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A sensitive antiviral neutralization bioassay for measuring antibodies to interferons
Journal of Immunological Methods, 127 (1990) 241- 248
241
Elsevier JIM05486
A sensitive antiviral neutralization bioassay for measuring antibodies to interferons M o r t e n Bagge H a n s e n , C h r i s t i a n R o s s a n d K u r t Berg The Interferon Laboratory, Institute of Medical Microbiology, University of Copenhagen, Juliane Maries Vej 22, DK-2100 Copenhagen O, Denmark
(Received 2 May 1989, revised received 2 October 1989, accepted 6 November 1989)
An improved bioassay for measuring neutralizing antibodies to interferons (IFN) is described. The assay is based upon an objective and precise quantification of the viral cytopathic effect. This effect is measured via the dehydrogenase-system in cells, and quantified spectrophotometrically. Virus-infected cells, in contrast to non-infected cells, possess low enzyme activity resulting in low O D signals. This fall in O D can be prevented by the addition of a small, but fixed amount of IFN before the addition of virus. Anti-IFN sera will neutralize the protective effect of IFN. This effect can be quantified by measurement of the reduction in the O D signals. Antibodies to recombinant IFN were found to cross-react with human leukocyte I F N although to a ten-fold lower degree. The assay requires no expensive reagents, it is performed in 96-well microtrays and the results can be measured in an ordinary ELISA scanner. The assay is highly reproducible, yielding inter- and intra-assay variability of less than 10%. The sensitivity is much higher than that reported previously for the CPE technique and that of ELISA techniques. Key words: Antibody to interferon; Antiviral neutralization bioassay; Microculture tetrazolium assay; MTT
Introduction
Interferons are known to have multiple effects on cells (antivirai, antiproliferative, etc.) and over the last 10 years their role in a wide variety of
Correspondence to: K. Berg, The Interferon Laboratory, Institute of Medical Microbiology, Juliane Maries Vej 22, DK-2100 Copenhagen B, Denmark. Abbreviations: ANB, antiviral neutralization bioassay; IFN, interferon; FCS, fetal calf serum; MTT, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PFU, plaque forming units; VSV, vesicular stomatitis virus; EMCV, enccphalomyocarditisvirus; MOI, multiplicityof infection; DMF, dimethylformamide; CIF, crude human leukocyte IFN; rHuIFN-a2~, recombinant human leukocyte IFN-a2a (Roferon); rHuIFN-a2b, recombinant human leukocyte IFN-azb (Intron A).
diseases has been studied (reviewed by Finter and Oidham, 1984). The-introduction of recombinant I F N has made it possible to further increase the numbers of such studies. However, in contrast to human leukocyte IFN, recombinant I F N has proved to be immunogenic in some patients since up to 60% of patients receiving r H u I F N - a 2 treatment have been reported to develop antibodies to the IFN (Steis, 1988). In addition, in recent years conflicting reports on the presence or absence of antibodies to IFN in patients receiving I F N treatment have appeared (Jakobs, 1988; Figlin, 1988; Freund, 1988; Von Wussow, 1988). These reports are based on two essentially different methods: (i) solid-phase immunoassays: the immunoradiomettic assay, I R M A (Protzmann, 1984) and the enzyme-linked immunosorbent assay, ELISA (Hennes, 1987), (ii) bioassays measuring the neutraliz-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
242 ing activity of sera for the antiviral action of IFNs. The former assays are fast and easy to perform, whereas the latter are of relatively low sensitivity and accuracy (Grossberg, 1984), which, in turn, can lead to ambiguities of interpretation. Recently, we have shown that the measurement of dehydrogenase enzyme activity permits the quantification of interferons (Berg et al., 1988). The method is based on the following principle: dehydrogenase enzyme activity in uninfected cells is high compared to that of virus-infected cells. A measure of the enzyme activity can be obtained spectrophotometrically by cellular transformation of a tetrazolium salt (MTT) into formazan (Mosmann, 1983). Uninfected cells will give high OD signals whereas virus-infected cells will yield significantly lower OD signals. IFN-treated virus-infected cells, however, will produce a signal which is 90% of that from uninfected cells, despite the addition of challenge virus. If antibody to IFN is present, the protective effect of IFN will be abrogated. This will lead to sharp decrease in the OD signal and make it possible to determine whether a neutralizing entity, for example antibody, is present or not. The above principle relies on a very precise quantification of the biological activity of IFN which, therefore, must be measured objectively. To establish such a method, the amounts of cells, virus and IFN must first be defined as precisely as possible. The experimental procedure has now been used for more than 5 months yielding inter- and intra-assay variations below 10%.
cells were grown in Eagle's MEM with the addition of 5% FCS, 1% penicillin, 1% streptomycin, 1% L-glutamine, and 5% N a H C O 3 in Nuncione plastic bottles. Cells were split twice weekly at different cell densities according to standard procedures using trypsin. Cell controls
Cells were seeded in microtrays in 100 ~1 medium including 5-10% serum and incubated for 72 h. The minimum seeding concentration yielding an OD signal > 0.5 was established, in this study 10,000 cells. Viruses
Vesicular stomatitis virus (VSV) was obtained from Dr. D. Burke (Warick University, U.K.); murine encephalomyocarditis virus (EMC) was kindly provided by Dr. S. Grossberg, WI, U.S.A. The viruses were propagated in murine L cells and supernatants were stored at - 7 0 o C. Viruses were plaque titrated before being frozen. Virus controls
After 48 h of incubation challenge virus was added to the wells at a concentration resulting in 90% cell destruction after 24 h of infection at which time the MTT assay was performed. Interferons
MTT, a tetrazolium salt (Sigma cat. no. M2128) was dissolved in autoclaved PBS (5 mg/ml), sterile filtered, and stored at 4 ° C in a dark bottle; sodium dodecyl sulfate, SDS (Sigma, practical grade) and dimethylformamide, D M F (Fluka cat. no. 40250, analytical grade).
Crude human leukocyte interferon (CIF), generously provided by Dr. Osther, Dallas, had been obtained from buffy coat suspensions to which Sendai virus had been added according to the method of Cantell (Berg, 1982); specific IFN activity was 104 I U / m g protein. Pure recombinant human leukocyte interferon (rHulFN-az), kindly provided by Essex-Pharma, Denmark, was produced in E. coli (Biogen, U.S.A.). The IFN assays described were calibrated using the 69/19 international reference preparation for human leukocyte IFN (from NIBSC, U.K.). All IFN activity was expressed in international units.
Cell lines
M T T assay
MDBK cells were from the American Type Culture Collection, and A549 cells from Dr. A. Meager, National Institute for Biological Standards and Control, London. M D B K and A549
Mosmann's method was modified as follows: 25 ~1 of a 5 m g / m i stock solution of MTT were added to each well and after incubation for 2 h at 37°C, 100 /.tl of extraction buffer (12.5% SDS,
Materials and methods Chemicals
243 45% D M F and p H 4.7) were added to dissolve formazan-protein complexes. After mixing, the ODs were measured at 570 nm using a Titer-Tech 96-well multiscanner, employing solubilizer as the blank probe. No medium was removed prior to the addition of any ingredient (Mosmann, 1983; Hansen, 1989).
Serum samples A polyclonal rabbit antiserum against crude human leukocyte IFN was produced by injecting a chinchilla rabbit subcutaneously with 100,000 IU per injection every second week as previously described (Berg, 1984). This was done for 18 months. The neutralization titer as based on the CPE method was approx. 6,000 I N U / m l (using 3 - 5 I U / m l of IFN during the period of incubation). Serum samples from two patients suffering from chronic hepatitis were collected and allowed to clot at room temperature; the sera were separated by centrifugation at 500 × g for 10 rain. Samples were stored at - 7 0 ° C until analyzed. Serum samples from Dr. Von Wussow, Hannover, F.R.G., had been collected from patients who were found to be positive in the ELISA procedure. Prior to the assay the sera were heat inactivated at 56 ° C for 30 min.
A ntioiral neutralization bioassay This was performed in 96-well tissue culture plates (Nunc, Roskiide, Denmark). Ten-fold serial dilutions of sera starting at 1/20, were incubated for 1 h at 3 7 ° C with 0.13-4 I U / m l IFN in a total volume of 100 /zl in Eagle's minimal essential medium, supplemented with 5% FCS (Gibco). The samples were transferred to cell monolayers grown from 24 h before (10,000 cells seeded in 100 #1 Eagle's MEM + 5% FCS). After overnight incubation at 3 7 ° C challenge virus was added. After further incubation for 24 h at 3 7 ° C 25 #! M T T were added to the wells, followed by 100 ~1 iysis buffer 2 h later, as described under the M T T assay. The neutralizing titer was defined as the reciprocal of the serum dilution causing a 50% decrease in the antiviral effect of 1.0 I U / m l of I F N (provided that neither serum toxicity nor antiviral activity were found).
Serum toxicity controls Serum samples diluted 1 / 2 0 were incubated with cells (neither I F N nor virus were added) and after 72 h the Mqq" technique was used and the O D signals compared to those of the cell control.
Antiviral controls (sample IFN-control) Serum samples diluted 1 / 2 0 were added to the cells and after 48 h of incubation, 100 /zl virus dilution were added. After another 24 h of incubation the M T T technique was used to evaluate cytotoxicity. The O D signals were compared to those of the virus controls.
Enzyme immunoassay for the detection of antibodies to interferon-a (ELISA kit from Anawa Laboratories, Switzerland) The assay comprised a solid-phase enzyme immunoassay based on the 'sandwich' principle. Beads sensitized with recombinant interferon-a 2 were incubated with serum samples. Interferona2-peroxidase conjugate was then added and the incubation continued (the conjugate being bound to the remaining free combining site of the captured antibodies). After washing, enzyme activity bound to the beads was determined by incubation with the usual substrate (OPD) and the results read at 492 nm in a Titer-Tech multiscanner.
Microscopic examination of CPE Cell layers were scored microscopically subsequent to the development of full CPE in virus controls. Scorings were as follows: 100% of cell destruction was given the number 4; 75% = 3, 5 0 % = 2 , 2 5 % = 1 and 0 % = 0 .
Neutral red procedure The medium was removed by inversion of the tray. The cell layer was washed twice with PBS p H 7.4, and the medium replaced by 100 #1 MEM containing 67 # g / m l of neutral red dye. After 2 h incubation at 37°C, the wells were emptied, washed twice with PBS p H 7.4, and the dye accumulated in the cells was extracted with 100 /~1 of 50% ethanol in 1% acetic acid. Color development in the microtrays was then quantified at 540 nm in a Titer-Tech multiscanner (Forti et al., 1985; Hansen et al., 1989).
244
Statistics Samples were assayed at least in triplicate and the results are presented as means and standard deviations. Each experiment was performed at least three times. The intra- and interassay variation was below 10%.
Results Since detection of IFN neutralization requires a delicate balance between cells, virus and IFN, the interplay of these factors on the sensitivity of the assay was first studied. Optimization of each parameter led to a laboratory protocol for performing a standard M'I-T antiviral neutralization bioassay (ANB). Examples illustrating this approach are given below.
neutral dye red method, making the former method more sensitive.
Interferon concentration Within the range 0.13-1.00 I U / m l an almost linear correlation between I F N concentration and O D readings (0.05-0.4) was seen (Fig. 1C), and the slope indicated that small changes in IFN concentration resulted in a large change in O D readings. Approximately 80% cell protection was obtained at an I F N level of 1.00 I U / m l , that was used in the experiments. Parallel experiments using the M D B K cell line (which is very sensitive to IFN alpha) and VSV were also performed. The parameters ensuring an optimal procedure were found to be: 10,000 cells/well (MDBK); infecting dose 10,000 p.f.u./well (VSV). The IFN titration reached an endpoint at 1.50 I U / m l with either C I F or rHulFN-a2a (data not shown).
Seeding cell density Cell densities from 1250 to 7500 cells/well yielded a linear correlation between the number of cells and O D readings (Fig. 1A). A saturation plateau was reached at 7500 cells/well. In order to minimize intra-assay variation yet still obtain high sensitivity (i.e., a steep slope in the dose-response curve) 10,000 cells/well were used.
The M T T antiviral neutralization bioassay (MTTANB) The results of titration of a rabbit antiserum against C I F in the M T T - A N B (using A549 cells and EMCV) showed the neutralization titer to be close to 160,000 IFN neutralizing units ( 1 N U ) / m i .
Virus concentration
The virus titration curve showed almost no CPE at a dilution of 10 -6 (Fig. 1B), but a distinct dose-response relationship within the dilution range 10-4-10 -3 . The lowest virus concentration (EMC) to cause maximum suppression of the O D signal was 10-2 corresponding to 25,000 P F U / m l or MOI 25; this virus concentration was subsequently used throughout the study. Three methods for the detection of virus cytopathic effects (microscopic examination, CPE; the neutral red and the M T T method) were examined and the results are summarized in Table I. The M T T method gave a 20-fold difference in the O D signal between non-infected/infected cells in contrast to the neutral red method which yielded a 3-4-fold difference. As expected, the CPE method was far less able to discriminate between the different levels of infection. It is obvious that small amounts of interferon influence the O D readings more readily in the M T T method than in the
TABLE 1 C O M P A R A T I V E Q U A N T I F I C A T I O N O F VSV BY T H R E E METHODS M D B K cells in suspension were seeded in 100 /tl medium supplemented with 5% FCS (10,000 cells/well). After 24 h of incubation at 37 ° C in 5% CO 2 the medium was replaced with 100 ~1 medium supplemented with 3% FCS containing two-fold dilutions of VSV starting at 50,000 plaque forming units (PFU) of challenge virus. After an additional 20 h of incubation at 37 ° C in 5% CO 2, the virus CPE was quantified. Results are shown as the median of quadriplicates (OD readings × 103). Preparation number
Microscopic examination
Neutral red-method
MTTmethod
1
4
2 3 4 5 6 7 8
4 4 4 3 2 1 0
120 119 153 131 214 317 339 425
35 60 136 228 414 531 632 650
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naHu IFN (CIF) iq4.nl 0.13 0.5 1.0 2.0 3.0 4.0 Fig. I. A: Seeding cell density. A549 cells were seeded in 100 pl volumes of medium containing 10% FCS and incubated in 5% CO 2 at 37 ° C for 72 h after which the MTT assay was performed. (Means of triplicates. SD < 10%, not shown). B: Virus titration. After seeding 10,000 A549 cells/well the microtray was further incubated for 48 h, and the medium was replaced with 100 #l fresh medium supplemented with 2% FCS containing EMC virus in serial ten-fold dilutions. The cells were further incubated overnight and the MTT assay performed as described. (Means of triplicates, SD < 10%, not shown). C: Interferon titration. A549 cells, 10,000/well, were seeded and after incubation for 24 h the medium was replaced by medium containing crude interferon-a (CIF), yielding the final concentrations of 0.13-4.00 1U/ml. After an additional 24 h of incubation the medium was replaced by 100 /zl medium containing EMC virus diluted 10- 2 and the MTT assay was performed after 24 h of infection. (Means of triplicates, SD < 10%, not shown.)
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In contrast to the virus control (OD = 0.024) the IFN-treated cells ( + virus) yielded an O D of 0.429 using CIF; 0.449 using rHulFN-%b (Intron A) and 0.404 using rHulFN-%a (Roferon) (Fig. 3). This indicates very good cell protection with all three IFNs. Serum from patient A was titrated against CIF, Intron A and Roferon. The neutralizing titer was found to be 40,000 I N U / m l against C I F and 400,000 I N U / m l against Intron A and Roferon, calculated as the reciprocal of the serum dilution which significantly abolished the antiviral activity of 1.0 I U / m l of IFN (Fig. 3A). The serum from patient B, which was antibody-negative in the ELISA, was titrated in a similar way and the neutralizing titer was 40 I N U / m l against CIF, 400 I N U / m l against Intron A and 4000 I N U / m l against Roferon (Fig. 3B). The minimum amount of serum necessary to perform the assay was 0.5 ml. However, by changing the dilution range it was possible to further modify the method and use sample volumes of only 100 ~1 (data not shown).
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o, IFN units/ml Fig. 2. ANB and titration of a rabbit serum. Titration of a
rabbit antiserum against CIF in a neutralization assay with A549 cells and EMCV. Two-folddilutions of serum starting at 40,000, were incubated for 1 h at 37°C with equal volumes (100 /~1) of medium supplemented with 5% FCS including 0; 0.50; 1.00 and 2.00 IU/ml (final concentration) crude IFN-a, after which any residual IFN in the samples was measured by the MTT method. The dashed curve represents IFN controls. C.C. = cell controls. T = x 103. Results are shown as means of triplicates (SD was less than 10%,not shown).
The data in Fig. 2 showed that the neutralizing titer was the same when using 0.5, 1.0 and 2.0 C I F I U / m l . The MTT-ANB was also applied using serum from two patients with chronic hepatitis who had been receiving IFN treatment (patients A and B). One of them (A) developed antibodies to IFN as measured by the ELISA, whereas the other (B) was consistently sero-negative in the anti-IFN ELISA, which is based on r H u l F N - a 2. At a dilution of 1/20, sera from both patients showed no antiviral activity, and no cell toxicity. The cell control yielded an O D signal of 0.454 compared with an OD signal of 0.455 for the standard bovine serum.
Discussion Since the tetrazolium salt MTT was first introduced (Mosmann, 1983) for use in biological assays for cytokines it has been extensively used. We have shown that the MTT method is suitable for the quantification of viral CPE and, in consequence, also for the quantification of IFN activity and neutralization. The method has proved superior to conventional antiviral neutralization bioassays which involve, for example, microscopy or neutral r e d / t r y p a n blue staining for quantification of, for example, viral CPE. It appears from Table I that the MT'T method yields a far more detailed grading of the results; microscopic examination gives the impression of a 100% CPE in those cases in which OD readings of the neutral red method vary from 0.119 to 0.153 whereas the corresponding O D readings of the MTT method vary from 0.035 to 0.228. This enables us to work with very small IFN concentrations and to detect very low levels of IFN neutralization. Precise quantification of IFN activity is crucial for a sensitive ANB. Traditionally, the neutralizing titer is defined as the reciprocal of the antiserum dilu-
247
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Fig. 3. MTT-ANB results from two patients suffering from chronic hepatitis. A: Serial dilutions of serum from the ELISA antibody-positive patient (patient A); and B: from the ELISA-negative patient (patient B) were prepared in triplicate using medium supplemented with 2% ( v / v ) fetal calf serum. To each well IFN was added to a final concentration of 1.00 I U / m l . The sera were titrated against three species of HulFN-a: IFN 1 = crude human leukocyte IFN-a (CIF); IFN 2 = rlFN-a2b (lntron A); and IFN 3 = rlFN-a2a (Roferon). C.C. = cell controls; V.C. = virus controls. T = × 103 (Means of triplicates, SD < 10%. not shown).
tion which will abolish the protective effect of, say, 10 I F N U / m l (Kawade, 1986). The drawbacks of the traditional CPE end-point neutralization method is its lack of precision and sensitivity, which is in part, attributable to the microscopic examinations. If, for example, the I F N level is raised to 20 U during the period of incubation, the CPE method becomes more reliable, but at the same time, the sensitivity of the assay is considerably reduced. This point was illustrated in one example: the rabbit anti-human | F N serum was titrated to 6000 I N U / m l using 4 - 8 I U / m l CIF. The same serum was found to have a titer of 1200-3000 if 10-14 I U / m l C I F were used (data not shown). However, with the MTT-ANB, using 1 I U / m l CIF, the serum had a titer of at least 160,000 N U / m l , reflecting an increase in sensitivity of more than 100. It is of interest to note that although the two patients with hepatitis had received Intron A only, the both developed antibodies to lntron A and
Roferon. These two recombinant I F N s only differ by a single amino acid at position 23 where they have arginine and lysine, respectively. It has been a point of discussion whether this change in amino acid position could result in a difference in immunogenicity (Figlin and Itri, 1988). The patients also developed antibodies to the human leukocyte IFN, which in contrast to the highly purified single molecular species of r H u I F N - a 2, is a mixture of several subspecies of IFNs, although at a ten-fold lower level. This can probably be ascribed to the fact that the neutralizing antibodies produced against r H u I F N - a 2 recognize the same or closely related epitopes present on the 15 subspecies of human leukocyte IFN. To the best of our knowledge, this is the first report which directly demonstrates that in certain patients the use of a single recombinant IFN species can also lead to the production of antibodies against human leukocyte IFN. The end-point of the MTT-ANB titer of a serum can be defined
248
by several approaches. For example: (a) as the reciprocal of the highest serum dilution which produces an OD signal significantly lower than the means of the OD signals from virus and IFN + virus controls; (b) as any OD signal which is 3 SD below that of IFN + virus control; (c) as any signal which is 50% below that of the 1FN + virus control. It is obvious that the mode of defining the end-point influences the sensitivity of the method greatly (Kawade, 1986). At present we have chosen suggestion (c) since the values obtained have been highly reproducible over periods of 5 months, with inter- and intra-assay variation below 10%. A more correct approach would be to apply nonparametric statistics using the Mann-Whitney test. The antiviral neutralization bioassay (ANB) is the standard method recommended by the World Health Organization for measuring neutralization of the biological activity of IFN. We would like to propose the use of the modified MTT-technique in the ANB, as it has proved a very reliable method.
Acknowledgements The authors are grateful to Dr. Allan Randrup Thomsen and Professor Charles J. Pfau for helpful comment and discussion. The expert help by Mrs. Beth Simonsen and Thorbj(3rg Sandquist is greatly acknowledged. This investigation was supported by The Danish Cancer Society (no. 87-022) and by The Danish Medical Research Council (no. 1276-88; no. 127947), and The Carlsberg Foundation (1989).
References Berg, K. (1982) Purification and characterisation of murine and human interferons. A review of the literature of the 1970s (Thesis). Acta Pathol. Microbiol. Immunol. Scand. Sect. C 279 (suppl.), 1-136. Berg, K. (1984) Identification, production, and characterization of murine monoclonal antibody (LO 22) recognising 12 native species of human alpha interferon. J. Interferon Res. 4, 481-491. Berg, K., Hansen, M.B. and Nielsen, S.E. (1988) A new sensitive bioassay for precise quantificatinns of interferon activity as measured via the mitochondrial dehydrogenase func-
tion in cells (M'lq-method). J. Interferon Res. 1 (suppl.), 67 (abstract). Figlin, R.A. and Itri, L.M. (1988) Anti-interferon antibodies: a perspective. Sere. Hematol. 25, 9-15. Figlin, R.A., DeKernion, J.B., Mukamel, E., Palleroni, A.V.. ltd, L.M. and Sarna, G.P. (1988) Recombinant interferon alfa-2a in metastatic renal cell carcinoma: assessment of antitumor activity and anti-interferon antibody formation. J. Clin. Oncol. 6, 1604-1610. Finter, N.B. and Oldham, R.K. (1984) Interferon 4. In Vivo and Clinical Studies. Elsevier. Amsterdam, pp. 1-363. Forti, R.L., Moldovan, R.A., Mitchel, W.M., Gallicoat, P., Schufman, S., Davies, H.A. and Smith, Jr., D.M. (1985) Application of an objective biological assay of human interferons to clinical specimens and a survey of a normal population. J. Clin. Microbiol. 21,689. Freund, M., Von-Wussov, P., Knuver, Hopf, J., Mohr, H., Pohl, U., Exeriede, G., Link, H., Wilke, H.J. and Polivoda, H. (1988) Treatment with natural human interferon alpha of a CML-patient with antibodies to recombinant interferon alpha 2b. Blur 57, 311-315. Grossberg, S.E. and Sedmak, J.J. (1984) Assay of Interferons. in: N. Finter (Ed.), Interferon 1. Elsevier, Amsterdam, pp. 189-213. Hansen, M.B., Nielsen,.S.E. and Berg, K. (1989) Re-examination and further developments of a precise and rapid method for measuring cell growth/cell kill. J. lmmunol. Methods 119, 203-210. Hennes, U., Jucker, W., Fischer, E.A., Krummenacher, T., Palleroni. A.V., Trown, P.W., Linder-Ciccolunghi, S. and Rainisio, M. (1987) The detection of antibodies to recombinant interferon alfa-2a in human serum. J. Biol. Stand. 15, 231-244. Jakobs, S.J., Sullivan, L.M., Salfi, M., Grossberg, H., Spiegel, R.J., Leibovitz, P.J., Oden, E.M., Kelsey, D.K. and Treuhaft, M.W. (1988) Antibodies to recombinant human interferon alpha 2b (Intron A) were detected in only a small number of 101 Intron A recipients. J. Biol. Response Modif. 7, 447-456. Kawade, Y. (1986) Quantitation of neutralization of interferon by antibody. Interferons Methods Enzymol. 119, 558-573. Mosmann, T. (1983) A rapid colorimetric assay for cellular growth and cytotoxicity assays. J. Immunol. Methods 65, 55 -63. Protzmann, W.P., Jacobs, S.L., Minnicozzi, M., Oden, E.M. and Kelsey, D.K. (1984) A radioimmunologic technique to screen for antibodies to alfa-2 interferon. J. Immunol. Methods 75, 317-323. Steis, R.G., Smith, J.W., Urba, W.J., Clark, J.W., Itri, L.M., Evans, L.M., Scoenberger, C. and Longo, D. (1988) Resistance to recombinant interferon alfa-2a in hairy-cell leukemia associated with neutralizing anti-interferon antibodies. New Engl. J. Med. 318, 1409-1413. Von Wussow, P., Freund, M., Dahle, S., Jakschies, D., Poliwode, H. and Deicher, H. (1988) Immunogenicity of different types of interferons in the treatment of hairy-cell leukemia. New Engl. J. Med. 319, 1226.
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Elsevier JIM05486
A sensitive antiviral neutralization bioassay for measuring antibodies to interferons M o r t e n Bagge H a n s e n , C h r i s t i a n R o s s a n d K u r t Berg The Interferon Laboratory, Institute of Medical Microbiology, University of Copenhagen, Juliane Maries Vej 22, DK-2100 Copenhagen O, Denmark
(Received 2 May 1989, revised received 2 October 1989, accepted 6 November 1989)
An improved bioassay for measuring neutralizing antibodies to interferons (IFN) is described. The assay is based upon an objective and precise quantification of the viral cytopathic effect. This effect is measured via the dehydrogenase-system in cells, and quantified spectrophotometrically. Virus-infected cells, in contrast to non-infected cells, possess low enzyme activity resulting in low O D signals. This fall in O D can be prevented by the addition of a small, but fixed amount of IFN before the addition of virus. Anti-IFN sera will neutralize the protective effect of IFN. This effect can be quantified by measurement of the reduction in the O D signals. Antibodies to recombinant IFN were found to cross-react with human leukocyte I F N although to a ten-fold lower degree. The assay requires no expensive reagents, it is performed in 96-well microtrays and the results can be measured in an ordinary ELISA scanner. The assay is highly reproducible, yielding inter- and intra-assay variability of less than 10%. The sensitivity is much higher than that reported previously for the CPE technique and that of ELISA techniques. Key words: Antibody to interferon; Antiviral neutralization bioassay; Microculture tetrazolium assay; MTT
Introduction
Interferons are known to have multiple effects on cells (antivirai, antiproliferative, etc.) and over the last 10 years their role in a wide variety of
Correspondence to: K. Berg, The Interferon Laboratory, Institute of Medical Microbiology, Juliane Maries Vej 22, DK-2100 Copenhagen B, Denmark. Abbreviations: ANB, antiviral neutralization bioassay; IFN, interferon; FCS, fetal calf serum; MTT, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PFU, plaque forming units; VSV, vesicular stomatitis virus; EMCV, enccphalomyocarditisvirus; MOI, multiplicityof infection; DMF, dimethylformamide; CIF, crude human leukocyte IFN; rHuIFN-a2~, recombinant human leukocyte IFN-a2a (Roferon); rHuIFN-a2b, recombinant human leukocyte IFN-azb (Intron A).
diseases has been studied (reviewed by Finter and Oidham, 1984). The-introduction of recombinant I F N has made it possible to further increase the numbers of such studies. However, in contrast to human leukocyte IFN, recombinant I F N has proved to be immunogenic in some patients since up to 60% of patients receiving r H u I F N - a 2 treatment have been reported to develop antibodies to the IFN (Steis, 1988). In addition, in recent years conflicting reports on the presence or absence of antibodies to IFN in patients receiving I F N treatment have appeared (Jakobs, 1988; Figlin, 1988; Freund, 1988; Von Wussow, 1988). These reports are based on two essentially different methods: (i) solid-phase immunoassays: the immunoradiomettic assay, I R M A (Protzmann, 1984) and the enzyme-linked immunosorbent assay, ELISA (Hennes, 1987), (ii) bioassays measuring the neutraliz-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
242 ing activity of sera for the antiviral action of IFNs. The former assays are fast and easy to perform, whereas the latter are of relatively low sensitivity and accuracy (Grossberg, 1984), which, in turn, can lead to ambiguities of interpretation. Recently, we have shown that the measurement of dehydrogenase enzyme activity permits the quantification of interferons (Berg et al., 1988). The method is based on the following principle: dehydrogenase enzyme activity in uninfected cells is high compared to that of virus-infected cells. A measure of the enzyme activity can be obtained spectrophotometrically by cellular transformation of a tetrazolium salt (MTT) into formazan (Mosmann, 1983). Uninfected cells will give high OD signals whereas virus-infected cells will yield significantly lower OD signals. IFN-treated virus-infected cells, however, will produce a signal which is 90% of that from uninfected cells, despite the addition of challenge virus. If antibody to IFN is present, the protective effect of IFN will be abrogated. This will lead to sharp decrease in the OD signal and make it possible to determine whether a neutralizing entity, for example antibody, is present or not. The above principle relies on a very precise quantification of the biological activity of IFN which, therefore, must be measured objectively. To establish such a method, the amounts of cells, virus and IFN must first be defined as precisely as possible. The experimental procedure has now been used for more than 5 months yielding inter- and intra-assay variations below 10%.
cells were grown in Eagle's MEM with the addition of 5% FCS, 1% penicillin, 1% streptomycin, 1% L-glutamine, and 5% N a H C O 3 in Nuncione plastic bottles. Cells were split twice weekly at different cell densities according to standard procedures using trypsin. Cell controls
Cells were seeded in microtrays in 100 ~1 medium including 5-10% serum and incubated for 72 h. The minimum seeding concentration yielding an OD signal > 0.5 was established, in this study 10,000 cells. Viruses
Vesicular stomatitis virus (VSV) was obtained from Dr. D. Burke (Warick University, U.K.); murine encephalomyocarditis virus (EMC) was kindly provided by Dr. S. Grossberg, WI, U.S.A. The viruses were propagated in murine L cells and supernatants were stored at - 7 0 o C. Viruses were plaque titrated before being frozen. Virus controls
After 48 h of incubation challenge virus was added to the wells at a concentration resulting in 90% cell destruction after 24 h of infection at which time the MTT assay was performed. Interferons
MTT, a tetrazolium salt (Sigma cat. no. M2128) was dissolved in autoclaved PBS (5 mg/ml), sterile filtered, and stored at 4 ° C in a dark bottle; sodium dodecyl sulfate, SDS (Sigma, practical grade) and dimethylformamide, D M F (Fluka cat. no. 40250, analytical grade).
Crude human leukocyte interferon (CIF), generously provided by Dr. Osther, Dallas, had been obtained from buffy coat suspensions to which Sendai virus had been added according to the method of Cantell (Berg, 1982); specific IFN activity was 104 I U / m g protein. Pure recombinant human leukocyte interferon (rHulFN-az), kindly provided by Essex-Pharma, Denmark, was produced in E. coli (Biogen, U.S.A.). The IFN assays described were calibrated using the 69/19 international reference preparation for human leukocyte IFN (from NIBSC, U.K.). All IFN activity was expressed in international units.
Cell lines
M T T assay
MDBK cells were from the American Type Culture Collection, and A549 cells from Dr. A. Meager, National Institute for Biological Standards and Control, London. M D B K and A549
Mosmann's method was modified as follows: 25 ~1 of a 5 m g / m i stock solution of MTT were added to each well and after incubation for 2 h at 37°C, 100 /.tl of extraction buffer (12.5% SDS,
Materials and methods Chemicals
243 45% D M F and p H 4.7) were added to dissolve formazan-protein complexes. After mixing, the ODs were measured at 570 nm using a Titer-Tech 96-well multiscanner, employing solubilizer as the blank probe. No medium was removed prior to the addition of any ingredient (Mosmann, 1983; Hansen, 1989).
Serum samples A polyclonal rabbit antiserum against crude human leukocyte IFN was produced by injecting a chinchilla rabbit subcutaneously with 100,000 IU per injection every second week as previously described (Berg, 1984). This was done for 18 months. The neutralization titer as based on the CPE method was approx. 6,000 I N U / m l (using 3 - 5 I U / m l of IFN during the period of incubation). Serum samples from two patients suffering from chronic hepatitis were collected and allowed to clot at room temperature; the sera were separated by centrifugation at 500 × g for 10 rain. Samples were stored at - 7 0 ° C until analyzed. Serum samples from Dr. Von Wussow, Hannover, F.R.G., had been collected from patients who were found to be positive in the ELISA procedure. Prior to the assay the sera were heat inactivated at 56 ° C for 30 min.
A ntioiral neutralization bioassay This was performed in 96-well tissue culture plates (Nunc, Roskiide, Denmark). Ten-fold serial dilutions of sera starting at 1/20, were incubated for 1 h at 3 7 ° C with 0.13-4 I U / m l IFN in a total volume of 100 /zl in Eagle's minimal essential medium, supplemented with 5% FCS (Gibco). The samples were transferred to cell monolayers grown from 24 h before (10,000 cells seeded in 100 #1 Eagle's MEM + 5% FCS). After overnight incubation at 3 7 ° C challenge virus was added. After further incubation for 24 h at 3 7 ° C 25 #! M T T were added to the wells, followed by 100 ~1 iysis buffer 2 h later, as described under the M T T assay. The neutralizing titer was defined as the reciprocal of the serum dilution causing a 50% decrease in the antiviral effect of 1.0 I U / m l of I F N (provided that neither serum toxicity nor antiviral activity were found).
Serum toxicity controls Serum samples diluted 1 / 2 0 were incubated with cells (neither I F N nor virus were added) and after 72 h the Mqq" technique was used and the O D signals compared to those of the cell control.
Antiviral controls (sample IFN-control) Serum samples diluted 1 / 2 0 were added to the cells and after 48 h of incubation, 100 /zl virus dilution were added. After another 24 h of incubation the M T T technique was used to evaluate cytotoxicity. The O D signals were compared to those of the virus controls.
Enzyme immunoassay for the detection of antibodies to interferon-a (ELISA kit from Anawa Laboratories, Switzerland) The assay comprised a solid-phase enzyme immunoassay based on the 'sandwich' principle. Beads sensitized with recombinant interferon-a 2 were incubated with serum samples. Interferona2-peroxidase conjugate was then added and the incubation continued (the conjugate being bound to the remaining free combining site of the captured antibodies). After washing, enzyme activity bound to the beads was determined by incubation with the usual substrate (OPD) and the results read at 492 nm in a Titer-Tech multiscanner.
Microscopic examination of CPE Cell layers were scored microscopically subsequent to the development of full CPE in virus controls. Scorings were as follows: 100% of cell destruction was given the number 4; 75% = 3, 5 0 % = 2 , 2 5 % = 1 and 0 % = 0 .
Neutral red procedure The medium was removed by inversion of the tray. The cell layer was washed twice with PBS p H 7.4, and the medium replaced by 100 #1 MEM containing 67 # g / m l of neutral red dye. After 2 h incubation at 37°C, the wells were emptied, washed twice with PBS p H 7.4, and the dye accumulated in the cells was extracted with 100 /~1 of 50% ethanol in 1% acetic acid. Color development in the microtrays was then quantified at 540 nm in a Titer-Tech multiscanner (Forti et al., 1985; Hansen et al., 1989).
244
Statistics Samples were assayed at least in triplicate and the results are presented as means and standard deviations. Each experiment was performed at least three times. The intra- and interassay variation was below 10%.
Results Since detection of IFN neutralization requires a delicate balance between cells, virus and IFN, the interplay of these factors on the sensitivity of the assay was first studied. Optimization of each parameter led to a laboratory protocol for performing a standard M'I-T antiviral neutralization bioassay (ANB). Examples illustrating this approach are given below.
neutral dye red method, making the former method more sensitive.
Interferon concentration Within the range 0.13-1.00 I U / m l an almost linear correlation between I F N concentration and O D readings (0.05-0.4) was seen (Fig. 1C), and the slope indicated that small changes in IFN concentration resulted in a large change in O D readings. Approximately 80% cell protection was obtained at an I F N level of 1.00 I U / m l , that was used in the experiments. Parallel experiments using the M D B K cell line (which is very sensitive to IFN alpha) and VSV were also performed. The parameters ensuring an optimal procedure were found to be: 10,000 cells/well (MDBK); infecting dose 10,000 p.f.u./well (VSV). The IFN titration reached an endpoint at 1.50 I U / m l with either C I F or rHulFN-a2a (data not shown).
Seeding cell density Cell densities from 1250 to 7500 cells/well yielded a linear correlation between the number of cells and O D readings (Fig. 1A). A saturation plateau was reached at 7500 cells/well. In order to minimize intra-assay variation yet still obtain high sensitivity (i.e., a steep slope in the dose-response curve) 10,000 cells/well were used.
The M T T antiviral neutralization bioassay (MTTANB) The results of titration of a rabbit antiserum against C I F in the M T T - A N B (using A549 cells and EMCV) showed the neutralization titer to be close to 160,000 IFN neutralizing units ( 1 N U ) / m i .
Virus concentration
The virus titration curve showed almost no CPE at a dilution of 10 -6 (Fig. 1B), but a distinct dose-response relationship within the dilution range 10-4-10 -3 . The lowest virus concentration (EMC) to cause maximum suppression of the O D signal was 10-2 corresponding to 25,000 P F U / m l or MOI 25; this virus concentration was subsequently used throughout the study. Three methods for the detection of virus cytopathic effects (microscopic examination, CPE; the neutral red and the M T T method) were examined and the results are summarized in Table I. The M T T method gave a 20-fold difference in the O D signal between non-infected/infected cells in contrast to the neutral red method which yielded a 3-4-fold difference. As expected, the CPE method was far less able to discriminate between the different levels of infection. It is obvious that small amounts of interferon influence the O D readings more readily in the M T T method than in the
TABLE 1 C O M P A R A T I V E Q U A N T I F I C A T I O N O F VSV BY T H R E E METHODS M D B K cells in suspension were seeded in 100 /tl medium supplemented with 5% FCS (10,000 cells/well). After 24 h of incubation at 37 ° C in 5% CO 2 the medium was replaced with 100 ~1 medium supplemented with 3% FCS containing two-fold dilutions of VSV starting at 50,000 plaque forming units (PFU) of challenge virus. After an additional 20 h of incubation at 37 ° C in 5% CO 2, the virus CPE was quantified. Results are shown as the median of quadriplicates (OD readings × 103). Preparation number
Microscopic examination
Neutral red-method
MTTmethod
1
4
2 3 4 5 6 7 8
4 4 4 3 2 1 0
120 119 153 131 214 317 339 425
35 60 136 228 414 531 632 650
245
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102
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naHu IFN (CIF) iq4.nl 0.13 0.5 1.0 2.0 3.0 4.0 Fig. I. A: Seeding cell density. A549 cells were seeded in 100 pl volumes of medium containing 10% FCS and incubated in 5% CO 2 at 37 ° C for 72 h after which the MTT assay was performed. (Means of triplicates. SD < 10%, not shown). B: Virus titration. After seeding 10,000 A549 cells/well the microtray was further incubated for 48 h, and the medium was replaced with 100 #l fresh medium supplemented with 2% FCS containing EMC virus in serial ten-fold dilutions. The cells were further incubated overnight and the MTT assay performed as described. (Means of triplicates, SD < 10%, not shown). C: Interferon titration. A549 cells, 10,000/well, were seeded and after incubation for 24 h the medium was replaced by medium containing crude interferon-a (CIF), yielding the final concentrations of 0.13-4.00 1U/ml. After an additional 24 h of incubation the medium was replaced by 100 /zl medium containing EMC virus diluted 10- 2 and the MTT assay was performed after 24 h of infection. (Means of triplicates, SD < 10%, not shown.)
246 OO.
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In contrast to the virus control (OD = 0.024) the IFN-treated cells ( + virus) yielded an O D of 0.429 using CIF; 0.449 using rHulFN-%b (Intron A) and 0.404 using rHulFN-%a (Roferon) (Fig. 3). This indicates very good cell protection with all three IFNs. Serum from patient A was titrated against CIF, Intron A and Roferon. The neutralizing titer was found to be 40,000 I N U / m l against C I F and 400,000 I N U / m l against Intron A and Roferon, calculated as the reciprocal of the serum dilution which significantly abolished the antiviral activity of 1.0 I U / m l of IFN (Fig. 3A). The serum from patient B, which was antibody-negative in the ELISA, was titrated in a similar way and the neutralizing titer was 40 I N U / m l against CIF, 400 I N U / m l against Intron A and 4000 I N U / m l against Roferon (Fig. 3B). The minimum amount of serum necessary to perform the assay was 0.5 ml. However, by changing the dilution range it was possible to further modify the method and use sample volumes of only 100 ~1 (data not shown).
2~0 "
o, IFN units/ml Fig. 2. ANB and titration of a rabbit serum. Titration of a
rabbit antiserum against CIF in a neutralization assay with A549 cells and EMCV. Two-folddilutions of serum starting at 40,000, were incubated for 1 h at 37°C with equal volumes (100 /~1) of medium supplemented with 5% FCS including 0; 0.50; 1.00 and 2.00 IU/ml (final concentration) crude IFN-a, after which any residual IFN in the samples was measured by the MTT method. The dashed curve represents IFN controls. C.C. = cell controls. T = x 103. Results are shown as means of triplicates (SD was less than 10%,not shown).
The data in Fig. 2 showed that the neutralizing titer was the same when using 0.5, 1.0 and 2.0 C I F I U / m l . The MTT-ANB was also applied using serum from two patients with chronic hepatitis who had been receiving IFN treatment (patients A and B). One of them (A) developed antibodies to IFN as measured by the ELISA, whereas the other (B) was consistently sero-negative in the anti-IFN ELISA, which is based on r H u l F N - a 2. At a dilution of 1/20, sera from both patients showed no antiviral activity, and no cell toxicity. The cell control yielded an O D signal of 0.454 compared with an OD signal of 0.455 for the standard bovine serum.
Discussion Since the tetrazolium salt MTT was first introduced (Mosmann, 1983) for use in biological assays for cytokines it has been extensively used. We have shown that the MTT method is suitable for the quantification of viral CPE and, in consequence, also for the quantification of IFN activity and neutralization. The method has proved superior to conventional antiviral neutralization bioassays which involve, for example, microscopy or neutral r e d / t r y p a n blue staining for quantification of, for example, viral CPE. It appears from Table I that the MT'T method yields a far more detailed grading of the results; microscopic examination gives the impression of a 100% CPE in those cases in which OD readings of the neutral red method vary from 0.119 to 0.153 whereas the corresponding O D readings of the MTT method vary from 0.035 to 0.228. This enables us to work with very small IFN concentrations and to detect very low levels of IFN neutralization. Precise quantification of IFN activity is crucial for a sensitive ANB. Traditionally, the neutralizing titer is defined as the reciprocal of the antiserum dilu-
247
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OD 570 t
C'C A
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Fig. 3. MTT-ANB results from two patients suffering from chronic hepatitis. A: Serial dilutions of serum from the ELISA antibody-positive patient (patient A); and B: from the ELISA-negative patient (patient B) were prepared in triplicate using medium supplemented with 2% ( v / v ) fetal calf serum. To each well IFN was added to a final concentration of 1.00 I U / m l . The sera were titrated against three species of HulFN-a: IFN 1 = crude human leukocyte IFN-a (CIF); IFN 2 = rlFN-a2b (lntron A); and IFN 3 = rlFN-a2a (Roferon). C.C. = cell controls; V.C. = virus controls. T = × 103 (Means of triplicates, SD < 10%. not shown).
tion which will abolish the protective effect of, say, 10 I F N U / m l (Kawade, 1986). The drawbacks of the traditional CPE end-point neutralization method is its lack of precision and sensitivity, which is in part, attributable to the microscopic examinations. If, for example, the I F N level is raised to 20 U during the period of incubation, the CPE method becomes more reliable, but at the same time, the sensitivity of the assay is considerably reduced. This point was illustrated in one example: the rabbit anti-human | F N serum was titrated to 6000 I N U / m l using 4 - 8 I U / m l CIF. The same serum was found to have a titer of 1200-3000 if 10-14 I U / m l C I F were used (data not shown). However, with the MTT-ANB, using 1 I U / m l CIF, the serum had a titer of at least 160,000 N U / m l , reflecting an increase in sensitivity of more than 100. It is of interest to note that although the two patients with hepatitis had received Intron A only, the both developed antibodies to lntron A and
Roferon. These two recombinant I F N s only differ by a single amino acid at position 23 where they have arginine and lysine, respectively. It has been a point of discussion whether this change in amino acid position could result in a difference in immunogenicity (Figlin and Itri, 1988). The patients also developed antibodies to the human leukocyte IFN, which in contrast to the highly purified single molecular species of r H u I F N - a 2, is a mixture of several subspecies of IFNs, although at a ten-fold lower level. This can probably be ascribed to the fact that the neutralizing antibodies produced against r H u I F N - a 2 recognize the same or closely related epitopes present on the 15 subspecies of human leukocyte IFN. To the best of our knowledge, this is the first report which directly demonstrates that in certain patients the use of a single recombinant IFN species can also lead to the production of antibodies against human leukocyte IFN. The end-point of the MTT-ANB titer of a serum can be defined
248
by several approaches. For example: (a) as the reciprocal of the highest serum dilution which produces an OD signal significantly lower than the means of the OD signals from virus and IFN + virus controls; (b) as any OD signal which is 3 SD below that of IFN + virus control; (c) as any signal which is 50% below that of the 1FN + virus control. It is obvious that the mode of defining the end-point influences the sensitivity of the method greatly (Kawade, 1986). At present we have chosen suggestion (c) since the values obtained have been highly reproducible over periods of 5 months, with inter- and intra-assay variation below 10%. A more correct approach would be to apply nonparametric statistics using the Mann-Whitney test. The antiviral neutralization bioassay (ANB) is the standard method recommended by the World Health Organization for measuring neutralization of the biological activity of IFN. We would like to propose the use of the modified MTT-technique in the ANB, as it has proved a very reliable method.
Acknowledgements The authors are grateful to Dr. Allan Randrup Thomsen and Professor Charles J. Pfau for helpful comment and discussion. The expert help by Mrs. Beth Simonsen and Thorbj(3rg Sandquist is greatly acknowledged. This investigation was supported by The Danish Cancer Society (no. 87-022) and by The Danish Medical Research Council (no. 1276-88; no. 127947), and The Carlsberg Foundation (1989).
References Berg, K. (1982) Purification and characterisation of murine and human interferons. A review of the literature of the 1970s (Thesis). Acta Pathol. Microbiol. Immunol. Scand. Sect. C 279 (suppl.), 1-136. Berg, K. (1984) Identification, production, and characterization of murine monoclonal antibody (LO 22) recognising 12 native species of human alpha interferon. J. Interferon Res. 4, 481-491. Berg, K., Hansen, M.B. and Nielsen, S.E. (1988) A new sensitive bioassay for precise quantificatinns of interferon activity as measured via the mitochondrial dehydrogenase func-
tion in cells (M'lq-method). J. Interferon Res. 1 (suppl.), 67 (abstract). Figlin, R.A. and Itri, L.M. (1988) Anti-interferon antibodies: a perspective. Sere. Hematol. 25, 9-15. Figlin, R.A., DeKernion, J.B., Mukamel, E., Palleroni, A.V.. ltd, L.M. and Sarna, G.P. (1988) Recombinant interferon alfa-2a in metastatic renal cell carcinoma: assessment of antitumor activity and anti-interferon antibody formation. J. Clin. Oncol. 6, 1604-1610. Finter, N.B. and Oldham, R.K. (1984) Interferon 4. In Vivo and Clinical Studies. Elsevier. Amsterdam, pp. 1-363. Forti, R.L., Moldovan, R.A., Mitchel, W.M., Gallicoat, P., Schufman, S., Davies, H.A. and Smith, Jr., D.M. (1985) Application of an objective biological assay of human interferons to clinical specimens and a survey of a normal population. J. Clin. Microbiol. 21,689. Freund, M., Von-Wussov, P., Knuver, Hopf, J., Mohr, H., Pohl, U., Exeriede, G., Link, H., Wilke, H.J. and Polivoda, H. (1988) Treatment with natural human interferon alpha of a CML-patient with antibodies to recombinant interferon alpha 2b. Blur 57, 311-315. Grossberg, S.E. and Sedmak, J.J. (1984) Assay of Interferons. in: N. Finter (Ed.), Interferon 1. Elsevier, Amsterdam, pp. 189-213. Hansen, M.B., Nielsen,.S.E. and Berg, K. (1989) Re-examination and further developments of a precise and rapid method for measuring cell growth/cell kill. J. lmmunol. Methods 119, 203-210. Hennes, U., Jucker, W., Fischer, E.A., Krummenacher, T., Palleroni. A.V., Trown, P.W., Linder-Ciccolunghi, S. and Rainisio, M. (1987) The detection of antibodies to recombinant interferon alfa-2a in human serum. J. Biol. Stand. 15, 231-244. Jakobs, S.J., Sullivan, L.M., Salfi, M., Grossberg, H., Spiegel, R.J., Leibovitz, P.J., Oden, E.M., Kelsey, D.K. and Treuhaft, M.W. (1988) Antibodies to recombinant human interferon alpha 2b (Intron A) were detected in only a small number of 101 Intron A recipients. J. Biol. Response Modif. 7, 447-456. Kawade, Y. (1986) Quantitation of neutralization of interferon by antibody. Interferons Methods Enzymol. 119, 558-573. Mosmann, T. (1983) A rapid colorimetric assay for cellular growth and cytotoxicity assays. J. Immunol. Methods 65, 55 -63. Protzmann, W.P., Jacobs, S.L., Minnicozzi, M., Oden, E.M. and Kelsey, D.K. (1984) A radioimmunologic technique to screen for antibodies to alfa-2 interferon. J. Immunol. Methods 75, 317-323. Steis, R.G., Smith, J.W., Urba, W.J., Clark, J.W., Itri, L.M., Evans, L.M., Scoenberger, C. and Longo, D. (1988) Resistance to recombinant interferon alfa-2a in hairy-cell leukemia associated with neutralizing anti-interferon antibodies. New Engl. J. Med. 318, 1409-1413. Von Wussow, P., Freund, M., Dahle, S., Jakschies, D., Poliwode, H. and Deicher, H. (1988) Immunogenicity of different types of interferons in the treatment of hairy-cell leukemia. New Engl. J. Med. 319, 1226.