Immunoprecipitation of apolipoprotein B-containing lipoproteins for isolation of HDL particles

Clin Chim Acta. 2014 Sep 25;436:348-50. doi: 10.1016/j.cca.2014.06.017. Epub 2014 Jun 30.

Abstract

Background: Immunoprecipitation (IP) of non-HDL particles with antisera provides the simplest and most specific method available for the separation of HDL. We compared the LipoSep™ IP reagent with the dextran sulfate/MgCl2 precipitation method (DS).

Methods: The IP reagent (200 μl) was added to an equal volume of serum, vortexed, incubated for 10 min at room temperature, and microcentrifuged at 12,000 rpm for 10 min.

Results: Equal volumes of a sample and the IP reagent precipitated apoB to 3.0 g/l without the coprecipitation of HDL. HDL-C measured in the supernatant after IP (Y) gave excellent agreement to DS precipitation (X) with a slope of 1.01, an intercept of 0.070 mmol/l (2.7 mg/dl), and a correlation of 0.99 (n=118; apoB 0.16-2.11 g/l). However, DS failed in most samples with moderate to elevated triglycerides. At triglyceride concentrations from 2.86 to 23.63 mmol/l (253-2091 mg/dl) the initial success rate was 65.4% for IP, while DS successfully precipitated only 5.8%. Success rate on repeat with additional reagent and/or sample dilution gave a success rate of 86.5% for IP and 40.4% for DS.

Conclusion: The IP reagent and protocol is a simple, effective and highly specific tool for isolating HDL particles in human serum and is effective with high triglyceride samples.

Keywords: Cholesterol; High density lipoprotein; Immunoprecipitation; Lipoproteins.

MeSH terms

  • Apolipoproteins B / chemistry*
  • Centrifugation
  • Humans
  • Immunoprecipitation / methods*
  • Lipoproteins, HDL / chemistry*
  • Lipoproteins, HDL / isolation & purification*
  • Time Factors

Substances

  • Apolipoproteins B
  • Lipoproteins, HDL