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Keywords:

  • multiplex flow cytometry;
  • TNF-α;
  • IL-8;
  • IL-1β;
  • pigs;
  • microspheres;
  • detection limit

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Background:

Multiplex flow cytometry is in widespread use for detection of cytokines in human samples. However, no report on the measurement of porcine cytokines using this method has previously been published. We report on the detection of the porcine proinflammatory cytokines TNF-α, IL-8, and IL-1β by the xMap-assay for multiplex flow cytometry.

Methods:

Commercially available antibodies to porcine cytokines were used as capture antibodies by attaching them to goat anti-mouse IgG coated microspheres with different fluorescent signatures. By the use of biotinylated detection antibodies and SAv-PE the amount of cytokines bound to the spheres were measured. Experiments were performed to determine the limits of detection and the amount of crossreactivity in buffer, serum, and plasma, using spiking with recombinant porcine cytokines.

Results:

The limit of detection ranged from 0.18 to 12 ng/ml. Generally, the detection limit was higher in serum and plasma, than in buffer. No crossreactivity between reagents was found.

Conclusions:

Porcine proinflammatory cytokines can be detected utilizing this method with satisfactory detection limits, and no crossreaction between the reagents involved. © 2006 International Society for Analytical Cytology

Secreted cytokines cannot be detected directly with flow cytometry and must therefore be captured on a target particle. This can be achieved by coupling antibodies against the analyte of interest to a microsphere. To acquire the amount of analyte, a secondary antibody, conjugated with a fluorophore, is added. In 1999, Carson and Vignali reported simultaneous quantification of 15 murine cytokines in a single sample by the use of multiplex flow cytometry by the xMap™-assay (formerly known as FlowMetrix and LabMap) by Luminex Corporation (Austin, TX) (1). Since then, several applications of multiplex particle based flow cytometry have been established. Reports of successful measurements of, for instance, apoptotic proteins, cancer markers, and different markers of immunological diseases in humans and mice have been described (2–5). Techniques such as multiplex PCR and multiplex flow cytometry make it possible to calculate the cytokine ratio in a sample, which often is considered more important than absolute concentrations of cytokines since the cytokines have abilities to alter each others functions (6, 7).

Infectious diseases of both viral and bacteriological origin are severe problems in the pig industry worldwide. Beside the animal well fare aspects, the economical losses are huge. For instance, production losses due to swine dysentery were estimated at approximately $100 per sow per year (8, 9). In general, porcine infections are controlled by antimicrobial treatments, eradication programmes, environmental measures, and vaccination. However, much of the shortcomings in early detection of an infection and the following treatments are due to lack of understanding of the interactions between the invading microorganism and host. In most cases, interaction between microorganisms and host elicits a cytokine response. The type of the response is not only dependent on the nature of the organism and genetics of the host, but also influenced by previous exposure to the infectious agent. Knowledge about a specific cytokine response indicates the type of immune response mounted, which is a prerequisite for the design of efficient prophylactic and therapeutic measures. The most important cytokines in the early response after an infection are the so-called proinflammatory cytokines. It is well known that proinflammatory cytokines play an important role in the pathogenesis of bacterial infections, e.g. in respiratory diseases caused by bacterial (10, 11) and viral (12) infections. In the case of bacterial infections the immediate cytokine response is dominated by TNF-α, IL-1, IL-6, and IL-8, whereas IFN-α is the main cytokine produced early after viral infections.

When performing studies in veterinary immunology a limiting factor is the lack of species-specific reagents (13). For porcine cytokines, however, the situation is more favorable than for many other species, and a number of antibodies directed against porcine cytokines are now commercially available. In spite of this, no reports on detection of multiple porcine cytokines by multiplex flow cytometry are, to our knowledge, available in the literature. The aim of the present study was to simultaneously quantify the three porcine cytokines, IL-1β, IL-8, and TNF-α by multiplex flow cytometry. To establish that detection was possible not only in buffer, but also in serum and plasma; standard curves were also obtained in these matrices. We report on the limits of detection in various matrices, as well as studies performed to establish the amount of cross-reaction between the antibodies used in the assays.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Reagents and Equipment

Microspheres coated with Goat anti-mouse IgG (GAM IgG) were purchased from R&D Systems (Minneapolis, MN). Regions used were region 07 (LDS002, TNF-α), region 32 (LDS004, IL-8) and region 37 (LDS005, IL-1β). Capture antibodies used were Mab 6811 (IL-1β), Mab 6902 (TNF-α), and Mab 5351 (IL-8), all from R&D Systems, as were the Biotinylated detection antibodies used, BAF 681 (IL-1β), BAF 690 (TNF-α), and BAF 535 (IL-8), all from R&D. The recombinant porcine cytokines used were IL-8 (535-IN), TNF-α (690-PT), and IL-1-β (681-PI), all from R&D Systems. Wash buffer (WB), Microparticle diluent (MPD), Biotin antibody diluent 2 (BAD), and Sav-PE were all from the multiplex assay accessory pack (LDS000, R&D Systems). Analyses were performed in 96-well-plates (MultiScreen 1.2 μm Durapore PVDF membrane, Millipore Corporate, Billerica, MA). For washing, a Multi Screen Resist Vacuum Manifold from Millipore Corporate was used. During incubations the samples were rotated on a Fischer Hematology Mixer (Fisher Scientific, Hampton, NH). The instrument used was the Luminex 100™ equipped with the XYPlatform. The software applied for collection and evaluation of data was Bioplex manager version 4.0 (Bio-Rad, Hercules, CA)

Samples

Serum and plasma were obtained from a healthy three-month old crossbred pig (Yorkshire × Swedish Landrace). The Ethical Committee for Animal Experiments, Uppsala, Sweden, approved the experimental design. Blood was taken from the jugular vein and transferred into vacuum tubes. Heparinized blood samples and blood without additives were centrifuged at 1,500g. Plasma and serum, respectively, were then stored at −80°C until analyzed. The samples were diluted 1:5 with WB prior to their addition to wells.

Preparation of Microspheres

GAM IgG-coated microspheres were transferred to the coupling vial provided with the microspheres. They were pelleted (1 min, 1,000g) and the supernatant was removed. To the microspheres, 65 μl of appropriate capture antibody in a concentration of 250 μg/ml, diluted in WB, was added. The vial was vortexed, and placed on gentle rotation in the dark at room temperature for 1 h. The microspheres were pelleted, and washed twice with 200 μl WB. The supernatant was removed, 65 μl WB was added, and the vial was vortexed. The microphere vials were kept in a refrigerator until use.

Standard Curves

The recombinant cytokines were diluted in WB, porcine serum diluted 1:5 with WB, or porcine plasma diluted 1:5 with WB. The highest concentration prepared with WB was 200 ng/ml. In plasma and serum, the highest concentrations prepared were 33 ng/ml, to correct for the dilution of the serum and plasma. The dilution factor used was four, and eight points were measured, yielding a lowest concentration of 12 pg/ml. The final concentration in the wells was half of this, due to dilution with equal amount of microparticle solution (see later). Samples without added cytokines were also analyzed.

Cross-Reaction Samples

Solutions were also prepared with only one recombinant cytokine present. These were prepared with a concentration of 200 ng/ml of cytokine in order to check if any cytokine was captured by antibodies to the other cytokines.

Multiplex Assay

A solution was prepared, with 0.4 μl of each microparticle solution and 50 μl MPD for each well in the assay. To a plate where the wells had been pre-wet with 100 μl WB, which was removed prior to adding the microparticles, 50 μl of the microsphere mix was added to each well. Then, 50 μl of sample, prepared as described earlier, was added. The plate was placed on gentle shaking for 1 h in the dark at room temperature. The solutions were removed, and the wells were washed three times with 100 μl WB, using a vacuum manifold. From a mix of the biotinylated detection antibodies, diluted to a concentration of 3 μg/ml with BAD, 50 μl was added to each well. The plate was placed on gentle shaking for 1 h in the dark at room temperature. The solutions were removed, and the wells were washed three times with 100 μl WB, using a vacuum manifold. Then, 50 μl of Sav-PE, diluted 1:100 in WB, was added to each well. The plate was placed on gentle shaking for 30 min in the dark at room temperature. The solutions were removed, and the wells were washed three times with 100 μl WB, using a vacuum manifold. Finally, 100 μl WB was added to each well and the samples were analyzed using the Luminex 100™ with the Bioplex manager software. From each well, 100 beads/region was analyzed.

Data Analysis

The results were analyzed using BioPlex Manager 4.0. The limit of detection (LOD) was determined by interpolation from standard curves obtained by logistic 5PL regression. Sample dilutions were taken into account when calculating the amount of cytokine in samples.

Limit of Detection

LOD for each cytokine was determined by analysis of ten wells respectively containing WB, serum, and plasma without added cytokines. The mean fluorescence intensity (MFI) and standard deviation (SD) were calculated and the LOD was set to the concentration correlating to the average MFI + 3SD. The LOD was calculated for all cytokines in all matrices.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Limit of Detection

In Table 1 the LOD for the three cytokines TNF-α, IL-1β, and IL-8 in different matrices are shown. The LOD was higher for cytokines diluted in serum and plasma than for cytokines diluted in WB. Lower LOD values were obtained for IL-8 and TNF-α than for IL-1β in all matrices, with the lowest LOD obtained being 0.18 ng/ml for IL-8 diluted in buffer, and the highest being 12 ng/ml for IL-1β diluted in plasma or serum.

Table 1. The Limit of Detection (LOD), Determined Using Ten Wells Containing the Different Matrices, was Calculated as Average MFI + 3SD
CytokineLOD bufferLOD serumLOD plasma
  1. Values given are in ng/ml.

IL-1β1.61212
IL-80.181.91.3
TNF-α0.230.890.92

Standard Curves

Representative standard curves are shown in Figure 1 for all the three cytokines diluted in wash buffer. The overall appearances of standard curves were similar in the different matrices.

thumbnail image

Figure 1. Standard curve for IL-1β (a, LOD = 1.6 ng/ml), IL-8 (b, LOD = 0.18 ng/ml), and TNF-α (c, LOD = 0.23 ng/ml), diluted in wash buffer. Added concentration of cytokine in ng/ml is given on the X-axis, mean fluorescence intensity on the Y-axis. Values shown are means of Duplicate samples.

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Cross-Reactivity Between Analytes

The results for samples spiked with one of the cytokines showed no detectable cross-reactivity between analytes (Table 2), since results for micropheres not designed to capture the added cytokine in all cases showed results below LOD, which, in this table, is expressed as MFI values, since we found no purpose in calculating concentrations below LOD.

Table 2. Obtained MFI Values in Different Matrices, When Samples Were Spiked with one of the Cytokines in a Final Concentration of 100 ng/ml
Cytokine used for spikingTNF-α spheresaIL-1β spheresbIL-8 spheresc
  • Results shown are means of duplicates. LOD is expressed as MFI value, determined as average MFI of blank samples + 3SD.

  • a

    LOD buffer = 362, LOD serum = 167, LOD plasma = 132.

  • b

    LOD buffer = 786, LOD serum = 373, LOD plasma = 388.

  • c

    LOD buffer = 187, LOD serum = 176, LOD plasma = 214.

Buffer
 TNF-α20,603421158
 IL-1β13414,519142
 IL-813237719,348
Serum
 TNF-α14,417231198
 IL-1β1003780192
 IL-89933812,146
Plasma
 TNF-α14,492259178
 IL-1β1004047163
 IL-88027417,727

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

In this study three porcine proinflammatory cytokines are simultaneously detected. The studies of the limits of detection show that cytokine levels around 1 ng/ml can be successfully measured using this assay. Further work on lengths of incubation, concentrations of antibodies and SAv-PE, and adjustments of numbers of beads used might further lower the limit of detection. The possibility to use GAM-IgG coated microspheres has eliminated the need for coupling capture antibodies covalently to microspheres, a procedure that we have found to be cumbersome, and to require extensive work on optimization. Using multiplex assays, it is now possible to measure many human cytokines simultaneously in plasma samples, e.g. Szodoray et al. measured 25 cytokines simultaneously in patients with primary Sjögren's syndrome (14). The commercially available kits for detection of human cytokines can detect concentrations down to a few pg/ml in blood (15). From the data presented in the present study, the possibility that contaminating mouse IgG might be present in the anti-porcine cytokine reagent, and thus affect the potency of the assay, cannot be ruled out. Even though the assay presented here does not detect that low concentrations, it is a valuable first step towards a multiplex panel of porcine biomarkers.

The present paper is, to our knowledge, the first paper written on multiplex detection of porcine cytokines by flow cytometry. Other options for detection of porcine cytokines also exist, one is to study cytokine mRNAs by PCR (16) or in situ hybridization (17). Another is to quantify the amount of intracellular cytokines using intracellular staining followed by flow cytometry (18). In comparison, the xMap assay presented here measures the actual amount of cytokines present in body fluids, while measurement of cytokine mRNAs measures an earlier event. The mRNA methods also involve various levels of tissue processing. Existing ELISA methods, although at present having lower limits of detection than the method presented here, suffer from the drawback of only being able to detect single cytokines. The interest of a versatile method for the detection of porcine cytokines is obvious, since the cytokine pattern is affected in various pig diseases (19–25).

The detection limit of the assay was lower in buffer than in serum and plasma. It is well-known that heterophile antibodies cause interferences in immunoassays performed in human serum and plasma (26). It is plausible to believe that this also is the case in serum and plasma from pigs. As mentioned previously, further work on the experimental setup will likely lower the detection. An option here would also be to include blocking steps in the incubation to lower the possibility of unspecific binding.

That no crossreactivity was found between the different antibodies used in the assay is a major benefit, enabling reliable detection of these cytokines in the same sample. It is especially a great benefit that no cross-reactivity was found in serum and plasma samples, ensuring us that such samples can be processed without the risk of false-positive results.

Crossreactivity can otherwise be a major obstacle to measuring multiple proteins in the same sample. This is especially true in veterinary research, where the lack of species-specific reagents often force scientists to use reagents originally developed for other species (10, 27).

The assay is very flexible, enabling rapid addition and withdrawal of new biomarkers depending on what disease or physiological event is to be studied. In the future, the addition of other markers can be foreseen. To obtain a complete view of the immune response, it is important not only to asses stimulating but also dampening factors. Also, other markers such as acute phase proteins and investigations of the presence of bacterial and viral proteins in samples are of interest. Our work will continue in this direction. We will now also progress towards measuring cytokine levels in pigs experimentally infected with Brachyspira hyodysenteriae.

To conclude, Porcine proinflammatory cytokines can be detected utilizing this method, with satisfactory detection limits and no crossreaction between the reagents involved.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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