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

Abstract

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.

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

“…These were markers of the IGF-axis (such as IGF-1 and IGFBP2) and bone markers (such as osteocalcin), known to be influenced by somatotropin and previously examined by the GH-2000 group for detecting somatotropin abuse in athletes [13], [32]. Furthermore, the immune response of cows treated with rbST was examined thoroughly and we used the presence of the specific endogenous antibodies against rbST as a biomarker for its abuse [4], [30], [33], [34]. Although PIIINP, a marker of collagen turnover, is known to show potential in human and bovine hormone abuse detection [10], [18], it has not been included into our biomarker panel yet because of the lack of a suitable commercially available standard protein and antibody.…”

“…A generic sample pre-treatment procedure which was crucial for removing non-specific interferences and making the candidate biomarkers accessible for detection was described previously [4], [28], [29], is depicted in Figure 1 and described in-depth in Materials S1.…”

“…Covalent coupling of 100 µg mL −1 Monsanto rbST standard, 100 µg mL −1 IGF-1 and 10 µg mL −1 IGFBP2 to seroMAP microspheres (sets 050, 025, 078 respectively) was described before [4], [28], [29]. Coupling 75 µg mL −1 osteocalcin to microspheres (set 084) was done following the same procedure.…”

“…Biomarker profiles are indicative for more than one administered agent as they reflect the physiological effect, hence, the abuse of unknown compounds can also be detected [1], [2]. Furthermore, in many cases, the analysis of biomarker profiles enables the detection of abused substances for a longer time period, because the biological effect lasts longer than the abused substance itself can be detected in the body [3], [4]. A lot of work was focused on the identification of indicative biomarkers and the development of assays for detection of those [2], [5]–[10].…”

“…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]. In a recent study, three candidate biomarkers were combined in one screening tool, but the <5% false-compliant rate target could not be achieved [31].…”