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Optimization, Comparison, and Application
of Colorimetric vs. Chemiluminescence
Based Indirect Sandwich ELISA for
Measurement of Human IL-23
Sridhar Samineni, Sitaram Parvataneni, and Caleb Kelly
Food Allergy & Immunology Laboratory, Department of Food Science &
Human Nutrition, Nutritional Immunology Program, Michigan State
University, East Lansing, Michigan, USA
Venu Gangur
National Food Safety & Toxicology Center, Food Allergy & Immunology
Laboratory, Department of Food Science & Human Nutrition, Nutritional
Immunology Program, Michigan State University, East Lansing,
Michigan, USA
Wilfried Karmaus and Kevin Brooks
Department of Epidemiology, Michigan State University, East Lansing,
Michigan, USA
Abstract: Currently, there is neither a published ELISA method nor it is clear whether
chemiluminescence substrates would provide better sensitivity vs. colorimetric sub-
strates for measuring human IL-23–a recently described Type-1 immunity associated
cytokine. Initially, we optimized a colorimetric ELISA using p-nitro-phenyl phosphate
substrate. Subsequently, we compared it with chemiluminescence substrates that
provided 5-fold enhanced sensitivity (mean sensitivity; 26.3 pg/mL vs. colorimetric
assay, 131 pg/mL; p , 0.01). Both methods were reliable, with ,10% inter- and
intra-assay variations. We then found that the chemiluminescence method was
useful in situations where human IL-23 was not readily measurable by a colorimetric
method.
Keywords: Cytokine, Human IL-23, Colorimetry, Chemiluminescence, ELISA
Address correspondence to Dr. Venu Gangur, 302-B, G. M. Trout Building,
Michigan State University, East Lansing, MI 48824, USA. E-mail: gangur@msu.edu
Journal of Immunoassay & Immunochemistry , 27: 183–193, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 1532-1819 print/1532-4230 online
DOI: 10.1080/15321810600573051
183
INTRODUCTION
Cytokines play a central role in inflammation, immune response, and
health.
[1,2]
Accurate quantification of cytokines in human samples is a vital
step in advancing basic and applied research in human health and disease.
Human cytokines with key function in Type-1 and Type-2 immune
responses have been classified broadly into at least three groups: Type-1
associated, Type-2 associated, and Type-3 or regulatory or suppressive
cytokines.
[1 – 3]
Specific examples of cytokines belonging to these groups
include: IFN-
g
, IL-12, TNF-
a
(Type-1 associated); IL-4, IL-5, IL-13, IL-9
(Type-2 associated); and IL-10, TGF-
b
(Type-3 or regulatory or suppressive
cytokines).
[1 – 3]
Whereas Type-1 cytokines are important in cell mediated
immunity (e.g., delayed hypersensitivity reactions), Type-2 cytokines are
implicated in allergic disorders. In contrast, Type-3 cytokines have been
proposed to be critical in control of excessive immune and inflammatory
responses that are thought to underlie allergy/asthma and autoimmune
disorders.
[3]
IL-23 is a heterodimeric cytokine containing IL-12 p40 and IL-23 specific
p19 subunits.
[4,5]
This cytokine was recently identified as a member of the
IL-12 related family of Type-1 immunity associated cytokines with IL-18
and IL-27 as other members.
[5,6]
Using IL-23 p19 knockout mice, it was
shown that IL-23 is critical for cell mediated immunity as well as humoral
immune responses.
[7]
It appears to participate in protection against intracellu-
lar infections and the pathogenesis of some autoimmune disorders.
[6,8,9]
One
property that distinguishes IL-23 from IL-12 is that it targets memory but
not naı
¨
ve T helper cells.
[8]
Thus, measurement of human IL-23 in clinical
samples will be very valuable for both basic and applied research in health
and disease.
Here, we describe optimization and comparison of an indirect sandwich
ELISA using colorimetric vs. chemiluminescence substrates for quantification
of human IL-23. We also demonstrate that the optimized chemiluminescence
method, as opposed to p-nitro-phenyl phosphate (PNPP) based colorimetric
assay, provides 5-fold enhanced sensitivity, but comparable reproducibility
for human IL-23 quantification. Furthermore, we apply these two assays to
measure IL-23 in human serum samples to demonstrate their utility.
EXPERIMENTAL
Materials
The following materials were purchased from sources as indicated in parenth-
eses. Capture antibody: Anti-human IL-23 p19 (R&D Systems, Minneapolis,
MN), Biotin labeled developing antibody: anti-human IL-12 p40/p70
(Biolegend, San Diego, CA), recombinant human IL-23 (R&D Systems,
Minneapolis, MN); p-nitro-phenyl phosphate (PNPP) (Sigma, St Louis, MO,
S. Samineni et al.184
USA); Streptavidin alkaline phosphatase (Jackson ImmunoResearch, West
Grove, PA); CSPD Sapphire and Emerald (Applied Bio-Systems, Foster
City, CA); ELISA plates: for both chemiluminescence assay as well as colori-
metric assay (Costar, Corning Inc., Corning, NY).
Colorimetric Assay for Human IL-23
An indirect sandwich enzyme linked immunosorbent assay (ELISA) was
optimized using similar principles as we have described previously.
[10]
All
reagents were used at a final volume of 50 mL/well, except for blocking
buffer that was used at 75 mL/well. Washing was done manually with
200 mL/well. Briefly, ELISA plates (96 well EIA/RIA plate, 96 well easy
wash
TM
, high binding, Corning Inc., NY) were coated with Anti-human
IL-23 p19 antibody diluted in carbonate buffer (0.05 M, pH 9.6) and
incubated at 48C, overnight. Unbound antibody was discarded and the
plates were blocked (0.17% BSA/PBS) at 378C. After washing (0.05%
Tween 20 in PBS), recombinant human IL-23 was added at various two-
fold dilutions from 4,000 pg/mL to 7.8 pg/mL in dilution buffer (0.085%
BSA, 0.05% Tween 20 in PBS). Following incubation, plates were washed
and a biotin labeled anti-human IL-23 p19 antibody added at the indicated
concentration. Plates were then washed and streptavidin alkaline phosphatase
conjugate was added at 1/4,000 (in dilution buffer). Subsequently, plates
were washed again and p-nitro phenyl phosphate (PNPP) substrate was
added (1 tablet per 5 mL substrate buffer, according to manufacturer’s
instruction; Sigma). Reactions were allowed to develop at room temperature
in the dark and absorbance was measured in a microplate reader with dual
mode of wavelength at 405 nm (peak) minus 690 nm (background) using a
KC4 software program (Synergy HT, Multifunction Reader, BioTek).
According to the manufacturer’s instructions, dual mode provides relatively
better measurements, since it adjusts the reading for background interference
(personal communication). Recombinant human IL-23 was used for optimiz-
ing these assays.
Chemiluminescence Assay for Human IL-23
The general method was similar to that described above, with the exception of
using opaque ELISA plates for the assay. After the addition of SAAP, 50 mL
of chemiluminescence substrate CSPD (Sapphire II or Emerald II) was used at
0.4 mM. The plates were read at the indicated times using a Synergy HT,
Luminescence reader (BioTek). The ELISA plates were covered with
aluminum foil at all times during the assay and in between readings. Plates
were manually washed four times after each step (with 200 mL/well;
washing buffer: 0.05% Tween 20 in PBS). Recombinant human IL-23 was
used for optimizing these assays.
Measurement of Human IL-23 185
Determination of Assay Sensitivity
The assay sensitivity was defined as the lowest amount of the analyte measur-
able using the assay.
Blood Collection
Blood was collected in glass tubes from subjects participating in the ongoing
study (PEACH study). Serum was harvested, stored at 2208C, and shipped on
dry ice to the laboratory where aliquots were made and stored at 2708C until
they were used in the assay.
Statistical Analysis
Student’s unpaired t-test and Mann-Whitney U-test were used to evaluate sig-
nificance, using the Analyse-It
TM
software program (Analyse-It Software Ltd,
Leeds, UK). The statistical significance level was set at 0.05.
RESULTS AND DISCUSSION
In order to determine the optimal capture (coating) antibody concentration for
human IL-23 detection, an indirect sandwich ELISA was set up using two
different amounts of capture antibody and developed with two different con-
centrations of biotin labeled antibody. As is evident from the results, a com-
bination of coating antibody concentration at 1 mg/mL yielded relatively
better profile. Since both biotin antibody concentrations provided comparable
results, keeping the cost of the reagents in mind, we chose the lower antibody
concentration (250 ng/mL) for further use. Incubation time of 2 hours after
addition of substrate was found optimal for reading the plates. Representative
data are shown in Figures 1A– C.
We used the optimized capture (coating) and biotin-labeled antibody
with buffer conditions of the colorimetric assay for the initial experiment
to optimize the chemiluminescence assay. As is evident, the conditions
provided an assay with a sensitivity of 31.25 pg/mL with 30 minutes of
developing time after addition of the substrate for reading the plate
(Figure 2A). We then tested the impact of different blocking buffers (0.17%
BSA vs. 0.5% BSA vs. 5% Gelatin) on chemiluminescence assay. As is
evident, blocking with 0.17% (Figure 2B) provided better results. Finally,
we tested whether different chemiluminescence substrates affect the assay
sensitivity. We found that the use of the CSPD substrate with Sapphire
yielded better sensitivity compared to the CSPD substrate with Emerald
(Figure 2C).
S. Samineni et al.186
We performed a series of experiments to compare the two types of sub-
strates in terms of their impact on assay sensitivity, time of incubation after
adding the substrate, and reproducibility of the assay (based on inter- and
intra-assay variations). As is evident from the results, whereas colorimetric
assay provided an average sensitivity of 131.25 pg/mL (range: 62.5–
250 pg/mL), chemiluminescence assay yielded 5-fold enhanced average
sensitivity of 26.3 pg/mL (Table 1). We found that the chemiluminescence
assay reads faster (30 minutes) relative to the colorimetric assay (2 hours)
to obtain optimal sensitivity. Results from the analysis of inter- and intra-
assay variation between the two assays is shown in Figures 3A, B, C, and
D. As is evident, both assays yielded comparable data and the coefficients
of variation were , 10% in each case.
We then used these assays to measure human IL-23 in subjects. Serum
samples collected from a group of adult humans were examined for the
Figure 1. Optimization of an indirect sandwich ELISA based on colorimetric method
for human IL-23. A) shows the assay that was performed using the capture antibody
(anti-IL23 p19 antibody) at 1 mg/mL and biotin labeled anti-p40/p70 antibody at
250 and 500 ng/mL. B) shows the assay that was performed using the capture antibody
at 0.5 mg/mL and biotin labeled anti-p40/p70 antibody at 250 and 500 ng/mL. Both
figures show the data read at 2 hour time point after adding the substrate that was
found optimal as evident in Figure 1C. C) shows the assay that was performed using
capture antibody at 1 mg/mL and biotin labeled anti-p40/p70 antibody at 250 ng/mL,
and read at different time periods (30, 60, 120 and 180 minutes) after addition of the
substrate. In these assays, recombinant human IL-23 was used as the standard. Data
is presented as mean + SE. Horizontal line indicates background þ3 SD values for
each condition tested in duplicates.
Measurement of Human IL-23 187
presence of circulating IL-23 levels. As is evident, IL-23 was measurable in 6
out of 10 subjects by colorimetric assay (Figure 4A). We then examined
whether the chemiluminescence assay with enhanced sensitivity would be
useful to measure IL-23 in those subjects where the colorimetric assay was
unsuccessful. As is evident, in 3 out of 4 subjects, IL-23 was measurable
using the chemiluminescence assay (Figure 4B).
The use of the chemiluminescence substrate provides an exciting
opportunity to improve current cytokine detection technology, since such
substrates have been reported to yield enhanced assay sensitivities for
certain cytokines.
[11]
However, one study reported that colorimetric assay
provides better sensitivity than chemiluminescence assay for other
Figure 2. Optimization of an indirect sandwich ELISA for human IL-23 using
chemiluminescence substrates. A) A chemiluminescence assay was optimized using
capture anti-IL23 p19 antibody (1 mg/mL) and biotin labeled anti-p40/p70 antibody
(250 ng/mL). Recombinant human IL-23 was used as the standard in these
assays and plates were read at 30 min after addition of the substrate. Data is shown
as mean + SE (n ¼ 4). B) This figure shows the chemiluminescence assay performed
using three different blocking buffers (0. 17% BSA; 0.5% BSA, 5% gelatin). Data is
shown as mean + SE. Recombinant human IL-23 was used the standard in these
assays and plates were read at 30 min after addition of the substrate. C) This figure
shows the chemiluminescence assay profile using two different substrates (Sapphire
II vs. Emerald). Data is shown as mean + SE. Recombinant human IL-23 was used
the standard in these assays and plates were read at 30 min after addition of the
substrate.
S. Samineni et al.188
cytokines.
[12]
For human IL-23 quantification, we are not aware of any pre-
viously published study comparing colorimetric vs. chemiluminescence
methods. In an effort to fill this need, we established the present method for
IL-23, a recently identified Type-1 immunity associated cytokine.
Previously, chemiluminescence based ELISA methods have been
described for other type-1 associated cytokines: TNF-
a
, IFN-
g
(human
study); IL-12 (murine study).
[11 – 13]
In one of the former studies (TNF-
a
),
the chemiluminescence assay was found to have similar or reduced sensi-
tivity compared to the colorimetric assay.
[12]
In the latter study, an
opposite conclusion was reached.
[11]
We also found that chemiluminescence
provides an enhanced sensitivity for IL-23, as well as for some type-2
cytokines, IL-5 and IL-13 (data not shown). Thus, the improved sensitivity
offered by the chemiluminescence method may not be generalizable to any
and every cytokine measured. Consequently, it is clear that one might need
to optimize methodology for each target cytokine protein in question, to
evaluate suitability of chemiluminescence vs. colorimetric substrates as
readouts.
There are previous reports examining human IL-23 p19 gene expression
by measuring either mRNA levels in macrophages or IL-23 p19 protein
detection by Western blotting.
[14]
Notably, interpretation of mRNA data
on cytokines, in general, becomes difficult due to the potential role of
post-transcriptional regulation of protein synthesis. Consequently, it is
difficult to accurately estimate the amount of protein secreted by cells
based on mRNA data.
[15]
Furthermore, the sample volume required, lower
assay sensitivity, and difficulty in accurate quantification are potential
problems for analysis of cytokine protein levels by Western blotting
analysis. We are not aware of previous published data on levels of human
IL-23 proteins present in serum/plasma samples or cultured cell super-
natants in health or disease. However, we have been able to measure
Table 1. Comparison of colorimetric assay vs. chemiluminescence assays for
measurement of human IL-23
Type of
assay
Assay properties
x/n
a
Average
sensitivity
(pg/mL)
Range of
sensitivity
(pg/mL)
ODT
b
(min)
Student’s
t-test
(P value)
Mann-
Whitney
U-test
(P value)
Colorimetric 12/5 131.25 62.25–250 120
Chemilumi-
nescence
12/5 26.3 7.8–62.5 30 ,0.01 ,0.01
a
Total number of replicates/number of experiments.
b
ODT- Optimal development time.
Measurement of Human IL-23 189
IL-23 in human serum samples using the combination of presently described
methods.
While chemiluminescence based substrates provide enhanced sensi-
tivity, there are a few limitations in terms of the cost involved for ELISA
plates and substrates. For development of 100 ELISA plates it costs
US$ 274 for PNPP based substrate vs. $1573 for Sapphire II based che-
miluminescence method. These estimates include plate plus substrate costs
(excluding cost of antibodies, recombinant protein and other reagents).
Thus, chemiluminescence assay are 6 times as expensive, relative to the
colorimetric assay. Another limitation is the cost of chemiluminescence
readers, which tend to be expensive compared to the traditional colorimetric
ELISA readers.
Figure 3. Inter- and intra-assay variations of colorimetric vs. chemiluminescence
assays for human IL-23. A) This Figure illustrates two experiments performed by
two different individuals using the optimized colorimetric method (inter-assay
variation). Data is shown as mean + SE (n ¼ 4). B) This Figure illustrates four
experiments performed by one individual using the optimized colorimetric method
(intra-assay variation). C) This Figure illustrates two experiments performed by two
different subjects using the optimized chemiluminescence method (inter-assay
variation). Data is shown as mean + SE (n ¼ 4). (D). This Figure illustrates four
experiments performed by one subject using the optimized chemiluminescence method
(intra-assay variation). Recombinant human IL-23 was used as the standard in these
assays.
S. Samineni et al.190
Conventionally, human cytokines have been measured using ELISA
based colorimetric assays. In many studies, the source of the human
specimens are invasive in nature (e.g., serum, peripheral blood cell stimulated
culture supernatants). A major attraction of the more recently developed tech-
nology of chemiluminescence is the potential for higher sensitivity of
detection. The enhanced sensitivity suggests potential application of these
assays for detection of disease relevant cytokines, especially in non-
invasive human samples where: (i) sample volumes might be limited (e.g.,
tears, sexual fluids, etc.), so that samples can be diluted and used for quanti-
fication; and (ii) where samples are naturally diluted (e.g., saliva, urine) and
cytokines are expected to be present in very low quantities.
[16 – 20]
Figure 4. Application of colorimetric and chemiluminescence assays for
measurement of human IL-23 in serum samples. A) This figure illustrates the utility
of colorimetric method for measurement of IL-23 in human serum samples. Each
bar represents one subject. Data is shown as mean + SE of duplicate analysis. B) In
this experiment, the serum samples from the experiment performed in Figure A
where IL-23 was not detectable by colorimetric method (subject ID #7–10) were
analyzed by the chemiluminescence method. This data illustrates the utility of the
chemiluminescence assay for measuring IL-23 in serum where colorimetric assay
was not useful. Each bar represents one subject. Data is shown as mean + SE of
duplicate analysis.
Measurement of Human IL-23 191
CONCLUSIONS
In summary, (i) we have optimized and compared indirect sandwich ELISA
methods using colorimetric vs. chemiluminescence substrates for quantifi-
cation of human IL-23; (ii) we have demonstrated that the optimized chemi-
luminescence method, as opposed to a PNPP based colorimetric assay,
provides enhanced sensitivity, but comparable reproducibility; and (iii) we
demonstrated that the chemiluminescence method is useful to measure
serum IL-23 in situations where it is not readily measurable by the colori-
metric method.
ABBREVIATIONS
OD, optical density; ELISA, enzyme linked immunosorbent assay; BSA, bovine
serum albumin; PBS, phosphate buffered saline; SD, standard deviation, SE,
standard error; PNPP, para-nitro-phenyl phosphate; CSPD, chloro-5-substituted
adamantyl-1,2-dioxetane phosphate; IL, Interleukin; TNF-
a
, tumor necrosis
factor-alpha; TGF-
b
, transforming growth factor-Beta; IFN-
g
,Interferon-
gamma; EIA, Enzyme immunoassay; RIA, radioimmunoassay.
ACKNOWLEDGMENTS
This work was supported by funding from Michigan State University (MSU),
East Lansing, MI, USA; the United States Environmental Protection Agency
(STAR Grant # R830825-01-0); and The Gerber Foundation. We would like to
thank Dr. James Pestka (MSU) for his encouragement and support including
access to instruments.
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Received June 10, 2005
Accepted July 9, 2005
Manuscript 3181
Measurement of Human IL-23 193
