A bioassay employing polyethylene glycol for measuring neutralization of interferon by specific antibodies

Journal of Immunological Methods, 13 (1976) 63--69 63 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

A BIOASSAY EMPLOYING POLYETHYLENE GLYCOL FOR M E A S U R I N G N E U T R A L I Z A T I O N O F I N T E R F E R O N BY SPECIFIC ANTIBODIES

ANNA D. INGLOT and TADEUSZ CHUDZIO Laboratory of Tumour Viruses, Department of Tumour Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland (Received 19 March 1976, accepted 18 May 1976)

A bioassay employing polyethylene glycol (PEG) for measuring the level of interferonneutralizing antibodies is described. The method is based on incubating a concentrated preparation of interferon with an appropriately diluted anti-interferon serum or globulin for 1 h at 37°C, precipitating the antigen--antibody complexes as well as some free serum protein with sterile PEG at a final concentration of 10% for 18 h at 4°C, separating the sediment by centrifugation, and titrating the residual interferon in the supernate. It is not necessary to remove PEG from the samples since it is not toxic for tissue culture at a final concentration of < 1%. The neutralization index is expressed as the number of interferon units neutralized in 1 ml of whole serum or by 1 mg of protein in a preparation of immunoglobulin.

INTRODUCTION H e t e r o l o g o u s a n t i - i n t e r f e r o n sera have been used with increasing f r e q u e n c y in i m m u n o l o g i c a l studies o n i n t e r f e r o n (Paucker, 1 9 6 5 ; F a u c o n n i e r , 1 9 6 7 ; L e v y - K o e n i g et al., 1 9 7 0 ; O g b u r n et al., 1 9 7 3 ; Berg et al., 1 9 7 5 ; Havell et al., 1 9 7 5 ) . T h e p r o d u c t i o n and assay o f the sera are c o s t l y and time-cons u m i n g d u e t o the w e a k a n t i g e n i c i t y o f i n t e r f e r o n and i m p e r f e c t i o n o f t h e titration methods. A t p r e s e n t the classical bioassay originally i n t r o d u c e d b y P a u c k e r ( 1 9 6 5 ) is c o m m o n l y used. It estimates the titre of a n t i - i n t e r f e r o n serum in t e r m s o f the highest d i l u t i o n o f the serum t h a t neutralizes a fixed a m o u n t o f i n t e r f e r o n (usually 10 units). The test is l a b o r i o u s and difficult because it requires p r e - t i t r a t i o n o f i n t e r f e r o n p r i o r t o every assay to d e t e r m i n e t h e e x a c t n u m b e r o f units available for n e u t r a l i z a t i o n b y a n t i b o d y , and use o f several graded dilutions o f i n t e r f e r o n . Moreover, t h e sera p r e p a r e d f o r this assay m u s t be t h o r o u g h l y a b s o r b e d t o r e m o v e a n t i b o d i e s t o various extran e o u s antigens e.g. c y t o t o x i c antibodies, w h i c h m a y interfere with p l a q u e f o r m a t i o n b y the challenge virus d u r i n g i n t e r f e r o n t i t r a t i o n . We describe here a new, simpler q u a n t i t a t i v e bioassay e m p l o y i n g PEG f o r

64 determining the neutralization index, which measures the degree of neutralization o f a c o n c e n t r a t e d preparation of interferon by a fixed volume of antiinterferon serum or by a fixed weight of immunoglobulin. MATERIALS AND METHODS

Cell cultures The LG (Glasgow) line of mouse fibroblasts originally obtained from the Institute o f Virology, Bratislava, Czechoslovakia has been maintained in our l a b o r ato r y for several years and was used routinely for the p r o d u c t i o n of interferon. Mouse L cells (L1, f r om the same source) were used for the assay. The cells were grown in Eagle's Minimal Essential Medium (MEM) with Earle's base, supplemented with 10% inactivated calf serum, 100 units/ml of penicillin and 100 pg/ml of s t r e pt om yci n. Primary cultures of mouse e m b r y o cells (MEC) were prepared f r om the whole Balb/c e m b r y o s by trypsinization using a standard pr oc e dur e r e c o m m e n d e d by Flow Laboratories, Ltd. in 1974.

In terferon Production The Gp (giant plaque) m u t a n t of Sinbis virus propagated in c o n c e n t r a t e d suspension culture of chick e m b r y o cells was used to induce interferon in 5--6 day old m o n o l a y e r cultures of LG cells in Roux bottles (Inglot et al., 1973; Chudzio and Inglot, 1975). Exposure to the virus at an infection rate o f a p p r o x i m a t e l y 500 pfu/cell, was for one h o u r at 37°C. After removal of the virus the cultures were washed twice with phosphate buffered saline (PBS), Eagle's MEM w i t h o u t serum was added and incubation cont i nued for 20 h at 37 ° C. The m e di um containing interferon was treated with zinc acetate at a final c o n c e n t r a t i o n of 0.02 M. The sediment was collected after centrifugation, suspended in a small a m o u n t of 0.01 N HC1 in deionized water and dialyzed against several changes of this solution at 4°C. Finally, it was dialyzed overnight against PBS, pH 7.2. Precipitate, if formed, was removed by centrifugation and the interferon-containing supernate was stored at 4 ° C. The whole p r o c e dur e was carried out under sterile conditions and the final p r o d u c t was tested f or bacterial contamination. Assay I n ter f er o n was titrated by the plaque inhibition test on m o n o l a y e r cultures in r o u n d b o t t o m tubes of L 1 cells (2 × 10 s cells/ml). One ml volumes o f serial two-fold dilutions of interferon in Eagle's MEM plus 2% calf serum pretreated with PEG (Inglot et al., 1975) were incubated overnight with the L 1 cells. The m edi um was then removed and the cultures were challenged with a p p r o x i m a t e l y 50 pfu per culture of Vesicular Stomatitis virus (VSV).

65 The CHR strain of VSV, Winnipeg was received from Dr J. Zavada, Institute o f Virology, Bratislava, Czechoslovakia. After adsorption of the challenge virus for one h at 37 ° the cultures were overlayed with Eagle's MEM containing 1% methylcellulose (British Drug Houses, Ltd. or Loba Chemie, Austria), and 2% o f PEG-treated calf serum. After incubation for two days at 37°C, 0.05 ml per culture of a 0.1% solution of neutral red in deionized water was added and the plaques c o u n t e d 5 h later. Titers of interferon are expressed as reciprocals of the highest dilutions giving a p p r o x i m a t e l y 50% inhibition of plaque formation. A lab o r ato r y interferon standard was included in each series of assays. I n t er f er o n units q u o t e d are as measured in our l a b o r a t o r y and one of our units equals 2 mouse interferon reference units. The reference mouse interferon standard G-002-904-511 (kindly provided in 1973 by Dr G.J. Gallasso, NIH, Bethesda, Md. U.S.A.) containing 12,000 research standard units/ml, was f o u n d to have a titre of 6000 by this m e t h o d . A n t i - i n t e r f e r o n sera

Rabbits were immunized once a week with a p p r o x i m a t e l y 10 000 units of crude interferon per intramuscular (i.m.) injection. Seven of 11 hyperi m m unized animals received 2--4 primary i.m. injections of the interferon covalently b o u n d to Enzacryt AH (Koch-Light, Ltd.). The linking to Enzacryl AH was carried out as r e c o m m e n d e d by the manufacturers for the preparation o f insoluble enzymes. The insoluble interferon retained 0.25--1% of the antiviral activity of the original preparation of interferon (Chudzio, Inglot and Szewczuk, unpublished results). It was e x p e c t e d t hat the elimination of the insoluble interferon from the b o d y fluids might be retarded and hence its i m m unogeni ci t y enhanced. All the animals received 13--28 weekly i.m. injections of the soluble interferon. All 11 rabbits sooner or later p r o d u c e d anti-interferon antibody, and were bled two weeks after the last injection. The sera were heated for 30 min at 56°C and stored at --20 ° C. Immunoglobulin fractions of the sera were prepared as follows. The absorption o f antibodies to various contaminants of the interferon used for immunization was carried out according to the m e t h o d of Ogburn et al. (1973). However, the best absorption of the antibodies (except anti-Sindbis virus) was obtained by incubating the sera with monolayers of living L cells in R o u x bottles for 3 h at 37°C and t hen for 18 h at 4°C. The absorbed sera were treated with a m m o n i u m sulfate (AS) at 50% saturation for 18 h at 4°C, the precipitated protein was centrifuged, washed three times with 50% AS and dialyzed against three changes of deionized water containing 0.02% sodium azide at 4°C. After centrifugation at 700 g for 30 min the supernate (containing mainly IgG) was dialyzed against PBS and the re-suspended sediment (containing mainly IgM and a-globulin) against 0.3 M NaC1 buffered with 0.02 M phosphate, for 3 days at 4°C. The

66 solutions o f immunoglobulin in PBS containing 1.5% to 3.8% protein were filtered through Selectron filter BA 83/6, PG 0.2 pm and stored in glass ampoules at --20°C f or several months. The degree o f pur i t y of the immunoglobulins was approxi m at el y 92%. T h e y were n o t f u r t h e r purified because for our purposes a high yield was m o r e i m p o r t a n t than high purity. For comparison, the preparation o f concentrated and electrophoretically-pure anti-interferon IgG, received through the co u r tes y of Dr L. Boreck:~, Bratislava, Czechoslovakia, was also tested.

Bioassay procedure One volume of the c o n c e n t r a t e d L cell interferon (titre ranging from 2,000 to 4,000 units) and one volume of the anti-interferon serum or immunoglobulin, b o t h usually diluted 1 : 2 or 1 : 4 with PBS, were incubated in a water bath at 37°C for 1 h. One volume of the 30% solution of PEG (Serva, mol. wt. 6,000) in deionized water was then added and the mixture held overnight at 4 °C. Final c o n c e n t r a t i o n of PEG in the reaction mixture was 10% unless otherwise stated. The samples were centrifuged at 700 g for 30 min at 4°C. The sediment was discarded and the supernate collected for titration of residual interferon activity.

Calculation o f results The neutralization index (NI) indicates the n u m b e r of interferon units neutralized by one ml of anti-interferon serum or by one mg of protein in the immunoglobulin preparation. It was calculated according to the following formula: log C - - l o g N = D anti-log D =R RXdSXdIF =NI where: log C is the titer of interferon expressed in log~0 in the controls (in the presence o f PBS, or normal rabbit serum, + PEG; b o t h values should be a p p r o x i m a t e l y equal); log N is the log~0 of the interferon titre after reaction with anti-interferon serum (or globulin) + PEG; R is the anti-logarithm of the difference D. NI is obtained by multiplying R by the initial dilution of the anti-interferon serum or globulin (dS) and by the final dilution of the interferon (dIF) in the m i xt ur e and in the tissue culture medium. Example: The titre of interferon in controls containing PBS + PEG, or PBS + PEG + normal rabbit serum, expressed in log~0 is 1.8, and after reaction with the anti-interferon serum A diluted 1 : 4 is 0.6. Thus, the difference is: 1.8 -- 0.6 = 1.2 anti-log10 1.2 = 16

67 Since the antiserum A reacting with interferon was diluted 1 : 4 and since t h e final dilution of i nt e r f e r on (in the reaction m i xt ure and in the tissue culture medium in the first dilution titrated) was 1 : 30, 16 X 4 X 30 = 1920. Hence, the NI of the anti-interferon serum A equals 1920 interferon units/ ml. When a preparation of immunoglobulin is titrated the calculated NI should be divided by the n u m b e r of mg of protein per 1 ml of the sample. RESULTS AND DISCUSSION The principle of the bioassay is as follows. The preparation of interferon (antigen) is mixed with the anti-interferon serum or globulin (antibody) and incubated for a given time. The antigen--antibody complexes are then precipitated u n d er sterile conditions. The precipitate is removed by centrifugation and the residual interferon in the supernate is titrated in a standard assay. The results are expressed as a neutralization index (NI). To develop the assay it was necessary to find o u t a convenient reagent to precipitate antigen--antibody complexes. P o l y e t h y l e n e glycol (PEG, mol. wt. 6000) was chosen for this purpose because it can be sterilized by autoclaving, is non-toxic for tissue cultures at concentrations < 1 % (Inglot et al., 1975), and in previous experiments we had found t hat during the purification o f Sindbis virus by precipitation with 6% PEG interferon is n o t precipitated with the virus but remains in the supernate (Inglot et al., 1973). A f u r t h e r advantage of PEG is its p r o p e r t y of precipitating from sera the majority of immunoglobulins and lipoproteins (Inglot et al., 1975). Thus, i m m u n e sera treated with PEG do n o t require absorption to remove various antibodies to extraneous antigens contaminating interferon preparations. The appropriate percentage of PEG was det erm i ned which gave maximal precipitation o f antigen--antibody complexes w i t h o u t simultaneous precipitation o f free interferon. The solubility of interferon was tested at PEG concentrations 5, 6, 10 and 15% in PBS alone or in the presence of several batches o f normal rabbit serum undiluted or diluted with PBS. It was f o u n d that interferon was almost com pl et el y soluble at PEG concentrations between 5 and 10% with or w i t h o u t normal rabbit serum. PEG at a c o n c e n t r a t i o n of 15% in PBS or diluted serum caused a reduct i on of the interferon titre by a b o u t a half. In the presence of undiluted serum this loss was doubled, p r oba bl y due to non-specific trapping of interferon by the precipitated protein. On the o th er hand PEG at concent r a t i on s ranging from 5--15% effectively removed the i n t e r f e r o n - - a n t i b o d y complexes from the reaction mixture as judged by r e d u c t i o n of the free interferon titre and did n o t interfere with virus plaque formation. Therefore, 10% PEG was routinely used for treatm e n t o f the i n t e r f e r o n - - a n t i b o d y m i xt ur e in order to obtain maximal precipitation o f i n t e r f e r o n - - a n t i b o d y complexes. The optimal a m o u n t of interferon in the reaction m i xt ure was determined on the basis o f the results of several experiments which indicated that the

68 t i t e r o f i n t e r f e r o n s h o u l d n o t b e l o w e r t h a n 1 : 2 0 0 0 . I t is n o t o t h e r w i s e p o s sible to titrate accurately the residual interferon since it becomes diluted with other reagents (antibody, PBS and PEG) and with the tissue culture m e d i a in t h e f i n a l b i o a s s a y . Various dilutions of the anti-interferon sera were titrated at a fixed concentration of interferon and PEG to determine the optimum ratio of antib o d y t o i n t e r f e r o n . I n all t e s t s , d i l u t i o n s o f s e r a o r g l o b u l i n s o l u t i o n in t h e range 1 : 2 to 1 : 8 proved most suitable; potent anti-interferon globulin f r a c t i o n s s h o u l d b e d i l u t e d 1 : 10 o r m o r e . I t w a s o f i n t e r e s t t o d e t e r m i n e h o w m u c h t h e r e s u l t s o f t h e t e s t s w e r e affected by the preparation of antibody employed and the choice of cells used f o r t h e t i t r a t i o n o f i n t e r f e r o n . O n t h e b a s i s o f s e v e r a l e x p e r i m e n t s it w a s c o n cluded that the new bioassay may be used equally well for measuring interf e r o n n e u t r a l i z i n g a n t i b o d y in a n i m a l s e r a a n d in i m m u n o g l o b u l i n f r a c t i o n s ( t a b l e s 1 a n d 2). A l t h o u g h b o t h L 1 a n d M E C cells a r e s u i t a b l e f o r t h e t i t r a tion of interferon, L 1 cells are preferred because they provide better reproducibility. The relation of our new bioassay to that of Paucker (1965) for measuring neutralization of interferon by specific antibodies has been studied. It should be pointed out, however, that the results cannot be directly compared because the assays are based on different principles. The new bioassay measures the quantity of interferon neutralized by a fixed volume of serum or a fixed weight of immunoglobulin while Paucker's method estimates the highest dilution of serum neutralizing a fixed amount of interferon. Table 1 shows the titers of three pools of the anti-interferon sera and their neutralization indices. It can be seen that sera with high anti-interferon titers a l s o h a v e h i g h n e u t r a l i z a t i o n i n d i c e s a n d v i c e versa.

TABLE 1 Activity of three different pools of anti-interferon sera in two bioassays. Serum

NRS anti-IF, A anti-IF, B anti-IF, C

New bioassay

Paucker's bioassay

NI a

S.D.

<

-907.7 558.1 280.2

60 1926.6 1391.4 426.7

b

N e

NT d

10 7 11 6

<1 1 1 1

: 20 : 1600 : 600 : 300

a Neutralization index = the number of IF units neutralized by 1 ml of the whole serum; serum reacting with IF was diluted 1 : 2 or 1 : 4 and 10% PEG was used for the precipitation of I F - - a n t i b o d y complex. b Standard deviation. c Number of titrations. d Neutralization titre = the highest dilution of the antiserum neutralizing approximately 10 units of IF, mean of two determinations. IF = interferon; NRS = normal rabbit serum.

69 TABLE 2 Activity of several batches of the rabbit anti-interferon globulin measured by the new method. Batch

Immunoglobulin

Protein a mg/ml

NI b

S.D. c

N d

A B C R A B C

IgG IgG IgG IgG IgM IgM IgM

13.6 25.5 27.6 12.8 64.0 43.2 13.6

67.1 56.5 11.6 1510.4 30.0 12.5 29.4

47.4 26.6 4.1 1290.1 26.0 8.5 8.3

5 5 5 3 3 3 4

a Determined spectrophotometrically at 260 mg or colorimetrically at 720 mp according to the method of Lowry et al. (1951). b NI = Neutralization index = number of interferon units neutralized by 1 mg of protein in the immunoglobulin preparation. c S.D. standard deviation. d N = number of titrations. Batch R of the highly purified anti-IF IgG was a gift from Dr. L. Boreck~, Institute of Virology, Bratislava, Czechoslovakia. A d v a n t a g e s o f t h e n e w b i o a s s a y a r e as f o l l o w s : a c c u r a c y , r a p i d i t y a n d r e l a tive simplicity. The sera or immunoglobulin solutions do not need to be abs o r b e d t o r e m o v e a n t i b o d i e s t o e x t r a n e o u s a n t i g e n s , eg. c y t o t o x i c a n t i bodies. We found it unnecessary to titrate the interferon prior to every test in o r d e r t o d e t e r m i n e t h e e x a c t n u m b e r o f u n i t s a v a i l a b l e f o r n e u t r a l i z a t i o n by antibody, or to use graded dilutions of interferon; the preparation of antib o d y m a y b e u s e d in o n e o r t w o d i l u t i o n s d e p e n d i n g o n its a c t i v i t y . Interferon--antibody complexes are eliminated by the treatment with PEG and are therefore not available to react with the cell surface during the titration of interferon. This seems to be particularly important because partially neutralized interferon may interfere with the titration. I t a p p e a r s p o s s i b l e t h a t t h i s t e c h n i q u e m a y h a v e a p p l i c a t i o n s in o t h e r interferon-neutralizing assays employing various cell systems and viruses. REFERENCES Berg, K., C.A. Ogburn, K. Paueker, E. Morgensen, and K. Cantell, 1975, J. Immunol. 114, 640. Chudzio, T. and A.D. Inglot, 1975, Arch. Immunol. Ther. Exp. 23, 113. Fauconnier, B., 1967, Ann. Inst. Pasteur 1 1 3 , 7 5 7 . Havell, E., A.B. Berman, C.A. Ogburn, K. Berg, K. Paucker and J. Vilcek, 1975, Proc. Natl. Acad. Sci. U.S. 72, 2185. Inglot, A.D., T. Chudzio and M. Albin, 1973, Acta Virol. 17,416. Inglot, A.D., H. Godziriska and T. Chudzio, 1975, Acta Virol. 1 9 , 2 5 0 . Levy-Koenig, R.E., M.J. Mundy and K. Paucker, 1970, J. Immunol. 104,785. Lowry, O.M., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. chem. 193, 265. Ogburn, C.A., K. Berg and K. Paucker, 1973, J. Immunol. 111, 1206. Paucker, K., 1965, J. Immunol. 94, 371.