George Jackowski

REMOVAL OF ENDOTOXIN FROM RECOMBINANT PROTEIN PREPARATIONS

OBJECTIVES To develop an effective method to remove endotoxin from large scale E. coli recombinant protein purifications. DESIGN AND METHODS Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay. The immunoactivity of these protein preparations was determined by BIAcore analysis using a panel of in-house generated monoclonal antibodies and by a Stratus Fluorometric Analyzer. In the case of troponin I, the BIAcore was also utilized to measure troponin C interactions. RESULTS Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography. With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90%. All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies. Troponin I also retained its ability to bind to troponin C in the presence of Ca2+. Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E. coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies. CONCLUSION Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.

Analytical Performance and Clinical Utility of a Sensitive Immunoassay for Determination of Human Cardiac Troponin I

OBJECTIVES To determine the serum and plasma level of human cardiac troponin I (cTnI) resulting from myocardial damage, we have developed a sensitive and specific one-step enzyme immunoassay to measure cardiac troponin I. DESIGN AND METHODS The COBAS cTnI assay is a semi-automated one-step solid phase immunoassay compatible with the COBAS Core. The assay is performed in a sandwich type format using a polyclonal goat antibody capture and two highly specific horseradish peroxidase conjugated monoclonal antibody detectors directed against different epitopes of the cTnI molecule. Calibrators were made with purified recombinant cTnI. RESULTS The level of cTnI was determined in 84 healthy donors with no evidence of myocardial injury, resulting in a lower limit of detection (LLD) of 0.09 microgram/L. The upper reference limit (URL) of the normal reference range was calculated as 0.20 microgram/L. The dynamic range of the consequent EIA was between 0.09 and 6.0 micrograms/L with a total assay time of 45 min. Intra-assay and inter-assay variances (CVs) were < or = 4%. Cross-reactivity with fast and slow skeletal troponin I was absent in concentrations up to 2.0 mg/L. Common interferents yielded negative results in the cTnI assay. Clinical utility was confirmed by measuring the circulating serum or plasma levels of cardiac troponin I in serial samples from marathon runners, clinical samples from trauma patients, and patients presenting to the Emergency Department with complaints of chest pain. Results were further evaluated using clinical diagnosis at discharge and quantified concentrations of other cardiac markers by a Stratus analyzer and ELISA procedures. CONCLUSIONS Results from normal and clinical samples assayed in house for cTnI concentrations indicate that the Spectral EIA is a highly sensitive means of quantifying cTnI levels in serum and plasma for acute cardiac syndrome. The cardiac specificity of cTnI over other well-known cardiac markers is reflected in experimental results and parallel clinical diagnosis.

Use of enzyme immunoassay for measurement of skeletal troponin-I utilizing isoform-specific monoclonal antibodies.

OBJECTIVE To determine the serum level of fast skeletal troponin I (fsTnl) resulting from skeletal muscle damage, we have developed a sensitive two-site enzyme immunoassay to measure skeletal troponin I. DESIGN AND METHODS Twelve monoclonal antibodies were raised against human fsTnl. Of these antibodies, 8 were fsTnl-specific and the remaining 4 reacted with both skeletal and cardiac troponin I (cTnl). Two monoclonals were utilized for a development of this fsTnl immunoassay. Standards were made with purified recombinant human fsTnl for the range of 0-25 micrograms/mL. RESULTS Total assay variance (CV) ranged from 1.7% to 9.6%. The upper limit of the normal reference range was established as 0.2 microgram/L by determining fsTnl concentration in sera of 108 healthy donors without evidence of muscle damage. Purified human cTnl up to 500 micrograms/L and cTnl-positive clinical serum samples yielded negative results in the fsTnl assay. The serum levels of fsTnl were determined in trauma patients, patients with chronic degenerative muscle disease, and marathon runners. In the study populations, the serum levels of fsTnl were correlated with other biochemical markers that are traditionally used to monitor striated muscle damage. CONCLUSIONS In the present preliminary studies, measuring the serum levels of fsTnl in patients with various forms of muscle damage is more accurate than using the classical non muscle-specific biochemical markers.

Oxidation levels of North American over-the-counter n -3 (omega-3) supplements and the influence of supplement formulation and delivery form on evaluating oxidative safety

The aim of the present study was to evaluate the oxidation status of North American n-3 (omega-3) PUFA nutritional supplements commercially available in Canada and evaluate the influence of product formulation and delivery form on oxidative safety. A total of 171 North American over-the-counter n-3 PUFA nutritional supplements were analysed for oxidation safety. Primary and secondary oxidation and total oxidation (TOTOX) were determined using the American Oil Chemists’ Society (AOCS) procedures. Comparisons between supplements’ final forms, oil source and n-3 PUFA concentration quartiles, as measures of product formulations and delivery forms, were compared using ANOVA. Of the products successfully tested, 50 % exceeded the voluntary recommended levels for markers of oxidation. Another 18 % of products were approaching the limits with 1–3 years before expiration. Encapsulated products without flavour additives had significantly lower secondary and TOTOX levels than bulk oils and flavoured products (P < 0·05). Childrens products had significantly higher primary, secondary and TOTOX levels compared with all other products (P < 0·05). Markers of oxidation did not differ between oil sources (P > 0·05), with the exception of krill oil products having higher secondary oxidation levels than plant-based products (P > 0·05). Markers of oxidation did not differ between n-3 PUFA supplement concentration quartiles. Consumers may be at risk of exposure to higher levels of oxidative products. New regulatory mandates need to be introduced to ensure that all n-3 PUFA products, used as nutritional supplements, regardless of their formulation or delivery form, can be tested for oxidative safety and compliance.