Biotinylation of human interleukin-2 for flow cytometry analysis of interleukin-2 receptors

Journal oflmmunologicalMethods, 122 (1989) 33-41 Elsevier

33

JIM05257

Biotinylation of human interleukin-2 for flow cytometry analysis of interleukin-2 receptors Shinsuke T a k i 1, T o s h i r o S h i m a m u r a a, M a s a a k i A b e 2, T o s h i k a z u Shirai 2 a n d Y o s h i y u k i T a k a h a r a a I Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan, and 2 Department of Pathology, Juntendo University, Hongo, Tokyo 113, Japan

(Received 9 September 1988, revised received 31 March 1989, accepted 3 April 1989)

We designed a method to analyze receptors for interleukin-2 (IL-2R) using biotinylated IL-2 (b-IL-2). To optimize the condition of biotinylation of IL-2 for flow cytometry, the degree of biotinylation was controlled by monitoring the relative biotin contents in b-IL-2 with a newly developed ELISA. The b-IL-2 prepared by incubating 150/~g IL-2 in 1 5 0 - 3 0 0 / ~ g / m l N-hydroxysuccinimidyl biotin retained biological activity and was appropriate for flow cytometry analysis. Positive fluorescence appeared in the IL-2Rbearing cell lines but not in those without IL-2R. This binding was inhibited by preincubation of the cells with unlabelled IL-2. The b-IL-2 bound to both low and high affinity IL-2Rs, but the binding to the latter was more intense. The advantage of this method is that expression of IL-2Rs of these two categories of affinity can be separately monitored. Key words." Interleukin-2; Interleukin-2 receptor complex; Avidin-biotin system; Flow cytometry; ELISA

Introduction

Interleukin-2 (IL-2) plays an important role as mediator in the immune system by binding to receptors on cells of various lineages (Mingari et al., 1984; Smith, 1984; Jenkinson et al., 1987). The disordered expression of the IL-2 receptor (IL-2R) is associated with some disease states (Yodoi and

Correspondence to: S. Taki, Central Research Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan. Abbreviations: IL-2, interleukin-2; IL-2R, interleukin-2 receptor; b-IL-2, interleukin-2 conjugated with biotin; BSF-2, B cell stimulatory factor 2; FITC-avidin, streptavidin conjugated with fluorescein isothiocyanate; NHS-biotin, N-hydroxysuccinimidyl biotin; Ab, antibody; RamIg, rabbit anti-mouse immunoglobulin; MFI, mean fluorescent intensity; PHA, phytohemmaglutinin; PBL, peripheral blood leukocytes.

Uchiyama, 1986; Ishida et al., 1987; Katagiri et al., 1987). Thus, it is important to investigate the mechanisms underlying the aberrant expression of this 'interaction' molecule. It has been suggested that the IL-2R are composed of, at least, two different polypeptides, the a chain ('Tac' molecule or p55) and the fl chain (p70), and that, if associated, these two chains form the high affinity IL-2R, although a and fl chains independently represent the low and intermediate affinity IL-2R, respectively (Sharon et al., 1986; Tsudo et al., 1986; Dukovich et al., 1987; Robb et al., 1987; Teshigawara et al., 1987). A method to assess the extent of expression of the fl chain on the cell surface by flow cytometry has not been available. The a chain can be detected using certain antibodies (Leonard et al., 1982; Osawa and Diamanstein, 1983). To distinguish the high, intermediate and low affinity states of IL-2R,

0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

34 the radioreceptor assay using radiolabelled IL-2 has been widely used (Robb et al., 1981, 1984). This assay is not applicable for a multiparameter analysis such as certain cell surface molecules or states in cell cycle versus IL-2R expression. We reported here an enzyme-linked immunosorbent assay (ELISA) which enables measurement of the relative extent of biotinylation of IL-2 and optimizes the condition of biotinylation for flow cytometry analysis. Such biotin-conjugated IL-2 (b-IL-2) can serve to detect the IL-2R or both high and low affinities on human cell lines.

Materials and methods

Reagents N-hydroxysuccinimidyl biotin (NHS-biotin) and fluorescein-labelled streptavidin (FITCavidin) were purchased from Boehringer-Mannheim (Mannheim, F.R.G.) and Vector Laboratory (Burlingame, CA), respectively. The alkaline phosphatase substrates, p-nitrophenyl phosphate (PNPP) from Sigma Chemical, St. Louis, MO, nitroblue tetrazolium (NBT) and 5-bromo-4chloro-3-indolyl phosphate (BCIP), from Promega, Madison, WI, were used. Cytokines and antibodies Monoclonal anti-IL-2 antibody (anti-IL-2 moAb) of IgG1 class produced from the hybridoma line, l l F 6 , established in the Ajinomoto Laboratory (N. Shimamura, unpublished), and purified on Protein A-Sepharose (Pharmacia, Uppsala, Sweden) column. The anti-Tac antibody (Leonard et al., 1982) was kindly provided by Dr. T. Uchiyama, Kyoto University, Japan. FITCconjugated rabbit anti-mouse immunoglobulin (FITC-Ramlg) was from Cappel (Cooper Biomedical, Malvern, PA). Recombinant human IL-2 was prepared according to Sano et al. (1987). The preparation used in this study had the specific activity of 5 X 1 0 7 U / m g of protein. The recombinant human B cell stimulatory factor 2 (BSF-2) were used was prepared as described (Taga et al., 1987). Cells An HTLV-I-positive cell fine, HUT102, was provided by Dr. H. Henmi, Sagami Chuken and

both of HTLV-I-transformed cell lines, MT-1 and thymoma cell line, YT, were provided by Dr. J. Yodoi, Kyoto University, Japan. These three cell lines were maintained in RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (Hyclone, Logan, UT). Human T leukemic cell lines, Jurkat and CCRF-CEM, human B lymphoblastoid cell line, RPMI 1788, and human Burkitt's lymphoma line, Raji, were also maintained in the same medium. Peripheral blood leukocytes (PBL) were obtained using Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation from healthy donors and cultured in the same medium containing 2% (v/v) phytohemagglutinin (PHA) (M-form, Gibco Laboratories, Grand Island, NY) for 2 days.

Biotinylation of IL-2 Varying amounts of NHS-biotin were dissolved in dimethyl sulfoxide, and 60/11 were mixed with 1 ml of 150 ~ g / m l IL-2 in 0.1 M N a H C O 3. After stirring at room temperature for 3 h, the b-IL-2 solution was dialyzed extensively against phosphate-buffered saline (PBS) to remove the unreacted NHS biotin. For the mock biotinylation, 60 /~1 of dimethyl sulfoxide with no NHS-biotin was added. The b-IL-2 was sterilized by passing through a 0.22 /~m filter (Millipore Corp., Bedford, MA) and was stocked at 4 ° C. Dot blot The nitrocellulose filter (Nitroplus 2000, Micron separation), on which the grids of 8 x 8 mm were marked, was first immersed in distilled water then in PBS and air-dried. The serially two-fold diluted b-IL-2 samples in PBS were dotted on the filter either directly or using Bio-Dot manifold (Bio-Rad Laboratories, Richmond, CA), and airdried. The filter was then blocked with 2% non-fat dry milk-containing PBS for 1 h. After washing with PBS containing 0.05% Tween 20 (TweenPBS), the complex of avidin and biotinyl alkaline phosphatase (Vectastain ABC-AP kits, Vector Laboratories), prepared according to instructions of the supplier, was loaded on the filter and this filter was incubated for 30 min at room temperature, washed with Tween-PBS and then loaded with the substrate buffer, 100 mM Tris-HC1, pH 8.8, 100 mM NaC1, 5 mM MgC12 containing NBT

35 (0.046 mM) and BCIP (0.4 mM). About 10 min later, the reaction was halted by washing the filter with distilled water.

ELISA Wells of microtiter plates (Titertek, Flow Laboratories, Rockville, MD) were coated with anti-IL-2 moAb (1 ~ g / m l ) overnight, washed with Tween-PBS and coated with 5% (v/v) bovine serum albumin (BSA)-containing PBS for 1 h. 50 /~1 o f varying dilutions of b-IL-2 were added and the preparation then left to settle at room temperature for 1 h. After washing the wells with TweenPBS, the avidin-biotinyl alkaline phosphatase complex (the same as that used in the dot blot) was added to each well (50 #l/well). After incubation at room temperature for 30 min, the wells were extensively washed with Tween-PBS, then 50 /~1 of substrate buffer (0.05 M NaHCO3, pH 9.8, 10 mM MgC12) containing 1 m g / m l of PNPP were added. Absorbance at 405 nm was measured every 10 min using a microplate reader (Toyo Sokki, Tokyo, Japan) until the absorbance of maximally reacted wells reached an appropriate value, normally 1.0. The dilution providing the half maximum absorbance was determined as the unit of biotinylation. Duplicated wells were usually set up for each dilution point.

Flow cytometry 10 6 cells were washed with PBS containing 0.5% ( w / v ) BSA and 0.05% ( w / v ) N a N 3 (BSAPBS) and 20 ttl of b-IL-2, anti-Tac antibody, both diluted with BSA-PBS, or BSA-PBS alone were added to each cell pellet. After incubation for 90 min on ice, the cells were washed with BSA-PBS, mixed with 20 /~1 of FITC-avidin (10 /~g/ml) or F I T C - R a m l g (10/xg/ml) and again incubated for 30 min on ice. The stained cells were fixed in 0.5% paraformaldehyde in PBS for 10 min on ice and were analyzed using an Epics V flow cytometer (Coulter Electronics, Hialeah, FL) or FACStar flow cytometer (Becton Dickinson, Mountain View, CA). To examine specificity of the binding, the cell pellet was mixed with 10 /~1 of unlabelled IL-2, BSF-2 or anti-Tac antibody, settled for 1 h on ice, and then stained with 10 /~1 of b-IL-2 followed by avidin-FITC. Percent inhibition of

binding in mean fluorescence intensity (MFI) was calculated as follows. % inhibition =100-

MFI with competitors- MFI without b-IL-2 MFI with b-IL-2 only- MFI without b-IL-2

x 100

CTLL assay The biological activity of b-IL-2 was determined using the murine IL-2-dependent cell line, CTLL-2, as described (Taniguchi et al., 1983).

Results

Optimal condition for biotinylation of IL-2 To optimize the condition for the biotyinylation of IL-2, we developed two different assay procedures to define the extent of biotinylation: one was the dot blot assay and the other was ELISA. In the former, the amounts of the biotinyl group on IL-2 molecules spotted on nitrocellulose filter were determined as the relative amount of bound avidin molecules. As shown in Fig. 1, the amount of biotin incorporated into the IL-2 molecules increased as the concentrations of NHS-biotin added were raised, and reached a plateau at amounts over 300/~g/ml. In case of ELISA which was more specific and quantitative, the amounts of the biotinyl group incorporated into the IL-2 molecules could be measured as those captured on microtiter wells coated with anti-IL-2 moAb, and expressed as the relative biotinylation units (see materials and methods section). As in the dot blot assay, the extent of biotinylation increased linearly with increase of the concentration of NHS-biotin added and reached a plateau at amounts of around 300 t t g / m l (Fig. 2). This event was not due to changes in the antigenicity of IL-2 upon biotinylation, because the result paralleled that of the dot blot which is independent of antigenicity.

Influence of biotinylation on the biological activity of IL-2 We carried out the CTLL assay to assess the influence of biotinylation on the biological activ-

36

A. ng b-lL-2/dot 15 7.5 3.8 1.9 0.9 0.5 (0) (15)

B-IL -2

(30) (60) (120)

B°

n 9 b-lL -2~dot 1.5

0.75 0.38 0.19 0.09 0.05

(1200) ( 600 )

B -IL-2

(3oo (150) (75) (0)

Fig. 1. Serially diluted b-IL-2 preparations in 1 #1 (B) or 100 txl (A) PBS were dotted on nitrocellulose filter. Dots were visualized with avidin-alkaline phosphatase complex. Amounts of b-IL-2 in each dot were shown as the final protein concentration. The concentration of NHS-biotin used for conjugation (ktg/ml) is shown in parentheses.

ity of IL-2. Fig. 2 shows the biological activities per mg of various preparations of IL-2 biotinylated with various a m o u n t s of NHS-biotin. The biological activity of IL-2 was retained u p o n m o d ification with concentrations of N H S - b i o t i n up to 150 /~g/ml, but was reduced progressively thereafter, in a dose-dependent manner. Therefore, the b - l L - 2 prepared with 150-300 / t g / r n l of N H S biotin was used in the following experiments.

Flow cytometry analysis of IL-2R lymphoid cell lines

on human

Biotinyl IL-2, p r e p a r e d u n d e r conditions described above, was used to stain the h u m a n

H T L V - I positive T cell line, H U T 1 0 2 cells k n o w n to express large a m o u n t of I L - 2 R on the cell surface (Lowenthal and Green, 1987). We used 20 /~1 of 1.67/xM (25 / z g / m l ) of b-IL-2 per 1 z 10 6 cells. As shown in Fig. 3A, a positive fluorescence was observed with H U T 1 0 2 cells stained with b-IL-2 followed by F I T C - a v i d i n F I T C - a v i d i n alone led to no specific staining. The optimal period of incubation with b-IL-2 was 90 rain and longer periods of incubation did not increase the intensity of fluorescence (data not shown). Fig. 3B shows that the m e a n fluorescence intensities ( M F I ) increased in a dose-dependent and saturating manner, as the a m o u n t s of b-IL-2 used for stain-

37 x 107 5-

~

-100

4-

o 3-

5o ~ b

2-

o

b

O-

,

0

-

,

30

,

and anti-Tac Ab. T h e HUT102 expressing both high and low affinity IL-2Rs and the MT-1 expressing only the low affinity IL-2R (Fujii et al., 1986) showed almost equal intensity of b-IL-2 binding whereas a higher expression of Tac molecules was observed on MT-1 than on HUT102. The YT cells which express relatively higher amounts of IL-2R of intermediate affinity than those of high affinity showed lower fluorescent intensity when stained with anti-Tac Ab than with b-IL-2. Preincubation in excess a m o u n t of

4.

0

120

480

1920

pg/ml NHS-biotin Fig. 2. The effect of biotinylation on the biological activity of IL-2. Relative biotinylation units (e) of the b-IL-2 preparation were the same as for Fig. 1 and were measured by ELISA and expressed as % of the maximum. The concentrations of NHSbiotin used for the conjugation are shown in the abscissa. (©), the biological activities were measured by CTLL assay.

43

E

0

'2

/

'3

Relative Fluorescence ing. b-IL-2 of 0.4 /~M (6.0 /~g/ml) and 0.04 ~ M (0.6/~g/ml) were sufficient to stain 100% and 50% of HUT102 cells, respectively. As shown in Fig. 4 a human HTLV-I-transformed cell line, MT-1, which is also known to express IL-2R (Fujii et al., 1986), was stained well with b-IL-2. The non-IL2R-bearing cell lines, R P M I 1788, C C R F - C E M , Jurkat and Raji, showed no positive fluorescence.

B 300-

-100

~200-

Specificity of b-IL-2 binding To examine the specificity of b-IL-2 staining, we carried out a competitive inhibition assay, using unlabelled IL-2. The BSF-2 was used as an unrelated cytokine control. As shown in Fig. 5, the unlabelled IL-2 (but not BSF-2) significantly inhibited the binding of b-IL-2 to HUT102. The inhibition was not complete, presumably because of the relatively low concentration of IL-2 used, compared with b - I L - 2 : 1 3 . 3 ~ M (200 t t g / m l ) of the competitor versus 1.67 /~M (25 # g / m l ) of b-IL-2.

Affinity status of 1L-2R accessible to binding of b-lL-2 Table I compares the intensities of binding to cell lines HUT102, Y T and MT-1 between b-IL-2

50

"~

loo-

O-

i

i

[lOl

i

i

lllili

r

o.1

i

i

irlliq

i

i

i

i

-0

1

b-lL-2 concentration ( ~M) Fig. 3. Binding of b-IL-2 on HUT102 cell lines. A: HUT102 cells were incubated with 1.67 /LM (25 t t g / m l ) of b-IL-2 prepared with 300 ~ g / m l NHS-biotin followed by FITCavidin. Stained cells were analyzed by Epics V with logarithmic amplification. Solid line: b-IL-2 and FITC-avidin. Broken line: FITC-avidin alone. B: relative fluorescence intensities of cells stained with b-IL-2 of given concentrations. HUT102 cells were stained with b-IL-2, as in A and analyzed with FACStar. The MFI expressed on a logarithmic amplification are shown as differences over background (o). Percent positively stained cells were shown as well (O).

38 TABLE I DIFFERENTIAL

BINDINGS OF b-IL-2 A N D ANTI-Tac Ab TO IL-2Rs OF DIFFERENT

AFFINITIES

Cell lines

Receptor number a

Binding b (MFI)

High

Intermediate

Low

(-)

b-IL-2

Anti-Tac

H UT102 YT MT-1

5 000 5000 (-)

(-) 20000 (-)

100 000 (-) 300000

18.4 29.8 20.8

738.9 195.1 816.2

1 271.7 175.0 3189.4

" I L - 2 R s of each affinity were s h o w n as I L - 2 - b i n d i n g sites per cell. b b - I L - 2 was used at 0.47/.tM (7 f f g / m l ) for H U T 1 0 2 or MT-1 and 1.83 /tM (27.5 # g / m l ) for YT. A n t i - T a c A b was used at 10 / ~ g / m l . The flow c y t o m e t e r used was F A C S t a r .

CCRFC -EM

\ PIT-~

RPM/1788

-Q

E (D

JURKTA.I~-~ .

RAJI

unlabelled IL-2 inhibited b-IL-2 binding to all these cell lines. The anti-Tac Ab inhibited b-IL-2 binding to YT cells in lesser extent than that to other cell lines, i.e., HUT102 and MT-1. The fluorescent profiles of YT cells stained with b-IL-2 with or without competitors are shown in Fig. 6. Unlabelled IL-2 inhibited the b-IL-2 binding on PHA-activated PBL slightly more intensely than anti-Tac antibody (Fig. 6). The M F I were as follows: FITC-avidin only, 9.09; b-IL-2, 39.01; IL-2 followed by b-IL-2, 10.65; anti-Tac antibody followed by b-IL-2, 12.72.

Discussion i

Relati~/e Fluorescence Fig. 4. B i n d i n g of b - I L - 2 on cell lines, C C R F - C E M , Jurkat, MT-1, R P M I 1 7 8 8 a n d Raji. Solid lines represent fluorescence profiles stained with b - I L - 2 followed b y F I T C - a v i d i n and b r o k e n lines represent those w i t h F I T C - a v i d i n alone. The c y t o m e t e r used was Epics V.

,.Q

~ L

The radioreceptor assay using radiolabelled IL-2 has been used to identify the expression of IL-2R T A B L E II COMPETITIVE INHIBITION OF b-IL-2 BINDING U N L A B E L L E D IL-2 O R A N T I - T a c A N T I B O D Y Cell lines

Competitors a (ffg/ml)

% inhibition b

HUT102

IL-2 Anti-Tac

(200) (80)

97.2 93.8

YT

IL-2 Anti-Tac

(200) (100)

88.4 12.5

MT-1

IL-2 Anti-Tac

(200) (80)

98.1 99.2

, • ~'

WITH

',

/

Relative

2

)

Fluorescence

Fig. 5. C o m p e t i t i v e i n h i b i t i o n of b-IL-2 binding. H U T 1 0 2 cells were p r e i n c u b a t e d w i t h u n l a b e l l e d IL-2 ( . . . . . ), BSF-2 ( . . . . . . ) or BSA-PBS ( ), then s t a i n e d and a n a l y z e d as in Fig. 3. The fluorescence profile of H U T 1 0 2 i n c u b a t e d w i t h F I T C - a v i d i n a l o n e is also s h o w n ( . . . . . . . . ).

a Cell lines were i n c u b a t e d in 10 /~1 of BSA-PBS, IL-2 or anti-Tac A b of the c o n c e n t r a t i o n indicated. A f t e r 1 h, 10 ~1 of b-IL-2 (the c o n c e n t r a t i o n s were the same as in T a b l e I) were a d d e d a n d i n c u b a t e d for a further 90 min. The flow c y t o m e t e r used was F A C S t a r . b Percent i n h i b i t i o n of b-IL-2 b i n d i n g w a s c a l c u l a t e d as in the m a t e r i a l s and m e t h o d s section.

39

YT

PHA-PBL

A

B. i~

;k

Fig. 6. Biotin IL-2 binding on YT cells and PHA-activated human PBL. A: ceils were stained with 1.83/.tM (27.5 ~g/ml) b-IL-2 alone. B: cells were preincubated with 13.3 /~M (200 t~g/ml) unlabelled IL-2, then stained with b-IL-2. C: cells were preincubated with 100 #g/ml of anti-Tac antibody, then stained with b-IL-2. Cells were analyzed on FACStar. Fluorescent profiles of the cells stained with FITC-avidin alone were also shown ( . . . . . . ).

of both high and low affinities (Robb et al., 1981, 1984). However, because the radioreceptor assay represents only a mean number of IL-2-binding sites on the cell surface, the amount of IL-2-binding sites on single cell level could not be estimated. In the present report, we described a novel ELISA technique to monitor the extents of biotinylation of IL-2, using anti-IL-2 moAb and we assessed the expression of IL-2R on the cell surface by flow cytometry, using b-IL-2. Highly biotinylated IL-2 loses its biological activity. Kuo and Robb (1986) showed that the regions covering amino acids 8-27 and 33-54 were candidates for the receptor binding site of the IL-2 molecule. These regions contain several lysine residues which are thought to be targets for the conjugation with NHS-biofin. Thus, it seems likely that the biotin molecules interfered with the IL-2-IL-2R interaction through steric hindrance in case of highly biotinylated IL-2. The most appropriate preparation of b-IL-2 for flow cytometry analysis was obtained when the IL-2 was modified with NHS-

biotin at concentrations ranging from 150 to 300 /~g/ml. Such a preparation was biotinylated enough, and the biological activity retained considerably well. Using this b-IL-2, the positive fluorescence was observed on IL-2R-bearing HUT102 cells (Fig. 3). Effective concentrations required for staining 100% and 50% of HUT102 cells were about 0.4/~M (6.0 /~g/ml) and 0.04 /~M (0.6 /~g/ml), respectively. These values fit well with those for low affinity IL-2R obtained from studies using radiolabelled IL-2 (Robb et al., 1987). In contrast, MFI did not reach its maximum level even when stained with 1.5 /~M (22.5 /~g/ml) of b-IL-2. Requirement of such a high concentration of b-IL-2 to obtain the maximum MFI suggests that only a small amount of b-IL-2 remained bound to IL-2R during subsequent washing and incubation with FITC-avidin. This is probably due to the rapid dissociation of b-IL-2 from the low affinity IL-2R (Lowenthal and Green, 1987; Wang and Smith, 1987). Alternatively, the interaction between FITC-avidin with the biotin residues on IL-2 may lead to a dissociation of b-IL-2 from its receptor, since the affinity between avidin and biotin ( K a = 10 -15 M) was much higher than that between IL-2 and low affinity IL-2R ( K a = 10 -8 M). The former seems more likely, since avidin at the same concentration as that used to stain the cells did not inhibit the b-IL-2-induced proliferation of CTLL-2 cells (unpublished observation). The binding of b-IL-2 to IL-2R appears to be specific, since the b-IL-2 bound to the cell lines bearing IL-2R but not to those without IL-2R. The binding could be inhibited with unlabelled IL-2 but not with BSF-2, which was shown to bind to the cellular receptor other than IL-2R (Taga et al., 1987). The HTLV-I-transformed cell fine, MT-1, expresses the low affinity IL-2R alone (Fujii et al., 1986), and the HTLV-I-positive cell line, HUT102, bears both high and low affinity IL-2Rs (Lowenthal and Green, 1987). The present study shows that the binding intensity of b-IL-2 to HUT102 was almost identical to that to MT-1. This is in contrast to the binding of anti-Tac Ab between these two cell lines, in that a higher intensity was observed on MT-1 than HUT102. Thus, b-IL-2 appears to bind to high affinity IL-2R more intensely than to low affinity. In

40 a d d i t i o n , b - I L - 2 a p p e a r e d to b i n d to the Y T cell line m a i n l y t h r o u g h t h e / 3 chain of IL-2R, b e c a u s e b - I L - 2 b i n d i n g was i n h i b i t e d with u n l a b e l l e d IL-2 b u t n o t with a n t i - T a c A b . T h e differential staining p a t t e r n s with b - I L - 2 b e t w e e n these three cell lines m a y facilitate differe n t i a t i o n of the three categories of I L - 2 R s a n d relative a m o u n t s of the high a n d low affinity I L - 2 R s can be e s t i m a t e d using b - I L - 2 a n d a n t i - T a c A b in c o m b i n a t i o n with the cell lines b e a r i n g I L - 2 R s of d e f i n e d n u m b e r s as reference. F r o m the result shown in Fig. 6, we can estim a t e the c o n t r i b u t i o n of the I L - 2 R /3 c h a i n to be a b o u t 7% ((12.72 - 10.65)/(39.01 - 10.65) × 100) of the total b - I L - 2 binding. A s s u m i n g that there are a b o u t 10000 I L - 2 R on P H A - a c t i v a t e d P B L a n d b o t h a a n d /3 c h a i n s c o n t r i b u t e e q u a l l y to I L - 2 b i n d i n g , there are an e s t i m a t e d 700 fl chains on P H A - a c t i v a t e d PBL. This value is a b o u t half of that o b t a i n e d f r o m r a d i o r e c e p t o r assay ( W o n g a n d Smith, 1987). This d i s c r e p a n c y is t h o u g h t to be d u e to the i n c o m p l e t e i n h i b i t i o n of b - I L - 2 b i n d i n g with u n l a b e l l e d IL-2. W e can e s t i m a t e there to be 1200 /3 chains on P H A - a c t i v a t e d hum a n PBL, if we used the value 9.09 in the p l a c e of 10.65 as b a c k g r o u n d fluorescence. T a k e n together, the t e c h n i q u e r e p o r t e d here a p p e a r s to b e useful for m o n i t o r i n g the expression of I L - 2 R s on cells of various lineages in association with o t h e r cell surface p a r a m e t e r s , in b o t h n o r m a l a n d p a t h o l o g i c a l clinical states in which the expression of I L - 2 R is d i s o r d e r e d . T h e b - I L - 2 can also be a p p l i e d to investigate the roles of IL-2 in d e v e l o p m e n t a n d r e g u l a t i o n in i m m u n e systems. T h e E L I S A system, d e v e l o p e d in our s t u d y to m o n i t o r the extent of b i o t i n y l a t i o n of IL-2 using a n t i - I L - 2 m o A b c a n also be a p p l i e d to o t h e r p r o teins or c a r b o h y d r a t e s to m e a s u r e the relative e x t e n t of b i o t i n y l a t i o n . T h e p r o c e d u r e , albeit less q u a n t i t a t i v e , is n o t c o m p l i c a t e d a n d is to be f a v o r e d over the c o n v e n t i o n a l s p e c t r o p h o t o m e t r i c a s s a y ( G r e e n , 1970). W h i l e the c o n v e n t i o n a l assay requires a large a m o u n t of b i o t i n y l proteins, i.e., of m i l l i g r a m order, this assay c a n be carried out with p r o t e i n s in p i c o g r a m ranges. W e used a highly p u r i f i e d p r e p a r a t i o n of IL-2, b u t this E L I S A , when used with specific antibodies, can also be a p p l i e d in case of less p u r e proteins.

Note added in proof D u r i n g the revision of this report, F o x w e l l et al. (1988) J. I m m u n o l . M e t h o d s 113, 221, a n d L i n et al. (1988) J. I m m u n o l . 141, 3847, r e p o r t e d the b i o t i n y l a t i o n of r e c o m b i n a n t I L - 2 of h u m a n a n d simian origin, respectively. They, however, presented n o d a t a c o n c e r n i n g the affinity of I L - 2 R to which b - I L - 2 b o u n d .

Acknowledgements W e are grateful to Drs. H. H e n m i a n d J. Y o d o i for the generous gifts of cell lines a n d to Dr. T. U c h i y a m a for the a n t i - T a c a n t i b o d y . W e also t h a n k Mr. N. K o n d o h for helpful suggestions a n d to M. O h a r a for critical c o m m e n t s .

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