| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Before developing an enzyme-linked immunosorbent assay of low density lipoproteins modified by degradation products of lipid peroxides in lipoproteins, methods for pretreatment of serum samples were studied and enzyme-linked immunosorbent assays of rat apolipoproteins E and A-I were established.
1) A sandwich enzyme-linked immunosorbent assay of rat apolipoprotein E (apo E) was developed. Apo E was purified from serum lipoproteins and antiserum was raised in rabbits. Diluted samples and standards were added into the wells of microtiter plates precoated with purified IgG and incubated. After washing, purified Fab'-horseradish peroxidase conjugate was added to each well and incubated. The bound enzyme was assayed by a colorimetric method. Samples and standards were pretreated with 6 M guanidine-HCI 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. The mean intra- and interassay coefficients of variation were 1.8 and 4.1%, respectively. Serum apo E concentrations were 21.2 <plus-minus> 2.1 and 61.3 <plus-minus> 17.0 mg/dl (mean <plus-minus> 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 and a small portion of apo E was present in a free
2) A sandwich enzyme-linked immunosorbent assay for rat apolipoprotein A-I (apo A-I) was also similarly developed. Treatment of serum samples with 6 M guanidine-HCl gave maximum exposure of the epitopes of apo A-I. The sensitivity reached to 1 pg, and the working range was 0.1 to 1.0 ng. The mean intra- and interassay coefficients of variation were 2.8 and 4.1%, respectively. Serum apo A-I concentrations in old (36-40 weeks old) rats (62.3 <plus-minus> 8.6 mg/dl, means <plus-minus> SD, n = 16) were significantly (p< 0.05) higher than those in young (8-12 weeks old) rats (55.1 <plus-minus> 4.3 mg/dl, n
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