Rapid, Quantitative, Semiautomated Assay for Virus-Induced and ...

Vol. 12, No. 3

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1980, p. 433-438 0095-1 137/80/09-0433/06$02.00/0

Rapid, Quantitative, Semiautomated Assay for Virus-Induced and Immune Human Interferons JON A.

GREEN,`*

TZE-JOU YEH,"3.AND JAMES C. OVERALL, JR.23

Howard Hughes Medical Institute Laboratories2 and Division of Infectious Diseases, Departments of Internal Medicine' and Pediatrics,3 University of Utah College of Medicine, Salt Lake City, Utah 84132

An improved human interferon (IF) assay is described. This procedure is based the ability of encephalomyocarditis virus to replicate in WISH cell microcultures with the production of discrete plaques in the presence of a liquid tissue culture medium. Performance of 50% plaque reduction endpoint assays in microculture required only 0.1 ml of specimen for determinations using duplicate dilutions beginning at 1:3. Semiautomated equipment facilitated simultaneous in situ dilution and distribution of multiple IF samples in cultures containing preformed WISH cell monolayers. An incubation period of 5 to 6 h was adequate for development of maximal antiviral activity by both virus- and immune-induced IF. Sensitivity of the encephalomyocarditis microplaque reduction assay was comparable to that of other commonly used techniques. The method is rapid, can be completed within 30 h from the beginning of the IF assay, and is able to accommodate as many as 40 to 50 samples at a single time. Encephalomyocarditis microplaque reduction is suitable for the quantitation of IF as an antiviral agent or a lymphokine. on

Interest in human interferon (IF) has intensified in recent years. Currently IF is being used in the prophylaxis and therapy of human diseases (8, 17). In addition, IF is increasingly recognized as a lymphokine (9) with immunomodulatory properties (3, 15), and as a potential means of increasing an understanding of the pathogenesis of viral and autoimmune diseases (14). There is a continuing need, therefore, for further development of improved methodologies for rapid IF determination (10). Advances in human IF determination have produced assay systems that are rapid, sensitive, and reproducible, but require only small quantities of specimen and a minimum of time and effort (10). One such assay uses a continuous line of human amnion (WISH) cells and endpoint determination based on 50% reduction in vesicular stomatitis virus (VSV) plaques which develop in the presence of a liquid culture medium overlay (12). This procedure is limited by formation of secondary microplaques and coalescence of primary microplaques within the 16- to 20-h incubation period. In addition, as originally described, only classical, virus-induced leukocyte or fibroblast IF was assayed using a 3- to 4h incubation period. Subsequent investigations indicated that immune-induced IF required a longer period of incubation to stimulate maximal antiviral activity (5). We have modified the previously described VSV-WISH cell microplaque reduction assay in 433

several ways. Encephalomyocarditis (EMC) virus was substituted for VSV, with elimination of secondary microplaque formation and a major reduction in microplaque coalescence. Simultaneously, machine dilution and distribution of multiple IF samples in cultures containing preformed WISH cell monolayers, as well as the use of a crystal violet stain for microplaque enumeration were also incorporated into the methodology. Finally, an abbreviated incubation period of 5 to 6 h was examined and found to be sufficient for the development of maximal antiviral activity by immune IF. MATERIALS AND METHODS Cell cultures. Stationary WISH amnion cell cultures, a continuous line established from a normal human placenta (13), were originally obtained from Flow Laboratories, Rockville, Md. Cultures were maintained in Eagle minimal essential medium (MEM) prepared with Earle salt solution and supplemented with 10% fetal bovine serum and antibiotics (streptomycin, 100 fug/ml, and penicillin, 100 ug/ml). Virus. EMC virus, obtained from Yvonne Bryson, was propagated in monolayer cultures of mouse L cells and had a titer of~107 plaque-forming units (PFU) per 0.1 ml when assayed in human WISH cell cultures as described below. EMC plaque formation in liquid-overlay WISH cell microcultures. Growth medium was removed, and a 5 x 10`' dilution of EMC, calculated to produce approximately 20 to 30 PFU, was added in 0.02-ml amounts of MEM with 2% fetal bovine serum by using an Eppendorf type of transfer pipette. One hour later,

434

GREEN, YEH, AND OVERALL

an additional 0.1 ml of MEM containing 2%c fetal bovine serum was dispensed to each microculture. At 22 to 24 h, the plates were fixed and stained, and plaques were counted as described below. IF preparations. Several IF preparations were used. An IF-containing pool was induced by Newcastle disease virus (NDV) in normal human peripheral blood leukocyte cultures. A second IF preparation was stimulated by phytohemagglutinin (PHA) (Burroughs Wellcome, no. HA 16) in human peripheral blood leukocyte cultures from healthy donors. Virus-induced IF (NI)V-IF), but not PHA-stimulated IF (PHA-IF), was resistant to exposure to pH 2 for 20 h and was neutralized by exposure to specific antiserum directed against virus-induced leukocyte IF. A National Institute of Health (NIH) human leukocyte IF standard, G023-901-527, had a predicted titer of 20.0 x 10: (range, 11.6 x 10" to 41.5 x 10') when assayed in human foreskin fibroblast cultures (data accompanying NIH leukocyte standard). IF preparations were stored in 0.5-ml volumes at -70°C at a 1:10 dilution of their original concentrations. Each sample was defrosted and used one time only. IF determinations by EMC plaque reduction in WISH cells with liquid medium overlay. WISH cells at a concentration of 7 x 104 were seeded in 0.1ml volumes in 96-well microculture plates (Falcon Plastics) 20 to 24 h before being used. Six IF-containing specimens at the appropriate dilution were added in duplicate 0.05-ml volumes to cultures in the first row of 12 micro-wells, each containing 0.1 ml of the original MEM growth medium. Six serial 1:3 (0.5 log,() dilutions were made with an automated diluter containing a row of 12 0.05-ml volume-calibrated wire loops (Titertek, Flow Laboratories). When more than one culture plate was used, two separate cassettes each containing 12 wire loops were used alternately so that cooling between flame sterilization could occur without appreciably delaying the procedure. IF-containing WISH cell cultures were placed in an unstacked fashion in a 5% C02, 100% water-saturated atmosphere at 37°C for 6 h. Supernatant media were decanted into an autoclavable receptable containing toweling to minimize splashing. Culture wells were washed one time by adding and decanting 0.1-ml volumes of MEM containing 2% fetal bovine serum. EMC virus was then added as described above. Culture plates were returned to 37°C for 1 h to allow virus adsorption, and 0.1 ml of MEM with 2% fetal bovine serum was added. Readily identifiable EMC microplaques developed within 22 to 24 h, at which time 20% Formalin was added for 30 min and the plates were washed under running tap water and stained with a 1% alcoholic solution of crystal violet (1). Plaque counting could be done with the unaided eye or with a dissecting microscope or other magnifying device. The IF titer was expressed as the reciprocal of the calculated dilution that reduced the number of microplaques by 50% (50% plaque reduction) (6).

RESULTS EMC plaque formation in WISH cell monolayer cultures with liquid medium

J. CLIN. MICROBIOL.

overlay. EMC plaques, visible to the unaided eye in stained monolayers, formed within 24 h in confluent WISH cell cultures containing liquid medium without solidifying agents (Fig. 1). Secondary plaque formation and coalescence of plaques were not appreciable during the first 24 h of incubation at 37°C. Reproducibility of plaque formation was examined on eight sepa-

FIG. 1. EMC virus microplaque production in WISH amnion cell culture monolayers. Rows A and B, Uninfected cells. Single defect in the two monolayers in row A was produced by the diluting loop. Rows C and D, Cultures 24 h after addition of approximately 20 PFU of EMC in an initial 0.02 ml of inoculum. All cultures were fixed in Formalin and stained with 1% crystal violet. Final magnification, 5x.

HUMAN INTERFERON ASSAY

VOL. 12, 1980

rate occasions in which a single EMC preparation was added to replicate microculture wells at a dilution estimated to contain 20 to 30 PFU. Data were obtained for a total of 407 individual cultures in which PFU were counted at 24 h (Table 1). In general, good correlation existed between the estimated and actual number of PFU per 0.02 ml of inoculum. Effect of IF on EMC plaque formation. Responsiveness of EMC plaque formation to inhibition by IF was examined under conditions in which PFU development was related to 0.5 log1o dilution of NDV-IF or PHA-IF (Fig. 2). For purposes of comparison, the data were "normalized" and actual plaque numbers at specific IF dilutions were converted to percentages of the TABLE 1. Reproducibility of EMC microplaque formation in WISH cells in the presence of liquid medium overlay Expt

No. of inoculated cultures"

No.ture of plaques per cul(avg ± SDb)

47 48 68 43 57 80 30 34

22.0 ± 7.7 25.4 ± 5.9 29.2±5.6 19.9 ± 6.3 23.1 ± 5.9 20.0 ± 5.6 18.4 ± 6.2 23.0± 3.4

1 2 3 4 5 6 7 8

22.6 ± 3.5' Total 407 "Each culture was an individual microculture well inoculated with a 0.02-ml volume of a 5 x 10-6 dilution of a separate sample of a single EMC preparation. h SD, Standard deviation. 'Mean ± standard error. o

E

o

il80

-90 o -80 E3 -70 oe 5-60 °

NDV-IF

5

70 z 60 E 50J o

-50

-40 E

o

-30 F-

1

Ô 20-

-20

Ir

2

10 h.l w

10

,ï 2.4

29

3.4

LOGIO

2.0 2.5 3.9 INTERFERON DILUTION

30

3.5

a-

FIG. 2. IF dose-response effect. Number of EMC microplaques developing in WISH cell cultures exposed to 0.5 logo dilutions of NDV-IF or PHA-IF. Data have been normalized such that each point is a percentage representing the number of plaques in duplicate, IF-treated cultures divided by the number ofplaques in virus control cultures.

435

number of plaques present in non-IF-containing virus control cultures. An almost linear reduction in EMC plaque number was noted to occur at four separate dilutions. The slopes of the curves for NDV-IF and PHA-IF were almost identical. Duration of IF and WISH cell incubation and the development of antiviral resistance. IFs produced by human leukocytes in response to NDV or PHA were compared with respect to the rate at which they induced the antiviral state in WISH cell cultures (Fig. 3). In each of four separate experiments, NDV-IF and PHA-IF were added to WISH cells for 0, 2, 4, 6, 8, 24, or 32 h. At the selected times, IF-containing dilutions were decanted, and monolayers were washed once and challenged with EMC virus. Both NDV-IF and PHA-IF induced a maximum antiviral state (synonymous with maximum IF titer) within 6 h of addition to WISH cells. By 2 h, NDV-IF and PHA-IF titers were 83 and 66% of their respective maxima. At 4 h the titer of NDV-IF was 92% and that of PHA-IF was 85% of peak values. Based on these data, a 6-h incubation was adopted for routine use in subsequent experiments. Comparison of the modes of sample dilution and distribution on the titer of IF preparations. These studies were done before the general use of the semiautomated mechanical diluter for the in situ dilution and distribution of IF preparations. Two methods were compared. Dilutions were prepared by sequential, manual pipetting of individual samples in glass tubes and were manually transferred to WISH cell monolayer cultures. Alternatively, machineoperated diluting loops were used for the simultaneous dilution and transfer of samples in flatbottom tissue culture wells containing preformed WISH cell monolayers. Development of antiviral activity was compared at four dilutions for each method. The results of multiple experiments are presented in Fig. 4. As measured by reduction in EMC microplaque formation, no appreciable difference existed between the two methodologies. PHA-IF was used in these experiments. Similar results (not shown) were obtained with NDV-IF. Number of replicate cultures necessary for accurate and reproducible IF titer. If three or four replicate cultures were necessary for an accurate and reproducible IF titer of a given specimen, fewer samples could be run in an assay (four and three samples per microculture plate, respectively). Six samples could be assayed in each microculture plate if only duplicate cultures were used. To examine this aspect of the assay system, all eight rows with 12 wells

436


J. CLIN. MICROBIOL.

O 2 4 6 8 24 32 HOURS OF INCUBATION

FIG. 3. Rate of onset of antiviral state in WISH cells exposed for various intervals to NDV-IF or PHA-IF. EMC virus was added after IF dilutions had been decanted and WISH cell monolayers had been washed once with MEM plus 2% fetal bovine serum. The heavy, solid line is the geometric mean of individual experiments represented by dashed lines. Vertical bars depict the standard errors of the geometric mean. Also shown is the percent of maximum IF titer.

each in a 96-well microculture plate were used to assay serial dilutions of NDV-IF. The 50% plaque reduction endpoint was calculated using a mean of the virus microplaques for: (i) all 12 cultures, and (il) duplicate, triplicate, or quadruplicate cultures for the serial IF dilutions. The IF titers using two, three, and four replicates were then compared to the titer based upon the mean plaque counts for 12 cultures. Results of two experiments indicated that the IF titers using duplicates varied a mean of 24.8 ± 15.8% (range, 3 to 47%) from the titer obtained when the plaque counts from all 12 cultures were used. Titers using triplicate cultures varied 29.1 ± 19.8% (range, 12 to 71%), and those using quadruplicate cultures varied 13.7 ± 10.4% (range, 3 to 25%). Similar results were obtained when PHA-IF was used. When all aspects of the assay system were considered (e.g., number of specimens that could be run in a given assay, amount of specimen required for each assay, technologist time, cost of materials), assays performed in duplicate were considered sufficient to provide an accurate and reproducible assessment of the IF titer. Using two rows per sample, repeated assay of our own human IF standard has resulted in IF titers that have varied 38.7 ± 27.8% (range, 4 to 108%) when the titers for 26 individual assays were compared with the mean titer for all assays.

Comparative sensitivity of EMC plaque reduction human IF determination. The NIH-supplied Sendai-induced human leukocyte IF standard (G023-901-527) was assayed on nine separate occasions as the 50% plaque reduction endpoint of Sindbis virus plaques in human foreskin fibroblast cultures by using the conventional agar overlay method. Results of these titrations were compared with those obtained by similar assay of the standard IF preparation on five separate occasions in the EMC WISH 50% plaque reduction liquid overlay procedure (Table 2). A high degree of accord existed between these two techniques and published results obtained when the NIH interferon standard was assayed by the VSV 50% plaque reduction technique with agar overlay in human BUD-8 cells. Comparison of titers for different techniques revealed a titer of 20.2 ± 12.1 x 103 units with the Sindbis-human foreskin fibroblast assay, 23.8 ± 10.7 x 103 units for the EMC 50% plaque reduction assay in WISH, and 20.0 x 103 units, previously reported, for VSV plaque reduction in 16 separate determinations in BUD-8 cells (data accompanying the NIH-IF standard). DISCUSSION The EMC-WISH microplaque reduction assay for human IF satisfies many of the criteria for an improved, rapid IF bioassay (10) and

VOL. 12, 1980

HUMAN INTERFERON ASSAY

437

TABLE 2. Comparison of conventional and semiautomated EMC- WISH microculture plaque reduction assay methods in titrating NIH human IF standard' Expt 1

2 3 4

5 6 7 8 9

Conventional agar overlay0 (Sindbis-HFF)

36.5 17.9 43.0 19.5 6.3 10.0 26.0

Microculture liquid overlay'

(EMC-WISH) 32.8 13.2 28.0 33.6 11.6

7.6 15.0

Mean + SEd

20.2 ± 12.1 23.8 ± 10.7 6.343.0 11.6-33.6 a Titer in VSV/BUD-8 50% plaque reduction assay: Mean, 20.0 x 10'; range, 11.641.5 when assay N = 6, 13.3-35.9 when N = 10 (3). b 0.5% agarose overlay with conventional 18- to 22h incubation period of IF on indicated human foreskin fibroblast (HFF) cell monolayer. e Liquid overlay technique with 6-h IF incubation period. d SE, Standard error.

Range

1.4

1.9 2.4 INTERFERON DLUTION(Logo)

2.9

FIG. 4. Comparison of manual and semiautomated, mechanical methods for the dilution and distribution of IF. Manual method (gray bars): Serial threefold dilutions were prepared with pipettes in tubes. Samples of these dilutions were transferred by use of micropipettes to WISH cell cultures. Semiautomated method (white bars): 12 simultaneous, serial threefold dilutions were made in microcultures containing preformed WISH monolayers. Transfer of dilutions (distribution) occurred automatically. Vertical lines represent standard errors ofeight separate experiments.

mercially available, continuous cell line (WISH) with stable IF sensitivity characteristics. Semiautomated mechanical equipment has been used previously to prepare IF dilutions in microculture plates (2). However, in this system the IF dilutions were performed before the addition of cells to form the monolayer. With the offers several advantages over previously re- EMC-WISH assay procedure, mechanical diluported assay systems (12, 16). EMC virus used tion is carried out in microcultures with prein this assay had a much more uniform rate of formed cell monolayers. This offers the advanmicroplaque formation, less coalescence of pri- tage of examination for quality of cell monomary plaques, and fewer secondary plaques durlayers before use, and eliminates potential intering the 22- to 24-h incubation period under the ference with growth of the monolayer by the liquid medium overlay than VSV used previ- anti-divisional effects of IF (17) or IF-containing ously for this purpose (12). EMC is as sensitive leukocyte preparations (11). In the EMC-WISH to human IF as VSV in WISH cells, and the procedure, the integrity of the indicator cell EMC-WISH system is equivalent in sensitivity monolayer is unaffected by in situ mechanical to the one used for the NIH reference human IF dilution, with the exception of an occasional (G023-901-527). Minimal quantities of specimen round, central defect caused by the calibrated are needed to perform an assay in duplicate, loop. even when beginning at a low dilution. The assay The current studies demonstrated efficient is rapid and has a high sample capacity capable mechanical dilution of IF in a medium containof accommodating as many as 40 to 50 specimens ing other macromolecules and some microscopic with results attainable approximately 30 h from cellular debris. The small dilution increments the beginning of the IF assay. The semiauto- used may have reduced the risk of inadvertent mated method of dilution saves considerable sample "carry-over." No evidence of this phetime and is as accurate as manual dilution. Re- nomenon was detected when the plaque counts quirements for media, plasticware, and other in serial dilutions of high-titered specimens were materials are reduced to a minimum with the examined. These findings support the earlier microculture system. Finally, a previously men- demonstration of the efficiency of mechanical tioned advantage arises from the use of a com- dilution of radiolabeled uridine (2).

438

J. CLIN. MICROBIOI,.


Activation of WISH cells to the maximal antiviral state was rapid, reaching a plateau by 6 to 8 h of incubation. No difference was noted in the kinetics of activation by IF induced by virus (NDV-IF) or by mitogen (PHA-IF). These data conflict with previous information which indicated that the onset of activation was longer with immune IF than with virus-induced IF (4, 5, 7). The current findings are supported indirectly by the similar dose-response curves of NDV-IF and PHA-IF assayed after 6 h of incubation in WISH cells (see Fig. 2). Delayed onset of activation with immune IF would have been anticipated to produce a relatively steeper curve, i.e., less inhibition of plaque formation at the higher dilutions, particularly with the short incubation period. The exact reasons for the difference in the observations reported here and those reported previously (4, 5, 7) are not known. This difference may be a function of the number of times IF-exposed monolayers are rinsed before the addition of the challenge inoculum (one in the current study, as opposed to three times in previous studies) (4, 5, 7). Alternatively, WISH cells were challenged with EMC at an approximate multiplicity of infection of 0.0003, which is much lower than the multiplicity of infection of 10 used in studies which demonstrated prolonged induction of the antiviral state by immune IF (4, 5, 7). Preliminary experiments in our laboratory support both of these hypotheses. Whatever the final explanation, the 5to 6-h incubation period used in the EMC-WISH assay system is sufficient to provide a rapid, quantitative determination of the titer of both virus- and immune-induced human IF.

3.

4. 5.

6.

7.

8.

9. 10.

11.

12.

13. 14.

ACKNOWLEDGMENT This work was supported, in part, by Public Health Service grant AI-15074 from the National Institutes of Health.

LITERATURE CITED 1. Armstrong, J. A. 1971. Semi-micro, dye-binding assay for rabbit interferon. Appl. Microbiol. 21:723-725. 2. Campbell, J. B., T. Grunberger, M. A. Kochman, and

15. 16. 17.

S. L. White. 1975. A microplaque reduction assay for human and mouse interferon. Can. J. Microbiol. 21: 1247-1253. De Maeyer, E., and J. De Maeyer-Guignard. 1977. Effect of interferon on cell-mediated immunity. Tex. Rep. Biol. Med. 35:370-374. Dianzani, F., and S. Baron. 1975. Unexpectedly rapid action of human interferon in physiologic conditions. Nature (London) 257:682-684. Dianzani, F., L. Salter, W. R. Fleischmann, Jr., and M. Zucca. 1978. Immune interferon activates cells more slowly than does virus-induced interferon. Proc. Soc. Exp. Biol. Med. 159:94-97. Epstein, L. B. 1976. Assay of human immune interferon from lymphocyte-macrophage cultures by a virus plaque reduction method, p. 120-128. In N. R. Rose and H. Freidman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C. Fleischmann, W. R., Jr., J. A. Georgiades, L. C. Osborne, and H. M. Johnson. 1979. Potentiation of interferon activity by mixed preparations of fibroblast and immune interferon. Infect. Immun. 26:248-253. Galasso, G. J., and J. K. Dunnick. 1977. Interferon, an antiviral drug for human use. Tex. Rep. Biol. Med. 35: 478-485. Gifford, G. E., and M. Tobey. 1977. Effect of interferon and lymphokines on lymphocytes. Tex. Rep. Biol. Med. 35:375-380. Green, J. A. 1977. Rapid as&say of interferon. Tex. Rep. Biol. Med. 35:167-172. Green, J. A., S. R. Cooperband, J. A. Rutstein, and S. Kibrick. 1970. Inhibition of target cell proliferation by supernatants from cultures of human peripheral lymphocytes. J. Immunol. 105:48-54. Green, J. A., G. J. Stanton, J. Goode, and S. Baron. 1976. Vesicular stomatitis virus plaque production in monolayer cultures with liquid overlay medium: description and adaptation to a one-day, human interferon-plaque reduction assay. J. Clin. Microbiol. 4:479485. Hayflick, L. 1961. The establishment of a line (WISH) of human amnion cells in continuous cultivation. J. Exp. Cell. Res. 23:14-20. Hooks, J. J., H. M. Moutsopoulos, S. A. Geis, N. I. Stahl, J. L. Decker, and A. L. Notkins. 1979. Immune interferon in the circulation of patients with autoimmune disease. N. Engl. J. Med. 300:5-8. Johnson, H. M. 1977. Effect of interferon on antibody formation. Tex. Rep. Biol. Med. 35:357-365. Oie, H. K. 1977. Conventional assay systems. Tex Rep. Biol. Med. 35:154-160. Strander, H. 1977. Anti-tumor effects of interferon and its possible use as an anti-neoplastic agent in man. Tex. Rep. Biol. Med. 35:429-435.