Anti-CK-BB monoclonal antibody, clone A027-11288

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(TM) analysis using a panel of in-house generated monoclonal antibodies and by a Stratus(R) Flu orometric Analyzer. In the case of troponin I, the BIAcore(TM) 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.

We evaluated the effectiveness of 5-day antibacterial therapy for bacterial meningitis in children. The study group included 26 children from 2 months to 15 years of age, admitted with microbiologically confirmed bacterial meningitis in 1990-1993 and treated for 5 days. A historical comparison group of 49 patients treated for 8 to 15 days was used. Penicillin monotherapy (300 mg/kg body weight) was used for meningococcal and pneumococcal meningitis and ampicillin (300 mg/kg body weight) for Haemophilus influenzas b meningitis. On day 5 of therapy the activity of aspartate aminotransferase (AST), lactic dehydrogenase (LDH), creatine phosphokinase (CPK) and gamma-glutamyl-transpeptidase (gamma GT) in the CSF was determined by photocolorimetric assay and the concentration of creatine kinase BE (CK-BB) by ELISA, IL-6 was analysed using EIA technique and a cerebral ultrasound was performed at the time of the termination of the antibacterial therapy, The mean follow-up time was 1.3 years for children in the study group and 3.2 in the control group, The time of hospitalisation was shorter in children treated for 5 days (p<0.005), Complete clinical recovery was 81% in the study group and 66% in the comparison group at the time of the termination of antibacterial therapy, No relapses occurred. The activity of AST, CPK, LDH, and gamma GT in the CSF had returned to normal by the 5th day of therapy, but almost a 7-fold higher concentration of CK-EB was registered. The concentration of IL-6 in the CSF decreased with the therapy from 1,800 pg/ml to 685 pg/ml but still remained high. Long term follow-up did not differ between the two groups. We conclude that 5 days of antibacterial therapy is adequate for the treatment of meningococcal meningitis in children.