Purification and characterisation of anti-pneumococcal capsular polysaccharide IgG immunoglobulins.

OBJECTIVES: The production of reference materials for quantifying pneumococcal antibody concentrations relies upon large scale vaccination. An alternative simple, reproducible protocol has been developed for the affinity purification of 23 serotype anti-pneumococcal capsular polysaccharide (PCP) IgG immunoglobulins. DESIGN & METHODS: The purification protocol utilised IgG fractionation, capsular polysaccharide (CPS) adsorption, and affinity chromatography using Pneumovax®-Sepharose. Purification efficiency and method reproducibility were assessed by comparison of 4 batches of anti-PCP IgG. Immunoglobulin composition was determined using nephelometry and functionality was evaluated using VaccZyme™ ELISAs. RESULTS: Anti-PCP IgG preparations were ≥95% pure by SDS-PAGE analysis with no contaminating IgA or IgM immunoglobulins or IgG antigen specific antibodies towards haemophilus influenzae b, diphtheria toxoid or tetanus toxoid. The predominant IgG subclass in the preparation was IgG2. CONCLUSIONS: This novel purification procedure produced highly specific anti-PCP IgG preparations that compared well to both Lot 89SF and 007sp international serum standards and could be used as an alternative method for the production of reference materials.

The ratio of parathyroid hormone as measured by third- and second-generation assays as a marker for parathyroid carcinoma.

BACKGROUND: Parathyroid carcinoma (PCa) is a rare disease that can be difficult to differentiate initially from severe benign parathyroid adenoma. PCa oversecrete the amino form of PTH, which is recognized by third-generation but not by second-generation PTH immunoassays. In normal individuals, the third-generation to second-generation PTH ratio should be less than 1. OBJECTIVE: Our objective was to study the utility of the third-generation to second-generation PTH ratio as a means of distinguishing PCa patients (n=24) from control groups with and without disorders of calcium secretion, including patients on renal hemodialysis (n=74), postrenal transplantation (n=60), and primary hyperparathyroidism (PHP; n=30). SETTING AND DESIGN: We conducted a retrospective, laboratory-based study at tertiary referral academic centers. RESULTS: The mean third-generation to second-generation ratio was 0.58+/-0.10 in the dialysis patients, 0.54+/-0.10 in the renal transplant group, 0.54+/-0.12 in the elderly healthy patients, and 0.68+/-0.11 in the PHP group. All 245 of these patients presented a PTH third-generation to second-generation ratio of less than 1. In contrast, we observed an inverted third-generation to second-generation PTH ratio of more than one in 20 PCa patients, whereas only four PCa patients had a normal ratio of less than 1. CONCLUSIONS: An inverted third-generation to second-generation PTH ratio occurred in the majority of patients with advanced PCa and was absent in all 245 relevant controls. A third-generation to second-generation PTH ratio higher than 1 had a sensitivity of 83.3% and a specificity of 100% among PHP patients as a marker for PCa. This ratio may be useful to identify patients with PCa earlier and to detect patients either at risk of developing PCa or those in whom recurrence is taking place.