Simultaneous detection of chicken cytokines in plasma samples using the Bio-Plex assay

Simultaneous detection of chicken cytokines in plasma samples using the Bio-Plex assay Abstract A chicken multiplex cytokine assay (Bio-Plex) to detect four different cytokines (IL-2, IL-12, IL-10, and interferon gamma) simultaneously in plasma samples was designed. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL, except for IFNγ with the range of 50 to 25,000 pg/mL. Such a chicken multiplex assay proved to be fast and reliable, and comparable in sensitivity, accuracy, and reproducibility to conventional enzyme-linked immunosorbent assays. Comparison of the multiplex assay with the ELISA technique using the same clones of detection and capture antibodies resulted in correlation coefficients for all cytokines ranging from 0.95 to 0.99. Lower limit of detection and limit of quantification values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. To reduce the risk of cross-reaction with other proteins, the Bio-Plex system was used, combining the principle of sandwich immunoassay with the Luminex bead-based technology. The cytokine standard recoveries for each cytokine varied between 86 and 118% in dynamic concentration ranges. A chicken multiplex cytokine assay (Bio-Plex) provided a more complete picture of differences between the Th1/Th2 cytokine profiles of the immunized via a new system of antigen delivery into chicken antigen-presenting cells and control groups. This multiplexed fluorescent-bead-based detection assay can be used as a quantitative or comparative tool for the study of the chicken ex vivo cellular immune response. INTRODUCTION Cytokines are soluble regulatory peptides secreted by cells of the immune system which act as extracellular signals between cells during the course of the immune response. Detection of cytokine profiles and their appearance serves as a tool to determine the type of preferentially activated immune response (Th1, Th2, or Th17). To quantify chicken cytokines in biological fluids and tissue culture samples, several methods have been developed. Using real-time PCR it is possible to detect the expression profile of cytokines at the mRNA expression level (Xing and Schat, 2000; Nang et al., 2011; Staines et al., 2016); intracellular proteins can be measured by flow cytometry of permeabilized cells (von Recum-Knepper et al., 2015) and secreted cytokines can be quantified with bioassays (Kaiser et al., 2006), ELISAs, radioactive immunosorbent assays, and microarrays (Sarson et al., 2007; Ranaware et al., 2016). However, each of these techniques has one or more significant limitations (ELISA measures only one cytokine at a time, PCR does not detect native proteins but only protein-coding transcripts). The multiplexed fluorescent-bead-based detection assay has been successfully used in several mammalian models, especially in humans and mice (Fulton et al., 1997; Carson and Vignali, 1999; Prabhakar et al., 2002). This method of multiple cytokine quantification has a clear advantage over the conventional ELISA, i.e., the ability to detect large numbers of cytokines simultaneously. The chicken immune response to vaccination, as measured by cytokine production, was proved to be very complex. Therefore, thorough evaluation of the cytokine profile after vaccination via a new system of antigen delivery into chicken antigen-presenting cells and immune challenge with infectious agents is of great importance for elucidation of the molecular mechanisms underlying the protective host response. The ability of Rous sarcoma virus (RSV) antigens fused with streptavidin (SA) and coupled with specific biotinylated monoclonal antibody anti-CD205 targeting chicken dendritic cells to induce virus-specific protective immunity has already been verified (Mucksová et al., 2017). The aim of this study was to optimize and comprehensively validate the multiplex fluorescent-bead-based detection assay for quantification of cytokine production by Th1 and Th2 cells in chicken plasma samples from immunized and control animals after RSV challenge. A comparative analysis of the Bio-Plex assay and enzyme-linked immunosorbent assay (ELISA) was also analyzed. MATERIALS AND METHOD Experimental Animals All immunization and tumor induction experiments were carried out with the highly inbred chicken line CB (B12/B12) homozygous in the B12 haplotype of the chicken MHC, also known as the B complex because it had originally been described as blood group B (Briles et al., 1982), maintained at the Institute of Molecular Genetics, Prague. All procedures were conducted in accordance with the EU Directive 2010/63/EU for animal experiments, complied with the ARRIVE guidelines and with the Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Commodities Dept. of the Ministry of Agriculture of the Czech Republic (43,229/2013-MZE-17,214). Assay Procedures For all measurements, fluorescent bead-based instrument Bio-Plex 200 (Bio-Rad, Hercules, CA) was used according to the manufacturer's protocol. All tested antibodies for IL-12, IL-2, IL-10, and interferon gamma (IFNγ) were purchased from Kingfisher Biotech, Inc. (Saint Paul, MN). For each cytokine, the recombinant protein, capture antibody, and detection antibody were obtained as a complete kit (Table 1) Bio-Plex instruments were validated using a Bio-Plex Validation kit (Bio-Rad, #171,203,001) within two weeks of each assay and calibrated on assay days using a Bio-Plex Calibration kit (Bio-Rad, #171,203,060). Fluorescently labeled carboxylated nonmagnetic beads (Biorad, cat # 171–506,033 number of region 33, cat # 171–506,036 number of region 36, cat # 171–506,037 number of region 37, and cat # 171–506,042 number of region 42) were used, each with a distinct color code or spectral address to permit discrimination of individual tests within a multiplex suspension. A Bio-Plex Amine Coupling kit (Bio-Rad, #171,406,001) was used for protein coupling. Streptavidin-HRP (R&D systems, Minneapolis, MN, #DY998) was used for ELISA detection. The standard ELISA protocol and recommendations from Kingfisher Biotech were used for ELISA. Table 1. Reagents used for Bio-Plex immunoassays. All used capture antibodies and biotinylated detection antibodies are directed against chicken cytokines. The reagents listed were obtained from the same source, Kingfisher Biotech, Inc. (Saint Paul, MN).   Recombinant protein  Capture Ab  Detection Ab  Chicken cytokine  Cat. no.  Source1  Cat. no.  Source1  Cat. no.  Source1  IL-2  RP0063C-005  KB  PB0387C-100  KB  PBB0395C-050  KB  IL-10  RP0018C-005  KB  KP1116C-100  KB  KPB1117C-050  KB  IL-12  RP0289C-005  KB  PB0435C-100  KB  PBB0436C-050  KB  IFN-γ  RP0115C-005  KB  PB0442C-100  KB  PBB0448C-050  KB    Recombinant protein  Capture Ab  Detection Ab  Chicken cytokine  Cat. no.  Source1  Cat. no.  Source1  Cat. no.  Source1  IL-2  RP0063C-005  KB  PB0387C-100  KB  PBB0395C-050  KB  IL-10  RP0018C-005  KB  KP1116C-100  KB  KPB1117C-050  KB  IL-12  RP0289C-005  KB  PB0435C-100  KB  PBB0436C-050  KB  IFN-γ  RP0115C-005  KB  PB0442C-100  KB  PBB0448C-050  KB  1KB: Kingfisher Biotech, Inc. (Saint Paul, MN). View Large Beads Preparation and Validation Beads of the selected region were mixed by vortexing and sonication for one minute. Ten million of beads in 80 μL of activation buffer were activated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Sigma-Aldrich, St. Louis, MO) (10 μL of 50 mg/ml EDC), and N-hydroxysuccinimide (NHS, Sigma-Aldrich) (10 μL of 50 mg/ml NHS) at room temperature for 20 minutes. After incubation, the activated beads were washed with PBS. One million of beads were coated with 10 μg of antibody in 0.5 mL of PBS overnight in a rotation shaker at 4°C. Subsequently, the beads were washed with storage buffer and counted manually in a Neubauer counting hemocytometer chamber (Shiran et al., 1995). Coupled beads were stored at 4°C. The stability of antibody on the beads was assayed by calibration prior to any assay and only beads not older than two months were used for the assays. Five thousand beads coated with a single antibody were used per single sample. When all five analytes were detected, 25,000 beads were present in 50 μL of buffer in a single well. Filter bottom 96-well plates (Merck, Kenilworth, NJ) were used for the multiplex. Three washing steps with PBS were used for washing beads in the plate before sample addition. Fifty μL of sample was added per well and the beads were incubated for 40 min with samples in a 96-well shaker (750 rpm). Three washing steps in wash buffer were done before adding the detection antibodies (50 μL, 200 ng/mL). A 10-minute incubation step in the dark with Streptavidin-PE (Bio-Rad, #171,304,501) was used, followed by three final washings. 120 μL of assay buffer was added per well. Measurements were done with the selected region of specific beads. Protein Coupling Validation Calibration with a mixed standard of all analytes (IL-12, IL-2, IL-10, and IFNγ), single analytes only, assay buffers only, or single missing analytes only were performed for both the single bead assay and the mixture of all beads. Concentration ranges 25,000 to 195 pg/mL for IL-10, 5000 to 19.5 pg/ml for IL-12, 15,000 to 4.2 pg/ml for IL-2, and 200 to 2.5 ng/ml for IFNγ were used. No signals above blank were obtained with an incomplete set; standard, capture, or detection antibodies for a particular analyte were not present in the mix. The cross-reactivity of capture and detection antibodies was tested three times in the presence of single cytokine standards at 12,500 pg/mL for IL-10, 2,500 pg/mL for IL-12, 7,500 pg/mL for IL-2, and 50 ng/mL for IFNγ. No signals above background were obtained when the standard for a specific capture and detection antibody was not added. All new bead preparations underwent this testing before use. Parameters of Bio-Plex Validation The major parameters evaluated during validation of the Bio-Plex method included: limit of detection/limit of quantification (LOD/LOQ) of the assay, upper limit of quantification (ULOQ), intra-assay variability (precision within an assay). The LOD, defined as the lowest concentration of analyte that can be detected, and LOQ, defined as the lowest concentration of analyte that can be quantified, were established by analyzing 28 wells of a sample matrix containing zero analyte as the sample. Statistical analyses Pearson correlation coefficient, where 1 is total positive linear correlation and 0 is no linear correlation, was used for estimation of the linear correlation between the signal intensity measured by multiplex and the absorbance obtained by ELISA. Statistical significance determined by Student t test: *P < 0.05; **P < 0.01; ***P < 0.001 was used for evaluation of the plasma cytokine profile of in vivo stimulated chickens. Immunization of Chickens Chickens were immunized with a dose of 100 nmol of the mixture of SA-RSV tetramers complexed with 200 nmol anti-CD205 in the presence of 200 μg poly I:C (Sigma-Aldrich, P1530) per chicken. The mixture of SA-RSV was composed of equal amounts of tetramers SA-vsrcA; SA- vsrcB; approximately 17 nmol (∼4 μg) of each per chicken. Determinations of the immunization dose and antigen preparation were described previously (Dong et al., 2013). The control group was immunized with 200 μg of poly I:C only per chicken. Chickens were inoculated with 0.2 mL of appropriately diluted freshly prepared antigenic solution subcutaneously into the left wing web (23 G × 25 mm needle). Chickens were immunized three times, first at the age of three weeks and then with two consecutive injections at 7-day intervals. RSV Challenge, Plasma Collection The Prague strain of RSV (PR-RSV-C) was used (Svoboda et al., 1992). Chickens were inoculated one week after the last immunization with a dose of virus corresponding to 100 focus-forming units (FFU) in 0.2 mL of cultivation medium (DMEM, Sigma-Aldritch, D6546), subcutaneously, into the outer area of pectoral muscle. The samples of plasma were collected on day 10 after the challenge. Samples of heparinized blood were centrifuged at 1,000 × g for 10 minutes. An additional centrifugation step of the plasma at 10,000 × g for 10 min at 4°C was included to completely remove platelets. All samples had the same freeze-thaw history at the time of testing. RESULTS Specific antibodies obtained from the kits (Table 1) were used for the development and validation of the 4-plex chicken cytokine assay. Several commercial cytokine antibody pairs suitable for use in ELISA did not perform well in the described multiplex system (data not shown). By combining twofold dilutions (concentration ranges: 25,000 to 195 pg/mL for IL-10, 5,000 to 19.5 pg/mL for IL-12, 15,000 to 4.2 pg/mL for IL-2, 200 to 2.5 ng/mL for IFNγ) of corresponding recombinant chicken cytokines, standard curves were generated. Using the Bio-Plex Manager software, curves with a five-parameter regression formula were calculated and the data were plotted (Figure 1). Large dynamic ranges were used to generate the curves so that, eventually, samples would not have to be concentrated or diluted. However, the dynamic ranges for the standard curves differ. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL (Table 2), except for IFNγ with the range of 50 to 25,000 pg/mL. Table 2 shows comparison of the limit of detection (LOD), limit of quantification (LOQ), and upper limit of quantification (ULOQ) values for all cytokines obtained by Bio-Plex and ELISA. Lower LOD and LOQ values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. The detection limit for IL-2, IL-10, and IL-12 was around 1 to 2 pg/mL, and for IFNγ it was 15.7 pg/mL by Bio-Plex, whereas LOD for IL-2 was 3.5 pg/mL, for IL12 it was 11.8 pg/mL, for IL-10 it was 58.9 pg/mL and for IFNγ 961 pg/mL by ELISA. Figure 1. View largeDownload slide Standard curves for recombinant chicken cytokines. Data were generated by combining a twofold dilution of the standards starting at concentration ranges: 25,000 to 195 pg/mL for IL-10; 5,000 to 19.5 pg/mL for IL-12; 15,000 to 4.2 pg/mL for IL-2; 200 to 2.5 ng/mL for IFNγ, coupled beads, and biotinylated antibodies in a 50 uL matrix solution incubated for 60 min. SA-PE was added after two washes and incubated for 10 min. After an additional wash, the median fluorescence intensity was measured. Data were obtained by measuring the fluorescent intensity with subtracted background in blank (AUI) of each standard concentration with the Bio-Plex system. Cytokine standards were analyzed in duplicates in five different plates with different bead preparations. Standard curves were calculated with Bio-Plex Manager software by a five-parameter regression formula. Figure 1. View largeDownload slide Standard curves for recombinant chicken cytokines. Data were generated by combining a twofold dilution of the standards starting at concentration ranges: 25,000 to 195 pg/mL for IL-10; 5,000 to 19.5 pg/mL for IL-12; 15,000 to 4.2 pg/mL for IL-2; 200 to 2.5 ng/mL for IFNγ, coupled beads, and biotinylated antibodies in a 50 uL matrix solution incubated for 60 min. SA-PE was added after two washes and incubated for 10 min. After an additional wash, the median fluorescence intensity was measured. Data were obtained by measuring the fluorescent intensity with subtracted background in blank (AUI) of each standard concentration with the Bio-Plex system. Cytokine standards were analyzed in duplicates in five different plates with different bead preparations. Standard curves were calculated with Bio-Plex Manager software by a five-parameter regression formula. Table 2. Cytokine dynamic concentration ranges of ELISA and the Bio-Plex system. To obtain the limit of detection (LOD), three standard deviations (SD) were added to the average mean fluorescence intensity of the blank wells n = 28. Dynamic concentration ranges are represented as the limit of quantification (LOQ) and upper limit of quantification (ULOQ) of the standard curve of each cytokine for ELISA and multiplex assay ( Bio-Plex system). The percent of linearity was between 80% and 120% in a given sample type for a minimum of eight points. For the LOQ, 6 SD were added to the mean MFI and concentration was determined by interpolation from the standard curve.   ELISA  Bio-Plex  Cytokine  LOD  LOQ  ULOQ  LOD  LOQ  ULOQ  IL-10  58.9  195  3125  2.2  7.3  6250  IL-12  11.8  39  5000  1.0  3.3  5000  IFNγ  961  3125  25,000  15.7  52.3  25,000  IL-2  3.5  11.7  937  0.8  2.6  4500    ELISA  Bio-Plex  Cytokine  LOD  LOQ  ULOQ  LOD  LOQ  ULOQ  IL-10  58.9  195  3125  2.2  7.3  6250  IL-12  11.8  39  5000  1.0  3.3  5000  IFNγ  961  3125  25,000  15.7  52.3  25,000  IL-2  3.5  11.7  937  0.8  2.6  4500  View Large To detect whether cross-reactivity occurred, a full-bead mixture with the detection antibodies for each bead was incubated three times in the presence of single cytokine standards at 12,500 pg/mL for IL-10, 2.500 pg/mL for IL-12, 7,500 pg/mL for IL-2, and 50 ng/mL for IFNγ. Although the background varied with each bead, none of the specific antibody sets gave readings above the background without the relevant cytokine standard, indicating that there was no detectable cross-reactivity with other primary or detection antibodies; only when a combination of primary labelled beads with the recombinant standard and the corresponding detection system were present, the fluorescent signal was obtained. Intra-assay variability, expressed as a coefficient of variation, was calculated based on the average of the diluted standard samples and measured twice in a multiplex assay repeated four times with different bead batches (Table 3). Table 3. Recombinant standard recovery. Cytokine standards were analyzed in duplicates in five different plates with different bead preparations. The accuracy of measured concentration is expressed as a percentage of expected and observed concentration. Expected concentration (pg/mL)  Accuracy (Obs/Exp) in %  IL-10  IL-12  IL-2  IFNγ  IL10  IL12  IL2  IFNγ  6,250  5,000  4,500  25,000  82  84  122  125  3,125  2,500  2,250  12,500  78  89  114  119  1,563  1,250  1,125  6,250  96  102  92  101  781  625  563  3125  103  97  114  105  391  313  281  1563  103  103  118  91  195  156  141  781  93  95  86  101  97.7  78.1  70.3  391  102  102  104  108  48.8  39.1  35.2  195  108  110  106  95  24.4  19.5  17.6  97.7  98  91  95  102  12.2  9.8  8.8  48.8  98  105  101  100  6.1  4.9  4.4  24.4  88  92  90  95  3.1  2.4  2.2  12.2  74  81  86  82  Expected concentration (pg/mL)  Accuracy (Obs/Exp) in %  IL-10  IL-12  IL-2  IFNγ  IL10  IL12  IL2  IFNγ  6,250  5,000  4,500  25,000  82  84  122  125  3,125  2,500  2,250  12,500  78  89  114  119  1,563  1,250  1,125  6,250  96  102  92  101  781  625  563  3125  103  97  114  105  391  313  281  1563  103  103  118  91  195  156  141  781  93  95  86  101  97.7  78.1  70.3  391  102  102  104  108  48.8  39.1  35.2  195  108  110  106  95  24.4  19.5  17.6  97.7  98  91  95  102  12.2  9.8  8.8  48.8  98  105  101  100  6.1  4.9  4.4  24.4  88  92  90  95  3.1  2.4  2.2  12.2  74  81  86  82  View Large To determine the standard recovery for each cytokine, the tested concentration of each standard was treated as unknown sample and measured in a multiplex assay as duplicate in different assays. In general, cytokine standard recoveries varied between 86 and 118 % in dynamic concentration ranges (Table 3). The results of Bio-Plex analysis were compared with those of ELISA for recombinant samples and plasma samples containing an unknown concentration of each cytokine. Correlations between both techniques were determined by measuring the levels of all cytokines with the Bio-Plex system and ELISA. All samples measured with the Bio-Plex system were assayed twice and at different time points so that the inter-assay variance could be determined. The concentrations of the majority of samples measured with ELISA did concur with the concentrations that were determined with the Bio-Plex system. High correlation coefficients (r2), ranging from 0.95 to 0.99, were found for all chicken cytokines (Figure 2). When comparing unknown samples, the absolute values were different since the multiplex assay is more sensitive, but when control (non-immunized) and treated (immunized) groups of samples were compared, the same significant differences between the groups were obtained, while absolute concentrations were higher when the multiplex was used. Figure 2. View largeDownload slide Correlation between Bio-Plex and ELISA results. Regression coefficients (r2) of the cytokine concentration express the correlation of four chicken cytokines detected in plasma samples by ELISA and a 4-plex assay run in the Bio-Plex system. Cytokine standards were analyzed in duplicates in three different plates with different bead preparations. Pearson correlation coefficient, where 1 is total positive linear correlation and 0 is no linear correlation, was used for estimation of the linear correlation between the signal intensity measured by multiplex and the absorbance obtained by ELISA. Figure 2. View largeDownload slide Correlation between Bio-Plex and ELISA results. Regression coefficients (r2) of the cytokine concentration express the correlation of four chicken cytokines detected in plasma samples by ELISA and a 4-plex assay run in the Bio-Plex system. Cytokine standards were analyzed in duplicates in three different plates with different bead preparations. Pearson correlation coefficient, where 1 is total positive linear correlation and 0 is no linear correlation, was used for estimation of the linear correlation between the signal intensity measured by multiplex and the absorbance obtained by ELISA. To establish the differences between the cytokine profiles of control (non-immunized) chickens and immunized chickens, the chickens were experimentally challenged with RSV(Figure 3). The data show that in response to viral infection, the levels of IL-12 (P ≤ 0.01), IL-2 (P≤ 0.001), and IFNγ (P ≤ 0.05) in plasma samples were significantly increased following RSV activation in immunized chickens compared with the non-immunized group. On the other hand, the IL-10 cytokine was not detected. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that the described system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity. Figure 3. View largeDownload slide Cytokine profile of in vivo stimulated chickens. The specific interleukin (IL-12, IL-2, IFNγ) responses were measured in chickens of the CB line using Bio-Plex system in the plasma samples (IL-10 cytokine was not detected). Horizontal bars indicate the mean values. The results represent at least two independent experiments. Non-immunized (n = 5): non-immunized control; Immunized (n = 5): samples from chickens immunized with the mixture of tetramers SA-vsrcA and SA-vsrcB with biotinylated anti-CD205. Statistical significance determined by Student's t test: *P < 0.05; **P < 0.01; ***P < 0.001. Figure 3. View largeDownload slide Cytokine profile of in vivo stimulated chickens. The specific interleukin (IL-12, IL-2, IFNγ) responses were measured in chickens of the CB line using Bio-Plex system in the plasma samples (IL-10 cytokine was not detected). Horizontal bars indicate the mean values. The results represent at least two independent experiments. Non-immunized (n = 5): non-immunized control; Immunized (n = 5): samples from chickens immunized with the mixture of tetramers SA-vsrcA and SA-vsrcB with biotinylated anti-CD205. Statistical significance determined by Student's t test: *P < 0.05; **P < 0.01; ***P < 0.001. DISCUSSION In this study we describe and validate the Bio-Plex detection system that can quantify multiple chicken cytokines simultaneously in a small sample volume of body fluid. This assay should prove to be a powerful tool in the quantitation of cytokines, or any other soluble product for which antibody pairs are available (Taylor et al., 2001; Yang et al., 2001; Jones et al., 2002; Martins, 2002). Because a large number of antibodies of different specificities are mixed in a single reaction in this multiplex assay, it is possible to envisage any nonspecific reaction that may be detected as a false positive result. To reduce the risk of cross-reaction with other proteins we used the Bio-Plex system, which combines the principle of sandwich immunoassay with the Luminex bead-based technology. The choice of (primary) antibodies is a critical parameter of this assay. Several studies have suggested good correlation between ELISA and multiplex; however, in absolute values these two assays are not comparable (Elshal and McCoy, 2006). One reason may be the different origin of the used antibodies: usually, a commercially available multiplex kit is compared with ELISA from a different provider, and therefore antibodies, which are crucial for all immunosorbent assays, differ in the targeting epitope and affinity to antigen. In this study, the ELISA and multiplex using the very same antibodies, serum blockers, and reporters were compared. However, the absolute values obtained by multiplex were higher; a good correlation in significance between the results obtained in experimental groups was shown. Similarly to previous observations (Vignali, 2000) we have proved that multiplex tests are easy to be performed and reproducible. Besides simultaneous measurement of more cytokines in the very same sample, this method is obviously faster (3.5 h vs. 24 h for ELISA) and cost-saving, especially when six or more cytokines are measured together. Additionally, these assays also minimize the sample volume requirements and significantly reduce the amount of reagents used per cytokine assay. The next important feature of this optimization/validation study is that the dynamic range of the immunoassay for the tested chicken cytokines was greatly improved by Bio-Plex. This resulted in a significant decrease in the LOD/LOQ values for these cytokines, permitting extremely low levels of these cytokines to be quantified in plasma samples that would previously be undetectable by ELISA. Using the multiplex analysis we were able to analyze cytokine production by Th1 and Th2 cells in plasma samples from both immunized and control chickens after the challenge with RSV. The cytokines of the classical Th1 immune profile (IL-12, IL-2, and IFNγ) were significantly increased in the immunized group compared with the control group. This is in accordance with the recent study (Mucksová et al., 2017) where was performed in vitro restimulation of chicken peripheral blood mononuclear cells of immunized and control groups in the same immunization model and evaluated four cytokines (IL-2, Il-10, IL-12, and IFNγ) in peripheral blood mononuclear cells culture supernatants. The cytokine levels of IL-2, IL-12, and IFNγ were also significantly increased in the immunized groups restimulated with v-src antigen. In conclusion, we designed and verified the Bio-Plex technique for practical use in the poultry for quantitative and comparative monitoring of the chicken immune cellular response, until now determined only by the ELISA technique. 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Simultaneous detection of chicken cytokines in plasma samples using the Bio-Plex assay

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Oxford University Press
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© 2017 Poultry Science Association Inc.
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0032-5791
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10.3382/ps/pex411
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Abstract

Abstract A chicken multiplex cytokine assay (Bio-Plex) to detect four different cytokines (IL-2, IL-12, IL-10, and interferon gamma) simultaneously in plasma samples was designed. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL, except for IFNγ with the range of 50 to 25,000 pg/mL. Such a chicken multiplex assay proved to be fast and reliable, and comparable in sensitivity, accuracy, and reproducibility to conventional enzyme-linked immunosorbent assays. Comparison of the multiplex assay with the ELISA technique using the same clones of detection and capture antibodies resulted in correlation coefficients for all cytokines ranging from 0.95 to 0.99. Lower limit of detection and limit of quantification values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. To reduce the risk of cross-reaction with other proteins, the Bio-Plex system was used, combining the principle of sandwich immunoassay with the Luminex bead-based technology. The cytokine standard recoveries for each cytokine varied between 86 and 118% in dynamic concentration ranges. A chicken multiplex cytokine assay (Bio-Plex) provided a more complete picture of differences between the Th1/Th2 cytokine profiles of the immunized via a new system of antigen delivery into chicken antigen-presenting cells and control groups. This multiplexed fluorescent-bead-based detection assay can be used as a quantitative or comparative tool for the study of the chicken ex vivo cellular immune response. INTRODUCTION Cytokines are soluble regulatory peptides secreted by cells of the immune system which act as extracellular signals between cells during the course of the immune response. Detection of cytokine profiles and their appearance serves as a tool to determine the type of preferentially activated immune response (Th1, Th2, or Th17). To quantify chicken cytokines in biological fluids and tissue culture samples, several methods have been developed. Using real-time PCR it is possible to detect the expression profile of cytokines at the mRNA expression level (Xing and Schat, 2000; Nang et al., 2011; Staines et al., 2016); intracellular proteins can be measured by flow cytometry of permeabilized cells (von Recum-Knepper et al., 2015) and secreted cytokines can be quantified with bioassays (Kaiser et al., 2006), ELISAs, radioactive immunosorbent assays, and microarrays (Sarson et al., 2007; Ranaware et al., 2016). However, each of these techniques has one or more significant limitations (ELISA measures only one cytokine at a time, PCR does not detect native proteins but only protein-coding transcripts). The multiplexed fluorescent-bead-based detection assay has been successfully used in several mammalian models, especially in humans and mice (Fulton et al., 1997; Carson and Vignali, 1999; Prabhakar et al., 2002). This method of multiple cytokine quantification has a clear advantage over the conventional ELISA, i.e., the ability to detect large numbers of cytokines simultaneously. The chicken immune response to vaccination, as measured by cytokine production, was proved to be very complex. Therefore, thorough evaluation of the cytokine profile after vaccination via a new system of antigen delivery into chicken antigen-presenting cells and immune challenge with infectious agents is of great importance for elucidation of the molecular mechanisms underlying the protective host response. The ability of Rous sarcoma virus (RSV) antigens fused with streptavidin (SA) and coupled with specific biotinylated monoclonal antibody anti-CD205 targeting chicken dendritic cells to induce virus-specific protective immunity has already been verified (Mucksová et al., 2017). The aim of this study was to optimize and comprehensively validate the multiplex fluorescent-bead-based detection assay for quantification of cytokine production by Th1 and Th2 cells in chicken plasma samples from immunized and control animals after RSV challenge. A comparative analysis of the Bio-Plex assay and enzyme-linked immunosorbent assay (ELISA) was also analyzed. MATERIALS AND METHOD Experimental Animals All immunization and tumor induction experiments were carried out with the highly inbred chicken line CB (B12/B12) homozygous in the B12 haplotype of the chicken MHC, also known as the B complex because it had originally been described as blood group B (Briles et al., 1982), maintained at the Institute of Molecular Genetics, Prague. All procedures were conducted in accordance with the EU Directive 2010/63/EU for animal experiments, complied with the ARRIVE guidelines and with the Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Commodities Dept. of the Ministry of Agriculture of the Czech Republic (43,229/2013-MZE-17,214). Assay Procedures For all measurements, fluorescent bead-based instrument Bio-Plex 200 (Bio-Rad, Hercules, CA) was used according to the manufacturer's protocol. All tested antibodies for IL-12, IL-2, IL-10, and interferon gamma (IFNγ) were purchased from Kingfisher Biotech, Inc. (Saint Paul, MN). For each cytokine, the recombinant protein, capture antibody, and detection antibody were obtained as a complete kit (Table 1) Bio-Plex instruments were validated using a Bio-Plex Validation kit (Bio-Rad, #171,203,001) within two weeks of each assay and calibrated on assay days using a Bio-Plex Calibration kit (Bio-Rad, #171,203,060). Fluorescently labeled carboxylated nonmagnetic beads (Biorad, cat # 171–506,033 number of region 33, cat # 171–506,036 number of region 36, cat # 171–506,037 number of region 37, and cat # 171–506,042 number of region 42) were used, each with a distinct color code or spectral address to permit discrimination of individual tests within a multiplex suspension. A Bio-Plex Amine Coupling kit (Bio-Rad, #171,406,001) was used for protein coupling. Streptavidin-HRP (R&D systems, Minneapolis, MN, #DY998) was used for ELISA detection. The standard ELISA protocol and recommendations from Kingfisher Biotech were used for ELISA. Table 1. Reagents used for Bio-Plex immunoassays. All used capture antibodies and biotinylated detection antibodies are directed against chicken cytokines. The reagents listed were obtained from the same source, Kingfisher Biotech, Inc. (Saint Paul, MN).   Recombinant protein  Capture Ab  Detection Ab  Chicken cytokine  Cat. no.  Source1  Cat. no.  Source1  Cat. no.  Source1  IL-2  RP0063C-005  KB  PB0387C-100  KB  PBB0395C-050  KB  IL-10  RP0018C-005  KB  KP1116C-100  KB  KPB1117C-050  KB  IL-12  RP0289C-005  KB  PB0435C-100  KB  PBB0436C-050  KB  IFN-γ  RP0115C-005  KB  PB0442C-100  KB  PBB0448C-050  KB    Recombinant protein  Capture Ab  Detection Ab  Chicken cytokine  Cat. no.  Source1  Cat. no.  Source1  Cat. no.  Source1  IL-2  RP0063C-005  KB  PB0387C-100  KB  PBB0395C-050  KB  IL-10  RP0018C-005  KB  KP1116C-100  KB  KPB1117C-050  KB  IL-12  RP0289C-005  KB  PB0435C-100  KB  PBB0436C-050  KB  IFN-γ  RP0115C-005  KB  PB0442C-100  KB  PBB0448C-050  KB  1KB: Kingfisher Biotech, Inc. (Saint Paul, MN). View Large Beads Preparation and Validation Beads of the selected region were mixed by vortexing and sonication for one minute. Ten million of beads in 80 μL of activation buffer were activated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Sigma-Aldrich, St. Louis, MO) (10 μL of 50 mg/ml EDC), and N-hydroxysuccinimide (NHS, Sigma-Aldrich) (10 μL of 50 mg/ml NHS) at room temperature for 20 minutes. After incubation, the activated beads were washed with PBS. One million of beads were coated with 10 μg of antibody in 0.5 mL of PBS overnight in a rotation shaker at 4°C. Subsequently, the beads were washed with storage buffer and counted manually in a Neubauer counting hemocytometer chamber (Shiran et al., 1995). Coupled beads were stored at 4°C. The stability of antibody on the beads was assayed by calibration prior to any assay and only beads not older than two months were used for the assays. Five thousand beads coated with a single antibody were used per single sample. When all five analytes were detected, 25,000 beads were present in 50 μL of buffer in a single well. Filter bottom 96-well plates (Merck, Kenilworth, NJ) were used for the multiplex. Three washing steps with PBS were used for washing beads in the plate before sample addition. Fifty μL of sample was added per well and the beads were incubated for 40 min with samples in a 96-well shaker (750 rpm). Three washing steps in wash buffer were done before adding the detection antibodies (50 μL, 200 ng/mL). A 10-minute incubation step in the dark with Streptavidin-PE (Bio-Rad, #171,304,501) was used, followed by three final washings. 120 μL of assay buffer was added per well. Measurements were done with the selected region of specific beads. Protein Coupling Validation Calibration with a mixed standard of all analytes (IL-12, IL-2, IL-10, and IFNγ), single analytes only, assay buffers only, or single missing analytes only were performed for both the single bead assay and the mixture of all beads. Concentration ranges 25,000 to 195 pg/mL for IL-10, 5000 to 19.5 pg/ml for IL-12, 15,000 to 4.2 pg/ml for IL-2, and 200 to 2.5 ng/ml for IFNγ were used. No signals above blank were obtained with an incomplete set; standard, capture, or detection antibodies for a particular analyte were not present in the mix. The cross-reactivity of capture and detection antibodies was tested three times in the presence of single cytokine standards at 12,500 pg/mL for IL-10, 2,500 pg/mL for IL-12, 7,500 pg/mL for IL-2, and 50 ng/mL for IFNγ. No signals above background were obtained when the standard for a specific capture and detection antibody was not added. All new bead preparations underwent this testing before use. Parameters of Bio-Plex Validation The major parameters evaluated during validation of the Bio-Plex method included: limit of detection/limit of quantification (LOD/LOQ) of the assay, upper limit of quantification (ULOQ), intra-assay variability (precision within an assay). The LOD, defined as the lowest concentration of analyte that can be detected, and LOQ, defined as the lowest concentration of analyte that can be quantified, were established by analyzing 28 wells of a sample matrix containing zero analyte as the sample. Statistical analyses Pearson correlation coefficient, where 1 is total positive linear correlation and 0 is no linear correlation, was used for estimation of the linear correlation between the signal intensity measured by multiplex and the absorbance obtained by ELISA. Statistical significance determined by Student t test: *P < 0.05; **P < 0.01; ***P < 0.001 was used for evaluation of the plasma cytokine profile of in vivo stimulated chickens. Immunization of Chickens Chickens were immunized with a dose of 100 nmol of the mixture of SA-RSV tetramers complexed with 200 nmol anti-CD205 in the presence of 200 μg poly I:C (Sigma-Aldrich, P1530) per chicken. The mixture of SA-RSV was composed of equal amounts of tetramers SA-vsrcA; SA- vsrcB; approximately 17 nmol (∼4 μg) of each per chicken. Determinations of the immunization dose and antigen preparation were described previously (Dong et al., 2013). The control group was immunized with 200 μg of poly I:C only per chicken. Chickens were inoculated with 0.2 mL of appropriately diluted freshly prepared antigenic solution subcutaneously into the left wing web (23 G × 25 mm needle). Chickens were immunized three times, first at the age of three weeks and then with two consecutive injections at 7-day intervals. RSV Challenge, Plasma Collection The Prague strain of RSV (PR-RSV-C) was used (Svoboda et al., 1992). Chickens were inoculated one week after the last immunization with a dose of virus corresponding to 100 focus-forming units (FFU) in 0.2 mL of cultivation medium (DMEM, Sigma-Aldritch, D6546), subcutaneously, into the outer area of pectoral muscle. The samples of plasma were collected on day 10 after the challenge. Samples of heparinized blood were centrifuged at 1,000 × g for 10 minutes. An additional centrifugation step of the plasma at 10,000 × g for 10 min at 4°C was included to completely remove platelets. All samples had the same freeze-thaw history at the time of testing. RESULTS Specific antibodies obtained from the kits (Table 1) were used for the development and validation of the 4-plex chicken cytokine assay. Several commercial cytokine antibody pairs suitable for use in ELISA did not perform well in the described multiplex system (data not shown). By combining twofold dilutions (concentration ranges: 25,000 to 195 pg/mL for IL-10, 5,000 to 19.5 pg/mL for IL-12, 15,000 to 4.2 pg/mL for IL-2, 200 to 2.5 ng/mL for IFNγ) of corresponding recombinant chicken cytokines, standard curves were generated. Using the Bio-Plex Manager software, curves with a five-parameter regression formula were calculated and the data were plotted (Figure 1). Large dynamic ranges were used to generate the curves so that, eventually, samples would not have to be concentrated or diluted. However, the dynamic ranges for the standard curves differ. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL (Table 2), except for IFNγ with the range of 50 to 25,000 pg/mL. Table 2 shows comparison of the limit of detection (LOD), limit of quantification (LOQ), and upper limit of quantification (ULOQ) values for all cytokines obtained by Bio-Plex and ELISA. Lower LOD and LOQ values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. The detection limit for IL-2, IL-10, and IL-12 was around 1 to 2 pg/mL, and for IFNγ it was 15.7 pg/mL by Bio-Plex, whereas LOD for IL-2 was 3.5 pg/mL, for IL12 it was 11.8 pg/mL, for IL-10 it was 58.9 pg/mL and for IFNγ 961 pg/mL by ELISA. Figure 1. View largeDownload slide Standard curves for recombinant chicken cytokines. Data were generated by combining a twofold dilution of the standards starting at concentration ranges: 25,000 to 195 pg/mL for IL-10; 5,000 to 19.5 pg/mL for IL-12; 15,000 to 4.2 pg/mL for IL-2; 200 to 2.5 ng/mL for IFNγ, coupled beads, and biotinylated antibodies in a 50 uL matrix solution incubated for 60 min. SA-PE was added after two washes and incubated for 10 min. After an additional wash, the median fluorescence intensity was measured. Data were obtained by measuring the fluorescent intensity with subtracted background in blank (AUI) of each standard concentration with the Bio-Plex system. Cytokine standards were analyzed in duplicates in five different plates with different bead preparations. Standard curves were calculated with Bio-Plex Manager software by a five-parameter regression formula. Figure 1. View largeDownload slide Standard curves for recombinant chicken cytokines. Data were generated by combining a twofold dilution of the standards starting at concentration ranges: 25,000 to 195 pg/mL for IL-10; 5,000 to 19.5 pg/mL for IL-12; 15,000 to 4.2 pg/mL for IL-2; 200 to 2.5 ng/mL for IFNγ, coupled beads, and biotinylated antibodies in a 50 uL matrix solution incubated for 60 min. SA-PE was added after two washes and incubated for 10 min. After an additional wash, the median fluorescence intensity was measured. Data were obtained by measuring the fluorescent intensity with subtracted background in blank (AUI) of each standard concentration with the Bio-Plex system. Cytokine standards were analyzed in duplicates in five different plates with different bead preparations. Standard curves were calculated with Bio-Plex Manager software by a five-parameter regression formula. Table 2. Cytokine dynamic concentration ranges of ELISA and the Bio-Plex system. To obtain the limit of detection (LOD), three standard deviations (SD) were added to the average mean fluorescence intensity of the blank wells n = 28. Dynamic concentration ranges are represented as the limit of quantification (LOQ) and upper limit of quantification (ULOQ) of the standard curve of each cytokine for ELISA and multiplex assay ( Bio-Plex system). The percent of linearity was between 80% and 120% in a given sample type for a minimum of eight points. For the LOQ, 6 SD were added to the mean MFI and concentration was determined by interpolation from the standard curve.   ELISA  Bio-Plex  Cytokine  LOD  LOQ  ULOQ  LOD  LOQ  ULOQ  IL-10  58.9  195  3125  2.2  7.3  6250  IL-12  11.8  39  5000  1.0  3.3  5000  IFNγ  961  3125  25,000  15.7  52.3  25,000  IL-2  3.5  11.7  937  0.8  2.6  4500    ELISA  Bio-Plex  Cytokine  LOD  LOQ  ULOQ  LOD  LOQ  ULOQ  IL-10  58.9  195  3125  2.2  7.3  6250  IL-12  11.8  39  5000  1.0  3.3  5000  IFNγ  961  3125  25,000  15.7  52.3  25,000  IL-2  3.5  11.7  937  0.8  2.6  4500  View Large To detect whether cross-reactivity occurred, a full-bead mixture with the detection antibodies for each bead was incubated three times in the presence of single cytokine standards at 12,500 pg/mL for IL-10, 2.500 pg/mL for IL-12, 7,500 pg/mL for IL-2, and 50 ng/mL for IFNγ. Although the background varied with each bead, none of the specific antibody sets gave readings above the background without the relevant cytokine standard, indicating that there was no detectable cross-reactivity with other primary or detection antibodies; only when a combination of primary labelled beads with the recombinant standard and the corresponding detection system were present, the fluorescent signal was obtained. Intra-assay variability, expressed as a coefficient of variation, was calculated based on the average of the diluted standard samples and measured twice in a multiplex assay repeated four times with different bead batches (Table 3). Table 3. Recombinant standard recovery. Cytokine standards were analyzed in duplicates in five different plates with different bead preparations. The accuracy of measured concentration is expressed as a percentage of expected and observed concentration. Expected concentration (pg/mL)  Accuracy (Obs/Exp) in %  IL-10  IL-12  IL-2  IFNγ  IL10  IL12  IL2  IFNγ  6,250  5,000  4,500  25,000  82  84  122  125  3,125  2,500  2,250  12,500  78  89  114  119  1,563  1,250  1,125  6,250  96  102  92  101  781  625  563  3125  103  97  114  105  391  313  281  1563  103  103  118  91  195  156  141  781  93  95  86  101  97.7  78.1  70.3  391  102  102  104  108  48.8  39.1  35.2  195  108  110  106  95  24.4  19.5  17.6  97.7  98  91  95  102  12.2  9.8  8.8  48.8  98  105  101  100  6.1  4.9  4.4  24.4  88  92  90  95  3.1  2.4  2.2  12.2  74  81  86  82  Expected concentration (pg/mL)  Accuracy (Obs/Exp) in %  IL-10  IL-12  IL-2  IFNγ  IL10  IL12  IL2  IFNγ  6,250  5,000  4,500  25,000  82  84  122  125  3,125  2,500  2,250  12,500  78  89  114  119  1,563  1,250  1,125  6,250  96  102  92  101  781  625  563  3125  103  97  114  105  391  313  281  1563  103  103  118  91  195  156  141  781  93  95  86  101  97.7  78.1  70.3  391  102  102  104  108  48.8  39.1  35.2  195  108  110  106  95  24.4  19.5  17.6  97.7  98  91  95  102  12.2  9.8  8.8  48.8  98  105  101  100  6.1  4.9  4.4  24.4  88  92  90  95  3.1  2.4  2.2  12.2  74  81  86  82  View Large To determine the standard recovery for each cytokine, the tested concentration of each standard was treated as unknown sample and measured in a multiplex assay as duplicate in different assays. In general, cytokine standard recoveries varied between 86 and 118 % in dynamic concentration ranges (Table 3). The results of Bio-Plex analysis were compared with those of ELISA for recombinant samples and plasma samples containing an unknown concentration of each cytokine. Correlations between both techniques were determined by measuring the levels of all cytokines with the Bio-Plex system and ELISA. All samples measured with the Bio-Plex system were assayed twice and at different time points so that the inter-assay variance could be determined. The concentrations of the majority of samples measured with ELISA did concur with the concentrations that were determined with the Bio-Plex system. High correlation coefficients (r2), ranging from 0.95 to 0.99, were found for all chicken cytokines (Figure 2). When comparing unknown samples, the absolute values were different since the multiplex assay is more sensitive, but when control (non-immunized) and treated (immunized) groups of samples were compared, the same significant differences between the groups were obtained, while absolute concentrations were higher when the multiplex was used. Figure 2. View largeDownload slide Correlation between Bio-Plex and ELISA results. Regression coefficients (r2) of the cytokine concentration express the correlation of four chicken cytokines detected in plasma samples by ELISA and a 4-plex assay run in the Bio-Plex system. Cytokine standards were analyzed in duplicates in three different plates with different bead preparations. Pearson correlation coefficient, where 1 is total positive linear correlation and 0 is no linear correlation, was used for estimation of the linear correlation between the signal intensity measured by multiplex and the absorbance obtained by ELISA. Figure 2. View largeDownload slide Correlation between Bio-Plex and ELISA results. Regression coefficients (r2) of the cytokine concentration express the correlation of four chicken cytokines detected in plasma samples by ELISA and a 4-plex assay run in the Bio-Plex system. Cytokine standards were analyzed in duplicates in three different plates with different bead preparations. Pearson correlation coefficient, where 1 is total positive linear correlation and 0 is no linear correlation, was used for estimation of the linear correlation between the signal intensity measured by multiplex and the absorbance obtained by ELISA. To establish the differences between the cytokine profiles of control (non-immunized) chickens and immunized chickens, the chickens were experimentally challenged with RSV(Figure 3). The data show that in response to viral infection, the levels of IL-12 (P ≤ 0.01), IL-2 (P≤ 0.001), and IFNγ (P ≤ 0.05) in plasma samples were significantly increased following RSV activation in immunized chickens compared with the non-immunized group. On the other hand, the IL-10 cytokine was not detected. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that the described system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity. Figure 3. View largeDownload slide Cytokine profile of in vivo stimulated chickens. The specific interleukin (IL-12, IL-2, IFNγ) responses were measured in chickens of the CB line using Bio-Plex system in the plasma samples (IL-10 cytokine was not detected). Horizontal bars indicate the mean values. The results represent at least two independent experiments. Non-immunized (n = 5): non-immunized control; Immunized (n = 5): samples from chickens immunized with the mixture of tetramers SA-vsrcA and SA-vsrcB with biotinylated anti-CD205. Statistical significance determined by Student's t test: *P < 0.05; **P < 0.01; ***P < 0.001. Figure 3. View largeDownload slide Cytokine profile of in vivo stimulated chickens. The specific interleukin (IL-12, IL-2, IFNγ) responses were measured in chickens of the CB line using Bio-Plex system in the plasma samples (IL-10 cytokine was not detected). Horizontal bars indicate the mean values. The results represent at least two independent experiments. Non-immunized (n = 5): non-immunized control; Immunized (n = 5): samples from chickens immunized with the mixture of tetramers SA-vsrcA and SA-vsrcB with biotinylated anti-CD205. Statistical significance determined by Student's t test: *P < 0.05; **P < 0.01; ***P < 0.001. DISCUSSION In this study we describe and validate the Bio-Plex detection system that can quantify multiple chicken cytokines simultaneously in a small sample volume of body fluid. This assay should prove to be a powerful tool in the quantitation of cytokines, or any other soluble product for which antibody pairs are available (Taylor et al., 2001; Yang et al., 2001; Jones et al., 2002; Martins, 2002). Because a large number of antibodies of different specificities are mixed in a single reaction in this multiplex assay, it is possible to envisage any nonspecific reaction that may be detected as a false positive result. To reduce the risk of cross-reaction with other proteins we used the Bio-Plex system, which combines the principle of sandwich immunoassay with the Luminex bead-based technology. The choice of (primary) antibodies is a critical parameter of this assay. Several studies have suggested good correlation between ELISA and multiplex; however, in absolute values these two assays are not comparable (Elshal and McCoy, 2006). One reason may be the different origin of the used antibodies: usually, a commercially available multiplex kit is compared with ELISA from a different provider, and therefore antibodies, which are crucial for all immunosorbent assays, differ in the targeting epitope and affinity to antigen. In this study, the ELISA and multiplex using the very same antibodies, serum blockers, and reporters were compared. However, the absolute values obtained by multiplex were higher; a good correlation in significance between the results obtained in experimental groups was shown. Similarly to previous observations (Vignali, 2000) we have proved that multiplex tests are easy to be performed and reproducible. Besides simultaneous measurement of more cytokines in the very same sample, this method is obviously faster (3.5 h vs. 24 h for ELISA) and cost-saving, especially when six or more cytokines are measured together. Additionally, these assays also minimize the sample volume requirements and significantly reduce the amount of reagents used per cytokine assay. The next important feature of this optimization/validation study is that the dynamic range of the immunoassay for the tested chicken cytokines was greatly improved by Bio-Plex. This resulted in a significant decrease in the LOD/LOQ values for these cytokines, permitting extremely low levels of these cytokines to be quantified in plasma samples that would previously be undetectable by ELISA. Using the multiplex analysis we were able to analyze cytokine production by Th1 and Th2 cells in plasma samples from both immunized and control chickens after the challenge with RSV. The cytokines of the classical Th1 immune profile (IL-12, IL-2, and IFNγ) were significantly increased in the immunized group compared with the control group. This is in accordance with the recent study (Mucksová et al., 2017) where was performed in vitro restimulation of chicken peripheral blood mononuclear cells of immunized and control groups in the same immunization model and evaluated four cytokines (IL-2, Il-10, IL-12, and IFNγ) in peripheral blood mononuclear cells culture supernatants. The cytokine levels of IL-2, IL-12, and IFNγ were also significantly increased in the immunized groups restimulated with v-src antigen. In conclusion, we designed and verified the Bio-Plex technique for practical use in the poultry for quantitative and comparative monitoring of the chicken immune cellular response, until now determined only by the ELISA technique. Additionally, this technique improves quantitative and comparative monitoring of the chicken immune cellular response and makes it possible to monitor the chicken immune system on daily basis if needed. Acknowledgements The authors would like to thank Věra Fialová for statistical analysis. Funding: This work was supported by the National Agency for Agricultural Research, the Ministry of Agriculture of the Czech Republic [grant no. QJ1210041] and by the Technological Agency of the Czech Republic [grant no. TA04010812]. REFERENCES Briles W. E., Bumstead N., Ewert D. L., Gilmour D. G., Gogusev J., Hála K., Koch C., Longenecker B. M., Nordskog A. W., Pink J. R., Schierman L. W., Simonsen M., Toivanen A., Toivanen P., Vainio O., Wick G.. 1982. Nomenclature for chicken major histocompatibility (B) complex. Immunogenetics  15: 441– 447. Google Scholar CrossRef Search ADS PubMed  Carson R. T., Vignali D. A.. 1999. 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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