Identification of Cows Treated with Recombinant Bovine Somatotropin

Breton, Marie-Hélène Le; Rochereau-Roulet, Sandrine; Chéreau, Sylvain; Pinel, Gaud; Delatour, Thierry; Bizec, Bruno Le

doi:10.1021/jf903032q

Cited by 20 publications

(9 citation statements)

References 12 publications

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“…For the three best-performing prediction models IA, AO and IAO, false-positive rates ranged from 10.6% to 14.7%, which was quite acceptable, since samples screened positive must be analysed by a subsequent confirmatory analysis method according to Commission Decision 2002/657/EC anyway [23]. The confirmation method is based on the detection of an N-terminal peptide of somatotropin, which has a different terminal amino acid in the recombinant form of the hormone [26].…”

Section: Resultsmentioning

confidence: 99%

“…Note that in practice, a whole stable and not an individual cow is treated with rbST, thus increasing the chance of detecting rbST use. And third, for final confirmation of rbST itself in serum samples of suspect individual cows, a highly sensitive mass spectrometric confirmatory method, which fulfils the 2002/657/EC confirmatory method requirements, is to be used [26].…”

Section: Discussionmentioning

confidence: 99%

“…Although biweekly injections containing slow-release formulations are used to prolong the presence of rbST in the cows' body, the protein levels in treated animals cannot be distinguished from the background level throughout the whole two-week inter-injection period and large inter-individual differences in blood rbST levels were reported [19], [20], [24]–[27]. Furthermore, rbST immunoassays were not capable to distinguish the almost identical recombinant and endogenous forms of bST [19], [20], [24], [25] and mass spectrometry-based methods on the other hand required very tedious sample preparation procedures [26], [27]. For screening of rbST in cattle, a few biomarker-based methods were developed, but focused on a single candidate biomarker only [4], [9], [28]–[30].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Ludwig¹,

Smits²,

Veer³

et al. 2012

PLoS ONE

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Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin – anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Ludwig¹,

Smits²,

Veer³

et al. 2012

PLoS ONE

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“…Direct detection of rbST, however, is problematic due to its short half‐life in blood, the similarity with the endogenous hormone (bST, also called growth hormone), the low concentrations of bST and rbST in serum, and strong fluctuations therein. Preliminary results regarding the development of a confirmation method for rbST detection based on liquid chromatography‐mass spectrometry (LC‐MS) show potential . But that method is rather laborious, time‐consuming, and requires highly trained personnel and expensive equipment.…”

Section: Introductionmentioning

confidence: 99%

Development of a flow cytometric immunoassay for recombinant bovine somatotropin‐induced antibodies in serum of dairy cows

Smits¹,

Bremer²,

Ludwig³

et al. 2011

Drug Testing and Analysis

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Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4 weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle.

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“…In contrast to MS‐based sports drug testing for hGH, several MS methodologies for identification of rGH in bovine and equine species have been developed (Blokland et al, 2003, ; Le Breton et al, 2008, ; Bailly‐Chouriberry et al, ). Identification of rGH in these species is possible by selective measurement of N‐terminal tryptic peptides that contain small amino acid substitutions (Fig.…”

Section: Bottom‐up Ms Detection Of Growth‐promoting Peptides and Protmentioning

confidence: 99%

Current trends in mass spectrometry of peptides and proteins: Application to veterinary and sports‐doping control

Broek

Blokland

Nessen

et al. 2013

Mass Spectrometry Reviews

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Detection of misuse of peptides and proteins as growth promoters is a major issue for sport and food regulatory agencies. The limitations of current analytical detection strategies for this class of compounds, in combination with their efficacy in growth-promoting effects, make peptide and protein drugs highly susceptible to abuse by either athletes or farmers who seek for products to illicitly enhance muscle growth. Mass spectrometry (MS) for qualitative analysis of peptides and proteins is well-established, particularly due to tremendous efforts in the proteomics community. Similarly, due to advancements in targeted proteomic strategies and the rapid growth of protein-based biopharmaceuticals, MS for quantitative analysis of peptides and proteins is becoming more widely accepted. These continuous advances in MS instrumentation and MS-based methodologies offer enormous opportunities for detection and confirmation of peptides and proteins. Therefore, MS seems to be the method of choice to improve the qualitative and quantitative analysis of peptide and proteins with growth-promoting properties. This review aims to address the opportunities of MS for peptide and protein analysis in veterinary control and sports-doping control with a particular focus on detection of illicit growth promotion. An overview of potential peptide and protein targets, including their amino acid sequence characteristics and current MS-based detection strategies is, therefore, provided. Furthermore, improvements of current and new detection strategies with state-of-the-art MS instrumentation are discussed for qualitative and quantitative approaches.

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Identification of Cows Treated with Recombinant Bovine Somatotropin

Cited by 20 publications

References 12 publications

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Development of a flow cytometric immunoassay for recombinant bovine somatotropin‐induced antibodies in serum of dairy cows

Current trends in mass spectrometry of peptides and proteins: Application to veterinary and sports‐doping control

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