High-sensitivity chemiluminescence immunoassays for detection of growth hormone doping in sports

23 Jan 2009

High-sensitivity chemiluminescence immunoassays for detection of growth hormone doping in sports / Martin Bidlingmaier, Jennifer Suhr, Andrea Ernst, Zida Wu, Alexandra Keller, Christian J. Strasburger, Andreas Bergmann. - (Clinical Chemistry 55 (2009) 3 (March); p. 445-453)
- PMID: 19168559.
- DOI: 10.1373/clinchem.2008.112458


Abstract

Background:
Recombinant human growth hormone (rhGH) is abused in sports, but adequate routine doping tests are lacking. Analysis of serum hGH isoform composition has been shown to be effective in detecting rhGH doping. We developed and validated selective immunoassays for isoform analysis with potential utility for screening and confirmation in doping tests.

Methods:
Monoclonal antibodies with preference for pituitary hGH (phGH) or rhGH were used to establish 2 pairs of sandwich-type chemiluminescence assays with differential recognition of rhGH (recA and recB) and phGH (pitA and pitB). We analyzed specimens from volunteers before and after administration of rhGH and calculated ratios between the respective rec- and pit-assay results.

Results:
Functional sensitivities were <0.05 microg/L, with intra- and interassay imprecision < or =8.4% and < or =13.7%, respectively. In 2 independent cohorts of healthy subjects, rec/pit ratios (median range) were 0.84 (0.09-1.32)/0.81 (0.27-1.21) (recA/pitA) and 0.68 (0.08-1.20)/0.80 (0.25-1.36) (recB/pitB), with no sex difference. In 20 recreational athletes, ratios (median SD) increased after a single injection of rhGH, reaching 350% (73%) (recA/pitA) and 400% (93%) (recB/pitB) of baseline ratios. At a moderate dose (0.033 mg/kg), mean recA/pitA and recB/pitB ratios remained significantly increased for 18 h (men) and 26 h (women). After high-dose rhGH (0.083 mg/kg), mean rec/pit ratios remained increased for 32 h (recA/pitA) and 34 h (recB/pitB) in men and were still increased after 36 h in women.

Conclusions:
Using sensitive chemiluminescence assays with preferential recognition of phGH or rhGH, detection of a single injection of rhGH was possible for up to 36 h.

CAS 1999_A_234 David Meca-Medina vs FINA

29 Feb 2000

CAS 1999/A/234 David Meca-Medina vs FINA
CAS 1999/A/235 Igor Majcen vs FINA

TAS 1999/A/234 David Meca-Medina v/ FINA
TAS 1990/A/235 Igor Majcen v/ FINA

Related case:
CAS 2000/A/270 David Meca-Medina & Igor Majcen vs FINA
May 1, 2001

On 8 August 1999 the FINA Doping Panel decided to impose a 4 year period of ineligibility on the Spanish swimmer David Meca-Medina and the Slovenian swimmer Igor Majcen after their A and B samples tested positive for the prohibited substance 19-norandrosterone (Nandrolone).

Hereafter in August 1999 both Athletes appealed the FINA decision with the Court of Arbitration for Sport (CAS).

The Athletes argued that there were departures of the standards for testing and laboratories; the substance in question was not prohibited; the positive tests were caused by the ingestion of pork offal; and the imposed sanction was too severe.

The Panel rejects the arguments that it was not their urine which was analysed and finds that the duration of the transport to the laborary had no effect on the validity of the test. The Panel holds that the chain of custody including the documentation was complete and satisfactory.

Further the Panel finds that the Athletes failed to establish that any alleged departure from the procedure could lead to genuine doubt on the reliability of any finding. They also failed to demonstrate that the positive tests was the result of the ingestion of meat injected with Nandrolone. The Panel establish that the substances in question were prohibited as part of the “related substances” before they were expressly included in the newer lists of anabolic androgenic steroids.


The Court of Arbitration for Sport decides on 29 February 2000 with respect to David Meca-Medina:

1.) The appeal is rejected as far as it is filed on behalf of David Meca-Medina.
2.) The suspension of David Meca-Medina is confirmed for a duration of 4 years from August 20, 1999 under deduction of 77 days of provisional suspension (May 14, 1999 to July 30, 1999).
3.) All results achieved by David Meca-Medina between January 31, 1999 and August 19, 1999 shall be cancelled.
4.) The award is pronounced without costs, except for the Court Office fee of CHF 500,- which shall be kept by the CAS (art. R65.2 of the Code)
5.) David Meca-Medina is ordered to pay an amount of CHF 4’000,- (together with interest at 5% from the date of the decision) to Respondent FINA as a contribution towards its legal fees and expenses (art. R65.3 of the Code).


The Court of Arbitration for Sport decides on 29 February 2000 with respect to Igor Majcen:

1.) The appeal is rejected as far as it is filed on behalf of Igor Majcen.
2.) The suspension of Igor Majcen is confirmed for a duration of 4 years from August 20, 1999 under deduction of 77 days of provisional suspension (May 14, 1999 to July 30, 1999).
3.) All results achieved by David Meca-Medina between January 31, 1999 and August 19, 1999 shall be cancelled.
4.) The award is pronounced without costs, except for the Court Office fee of CHF 500,- which shall be kept by the CAS (art. R65.2 of the Code)
5.) Igor Majcen is ordered to pay an amount of CHF 4’000,- (together with interest at 5% from the date of the decision) to Respondent FINA as a contribution towards its legal fees and expenses (art. R65.3 of the Code).

A robust test for growth hormone doping - present status and future prospects.

1 May 2008

A robust test for growth hormone doping : present status and future prospects / Anne E Nelson. Ken K Ho. - (Asian Society of Andrology 10 (2008) 3 (May); p. 416-425).
- PMID: 18385903.
- DOI: 10.1111/j.1745-7262.2008.00395.x


Abstract

Although doping with growth hormone (GH) is banned, there is anecdotal evidence that it is widely abused. GH is reportedly used often in combination with anabolic steroids at high doses for several months. Development of a robust test for GH has been challenging because recombinant human 22 kDa (22K) GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22K and other GH isoforms. Administration of 22K GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method has been implemented; however, its utility is limited because of the short window of opportunity of detection. The second approach, which will extend the window of opportunity of detection, is based on the detection of increased levels of circulating GH-responsive proteins, such as insulin-like growth factor (IGF) axis and collagen peptides. Age and gender are the major determinants of variability for IGF-I and the collagen markers; therefore, a test based on these markers must take age into account for men and women. Extensive data is now available that validates the GH-responsive marker approach and implementation is now largely dependent on establishing an assured supply of standardized assays. Future directions will include more widespread implementation of both approaches by the World Anti-Doping Agency, possible use of other platforms for measurement and an athlete's passport to establish individual reference levels for biological parameters such as GH-responsive markers. Novel approaches include gene expression and proteomic profiling.

Discrimination of recombinant from natural human growth hormone using DNA aptamers

1 Apr 2011

Discrimination of recombinant from natural human growth hormone using DNA aptamers / John G. Bruno, Maria P. Carrillo, Taylor Phillips, Allison Edge. - (Journal of Biomolecular Techniques 22 (2011) 1 (April) ; p. 27-36)

  • PMID: 21455479
  • PMCID: PMC3059541

Abstract

Detection of athletes who use synthetic human growth hormone (hGH; or somatotropin) to enhance physical strength and obtain an advantage in competitive sports is a formidable problem, as rhGH is virtually identical to the natural pituitary hormone. However, some post-translational and other modifications have been documented by chromatographic separation and mass spectrometry (MS) in a small percentage of rhGH. In the present work, development of DNA aptamers against research-grade rhGH and natural hGH with adsorption of the rhGH aptamers against natural hGH was shown to produce a small family of aptamer sequences that bound consistently with greater affinity to rhGH over a low nanogram-to-microgram range in ELISA-like microplate assays. This collection of rhGH discriminatory aptamer sequences shared some short sequence segments and secondary structural features. The top rhGH discriminatory aptamers also appeared to cross-react with human myoglobin and BSA but not with bone collagen peptides and an unrelated viral envelope peptide. The cross-reactivity results suggested several strings of up to five consecutive amino acids that might serve as common epitopes for aptamer binding. SDS-PAGE revealed that the rhGH existed largely as a 45-kDa dimer, and the natural hGH was almost exclusively monomeric. The existence of the rhGH dimer suggests that a discontinuous "bridge" epitope may exist on the rhGH, which spans the subunits, thereby accounting somewhat for the difference in detection. Overall, these results suggest that aptamers might be useful for routine, presumptive laboratory screening to identify athletes who are potentially cheating by administration of rhGH.

Detection of GH abuse in sport: Past, present and future

30 May 2009

Detection of GH abuse in sport: Past, present and future / Osquel Barroso, Patrick Schamasch, Olivier Rabin. - (Growth Hormone & IGF Research 19 (2009) 4 (August) ; p. 369-374)

  • PMID: 19482501
  • DOI: 10.1016/j.ghir.2009.04.021

Abstract:

Due to its considered performance enhancing effects, human growth hormone (hGH) is abused as a doping agent in sport. Its misuse also carries potentially serious side effects to a person’s health. Consequently, hGH and its releasing factors are prohibited in sport, as established in the Prohibited List which is updated and published yearly by the World Anti-Doping Agency (WADA). In order to fight the menace that hGH doping poses to the spirit of sport and to the health of athletes, the sport movement and the anti-doping authorities, initially led by the International Olympic Committee (IOC) and later by WADA, have put substantial efforts into developing tests for its detection. Currently, a primary analytical approach, the isoform differential immunoassay, has been implemented in WADA-accredited laboratories. In parallel, a second, indirect approach for the detection of hGH abuse, based on the quantification of hGH-associated biological markers, has been developed. The final aim is to combine both methodologies to improve the sensitivity and expand the time window to detect doping with hGH. In addition, novel analytical procedures, based on proteomic and genomic technologies as well as the use of mass spectrometry-based methods of detection, are being investigated for future application in hGH anti-doping tests.

CAS 2003_A_484 Kicker Vencill vs USADA - Interim award

18 Nov 2003

CAS 2003/A/484 Kicker Vencill vs USADA - Interim award

The Court of Arbitration Hereby rules:
1. The Jurisdiction oif CAS is affirmed;
2. Ibe appeal filed by mr. Vencill on 14 July 2003 is dismissed;

CAS 2003_A_484 Kicker Vencill vs USADA - Final award

11 Mar 2004

CAS 2003/A/484 Kicker Vencill vs USADA

Related documents:
AAA No. 30 190 00291 03 USADA vs Kicker Vencill
July 24, 2003
CAS 2003_A_484 Kicker Vencill vs USADA - Interim award
November 18, 2003
USADA - Supplement 411 - Kicker Vencill, Introduction Video
May 24, 2012

On 23 June 2003 the North American Court of Arbitration (NACAS) decided to impose a 4 year period of ineligibility on the American swimmer Kicker Vencill after his A and B samples tested positive for the prohibited substance 19-norandrosterone (Nandrolone).

Hereafter in July 2003 the Athlete appealed the NACAS decision with the Court of Arbitration for Sport (CAS). The Athlete requested for a reduced sanction on the basis of No Significant Fault or Negligence.

The Athlete argued a number of issues in support of his appeal, ranging from questions concerning the chain of custody of his sample, alleged violations of his right to be present for the testing of his B sample, supposed inaccuracies in the results reported by the UCLA Lab and allegations to the effect that the low concentration of 19-norandrosterone found in the athlete's sample is consistent with
endogenous production as opposed to exogenoμs administration or ingestion of a prohibited substance.

The Panel finds that there is no question that the Athlete is guilty of committing an anti-doping rule violation and he failed to establish that the chain of custody of his sample was anything other than intact.
The Panel concludes that the laboratory analysis was correctly conducted, the Athlete’s samples had not deteriorated or been contaminated and the proper laboratory procedures had been followed.

The Panel accepts that the violation was not intentional and that laboratory analysis revealed that the supplement in question was contaminated. However the Athlete showed also a total disregard of his positive duty to ensure that no prohibited substance enters his body. As a result the Panel holds that the Athlete’s Fault or Negligence in the circumstances is exceptionally significant in relation to the doping violation.

Therefore the of Court of Arbitration for Sport decides on 11 March 2004 that:

1.) The jurisriction of the CAS is affirmed;
2.) The appeal filed by Mr. Vencill on 14 July 2003 is dismissed;
3.) Save for the applicable period of ineligibility as specified in paragraph 4 below, the decision in this matter issued by the North American Court of Arbitration for Sport Panel dated 23 June 2003 is upheld;
4.) Kicker Vencill shall be declared ineligible for competition for two years commencing as of 22 May 2003:
5.) The Court Office fee of CHF 500 already paid by Mr. Vencill shall be retained by the CAS;
6.) Each party shall bear its own costs.

A false start in the race against doping in sport: concerns with cycling's biological passport

1 Nov 2011

A false start in the race against doping in sport: concerns with cycling's biological passport / Nicolas Hailey. - (Duke law journal 61 (2011) 2 (November) ; 393-432).

  • PMID: 22069854

Abstract:

Professional cycling has suffered from a number of doping scandals. The sport's governing bodies have responded by implementing an aggressive new antidoping program known as the biological passport. Cycling's biological passport marks a departure from traditional antidoping efforts, which have focused on directly detecting prohibited substances in a cyclist's system. Instead, the biological passport tracks biological variables in a cyclist's blood and urine over time, monitoring for fluctuations that are thought to indirectly reveal the effects of doping. Although this method of indirect detection is promising, it also raises serious legal and scientific concerns. Since its introduction, the cycling community has debated the reliability of indirect biological-passport evidence and the clarity, consistency, and transparency of its use in proving doping violations. Such uncertainty undermines the legitimacy of finding cyclists guilty of doping based on this indirect evidence alone. Antidoping authorities should address these important concerns before continuing to pursue doping sanctions against cyclists solely on the basis of their biological passports.

Sports doping : Racing just to keep up.

15 Jul 2011

Sports doping : Racing just to keep up / Ewen Callaway. - (Nature 475 (2011) 7356 (21 July) ; p. 283-285)
- PMID: 21776058.
- DOI: 10.1038/475283a


Anti-doping researchers are looking for new ways to catch cheaters. Can a biological passport help to save the sport?

Gene doping [2006]

1 Apr 2006

Genetische doping / H.J. Haisma, Olivier de Hon. - (Nederlands Tijdschrift voor Klinische Chemie en Laboratoriumgeneeskunde 31 (2006) 1 : p. 261-263)

Genetische doping zal zeer waarschijnlijk binnen 5 jaar zijn intrede doen in de sportwereld. Bepaalde genen kunnen de sportieve prestatie immers verbeteren. Deze genen worden thans onderzocht in klinische studies voor de behandeling van ziektes. Het niet-therapeutische en ongecontroleerde gebruik van gendoping door sporters kan resulteren in gezondheidsschade.
Preventieve maatregelen om het gebruik van gendoping te voorkomen zijn dus noodzakelijk. Een uitvoerig voorlichtingsprogramma t.b.v. de sportbegeleiders en de sporters zelf, een evaluatie van de huidige regelgeving en de ontwikkeling van een geavanceerde detectiemethode op basis van proteomische technieken lijken de meest veelbelovende preventieve maatregelen.

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