Development of an immunoassay for the quantification of soluble LR11, a circulating marker of atherosclerosis.
Circulating sLR11 can be immunologically detected in serum by use of specific antibodies against LR11 (6). Circulating concentrations of LR11 were positively correlated with intimal-media thickness in dyslipidemic individuals, and the correlation was independent of other classical risk factors for atherosclerosis (6). In addition, neuronal LR11 expression is characteristically reduced in mild cognitive impairment and in the brains of individuals with Alzheimer disease (AD) (10-13). Single nucleotide polymorphism analysis of the gene for LR11 [sortilin-related receptor, L(DLR class) A repeats-containing (SORL1)] has been used to predict AD onset (14, 15).
In this study, we produced specific monoclonal antibodies (MAbs) that bind to intact sLR11 without prior purification. Using these antibodies, we developed a novel sandwich ELISA method to quantify circulating sLR11 concentrations in both serum and cerebrospinal fluid (CSF). This technique provides a means for quantifying sLR11 to be used potentially as a marker for atherosclerosis and a predictor of AD and other neurodegenerative diseases.
Materials and Methods
Human and animal sera were purchased from Tennessee Blood Services and Cosmo Bio, respectively. Commercial human CSF samples (n = 13) were obtained from Scipac. To evaluate the normal concentration range of circulating sLR11, human serum was obtained from 87 healthy normolipidemic individuals (41 males and 46 females), who gave informed consent for participation in this study, which was approved by the Human Investigation Review Committee of the Chiba University Graduate School of Medicine.
EXTRACTION OF sLR11 WITH A RECEPTOR-ASSOCIATED PROTEIN AFFINITY RESIN
Recombinant human receptor-associated protein (RAP) was prepared as a glutathione S-transferase fusion protein (16) and applied to a glutathione-Sepharose resin (GE Healthcare), which was used to extract sLR11 from serum. Briefly, samples were incubated overnight at 4[degrees]C at a RAP affinity resin-to-sample volume ratio of 1:20, and the resin was packed into a separation column. The column was washed with 20 mmol/L Na,K-phosphate buffer (pH 7.2) containing 150 mmol/L NaCL, and sLR11 was eluted with 50 mmol/L sodium citrate buffer (pH 5.0) containing 150 mmol/L NaCl. The sLR11 from cultured human IMR32 cells was extracted with a RAP affinity resin as described previously (5).
PREPARATION OF MAb BY SYNTHETIC PEPTIDE IMMUNIZATION
Anti-LR11 MAb for use in the immunoblot analyses of human and animal sera was prepared by immunizing mice with a synthetic peptide (SMNEENMRSVITFDKG) corresponding to amino acid residues 432-447 of LR11 (2,17) coupled to keyhole-limpet hemocyanin. The peptide-keyhole-limpet hemocyanin complex was emulsified with complete Freund's adjuvant (Gibco) and subcutaneously injected into BALB/c mice, 4 times at 2-week intervals. The spleen cells extracted from the immunized mice were fused with mouse myeloma cells (Sp2/0) in the presence of 50% polyethylene glycol. A single clone was selected to yield MAb A2-2-3 (IgG1,k), which reacted with both human and rabbit sLR11 in immunoblot analyses.
[FIGURE 1 OMITTED]
Before immunoblot analysis, serum proteins were boiled in SDS-Tris buffer, with or without [beta]-mercaptoethanol (reducing or nonreducing condition, respectively), and then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 1% BSA in PBS containing 0.05% Tween 20 (PBST), incubated with MAb A2-2-3, reacted with horseradish peroxidase-conjugated rabbit antimouse IgG using a VECTASTAIN ABC kit (Vector Laboratories) according to the manufacturer's instructions, and subsequently stained with diaminobenzidine.
PREPARATION OF MAbs BY DNA IMMUNIZATION
Anti-LR11 MAbs for the sandwich ELISA were prepared via DNA immunization at Nosan Corporation (18 -20). Briefly, cDNA encoding amino acid residues 1000-1550 of LR11 (2, 17) was cloned into an expression plasmid (in-house vector, Nosan), and we immunized BALB/c mice or Wistar rats by intradermal application of DNA-coated gold particles, using a hand-held device for particle bombardment (Gene Gun, Bio-Rad). Antibody-producing cells were isolated and fused with Sp2/0 myeloma cells by use of polyethylene glycol, according to standard procedures. Five mouse and 5 rat MAbs were selected based on their reactivity with extracted rabbit sLR11, and preliminary sandwich ELISAs were performed using various combinations of these MAbs and A2-2-3. Mouse MAb M3 (IgG2a,k) and rat MAb R14 (IgG2b,k) were identified as the most sensitive for rabbit and human sLR11, respectively, and the combination of these antibodies gave the strongest reactivity against serum sLR11 in our ELISA system. Rat MAb R14 was then conjugated with sulfo-NHSLC-biotin (Pierce), according to the manufacturer's instructions.
[FIGURE 2 OMITTED]
PURIFICATION OF sLR11 FROM HUMAN AND RABBIT SERA
The RAP affinity resin described above was used to extract sLR11 from 2.5 L of human serum or 1.0 L of rabbit serum. The eluted proteins were concentrated and applied to a HiLoad Superdex 200 gel filtration column (GE Healthcare) equilibrated with PBS. The fractions containing immunologically detected sLR11 were pooled, concentrated, and incubated overnight at room temperature with anti-LR11 MAb M3-Sepharose resin. After the resin was rinsed with PBS, immunologically bound sLR11 was eluted with 100 mmol/L sodium citrate buffer (pH 3.0). The sLR11 content was quantified by comparison with BSA standards on silver-stained gels.
The wells of a polystyrene microtiter plate (Nunc) were coated with 100 of MAb M3 (10 mg/L in PBS) and incubated for 2 h. After extensive washing with PBST, the wells were blocked by incubation with 200 [micro]L of 1% BSA-PBST for 1 h. The samples (10 [micro]L) were diluted with 100 AL of sample buffer, which consisted of 5.25% n-nonanoyl-N-methyl-d-glucamine (MEGA-9; Dojindo) and 25% heterophilic blocking reagent (Scantibodies Laboratory) in PBS. The calibration samples (0-4.0 [micro]g/L rabbit sLR11) serially diluted in sample buffer together with the above diluted samples (100 [micro]L) were placed into wells and then incubated for 6 -16 h. After extensive washing with PBST, 100 [micro]L of biotinylated MAb R14 was added to each well, and the plate was subsequently incubated for 4 h. After extensive washing with PBST, the LR11-MAb complex was reacted with horseradish peroxidase-conjugated streptavidin (Pierce) for 1 h. The trapped complexes were washed and incubated with 100 [micro]L of substrate solution (tetramethyl-benzidine in citrate buffer, pH 3.65, containing hydrogen peroxide) for 30 min. The chromogenic reaction was stopped with 100 [micro]L[H.sub.2]S[O.sub.4], and the absorbance of each sample was determined at 450 nm. All steps were performed at room temperature. ELISA data [micro]g/L) were significantly and positively correlated with the immunoblotting data (U) previously observed following purification with RAP affinity chromatography (r = 0.781, P < 0.001, y = 1.31 x + 8.34) (6).
[FIGURE 3 OMITTED]
Statistical analyses were performed with commercial software (Stat Flex, Ver. 5.0). The effect of sample dilution with various concentrations of MEGA-9 on the ELISA results was examined using a paired t-test, with P < 0.05 considered significant. The correlation between variables was evaluated using Pearson correlation analysis. Furthermore, sLR11 concentrations in individuals with atherosclerosis vs those in normal individuals were compared by use of box plot analysis.
IDENTIFICATION OF sLR11 IN VARIOUS SERA AND HUMAN CSF
sLR11 was isolated as a 250-kDa protein from rabbit SMCs and human IMR32 cells by use of immunoblot techniques under reducing conditions and an antibody against a recombinant protein corresponding to a partial amino acid sequence of rabbit LR11 (5). For comparison, sLR11 was extracted from both human and animal sera, using RAP-glutathione S-transferase resin. A single 250-kDa protein was detected in human serum by use of MAb A2-2-3 (Fig. 1A). The migration distance of the protein during electrophoresis was consistent with that of sLR11 from rabbit SMCs and human IMR32 cells (5). A single protein band, similar in size to that obtained from human serum, was detected immunologically by MAb A2-2-3 in mouse, rat, rabbit, goat, and porcine sera. The relative intensities of the immunological signals suggested that sLR11 was most abundant in rabbit serum.
We also assessed the presence of sLR11 in human CSF (Fig. 1B), where it was identified by MAb A2-2-3 without the need for RAP extraction. Although sLR11 obtained from CSF was slightly larger than that in serum, the protein appeared as a single band at 250-kDa in both cases.
PURIFICATION OF sLR11 FROM HUMAN AND RABBIT SERA
Using DNA immunization, we established 2 MAbs, M3 and R14, against different epitopes of human sLR11; these MAbs were then used to purify intact sLR11 from serum and construct a sandwich ELISA assay. Intact sLR11 protein was first purified from human and rabbit sera using RAP affinity resin and was released from the resin with an eluting buffer, without decoupling from RAP-glutathione S-transferase (Fig. 2A, lanes 1 and 4). The eluted samples were treated with anti-LR11 MAb M3-Sepharose resin. Silver staining after electrophoresis indicated that the M3-reactive samples contained sLR11 as a single protein at 250 kDa as well as low molecular weight proteins, in both human and rabbit sera (lanes 2 and 5). The purified sLR11, but no other low molecular weight protein, was specifically bound to MAb A2-2-3 (lanes 3 and 6). The migration distance of sLR11 from human and rabbit sera was not different from that of sLR11 in the culture medium of IMR32 cells (lane 7). Therefore, 2-step affinity chromatography with RAP and MAb M3 can be used to specifically purify serum sLR11 as a soluble protein identical to that released from cultured cells. R14, as well as A2-2-3 and M3, showed reactivity against the purified sLR11, but did not bind any other low molecular weight protein (lanes 8-11). Notably, R14 reacted with sLR11 under both reducing and nonreducing conditions, whereas M3 reacted with sLR11 under nonreducing condition only.
Silver staining of the purified protein after gel filtration chromatography showed that the position to which purified sLR11 eluted corresponded to an estimated molecular weight > 398 kDa (Fig. 2B). Notably, no other distinct protein proportional to the level of the stained sLR11 protein was detected in these fractions. The apparent molecular weight of sLR11 estimated from gel filtration was greater than that determined by use of gel electrophoresis (see Figs. 1 and 2A).
PREPARATION OF SAMPLES WITH MEGA-9 FOR SANDWICH ELISA
Sample conditions for the sandwich ELISA were determined using the above MAbs and purified samples. The absorbance level of immunologically detected sLR11 was proportional to the volume of extracted human sLR11 when diluted with PBS. However, the expected change in absorbance was not observed when samples were diluted with human serum instead of PBS (Fig. 3A). To measure sLR11 in human serum accurately, the matrix effects were mitigated by the addition of MEGA-9 detergent. The effects of MEGA-9 on absorbance recovery increased with increasing amounts of MEGA-9, up to 4.5%. No significant differences were observed at higher concentrations (Fig. 3B). These results suggest that human serum contains unknown factors that interfere with sLR11 quantification, and that this interference could be diminished by the presence of MEGA-9. Therefore, samples were diluted with 5.25%, which was chosen as the middle concentration of 4.5% and 6.0%.
[FIGURE 4 OMITTED]
CHARACTERIZATION OF sLR11 IN SERUM AND CSF BY GEL FILTRATION
To assess whether ELISA can specifically detect naturally occurring sLR11, each fraction of human serum and CSF was analyzed for sLR11, following to separation by gel filtration chromatography; the results were then compared with the immunologically purified sLR11 protein as a quantitative calibrator. The ELISA of serum and CSF samples that had been diluted with or without MEGA-9 showed abundant sLR11 in fractions with molecular weights > 398 kDa (Fig. 4), similar to the results of the purified protein (see Fig. 2B). Immunoblot analyses showed that the concentration of sLR11 was proportional to the signal intensity of the gel-filtered 250-kDa proteins in both the serum and CSF samples. These results strongly suggest that this ELISA based on the immunologically purified 250-kDa sLR11 protein is also appropriate for quantifying the naturally occurring sLR11 in serum and CSF, although the gel filtration analyses suggested that the naturally occurring protein may be involved in a high molecular weight complex.
[FIGURE 5 OMITTED]
ELISA PERFORMANCE: ASSAY CHARACTERISTICS
A representative calibration curve is shown in Fig. 5A. The working range of this ELISA was 0.25-4.0 [micro]/L. A quadratic equation was applied to the calibration curve in the working range. The sensitivity, defined as the mean back-fit value for the lowest standard giving acceptable precision (CV = 10%), was 0.25 [micro]/L. With this ELISA method the lower limit of detection for sLR11 was 0.1 [micro]g/L, which corresponds to the mean blank signal plus 3 SDs. The intraassay CVs (n = 10) were 3.0% and 3.7% at sLR11 concentrations of 7.6 [micro]g/L in serum and 4.4 [micro]g/L in CSF, respectively. The interassay CVs (n = 4) were 3.9% and 10.5% at sLR11 concentrations of 7.6 [micro]g/L in serum and 4.1 [micro]g/L in CSF, respectively. When we used samples containing 5%-16% human serum, percentage recovery ranged from 96.5% to 102.6% (Fig. 5B).
VARIATIONS OF sLR11 IN SERUM AND CSF
Measurements of sLR11 in 87 serum samples and 13 CSF samples obtained from normal individuals gave mean (SD) sLR11 concentrations of 8.7 (2.1) [micro]g/L (range, 4.5-14.2 [micro]g/L) and 8.5 (3.5) [micro]g/L (range, 3.7-13.0 [micro]g/L) in serum and CSF, respectively. We observed no significant difference in serum sLR11 concentrations between males [8.4 (1.9) [micro]g/L, n = 41] and females [9.8 (5.8) [micro]g/L, n = 46].
sLR11 CONCENTRATIONS IN INDIVIDUALS WITH ATHEROSCLEROSIS
To evaluate whether this ELISA method is useful for detecting variation in circulating sLR11 under pathophysiological conditions, we measured sLR11 concentrations in individuals with atherosclerosis. The sLR11 concentrations determined by immunoblotting after RAP affinity chromatography were positively correlated with the degrees of atherosclerosis in the carotid arteries of individuals with dyslipidemia (6). The sLR11 concentrations in individuals with atherosclerosis were compared to those of healthy individuals (see previous section on variation of sLR11 in serum and CSF). The sLR11 concentrations [14.2 (6.0) [micro]g/L] in individuals with atherosclerosis were significantly higher than those in healthy individuals (Fig. 6). The variation in circulating sLR11 concentrations in individuals with atherosclerosis was within the dynamic range of the ELISA.
[FIGURE 6 OMITTED]
Three MAbs were established against different epitopes of human sLR11, and an ELISA method was developed for the quantitative measurement of sLR11 in serum and CSF. One of the MAbs (M3), in combination with RAP affinity extraction, enabled the purification of sLR11 from human and rabbit sera. The purified human and rabbit sLR11 was immunologically identical to sLR11 released from cultured cells, strongly suggesting that circulating sLR11 corresponds to the soluble form of membrane-bound LR11. This soluble form has been identified in the media of IMR32 and SMC cultures (5, 6, 9). The combination of MAb M3 and MAb R14 yielded an ELISA that is highly specific for sLR11 in serum and CSF, without the need for prior RAP affinity extraction.
Strong matrix effects interfered with the accurate determination of sLR11 in serum by ELISA. However, these effects were diminished by pretreatment with MEGA-9 detergent (see Fig. 3). This pretreatment may dissociate complexes of sLR11 and serum components or may induce a conformational change in sLR11 such that it more efficiently interacts with the MAbs. Previous studies have shown that several serum components, including apolipoprotein E-containing lipoproteins, urokinase plasminogen activator-plasminogen activator inhibitor type 1 complex, and amyloid-[beta], can interact with membrane-bound LR11 (1 , 16, 21). The observation that MEGA-9 increased the absorbance of the isolated protein at 450 nm in gel filtration fractions obtained from both serum and CSF, and did so in proportion to the signal intensity of the sLR11 protein detected immunologically in the absence of MEGA-9 (see Fig. 4), suggests that epitope recognition by MAbs was strengthened by MEGA-9. The mechanism of MEGA-9-mediated absorbance enhancement requires further elucidation, specifically with regard to the interaction between naturally occurring sLR11 and various matrices in serum and with homomeric or heteromeric complexes under various column conditions (see Fig. 2B).
Using the established ELISA conditions, we investigated the mean sLR11 concentrations in serum and CSF. In 74% of healthy individuals, serum sLR11 concentrations were < 10 [micro]g/L. The sLR11 concentrations in the sera of individuals with atherosclerosis ranged from 6 to 30 [micro]g/L. Therefore, the ELISA technique described here provides sufficient sensitivity for detecting circulating sLR11 concentrations in individuals with atherosclerosis and in normal populations.
Given that sLR11 is abundantly expressed in intimal SMCs (3) and that circulating sLR11 concentrations are positively correlated with the carotid intimamedia thickness in dyslipidemic individuals (6), variation in the circulating sLR11 concentration may be indicative of the condition of intimal SMCs. Metabolic disorders such as dyslipidemia and diabetes can cause pathological changes in intimal SMC function, possibly leading to accelerated progression of atherosclerosis (22-24). The expression level of LR11 is drastically higher in intimal SMCs relative to that in medial SMCs (3), and a large proportion of the LR11 in the cell membrane is released into the culture medium of SMCs (5). Therefore, the concentration of circulating sLR11, rather than the LR11 expression level in intimal SMCs, may be more effective as a novel marker for pathogenic changes in SMCs.
Recent studies have highlighted the pathological function of sLR11 in neurodegenerative diseases. Immunological analyses indicate that sLR11 exists in CSF at concentrations similar to those in serum. Neuronal LR11 expression is significantly reduced in individuals with mild cognitive impairment and AD (10-13), and polymorphism of the gene for LR11 is highly associated with the onset of AD (14). Therefore, methods for determining sLR11 concentrations in CSF may be vital for future research into neuronal diseases, particularly AD.
In conclusion, we established a sensitive ELISA method for determining sLR11 concentrations in serum and CSF. This ELISA method constitutes a useful tool for monitoring the pathological condition of intimal SMCs and the progression of atherosclerosis (25). Use of this ELISA method to measure sLR11 as an indicator of intimal SMC function may enable novel strategies for treating atherosclerosis and help to determine risk factors for vascular disease. Furthermore, the ELISA described here has adequate sensitivity and dynamic range for determining sLR11 concentrations in CSF and may allow significant progress in AD-related research.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
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Masanao Matsuo,  Hiroyuki Ebinuma,  * Isamu Fukamachi,  Meizi Jiang,  Hideaki Bujo,  and Yasushi Saito 
 Tsukuba Research Institute, Sekisui Medical, Ibaraki, Japan;  Department of Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba, Japan;  Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, Chiba, Japan.
 Nonstandard abbreviations: LR11, LDL receptor relative with 11 ligand-binding repeats; SMC, smooth muscle cells; sLR11, soluble form of LR11; AD, Alzheimer disease; MAb, monoclonal antibody; CSF, cerebrospinal fluid; RAP, receptor-associated protein; PBST, PBS with Tween.
* Address correspondence to this author at: Tsukuba Research Institute, Sekisui Medical, 3-3-1, Koyodai, Ryugasaki, Ibaraki 301-0852, Japan. Fax +81-29762-8635; e-mail email@example.com.
Received March 9, 2009; accepted July 9, 2009.
Previously published online at DOI: 10.1373/clinchem.2009.127027
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|Title Annotation:||Proteomics and Protein Markers|
|Author:||Matsuo, Masanao; Ebinuma, Hiroyuki; Fukamachi, Isamu; Jiang, Meizi; Bujo, Hideaki; Saito, Yasushi|
|Date:||Oct 1, 2009|
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