Related Experiment Videos
Enzyme-linked immunosorbent assay for human plasma apolipoprotein B
J M Ordovas1, J P Peterson, P Santaniello
1USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA.
Journal of Lipid Research
|October 1, 1987
Summary
A new assay accurately measures apolipoprotein B (apoB) in plasma. This method revealed significant differences in apoB levels based on age and sex in study participants.
Area of Science:
- Biochemistry
- Immunology
- Clinical Chemistry
Background:
- Apolipoprotein B (apoB) is a critical component of lipoproteins, playing a key role in lipid metabolism and cardiovascular disease.
- Accurate and reliable measurement of plasma apoB is essential for assessing cardiovascular risk.
- Existing methods for apoB quantification may have limitations in terms of precision or accessibility.
Purpose of the Study:
- To develop and validate a noncompetitive enzyme-linked immunosorbent assay (ELISA) for the accurate measurement of total plasma apolipoprotein B (apoB).
- To assess the performance characteristics of the developed ELISA, including antibody selection and optimal assay conditions.
- To determine plasma apoB levels in a large cohort and investigate potential associations with demographic factors.
Main Methods:
- Development of a noncompetitive ELISA utilizing affinity-purified polyclonal and monoclonal antibodies against apoB.
- Screening of microtiter plates for optimal IgG binding characteristics to ensure assay consistency.
- Optimization of plasma dilution (1:3000) and use of alkaline phosphatase-conjugated secondary antibodies for signal detection.
- Colorimetric detection using p-nitrophenyl phosphate substrate and absorbance readings at 410 nm, with data processed via a microcomputer.
Main Results:
- A highly consistent ELISA was established, with coefficients of variation below 5% using a selected microtiter plate.
- The assay demonstrated optimal performance with a plasma dilution of 1:3000.
- Plasma apoB levels were determined in 1115 participants of the Framingham Offspring Study, with a mean concentration of 89 +/- 28 mg/dl.
- Significant differences in plasma apoB levels were observed in relation to participant age and sex.
Conclusions:
- A robust and reproducible noncompetitive ELISA for total plasma apoB has been successfully developed.
- The assay provides a reliable tool for large-scale epidemiological studies investigating apoB.
- The findings highlight the influence of age and sex on plasma apoB concentrations, contributing to a better understanding of lipid metabolism and cardiovascular risk factors.