Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf).

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.

Inhibition of erythropoietin activity by cyanate.

OBJECTIVE: Increased urea concentration is a measure of advanced renal failure and the adequacy of renal replacement therapy in end-stage renal disease (ESRD). Altered biologic activity due to changes in protein structure occurs when cyanate, formed spontaneously from urea, reacts with proteins. Carbamylation results in impaired erythropoietin (EPO) activity when high concentrations of cyanate react with EPO. In this study, the activity of carbamylated EPO (C-EPO), formed at a cyanate concentration which may occur in vivo, was studied in Sprague-Dawley rats. MATERIAL AND METHODS: The extent of carbamylation, causing loss of free amino groups, was monitored using trinitrobenzenesulfonic acid. Erythrocyte, hemoglobin, hematocrit and leukocyte levels were measured after either EPO, incubated EPO, C-EPO, physiologic saline or cyanate (1.5 microM; 0.2 ml) were injected subcutaneous twice weekly for 3 weeks in rats. RESULTS: In vitro carbamylation of EPO was time- and concentration-dependent. C-EPO concentration increased as the duration of exposure to cyanate increased from 6 to 72 h, or as cyanate concentration increased from 15 nM to 1.5 microM. Injections of EPO caused significant increases in vivo in all erythropoietic measures. In contrast, injections of C-EPO, physiologic saline or 1.5 microM cyanate caused no change from baseline. CONCLUSIONS: These results demonstrated diminished biologic activity in healthy rats by C-EPO formed in vitro at cyanate concentrations that may be found in vivo. C-EPO and high urea-derived cyanate levels may contribute to suboptimal erythropoietic responses to EPO therapy for chronic renal failure and ESRD, and may provide another measurement indicating inadequate dialysis.