Research on spiking rat EPO as internal standard in doping control samples for detection of EPO using SAR-PAGE analysis with biotinylated primary antibody.

According to the current Technical Document (TD) for erythropoietin (EPO), SAR-PAGE is the most commonly applied method for both screening and confirmation procedures. Although this method is effective and robust, it lacks an internal standard (IS) to monitor the efficiency of analysis for each sample covering every step of the whole procedure, including preparation, immunopurification, and western blotting. This internal standard needs to be recognized by both anti-EPO antibodies used for immunopurification and western blotting, respectively. Besides that, the band of IS could not be allowed to interfere with the recognition of all types of targeted EPO and analogs. To meet these two principles, rat EPO was selected. In this study, rat EPO was used to spike both urine and blood samples at the beginning of analysis. After preparation and immunopurification, single blotting was performed with biotinylated AE7A5 as the primary antibody, followed by incubation with streptavidin-coupled HRP. Based on the comparison of different immunopurification methods, the AB-286-NA antibody coupled to M-280 magnetic beads was the better choice for urine samples, whereas the MAIIA column was suitable for blood samples. All these methods were validated for selectivity, repeatability, and sensitivity. The modified method in this study could not only eliminate the cross-reactivity between antibodies but also monitor the whole procedure of the analysis of EPO with spiked rat EPO. Besides that, rat EPO could also be used as an indicator for monitoring the presence of protease(s) in urine samples.

Comparison and optimization of SAR-PAGE tests for erythropoietins in doping analysis.

Erythropoietins (EPOs) are substances listed in S2 of the World Anti-Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR-PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two-step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H](+) as precursor ion in full-scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N-dimethyl, N-desmethyl and N,N-didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono- and di-hydroxylation, methoxylation, N-desmethylation, N,N-didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week.

Biomarker detection of rhGH doping: an excretion study.

The purpose of this research is to validate the biomarker-based approach for the detection of doping with recombinant human growth hormone (rhGH) in sport. The GH-2000 project proposed an indirect method for the detection of exogenously administered growth hormone (GH) based on the measurement of the GH-dependent markers: insulin-like growth factor-I (IGF-I) and Type III pro-collagen (P-III-P). These markers rise in a dose-dependent manner after GH application. In this study, the concentrations of IGF-I, IGF-BP3, and P-III-P in serum were determined to provide further incentives for the implementation of this detection assay in modern anti-doping programmes. This paper reports on an administration study of rhGH involving 25 Chinese male volunteers at a dose of 0.1 IU /kg/day for a continuous 14-day period. We observed that the serum IGF-I concentration increased rapidly in the rhGH treatment group and showed significantly higher levels compared to baseline between days 4 and day 16 after administration. Although the response of P-III-P to rhGH administration was delayed compared to the IGF-I axis, the P-III-P concentration remained increased for a longer period (from day 4 to day 28). Statistical analysis was carried out to establish a discriminant formula with Statistical Product and Service Solutions (SPSS) concluding that the biomarker methodology is valid and universally applicable.

Immunological screening and characterization of highly specific monoclonal antibodies against 20 kDa hGH.

BACKGROUND: hGH has been widely abused as a doping agent in sports for many years. There are some important approaches for the detection of hGH doping, and the ratio of 22:20 kDa GH was considered one of the most suitable detection indicators of GH abuse. Currently, effective anti-GH antibodies and related reagents are needed to develop a detection method, in particular, highly specific anti-20 kDa hGH monoclonal antibodies are a prerequisite. Herein we constructed the expression vector of 20 kDa hGH and prepared the corresponding antibodies by the immunization of the recombinant human 20 kDa into mice. Positive clones that can specifically recognize 20 kDa hGH were screened and characterized by enzyme immunoassay, Dot-ELISA and surface plasmon resonance. In total, 14 specific monoclonal cell lines were screened out. RESULTS: By a series of characterization, it was found that the 6C8, 44H3, 12G7 and 33Y19 clones were showing much higher specificity and affinity to 20 kDa hGH, and P3H9 could recognize both 20 and 22 kDa hGH isoforms. 6C8 and 44H3 matched well with P3H9 in the surface plasmon resonance testing. The 12G7 clone had the best surface properties with an association constant of 3.4 × 10(9) M(-1) and a dissociation constant of 2.95 × 10(10) M. CONCLUSION: Highly specific monoclonal antibodies against 20 kDa hGH were generated, and also two paired antibodies (P3H9 and 6C8 or P3H9 and 44H3) were characterized, which can serve as the potential components for 22:20 kDa detection kit.

Detection of doping with rhGH: excretion study with WADA-approved kits.

The detection of recombinant human growth hormone (rhGH) doping using the World Anti-Doping Agency (WADA) approved kits is reported in this research. Twenty-five young male students were selected and divided randomly into two groups with six belonging to the placebo and nineteen to the administration group. Thirteen volunteers in one group were administered with a Chinese preparation of rhGH while six volunteers included in the other group were given rhGH made in Switzerland. Both preparations were administered at a dose of 0.1 IU/kg body weight, one injection per day for 14 consecutive days. Blood samples were collected using WADA guidelines and all blood samples were analyzed with WADA-approved Kits 1 and 2. The time window for detection of rhGH doping using WADA-approved kits and criteria are discussed. Based on the comparison of the data obtained from this excretion study and from our routine (Chinese population as reference), consideration of the recent WADA criteria for rhGH AAF (Analytical Adverse Findings) is reported statistically. A comparison of data obtained from the two sample groups administered with pharmaceutical preparations, one Chinese rhGH (GenHeal®, S19990019, 1.6 mg (4 IU), Shanghai, China) obtained from prokaryotic cells and the other (Saizen®, S20080036, 1.33 mg (4 IU), Laboratoires Serone S.A., Switzerland) from eukaryotic cells is reported and did not show any significant difference for the detection of doping with rhGH.

Detection of doping with recombinant human growth hormone.

Detection of doping with recombinant human growth hormone is one of the challenges for antidoping analysis. This review focuses on the most important relevant publications that provide insight into the laboratory measurement of human growth hormone (hGH), antibodies and standards, the isoform approach and the biomarker approach. The isoform approach monitors the changes of hGH molecular isoform composition in serum and was applied at the Olympic Games in Athens in 2004, Turin in 2006 and Beijing in 2008. The markers approach detects a formula score, which reflects the changes in concentration of IGF-1 and P-III-P. All these methodologies measure the concentrations of growth hormone and its isoforms for isoform approach, or the concentrations of IGF-1 and P-III-P. All factors that affect these measurements should be taken into account for the development of methods to detect doping with recombinant hGH.