Development of an Amplified Enzyme-Linked Immunosorbent Assay for Sensitive Measurement of Apolipoprotein B in Plasma and Tissue Culture Media
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We have developed a non-competitive sandwich enzyme-linked immunosorbent assay for the quantitation of apolipoprotein B, which utilizes the biotin-avidin amplification system. All components were optimized with respect to either concentration or incubation time. The assay uses microtitre wells as a solid phase and polyclonal, affinity-purified capture antibody. The amplification system was composed of hydroxy-succinimide biotin conjugated to an affinity-purified anti-apolipoprotein B IgG and either ExtrAvidin-alkaline phosphatase or streptavidin-alkaline phosphatase. The within-run precision (n = 10) of the apolipoprotein B control (1.39 g/l) was 3%, while the between-run precision of the assay (n = 6) for 9 consecutive assays was determined to be 8%. A sensitivity of 3 ng was attained, with a mean analytical recovery of 110%. Comparison of the assay with an established immunoturbidometric method resulted in a correlation of 0.92 using patient plasma samples (n = 19). The use of the biotin-avidin amplification system provides the sensitivity required for measuring apolipoprotein B in tissue culture media samples and circumvents the many problems associated with the direct conjugation of enzymes to antibodies. Minimal amounts of reagents are required and the assay can be performed in 5 h, making it both economical and practical for clinical laboratories.
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