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Technical Briefs |
1
Clinical Pathology Department, Clinical Center, National Institutes of Health, Bldg. 10, Rm. 2C-407, Bethesda, MD 20892-1508
a author for correspondence: fax 301-402-1885, e-mail aremaley{at}nih.gov
Serum lipoprotein analysis frequently is used in assessing the risk for coronary artery disease and for monitoring cholesterol-lowering therapy (1)(2). A recent improvement in the analysis of lipoproteins is the development of homogeneous assays for HDL-cholesterol (HDL-C) (3)(4) that are easier to perform because they do not require the physical separation of the apolipoprotein B (apoB)-containing lipoproteins by precipitation and centrifugation. The measurement of LDL-cholesterol (LDL-C) and apoB, the principal protein on LDL, is also useful for estimating the risk for cardiovascular risk (1)(2). An isolated increase in apoB in the absence of increased total cholesterol and LDL-C is diagnostic for a risk condition called hyperapoB-lipoproteinemia (5). We describe a simple modification of an antibody-based commercial homogeneous assay for HDL-C (EZ-HDL; Sigma Diagnostics) that in addition to measuring HDL-C, also provides an estimate of the apoB concentration.
In step 1 of the EZ-HDL assay, an anti-apoB antibody is added, which
binds to the surface of the apoB-containing lipoproteins. In step 2,
the reagents for the enzymatic detection of "accessible"
cholesterol are added, which produces an absorbance (600 nm) change in
step 2 that is proportional to the concentration of HDL-C. The assay is
specific for HDL-C because the anti-apoB antibody sterically blocks
cholesterol oxidase from reacting with cholesterol on the
apoB-containing lipoproteins. While routinely using the EZ-HDL assay,
we often observed, particularly in specimens with high LDL-C, an upward
shift in the absorbance at the end of the 4-min antigen-antibody
incubation, just before the addition of the reagents in step 2 for the
enzymatic detection of cholesterol (Fig. 1
A). A similar change in absorbance was observed upon the
addition of purified LDL but not HDL (Fig. 1A
), which indicates that
the increase in turbidity in step 1 is the result of the binding of the
anti-apoB antibody to the apoB-containing lipoproteins. Because it is a
relatively abundant serum protein, apoB is routinely measured by
turbidimetric methods (6) that depend on the physical
aggregation of apoB-containing lipoproteins after incubation with an
anti-apoB antibody. Taking advantage of this phenomenon, we assessed
the turbidity change at 600 nm during the first incubation step of the
EZ-HDL assay as a possible measure of serum apoB.
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In Fig. 1B
, the measurement of apoB by the turbidimetric change in the
EZ-HDL assay was compared with a standard nephelometric assay for apoB
(Protein Array; Beckman). An endpoint calculation was used in measuring
the turbidity of the EZ-HDL assay by subtracting the background
absorbance reading at 0.5 s from the absorbance reading at 4 min
at the end of step 1. A high apoB (1.43 g/L) and a low apoB (0.51 g/L)
serum pool, as measured by the standard assay, were used to calibrate
the first step of the EZ-HDL assay. The turbidimetric measurement from
the EZ-HDL assay correlated relatively well (r = 0.92)
with the standard assay for nonlipemic specimens (Fig. 1B
, 
). The
assay also had an acceptable (7) intraassay CV of 2.9% at
0.59 g/L (n = 20) and an interassay CV of 4.6% at 1.23 g/L
(n = 10). Lipemic specimens (triglycerides, 3.05–9.83 mmol/L;
total cholesterol, 4.95–7.69 mmol/L), which had a baseline absorbance
>0.016 (Fig. 1B
, •), often deviated from the standard assay and
typically showed a negative bias. Although this is a limitation of the
assay, the ability to identify specimens that are not suitable for
analysis by the baseline absorbance should be helpful.
The modified EZ-HDL assay potentially represents a cost-effective procedure for performing lipoprotein analysis because it provides a simultaneous measure of HDL-C and apoB in a single test. The modification of the assay involves calibrating the assay for apoB and monitoring the absorbance during the first incubation step, and it does not require any additional reagents. Unlike HDL-C, apoB often is not measured initially in the screening for hyperlipidemia. The modified EZ-HDL assay may, therefore, provide a convenient way for identifying patients with hyperapoB-lipoproteinemia (5) who might not otherwise be diagnosed. Given the limitations of the assay in regard to standardization and interference by lipemia, however, it should not be viewed, however, as a replacement for a standard assay of apoB. Any specimen found to have increased apoB by the EZ-HDL assay should be confirmed with a standard apoB test.
References
The following articles in journals at HighWire Press have cited this article:
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  M. L. Sampson, A. Aubry, G. Csako, and A. T. Remaley Triple Lipid Screening Test: A Homogeneous Sequential Assay for HDL-Cholesterol, Total Cholesterol, and Triglycerides Clin. Chem., March 1, 2001; 47(3): 532 - 539. [Abstract] [Full Text] [PDF]
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