Sensitivity of testing increased 100-1,000 fold, virtually overnight, for 20+ drugs
  • Multiple identifications of narcotics, stimulants, tranquilizers, bronchodilators
33
ELISA Testing
  • Enzyme Linked ImmunoSorbent Assay (ELISA)
  • Simple, fast, high sensitivity testing
  • ~1,000 times more sensitive than thin layer chromatography
  • In 1985, UK started a research project that rapidly evolved into the ELISA testing program
34
Enzyme-Linked ImmunoSorbent Assay (ELISA) Testing

Thomas Tobin, Daniel Tai, and David Watt, 1985-1992
  • 1985: Kentucky Racing Commission requests control of high potency narcotics
  • 1986: RIA tests for fentanyl and etorphine (“Elephant     juice”)
  • 1987: Program develops ELISA tests
  • 1988: ELISA tests dramatically effective: POLITICS
  • 1989: Program reorganized – development of tests continues
  • 1992: 50 ELISA tests for high potency drugs, yielding ELISA Technologies, a division of Neogen    Corporation
35
 
36
 
37
How to make an ELISA test
  • Obtain drug or drug structure
  • Derivatize drug: add COOH or NH2 group: hapten
  • Link derivatized drug (hapten) to BSA
  • Inject into rabbit, re-inject, wait
  • Link hapten to Horse Radish Peroxidase (HRP)
  • Check rabbit serum for antibodies
  • Construct and validate ELISA test


38
 
39
 
40
 
41
 
42
 
43
 
44
 
45
ELISA Screening of Post-Race Urine Samples Followed by Mass Spectral Analysis
46
Efficacy of PCFIA/ELISA Tests, 1987-1988
47
Fall 1987
  • ELISA testing of frozen post-race samples in New Mexico
  • First panel of ELISA tests puts down 40 plus trainers in New Mexico
  • A problem that had been in place, in one form or another for 100, years had been controlled
48
The 1980’s: Impact of ELISA Testing
  • Probably the biggest single “across the board” increase in the sensitivity of equine drug screening
  • Stopped patterns of medication that had been in place in one form or another for the better part of 100 years
  • Also greatly increased the sensitivity of testing for therapeutic medications
49
ELISA Testing
  • Offers virtually complete control of the abuse of high potency medications
  • Also offers super-sensitive testing for therapeutic medications
  • Has accentuated a long-standing problem in equine medication control: Detection of trace residues of therapeutic medications and environmental and endogenous substances
50
Where To Go, 1987?
  • 1987  -- Five ELISA tests   
                  (but 100’s of drugs)
  • 2002  -- Neogen’s nearly 100 ELISAs,   can screen for more than    250 agents
  • Many current ELISA tests are of interest in HUMAN FORENSICS and the FOOD SAFETY INDUSTRIES
51
Neogen’s ELISA Tests
for Use in Racing (May 1st, 2002)
  • Alfentanil, amphetamine, amphetamine ultra, anileridine, apomorphine, azaperone, barbiturate group, benzodiazepine group, boldenone, bronchodilator group, bumetanide, buprenorphine, butorphanol, caffeine/pentoxifylline, carfentanil, clenbuterol, cocaine/benzoylecgonine, corticosteroid group, cromoglycate, dantrolene, detomidine, dexamethasone, diprenorphine, doxapram, droperidol, ethacrynic acid, etorphine, fentanil group, fentanyl, flunitrazepam, flunixin, fluoxetine, fluphenazine, furosemide, glycopyrrolate, guanabenz, haloperidol…
52
Neogen’s ELISA Tests, cont’d
  • Haloperidol metabolite, hydromorphone, isoxsuprine, ketamine, ketorolac, levallorphan, lidocaine, lofentanil, mazindol, mazindol metabolite, meperidine, mephentermine, mepivacaine, methadone, methamphetamine/MDMA, methocarbamol, methotrexate, methylphenidate, methylprednisolone, nalbuphine, nandrolone, opiate group, oxymorphone/oxycodone, pentazocine, phenylbutazone, procaine, promazine group, propanolol, pyrilamine, reserpine, stanozolol, sufentanil, sulfamethazine, THC metabolite, theophylline, trenbolone, triamcinolone acetonide, tricyclics group, zomepirac


53
Neogen’s Forensic Market
 ELISA Tests
  • Alfentanil, Amphetamine, Barbiturate group, Benzodiazepine group, Boldenone, Buprenorphine, Butorphanol,Carfentanil, Cocaine/BZE, Etorphine, Fentanil group, Flunitrazepam, Hydromorphone, Lofentanil, Ketamine, LSD, Meperidine, Methadone, Methylphenidate, Methamphetamine/MDMA, Nandrolone, Opiate group, Oxymorphone/Oxycodone, Sufentanil, THC Metabolite
54
NEOGEN AND FORENSIC CHEMISTRY
  • Neogen has available a large and expanding range of high quality forensic tests
  • Largest selection of high quality forensic ELISA tests available worldwide
  • Available to Equine and Human Forensic Labs.

    -AD INFINITUM?-
55
Efficacy of Equine Drug Testing
  • Rate of detection of performance altering agents in racing is relatively low
  • “Positives” in about 1-1,000 samples tested.
  • All identifications confirmed by mass spectrometry in the primary and the referee laboratory.
  • “Top-end” of racing is actually very clean
  • Thanks in part to the technology I have just described
56
SUMMARY
  • ELISA testing technology is highly effective: the Worldwide Standard in Racing
  • Simple,fast,inexpensive and highly sensitive
  • Technology is highly adaptable
  • Single, portable “Horse Side” tests
  • Inexpensive automated systems
  • High end, high volume, high throughput completely automated systems
57
THREE CUSTOMIZED LEVELS OF ELISA TEST UTILIZATION
  • Limited Throughput:  10K samples/year
         5-10 tests/sample
  • Medium Throughput: 20K samples/year
         10-15 tests/sample
  • High Throughput:      100K samples/year
          25-30 tests/sample
58
LIMITED VOLUME THROUGHPUT
  • Contract to buy a certain annual volume of ELISA tests (100K tests/year, or so)
  • Make available fully automated $50K Thermolabsystems ‘DSX’ Analyzer to perform the ELISA analyses
  • Information: scottcongleton@Neogen.com
59
 
60
 
61
 
62
MEDIUM VOLUME THROUGHPUT
  • Contract to buy a certain annual volume of ELISA tests (300K tests/year, or so)
  • Make available fully automated $100K Tecan Genesis System to perform the ELISA analyses
  • Information: scottcongleton@Neogen.com


63
 
64
HIGH VOLUME THROUGHPUT
  • Contract to buy a certain annual volume of ELISA tests (3,000K tests/year, or so)

    Make available fully automated $200K Beckman Coulter Orca System to perform the ELISA analyses

    Information: scottcongleton@Neogen.com


65
 
66
 
67
 
68
 
69
 
70
 
71
THANKS TO THE FOLLOWING
  • Thanks to my longtime colleagues at Neogen Corp, for their ongoing support in expanding and commercializing this technology
  • To all the members of my research group,and  especially Drs. W. Karpiesiuk, S. Kwiatkowski and Mr. W. E. Woods.
  • The Kentucky Racing Commission and the Kentucky Equine Drug Council for support
  • To the Director of the Gluck Equine Research Center and to my colleagues there and at UK for their support over the years
72
Finally, in closing, thank you very much,Ladies and Gentleman, for your kind  attention this morning
73
LITERATURE REFERENCES
  • 1/ Remifentanil in the Horse: Identification and    Detection of its Major Urinary Metabolite; Lehner et al., J. Anal. Tox. Vol 24, July/August, 2000
  • 2/ “Kentucky Laboratory Takes Lead Against Horse Dopers,” Tobin, Clinical Chemistry News, Vol. 18, Issue 1, January 1992
  • 3/ Non-isotopic Immunoassay Drug Tests in Racing Horses: A Review of their Application to Pre- and Post-Race Testing, Drug Quantitation and Human Testing; Tobin et al., Res. Comm. Chem. Path. Pharmacol. Vol 62, #3, P371-396, 1988
74