- Email: [email protected]
Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis synovial fluid
Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis synovial fluid Ulrike Hampel, Stefan Sesselmann, Pavel Iserovich, Saadettin Sel, Friedrich Paulsen, Robert Sack PII: DOI: Reference:
S0022-1759(13)00232-9 doi: 10.1016/j.jim.2013.08.007 JIM 11711
To appear in:
Journal of Immunological Methods
Received date: Revised date: Accepted date:
28 June 2013 3 August 2013 12 August 2013
Please cite this article as: Hampel, Ulrike, Sesselmann, Stefan, Iserovich, Pavel, Sel, Saadettin, Paulsen, Friedrich, Sack, Robert, Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis synovial fluid, Journal of Immunological Methods (2013), doi: 10.1016/j.jim.2013.08.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis
T
synovial fluid
IP
Ulrike Hampel1, Stefan Sesselmann2, Pavel Iserovich3, Saadettin Sel4, Friedrich
1
SC R
Paulsen1, Robert Sack3
Department of Anatomy II, Friedrich Alexander University Erlangen Nürnberg,
Division of Molecular Immunology, Friedrich Alexander University Erlangen
MA
2
NU
Erlangen, Germany
Nürnberg, Erlangen, Germany 3
Department of Biological Sciences, SUNY College of Optometry New York, New
TE
4
D
York, USA
Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany,
CE P
Erlangen, Germany
AC
Corresponding author: Ulrike Hampel, Department of Anatomy II, Friedrich Alexander University Erlangen Nürnberg, Erlangen, Germany
Keywords: ELISA, interference, heterophilic antibody, chemokine, cytokine, arthritis
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Abstract
T
To develop a method of the assay of chemokine and cytokine signaling in synovial
IP
fluid from patients suffering from osteoarthritis (OA) or rheumatoid arthritis (RA) and
SC R
evaluate the effect of heterophilic antibodies on the reliability of the data. 21 synovial fluid samples from OA and 16 synovial fluid samples from RA patients were analyzed using a unique 2 step dot sandwich ELISA based micro-well protein
NU
array designed to detect heterophilic antibody signaling in the presence or absence
MA
of an effective heterophilic blocking reagent with assays carried out for Eotaxin, hGROa, Interleukin (IL)-8, IP10, MCP-1, MCP-2, MIG, RANTES, TARC and IL-6. Array analysis reveals that the selective presence of heterophilic antibodies interferes
TE
D
with the accurate assay of synovial fluid samples from a minority of RA patients but not OA synovia. Using a commercial blocking diluent OA and RA synovial fluids
CE P
reveal significant differences in chemokine content (IL-6, Eotaxin, hGROa, MCP-2, MIG, TARC, IL-8, RANTES).
AC
Using a two-step assay protocol it is possible to readily detect inappropriate antibody signaling due to heterophilic antibodies and devise a protocol designed to eliminate this problem thereby more accurately quantify cytokines and chemokines specific to both RA and OA fluids.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
1 Introduction
T
A gradual loss of cartilage results in the destruction of the joints in osteoarthritis (OA).
IP
In contrast, rheumatoid arthritis (RA) is an autoimmune disease that leads to
SC R
deformity of the joints through chronic inflammation (Andreas et al., 2008). Inflammation is accompanied by infiltration by neutrophils and lymphocytes leading to elevated proinflammatory cytokine, chemokine and growth factor levels (Gorman and
NU
Cope, 2008). Chemokines play a key role in the perpetuation of the inflammation by
MA
attracting proinflammatory cells to the inflamed joint. Chemokines are classified into CXC, CC, C and CX3C supergene families (Koch, 2005). In most cases the cause for the rheumatic disorder is unknown. RA therapy is intended to inhibit the inflammation
TE
D
with the help of non-steroidal anti-inflammatory drugs (NSAID), corticosteroids, disease-modifying anti-rheumatic drugs (DMARDs) and drugs directed against TNFα
CE P
(Hoff et al., 2009). Furthermore, recent advances focused on chemokine and chemokine receptor blocking to prevent or diminish chemokine recruitment of
AC
inflammatory cells into RA tissue (Szekanecz et al., 2011). The effectiveness of these drugs could be monitored by the measurement of inflammatory markers, cytokines and chemokines in serum and synovial fluids. Synovial fluid is produced by synovial membrane and secreted into the joint cavity and can be used for diagnostic purposes. Several authors point out that the measurement of serum and synovial fluids derived from RA patients needs critical investigation, because heterophilic antibodies are a well-known source of interference in immunoassay of proteins (Bartels et al., 2011), which can cause false-positive or false-negative results (Kricka, 1999). Aly et al. defined heterophilic antibodies as antibodies directed against animal antigens that arise in humans. Most commonly the cause is unknown, but heterophilic antibodies might be the result of intravenous administration of animal
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
proteins (Aly et al., 2004). This is particularly common in autoimmune diseases such as RA. In order to obtain accurate data it is therefore crucial to eliminate interfering
T
factors from ELISA assays.
IP
The aim of this study was to establish a chemokine assay that allows one to detect
SC R
and minimize or eliminate this interference and more accurately determine differences in chemokine levels in synovial fluid from patients suffering from OA or RA. Our investigations focused on the CXC chemokines interleukin (IL)-8, human
NU
growth related oncogene-alpha (hGROα), interferon gamma induced protein 10 (IP-
MA
10) and Monokine induced by gamma interferon (MIG) and the CC chemokines eotaxin, regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), MCP-2 and thymus and activation-
TE
D
regulated chemokine (TARC) as well as on the cytokine IL-6. Those chemokines have proinflammatory and angiogenic in the OA and RA context (Szekanecz et al.,
AC
CE P
2003).
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
2 Materials and methods
T
2.1 Subjects
IP
All subjects provided informed consent for synovial fluid collection in accordance with
SC R
the Declaration of Helsinki. Synovial samples were obtained from 37 subjects (21 with OA and 16 with RA). The demographic data of the subjects, including age, sex, leukocyte and CPR levels on the day before surgery are summarized in table A. 62%
NU
of the RA patients received corticoids, 62% DMARD, 31% anti-TNFα therapy and
MA
19% NSAID. The average number of medications for RA patients was 1.7 (range 1-
TE
2.2 Synovial fluid collection
D
3); OA patients did not receive any of these drugs.
All synovial fluid samples (10 to 20 ml) were taken during total knee alloarthroplasty
CE P
after preparation of the subcutaneous tissue and before opening of the articular capsule using a 20 ml syringe with 18 gauge needle, with care taken to prevent blood
AC
and intra-articular tissue contamination. All samples were immediately frozen and stored at -800C until the analyses were performed.
2.3 Micro-well protein arrays for quantitative protein assays Prior to assaying the samples were centrifuged at 5000 rpm for 20 min at 4°C and the supernatant has been used for further procedures. The studies were carried out using the components of multiplex micro well protein array kits that were custom manufactured and purchased from Quansys Biosciences (Logan, Utah USA). Each kit contained a single 96 micro well plate with individual wells imprinted with the same matrix of capture antibodies for the targeted proteins and all of the reagents necessary to calibrate and assay samples using classic
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
sandwich ELISA dot assays. The assay protocols employ in common an avidin-biotin amplification step with horseradish peroxidase serving as the reporter enzyme and
T
detection and quantification carried out using a chemiluminescent substrate.
IP
The kits were custom manufactured such that each of the relevant biotinylated
SC R
secondary antibodies and the complement of recombinant protein standards were supplied as individual reagents. This allowed us to independently verify the specificity of each of the assays by varying the composition of protein standards employed in
NU
calibration and the composition of the employed secondary antibody probes.
MA
For the chemokine array this assay consisted of an incomplete cocktail of biotinylated secondary antibodies specific for 8 of the 9 probed chemokines with the biotinylated antibody specific for IL-8 supplied separately. In this study the chemokine arrays
TE
D
were developed in a sequential manner first probing for the 8 chemokines and then re-probing for IL-8. Therefore, 50 µl of diluted samples (dilution factor 1:10) were
CE P
incubated in the wells and the residual fluid removed with the wells washed as directed by the array manufacturer. The wells were then incubated with the partial
AC
cocktail of biotinylated antibodies specific for 8 chemokines. After removal of the residual reagent and further washing the substrate was added and the plate imaged for an extended period of time allowing the detection of a relatively weak set of signals for some of the 8 probed chemokines. The exclusion of the biotinylated antibody to IL-8 in this step of the assay allows the detection of a false positive signal for IL-8 in the absence of the appropriate secondary antibody. The array is then washed repeatedly with the kit-supplied buffer and re-probed using this time the biotinylated antibody specific to IL-8. The substrate is then added and the plate then re-imaged for a short period of time. The capacity to screen arrays for non-specific cross reactivity proved critical for the detection of heterophilic antibodies. Different methods were tested to eliminate the
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
interference of heterophilic antibodies. The samples were either diluted with human sample diluent (HSD, Quansys Biosciences), tear dilution buffer (Quansys
T
Biosciences), LowCross Buffer (Candor Biosciences GmbH, Wangen, Germany) or
IP
pre-incubated on protein-covered plates (Thermo Fisher Scientific Inc., Rockford,
SC R
USA) for 3 hours at room temperature following a dilution with tear dilution buffer. A 9-plex chemokine array (capture antibodies: Eotaxin, hGROa, I-309, IL-8, IP-10, MCP-1, MCP-2, RANTES, TARC) with a detection antibody mix without IL-8 was
NU
used. Further investigations were carried out using a dilution with HSD.
MA
Synovial fluids were assayed using arrays specific for 17 proteins. The 9-plex chemokine array (purchased from Quansys Biosciences) contained Eotaxin, hGROa, IL-8, IP-10, MCP-1, MCP-2, MIG, RANTES and TARC. The 5-plex cytokine array
TE
D
(purchased from Quansys Biosciences, Logan, USA) contained IL-6. The intra-assay coefficient of variation ranged from 4% to 10% (table B). The inter-assay coefficient
CE P
of variation varied from 7% to 13%. Samples were run in duplicate and at least on
AC
two different plates.
2.4 Statistical Analysis Data are presented as the mean ± standard deviation (SD) and analyzed using IBM SPSS Statistics 20. If a datum was below or above the detection limit, the lower and upper detection limit value would be used for calculation, respectively. For comparisons between OA and RA synovial fluid groups, the differences in continuous data including age, leukocyte number, CRP, chemokine and cytokine concentrations, and in categorical data including sex were calculated using the Student´s t test, Mann-Whitney U-test and Chi square test, respectively. Correlations between chemokine and cytokine concentrations were calculated using Spearman’s rho correlation test or Pearsons correlation test.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
For the correction of multigroup comparisons, P values of 0.0167 and 0.0033 for the Mann-Whitney U-test or Fisher’s exact probability test and 0.0071 and 0.0014 for
T
Spearman’s correlation test were considered statistically significant with significance
AC
CE P
TE
D
MA
NU
SC R
IP
levels of 5% and 1%, respectively, based on Bonferroni’s methods.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
3 Results
T
The interference by heterophilic antibodies in ELISA arrays has been previously
IP
described (Kricka, 1999). When using a 9-plex chemokine array and an antibody
SC R
detection mix without IL-8, we found a false positive signal for IL-8 in three out of 37 samples. Figure A represents a digital image of the used 9-plex chemokine array. Each spot is separately visible without any overlapping of spots. Furthermore, no
NU
comet effect or spot bleeding over into an adjacent spot can be found. To prevent
MA
interference by heterophilic antibodies, several suggestions are found in the literature (Hennig et al., 2000; Warren et al., 2005; Bartels et al., 2011). We investigated the influence of different buffers as well as pre-incubation of the sample on protein-
TE
D
covered plates. The substitution of HSD (Quansys Biosciences, Login, Ut) for the kit dilution buffer in all of the assays prevented the interference by heterophilic
CE P
antibodies (figure A).
AC
The mean age in the OA group was 73.1 years; in the RA group it was 64.2 years. In both groups more women suffered from OA or RA. Leukocyte number (7075 ± 19395 n/µl vs. 9923 ± 2553; P value <0.0001) and CRP concentration (0.33 ± 0.36 vs. 1.19 ± 1.08; P value 0.004) were significantly higher in the RA group.
All measurements of MCP-2, MIG, TARC, and IL-6 were within the detection range of the used arrays. Samples below the detection limit were found for Eotaxin (n=3), hGROa (n=4), IL-8 (n=2), IP-10 (n=7) and RANTES (n=6) and above the detection limit for hGROa (n=2), IP-10 (n=1), MCP-1 (n=1) (table B). Results of synovial fluid concentrations are shown in table C. Compared to the OA group, the concentrations of eotaxin (P=0.003), hGROα (P=0.033), IL-8 (P<0.0001), MCP-2 (P=0.001),
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
RANTES (P=0.037), TARC (P=0.048) and IL-6 (P=0.018) were significantly higher in
T
the RA group and MIG (P=0.021) was significantly lower.
IP
In OA synovial fluid, the correlations among the chemokine, IL-6, CRP and leukocyte
SC R
concentrations are shown in table D. Significant positive correlations with CXC chemokines were found between IL-8 and IP-10 (P = 0.032), MCP-1 (P = 0.039), IL-6 (P = 0.001); hGROα and IL-8 (P < 0.001), MCP-1 (P =0.009), MCP-2 (P = 0.032), IL-
NU
6 (P < 0.001); IP-10 and MIG (P < 0.001), RANTES (P = 0.004), IL-6 (P= 0.029); MIG
MA
and RANTES (P < 0.001).
Significant positive correlations with CC chemokines were found between eotaxin and IL-8 (P = 0.009), IP10 (P = 0.036), MCP-1 (P =0.001), MIG (P = 0.022), TARC (P
TE
D
< 0.001); MCP-1 and MCP-2 (P = 0.007), IL-6 (P = 0.039); MCP-2 and IL-6 (P =
CE P
0.007). A significant negative correlation was not found.
In RA synovial fluid, the correlations among the chemokine, IL-6, CRP and leukocyte
AC
concentrations are shown in table E. Significant positive correlations with CXC were found between IL-8 and IP10 (P = 0.028), RANTES (P = 0.027), IL-6 (P = 0.001); hGROα and IL-8 (P < 0.001), IP10 (P = 0.011), RANTES (P = 0.035), IL-6 (P = 0.002), CRP (P = 0.038); IP10 and MCP-2 (P = 0.003), RANTES (P = 0.001), IL-6 (P= 0.002); MIG and RANTES (P < 0.001), IL-6 (P = 0.001). Significant positive correlations with CC chemokines were found between eotaxin and MCP-2 (P =0.005), RANTES (P = 0.017); MCP-1 and MCP-2 (P = 0.005); MCP2 and MIG (P = 0.018); RANTES and IL-6 (P = 0.001). Furthermore, in RA synovial fluids significant positive correlations are between CRP and leukocyte number (P = 0.011). A significant negative correlation was not found.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
5 out of 16 RA patients received an anti-TNFα therapy (Etanercept, Adalimumab or Infliximab). In this small study there was no significant difference between chemokine
AC
CE P
TE
D
MA
NU
SC R
IP
T
levels from patients with or without anti-TNFα antibody therapy (data not shown).
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
4 Discussion
T
The presence of autoantibodies in RA is a well-known phenomenon (Mewar and
IP
Wilson, 2006). The autoantibodies include rheumatoid factor (RF) and anti-
SC R
citrullinated protein antibodies (anti-CCP), which are used for laboratory diagnosis of RA (Lee et al., 2008). RFs are defined as Ig autoantibodies that bind via variable sequences of their Fab region to the Fc region of an IgG (Mannik et al., 1988). There
NU
are autoantibodies beside RF and anti-CCP that react with antigens from other
MA
species, but with weak avidity (Bartels et al., 2011). The most prevalent “heterophilic” human anti-animal immunoglobulins are human anti-mouse antibodies (HAMA). Circulating human antibodies reactive with animal proteins are an often unrecognized
TE
D
and unsuspected source of interference in immunological assays (Kricka, 1999). Especially, in two-site (sandwich) immunoassays heterophilic antibodies can cause
CE P
both positive and negative interferences. The frequency of interference from heterophilic antibodies has been investigated in several studies, with a prevalence
AC
that ranges from 0.5% to 12% up to 52% (Warren et al., 2005). In this study we made use of a laboratory designed two step assay protocol with the methodology designed specifically to detect inappropriate signaling for pseudo IL-8 as marker. The methodology also allowed us to rapidly visually evaluate the effectiveness of varies modes of sample treatment in eliminating this problem with similar problems encountered in the assay of other biological fluids (de Jager et al., 2005; Sehlin et al., 2010). In this study using a non-specific blocking protocol we detected false positive signals for chemokines in the assay of ~17 % of the RA samples but none of the OA samples. This is not meant to infer that this problem is restricted solely to the RA population. Heterophilic antibodies are clearly present in normal synovial fluid but at low levels that have minimal impact upon the reliability of
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
the employed assays. Several methods can be developed to inhibit heterophilic antibody interference. Bartels and Coworkers suggest PEG 6000 precipitation to
T
abolish RF interference (Bartels et al., 2011). Another suggestion is the use of the
IP
precipitation agent protein L (de Jager et al., 2005) or a blocking copolymer of
SC R
ethylene and propylene (HeterBlock) and/or protein L, but the results were described as variable and inconclusive (Bartels et al., 2011). Our results show that by using a proprietary diluent that is designed to block heterophilic antibody reactivity as well as
NU
limiting the volume of sample added to each assay this artifact can be avoided as
MA
documented using a two-step assay protocol. The composition of the diluent is unclear, but presumably it contains mouse IgG to block unspecific binding. Our procedure allowed us to obtain a more accurate set of data in the assay of all of the
TE
D
samples. This methodology can be readily adapted for the assay of other biological fluids such as tear fluid thereby eliminating a similar artifact in the study of Sjögren’s
CE P
syndrome as we will detail elsewhere. The hallmark of RA as compared to OA is an inflammatory process characterized by
AC
infiltration of neutrophils and lymphocytes into the synovial tissue and joint fluid (Paquet et al., 2012). Immune cells are attracted by chemokines to the site of inflammation. Using HSD (Quansys Biosciences, Login, Ut), we found statistically significant elevations in the levels of various chemokines related to an immune reaction or inflammation in the RA as compared to OA synovial fluid. These include eotaxin, hGROα, IL-8, MCP-2, RANTES, TARC, and IL-6. Many of these findings corroborate data from earlier studies (Szekanecz et al., 2003; Kokkonen et al., 2010). The data from individual ELISA and multiplex analytical studies, however, have yielded surprisingly wide ranges of values for the concentration of various chemokines and cytokines in synovial fluids making the integration of data from different studies problematic. Moreover, some of our data contradicts the literature.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
For example, unlike Patel and coworkers we found no significant differences in MCP1 levels between OA and RA synovial fluid and obtained contradictory data in regard
T
to IP-10 and MIG (Patel et al., 2001). Part of this difference could be due the
IP
differences in the effectiveness of the methodologies in correcting for the presence of
SC R
heterophilic antibodies. Patel and his group established their own ELISA assays using 1% BSA (bovine serum albumin) as a blocking reagent. From our knowledge BSA is not sufficient to block heterophilic antibody signaling. Interestingly, we found a
NU
decrease in MIG in RA synovial fluid whereas Patel and coworkers found an increase
MA
of MIG (Patel et al., 2001). MIG as well as IP-10 lack of the glutamic acid-leucinearginine (ELR) amino acid motif in the protein sequence of CXC chemokines and function as angiostatic and anti-inflammatory agents. In contrast the ELR containing
TE
D
chemokines hGROα and IL-8 promote angiogenesis (Szekanecz et al., 2011). Our findings of a decrease in the level of MIG and an increase the levels of hGROα and
CE P
IL-8 in the RA samples would suggest that these chemokines contribute to stimulate ongoing neovascularsation in the RA tissue. The chemokine IL-8 is produced by
AC
synovial tissue macrophages (Koch et al., 1991), which might lead to the 20-fold measured increase of IL-8 in RA synovial fluid (161.2 ± 187.4 pg/ml). Surprisingly a wide range of values for the concentration of IL-8 in synovial fluid can be found in literature. For example, the concentration of IL-8 has been reported to range from 1330 ± 431 pg/ml (Patel et al., 2001) to 14.37 ± 5.8 ng/ml (Koch et al., 1991), maybe due to unsatisfying blocking by BSA. New advancement in technology led to various forms of multiplex protein analysis. Multiplex assays employing the Luminex system have been used by Kokkonen and coworkers (Kokkonen et al., 2010). Prevention of the interference of RF has been investigated using different approaches e.g. HeterBlock (Omega Biologicals, Bozeman, MT) and/or protein L (Pierce, Rockford, IL). We can confirm the effect of
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
both is unsatisfying. In plasma from RA patients IL-6, eotaxin and IP-10 levels are elevated compared to healthy controls (Kokkonen et al., 2010), which is consistent
T
with our results showing that IL-6, eotaxin and IP-10 in RA synovial fluid.
IP
Reports on anti-TNFα therapy using Infliximab or Etanercept describe a suppression
SC R
of GROα, IL-8, IP-10, MCP-1 and RANTES (Taylor et al., 2000; Klimiuk et al., 2006; Torikai et al., 2007; Kageyama et al., 2009). Chemokine levels in these studies have all been measured using commercial ELISA assays without mentioning specific
NU
procedures to prevent heterophilic antibody interference. In contrast to the cited references in this small pilot study (N=5) we could not find a significant influence of
MA
anti-TNFα therapy on the chemokines levels which might be due to the prevention of false positive signaling of heterophilic antibodies. Given the importance of this
AC
CE P
signaling documented.
TE
D
information larger studies are warranted with accurate control of heterophilic antibody
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Authors’ contributions
T
Ulrike Hampel: Analysis and interpretation of the data; drafting the article.
SC R
Pavel Iserovich: Analysis and interpretation of the data
IP
Stefan Sesselmann: Provision of study material.
Saadettin Sel: Statistical expertise
Friedrich Paulsen: critical revision of the article for important intellectual content.
NU
Robert Sack: concept and design; administrative, technical, or logistic support
MA
All authors gave final approval of the article.
TE
D
Funding sources
CE P
This work was supported by a Boehringer Ingelheim Fonds travel grant.
AC
Conflict of interest
The authors have nothing to disclose.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
References
AC
CE P
TE
D
MA
NU
SC R
IP
T
Aly, T., Devendra, D., Barker, J., Liu, E., Yu, L. and Eisenbarth, G.S., 2004, Heterophile antibodies masquerade as interferon-alpha in subjects with new-onset type 1 diabetes. Diabetes Care 27, 1205-6. Andreas, K., Lubke, C., Haupl, T., Dehne, T., Morawietz, L., Ringe, J., Kaps, C. and Sittinger, M., 2008, Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study. Arthritis Res Ther 10, R9. Bartels, E.M., Falbe Watjen, I., Littrup Andersen, E., Danneskiold-Samsoe, B., Bliddal, H. and RibelMadsen, S., 2011, Rheumatoid factor and its interference with cytokine measurements: problems and solutions. Arthritis 2011, 741071. de Jager, W., Prakken, B.J., Bijlsma, J.W., Kuis, W. and Rijkers, G.T., 2005, Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies. J Immunol Methods 300, 124-35. Gorman, C.L. and Cope, A.P., 2008, Immune-mediated pathways in chronic inflammatory arthritis. Best Pract Res Clin Rheumatol 22, 221-38. Hennig, C., Rink, L., Fagin, U., Jabs, W.J. and Kirchner, H., 2000, The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods 235, 71-80. Hoff, M., Kvien, T.K., Kalvesten, J., Elden, A. and Haugeberg, G., 2009, Adalimumab therapy reduces hand bone loss in early rheumatoid arthritis: explorative analyses from the PREMIER study. Ann Rheum Dis 68, 1171-6. Kageyama, Y., Kobayashi, H., Kato, N. and Shimazu, M., 2009, Etanercept reduces the serum levels of macrophage chemotactic protein-1 in patients with rheumatoid arthritis. Mod Rheumatol 19, 372-8. Klimiuk, P.A., Sierakowski, S., Domyslawska, I. and Chwiecko, J., 2006, Regulation of serum chemokines following infliximab therapy in patients with rheumatoid arthritis. Clin Exp Rheumatol 24, 529-33. Koch, A.E., 2005, Chemokines and their receptors in rheumatoid arthritis: future targets? Arthritis Rheum 52, 710-21. Koch, A.E., Kunkel, S.L., Burrows, J.C., Evanoff, H.L., Haines, G.K., Pope, R.M. and Strieter, R.M., 1991, Synovial tissue macrophage as a source of the chemotactic cytokine IL-8. J Immunol 147, 2187-95. Kokkonen, H., Soderstrom, I., Rocklov, J., Hallmans, G., Lejon, K. and Rantapaa Dahlqvist, S., 2010, Upregulation of cytokines and chemokines predates the onset of rheumatoid arthritis. Arthritis Rheum 62, 383-91. Kricka, L.J., 1999, Human anti-animal antibody interferences in immunological assays. Clin Chem 45, 942-56. Lee, A.N., Beck, C.E. and Hall, M., 2008, Rheumatoid factor and anti-CCP autoantibodies in rheumatoid arthritis: a review. Clin Lab Sci 21, 15-8. Mannik, M., Nardella, F.A. and Sasso, E.H., 1988, Rheumatoid factors in immune complexes of patients with rheumatoid arthritis. Springer Semin Immunopathol 10, 215-30. Mewar, D. and Wilson, A.G., 2006, Autoantibodies in rheumatoid arthritis: a review. Biomed Pharmacother 60, 648-55. Paquet, J., Goebel, J.C., Delaunay, C., Pinzano, A., Grossin, L., Cournil-Henrionnet, C., Gillet, P., Netter, P., Jouzeau, J.Y. and Moulin, D., 2012, Cytokines profiling by multiplex analysis in experimental arthritis: which pathophysiological relevance for articular versus systemic mediators? Arthritis Res Ther 14, R60. Patel, D.D., Zachariah, J.P. and Whichard, L.P., 2001, CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin Immunol 98, 39-45.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
AC
CE P
TE
D
MA
NU
SC R
IP
T
Sehlin, D., Sollvander, S., Paulie, S., Brundin, R., Ingelsson, M., Lannfelt, L., Pettersson, F.E. and Englund, H., 2010, Interference from heterophilic antibodies in amyloid-beta oligomer ELISAs. Journal of Alzheimer's disease : JAD 21, 1295-301. Szekanecz, Z., Kim, J. and Koch, A.E., 2003, Chemokines and chemokine receptors in rheumatoid arthritis. Semin Immunol 15, 15-21. Szekanecz, Z., Koch, A.E. and Tak, P.P., 2011, Chemokine and chemokine receptor blockade in arthritis, a prototype of immune-mediated inflammatory diseases. Neth J Med 69, 356-66. Taylor, P.C., Peters, A.M., Paleolog, E., Chapman, P.T., Elliott, M.J., McCloskey, R., Feldmann, M. and Maini, R.N., 2000, Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor alpha blockade in patients with rheumatoid arthritis. Arthritis Rheum 43, 3847. Torikai, E., Kageyama, Y., Suzuki, M., Ichikawa, T. and Nagano, A., 2007, The effect of infliximab on chemokines in patients with rheumatoid arthritis. Clin Rheumatol 26, 1088-93. Warren, D.J., Bjerner, J., Paus, E., Bormer, O.P. and Nustad, K., 2005, Use of an in vivo biotinylated single-chain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies. Clin Chem 51, 830-8.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Table A. Demographic subject data Groups
73.1 ± 8.0 59 - 87
64.2 ± 12.8 38 - 82
5 16
2 14
0.3842
7075 ± 1939
9923 ± 2553
<0.00011
0.33 ± 0.36
1.19 ± 1.08
0.0043
IP
T
16
SC R
0.0141
CE P
TE
D
Student´s t test; 2 Chi square test; 3 Mann-Whitney U test
AC
1
21
MA
Age (yrs) Mean ± SD Range Sex Men Women Leukocyte (n/µl) Mean ± SD CRP (mg/dl) Mean ± SD
RA
NU
Number
P value
OA
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Inter-assay CV (%)
Cross reactivity (%)
range (pg/ml)
LLD (pg/ml)
IL-6
8
11
1
2.4-2500
3.81
Eotaxin
9
11
1
1.8-1900
0.4
hGROa
9
12
1
1.6-1700
1.49
IL-8
6
8
IP-10
9
10
MCP-1
10
13
MCP-2
9
9
MIG
10
12
RANTES
9
TARC
10
SC R
0.9-1000
0.24
1
1.6-1700
1.09
1
1.2-2200
0.37
1
1.2-1300
2.03
1
3.1-3200
1.25
10
1
1.7-1800
0.78
11
1
1.1-1200
0.39
D
MA
NU
1
TE CE P AC
IP
Intra -assay CV (%)
T
Table B. Intra- and Interassay CV, cross reactivity, standard range, lowest detection limit (LLD)
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Table C. Detection of IL-6 and chemokines in synovial fluids Groups
Eotaxin
1.5 ± 1.0
2.6 ± 0.9
0.0031
hGROα
4.9 ± 6.6
101.4 ± 164.0
0.0331
IL-8
8.2 ± 14.5
161.2 ± 187.4
IP-10
9.1 ± 10.5
61.7 ± 115.7
0.0901
MCP-1
30.3 ± 18.9
75.7 ± 147.1
0.0732
MCP-2
2.6 ± 0.8
3.9 ± 1.3
0.0011
MIG
7.0 ± 5.7
1.4 ± 37.9
0.0211
RANTES
3.5 ± 5.7
7.0 ± 6.6
0.0372
TARC
1.4 ± 0.5
IL-6
8.3 ± 11.1
D
TE CE P
IP
SC R
19.3 ± 16.9
0.0182
MA
0.0481
Student´s t test; 2 Mann-Whitney U test
AC
<0.00012
2.5 ± 1.9
Concentrations are expressed as mean ± SD (pg/ml). 1
T
RA
NU
P value
OA
ACCEPTED MANUSCRIPT Chemokines and cytokines in synovial fluids.
RANTES
TARC
IL-6
CRP
leukocyte
.727**1
.4012
-.1962
-.2641
.0781
.2232
.1521
.770**2
-.0612
.2471
.3482
.2182
.2092
.3262
.688**2
-.1212
.1782
.3551
.705**1
.595**2
.1311
.476*2
-.1102
-.1071
.569**2
.1432
-.0882
.2322
.452*2
-.0872
-.3452
.4001
.0552
.3251
.571**2
.0712
-.1461
.747**2
.2411
.3192
-.2072
-.2411
.0102
.3302
-.0102
-.1842
.3772
-.1782
-.1951
.0072
-.1182
IP10
MCP-1
MCP-2
.552**2
.459*1
.687**2
.4221
.812**2
.0521
.553**2
.470*1
.470*2
.453*2 .4182
.000
IP10
.036
.824
.032
MCP-1
.001
.009
.039
.060
MCP-2
.057
.032
.122
.114
MIG
.022
.738
.342
<0.001
RANTES
.579
.330
.363
.004
TArC
<0.001
.510
.150
IL-6
.072
<0.001
CRP
.394
leukocyte
.248
US
.009
MA N
IL-8
TE D
hGROα
CR
IL-8
IMIG PT
.1292
.2341
ρ P value .308
.007
.072
.703
.814
<0.001
.572
.311
.151
.292
.964
.001
.029
.039
.007
.159
.145
.092
.794
.601
.635
.707
.759
.367
.966
.440
.976
.280
.440
.644
.125
.529
.292
.424
.397
.610
CE P
.537
AC
Eotaxin
.497*1
hGROα
OA
Eotaxin
Table D. Correlation among Chemokines in OA synovial fluid samples
Correlation coefficient (ρ) and P values are calculated by Pearson´s1 or Spearman´s rho 2 correlation test. **. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).
-.0132 .955
ACCEPTED MANUSCRIPT Chemokines and cytokines in synovial fluids.
.795 .132 .440 .035 .826 .002
.753 .485 .055 .027 .244 .001
CRP leukocyte
.114 .777
.038 .589
.076 .240
.412 .003 .336 .001 .804 .002
CE P
.074 .005 .098 .017 .212 .210
.174 .452
leukocyte
TE D
.028
MCP-1 MCP-2 MIG RANTES TARC IL-6
CRP
.0712 -.0852 .2212
IL-6
.618*1 .547*
TARC
.932**2
RANTES
.4592
US MCP-2 CR MIGIP T
MCP-1
.3141
.4281
.588*2
.3301
.3312
.4112
-.0771
.3931 .1882 .697**1
.2081 .4882 .2571
.529*2 .550*2 .744**2
-.0601 .309 -.0671
.723**2 .736**2 .723**2
.522*2 .4562 .3582
.1461 .3122 -.2031
.662**2
.3882 .581*1
-.0292 .2752 .788**2
-.344 .0541 .1231 .388
-.1322 .2002 .731**2 .739**2 .2402
.4342 .3342 .4302 .4492 -.0412 .3052
.1502 -.0761 .4641 .1562 -.1811 .1522
.669**1
MA N
IP10
.1652
AC
hGROα IL-8 IP10
ρ -.0961 P value .725 .542 <0.001 .236 .011
IL-8
Eotaxin
hGROα
RA
Eotaxin
Table E. Correlation among Chemokines in RA synovial fluid samples
.005 .137 .914 .192 .625
.018 .302 .844 .457
<0.001 .650 .001
.137 .001
.371
.093 .579
.207 .780
.097 .070
.081 .564
.880 .503
.251 .575
Correlation coefficient (ρ) and P values are calculated by Pearson´s1 or Spearman´s rho 2 correlation test. **. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).
.615*2 .011
ACCEPTED MANUSCRIPT
SC R
IP
T
Chemokines and cytokines in synovial fluids.
AC
CE P
TE
D
MA
NU
Figure A
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Figure legends
T
Figure A. Testing of buffers - Three out of 37 samples gave a false-positive signal for
IP
IL-8. Different buffers were tested to prevent the interference of heterophilic
SC R
antibodies in this sample (tear dilution buffer (TDB), human sample diluent (HSD), LowCross Buffer (LCB) or preincubation on protein plates). A false-positive signal for IL-8 occurs when using TDB, LCB and preincubation on proteinL plates, but not HSD.
NU
A false-negative signal for IL-8 occurs when using LCB. Further investigations were
AC
CE P
TE
D
MA
performed with HSD. ab (antibody)
S0022-1759(13)00232-9 doi: 10.1016/j.jim.2013.08.007 JIM 11711
To appear in:
Journal of Immunological Methods
Received date: Revised date: Accepted date:
28 June 2013 3 August 2013 12 August 2013
Please cite this article as: Hampel, Ulrike, Sesselmann, Stefan, Iserovich, Pavel, Sel, Saadettin, Paulsen, Friedrich, Sack, Robert, Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis synovial fluid, Journal of Immunological Methods (2013), doi: 10.1016/j.jim.2013.08.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis
T
synovial fluid
IP
Ulrike Hampel1, Stefan Sesselmann2, Pavel Iserovich3, Saadettin Sel4, Friedrich
1
SC R
Paulsen1, Robert Sack3
Department of Anatomy II, Friedrich Alexander University Erlangen Nürnberg,
Division of Molecular Immunology, Friedrich Alexander University Erlangen
MA
2
NU
Erlangen, Germany
Nürnberg, Erlangen, Germany 3
Department of Biological Sciences, SUNY College of Optometry New York, New
TE
4
D
York, USA
Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany,
CE P
Erlangen, Germany
AC
Corresponding author: Ulrike Hampel, Department of Anatomy II, Friedrich Alexander University Erlangen Nürnberg, Erlangen, Germany
Keywords: ELISA, interference, heterophilic antibody, chemokine, cytokine, arthritis
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Abstract
T
To develop a method of the assay of chemokine and cytokine signaling in synovial
IP
fluid from patients suffering from osteoarthritis (OA) or rheumatoid arthritis (RA) and
SC R
evaluate the effect of heterophilic antibodies on the reliability of the data. 21 synovial fluid samples from OA and 16 synovial fluid samples from RA patients were analyzed using a unique 2 step dot sandwich ELISA based micro-well protein
NU
array designed to detect heterophilic antibody signaling in the presence or absence
MA
of an effective heterophilic blocking reagent with assays carried out for Eotaxin, hGROa, Interleukin (IL)-8, IP10, MCP-1, MCP-2, MIG, RANTES, TARC and IL-6. Array analysis reveals that the selective presence of heterophilic antibodies interferes
TE
D
with the accurate assay of synovial fluid samples from a minority of RA patients but not OA synovia. Using a commercial blocking diluent OA and RA synovial fluids
CE P
reveal significant differences in chemokine content (IL-6, Eotaxin, hGROa, MCP-2, MIG, TARC, IL-8, RANTES).
AC
Using a two-step assay protocol it is possible to readily detect inappropriate antibody signaling due to heterophilic antibodies and devise a protocol designed to eliminate this problem thereby more accurately quantify cytokines and chemokines specific to both RA and OA fluids.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
1 Introduction
T
A gradual loss of cartilage results in the destruction of the joints in osteoarthritis (OA).
IP
In contrast, rheumatoid arthritis (RA) is an autoimmune disease that leads to
SC R
deformity of the joints through chronic inflammation (Andreas et al., 2008). Inflammation is accompanied by infiltration by neutrophils and lymphocytes leading to elevated proinflammatory cytokine, chemokine and growth factor levels (Gorman and
NU
Cope, 2008). Chemokines play a key role in the perpetuation of the inflammation by
MA
attracting proinflammatory cells to the inflamed joint. Chemokines are classified into CXC, CC, C and CX3C supergene families (Koch, 2005). In most cases the cause for the rheumatic disorder is unknown. RA therapy is intended to inhibit the inflammation
TE
D
with the help of non-steroidal anti-inflammatory drugs (NSAID), corticosteroids, disease-modifying anti-rheumatic drugs (DMARDs) and drugs directed against TNFα
CE P
(Hoff et al., 2009). Furthermore, recent advances focused on chemokine and chemokine receptor blocking to prevent or diminish chemokine recruitment of
AC
inflammatory cells into RA tissue (Szekanecz et al., 2011). The effectiveness of these drugs could be monitored by the measurement of inflammatory markers, cytokines and chemokines in serum and synovial fluids. Synovial fluid is produced by synovial membrane and secreted into the joint cavity and can be used for diagnostic purposes. Several authors point out that the measurement of serum and synovial fluids derived from RA patients needs critical investigation, because heterophilic antibodies are a well-known source of interference in immunoassay of proteins (Bartels et al., 2011), which can cause false-positive or false-negative results (Kricka, 1999). Aly et al. defined heterophilic antibodies as antibodies directed against animal antigens that arise in humans. Most commonly the cause is unknown, but heterophilic antibodies might be the result of intravenous administration of animal
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
proteins (Aly et al., 2004). This is particularly common in autoimmune diseases such as RA. In order to obtain accurate data it is therefore crucial to eliminate interfering
T
factors from ELISA assays.
IP
The aim of this study was to establish a chemokine assay that allows one to detect
SC R
and minimize or eliminate this interference and more accurately determine differences in chemokine levels in synovial fluid from patients suffering from OA or RA. Our investigations focused on the CXC chemokines interleukin (IL)-8, human
NU
growth related oncogene-alpha (hGROα), interferon gamma induced protein 10 (IP-
MA
10) and Monokine induced by gamma interferon (MIG) and the CC chemokines eotaxin, regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), MCP-2 and thymus and activation-
TE
D
regulated chemokine (TARC) as well as on the cytokine IL-6. Those chemokines have proinflammatory and angiogenic in the OA and RA context (Szekanecz et al.,
AC
CE P
2003).
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
2 Materials and methods
T
2.1 Subjects
IP
All subjects provided informed consent for synovial fluid collection in accordance with
SC R
the Declaration of Helsinki. Synovial samples were obtained from 37 subjects (21 with OA and 16 with RA). The demographic data of the subjects, including age, sex, leukocyte and CPR levels on the day before surgery are summarized in table A. 62%
NU
of the RA patients received corticoids, 62% DMARD, 31% anti-TNFα therapy and
MA
19% NSAID. The average number of medications for RA patients was 1.7 (range 1-
TE
2.2 Synovial fluid collection
D
3); OA patients did not receive any of these drugs.
All synovial fluid samples (10 to 20 ml) were taken during total knee alloarthroplasty
CE P
after preparation of the subcutaneous tissue and before opening of the articular capsule using a 20 ml syringe with 18 gauge needle, with care taken to prevent blood
AC
and intra-articular tissue contamination. All samples were immediately frozen and stored at -800C until the analyses were performed.
2.3 Micro-well protein arrays for quantitative protein assays Prior to assaying the samples were centrifuged at 5000 rpm for 20 min at 4°C and the supernatant has been used for further procedures. The studies were carried out using the components of multiplex micro well protein array kits that were custom manufactured and purchased from Quansys Biosciences (Logan, Utah USA). Each kit contained a single 96 micro well plate with individual wells imprinted with the same matrix of capture antibodies for the targeted proteins and all of the reagents necessary to calibrate and assay samples using classic
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
sandwich ELISA dot assays. The assay protocols employ in common an avidin-biotin amplification step with horseradish peroxidase serving as the reporter enzyme and
T
detection and quantification carried out using a chemiluminescent substrate.
IP
The kits were custom manufactured such that each of the relevant biotinylated
SC R
secondary antibodies and the complement of recombinant protein standards were supplied as individual reagents. This allowed us to independently verify the specificity of each of the assays by varying the composition of protein standards employed in
NU
calibration and the composition of the employed secondary antibody probes.
MA
For the chemokine array this assay consisted of an incomplete cocktail of biotinylated secondary antibodies specific for 8 of the 9 probed chemokines with the biotinylated antibody specific for IL-8 supplied separately. In this study the chemokine arrays
TE
D
were developed in a sequential manner first probing for the 8 chemokines and then re-probing for IL-8. Therefore, 50 µl of diluted samples (dilution factor 1:10) were
CE P
incubated in the wells and the residual fluid removed with the wells washed as directed by the array manufacturer. The wells were then incubated with the partial
AC
cocktail of biotinylated antibodies specific for 8 chemokines. After removal of the residual reagent and further washing the substrate was added and the plate imaged for an extended period of time allowing the detection of a relatively weak set of signals for some of the 8 probed chemokines. The exclusion of the biotinylated antibody to IL-8 in this step of the assay allows the detection of a false positive signal for IL-8 in the absence of the appropriate secondary antibody. The array is then washed repeatedly with the kit-supplied buffer and re-probed using this time the biotinylated antibody specific to IL-8. The substrate is then added and the plate then re-imaged for a short period of time. The capacity to screen arrays for non-specific cross reactivity proved critical for the detection of heterophilic antibodies. Different methods were tested to eliminate the
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
interference of heterophilic antibodies. The samples were either diluted with human sample diluent (HSD, Quansys Biosciences), tear dilution buffer (Quansys
T
Biosciences), LowCross Buffer (Candor Biosciences GmbH, Wangen, Germany) or
IP
pre-incubated on protein-covered plates (Thermo Fisher Scientific Inc., Rockford,
SC R
USA) for 3 hours at room temperature following a dilution with tear dilution buffer. A 9-plex chemokine array (capture antibodies: Eotaxin, hGROa, I-309, IL-8, IP-10, MCP-1, MCP-2, RANTES, TARC) with a detection antibody mix without IL-8 was
NU
used. Further investigations were carried out using a dilution with HSD.
MA
Synovial fluids were assayed using arrays specific for 17 proteins. The 9-plex chemokine array (purchased from Quansys Biosciences) contained Eotaxin, hGROa, IL-8, IP-10, MCP-1, MCP-2, MIG, RANTES and TARC. The 5-plex cytokine array
TE
D
(purchased from Quansys Biosciences, Logan, USA) contained IL-6. The intra-assay coefficient of variation ranged from 4% to 10% (table B). The inter-assay coefficient
CE P
of variation varied from 7% to 13%. Samples were run in duplicate and at least on
AC
two different plates.
2.4 Statistical Analysis Data are presented as the mean ± standard deviation (SD) and analyzed using IBM SPSS Statistics 20. If a datum was below or above the detection limit, the lower and upper detection limit value would be used for calculation, respectively. For comparisons between OA and RA synovial fluid groups, the differences in continuous data including age, leukocyte number, CRP, chemokine and cytokine concentrations, and in categorical data including sex were calculated using the Student´s t test, Mann-Whitney U-test and Chi square test, respectively. Correlations between chemokine and cytokine concentrations were calculated using Spearman’s rho correlation test or Pearsons correlation test.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
For the correction of multigroup comparisons, P values of 0.0167 and 0.0033 for the Mann-Whitney U-test or Fisher’s exact probability test and 0.0071 and 0.0014 for
T
Spearman’s correlation test were considered statistically significant with significance
AC
CE P
TE
D
MA
NU
SC R
IP
levels of 5% and 1%, respectively, based on Bonferroni’s methods.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
3 Results
T
The interference by heterophilic antibodies in ELISA arrays has been previously
IP
described (Kricka, 1999). When using a 9-plex chemokine array and an antibody
SC R
detection mix without IL-8, we found a false positive signal for IL-8 in three out of 37 samples. Figure A represents a digital image of the used 9-plex chemokine array. Each spot is separately visible without any overlapping of spots. Furthermore, no
NU
comet effect or spot bleeding over into an adjacent spot can be found. To prevent
MA
interference by heterophilic antibodies, several suggestions are found in the literature (Hennig et al., 2000; Warren et al., 2005; Bartels et al., 2011). We investigated the influence of different buffers as well as pre-incubation of the sample on protein-
TE
D
covered plates. The substitution of HSD (Quansys Biosciences, Login, Ut) for the kit dilution buffer in all of the assays prevented the interference by heterophilic
CE P
antibodies (figure A).
AC
The mean age in the OA group was 73.1 years; in the RA group it was 64.2 years. In both groups more women suffered from OA or RA. Leukocyte number (7075 ± 19395 n/µl vs. 9923 ± 2553; P value <0.0001) and CRP concentration (0.33 ± 0.36 vs. 1.19 ± 1.08; P value 0.004) were significantly higher in the RA group.
All measurements of MCP-2, MIG, TARC, and IL-6 were within the detection range of the used arrays. Samples below the detection limit were found for Eotaxin (n=3), hGROa (n=4), IL-8 (n=2), IP-10 (n=7) and RANTES (n=6) and above the detection limit for hGROa (n=2), IP-10 (n=1), MCP-1 (n=1) (table B). Results of synovial fluid concentrations are shown in table C. Compared to the OA group, the concentrations of eotaxin (P=0.003), hGROα (P=0.033), IL-8 (P<0.0001), MCP-2 (P=0.001),
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
RANTES (P=0.037), TARC (P=0.048) and IL-6 (P=0.018) were significantly higher in
T
the RA group and MIG (P=0.021) was significantly lower.
IP
In OA synovial fluid, the correlations among the chemokine, IL-6, CRP and leukocyte
SC R
concentrations are shown in table D. Significant positive correlations with CXC chemokines were found between IL-8 and IP-10 (P = 0.032), MCP-1 (P = 0.039), IL-6 (P = 0.001); hGROα and IL-8 (P < 0.001), MCP-1 (P =0.009), MCP-2 (P = 0.032), IL-
NU
6 (P < 0.001); IP-10 and MIG (P < 0.001), RANTES (P = 0.004), IL-6 (P= 0.029); MIG
MA
and RANTES (P < 0.001).
Significant positive correlations with CC chemokines were found between eotaxin and IL-8 (P = 0.009), IP10 (P = 0.036), MCP-1 (P =0.001), MIG (P = 0.022), TARC (P
TE
D
< 0.001); MCP-1 and MCP-2 (P = 0.007), IL-6 (P = 0.039); MCP-2 and IL-6 (P =
CE P
0.007). A significant negative correlation was not found.
In RA synovial fluid, the correlations among the chemokine, IL-6, CRP and leukocyte
AC
concentrations are shown in table E. Significant positive correlations with CXC were found between IL-8 and IP10 (P = 0.028), RANTES (P = 0.027), IL-6 (P = 0.001); hGROα and IL-8 (P < 0.001), IP10 (P = 0.011), RANTES (P = 0.035), IL-6 (P = 0.002), CRP (P = 0.038); IP10 and MCP-2 (P = 0.003), RANTES (P = 0.001), IL-6 (P= 0.002); MIG and RANTES (P < 0.001), IL-6 (P = 0.001). Significant positive correlations with CC chemokines were found between eotaxin and MCP-2 (P =0.005), RANTES (P = 0.017); MCP-1 and MCP-2 (P = 0.005); MCP2 and MIG (P = 0.018); RANTES and IL-6 (P = 0.001). Furthermore, in RA synovial fluids significant positive correlations are between CRP and leukocyte number (P = 0.011). A significant negative correlation was not found.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
5 out of 16 RA patients received an anti-TNFα therapy (Etanercept, Adalimumab or Infliximab). In this small study there was no significant difference between chemokine
AC
CE P
TE
D
MA
NU
SC R
IP
T
levels from patients with or without anti-TNFα antibody therapy (data not shown).
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
4 Discussion
T
The presence of autoantibodies in RA is a well-known phenomenon (Mewar and
IP
Wilson, 2006). The autoantibodies include rheumatoid factor (RF) and anti-
SC R
citrullinated protein antibodies (anti-CCP), which are used for laboratory diagnosis of RA (Lee et al., 2008). RFs are defined as Ig autoantibodies that bind via variable sequences of their Fab region to the Fc region of an IgG (Mannik et al., 1988). There
NU
are autoantibodies beside RF and anti-CCP that react with antigens from other
MA
species, but with weak avidity (Bartels et al., 2011). The most prevalent “heterophilic” human anti-animal immunoglobulins are human anti-mouse antibodies (HAMA). Circulating human antibodies reactive with animal proteins are an often unrecognized
TE
D
and unsuspected source of interference in immunological assays (Kricka, 1999). Especially, in two-site (sandwich) immunoassays heterophilic antibodies can cause
CE P
both positive and negative interferences. The frequency of interference from heterophilic antibodies has been investigated in several studies, with a prevalence
AC
that ranges from 0.5% to 12% up to 52% (Warren et al., 2005). In this study we made use of a laboratory designed two step assay protocol with the methodology designed specifically to detect inappropriate signaling for pseudo IL-8 as marker. The methodology also allowed us to rapidly visually evaluate the effectiveness of varies modes of sample treatment in eliminating this problem with similar problems encountered in the assay of other biological fluids (de Jager et al., 2005; Sehlin et al., 2010). In this study using a non-specific blocking protocol we detected false positive signals for chemokines in the assay of ~17 % of the RA samples but none of the OA samples. This is not meant to infer that this problem is restricted solely to the RA population. Heterophilic antibodies are clearly present in normal synovial fluid but at low levels that have minimal impact upon the reliability of
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
the employed assays. Several methods can be developed to inhibit heterophilic antibody interference. Bartels and Coworkers suggest PEG 6000 precipitation to
T
abolish RF interference (Bartels et al., 2011). Another suggestion is the use of the
IP
precipitation agent protein L (de Jager et al., 2005) or a blocking copolymer of
SC R
ethylene and propylene (HeterBlock) and/or protein L, but the results were described as variable and inconclusive (Bartels et al., 2011). Our results show that by using a proprietary diluent that is designed to block heterophilic antibody reactivity as well as
NU
limiting the volume of sample added to each assay this artifact can be avoided as
MA
documented using a two-step assay protocol. The composition of the diluent is unclear, but presumably it contains mouse IgG to block unspecific binding. Our procedure allowed us to obtain a more accurate set of data in the assay of all of the
TE
D
samples. This methodology can be readily adapted for the assay of other biological fluids such as tear fluid thereby eliminating a similar artifact in the study of Sjögren’s
CE P
syndrome as we will detail elsewhere. The hallmark of RA as compared to OA is an inflammatory process characterized by
AC
infiltration of neutrophils and lymphocytes into the synovial tissue and joint fluid (Paquet et al., 2012). Immune cells are attracted by chemokines to the site of inflammation. Using HSD (Quansys Biosciences, Login, Ut), we found statistically significant elevations in the levels of various chemokines related to an immune reaction or inflammation in the RA as compared to OA synovial fluid. These include eotaxin, hGROα, IL-8, MCP-2, RANTES, TARC, and IL-6. Many of these findings corroborate data from earlier studies (Szekanecz et al., 2003; Kokkonen et al., 2010). The data from individual ELISA and multiplex analytical studies, however, have yielded surprisingly wide ranges of values for the concentration of various chemokines and cytokines in synovial fluids making the integration of data from different studies problematic. Moreover, some of our data contradicts the literature.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
For example, unlike Patel and coworkers we found no significant differences in MCP1 levels between OA and RA synovial fluid and obtained contradictory data in regard
T
to IP-10 and MIG (Patel et al., 2001). Part of this difference could be due the
IP
differences in the effectiveness of the methodologies in correcting for the presence of
SC R
heterophilic antibodies. Patel and his group established their own ELISA assays using 1% BSA (bovine serum albumin) as a blocking reagent. From our knowledge BSA is not sufficient to block heterophilic antibody signaling. Interestingly, we found a
NU
decrease in MIG in RA synovial fluid whereas Patel and coworkers found an increase
MA
of MIG (Patel et al., 2001). MIG as well as IP-10 lack of the glutamic acid-leucinearginine (ELR) amino acid motif in the protein sequence of CXC chemokines and function as angiostatic and anti-inflammatory agents. In contrast the ELR containing
TE
D
chemokines hGROα and IL-8 promote angiogenesis (Szekanecz et al., 2011). Our findings of a decrease in the level of MIG and an increase the levels of hGROα and
CE P
IL-8 in the RA samples would suggest that these chemokines contribute to stimulate ongoing neovascularsation in the RA tissue. The chemokine IL-8 is produced by
AC
synovial tissue macrophages (Koch et al., 1991), which might lead to the 20-fold measured increase of IL-8 in RA synovial fluid (161.2 ± 187.4 pg/ml). Surprisingly a wide range of values for the concentration of IL-8 in synovial fluid can be found in literature. For example, the concentration of IL-8 has been reported to range from 1330 ± 431 pg/ml (Patel et al., 2001) to 14.37 ± 5.8 ng/ml (Koch et al., 1991), maybe due to unsatisfying blocking by BSA. New advancement in technology led to various forms of multiplex protein analysis. Multiplex assays employing the Luminex system have been used by Kokkonen and coworkers (Kokkonen et al., 2010). Prevention of the interference of RF has been investigated using different approaches e.g. HeterBlock (Omega Biologicals, Bozeman, MT) and/or protein L (Pierce, Rockford, IL). We can confirm the effect of
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
both is unsatisfying. In plasma from RA patients IL-6, eotaxin and IP-10 levels are elevated compared to healthy controls (Kokkonen et al., 2010), which is consistent
T
with our results showing that IL-6, eotaxin and IP-10 in RA synovial fluid.
IP
Reports on anti-TNFα therapy using Infliximab or Etanercept describe a suppression
SC R
of GROα, IL-8, IP-10, MCP-1 and RANTES (Taylor et al., 2000; Klimiuk et al., 2006; Torikai et al., 2007; Kageyama et al., 2009). Chemokine levels in these studies have all been measured using commercial ELISA assays without mentioning specific
NU
procedures to prevent heterophilic antibody interference. In contrast to the cited references in this small pilot study (N=5) we could not find a significant influence of
MA
anti-TNFα therapy on the chemokines levels which might be due to the prevention of false positive signaling of heterophilic antibodies. Given the importance of this
AC
CE P
signaling documented.
TE
D
information larger studies are warranted with accurate control of heterophilic antibody
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Authors’ contributions
T
Ulrike Hampel: Analysis and interpretation of the data; drafting the article.
SC R
Pavel Iserovich: Analysis and interpretation of the data
IP
Stefan Sesselmann: Provision of study material.
Saadettin Sel: Statistical expertise
Friedrich Paulsen: critical revision of the article for important intellectual content.
NU
Robert Sack: concept and design; administrative, technical, or logistic support
MA
All authors gave final approval of the article.
TE
D
Funding sources
CE P
This work was supported by a Boehringer Ingelheim Fonds travel grant.
AC
Conflict of interest
The authors have nothing to disclose.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
References
AC
CE P
TE
D
MA
NU
SC R
IP
T
Aly, T., Devendra, D., Barker, J., Liu, E., Yu, L. and Eisenbarth, G.S., 2004, Heterophile antibodies masquerade as interferon-alpha in subjects with new-onset type 1 diabetes. Diabetes Care 27, 1205-6. Andreas, K., Lubke, C., Haupl, T., Dehne, T., Morawietz, L., Ringe, J., Kaps, C. and Sittinger, M., 2008, Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study. Arthritis Res Ther 10, R9. Bartels, E.M., Falbe Watjen, I., Littrup Andersen, E., Danneskiold-Samsoe, B., Bliddal, H. and RibelMadsen, S., 2011, Rheumatoid factor and its interference with cytokine measurements: problems and solutions. Arthritis 2011, 741071. de Jager, W., Prakken, B.J., Bijlsma, J.W., Kuis, W. and Rijkers, G.T., 2005, Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies. J Immunol Methods 300, 124-35. Gorman, C.L. and Cope, A.P., 2008, Immune-mediated pathways in chronic inflammatory arthritis. Best Pract Res Clin Rheumatol 22, 221-38. Hennig, C., Rink, L., Fagin, U., Jabs, W.J. and Kirchner, H., 2000, The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods 235, 71-80. Hoff, M., Kvien, T.K., Kalvesten, J., Elden, A. and Haugeberg, G., 2009, Adalimumab therapy reduces hand bone loss in early rheumatoid arthritis: explorative analyses from the PREMIER study. Ann Rheum Dis 68, 1171-6. Kageyama, Y., Kobayashi, H., Kato, N. and Shimazu, M., 2009, Etanercept reduces the serum levels of macrophage chemotactic protein-1 in patients with rheumatoid arthritis. Mod Rheumatol 19, 372-8. Klimiuk, P.A., Sierakowski, S., Domyslawska, I. and Chwiecko, J., 2006, Regulation of serum chemokines following infliximab therapy in patients with rheumatoid arthritis. Clin Exp Rheumatol 24, 529-33. Koch, A.E., 2005, Chemokines and their receptors in rheumatoid arthritis: future targets? Arthritis Rheum 52, 710-21. Koch, A.E., Kunkel, S.L., Burrows, J.C., Evanoff, H.L., Haines, G.K., Pope, R.M. and Strieter, R.M., 1991, Synovial tissue macrophage as a source of the chemotactic cytokine IL-8. J Immunol 147, 2187-95. Kokkonen, H., Soderstrom, I., Rocklov, J., Hallmans, G., Lejon, K. and Rantapaa Dahlqvist, S., 2010, Upregulation of cytokines and chemokines predates the onset of rheumatoid arthritis. Arthritis Rheum 62, 383-91. Kricka, L.J., 1999, Human anti-animal antibody interferences in immunological assays. Clin Chem 45, 942-56. Lee, A.N., Beck, C.E. and Hall, M., 2008, Rheumatoid factor and anti-CCP autoantibodies in rheumatoid arthritis: a review. Clin Lab Sci 21, 15-8. Mannik, M., Nardella, F.A. and Sasso, E.H., 1988, Rheumatoid factors in immune complexes of patients with rheumatoid arthritis. Springer Semin Immunopathol 10, 215-30. Mewar, D. and Wilson, A.G., 2006, Autoantibodies in rheumatoid arthritis: a review. Biomed Pharmacother 60, 648-55. Paquet, J., Goebel, J.C., Delaunay, C., Pinzano, A., Grossin, L., Cournil-Henrionnet, C., Gillet, P., Netter, P., Jouzeau, J.Y. and Moulin, D., 2012, Cytokines profiling by multiplex analysis in experimental arthritis: which pathophysiological relevance for articular versus systemic mediators? Arthritis Res Ther 14, R60. Patel, D.D., Zachariah, J.P. and Whichard, L.P., 2001, CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin Immunol 98, 39-45.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
AC
CE P
TE
D
MA
NU
SC R
IP
T
Sehlin, D., Sollvander, S., Paulie, S., Brundin, R., Ingelsson, M., Lannfelt, L., Pettersson, F.E. and Englund, H., 2010, Interference from heterophilic antibodies in amyloid-beta oligomer ELISAs. Journal of Alzheimer's disease : JAD 21, 1295-301. Szekanecz, Z., Kim, J. and Koch, A.E., 2003, Chemokines and chemokine receptors in rheumatoid arthritis. Semin Immunol 15, 15-21. Szekanecz, Z., Koch, A.E. and Tak, P.P., 2011, Chemokine and chemokine receptor blockade in arthritis, a prototype of immune-mediated inflammatory diseases. Neth J Med 69, 356-66. Taylor, P.C., Peters, A.M., Paleolog, E., Chapman, P.T., Elliott, M.J., McCloskey, R., Feldmann, M. and Maini, R.N., 2000, Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor alpha blockade in patients with rheumatoid arthritis. Arthritis Rheum 43, 3847. Torikai, E., Kageyama, Y., Suzuki, M., Ichikawa, T. and Nagano, A., 2007, The effect of infliximab on chemokines in patients with rheumatoid arthritis. Clin Rheumatol 26, 1088-93. Warren, D.J., Bjerner, J., Paus, E., Bormer, O.P. and Nustad, K., 2005, Use of an in vivo biotinylated single-chain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies. Clin Chem 51, 830-8.
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Table A. Demographic subject data Groups
73.1 ± 8.0 59 - 87
64.2 ± 12.8 38 - 82
5 16
2 14
0.3842
7075 ± 1939
9923 ± 2553
<0.00011
0.33 ± 0.36
1.19 ± 1.08
0.0043
IP
T
16
SC R
0.0141
CE P
TE
D
Student´s t test; 2 Chi square test; 3 Mann-Whitney U test
AC
1
21
MA
Age (yrs) Mean ± SD Range Sex Men Women Leukocyte (n/µl) Mean ± SD CRP (mg/dl) Mean ± SD
RA
NU
Number
P value
OA
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Inter-assay CV (%)
Cross reactivity (%)
range (pg/ml)
LLD (pg/ml)
IL-6
8
11
1
2.4-2500
3.81
Eotaxin
9
11
1
1.8-1900
0.4
hGROa
9
12
1
1.6-1700
1.49
IL-8
6
8
IP-10
9
10
MCP-1
10
13
MCP-2
9
9
MIG
10
12
RANTES
9
TARC
10
SC R
0.9-1000
0.24
1
1.6-1700
1.09
1
1.2-2200
0.37
1
1.2-1300
2.03
1
3.1-3200
1.25
10
1
1.7-1800
0.78
11
1
1.1-1200
0.39
D
MA
NU
1
TE CE P AC
IP
Intra -assay CV (%)
T
Table B. Intra- and Interassay CV, cross reactivity, standard range, lowest detection limit (LLD)
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Table C. Detection of IL-6 and chemokines in synovial fluids Groups
Eotaxin
1.5 ± 1.0
2.6 ± 0.9
0.0031
hGROα
4.9 ± 6.6
101.4 ± 164.0
0.0331
IL-8
8.2 ± 14.5
161.2 ± 187.4
IP-10
9.1 ± 10.5
61.7 ± 115.7
0.0901
MCP-1
30.3 ± 18.9
75.7 ± 147.1
0.0732
MCP-2
2.6 ± 0.8
3.9 ± 1.3
0.0011
MIG
7.0 ± 5.7
1.4 ± 37.9
0.0211
RANTES
3.5 ± 5.7
7.0 ± 6.6
0.0372
TARC
1.4 ± 0.5
IL-6
8.3 ± 11.1
D
TE CE P
IP
SC R
19.3 ± 16.9
0.0182
MA
0.0481
Student´s t test; 2 Mann-Whitney U test
AC
<0.00012
2.5 ± 1.9
Concentrations are expressed as mean ± SD (pg/ml). 1
T
RA
NU
P value
OA
ACCEPTED MANUSCRIPT Chemokines and cytokines in synovial fluids.
RANTES
TARC
IL-6
CRP
leukocyte
.727**1
.4012
-.1962
-.2641
.0781
.2232
.1521
.770**2
-.0612
.2471
.3482
.2182
.2092
.3262
.688**2
-.1212
.1782
.3551
.705**1
.595**2
.1311
.476*2
-.1102
-.1071
.569**2
.1432
-.0882
.2322
.452*2
-.0872
-.3452
.4001
.0552
.3251
.571**2
.0712
-.1461
.747**2
.2411
.3192
-.2072
-.2411
.0102
.3302
-.0102
-.1842
.3772
-.1782
-.1951
.0072
-.1182
IP10
MCP-1
MCP-2
.552**2
.459*1
.687**2
.4221
.812**2
.0521
.553**2
.470*1
.470*2
.453*2 .4182
.000
IP10
.036
.824
.032
MCP-1
.001
.009
.039
.060
MCP-2
.057
.032
.122
.114
MIG
.022
.738
.342
<0.001
RANTES
.579
.330
.363
.004
TArC
<0.001
.510
.150
IL-6
.072
<0.001
CRP
.394
leukocyte
.248
US
.009
MA N
IL-8
TE D
hGROα
CR
IL-8
IMIG PT
.1292
.2341
ρ P value .308
.007
.072
.703
.814
<0.001
.572
.311
.151
.292
.964
.001
.029
.039
.007
.159
.145
.092
.794
.601
.635
.707
.759
.367
.966
.440
.976
.280
.440
.644
.125
.529
.292
.424
.397
.610
CE P
.537
AC
Eotaxin
.497*1
hGROα
OA
Eotaxin
Table D. Correlation among Chemokines in OA synovial fluid samples
Correlation coefficient (ρ) and P values are calculated by Pearson´s1 or Spearman´s rho 2 correlation test. **. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).
-.0132 .955
ACCEPTED MANUSCRIPT Chemokines and cytokines in synovial fluids.
.795 .132 .440 .035 .826 .002
.753 .485 .055 .027 .244 .001
CRP leukocyte
.114 .777
.038 .589
.076 .240
.412 .003 .336 .001 .804 .002
CE P
.074 .005 .098 .017 .212 .210
.174 .452
leukocyte
TE D
.028
MCP-1 MCP-2 MIG RANTES TARC IL-6
CRP
.0712 -.0852 .2212
IL-6
.618*1 .547*
TARC
.932**2
RANTES
.4592
US MCP-2 CR MIGIP T
MCP-1
.3141
.4281
.588*2
.3301
.3312
.4112
-.0771
.3931 .1882 .697**1
.2081 .4882 .2571
.529*2 .550*2 .744**2
-.0601 .309 -.0671
.723**2 .736**2 .723**2
.522*2 .4562 .3582
.1461 .3122 -.2031
.662**2
.3882 .581*1
-.0292 .2752 .788**2
-.344 .0541 .1231 .388
-.1322 .2002 .731**2 .739**2 .2402
.4342 .3342 .4302 .4492 -.0412 .3052
.1502 -.0761 .4641 .1562 -.1811 .1522
.669**1
MA N
IP10
.1652
AC
hGROα IL-8 IP10
ρ -.0961 P value .725 .542 <0.001 .236 .011
IL-8
Eotaxin
hGROα
RA
Eotaxin
Table E. Correlation among Chemokines in RA synovial fluid samples
.005 .137 .914 .192 .625
.018 .302 .844 .457
<0.001 .650 .001
.137 .001
.371
.093 .579
.207 .780
.097 .070
.081 .564
.880 .503
.251 .575
Correlation coefficient (ρ) and P values are calculated by Pearson´s1 or Spearman´s rho 2 correlation test. **. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).
.615*2 .011
ACCEPTED MANUSCRIPT
SC R
IP
T
Chemokines and cytokines in synovial fluids.
AC
CE P
TE
D
MA
NU
Figure A
ACCEPTED MANUSCRIPT
Chemokines and cytokines in synovial fluids.
Figure legends
T
Figure A. Testing of buffers - Three out of 37 samples gave a false-positive signal for
IP
IL-8. Different buffers were tested to prevent the interference of heterophilic
SC R
antibodies in this sample (tear dilution buffer (TDB), human sample diluent (HSD), LowCross Buffer (LCB) or preincubation on protein plates). A false-positive signal for IL-8 occurs when using TDB, LCB and preincubation on proteinL plates, but not HSD.
NU
A false-negative signal for IL-8 occurs when using LCB. Further investigations were
AC
CE P
TE
D
MA
performed with HSD. ab (antibody)