Veterinary Immunology and Immunopathology, 30 (1992) 319-327
Elsevier Science Publishers B.V., Amsterdam
A sensitive immunoassay for porcine interferon-a H. Diaz de Arcea'c, K. Artursson a, R. L'Haridon d, A. Perers b, C. La Bonnardiere d and G.V. Alm a'b'l aDepartment of Veterinary Microbiology, Division of Immunology, Swedish Universityof Agricultural Sciences, Uppsala, Sweden blnterferon Laboratory, Uppsala University, Uppsala, Sweden CDepartment of Virology, CENSA, San Jose de Las Lajas, Cuba dDepartment of Virology and Molecular Immunology, INRA, Jouy-en-Josas, France (Accepted 10 July 1991 )
ABSTRACT Diaz de Arce, H., Artursson, K., L'Haridon, R., Perers, A., La Bonnardiere, C. and Aim, G.V., 1992. A sensitive immunoassay for porcine interferon-a. Vet. Immunol. Immunopathol., 30:319-327. Two murine monoclonal antibodies (mAbs) directed against different epitopes on recombinant porcine interferon-or (IFN-a) were selected and used to construct a two-site ELISA. This ELISA, when performed in a one-step version, detected about 0.5 units ml-1 of IFN-a and showed similar sensitivity but better precision than a cytopathic effect inhibition bioassay. Estimates of IFN-a in tissue culture medium by the two assays correlated well. In contrast, one or several factors in porcine serum reduced the sensitivity of the ELISA. Measurements of IFN-a in porcine serum was, however, possible in a two-step version of the ELISA, with a sensitivity of about 1 unit IFN-a ml- t. Results of ELISA and bioassay agreed, except that the ELISA possibly produced false positive results in two out of a total of 91 sera negative in the bioassay. In addition, one of 23 sera positive in the bioassay was negative in the ELISA.
Interferon-a and interferon-fl (IFN) are potent broad-spectrum antiviral proteins made early in viral diseases and recognized as important in limiting infection (Gresser, 1984 ). Detection of them in peripheral blood, for example, can be utilised as a signal of ongoing viral infection (Skidmore and Jarlow, 1987). During the breeding of pigs for slaughter in units containing a large number of animals, the stress to the animals is considerable, as is the incidence of infections. We found in a previous study (Artursson et al., 1989) that a large proportion of pigs, about 1 week after transfer to a fattening unit, despite the 'Author to whom correspondence should be addressed at: Immunology (V), Biomedical Center, Box 582, 751 23 Uppsala, Sweden.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
H. DIAZ DE ARCE ET AL.
absence of signs of disease had significant levels of IFN-a in serum detected by a cytopathic effect inhibition bioassay. Consequently, viral infections among the animals may be more c o m m o n than anticipated. Further, some leukocyte functions such as the ability to produce IFN-o~ in vitro seem to be impaired during stressful conditions (Artursson et al., 1989). In order to further evaluate the contribution of genetic and environmental factors to this suggested high incidence of infections as well as to measure their effect on the capacity of leukocytes to produce IFN, it was important to have access to an immunoassay allowing the processing of large numbers of samples with precise determination of IFN-c~ concentrations. Several murine monoclonal antibodies (mAbs) directed to recombinant porcine IFN-a described elsewhere (Lefevre et al., 1990) were therefore evaluated and two selected in order to construct a two-site ELISA. We describe here this immunoassay and its application for determination of IFN-a levels in tissue culture m e d i u m and porcine serum. MATERIALS AND METHODS
Monoclonal antibodies The preparation and properties of the murine mAbs to porcine IFN-ot have been described before (Lefevre et al., 1990). The combination that was selected in the present study was mAb FI 7 on solid phase and mAb K9 conjugated by horseradish peroxidase ( H R P ) . Both mAbs were of IgG j subclass. The mAbs were precipitated from ascitic fluid by a m m o n i u m sulphate at 50% saturation and further purified by fast protein liquid chromatography (FPLC) using a mono S ion exchange column (Pharmacia, Uppsala, Sweden). The IgG concentrations were determined by ELISA. The IgG fractions were dialysed against 10 m M sodium carbonate buffer (pH 9.5 ) overnight and conjugated with H R P (Boehringer, Mannheim, Germany), according to Wilson and Nakane ( 1978 ). The conjugated antibodies were purified by gel filtration on Sephacryl-300 (Pharmacia) and bovine serum albumin (BSA; Sigma, St. Louis, MO ) was added to a final concentration of 10 mg ml-J. All mAbs were stored at - 8 0 ° C .
One-step ELISA for detection of lFN-o~ in tissue culture medium Flat-bottomed 96-well microtitreplates ( I m m u n o plate maxisorp; Nunc, Roskilde, Denmark) were coated overnight at room temperature (RT) with F17 mAb at 0.3/tg m1-1 in 50 m M Tris-HC1 buffer (pH 9.5), using 0.1 ml per well. Non-specific binding sites were then blocked by incubating plates with 0.15 ml per well of diluent buffer consisting of 0.1 M phosphate buffer (pH 6.8) with 0.5% BSA and 0.05% Tween 20 for 1 h at 37°C. Plates were
IMMUNOASSAY FOR PORCINE INTERFERON-o~
washed five times with 0.05% Tween 20 in deionized water. To each triplicate well was then added 0.05 ml of IFN standards or samples and 0.05 ml of HRP-conjugated K9 mAb at a concentration of 0.02/lg m l - ~ diluent buffer. Plates were incubated overnight at RT. After washing as described above, each well of the plates received 0.2 ml substrate solution consisting of 1 m M tetramethylbenzidine (Fluka, Buchs, Switzerland) in 0.1 M potassium citrate buffer (pH 4.25 ) with 2 m M H202. The reaction was stopped by adding 0.1 ml 1 M H 2 S O 4 per well and the absorbance at 450 n m read in a Titertek Multiskan spectrophotometer (Flow Laboratories, Irvine, UK).
Two-step ELISA for detection of lFN-o~ in pig serum In order to measure IFN-ot levels in pig serum, the protocol for the one-step ELISA was modified. The incubation of the samples and HRP-labelled K9 mAb was performed in two steps, i.e. the plates were first incubated overnight at RT with 0.1 ml volumes of either 50% pig serum sample in dilution buffer or standard IFN-c~ in dilution buffer with 50% IFN-negative control pig serum. Plates were then washed five times, and incubated overnight at RT with 0.1 ml dilution buffer containing 0.01/~g m l - i of HRP-labelled K9 mAb per well. The plates were then washed and developed as described above.
Bioassay for IFN The cytopathic effect inhibition bioassay for IFN has been described before (Artursson et al., 1989 ). In brief, monolayer cultures of bovine MDBK cells in 96-well microtitre plates were incubated with twofold serial dilutions of samples for 24 h, and then challenged with Vesicular stomatitis virus (VSV). After a further 24 h, when complete cytopathic effect developed in cultures without IFN, the plates were washed and stained by a crystal violet solution. A laboratory standard consisting of IFN-containing culture m e d i u m from Sendai virus (SV)-stimulated porcine leukocytes was dialysed at pH 2 to inactivate the virus, neutralised and used both in the ELISA and bioassay. This standard was assigned a titre value expressed as antiviral units (U). One unit per millilitre is here defined as that concentration which in the bioassay protects 50% of the cells in a culture against the VSV.
Interferon-containing samples The ELISA was evaluated on several types of samples. Tissue culture medium containing different concentrations of IFN was collected from cultures of porcine mononuclear leukocytes stimulated by SV or by PK15 cells infected with Pseudorabies virus (PRV) as described previously (Artursson et al., 1989).
A R C E
A total of 114 swine sera were available from a previous study (Artursson et al., 1989), of which 23 were defined to contain IFN-o~ by bioassay and subsequent neutralisation of the cytopathic effect inhibition by polyclonal bovine anti-human IFN-ot antibodies that cross-react with porcine IFN. In some experiments, sera were supplemented by the protease inhibitors 4amidinophenyl-methanesulphonylfluoride (APMSF), Na-EDTA, leupeptin and pepstatin (all from Boehringer) at concentrations recommended by the supplier. This was done in an attempt to prevent possible degradation oflFN.
Evaluation of the results All samples were assayed in triplicate. Standard curves were fitted, regression analysis performed and coefficient of variations (CV) calculated using the StatView II program for the Macintosh computer (Abacus Concepts, Berkeley, CA). RESULTS
Correlation between bioassay and ELISA estimates of lFN in culture medium Initial experiments (results not shown) demonstrated that porcine PRVor SV-induced IFN with similar antiviral activity (U ml -] ) in the bioassay were equally well detected at both high and low IFN concentrations in the one-step ELISA. As little as 0.5 U ml-1 produced an absorbance three times the background level caused by tissue culture medium from non-stimulated leukocyte cultures (results not shown). The coefficient of variation (CV) 1000"
400' E E
Fig. 1. Relation between I F N - a concentrations determined by ELISA and by cytopathic effect inhibition bioassay in a series of 23 individual samples of medium from porcine leukocyte cultures stimulated by Pseudorabies virus.
within assays was less than 9.0% over the concentration range 0.5-50 U IFN m l - 1, and less than 10.0% between assays. Samples of tissue culture m e d i u m containing IFN induced by PRV in porcine leukocytes were assayed by both the one-step ELISA and by the conventional bioassay. As shown in Fig. 1 there was a significant correlation ( r = 0.93 ) and a linear relation with a slope of 0.67 between results obtained by the two assays. The numerical values (U m l - 1) produced by the two assays were thus somewhat lower in the ELISA than in the bioassay. Measurements o f IFN in serum
To evaluate the ability of the ELISA to detect IFN in porcine serum, Sendai virus-induced IFN in diluent buffer was mixed with equal volumes of either porcine serum or diluent buffer in the one-step ELISA. As shown in Fig. 2 (A), the presence of porcine serum markedly reduced the sensitivity of the assay. A similar inhibitory effect of porcine serum was not seen in the cytopathic effect inhibition bioassay for IFN, and the protease inhibitors APMSF, NaEDTA, leupeptin and pepstatin did not prevent the serum-mediated inhibition of the ELISA (results not shown). Therefore, porcine serum probably did not degrade IFN. Assuming that porcine serum non-specifically blocked the binding of mAbs to IFN, we attempted a two-step version of the ELISA. Here, mAbs on solid phase were first incubated with IFN in the presence or absence of 50% porcine serum, washed and then incubated with the HRP-labelled mAb to IFNa. When the concentration of the capturing antibodies on the solid phase was sufficiently high, achieved by coating with F17 mAb at or more than 2-
20 IFN U/ml
Fig. 2. Comparison of the one-step and two-step versions of the two-site ELISA for IFN-a performed in the presence ( • • ) or absence ( O O ) of 50% porcine serum. (A) One-step version, with simultaneous incubation of HRP-conjugated mAbs and IFN-a. (B) Two-step version with incubation ofmAb on solid phase and IFN-a with or without porcine serum, followed by washing and incubation with HRP-conjugated mAb.
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0.3 pg m l - 1, this two-step procedure largely prevented the negative effect of the porcine serum (Fig. 2(B) ). By the two-step ELISA, as little as 1 U ml -I of IFN in serum could be detected with an intra- and inter-assay CV of approximately 10% for samples containing 6 U IFN m l - 1 serum.
Correlation between bioassay and ELISA estimates of lFN in porcine sera The IFN levels in a series of serum samples previously analysed by bioassay (Artursson et al., 1989) were in the present study determined by the two-step ELISA. Figure 3 shows the comparison of all sera that were defined as positive for IFN by both assays. There was a good correlation ( r = 0.94) between results of the two assays. However, the slope of the regression line fitted to the data was 0.58 and the apparent IFN concentrations in the ELISA were therefore about 60% of those determined in the bioassay. This was seen despite the fact that the standard curve in the ELISA was constructed using dilutions of the reference IFN in normal porcine serum. As summarised in Table 1, using cutoffs of 1 U m l - 1 in the ELISA and 3 U ml-1 in the bioassay, of the 23 porcine serum samples positive in bioassay, 22 were positive and one was negative in the ELISA. The latter sample contained 4.6 U ml-1 by bioassay. Of the 91 sera that were negative in the bioassay, two were positive in the ELISA (2.4 and 1.1 U m l - 1). We found that the specificity of positive reactions in the ELISA could be tested, because addition of polyclonal bovine anti-IFN-a antibodies simultaneously with the HRP-conjugated mAb in the second step blocked specific reactions (results not shown). Unfortunately, the two potentially false positive sera in the ELISA were not available for such confirmatory testing. 3O 25 O
Fig. 3. Relation betweenIFN-a concentrationsdeterminedby ELISAand cytopathiceffectinhibition bioassayin a seriesof 22 individualporcineserum samples,definedas positive in both assays (cf. Table 1).
IMMUNOASSAY FOR PORCINE INTERFERON-~
TABLE 1 Proportion of porcine sera with apparent content of IFN-a as determined by EL1SA and by cytopathic effect inhibition bioassay ELISA
IA total of 114 serum samples obtained from normal pigs in a fattening unit were analysed. Sera with
1 U m l - l or more in the ELISA or 3 U m l - ~or more in the cytopathic effect inhibition bioassay were taken as positive for IFN-a. DISCUSSION
In the present communication we describe a convenient two-site ELISA for porcine IFN-ot, based on two murine anti-IFN-ol mAbs. This ELISA was sensitive, detecting as little as 0.5 units of IFN per millilitre of tissue culture medium, and produced results that correlated well with those of a cytopathic effect inhibition bioassay for IFN, but showed superior precision. The IFN produced in cultures of leukocytes stimulated by either Pseudorabies or Sendai virus were detected to the same extent, although these viruses might stimulate production of different spectra of the many IFN-a subtypes demonstrated at the gene level in the pig (Lefevre and La Bonnardiere, 1986). The ELISA should therefore be useful for determinations of levels of IFN in cultures of leukocytes stimulated by various microorganisms. The mAbs utilised in the ELISA may also be used in other assays of IFN, including the immunohistochemical staining for intracellular IFN and the detection of IFN-secreting cells by the Elispot technique (Cederblad and Alm, 1990). An important goal was to use the ELISA to measure IFN-a in porcine serum. We found, however, that porcine serum interfered with the one-step variant of the assay. This appeared not to be due to degradation of IFN by proteases. More likely, components in porcine serum interfered with the binding of the murine mAbs to IFN. Similar problems have been noted for a two-site ELISA for detection of IFN-y in h u m a n serum, and was probably mediated by IgG antibodies (Andersson et al., 1989). Another possibility, also described before (Place and Schroeder, 1982 ) is that certain sera can displace antibodies from plastic surfaces. The technical problems were alleviated by having a sufficiently high mAb concentration on the solid phase and by performing the ELISA in two steps, i.e. first incubating IFN-containing sera with mAbs on the solid phase, washing and then incubating with the HRP-labelled mAb. The modified ELISA measured IFN-a in porcine serum with good precision,
H. DIAZ DE ARCE ET AL.
better than that possible with the bioassay and with similar sensitivity. It was noted, however, that serum levels of IFN were about 40% lower as judged by ELISA than by bioassay. The reason for this discrepancy remains to be determined, but could be caused by IFN-a subtypes or IFN-fl not detected by the ELISA. More likely, factors in serum such as cytokines may potentiate the antiviral activity of IFN in the bioassay, without influencing the immunoassay results. The IFN-7 does not seem to be involved, however, because this lymphokine is not detected in the bioassay used in the present study. In 114 serum samples from different pigs (Artursson et al., 1989 ), concordant results were obtained with the ELISA and the bioassay for IFN. Some discrepancies were seen, because the ELISA was negative for one out of 23 bioassay-positive samples. The reason for this may be the presence of lower than immunologically detectable levels of IFN-a, which for various reasons still can be measured in the bioassay, e.g. because of potentiation by additional cytokines. In contrast, the ELISA was positive for two out of 91 bioassay-negative samples. The latter result may be due to IFN not measured by the bioassay, e.g. because of degradation. Alternatively, the two sera may be false positives caused by anti-murine antibodies crosslinking the mAbs in the ELISA. This p h e n o m e n o n was observed with occasional h u m a n sera in a twosite ELISA for IFN-y in previous work (Andersson et al., 1989), and could be eliminated by including either mouse serum or an irrelevant murine mAb as supplement in the diluent buffer of the ELISA. Although we found that truly positive ELISA reactions for I F N - a could be inhibited by polyclonal bovine antibodies to IFN-a, none of the potentially false positives were available for such verification. To conclude, we have described an ELISA which should be instrumental to further explore the use of serum levels of I F N - a as an acute phase reactant defining the incidence of infections, particularly of viral origin, in pig populations. ACKNOWLEDGEMENTS
This work was supported by grants from the Swedish Council for Forestry and Agricultural Research, the Swedish Agency for Research Cooperation with Developing Countries, and the French Agence Nationale pour la Valorisation de la Recherche. We thank Lisbeth Fuxler and Charlotte Persson for performing bioassays.
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Artursson, K., Wallgren, P. and Aim, G.V., 1989. Appearance ofinterferon-a in serum and signs of reduced immune function in pigs after transport and installation in a fattening farm. Vet. Immunol. Immunopathol., 23: 345-353. Cederblad, B. and Alm, G.V., 1990. Infrequent but efficient interferon-a-producing human mononuclear leukocytes induced by herpes simplex virus in vitro studied by immuno-plaque and limiting dilution assays. J. Interferon Res., 10: 65-73. Gresser, I., 1984. Role of interferon in resistance to viral infections in vivo. In: J. Vilcek and E. de Maeyer (Editors), Interferons, Vol. 2: Interferons and the Immune System. Elsevier, Amsterdam, pp. 221-247. Lefevre, F. and La Bonnardiere, C., 1986. Molecular cloning and sequencing of a gene encoding biologically active porcine a-interferon. J. Interferon Res., 6: 349-360. Lefevre, F., L'Haridon, R., Borras-Cuesta, F. and La Bonnardiere, C., 1990. Production, purification and biological properties of an Escherichia col#derived recombinant porcine alpha interferon. J. Gen. Virol., 71: 1057-1063. Place, J.D. and Schroeder, H.R., 1982. The fixation of anti-HBsAg on plastic surfaces. J. Immunol. Methods, 48:251-260. Skidmore, S. and Jarlow, M.J., 1987. Interferon as a viral diagnostic test. J. Virol. Methods, 16: 155-158. Wilson, M.B. and Nakane, P., 1978. Recent developments in the periodate method of conjugating horseradish peroxidase (HRP) to antibodies. In: W. Knapp, K. Holubar and G. Wick, (Editors), Immunofluorescence and Related Staining Techniques. Elsevier-North Holland Biomedical Press Amsterdam, pp. 215-224.