Procedure for development of an enzyme-linked immunosorbent assay. Development of an assay for human apolipoprotein A-I.
A set of criteria for selection of antibodies during the development of enzyme-linked immunosorbent assay (ELISA) is described. Using these criteria, a competitive ELISA for human apo A-I using a polyclonal goat antibody was developed. The assay recognizes apo A-I from plasma and high-density lipoprotein (HDL), as well as the pure delipidated apo A-I, equally. Intra- and inter-assay variations were 5.2% and 3.5%, respectively. Recovery rate, as determined by spiking a known quantity of pure delipidated apo A-I into a reference plasma, was determined to be 101.3%. The assay was validated by comparing the concentration of apo A-I in HDL with the dye elution method. The apo A-I ELISA to apo A-I dye elution ratio was 1.01. Apo A-I concentration in Centers for Disease Control reference material determined by this method was in agreement with the reported consensus value. Repeated freezing and thawing of the samples (three freeze-thaw cycles at -20 degrees C) as well as long-term freezing (up to 1 year at -70 degrees C) did not affect the concentration of apo A-I in the samples. The assay was applicable both to normolipidemic and dyslipidemic plasmas. No immunologic difference was noted when plasma from dyslipidemic subjects was assayed. A frequent problem of long-term storage is deamidation. The values found for apo A-I in a deamidated plasma were the same as those for the corresponding fresh plasma. Plasma apo A-I values were also positively correlated with that of HDL-cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)
Gaver
A
A
Louisiana State University Medical Center, Department of Physiology, New Orleans 70112-2822.
Weedy
N K
NK
Maldonado
B R
BR
Huang
S
S
Wong
L
L
D000906:Antibodies; D016632:Apolipoprotein A-I / Q000032:analysis* / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D012016:Reference Values
D000906:Antibodies; D016632:Apolipoprotein A-I