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Technical Briefs |
1
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece
a author for correspondence: fax 301 7231 608, e-mail lianidou{at}nestor.dc.uoa.gr
Interleukin 6 (IL-6) is a cytokine that is produced by a wide variety of cells and has pleiotropic biological functions, such as the induction of acute phase proteins in hepatocytes, the terminal differentiation and growth promotion of B cells, the differentiation and activation of T cells and macrophages, and the regulation of expression of other cytokines (1). IL-6 has been implicated in the pathology of several diseases, and its role in inflammation, viral infection, autoimmunity, and cancer has been reviewed recently (1)(2).
Serum IL-6 increases markedly in many pathological conditions, and its accurate and precise determination in biological fluids is of great importance for the early diagnosis of many diseases (3)(4)(5)(6)(7). Measurement of IL-6 in biological fluids is mainly based on bioassays and immunoassays (8)(9)(10)(11)(12)(13). The presence of natural compounds known to bind IL-6 in biological fluids (14)(15)(16)(17)(18) and the effect of preanalytical factors (19), as well as the use of an internationally accepted IL-6 standard preparation (20)(21), is of great importance for the performance of IL-6 immunoassays.
Here we describe a highly sensitive enzyme immunoassay for the determination of IL-6 in serum and plasma, based on the enzyme-amplified lanthanide luminescence (EALL) detection approach (22)(23)(24)(25). An ultra-low detection limit is obtained by the powerful detection system that is based on the combination of enzymatic amplification introduced by the enzyme alkaline phosphatase (ALP) and the formation of a highly fluorescent terbium complex (26)(27), which is monitored by time-resolved or conventional fluorometry.
A monoclonal antibody (anti-IL-6, CLB IL-6/16) and a biotinylated goat polyclonal antibody against human IL-6 were kindly donated by Prof. L. Aarden (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). The IL-6 stock solution (100 µg/L) was prepared by dissolving the lyophilized human recombinant IL-6 international standard, 88/514 (National Institute of Biological Standards and Controls), in 10 g/L bovine serum albumin, in a 50 mmol/L Tris buffer, pH 7.40, containing 0.5 g/L NaN3 and 9 g/L NaCl, and was kept in aliquots at -20 °C. IL-6 calibrators at a concentration range of 1–2000 ng/L were prepared by appropriate dilutions of the IL-6 stock solution in assay buffer and in IL-6-free plasma. Coating, wash, and blocking solutions and the substrate buffer of ALP were prepared as described previously (25). Assay buffer was 60 g/L bovine serum albumin in 50 mmol/L Tris buffer, pH 7.40, containing 0.5 g of NaN3, 9 g of NaCl, 50 mL of mouse serum, and 50 mL of goat serum per liter. Developing solution was 0.100 mol/L 3-[cyclohexylamino]-1-propanesulfonic acid, pH 12.60, containing 3 mmol/L Tb3 and 3 mmol/L EDTA. The phosphate ester of diflunisal (DIFP) stock solution (10 mmol/L) was prepared in 0.1 mol/L NaOH and kept at 4 °C, whereas the DIFP working solution (500 µmol/L) was prepared just before use by a 20-fold dilution of the stock solution in ALP substrate buffer (23).
For serum, blood samples were collected in clean glass tubes, left at room temperature for 1 h, and centrifuged at 4 °C. For plasma, blood samples were collected in EDTA-containing Vacutainer Tubes (Becton Dickinson) and centrifuged at 4 °C. Serum and plasma samples were stored at -20 °C until assayed. All samples were kindly provided by Dr. E. P. Diamandis (Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada).
A typical "sandwich type" immunoassay was used. Assay conditions involving all steps, such as antibody concentrations and incubation times, were optimized in respect to the best signal-to-noise ratios, sensitivity, and rapidity. The final optimized assay protocol was the following: (a) immobilization of the anti-IL-6 monoclonal (stock solution, 1 g/L), diluted 400-fold (100 µL/well) in coating solution, overnight, wash; (b) blocking with blocking solution (200 µL/well) for 2 h, wash; (c) addition of the analyte (100 µL/well) diluted with assay buffer (50 µL/well), wash twice (incubation time of 2 h was found to be optimum when time-resolved fluorescence was used as a detection technique, whereas 3.5 h were required to achieve the same sensitivity with conventional fluorescence); (d) addition of the biotinylated anti-IL-6 goat polyclonal antiserum (stock solution, 1 g/L) diluted 3000-fold in assay buffer (100 µL/well), incubation for 30 min, wash four times; (e) addition of SA-ALP conjugate (stock solution, 1 g/L), diluted 20 000-fold in assay buffer (100 µL/well), incubation for 15 min, wash four times; and (f) addition of the DIFP working solution (100 µL/well), incubation for 30 min. The enzymatic reaction was stopped by adding developing solution (100 µL/well). All incubation steps were performed at room temperature.
Time-resolved fluorescence measurements were performed in the
Cyber-Fluor 615 Time Resolved Fluorometer/Immunoanalyzer (
exc =
337.1 nm, em = 615 nm). Conventional fluorescence measurements
were performed in the Perkin-Elmer M 512-A fluorescence
spectrophotometer (exc = 284 nm, em = 546 nm). In this
case, aliquots of 180 µL of the DIF-Tb3-EDTA
ternary complex, developed finally in each well, and 400 µL of
developing solution were brought into a 1.00-mL cuvette and mixed
thoroughly before measurement.
The calibration curve for IL-6 has a reportable assay range up to 2000
ng/L. However, when IL-6 calibrators were diluted in IL-6-free plasma,
the calibration curve gave a 30% lower slope than that obtained by
dilutions in assay buffer. This could be explained by the presence of
various IL-6 binding proteins (sIL-6R, 
2-macroglobulin, C-reactive
protein) in serum and plasma (14)(15)(16)(17)(18), which can compete
with the assay antibodies for binding to IL-6.
The accuracy of the assay was tested by performing analytical recovery
and dilution experiments. To assess analytical recovery, three plasma
and three serum samples supplemented with recombinant human IL-6
(rhIL-6) at three different concentrations were assayed before and
after the addition of rhIL-6. When IL-6 calibrators were diluted in a
pool of IL-6-free plasma, satisfactory analytical recoveries were
obtained (Table 1
). Serum dilution experiments showed that both recombinant and
native IL-6 are equally recognized by the assay. When we serially
diluted three pathological serum samples with high concentrations of
IL-6 and one serum sample with rhIL-6, added to a final concentration
of 1000 ng/L with a pool of negative control plasmas, the dilution
curves were parallel in all cases.
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To determine within-run precision of the assay, three serum samples
were assayed in six parallel determinations. To determine between-run
precision of the assay, aliquots of three serum samples were stored
frozen (-20 °C) and analyzed over a period of 2 weeks in three
parallel determinations, each in six separate assays. Within-run CVs
were 3% to 9% (n = 6) at 10–100 ng/L, and between-run CVs were
5% to 12% (n = 6 over 2 weeks) at 6–85 ng/L. The precision
profile of the method in assay buffer and serum, as measured by time
resolved fluorescence, is shown in Fig. 1
%The detection limit, defined as the minimal concentration of IL-6 that
produces a fluorescent signal equal to the nonspecific background plus
2 SD, was as low as 0.15 ng/L when rhIL-6 calibrators were diluted in
assay buffer (n = 12) and 0.5 ng/L when diluted in human plasma
(n = 12).
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The specificity of the assay is based on the use of well-characterized and selected antibodies (10). However, when 22 apparently positive sera (IL-6, ~20–10 000 ng/L) were assayed after the addition of mouse and goat serum (5%) to the assay buffer, 12 of them were found to contain markedly lower amounts of IL-6 (1–3 orders of magnitude). Therefore, the addition of mouse and goat serum in the assay buffer was shown to abolish false-positive interferences by rheumatoid factors and heterophilic antibodies, whereas the IL-6 calibration curve or the IL-6-positive sera were not affected. These results are in agreement with those reported previously (10).
The mean serum concentration of IL-6 in 32 apparently healthy
individuals was 3.0 ng/L (SD, 2.7 ng/L), whereas the mean serum
concentration of IL-6 in 27 patients with various inflammatory diseases
was 72 ng/L (SD, 43 ng/L). We compared the results of our assay with
those obtained with a commercially available immunoassay for IL-6,
Medgenix Diagnostics ELISA. For 21 serum samples from patients with
inflammatory diseases and healthy blood donors analyzed by our EALL
immunoassay (x) and by Medgenix Diagnostics ELISA
(y), the regression equation was: y =
1.48x 3.4 ng/L, r = 0.948,
Sy
x = 23.1. These results indicate that EALL
gives lower values for IL-6 than this particular ELISA kit. There is no
simple explanation for this, because it has been clearly stated in a
recent study (20) that even when the same international
standards provided by the National Institute of Biological Standards
and Controls were used, a wide variation in IL-6 values was obtained
for the same samples when analyzed by several commercial ELISA kits.
In conclusion, the EALL immunoassay for IL-6 in serum demonstrates exceptional sensitivity and a much lower detection limit (~0.5 ng/L in serum) in a very short incubation time (~3 h) in comparison with other immunoassays for IL-6 (8)(9)(10)(11)(12)(13).
Acknowledgments
We thank L. Aarden for the generous supply of the IL-6 antibodies and E. P. Diamandis for the use of the Cyber-Fluor 615 Time Resolved Fluorometer/Immunoanalyzer and for providing the serum and plasma samples used in this study. This work was supported by a research grant from the Secretariat of the Research Committee of the University of Athens.
References
2 macroglobulin as a carrier protein for interleukin 6. J Immunol 1989;142:148-152.
[Abstract]
-fetoprotein. Anal Chim Acta 1996;335:177-184.
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), assay buffer and (
), serum.