Sandwich enzyme-linked immunosorbent assay of rat apolipoprotein E.
A sandwich ELISA for rat apo E was developed. Blood was drawn from rats that had been administered with triamcinolone diacetate to increase apo E-rich HDL in serum. Apo E was purified from the d less than 1.225 g/ml lipoproteins, and antiserum was raised in rabbits. Diluted samples and standards were added into the wells of polystyrene microtiter plates precoated with immunoaffinity-purified IgG and incubated for 90 min. After washing, immunoaffinity-purified Fab'-horseradish peroxidase conjugate was added to each well and incubated for 90 min. The bound enzyme was assayed by a colorimetric method. Samples and standards were pretreated with 6 M guanidine. HCl to maximize the antigenic response of apo E. The sensitivity lies around 1 pg apo E, and the working range was 0.1 to 1.0 ng. All assay procedures were completed within 4-5 hr. The mean intra- and interassay coefficients of variation were 1.8 and 4.1%, respectively. Serum apo E concentrations were 21.2 +/- 2.1 and 61.3 +/- 17.0 mg/dl (mean +/- SD) for young (8-12 weeks old, n = 9) and old (36-40 weeks old, n = 16) rats, respectively. As determined by gel filtrations, most of the apo E in fasted rat serum was associated with larger HDL particles (or HDL1) and a small portion of apo E was present in a free form.
Chiba
H
H
Department of Laboratory Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
Mitamura
T
T
Matsumiya
H
H
D000375:Aging / Q000378:metabolism; D000818:Animals; D001057:Apolipoproteins E / Q000032:analysis*; D001835:Body Weight; D004797:Enzyme-Linked Immunosorbent Assay; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis; D008055:Lipids / Q000097:blood; D008297:Male; D011817:Rabbits; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
D001057:Apolipoproteins E; D007074:Immunoglobulin G; D008055:Lipids