Solid-phase extraction and simultaneous determination of tetracycline residues in edible cattle tissues using an HPLC-FL method

In this assay, edible cattle tissues from local markets of Ardabil, a Province of Iran, were examined for residues of tetracycline antibiotics [tetracycline, oxytetracycline and chlortetracycline]. In total, 110 samples of triceps, gluteal muscle, diaphragm, kidney and liver were randomly obtained from the local markets of the city of Ardabil. Solid-phase extraction [SPE] and high-performance liquid chromatography [HPLC] methods were used to extract and analyze tetracycline antibiotic [TC] residues, respectively. The mean amount of total TC residues in all tested samples was 226.3 +/- 112.5 ng/g and the mean amount of the total TC residues in triceps, gluteal muscle, diaphragm, kidney and liver samples were 176.3 +/- 46.8, 405.3 +/- 219.6, 96.8 +/- 26.9, 672.4 +/- 192.0 and 651.3 +/- 210.1 ng/g, respectively. Additionally, 25.8% of muscle samples, 31.8% of liver samples and 22.7% of kidney samples contained amounts of TC residues beyond the maximum residue limit [MRLs]. To reduce the TC residues found in edible cattle tissues, regulatory authorities should ensure that the cattle would undergo the proper withdrawal period from TCs before the slaughtering

Optimizing refolding condition for recombinant tissue plasminogen activator

Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro refolding to increase recombinant proteins production. Additives increase protein refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process. In this work, a comparative study was performed on refolding of recombinant plasminogen activator [rPA] in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride [Gn-HCl] and Dithiothreitol [DTT] on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10.In refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM. Glutathione pairs [GSH-GssG] showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10.In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the presence of higher concentration of L-arginine [>500 mM] or pH [>10] is not associated with higher activity

Radioiodine D amino acids labeling of rituximab, a new method for enhancing the radiopharmaceutical targeting and biostability

Radioimmunotherapy [RIT] is a very promising new therapy for the treatment of recurrent B-Cell non-Hodgkin's lymphoma [NHL]. Iodine-131 is the most frequently used nuclide in clinical RIT, but its usefulness has been limited by dehalogenation of monoclonal antibodies labeled via conventional methods. To circumvent this problem, we have synthesized a tri-peptide consisting of non-metabolizable D amino acids attached to N-Hydroxysuccinimide [NHS]. Tri-peptide was synthesized by standard Fmoc solid phase synthesis on tritylchloride resin. Labeling of tri-peptide was performed using the chloramine-T method and the conventional extraction. Radioiodination of tri-peptide was followed by conjugation to anti-CD20 antibody. In vitro stability of labeled antibody in serum and phosphate buffered saline [PBS] was measured for 48hr by [thin layer chromatography] TLC. Raji cell line was used to test cell binding of the labeled anti-CD20. The chemical purity of synthesized peptide as assessed by analytical [high performance liquid chromatography] HPLC was 95%. Labeling of tri-peptide resulted in a radiochemical yield of 50-71% with radiochemical purity of > 95%. At Rituximab concentration of 10mg/ml, coupling efficiencies of 65-80% was obtained with radiochemical purity of 95% and Specific activity [SA] of 185MBq/mg [5mCi/mg]. This study showed that labeling monoclonal antibodies with radioiodine by non-metabolizable D amino acids will improve bio-stability of the product

Evaluation of tumor targeting with radiolabeled F [AB'] 2 fragment of a humanized monoclonal antibody

Humanized monoclonal antibody U36 and its F[ab']2 fragment, radio labeled with 125I, were tested for tumor localization in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma. Monoclonal antibody IgG or F[ab']2 fragment were injected in parallel and at days 1, 2 and 3, mice were dissected for determination of isotope biodistribution. IgG as well as F[ab']2 showed highly specific localization in tumor tissue. The mean tumor uptake [n=3] is expressed as the percentage of the injected dose per gram of tumor tissue [%ID/g].%ID/g of IgG was 11.7% at day 1 and decreased to 10.9% at day 3 whereas%ID/g of F[ab']2 was 2.9% at day 1 and decreased on following days. Tumor to blood ratios [T/B] at day 1 were 0.86 for IgG and 1.32 for F[ab']2 and reached a maximum at day 3 with values of 4.41 and 1.84 respectively. These findings suggest that the superior tumor to non-tumor ratios in the day of 1 render the F[ab']2 fragment more qualified for specific targeting radioisotopes to tumor xenografts in this exprimental setting