Procedure for development of an enzyme-linked immunosorbent assay. Development of an assay for human apolipoprotein A-I

Anat Gaver, Nora K. Weedy, Blanca R. Maldonado, Shiqi Huang, Laurence Wong*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

A set of criteria for selection of antibodies during the development of enzymelinked 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°C) as well as long-term freezing (up to 1 year at -70°C) did not affect the concentration of apo A-I in the samples. The assay was applicable both to normolipidemic and dyslipidemic plasmas. No immunologie 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. It is concluded that with the proper selection of antibody, the assay can be robust and differences in quantification could be minimized.

Original languageEnglish
Pages (from-to)23-37
Number of pages15
JournalClinica Chimica Acta
Volume208
Issue number1-2
DOIs
StatePublished - 15 Jun 1992
Externally publishedYes

Funding

FundersFunder number
National Heart, Lung, and Blood InstituteP01HL025596
National Institute of Diabetes and Digestive and Kidney DiseasesR01DK035278

    Keywords

    • Apolipoprotein A-I
    • Deamidation
    • Enzyme-linked immunosorbent assay

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