Rapid screening method for polymorphism of group A apolipoproteins

Polymorphism of apolipoproteins Al and All (ap Al and apo All) can be easily investigated in plasma by a simple method involving a 30‐min incubation of EDTA plasma in the presence of urea, dithiothreitol, and Nonidet P‐40 followed by subsequent isoelectric focusing (IEF). The sample (2 μL) was applied to an ultrathin flat acrylamide gel of pH range 4–6, and focused using a Bio‐Rad Mini IEF Cell for 1.5 h at a maximum of 500 V. Coomassie Blue R‐250 was used to visualize the apolipoproteins. To verify the identity of the different apolipoproteins aftr IEF, the gel was immunofixed directly with anti‐apo Al, or immunoblotted on polyvinylidene difluoride (PVDF) membrane using monospecific antibodies to apo Al and ap All and an antiimmunoglobulin‐alkaline phosphatase conjugate. High‐density lipoprotein (HDL) was used as a standard for Apo Al variants. Employing these techniques, human plasma apo Al was resolved into one major band (apo Al 0 pl 5.54), and four minor bands identified as apo Al +2 (pl 5.75), apo Al +1 (pl 5.66), apo Al −1 (pl 5.45), and apo Al −2 (pl 5.34). Apo All was resolved into one major isoprotein designated as apo All +1 and apo All −1 which focused at pls of 5.18 and 4.58, respectively. The results showed that these methods can be used to identify apo Al and All isoforms without prior ultracentrifugation to isolate the HDL. The entire procedure, including IEF, Fixation (chemical or immunofixaiton). and staining, can be accomplished in 5 h compared to 2 days using previously reported technique. The identification and characterization of human apolipoprotein Al and All isoforms is important in clinical practice, e.g., diagnosis of tangier disease, and may be useful in studying structure‐function relationships of these apoproteins. © 1992 Wiley‐Liss, Inc.