A method for the separation of delta bilirubin using Cibacron Blue affinity chromatography

Abstract We developed and evaluated a method for the separation of delta bilirubin (Bδ) by microcolumn affinity chromatography based on Cibacron Blue 3G-A-Agarose. Untreated serum was applied to affinity columns and free non-protein bound bilirubins were eluted with phosphate buffer containing 20 g/l Triton X-100. Retained albumin was eluted using caffeine-benzoated reagent and bilirubin associated with this fraction (Bδ) quantitated by the method of Jendrassik and Grof modified by Doumas et al (Clin Chem 1985;31:1779–1789); results correlated well with the high performance liquid chromatography (HPLC) method ( n = 35, γ (affinity) = 1.009 x (HPLC) − 5.49; r = 0.959) described by Lauff et al. (J. Chromatogr 1981;226:391–402). Two controls analyzed with each batch gave between-batch imprecision less than 4.0% ( n = 10; Control 1, mean = 20.05 μ mol/l; contron 2, mean = 74.82 μ mol/l). Within-batch imprecision was less than 3.3% for both levels. Specimens collected from 25 neonates less than 20 days of age showed a bδ concentration of 1.7 ± 0.7 μ mol/l (mean ± 1 S.D.) and percent Bδ of 2.2 ± 1.9 (mean total bilirubin ± 1 S.D. = 118 ± 79 μ mol/l). Although time consuming, this simple and precise method allows the measurement of Bδ in laboratories without the need for specialized instruments.