A METHOD FOR THE SEPARATION OF DELTA-BILIRUBIN USING CIBACRON-BLUE AFFINITY-CHROMATOGRAPHY

We developed and evaluated a method for the separation of delta bilirubin (B delta) 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-benzoate reagent and bilirubin associated with this fraction (B delta) 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, gamma (affinity)= 1.009x (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 mu mol/l; Control 2, mean = 74.82 mu 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 B delta concentration of 1.7 +/- 0.7 mu mol/l (mean +/- 1 S.D.) and percent B delta of 2.2 +/- 1.9 (mean total bilirubin +/- 1 S.D. = 118 +/- 79 mu mol/l). Although time consuming, this simple and precise method allows the measurement of B delta in laboratories without the need for specialized instruments.