Multiplexed analysis of biomarkers related to obesity and the metabolic syndrome in human plasma, using the Luminex-100 System.Obesity has reached epidemic proportions in the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. ; ~30% of US adults are obese, and the percentage is increasing (1). Obesity increases the risk for the metabolic syndrome, diabetes, hypertension, atherosclerosis atherosclerosis (ăth'ərōsklərō`sĭs): see arteriosclerosis.
or hardening of the arteries , and thrombosis (2-4). The National Cholesterol Education Program Adult Treatment Panel III (ATP ATP: see adenosine triphosphate.
in full adenosine triphosphate
Organic compound, substrate in many enzyme-catalyzed reactions (see catalysis) in the cells of animals, plants, and microorganisms. III)  defined the metabolic syndrome using clinical criteria for abdominal obesity, triglyceride and HDL-cholesterol concentrations, blood pressure, and fasting glucose fasting glucose Fasting blood sugar, fasting plasma glucose Endocrinology Glucose obtained from a Pt who has had nothing–except water by mouth for 8+ hrs; FG is used in evaluating Pts for possible DM Ref range 65-115 mg/dL non-diabetic; 110-140 mg/dL, (5), and >20% of adults in the United States meet these clinical criteria (6). Novel technologies such as gene expression arrays have identified a large number of molecules that have altered expression in adipose tissue adipose tissue (ăd`əpōs'): see connective tissue.
or fatty tissue
Connective tissue consisting mainly of fat cells, specialized to synthesize and contain large globules of fat, within a and may be relevant to the development of the metabolic syndrome, diabetes, and cardiovascular disease Cardiovascular disease
Disease that affects the heart and blood vessels.
Mentioned in: Lipoproteins Test
cardiovascular disease with obesity. In contrast to the ability to simultaneously examine the concentrations of thousands of mRNA transcripts, plasma concentrations of cytokines, chemokines, or other biomarkers are usually measured one at a time by ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.
n. . Measurement of plasma concentrations of each putative biomarker with individual ELISAs incurs considerable time, cost, and sample volume, which thus limits the ability to systematically examine the effects of a clinical intervention.
Multiplexed proteomics research may offer a major advance for clinical research. Protein microarrays are the most commonly used for simultaneous determination of multiple proteins in a biological fluid. The technique uses primary antibodies Primary antibodies are antibodies raised against an antigenic target of interest (a protein, peptide, carbohydrate, or other small molecule) and are typically unconjugated (unlabelled). as the immobilized probe on a solid surface, and protein antigens labeled with fluorophores with or without bound secondary antibodies are recognized and detected. However, binding of antibodies and antigens to a solid support can cause denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. or drying of proteins. The Luminex-100 bead-based system is a recently developed platform that provides multiplexing in a solution phase and thus is particularly flexible and nondestructive for protein analysis. Each set of up to 100 uniquely color-coded polystyrene microspheres is anchored with a different capture antibody. The use of detection antibodies labeled with the fluorochrome fluorochrome /flu·o·ro·chrome/ (-krom) a fluorescent compound used as a dye to mark protein with a fluorescent label.
n. R-phycoerythrin allows quantification of antigen-antibody reactions that occur on the microsphere Not to be confused with Glass microphere.
This article largely refers to micropheres or protein protocells as small spherical units postulated by some scientists as a key stage in the origin of life. surface by measurement of the relative fluorescence intensity. The system is capable of measuring potentially up to 100 analyses simultaneously in a small sample volume (25-50 [micro]L). In theory, the Luminex platform may also provide a wider dynamic range than conventional ELISA methods because of the greater linear range of fluorescence intensity compared with absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .
2. . The increased dynamic movement during antibody-antigen reactions that occurs in solution also may increase its assay efficiency (7-11). The purpose of this study was to compare, in a clinical research study, several commercially available Luminex multiplex panels with conventional commercial ELISAs for measurement, in human plasma, of biomarkers associated with obesity and inflammation.
Materials and Methods
From September 2001 to September 2002, a total of 80 obese individuals [56 women and 24 men; mean (SD) age, 47.1 (0.9) years; mean (SD) body mass index, 38.3 (0.7) kg/[m.sup.2]] were enrolled in a protein-sparing, very low calorie diet-induced weight loss program. Written informed consent was obtained from each participant. The weight loss program was approved by the Baylor College of Medicine Baylor College of Medicine is a private medical school located in Houston, Texas, USA on the grounds of the Texas Medical Center. It has been consistently rated the top medical school in Texas and among the best in the United States. Institutional Review Board (12). All participants were obese (body mass index >30 kg/[m.sup.2]), and one-half had the metabolic syndrome based on the ATP III criteria (5). Analytes were measured in plasma at baseline for a1180 participants and after 4-6 weeks of rapid weight loss for the 40 individuals with metabolic syndrome (mean weight loss, 7%). During weight loss, participants were not started on any medication that would influence glucose tolerance, insulin secretion, or insulin sensitivity insulin sensitivity The systemic responsiveness to glucose, which can be measured by 1. The insulin sensitivity index–measures the ability of endogenous insulin to ↓ glucose in extracellular fluids by inhibiting glucose release from the liver and .
COLLECTION AND STORAGE OF BLOOD SAMPLES
Blood samples were collected by venipuncture venipuncture /veni·punc·ture/ (ven?i-pungk´chur) surgical puncture of a vein.
ve·ni·punc·ture or ve·ne·punc·ture
n. from the participants into EDTA-containing Vacutainer Tubes after an overnight fast. The freshly drawn blood was centrifuged at 30008 for 20 min at 4 [degrees] C. Plasma was then separated and subsequently stored at -70 [degrees] C in small aliquots. All analyses were performed on frozen plasma samples.
LUMINEX MULTIPLEX ASSAYS
Multianalyte profiling was performed on the Luminex-100 system and the XY Platform (Luminex Corporation). Calibration microspheres for classification and reporter readings as well as sheath fluid were also purchased from Luminex Corporation. Acquired fluorescence data were analyzed by the MasterPlex[TM] QT software (Ver. 1.2; MiraiBio, Inc.). Plasma concentrations of leptin Leptin
A protein hormone that affects feeding behavior and hunger in humans. At present it is thought that obesity in humans may result in part from insensitivity to leptin. , insulin, and C-peptide were determined by the Lineo Human Endocrine 3-Plex Panel (Lineo Research, Inc.). Chemokines [monocyte monocyte /mono·cyte/ (mon´o-sit) a mononuclear, phagocytic leukocyte, 13µ to 25µ in diameter, with an ovoid or kidney-shaped nucleus, and azurophilic cytoplasmic granules. chemoattractant chemoattractant /che·mo·at·trac·tant/ (ke?mo-ah-trak´tant) a chemotactic agent that induces an organism or a cell (e.g., a leukocyte) to migrate toward it. protein-1 (MCP-1) and eotaxin] were measured by both the Biosource Chemokine chemokine /che·mo·kine/ (ke´mo-kin) any of a group of low molecular weight cytokines identified on the basis of their ability to induce chemotaxis or chemokinesis in leukocytes (or in particular populations of leukocytes) in inflammation. 5-Plex Panel (Biosource International, Inc.) and the Upstate Beadlyte Human Cytokine Cytokine
Any of a group of soluble proteins that are released by a cell to send messages which are delivered to the same cell (autocrine), an adjacent cell (paracrine), or a distant cell (endocrine). 8-Plex Panel (Upstate USA, Inc.). Cytokines [tumor necrosis tumor necrosis Death of tumor tissue, a common event in aggressive CAs in which the tumor rapidly outgrows its blood supply, resulting in tumor cell death. Cf Apoptosis. factor-[alpha] (TNF-[alpha]), interleukin-8 (IL-8), and IL-6] were analyzed by the Lineo Human Cytokine 6-Plex Panel (Lineo), the Upstate Beadlyte Human Cytokine 8-Plex Panel (Upstate), and the R&D Fluorokine MAP Human Cytokine 6-Plex Panel (R&D Systems, Inc.). All analyses were performed according to according to
1. As stated or indicated by; on the authority of: according to historians.
2. In keeping with: according to instructions.
3. the manufacturers' protocols.
All corresponding ELISAs were performed with commercially available reagent sets. Plasma leptin and C-peptide were analyzed by use of the Human Leptin ELISA Kit and the Human C-Peptide ELISA Kit (Lineo). Insulin was assayed by the 1-2-3 Ultrasensitive Human Insulin human insulin
A protein that has the normal structure of insulin produced by the human pancreas but that is prepared by recombinant DNA techniques and by semisynthetic processes. ELISA (Alpco Diagnostics, American Laboratory Products Company). MCP-1 and eotaxin were measured by the Quantikine Human Eotaxin Immunoassay Immunoassay
An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus. and Quantikine Human MCP-1 Immunoassay (R&D Systems). TNF-[alpha] and IL-6 were measured by the Quantikine HS Human TNF-[alpha] Immunoassay and the Quantikine HS Human IL-6 Immunoassay (R&D Systems). The circulating IL-8 concentration was measured by the Biosource Human IL-8 Immunoassay (Biosource International).
DETECTION LIMITS AND RECOVERY STUDY
To determine the detection limits for the Luminex assays, 3 SD were added to the mean fluorescence intensity of 2 zero calibrator calibrator
an instrument for dilating a tubular structure or for determining the caliber of such a structure. replicates. The respective analyte concentrations were then calculated from the calibration curve In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. . Similarly, to evaluate the detection limits for the ELISAs, 3 SD were added to the mean absorbance value of 2 zero calibrator replicates, and the respective analyte concentrations were calculated from the calibration curves. The recombinant protein recombinant protein Molecular biology A protein encoded by recombinant DNA or generated from a recombinant gene. See Recombinant pharmacology. MCP-1 and IL-6 calibrators provided with the ELISA and Luminex reagents were added into normal healthy human plasma. The plasma was manufacturer pooled with specific concentrations of lipids requested (lot no. BC0208A; Pacific Biometrics, Inc.).
Statistical analyses were performed with SP55 data analysis software (Ver. 11.5; SP55 Inc.). Analyze-it software (Ver. 1.71; Analyze-it Software Ltd.) was used for Deming regression In statistics, Deming regression, named after W. Edwards Deming, is a method of linear regression that finds a line of best fit for a set of related data. It differs from simple linear regression in that it accounts for error in both the x- and the y-axes. and Passing-Bablok analyses for method comparisons to account for imprecision in both the ELISA and Luminex methods. The Wilcoxon signed-rank test The Wilcoxon signed-rank test is a non-parametric alternative to the paired Student's t-test for the case of two related samples or repeated measurements on a single sample. was used to analyze the effect of rapid weight loss on leptin and insulin. Bland-Altman analyses (13) were performed to calculate limits of agreement and systematic errors, and the results were plotted with y as the value of the difference ([DELTA]) between Luminex and ELISA, x as the mean of the Luminex and ELISA values, and the upper and lower limits defined by the mean [DELTA] [+ or -] 2 SD.
DETECTION LIMITS OF LUMINEX ASSAYS AND ELISAS
A comparison of detection limits for the Luminex assays and the ELISAS is shown in Table 1. Some analyte concentrations were also converted to the National Institute for Biological Standards and Control (NIBSC NIBSC National Institute for Biological Standards and Control (UK) )/WHO international reference standard unit (N/L) if calibrations of the assays were performed by the manufacturers. In the Lineo Endocrine multiplex panel, leptin and C-peptide had significantly lower detection limits by the Luminex assay. The Alpco ultrasensitive insulin ELISA was more sensitive for detection of insulin than the Lineo Endocrine multiplex.
Two chemokine panels (Biosource and Upstate) were chosen to compare with ELISAS for the analysis of MCP-1 and eotaxin. Because the MCP-1 assay in the Biosource panel was not yet calibrated against the WHO standard and no WHO standard for eotaxin is currently available, it was not possible to compare the detection limits of the Biosource panel with ELISAS. Unlike the Biosource panel, the Upstate multiplex panel has a significantly higher detection limit for MCP-1 than the ELISA from R&D.
Cytokines TNF-[alpha], IL-8, and IL-6 in 3 different multiplex panels from different manufacturers (Lineo, Upstate, and R&D) were compared with ELISAS. In general, the ELISAS tended to have lower detection limits.
PERCENTAGE OF SAMPLES WITH UNDETECTABLE MCP-1, TNF-[alpha], IL-6, AND IL-8 CONCENTRATIONS IN THE LUMINEX ASSAYS AND ELISAS
We compared the percentage of samples in which 4 representative analyses could not be detected by the Luminex and ELISA methods; the results are shown in Table 2. For the ELISAS, generally the percentage of samples with undetectable analyte concentrations was 0%, except for IL-8 in the Biosource assay, which is not a high-sensitivity assay. For the Luminex assay, in the Upstate multiplex panel, nearly 50% of samples had undetectable concentrations of IL-6 and TNF-[alpha]; in the R&D multiplex panel, nearly 100% of samples had undetectable IL-6, TNF-[alpha], and IL-8; and in the Biosource multiplex panel, 61% of samples had undetectable IL-8.
ASSAY PRECISION OF LUMINEX AND ELISA METHODS
The between- and within-run imprecision, as the CV (CV = SD/mean), for the Luminex assays is shown in Table 1 of the Data Supplement (available at http:// www.clinchem.org/content/vol51/issue7/) and in Table 3. The Upstate multiplex panel and 3 plasma pools from patients with metabolic syndrome with low, medium, and high concentrations of added analyte were chosen to test the between-run precision. The 2 normal control plasma samples (QA and QC) were not selected because they were undetectable by the multiplex panel. The MCP-1 between-run imprecision, determined from 7 multiplex assays with each sample analyzed in duplicate, was >15%. The within-run precision represents the mean values of 3 samples with low, medium, and high concentrations of the analyte that were analyzed 20 times in 1 assay. The within-run CVs for all analytes ranged from 6.6% to 12% and were within the acceptable limit. The between- and within-run precision of the ELISAs are shown in Tables 1 and 3 of the online Data Supplement.
The between-run CV was determined from 2 plasma control samples (QA and QC) because they were detectable by the high-sensitivity ELISAs. For each sample, the CV was determined from the means of 6, 8, and 5 assays for MCP-1, TNF-[alpha], and IL-6, respectively, and were analyzed in duplicate. The within-run precision was calculated from the mean CVs of n samples analyzed in duplicate. Overall, the within-run CVs were within reasonable limits, ranging from 5.0% to 15%, except for IL-6 (21%). For the high-sensitivity ELISAs, the between-run imprecision generally indicated a >15% CV of random errors, and the within-run imprecision showed a >15% CV of random errors.
VALIDATION OF THE LUMINEX METHOD IN HUMAN PLASMA
We chose ELISAs for comparison with the newer Luminex technique because the ELISA is a generally accepted method for analyzing biomarkers in human serum or plasma. Two methods of comparison between Luminex assays and ELISAs were carried out. We first used linear correlation to evaluate the relationship between Luminex assays and ELISAs. We then applied the Bland-Altman method (13) to assess the agreement between the 2 measurement methods.
Correlation. The results of the Deming regression analysis comparing the Luminex and ELISA methods are shown in Table 4. The correlation coefficient Correlation Coefficient
A measure that determines the degree to which two variable's movements are associated.
The correlation coefficient is calculated as: indicates scatter of the measured data about the Deming regression line, and it is determined by the precision of the 2 assay methods. Table 4 shows the results for the Linco endocrine panel. The correlation coefficients (R) were 0.812, 0.895, and 0.582 for leptin, insulin, and C-peptide, respectively. The correlation coefficient for C-peptide was 0.67 after omission of the 2 outliers (defined as values outside 3 SD). In the Deming regression analysis, the slope indicates a proportional error that might have originated from incomplete recovery in one of the methods or from different accuracies of the calibrators used. The slopes were 1.28, 2.38, and 0.68 for leptin, insulin, and C-peptide, respectively. The smallest proportional error was for leptin, i.e., the values obtained from the 2 assay methods were the least different. The intercept indicates a systematic error that might be caused by interference, such as insufficient blank compensation. The intercepts were 2.26 [micro]g/L, -3.03 kIU/L, and -1.83 [micro]g/L for leptin, insulin, and C-peptide, respectively. The smallest systematic error was for C-peptide.
The correlation coefficients were 0.263 and 0.899 for MCP-1 in the Biosource panel with and without the 2 outliers, respectively; outliers were defined as values outside 3 SD. For eotaxin, the correlation coefficient was 0.711 for the Biosource panel. The slopes for MCP-1 without the outliers and for eotaxin were 4.97 and 7.71 for the Biosource panel, respectively, and the intercepts were 79.60 and -49.85 ng/L, respectively. Passing-Bablok analysis revealed both proportional and constant bias for MCP-1 and eotaxin (data not shown).
We also analyzed the correlations between the Luminex assays and ELISAs for the cytokines TNF-[alpha], IL-8, and IL-6. The correlation coefficients were 0.104 and -0.107 for TNF-[alpha] and 0.266 and 0.318 for IL-8 in the Linco and Upstate panels, respectively. The correlation coefficient was 0.298 for IL-6 in the Upstate panel. Overall, the correlations between Luminex assays and ELISAs for the 3 cytokines analyzed were poor in all the multiplex panels.
Bland-Altman analysis. The difference between the Luminex assay and the ELISA was plotted against the mean of the Luminex assay and ELISA for each plasma sample measured. The Bland-Altman plots for several analytes are shown in Fig. 1. The Bland-Altman analysis was carried out only for (a) analytes that showed good correlation and used the same calibrators for calibration curves in both the Luminex assays and ELISAs (leptin, insulin, and C-peptide) or (b) assays already calibrated against the WHO standards (MCP-1 and insulin).
The Bland-Altman plots for leptin, insulin, and C-peptide in the Linco endocrine panel and MCP-1 in the Upstate panel are shown in Fig. 1. The systematic differences between the 2 assay methods as calculated by the Bland-Altman analysis were 11.0 [micro]g/L, 13.8 kIU/L, 3.9 [micro]g/L, and 0.21 kIU/L for leptin, insulin, C-peptide, and MCP-1, respectively. The 2 methods agreed fairly well for leptin values <50 [micro]g/L. Above 50 [micro]g/L, however, the Luminex assays gave both higher and lower values than the ELISA; the discrepancy for some measurements was as large as 75%. For leptin, 7.5% of the patients had values that differed by >2 SD. Agreement between 2 methods is generally acceptable if <5% of values differ by >2 SD. For insulin, the 2 assays agreed very well at concentrations <30 mN/L, but above 30 mN/L, the Luminex assay gave higher values than the ELISA, although only 2.5% of the values differed by >2 SD. For C-peptide, most of the Luminex measurements gave lower values than the ELISA (mean difference, 3.9 [micro]g/L). At concentrations <4 [micro]g/L, the 2 methods agreed very well, and only 2.5% of the values differed by >2 SD. For MCP-1 in the Biosource panel, the 2 methods agreed well at concentrations <0.5 kIU/L, and 5.5% of patients had values that differed by >2 SD. For insulin, C-peptide, and MCP-1, the only measurements that differed by >2 SD were the extreme highest values of the analytes for patients in this study.
Recovery experiments detect inaccuracy of a method resulting from systematic errors. The results of the recovery study for MCP-1 in both the ELISA and Luminex assay are shown in Table 2 of the online Data Supplement. The recombinant protein calibrators provided with the ELISA and the Luminex assay were added at 3 different concentrations in pooled normal healthy human serum for measurement of recovery with 10 replicates or more. The 3 concentrations (32, 128, and 512 ng/L) of MCP-1 were selected because they were commonly seen in our study in healthy individuals and in patients with metabolic syndrome. The pools containing added Luminex calibrator were measured by the Luminex method, and the pools containing the added ELISA calibrator were measured by ELISA. As shown in Table 2 of the online Data Supplement, for the pools containing 128 and 512 ng/L MCP-1, the ELISA gave mean recoveries of 98.8% and 102.1%, respectively. However, in the Luminex assay, the mean recoveries of MCP-1 were 58.2%, 49.1%, and 54.6%, respectively, for the 3 concentrations.
EFFECTS OF ACTIVE WEIGHT LOSS ON LEPTIN AND INSULIN IN INDIVIDUALS WITH THE METABOLIC SYNDROME
The patients with metabolic syndrome lost 7% of their initial weight [8.0 (0.55) kg, or 17.6 (1.2) pounds] after 4-6 weeks of the diet-induced weight loss program. Plasma concentrations of leptin and insulin at baseline and after weight loss were measured by both the Luminex and ELISA methods, and the results were compared. Mean (SD) leptin concentrations measured by ELISA were significantly higher before weight loss compared with after weight loss [36.3 (21.5) vs 19.8 (18.7) [micro]g/L; n = 40; P <0.001, Wilcoxon signed-rank test]. The Luminex assay also showed a significant mean (SD) difference after weight loss [47.4 (25.6) vs 27.6 (22.1) [micro]g/L; n = 40; P <0.001]. Insulin concentrations were also significantly higher before weight loss than after weight loss whether measured by ELISA [19.2 (17.3) vs 9.4 (7.8) mN/L; n = 13; P = 0.039] or the Luminex assay [37.4 (41.1) vs 14.7 (16.0) mIU/L; n = 13; P = 0.028]. The percentage changes in leptin and insulin after weight loss are shown in Fig. 1 of the online Data Supplement. The median percentage changes in leptin were -54% and -46% as measured by the ELISA and Luminex assay, respectively, and the median percentage changes in insulin were -41% and -56%, respectively.
[FIGURE 1 OMITTED]
COMPARISON OF MCP-1 BETWEEN HEALTHY CONTROLS AND METABOLIC SYNDROME PATIENTS WITH 4 OR MORE RISK FACTORS
We compared plasma MCP-1 concentrations in healthy controls (n = 14) and metabolic syndrome patients with 4 or more risk factors (n = 20). The MCP-1 concentrations measured by ELISA and the Luminex assay are shown in Fig. 2 of the online Data Supplement. The mean (SD) MCP-1 concentrations measured by ELISA were significantly higher in the metabolic syndrome patients than in healthy controls [536.5 (316.1) vs 263.3 (96.5) ng/L; P <0.001, Wilcoxon signed-rank test]. The median MCP-1 values measured by ELISA were 414.6 and 231.4 ng/L for patients and controls, respectively. The results obtained with the Luminex assay also indicated that the mean (SD) MCP-1 concentrations in the metabolic syndrome patients were significantly higher than those in healthy controls [83.5 (101.1) vs 20.4 (8.6) ng/L; P <0.001, Wilcoxon signed-rank test]. In the Luminex assay, the median MCP-1 values were 63.9 and 18.4 ng/L for patients and controls, respectively.
Multiplex immunoassays using a small sample volume could facilitate clinical research in complex disorders such as the metabolic syndrome and could be extremely helpful in the evaluation of changes in biomarkers with therapeutic interventions. The detection limits shown in Table 1 allow for a potentially high degree of detectability in the patients' samples. However, because the focus of this study was to compare the validity of Luminex-based assays vs traditional ELISAs as a tool for clinical research, the use of functional sensitivity is a more meaningful index than limit of detection, and in our clinical study, many cytokines were not detectable in a high percentage of patient plasma samples by the Luminex multiplex method. The inability to detect these analytes by the multiplex method may be explained in part by interferences, such as heterophilic antibodies, that are present in complex sample matrices such as plasma.
The within-run imprecision (CV) values obtained for the multiplex method were generally <15%, whereas the between-run imprecision for the multiplex method for MCP-1 was >15%. The large interassay variations seen with MCP-1 should be considered when using multiplex panels for this analyte (and potentially others) in clinical research, for example, by measuring all samples in 1 run (or at least all the samples from a specific individual before and after an intervention), and by including control sample pools in all runs.
The poor correlations between the Luminex assay and ELISAs for the 3 cytokines analyzed in our study are most likely attributable to the extremely low plasma concentrations of these cytokines and the presence in blood of interfering substances such as heterophilic antibodies, which are known to interfere with immunoassays (14,15). Furthermore, our results show an increase in variability at lower concentrations for both methods (Luminex and ELISA), which also may partially explain the poor correlations between the 2 methods.
Our studies demonstrate that in our laboratory the multiplexed assays for leptin (Lineo), insulin (Lineo), MCP-1 (Biosource and Upstate), and eotaxin (Biosource) showed good correlations of >0.7 (correlation coefficients ranging from 0.711 to 0.895); eotaxin (Upstate) and C-peptide (Lineo) showed fair correlations of 0.5-0.7 (correlation coefficients ranging from 0.496 to 0.582); and TNF-[alpha], IL-8, and IL-6 (Lineo, Biosource, Upstate, and R&D) showed very poor correlations (<0.5) with ELISAs (correlation coefficients ranging from -0.107 to 0.318). The changes in plasma concentrations of leptin and insulin after diet-induced weight loss showed similar percentage reductions as assessed by multiplex assays or ELISA (Fig. 1 of the online Data Supplement). The relative difference in plasma concentrations of MCP-1 between controls and metabolic syndrome patients also were similar in the 2 methods (Fig. 2 of the online Data Supplement). The total error acceptable for an assay depends on the clinical setting in which the assay is being used. For example, in clinical research, studies are frequently designed to examine the association of plasma concentrations of analytes with the disease phenotype (in this case, obese patients with the metabolic syndrome vs lean controls) and the impact of a therapeutic intervention on the disease state (such as weight loss). In the present study, measurements of MCP-1 by ELISA or Luminex assay provided similar information about the relative increase in plasma concentrations associated with obesity and the metabolic syndrome compared with lean controls, and the relative changes in leptin and insulin concentrations before and after weight loss were similar by either ELISA or Luminex assay. However, the "total error[degrees] of an assay that is acceptable to assess relative change in a clinical research setting may be far greater than what is acceptable in a clinical practice setting, which is focused on diagnosis and treatment of disease.
Our study has important differences in design compared with previous reports that have validated multiplexed immunoassays, which measured IgG antibodies (16), lipopolysaccharide-stimulated human plasma samples (8), or supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material.
the liquid lying above a layer of precipitated insoluble material. from stimulated cells (17). A recent study reported the use of Luminex-based multiplex analysis to measure 9 cytokines in sera from 30 children infected by rotavirus rotavirus /ro·ta·vi·rus/ (ro´tah-vi?rus) any member of the genus Rotavirus. ro´taviral
Rotavirus /Ro·ta·vi·rus/ (ro´tah-vi?rus and 9 healthy control children, but the investigators did not validate their findings against conventional ELISAs (18). A different study used dried blood, spotted on paper, from children with cerebral palsy cerebral palsy (sərē`brəl pôl`zē), disability caused by brain damage before or during birth or in the first years, resulting in a loss of voluntary muscular control and coordination. and showed rather poor correlation between the Lineo and Upstate multiplex panels (19).
In conclusion, although this technology appears useful for analytes present in a wide range of concentrations in plasma, low detectability and poor correlations with conventional ELISA methods for some analytes when present in very low concentrations in plasma should be considered when using the multiplexed assay platform in clinical studies. Furthermore, improvement in the detection limits for some of the analytes that have very low plasma concentrations and are currently commercially available for Luminex-based multiplex assays is crucial for their use in analysis of unstimulated blood samples. Issues such as the effects of interfering heterophilic antibodies, efficiency of capture antibody-coupling techniques, development of high-sensitivity multiplex assays, and optimization of capture/detection antibody pairing need to be studied further to improve the efficacy of this technology for use in clinical studies. Manufacturers should calibrate To adjust or bring into balance. Scanners, CRTs and similar peripherals may require periodic adjustment. Unlike digital devices, the electronic components within these analog devices may change from their original specification. See color calibration and tweak. their secondary standards to the primary NIBSC/WHO standards, consistent with the recommendations for immunoassay standardization described in the report of Wadhwa and Thorpe (20). However, our data, as well as results from a growing number of research studies, demonstrate that multiplex technology may be useful in clinical research to measure a large number of analytes to examine the association with a clinical phenotype and the effects of therapeutic interventions, and that this technology may be particularly useful when sample volume is limited, such as in large epidemiologic studies and clinical trials.
This study was supported by Applied Technology Grant No. 004949-0093-2001 from the state of Texas and an American Diabetes Association Research Award. The lipid laboratory is supported by donations from George and Cynthia Mitchell, Nijad Fares, and Jeffrey Hines. The Upstate Beadlyte Human Cytokine 8-Plex Panel was kindly provided by Upstate USA, Inc. (Lake Placid Lake Placid, village (1990 pop. 2,485), Essex co., NE N.Y.; settled 1850, inc. 1900. In the Adirondack Mts. at an altitude of 1,800 ft (549 m), the village surrounds Mirror Lake. It is a famous resort and sports center. , NY), The IL-8 ELISA was a gift from Biosource International, Inc. (Camarillo, CA). We also appreciate the technical assistance and useful discussions of Dr. Laurie Stephen of Upstate USA, Inc., and the helpful comments of Henry Pownall, PhD, of the Section of Atherosclerosis, Baylor College of Medicine.
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Of or relating to a chemical bond characterized by one or more pairs of shared electrons. microsphere immunoassay and an enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA)
A diagnostic blood test used to screen patients for AIDS or other viruses. for measurement of human immunoglobulin G immunoglobulin G
n. Abbr. IgG
The most abundant class of antibodies found in blood serum and lymph and active against bacteria, fungi, viruses, and foreign particles. Immunoglobulin G antibodies trigger action of the complement system. antibodies to anthrax anthrax (ăn`thrăks), acute infectious disease of animals that can be secondarily transmitted to humans. It is caused by a bacterium (Bacillus anthracis toxins. Clin Diagn Lab Immunol 2004;11: 50-5.
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MINE Y. LIU LIU Linköpings Universitet (Sweden)
LIU Long Island University (New York)
LIU Line Interface Unit
LIU Lightguide Interconnection Unit (AT&T)
LIU Laugh It Up ,  ANTONIOS M. XYDAKIS,  RON C. HOOGEVEEN,  PETER H. JONES,  E. O'BRIAN SMITH,  KATHLEEN W. NELSON,  and CHRISTIE M. BALLANTYNE  *
 Section of Atherosclerosis, Department of Medicine,  Division of Endocrinology, Diabetes and Metabolism, and  Section of Nutrition, Department of Pediatrics, Baylor College of Medicine, Houston, TX.
 Methodist Wellness Services, The Methodist Hospital Methodist Hospital is the name of numerous medical institutions.
 Nonstandard non·stan·dard
1. Varying from or not adhering to the standard: nonstandard lengths of board.
2. abbreviations: ATP III, Adult Treatment Panel III; MCP-1, monocyte chemoattractant protein-1; TNF-[alpha], tumor necrosis factor-a; IL, interleukin interleukin
Any of a class of naturally occurring proteins important in regulation of lymphocyte function. Several known types are recognized as crucial constituents of the body's immune system (see immunity). ; and NIBSC, National Institute for Biological Standards and Control.
* Address correspondence to this author at: Baylor College of Medicine, 6565 Fannin, M.S. A-601, Houston, TX 77030. Fax 713-798-3057; e-mail firstname.lastname@example.org.
Received December 17, 2004; accepted April 6, 2005.
Previously published online at DOI (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.1373/clinchem.2004.047084
Table 1. Comparison of detection limits for the Luminex multiplexed assays and the individual ELISAs. Multiplex panels Analytes Luminex Linco Endocrine Leptin 0.13 [micro]g/L Insulin 0.31 mIU/L C-peptide 0.037 [micro]g/L Biosource Chemokine MCP-1 2.35 ng/L Eotaxin 4.37 ng/L Upstate Chemokine MCP-1 20.5 ng/L (0.023 kIU/L) Eotaxin 27.72 ng/L Linco Cytokine TNF-[alpha] 1.61 ng/L (0.249 kIU/L) IL-8 2.52 ng/L (0.026 kIU/L) Upstate Cytokine TNF-[alpha] 0.065 ng/L (0.0019 kIU/L) IL-8 0.70 ng/L (0.0003 kIU/L) IL-6 0.75 ng/L (0.1029 kIU/L) R&D Cytokine TNF-[alpha] 1.53 ng/L (0.052 kIU/L) IL-6 0.977 ng/L (0.1280 kIU/L) Multiplex panels ELISA ELISA manufacturer Linco Endocrine 1.39 g/L Linco 0.14 mIU/L Alpco 0.132 g/L Linco Biosource Chemokine 2.57 ng/L R&D 3.80 ng/L R&D Upstate Chemokine 2.57 ng/L (0.002 kIU/L) R&D 3.80 ng/L R&D Linco Cytokine 0.092 ng/L (0.0028 kIU/L) R&D 0.26 ng/L (0.0018 kIU/L) Biosource Upstate Cytokine 0.092 ng/L (0.0028 kIU/L) R&D 0.26 ng/L (0.0018 kIU/L) Biosource 0.068 ng/L (0.0084 kIU/L) R&D R&D Cytokine 0.092 ng/L (0.0028 kIU/L) R&D 0.068 ng/L (0.00843 kIU/L) R&D Table 2. Samples with undetectable concentrations of MCP-1, TNF-[alpha], IL-6, and IL-8 in the ELISAs and the Luminex multiplexed assays. IL-6 TNF-[alpha] Assay manufacturer % undetectable n % undetectable n ELISAs R&D 0 40 0 40 Biosource NA NA NA NA Luminex system Upstate 47.4 154 51.7 114 R&D 100 40 95.2 40 Linco 16.7 40 7.1 40 Biosource NA NA NA NA MCP-1 IL-8 Assay manufacturer % undetectable n % undetectable n ELISAs R&D 0 120 NA (a) NA Biosource 40.5 40 Luminex system Upstate 10.3 154 0 120 R&D NA NA 100 40 Linco NA NA 21.4 40 Biosource 2.4 40 61 40 (a) NA, not applicable. Table 3. Within-run assay imprecision. Analytes Within-run n CV, % Multiplexed panels Linco Endocrine Leptin 11 120 Insulin 6.6 120 C-Peptide 10 80 Biosource Chemokine MCP-1 12 40 Eotaxin 10 40 Upstate Chemokine MCP-1 8.1 40 Eotaxin 11 40 ELISAs Linco Leptin 5.9 120 Alpco Insulin 15 40 Linco C-Peptide 10 40 R&D MCP-1 5.4 120 R&D Eotaxin 5.0 120 Table 4. Deming regression results for comparison of the multiplexed assays performed on the Luminex systems (y) and individual ELISAs (x). Analyte (a) Deming R [S.sub.y|x] Leptin ([micro]g/L) 9.90 0.812 Insulin (mIU/L) 5.62 0.895 C-Peptide ([micro]g/L) 2.32 0.582 Eotaxin (ng/L) 20.44 0.711 MCP-1 (c) (ng/L) With outliers 110.14 0.263 Without outliers 49.99 0.899 Slope Analyte (a) Coefficient SE 95% CI (b) Leptin ([micro]g/L) 1.28 1.28 1.11-1.44 Insulin (mIU/L) 2.38 0.19 1.99-2.77 C-Peptide ([micro]g/L) 0.68 0.15 0.37-0.99 Eotaxin (ng/L) 7.71 1.29 5.10-10.32 MCP-1 (c) (ng/L) With outliers 40.44 25.09 -10.49 to 91.36 Without outliers 4.97 0.42 4.11-5.82 Intercept Analyte (a) Mean SE 95% CI Leptin ([micro]g/L) 2.26 3.19 -4.05 to 8.57 Insulin (mIU/L) 3.03 3.47 -10.06 to 4.00 C-Peptide ([micro]g/L) -1.83 1.20 -4.25 to 0.59 Eotaxin (ng/L) -49.85 80.64 -213.55 to 113.86 MCP-1 (c) (ng/L) With outliers -7829.64 6421.28 -20 865.54 to 5206.25 Without outliers 79.60 109.36 -142.90 to 302.09 (a) The manufacturers of the assays for the Luminex multiplex systems (y) and the individual ELISAs (x) were as follows: for leptin, Linco for both; for insulin, Linco and Alpco, respectively; for C-peptide, Linco for both; for eotaxin, Biosource and R&D, respectively; and for MCP-1, Biosource and R&D, respectively. (b) CI, confidence interval. (c) Outliers are defined as values outside 3 SD of the mean for 2 replicates of the zero calibrators.