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 micro-column 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 Gróf modified by Doumas et al (Clin Chem 1985;31:1779-1789); results correlated well with the high performance liquid chromatography (HPLC) method (n = 35, y (affinity) = 1.009x (HPLC)-5.49; r = 0.959) described by Lauff et al. (J Chromatography 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 mumol/l; Control 2, mean = 74.82 mumol/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 mumol/l (mean +/- 1 S.D.) and percent B delta of 2.2 +/- 1.9 (mean total bilirubin +/- 1 S.D. = 118 +/- 79 mumol/l). Although time consuming, this simple and precise method allows the measurement of B delta in laboratories without the need for specialized instruments.
Burnett
J R
JR
Department of Laboratory Services, Wellington Hospital, New Zealand.
Lim
C W
CW
Mahoney
G N
GN
Crooke
M J
MJ
D000328:Adult; D001565:Benzoates; D001663:Bilirubin / Q000097:blood*; D002110:Caffeine; D002846:Chromatography, Affinity / Q000706:statistics & numerical data; D002851:Chromatography, High Pressure Liquid; D004338:Drug Combinations; D004587:Electrophoresis, Agar Gel; D006801:Humans; D006932:Hyperbilirubinemia / Q000097:blood*; D007231:Infant, Newborn; D012016:Reference Values; D012680:Sensitivity and Specificity; D014227:Triazines
D001565:Benzoates; D004338:Drug Combinations; D014227:Triazines; D002110:Caffeine; C012842:Cibacron Blue F 3GA; C025741:caffeine, sodium benzoate drug combination; D001663:Bilirubin