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Clinical Chemistry 43: 669-674, 1997;
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(Clinical Chemistry. 1997;43:669-674.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Automated measurement of mouse apolipoprotein B: convenient screening tool for mouse models of atherosclerosis

Daniel M. Levine1,a and Kevin Jon Williams2

1 The Rogosin Institute and Department of Biochemistry, The New York Hospital–Cornell Medical Center, 505 East 70th St., New York, NY 10021.

2 The Dorrance H. Hamilton Research Laboratories, Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, 1020 Locust St., Suite 349, Philadelphia, PA 19107-6799.
a Author for correspondence. Fax 212-327-7331; e-mail dmlevine{at}mail.med.cornell.edu


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although mice are commonly used for studies of atherosclerosis, investigators have had no convenient way to quantify apolipoprotein (apo) B, the major protein of atherogenic lipoproteins, in this model. We now report an automated immunoturbidimetric assay for mouse apo B with an NCCLS imprecision study CV <5%. Added hemoglobin up to 50 g/L did not interfere with the assay, nor did one freeze–thaw cycle of serum samples. Assay linearity extends to apo B concentrations of 325 mg/L. We have used the assay to determine serum apo B concentrations under several atherogenic conditions, including the apo E "knock-out" genotype and treatment with a high-cholesterol diet. Our assay can be used to survey inbred mouse strains for variants in apo B concentration or regulation. Moreover, the mouse can now be used as a convenient small-animal model to screen compounds that may lower apo B concentrations.


Key Words: indexing terms: animal models of disease • apo E • LDL receptor • diet, effects of • lipoproteins • mice, transgenic


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mouse has become an important experimental model in lipoprotein and atherosclerosis research for three reasons. First, many inbred strains are available that differ in susceptibilities to atherosclerosis. These strains have been used in cross-breeding studies to find new genetic loci that influence this disease (1)(2)(3)(4)(5)(6). Second, sophisticated techniques of genetic manipulation, such as the introduction of artificial transgenes (7)(8)(9) and the disruption of endogenous genes through homologous recombination (10), have been best adapted to the mouse. Murine strains have been created with overexpression or disruption of nearly the entire list of apolipoproteins and of the known enzymes that act on lipoproteins (11). Third, the mouse is attractive for experimental work because of its small size and short generation time.

Nevertheless, no reports have presented a convenient method to measure plasma concentrations of mouse apolipoprotein (apo) B, the major protein component of atherogenic lipoproteins (12)(13)(14)(15) and a major influence on arterial lesion development (see reference 16 for a review).1 Here, we report the development of a simple, inexpensive, and automated immunoturbidimetric assay that is sensitive to both full-length apo B-100, the major protein of murine low-density lipoprotein (LDL) (12), and truncated apo B-48, the N-terminal 48% of apo B-100 and the major protein of atherogenic remnant lipoproteins (13)(14)(15). Our assay should have broad applications in this important small-animal model.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
antigen and antibody production
Mouse apo B-100 was purified by the method of Sparks and Marsh (14). In brief, apo B-rich lipoproteins were isolated from 60 mL of mouse plasma (Pel-Freez, Rogers, AK) by flotation at a density of 1.063 kg/L and dialyzed against EDTA–saline (NaCl 150 mmol/L; EDTA, 1 mmol/L; pH 7.4) to remove the KBr used in flotation. Apolipoproteins were solubilized by an overnight incubation in lithium dodecyl sulfate (20 g/L) at 4 °C, and fractionated by size by passage over a 150 x 1.5 cm column of Sepharose CL-6B (Pharmacia, Piscataway, NJ) equilibrated with sodium dodecyl sulfate (SDS, 10 g/L). The first peak, which eluted immediately after the void volume, was pooled, concentrated, and verified by SDS–polyacrylamide gel electrophoresis (PAGE) to contain only apo B-100 (17). This material was divided into two aliquots, one used to immunize a goat, and one for the initial standardization of the immunoturbidimetric assay.

Goat antiserum was prepared by a standard protocol (18), i.e., primary immunization with 500 µg of mouse apo B-100 mixed with Freund's Complete Adjuvant and administered subcutaneously, followed by two monthly boosts administered intramuscularly, each consisting of 250 µg of apo B-100 mixed with Freund's Incomplete Adjuvant. From 10 to 20 days after the second boost, the goat was serially plasmapheresed, being exsanguinated on the final session, to obtain a large volume of antiserum (raised under contract by Hazelton Research Products, Denver, PA). These sera were pooled to obtain a single lot.

High background signals in our initial attempt to develop the immunoturbidimetric assay led us to partially purify the IgG fraction by ammonium sulfate precipitation. The precipitate was resuspended in two volumes of antibody diluent (available from Incstar, Stillwater, MN) of proprietary composition and then filtered through a 0.22-µm (pore size) filter, aliquoted, and stored at -70 °C. Background readings in the immunoturbidimetric assays carried out with this partially purified antibody have been consistently low.

The assay is specific for apo B. Western blotting after electrophoresis (17) of whole plasma from an apo E "knock-out" mouse (19) through an SDS–glycerol gel of 3.5% acrylamide demonstrated that the antiserum reactivity is limited to apo B-100 and apo B-48 (Fig. 1 ). The dilution of our resuspended antibody preparation was 1:2500 (i.e., 1:5000 relative to the original antiserum), and bands were detected with the Vectastain ABC system (Vector Labs., Burlingame, CA), which uses biotinylated rabbit anti-goat IgG and avidin–horseradish peroxidase complexes. Moreover, the immunoturbidimetric assay showed no reactivity for the density >1.063 kg/L fraction of mouse serum, which contains essentially no apo B-rich lipoproteins and had been dialyzed to remove KBr; in contrast, strong reactivity was observed in the <1.063 kg/L fraction.



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  		Figure 1. Specificity of the goat anti-mouse apo B antibody.

Western blot (left lane) and Coomassie stain (right lane) after electrophoresis (SDS–glycerol gel of 3.5% acrylamide) of whole plasma from an apo E knock-out mouse. The proteins shown were identified from comparison with standards.

To verify reactivity against apo B-100 and apo B-48 in the assay, we measured the apo B concentrations in chow-fed LDL receptor knock-out mice (20) and apo E knock-out mice (19) (both murine lines from Jackson Labs., Bar Harbor, ME). Atherogenic lipoproteins from these mice contain mostly apo B-100 and apo B-48, respectively ( (19)(20)(21) and Fig. 1Up ). The relative reactivities of purified apo B-100 and apo B-48 in our assay were also quantified.

preparation of calibrators and controls
The preparation of purified apo B-100 described above was assayed by the SDS–Lowry procedure (22) and was used in the initial calibration of our immunoturbidimetric assay. Purified apo B-100 performed well at concentrations >50–70 mg/L. At lower concentrations, absorbance values were too close to background to allow adequate quantification, a typical problem in assaying non-serum-based calibrators with automated analyzers. Thus, this calibrator was unsuitable for assessing low plasma concentrations of apo B. Further, the purified apo B-100 could not be frozen and thawed without substantially altering its reactivity in the assay. Hence, we prepared a second-generation (lipoprotein-based) calibrator consisting of apo B-rich lipoproteins (density <1.063 kg/L fraction of mouse plasma). Using a five-point dilution curve of the pure apo B-100, we determined that the apo B concentration in the mouse lipoprotein-based calibrator was 125.3 mg/L. This step was necessary because, unlike human LDL, no mouse lipoprotein contains essentially only apo B (12). Serial dilutions of this lipoprotein-based calibrator reacted well at apo B concentrations as low as 12 mg/L but also failed to survive a freeze–thaw cycle. The rate of complex formation, as assessed by absorbance, at concentrations >50–70 mg/L was virtually identical between purified apo B-100 and this lipoprotein-based calibrator, indicating similar antigenic reactivity (see also below).

Finally, we prepared a third-generation (serum-based) calibrator as follows. LDL receptor knock-out mice were fed for 4 weeks on a semisynthetic, atherogenic diet that contained cholesterol, 10 g/kg, and sodium cholate, 5 g/kg (23). Then, after an 8-h fast, these mice were exsanguinated and their sera pooled to obtain a single lot enriched in both apo B-100 and B-48 (21). The apo B concentration in the pooled serum was determined by using a five-point dilution curve constructed with the lipoprotein-based calibrator. We then serially diluted the serum pool with a custom-manufactured bovine serum albumin-based calibrator/control diluent (proprietary; Incstar) to obtain a set of serum-based calibrators with the following apo B concentrations: 12.5, 24.9, 49.9, 74.8, 99.8, and 124.7 mg/L. A single freeze–thaw cycle had no detectable effect on these calibrators.

To verify that the reactivity of the purified apo B was similar to that of the apo B in serum samples, we compared rates of antigen–antibody complex formation. Starting 5 s after addition of antibody, we read the absorbance at 340 nm (A340) eight times, at 1-min intervals. The A340 value at each time point was subtracted from the endpoint A340 value, and the natural logarithm of this difference was plotted vs time. Dividing ln2 by the absolute value of the slope, calculated by linear regression, gives the t1/2 value, the time it takes to progress halfway to the end-point absorbance. For a sample of purified mouse apo B (121.9 mg/L), t1/2 was 0.760 min (r = -0.996); for a serum-based calibrator (124.7 mg/L), it was 0.753 min (r = -0.995; Fig. 2 ), indicating similar reactivity.



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  		Figure 2. Rates of antigen–antibody complex formation for six concentrations of apo B calibrators.

The control sera were prepared as follows. Control 1 was prepared from mouse plasma that had never been frozen, obtained from Pel-Freez in citrate; it was converted to serum by adding calcium, centrifuging at low speed to remove fibrin, and dialyzing to remove excess calcium. We added sucrose, 100 g/L, as a cryoprotectant because our preliminary measurements of frozen aliquots of this control showed considerable variation. Control 2 was pooled from chow-fed LDL receptor knock-out mice, assayed, and diluted fourfold with the Incstar calibrator/control diluent. This material was considered to contain mainly apo B-100, because the LDL receptor knock-out mice were fed on a regular diet, without added cholesterol or cholic acid (20)(21). Preliminary measurements of control 2, which had been manipulated less than control 1, indicated that a cryoprotectant was not necessary. Both controls were aliquoted and frozen at -70 °C.

immunoturbidimetric assay of apo b
This assay was developed with use of a Cobas Fara II (Roche Diagnostics, Sommerville, NJ) clinical chemistry analyzer, polyethylene glycol (8000 Mr, 40 g/L) buffer in phosphate-buffered saline containing sodium azide (1 g/L), partially purified goat anti-mouse apo B antiserum (IgG), six serum-based calibrators (apo B 12.5–124.7 mg/L), and two control sera. The analyzer delivers 10 µL of sample and 250 µL of buffer, incubates the reaction mixture at 37 °C for 30 s, delivers 15 µL of anti-apo B antibody to each cuvette, and incubates for an additional 8 min. The apo B concentration of samples is calculated by the Cobas DENS (Data Evaluation for Nonlinear Standard Curves) option by fitting the calibration curve values to a four-parameter logit-log curve after subtracting a background blank (buffer plus sample).

Assay performance studies.
NCCLS imprecision studies were conducted over 20 days with the two control serum pools described above according to protocol EP5-T2 (24). Interference studies were conducted by adding known amounts of hemoglobin from a freshly prepared hemolysate to the two control pools; two hemoglobin concentration ranges were studied in each pool, 1–20 g/L and 25–50 g/L, and compared with samples containing no added hemoglobin. The linearity study was performed with serial dilutions in saline of serum from an apo E knock-out mouse (yielding samples with expected apo B concentrations of 10–325 mg/L).

Reactivity to mouse apo B-48.
Because apo B-48 concentrations in mice vary according to diet, feeding state, and genetic strain (21)(23), we studied its reactivity in our assay. Mouse apo B-48 was purified from apo E knock-out plasma essentially as described above for apo B-100 (14). The resulting preparation contained pure apo B-48, as demonstrated by SDS-PAGE; its protein concentration was 104 mg/L measured by the SDS–Lowry assay, whereas its apo B content was 219 mg/L measured by our mouse apo B assay. To completely characterize the reactivity of mouse apo B-48 in our assay, we prepared a dilution series of the purified material in saline, assaying each dilution for protein with the SDS–Lowry assay and for apo B with the immunoturbidimetric assay. We then plotted mouse apo B concentration as a function of SDS–Lowry protein concentration.

Reactivity to apo B-100 from rat.
We also purified apo B-100 from rat plasma, verifying its purity by SDS-PAGE, and analyzed samples for protein by the SDS–Lowry assay and for immunoreactive apo B by the immunoturbidimetric assay. In addition, to determine whether non-apo B proteins would react in our assay, we dialyzed the >1.063 kg/L fraction of rat plasma to remove KBr and assayed the dialysate with our immunoturbidimetric assay.

apo b concentrations in mouse strains on different diets
Apo B concentrations in individual serum samples from five groups of mice were determined immunoturbidimetrically. Unless otherwise indicated, all of these mice, including the genetically manipulated strains, were of C57BL/6J background.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Program settings for the Cobas Fara II are shown in Table 1 . Notice that the sample volumes are appropriate to the mouse. After calibration, the assay throughput is 28 tests in ~20 min. Fig. 2Up shows reaction rates of the six calibrators (apo B range 12.5–124.7 mg/L).


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  		Table 1. Program settings for the Roche Cobas Fara II.

assay performance studies
Results of NCCLS imprecision tests are shown in Table 2 . Imprecision (CV) was <5% for all estimates for both controls. Control 1 was extensively manipulated during preparation and had to be stabilized with sucrose before freezing. The greater imprecision observed for this pool is probably related to the handling procedures as well as matrix differences, i.e., calcium addition, dialysis, and sucrose addition. Control 2 was prepared from fresh serum that had been diluted but not otherwise manipulated. The use of fresh serum for Control 2 and the calibrators, along with the Incstar bovine serum albumin-based diluent, precluded the need to use sucrose as a cryoprotectant. Imprecision was considerably less for this control.


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  		Table 2. Results of NCCLS imprecision studies.1

Results of the linearity study are shown in Fig. 3 . The assay is linear to mouse apo B-100 concentrations of 325 mg/L, more than twice the calibrated range.



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  		Figure 3. Linearity of the mouse apo B immunoturbidimetric assay assessed with stepwise-diluted serum samples.

Because serum samples drawn from mice are often hemolyzed, we added hemoglobin to serum at final concentrations of 20, 40 and 50 g/L. Each addition produced <=1.0% deviation in assay results (n = 2 each). Also, serum samples from mice are often frozen once for convenience. A single freeze–thaw cycle produced <5% deviation in assay results (n = 5).

assay reactivity
Reactivity to mouse apo B-48.
Per unit of protein mass (determined by SDS–Lowry), apo B-48 is almost exactly twice as reactive as apo B-100 in our immunoturbidimetric assay (Fig. 4 ). Because 1 g of apo B-48 contains twice as many molecules as 1 g of apo B-100, we conclude that each molecule of apo B-48 gives almost exactly the same signal in the assay as a molecule of apo B-100.



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  		Figure 4. Mouse apo B-48 measured by the immunoturbidimetric assay as a function SDS–Lowry protein results.

Reactivity to rat apo B-100.
In this assay, purified rat apo B-100 gave values only 29.6% ± 4.3% (mean ± SE, n = 3) of the concentrations measured by the SDS–Lowry assay. However, the density >1.063 kg/L fraction of rat serum showed no reactivity. Thus, the assay can be used to measure apo B in rat samples but with use of a correction factor of 3.38.

apo b concentrations in mouse strains on different diets
Screening studies for five groups of mice are summarized in Table 3 . The only one of these groups for which apo B values have been previously reported is chow-fed C57BL/6J; their values, 65 mg/L by SDS-gel scanning (23), were close to ours. As expected, serum apo B concentrations were substantially increased by cholesterol feeding or by disruption of the LDL receptor or apo E genes by homologous recombination.


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  		Table 3. Screening results for five groups of mice.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The traditional method for measuring mouse apo B concentrations involves SDS-PAGE, followed by scanning densitometry (23)(25). That method is labor intensive, expensive, and slow. In contrast, our method is automated and inexpensive and, after calibration, can assay 28 tests every 20 min. Alternatively, because this is an end-point assay (Fig. 2Up ), the method could be run manually, the absorbance being read with a spectrophotometer or a plate reader just before and 8 min after addition of the antibody.

Although the assay was calibrated with pure mouse apo B-100, we also quantified the reactivity of mouse apo B-48 in the assay (Fig. 4Up ). Thus, the assay is suitable to detect abnormal plasma concentrations of either form of mouse apo B (Table 3Up ). Mouse serum containing both apo B-100 and B-48 can be analyzed by gel electrophoresis to determine the relative percentages of each form, taking into account their different chromogenicities when stained with Coomassie Blue (23)(25). Because each molecule of apo B-100 or apo B-48 generates the same signal in our assay, results when both forms of apo B are present would be best expressed as moles, not mass, per volume. Our original calibrator was mouse apo B-100, 1 mol of which would contain ~514 000 g of protein. To our knowledge, incidentally, the reactivity of human apo B-48 in the clinical immunoturbidimetric assay for human apo B has not been reported.

In conclusion, the assay described overcomes many of the difficulties associated with mouse samples. First, the small sample volumes typically available from mice are sufficient for this assay, even when apo B concentrations are low (12–50 mg/L). Second, the assay is unaffected by hemolysis, a frequent problem with mouse samples. Third, triglyceride interference is not an issue because hypertriglyceridemia is uncommon in mice; moreover, this assay format is not sensitive to triglyceride interference <=5.0 g/L (26)(27). Finally, the assay results are not affected by one freeze–thaw cycle of samples, thus allowing storage of sera for later measurement. The development of this assay makes it practical to survey large numbers of inbred mouse strains for variants in apo B concentration or regulation. Genetic analysis of such variants may lead to new insights into the atherosclerotic process. Assessments of mouse apo B concentrations may also reduce variability in studies of atherosclerosis. Moreover, the mouse can now be used as a convenient small-animal model to screen compounds that might lower plasma apo B concentrations.


   Acknowledgments
 
We thank Beverly Paigen of Jackson Labs. (Bar Harbor, ME) for the serum samples listed in Table 3Up and for discussion of results; Roche Diagnostic Systems (Sommerville, NJ) for the use of the Roche Cobas Fara II; Wendi V. Rodrigueza of the Medical College of Pennsylvania (Philadelphia, PA) for assisting with the preparation of the cholesterol-fed LDL receptor knock-out serum; and Evelyne Ribary and Jocelyn Robles of the Rogosin Institute for conducting the NCCLS imprecision studies. This work was supported in part by a Grant-in-Aid from the American Heart Association (K.J.W). K.J.W. is also an Established Investigator of the American Heart Association and Genentech.


   Footnotes
 
Portions of this work have been published in abstract form (Clin Chem 1995;41:S139).

1 Nonstandard abbreviations: apo, apolipoprotein; SDS, sodium dodecyl sulfate; and PAGE, polyacrylamide gel electrophoreses.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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