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A highly sensitive enzyme-linked immunosorbent assay for the measurement of interleukin-8 in biological fluids
Journal of lmmunological Methods, 156 (1992) 27-38 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
27
JIM 06485
A highly sensitive enzyme-linked immunosorbent assay for the measurement of interleukin-8 in biological fluids N o b u o Ida a, Shingou Sakurai b, Kazuo Hosoi b and Tetsunosuke Kunitomo a a Medical Devices and Diagnostics Research Laboratories, Toray Industries, Inc., ! 1! 1 Tebiro, Kamakura, Kanagawa 248, Japan, and b Mishima Plants, Toray Industries, Inc., 4845 Mishirna, Shizuoka 411, Japan (Received 3 March 1992, revised received 5 May 1992, accepted 29 May 1992)
Interleukin-8 (IL-8) is a chemotactic and activating cytokine for neutrophils, which plays an important role in acute inflammatory responses. We aimed to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for IL-8 and established 18 clones of anti-IL-8 monoclonal antibodies (mAbs). These mAbs were evaluated in terms of their antigen-binding affinities, and five clones were selected and used for the comparative study of various combinations of antibodies in sandwich ELISA. Affinity purified rabbit polyclonal antibody was also used in this study. One antibody pair, which showed relatively high sensitivity and which was not severely interfered with blood components, was selected and the assay conditions were optimized by choosing the appropriate buffer for sample dilution and by directly labeling the second antibody with enzyme. The finalized ELISA, using polyclonal antibody as first (coated) antibody and horseradish peroxidase-labeled mAb (clone EL139) Fab' fragment as second antibody, could detect as low as 2.5 pg/ml (0.125 pg/well) of IL-8 by in total 2 h incubation, without being affected by body fluid components. The ELISA was specific to IL-8, showing no cross-reactivity with other cytokines or various IL-8 family proteins which share some amino acid sequence homology with IL-8. As an example of its application to clinical specimens, plasma samples from patients with septic shock were measured. The results showed that sepsis patients contain significantly higher levels of plasma IL-8 compared to normal controls. When analyzed by gel-filtration chromatography, IL-8 in sepsis plasma was eluted in a molecular weight (M r) region corresponding to the monomer form. The ELISA established here is expected to be effectively used for further investigations on the relationship between IL-8 and various diseases. Key words: Interleukin-8; ELISA; Sepsis
Introduction Correspondence to: N. Ida, Medical Devices and Diagnostics Research Laboratories, Toray Industries, Inc., 1111 Tebiro, Kamakura, Kanagawa 248, Japan. Tel.: 81-467-322111. Fax: 81-467-32-4791. Abbreviations: Ab, antibody; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; IL-8, interleukin-8; mAb, monoclonal antibody; Mr, molecular weight; PBS, phosphate-buffered saline.
Interleukin-8 (IL-8), which is also known as monocyte-derived neutrophil chemotactic factor (MDNCF) (Yoshimura et al., 1987), neutrophil activating factor (NAF) (Waits et al, 1987), neutrophil activating peptide-1 (NAP-l) (Schr6der et al., 1987), or T cell chemotactic factor (TCF)
28 (Larsen et al., 1989), is a potent chemotactic and activating cytokine for neutrophils. It is produced by various types of cells such as monocytes (Yoshimura et al., 1987; Larsen et al., 1989), lymphocytes (Gregory et al., 1988), endothelial cells (Strieter et al., 1989a), hepatocytes (Thronton et al., 1990), and fibroblasts (Strieter et al., 1989b), and believed to play an important role as a mediator of acute inflammatory reactions. It belongs to a family of low molecular weight (8-10 kDa) proteins including connective tissue activating peptide III (CTAP-III) (Castor et al., 1983), /3-thromboglobulin (/3-TG) (Begg et al., 1978), platelet factor 4 (PF4) (Deuel et al., 1977), interferon-y inducible protein (IP-10) (Luster et al., 1985) and growth related oncogene (GRO) (Richmond et al., 1988), with 29-46% sequence homology at the protein level. Structural and functional analyses of IL-8 have been advancing rapidly, since its cDNA was cloned and sequenced (Matsushima et al., 1988). For example, three-dimensional structure was determined by NMR (Clore et al., 1990) and X ray crystallographic analysis (Baldwin et al., 1991), and two types of its receptor gene were isolated recently (Holmes et al., 1991; Murphy et al., 1991). As expected from its important role in acute inflammatory responses, IL-8 is also reported to be involved in the pathogenesis of several diseases including rheumatoid arthritis (Brennan et al., 1990), psoriasis (Sticherling et al., 1991), and sepsis (Martich et al., 1991; Van Zee et al., 1991). In order to further clarify the relationship between IL-8 and these various disorders, accurate determination of IL-8 levels in body fluids is an important technique. Widely used bioassays, which measure the chemotactic activity or the release of lysosomal enzymes from activated neutrophils (Yoshimura et al., 1987; Schr6der et al., 1987), are not easily applied to clinical samples, because these assays are not sensitive enough, time-consuming, and most importantly, results are affected by other bioactive substances contained in the body fluids. Several immunological assays have also been developed and successfully used for the determination of IL-8 levels in culture supernatants or in plasma samples (Ceska et al., 1989; Sticherling et al., 1989; Sylvester et al., 1990; DeForge et al., 1991). In these assays, how-
ever, interference of body fluid components have not been fully examined. In this paper, we describe the establishment of anti-IL-8 mAbs with high antigen-binding affinity, and the development of a highly sensitive ELISA for IL-8 which is directly applicable to clinical samples.
Materials and methods
IL-8 preparation IL-8, used as the immunogen and the standard sample for ELISA, was produced from poly I : Cstimulated human fibroblast cell, DIP-2, and purified to homogeneity by silica column chromatography, heparin column chromatography and reverse-phase HPLC. This preparation was a mixture of three isoforms of IL-8 with 72, 77, and 79 amino acid residues, which are known to be caused by differential processings from the same precursor (Yoshimura et al., 1989), and the contents of these three isoforms were about 50, 40 and 10%, respectively. Concentration of IL-8 in the purified preparation was determined by amino acid analysis.
Polyclonal antibody Two New Zealand White rabbits were immunized s.c. with 350 /.Lg each of IL-8 in Freund's complete adjuvant. The rabbits were boosted twice with the same amount of IL-8 in Freund's incomplete adjuvant at intervals of 2 and 3 weeks. 10 days after the final immunization, they were bled and the antiserum (110 ml total) was separated. The antiserum obtained was diluted twice with PBS and applied to IL-8 immobilized affinity column to which 10 mg of IL-8 was covalently coupled, and the bound antibody was eluted with 0.1 M glycine-HCl buffer (pH 2.4). By this purification, 7.5 mg of antibody was obtained from 30 ml of antiserum.
Establishment bridoma
of anti-IL-8 mAb-producing hy-
Ten female B A L B / c mice (8 weeks of age) were immunized i.p. with 10-20 ~g of IL-8 in an adjuvant of aluminum potassium sulfate with inactivated Bordetella pertussis (Wako Pure Chemical, Osaka, Japan). They were boosted twice or
29 three times with 10 ~g of antigen (first with aluminum potassium sulfate adjuvant, second and third in phosphate buffered saline (PBS)) at the intervals of 10-40 days. 3 days after the last immunization, the spleen cells were isolated, mixed with P3U1 myeloma cells at a ratio of 10: 1, fused in 50% polyethylene glycol 1500 (Boehringer Mannheim, Germany) and seeded into 96-well microplate in HAT medium (RPMI 1640) containing 15% fetal calf serum and 2 ng/ml human IL-6. The anti-IL-8 mAb secreting hybridomas were screened by ELISA method. Microtiter plates (Nunc, Denmark) were coated with IL-8 (0.5 /zg/ml in PBS 100/xl/well) at 4°C overnight, and blocked with 0.5% BSA in PBS. Hybridoma culture supernatant (100 ~1) was dispensed into each well and incubated for 1 h at room temperature, followed by sequential incubations with biotinylated goat anti-mouse IgG (Zymed, San Francisco, CA), streptavidin-horseradish peroxidase (HRP) conjugate (Zymed, San Francisco, CA), and the enzyme substrate (0.1 M sodium acetate-citrate buffer (pH 5.5) containing 0.006% H 2 0 z and 0.2 mg/ml 3,3',5,5'-tetramethyl benzidine (TMB)). The plate was washed with the washing solution (2-fold diluted PBS containing 0.025% Tween 20) three times between each incubation step. Finally, 1 N H 2 S O 4 was added to stop the reaction, and the color development was measured at 450 nm. Cells in positive wells were further cultured in HT medium overnight, and on the next day, the supernatants were withdrawn again, serially diluted in PBS containing 0.25% BSA and 0.05% Tween 20, and tested for the reactivity with solid phase IL-8 as described above. Positive wells which showed the reactivity at relatively higher dilution rate (with lower concentration of mAb) were selected and cloned twice by limiting dilution.
m,4b production and purification Hybridomas were cultured in serum-free medium (Celgrosser-H, Sumitomo Pharmaceuticals, Osaka, Japan) and the supernatants were used for the characterization of mAbs. Purified mAbs, used in IL-8 ELISA, were purified from mouse ascites by ammonium sulfate precipitation and protein A column chromatography.
Isotyping of mAb The subclasses of mAbs were determined using commercially available ELISA kit (Zymed, San Francisco, CA).
Western blotting Western blotting was performed according to the method of Towbin et al. (1979). Briefly, IL-8 sample (75/xg/ml, 100/zl) was separated by 20% gel SDS-PAGE, and electrically blotted to a membrane (Clear Blot Membrane-P, Atto, Tokyo, Japan). After the sheet was blocked with 5% skim milk, 2 p.g/ml of anti-IL-8 mAbs, biotinylated goat anti-mouse IgG or biotinylated goat anti-mouse IgM (Cappel, West Chester, PA), and streptavidin-HRP conjugate were sequentially added and incubated. Finally, the enzyme activity was detected with color developing kit (Immunostaining H R P kit, Konica, Tokyo, Japan).
Epitope analysis Epitopes of MAbs were examined using eight synthetic peptides (15 amino acid length each), which cover the whole sequence of IL-8 with overlapping of six or seven amino acid residues between each peptide. The peptides, Serl-Lys15 (SAKELRCQCIKTYSK), Cys9-Lys23 (CIKTYSKPFHPKFIK), Phe17-Gly31 (FHPKFIKELRVIESG), Leu25-Ile39 (LRVIESGPHCANTEI), His33-Arg47 ( H C A N T E I I V K L S G D R), V a 1 4 1 - G l u 5 5 (VKLSDGRELCLDPKE), Leu49-Glu63 ( L C L D P K E N W V Q R V V E ) and Val58-Ser72 (VQRVVEKFLKRAENS), were synthesized by solid phase method and covalently attached to microplate wells ('Co-Bind' Micromenbranes, Newark, N J) according to the manufacturer's instruction. After blocking, serially diluted MAbs (2-0.016 /xg/ml) were dispensed into each well and incubated for 1 h. The bound mAbs were detected by the sequencial reaction of biotinylated goat anti-mouse IgG or biotinylated goat anti-mouse IgM, streptavidin-HRP, and the enzyme substrate.
Evaluation of the affinity of mAb The affinity of each mAb to the antigen IL-8 was estimated by two different sandwich ELISA methods as follows. In evaluation (1), the plate
30 was c o a t e d with r a b b i t p o l y c l o n a l a n t i - I L - 8 antib o d y a n d b l o c k e d . Serially d i l u t e d IL-8 was a d d e d a n d i n c u b a t e d at 25°C for 1 h. T h e n , 2 / . ~ g / m l o f each mAb, biotinylated rabbit anti-mouse IgG (Tago, B u r l i n g a m e , CA), s t r e p t a v i d i n - H R P conjugate, a n d t h e e n z y m e s u b s t r a t e w e r e s e q u e n t i a l l y d i s p e n s e d a n d i n c u b a t e d . A f t e r t h e r e a c t i o n was s t o p p e d with 1 N H 2 8 0 4 , t h e a b s o r b a n c e at 450 n m was m e a s u r e d . A O D v a l u e ( d i f f e r e n c e b e t w e e n t h e a b s o r b a n c e o f 1 n g / m l IL-8 s a m p l e a n d o f 0 n g / m l IL-8) was c a l c u l a t e d f r o m t h e s l o p e o f the s t a n d a r d curve, a n d u s e d as an index o f t h e affinity o f m A b . I n e v a l u a t i o n (2), t h e p l a t e was c o a t e d with e a c h m A b (2 / ~ g / m l ) o v e r n i g h t at 4°C, b l o c k e d , a n d t h e n serially d i l u t e d IL-8 was a d d e d a n d i n c u b a t e d at 25°C for 1 h. B i o t i n y l a t e d rabbit anti-IL-8 polyclonal antibody, streptavidinHRP conjugate, and the enzyme substrate were s e q u e n t i a l l y d i s p e n s e d a n d i n c u b a t e d f o l l o w e d by a d d i t i o n o f 1 N H 2 5 0 4 , a n d t h e a b s o r b a n c e at 450 m was m e a s u r e d . A O D was c a l c u l a t e d as d e s c r i b e d in e v a l u a t i o n (1). B i o t i n y l a t i o n o f t h e a n t i b o d y was p e r f o r m e d by using N H S - L S - b i o t i n (Pierce, R o c k f o r d , IL).
Pepsin digestion and HRP labeling of rnAb 5 m g o f E L 1 3 9 m A b , in 5 ml o f 0.1 M s o d i u m c i t r a t e b u f f e r ( p H 3.5), was d i g e s t e d with 50 /zg o f p e p s i n (Sigma, St. Louis, M O ) at 37°C for 1 h. G e n e r a t e d F ( a b ' ) 2 f r a g m e n t was l a b e l e d with H R P ( B o e h r i n g e r M a n n h e i m , G e r m a n y ) by male i m i d e - h i n g e m e t h o d ( Y o s h i t a k e et al., 1982). P u r i f i c a t i o n o f H R P - E L 1 3 9 F a b ' c o n j u g a t e was p e r f o r m e d with h y d r o x y a p a t i t e c o l u m n H P L C .
ELISA for IL-8 A m i c r o t i t e r p l a t e was c o a t e d with r a b b i t anti° IL-8 p o l y c l o n a l a n t i b o d y (0.5 / x g / m l in PBS) at 4°C overnight, a n d b l o c k e d with 0.5% B S A in PBS at 25°C for 2 h. A f t e r e a c h well was w a s h e d with 400 ~1 o f w a s h i n g s o l u t i o n (2-fold d i l u t e d PBS c o n t a i n i n g 0.025% T w e e n 20), 100 ~1 o f t h e r e a c t i o n b u f f e r (0.1 M T r i s - H C l p H 8.0 c o n t a i n ing 0.25% B S A , 0.05% T w e e n 20, 0.5% e a c h o f n o r m a l m o u s e a n d r a b b i t s e r u m ) was d i s p e n s e d , a n d 5 0 / z l o f s a m p l e s o r s t a n d a r d IL-8 was a d d e d a n d i n c u b a t e d at 25°C for 1 h. T h e p l a t e was s h a k e n on a m i c r o p l a t e m i x e r t h r o u g h o u t the i n c u b a t i o n time. H R P - l a b e l e d EL139 F a b ' m A b
TABLE I CHARACTERIZATION OF mAbs AOD represents the difference of the absorbance values of 1 ng/ml IL-8 standard and 0 ng/ml IL-8 standard, when measured by sandwich ELISA in combination with each mAb and rabbit polyclonal antibody. Clone
EC81 EC226 EF79 EI7 EIl80 EK21 EK35 EK50 EK56 EK57 EK60 EL138 EL139 EL140 EL152 EM12 EM24 EM30
Subclass
IgG1,K IgM,K IgG1,K IgG1,K IgG1,K IgG1,A IgG1,K IgG1,K IgG1,K IgG1,K IgG1,K IgG2a,x IgG1,K IgG1,K IgG1,K IgG1,K IgG1,A IgG1,K
a Not determined.
Affinities Evaluation (1) AOD
Evaluation (2) AOD
1.76 n.d. a 0 1.75 0.70 0.01 2.78 0.53 1.48 0.37 0.13 0.02 2.65 0.03 1.59 0.59 0 0.75
6.29 0 0.01 0.42 0.22 0.01 2.74 1.29 1.68 1.21 0.02 0.32 1.48 0 1.96 2.54 0.03 3.78
Western blot
Reactivity with synthetic peptide
+ + + + + + + + + + + + + -
Cys9-Lys23 Leu49-Glu63 Va158-Ser72 --
31 (0.5 /~g/ml in 100 tzl of PBS containing 0.25% BSA, 0.05% Tween 20 and 0.3 M NaCI) and the substrate solution (100 tzl of 0.1 M sodium acetate-citrate (pH 5.5) containing 0.006% H 2 0 2 and 0.2 mg/ml TMB) were sequentially added and incubated at 25°C for 30 min each, with three washing steps between each incubation. The colorimetric reaction was stopped by adding 100/xl of 1 N H2SO 4 and the absorbance at 450 nm (reference at 595 nm) was measured. The assay was usually performed in duplicate.
Results
Characterization of mAb Based on the results of ELISA screening, 18 hybridomas were selected and established. Table I summarizes the results of the characterization of mAbs. The most abundant isotype was IgG1,K (14 among 18 clones). The other four clones were IgGl,h (two clones), IgG2a,K (one clone) and IgM,K (one clone). By Western blotting analysis, 13 mAbs showed reactivity with membrane blot-
B
C
2.0
1.5
r -u-
1.0
.fl
0.5
<
o "-'q" . . . . . 20
40
"q 80
20
40
80 0
I 20
I 40
l
80
[m-s] (pe/ma) Fig. 1. Measurement of IL-8 in buffer and serum by various combinations of antibodies. 96 well microplate was coated with 100 ~1 of EC81 (A), EM30 (B) or rabbit polyclonal antibody (C), 1 / z g / m l each at 4°C overnight, and blocked with 0.5% BSA. Various concentrations of IL-8, diluted in either buffer (e, PBS containing 0.25% BSA and 0.05% Tween 20) or serum ( A , pool of two healthy subjects), 100/zl, were reacted at 25°C for 1 h. Biotinylated EL139 (1 /xg/ml), streptavidin-HRP conjugate and the enzyme substrate were serially reacted at 25°C for 30, 15 and 30 min, respectively, with a washing step between each incubation. Finally, the color development was stopped by the addition of 1 N HzSO4, and the absorbance at 450 nm (reference at 595 nm) was measured.
ted IL-8, while five clones were negative in the assay. The 13 positive mAbs all stained the adjacent two bands, which were thought to be two isoforms of IL-8 (72 amino acid residues and 77 amino acid residues), with nearly equal densities. The epitope of each mAb was analyzed by using synthetic peptides, and EK50, EK56 and EK60 showed reactivities against the peptides corresponding to Cys9-Lys23 (CIKTYSKPFHPKFIK), Leu49-Glu63 (LCLDPKENWVQRVVE) and Va158-Ser72 (VQRVVEKFLKRAENS), respectively. The other 15 clones, however, reacted with none of the eight peptides. Results of the estimation of mAb affinities are also shown in Table I. In these evaluations, each MAb was used either as capture (coated) antibody (evaluation (2)) or reporter (second) antibody (evaluation (1)) in combination with rabbit polyclonal antibody, and serially diluted standard IL-8 was measured by sandwich ELISA. The slope of the standard curves was compared and the promising five clones, EC81, EK35, EL139, EM12 and EM30, were selected for the further studies. Comparison of antibody combinations and the examinations on the inhibitory effects of serum components The five selected mAbs (see above) and the affinity purified rabbit polyclonal antibody were used as coated (first) and biotinylated (second) antibody, and the possible 36 antibody combinations were compared. In each combinations, IL-8 samples diluted in buffer (PBS containing 0.25% BSA and 0.05% Tween 20) and in normal human serum (pool of two healthy subjects) were assayed and the standard curve was constructed for each sample. Figs. 1A and 1B indicate the results of two combinations (EC81/biotin-EL139 and EM30/biotin-EL139) which gave especially good performance (high sensitivity) for a buffer sample. As seen in these systems, however, serum samples generally gave lower color developments compared to buffer samples, which suggest the inhibitory effects of serum components. In case of EC81/biotin-EL139, inhibition turned out to be caused by some cross-reactive material to coated mAb (EC81) (data not shown), so we purified the inhibitory substance by Sephacryl S-200 gel-filtration column chromatography and
32 the affinity chromatography using EC81 mAb-immobilized column, and analyzed its N terminal sequence. The sequence obtained, N L A K G K E E S L D S - , exactly corresponded with that of C T A P - I I I (Castor et al., 1983). In case of E M 3 0 / E L 1 3 9 , we could not clarify the inhibitory mechanism, although the inhibition seemed to be caused by some rather heat-unstable (destroyed by 30 rain incubation at 56°C) substance. A m o n g the antibody combinations tested, polyclonal antibody/biotin-EL139 gave relatively good performance in terms of sensitivity and also the serum inhibition (Fig. IC), so we focused on this antibody pair and tried to improve the assay system.
2.0
/ 1.5
Recovery from various biological samples In order to further examine the interference of body fluid components, recovery tests from nor-
]
H ~r
1.0
.o
<
0.05 0.5
Optimization of the ELISA conditions and the evaluations of minimun detection limit and intraassay variation By diluting the samples three-fold with 0.1 M Tris-HC1 buffer (pH 8.0) containing 0.25% BSA and 0.05% Tween 20 (mixing 100/~1 of the buffer with 50 ~1 of sample in the assay well), we could avoid serum inhibition, and by labeling EL139 mAb (after pepsin digestion) directly with H R P instead of biotin, a simple and rapid (2 h total) protocol was established. Fig. 2 shows the standard curve and the result of the estimation of minimum detection limit by this finalized protocol. The standard curve was linear in the range from 0 to 300 p g / m l IL-8. Absorbance of 600 p g / m l standard was above the measurable range (0-3.0) of the microplate reader (data not shown). A similar standard curve was obtained when standards diluted in normal pool plasma was measured. The minimum detection limit was estimated to be 2.5 p g / m l , since mean + 2 SD value of 0 p g / m l standard was lower than the mean - 2 SD value of 2.5 p g / m l standard, both for the buffer and plasma samples. Table II shows the intra-assay variations of three plasma specimens each containing various concentrations of exogeneously added IL-8. Coefficient of variation (CV) value of 16 wells were all less than 6%.
5
In 0
0
O0 2.5 5 25 50
100
200
12.5 300
[IL-8] (pg/ml)
Fig. 2. Standard curve and the estimation of minimum detection limit. Standard IL-8, diluted either in buffer (e, PBS containing 0.25% BSA and 0.05% Tween 20) or plasma (A, pool of four citrated plasmas from healthy subjects) were measured accordingto the ELISAprocedure described in the materials and methods section. Each point represents mean+_ 2 SD value of n = 6 (1.25-300 pg/ml 1L-8)or n = 12 (0 pg/ml 1L-8) measurements.
mal serum, plasma, urine and culture medium (RPMI 1640 containing 10% FCS) samples were performed. As shown in Table III, mean recovery rates were 91.5, 93.1, 105.0 and 108.0%, respectively, which indicated that these specimens do
TABLE II INTRA-ASSAYVARIATIONSOF PLASMASAMPLES Samples are a pool of four plasma specimens (containing citrate as anticoagulant) obtained from healthy volunteers. IL-8 was added to give final concentrations of 20 (L), 60 (M) and 180 pg/ml (H), respectively. Sample
n
Mean (pg/ml)
SD
CV (%)
L M H
16 16 16
19.3 54.6 157.8
1.13 2.91 6.83
5.8 5.3 4.5
Mean
5.2
33 not i n t e r f e r e with t h e e s t a b l i s h e d E L I S A . Since t h e r e existed s o m e positive s a m p le s ( e x c e e d 2.5 p g / m l ) in s e r u m a n d urine, we p e r f o r m e d t h e a n t i b o d y - a d s o r p t i o n e x p e r i m e n t s . W h e n t h e samples w e r e p r e - i n c u b a t e d with a n t i- I L - 8 polyclonal antibody, all s am p l e s s h o w e d a l m o s t n e g a t i v e resuits, while t h e p r e - i n c u b a t i o n with t h e s a m e c o n c e n t r a t i o n o f c o n t r o l ( n o n - i m m u n e ) rabbit I g G did not affect t h e assay. T h e s e results i n d i c a t e that the positive s a m p l e s f r o m h e a l t h y subjects i n d e e d c o n t a i n e n d o g e n e o u s IL-8, or at least s o m e s u b s t an ce r e a c t i v e with a n ti - I L - 8 rabbit
polyclonal a n t i b o d y (and m A b c l o n e EL139), b u t not with rabbit IgG.
Cross-reacticity with other cytokines V a r i o u s cytokines, h u m a n IFN-o~, IFN-/3, I F N 3', IL-2, IL-6, G - C S F an d G M - C S F , w e r e d i l u t e d in P BS c o n t a i n i n g 0.25% B S A an d 0.05% T w e e n 20 at the c o n c e n t r a t i o n s o f 1 n g / m l , an d m e a s u r e d by this E L I S A . A s shown in T a b l e IV, results w e r e all b e l o w the d e t e c t i o n limit, 2.5 p g / m l . T a b l e I V also shows the cross-reactivity and t h e inhibitory effects o f v ar i o u s IL-8 family
TABLE III RECOVERY OF EXOGENEOUSLY ADDED IL-8 FROM VARIOUS BIOLOGICAL SAMPLES IL-8 was added to serum, plasma, urine and culture medium (RPMI 1640 containing 10% FCS) sample at the final concentrations of 100 pg/ml, left for 1 h at room temperature, and assayed. For antibody adsorption test, affinity purified rabbit anti-lL-8 polyclonal antibody or control (non-immune) rabbit IgG, final concentrations 20 ~g/ml each, were added to each sample, left for 1 h at room temperature, and assayed. Values below detection limit (2.5 pg/ml) were assumed to be 0 pg/ml for the calculation of recovery rate. Sample
[IL-8] (pg/ml) Without addition
Serum 1 2 3 4 5 6 7 8 9 10 11
15.5 61.0 5.0 4.0 10.0 11.5 8.0 17.0 3.5 6.5 9.5
% recovery 20/zg/ml Anti-IL-8 added
20/~g/ml Rabbit IgG added
100 pg/ml IL-8 added
< 2.5 < 2.5 < 2.5 < 2.5 < 2.5 < 2.5 < 2.5 3.0 < 2.5 < 2.5 < 2.5
19.0 55.0 4.5 < 2.5 11.0 10.0 11.5 15.0 3.5 9.0 8.5
106.5 152.0 96.5 86.5 101.1 108.5 105.5 94.5 103.0 115.0 89.0
Mean
91.0 91.0 91.5 82.5 91.0 97.0 97.5 77.5 99.5 108.5 79.5 91.5
Plasma 1 2 3 4 5
< 2.5 4.5 3.0 < 2.5 4.5
87.0 100.5 93.0 94.5 102.5
Mean
87.0 96.0 90.0 94.5 98.0 93.1
Urine 1 2 3 4
< 2.5 < 2.5 16.0 4.0
< 2.5 < 2.5 < 2.5 < 2.5
< 2.5 < 2.5 17.5 3.5
104.0 104.0 118.5 113.5
Mean
Culture medium
104.0 104.0 102.5 109.5 105.0
< 2.5
< 2.5
< 2.5
108.0
108.0
34 T A B L E IV C R O S S R E A C T I V I T Y W I T H V A R I O U S C Y T O K I N E S A N D IL-8 F A M I L Y P R O T E I N S Sample
[IL-8] ( p g / m l )
% recovery
Without IL-8 addition IFN-a IFN-/3 IFN-y IL-2 IL-6 G-CSF GM-CSF /3-TG CTAP-III PF-4 IP-10 GRO
(1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (45 n g / m l ) (100 n g / m l ) (100 n g / m l ) (100 n g / m l ) (100 n g / m l )
< < < < < < < < < < <
50 p g / m l IL-8 added
2.5 2.5 2.5 2.5 2.5 2.5 2.5 3.0 2.5 2.5 2.5 2.5
proteins,/3-TG, CTAP-III, PF-4, IP-10 and GRO. Neither protein, 45-100 ng/ml, showed cross-reactivity, or interfered with the assay of 50 pg/ml IL-8.
Comparison between serum and plasma specimens Results of Table III and some other preliminary investigations indicated that serum (but not plasma) samples from normal subjects often show positive results. Furthermore, the serum levels were collectively high or low, depending on the source of the sample, namely the places and periods they were collected, suggesting the possibility that the results were varied by the conditions of blood separation. To check this possibility, we prepared plasma and serum samples from the same blood specimens at the same time, and compared their IL-8 levels by ELISA. As indicated in Table V, serum samples showed obviously higher values than corresponding plasma samples (containing EDTA as anticoagulant). The values of serum samples declined to undetectable levels by pre-incubation with anti-IL-8 polyclonal antibody but not with the same concentrations of non-immune rabbit IgG (data not shown). These results suggest that IL-8 is produced from blood cells during the separation of serum samples. On the other hand, the recovery rate of exogenously spiked IL-8 was reasonable both for serum samples (89-103%) and plasma samples (92-109%).
46.0 44.5 50.5 52.5 50.0
86.0 89.0 101.0 105.0 100.0
TABLE V COMPARISON OF SIMULTANEOUSLY PREPARED SERUM AND PLASMA SAMPLES FROM HEALTHY SUBJECTS Blood samples of seven healthy volunteers were collected each into two commercially available sterile glass tubes (Nipro, Osaka, Japan), one of which contains E D T A - 2 K as anticoagulant. The tubes were left at room temperature for about 1 h (plasma samples) or 2 h (serum samples) and centrifuged at 4°C for 15 min. Separated samples were stored frozen until assayed. Sample
[IL-8] ( p g / m l ) Without IL-8 addition
Serum 1 2 3 4 5 6 7
23.0 31.0 9.0 57.0 7.5 12.5 7.0
% recovery 100 p g / m l IL-8 added 124.5 131.5 107.5 146.5 98.0 115.5 100.5
Mean
Plasma 1 2 3 4 5 6 7 Mean
101.5 100.5 98.5 89.5 91.5 103.0 93.5 96.9
< < < < < < <
2.5 2.5 2.5 2.5 2.5 2.5 2.5
98.5 94.0 108.5 92.0 92.0 95.0 93.5
98.5 94.0 108.5 92.0 92.0 95.0 93.5 96.2
35 In order to examine the effects of various anticoagulants, we also examined the citrated-plasma (Table III) and heparinized plasma samples (data not shown), besides EDTA-plasma indicated in this table. In all cases, most of the samples contained undetectable levels of IL-8, and no serious problems were observed as to the recovery rates. (Mean recovery rate was 86.6% for heparinized plasma, 96.2% for EDTA plasma, and 93.1% for citrated plasma, respectively.)
Application to clinical samples Plasma samples from patients with septic shock were collected and assayed. Although differences between individuals were prominent, the mean IL-8 concentration of 16 sepsis patients (157.6 pg/ml) was significantly higher than that of 29 healthy subjects (0.8 pg/ml), as shown in Fig. 3. In order to obtain a clue about how IL-8 exists in the blood circulation, we separated IL-8 in sepsis plasma by Sephacryl S-200 column chromatography and measured each fraction by the ELISA. As shown in Fig. 4, the ELISA reactive material was eluted in a M r region of 5-10 kDa as a single peak, which suggested that IL-8 in the plasma exists as monomer, free from any carrier proteins.
Discussion Cytokines play key roles in many types of host defence reactions through their diverse biological activities. Recently, they are also attracting much attention as pathogenic or marker substances in various diseases. This is also the case for IL-8, an important mediator of proinflammatory responses, which is reported to have relationship with rheumatoid arthritis (Brennan et al., 1990), psoriasis (Sticherling et al., 1991) and sepsis (Martich et al., 1991; Van Zee et al., 1991). For further investigation of its relationship to various diseases, accurate quantitation of IL-8 levels in body fluid samples is a basic and indispensable technique. Since the reported IL-8 assays, based on biological or immunological methods, are not ideal for this purpose, we aimed to develop a sensitive and simple ELISA, which is directly applicable to clinical specimens.
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Fig. 3. Plasma IL-8 levels in healthy volunteers and sepsis patients. T h e horizontal dotted line indicates the detection limit (2.5 p g / m l ) . At the time of blood sampling, most of the sepsis patients had fever of higher than 38°C, systolic blood pressure of less than 100 m m H g and blood endotoxin levels of higher than 10 p g / m l . Septic shock was due to the co-infection of gram-positive and gram-negative bacteria in two patients (out of 16), gram-positive bacteria in four cases, gramnegative bacteria in four cases and fungus in one case. In five patients, the etiological agent was not identified.
The key factor determining the sensitivity of ELISA is the antigen-binding affinity of antibodies. For the purpose of obtaining high affinity clones, we roughly checked the mAb affinities in the first screening of hybridomas by measuring the reactivity of serially diluted mAbs with the coated antigen. 18 established clones of mAbs were further analyzed precisely in terms of their affinity to IL-8 by another evaluation method, sandwich ELISA in combination with polyclonal antibody. Although this evaluation seems to be better than the first screening method, which would be affected by the conformational change of IL-8 induced by the adsorption to the solid phase, it has another drawback. Namely, it could be affected by the overlapping of the epitope of
36
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Fig. 4. Gel-filtration column chromatographyof IL-8 in sepsis plasma. 200 /xl of plasma sample from sepsis patient, which contained 790 pg/ml of IL-8, was subjected to gel filtration on a column of Sephacryl S-200 (15 x420 mm) equilibrated with PBS containing 0.1% BSA. Fractions of 2 ml were collected and the IL-8 level in each fraction was measured by ELISA. Arrows at the top indicate the elution position of the following Mr markers: 670 (thyroglobulin, 670 kDa), 158 (gamma globulin, 158 kDa), 44 (ovalbumin, 44 kDa), 17 (myoglobin, 17 kDa), 1.35 (vitamin B-12, 1.35 kDa).
each mAb and the dominant epitope of the polyclonal antibodies. In fact, our preliminary analysis using synthetic peptides indicated that the epitopes of polyclonal antibodies are not uniformly distributed along the IL-8 sequence but rather limited in the C terminal peptide (Va158-Ser72), which would be in accordance with the results that a combination of polyclonal antibody and EK60 (which recognizes Va158-Ser72 peptide) showed poor performance. We think there remains a possibility that combination of mAbs other than the selected five clones give good results, when used as m A b / m A b sandwich ELISA. In some mAb combinations examined, such as E C 8 1 / E L 1 3 9 or E M 3 0 / E L 1 3 9 (Figs. 1A and 1B), extremely high sensitivity was obtained for the IL-8 sample in the buffer, while most of these ELISAs were severely interfered with serum components, resulting in low sensitivity for IL-8 in serum. We tried to clarify the inhibitory mechanism and identified the interfering substance in E C 8 1 / E L 1 3 9 ELISA as CTAP-III, one of the IL-8 related peptides existing in serum, which cross-react with coated EC81. Similar cross-reactivity of anti-IL-8 mAbs with this protein or/3-TG, which is the N terminally processed form of
CTAP-III, was also reported by other groups (Sticherling et al., 1898; Sylvester et al. 1990). We must pay careful attention to these cross-reactivities in the immunological assay for IL-8. As indicated in Table V, normal serum samples contained higher levels of IL-8 compared to plasma samples prepared from the same blood. Since the value was lowered to a undetectable level by preincubation with anti-IL-8 polyclonal antibody, the presence of internal IL-8 in the serum samples, possibly produced by blood cells during the separation of serum, was suggested. When plasma samples were measured, levels in normal subjects were generally low and the recovery of exogenously added IL-8 was good (mean 96.2%), so we decided to use plasma for the IL-8 determination. It would be necessary to interpret the results carefully, when measuring the blood cell-derived substances in serum samples. Very recently, Darbonne et al. (1991) reported that IL-8 is effectively trapped onto the surface of red blood cells in physiological conditions. Existence of internal IL-8 after serum separation could be explained by this phenomenon. As an example of the ELISA application to clinical samples, we measured IL-8 in the plasma of patients with septic shock, and detected much higher levels compared to healthy subjects. This result is in accordance with the former informations that IL-8 is induced in vitro and in vivo by the stimulation with LPS or bacterial infusion (Yoshimura et al., 1987; Martich et al. 1991; Van Zee et al., 1991). We further analyzed one of the IL-8 positive sepsis plasmas by gel-filtration column chromatography and found out that ELISA-detectable IL-8 in the plasma exists entirely as monomer form. Although the result is rather preliminary, it provided the first information as to how IL-8 exists in the human blood circulation. The minimum detection limit of the established ELISA, 2.5 p g / m l , is much lower than the previously reported assays, and the results could be obtained in totally 2 h incubation without being affected by body fluid components. Through this high sensitivity and specificity, the ELISA is expected to be effectively used for further investigation on the involvement of IL-8 in various diseases.
37
Acknowledgements We would like to thank Dr. K. Matsushima, Kanazawa University, for supplying recombinant 1P-10, Dr. S. Kanesaka, Showa University Fujigaoka Hospital, and Dr. Y. Yamamoto, Nippon Medical School, for supplying plasma samples from sepsis patients, and Dr. O. Ishida, SMI Bristol Ltd., for collecting serum and plasma samples from healthy subjects. We also thank Dr. T. Kanno for preparing rabbit antiserum to IL-8, Mr. T. Maruyama for peptide synthesis, Dr. T. Sasagawa for amino acid sequencing, Ms. T. Hosaka for helping in IL-8 ELISA, and Dr. P.B. Sehgal, New York Medical College, for valuable discussions.
References Baldwin, E.T., Weber, I.T., St. Charles, R., Xuan, J.C., Appella, E., Yamada, M., Matsushima, K., Edwards, B.F.P., Clore, G.M., Gronenborn, A.M. and Wlodawer, A. (1991) Crystal structure of interleukin 8: symbiosis of NMR and crystallography. Proc. Natl. Acad. Sci. USA 88, 502. Begg, G.S., Pepper, D.S., Chestermann, C.N. and Morgan, F.G. (1978) Complete covalent structure of human /3thromboglobulin. Biochemistry 17, 1739. Brennan, F.M., Zachariae, C.O.C., Chantry, D., Larsen, C.G., Turner, M., Maini, R.N., Matsushima, K. and Feldmann, M. (1990) Detection of intrleukin 8 biological activity in synovial fluids from patients with rheumatoid arthritis and production of interleukin 8 mRNA by isolated synovial cells. Eur. J. Immunol. 20, 2141. Castor, C.W., Miller, J.W. and Walz, D.A. (1983) Structural and biological characteristics of connective tissue activating peptide (CTAP-III), a major human platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 80, 765. Ceska, M., Effenberger, F., Peichl, P. and Pursch, E. (1989) Purification and characterization of monoclonal and polyclonal antibodies to neutrophil activation peptide (NAP-I). The development of highly sensitive ELISA methods for the determination of NAP-1 and anti-NAP-1 antibodies. Cytokine 1, 136. Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, M. (1990) Three-dimensional structure of interleukin 8 in solution. Biochemistry 29, 1689. Darbonne0 W.C., Rice, G.C., Mohler, M.A., Apple, T., H6bert, C.A., Valente, A.J. and Baker, J.B. (1991) Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin. J. Clin. Invest. 88, 1362. DeForge, L.E. and Remick D.G. (1991) Sandwich ELISA for detection of picogram quantities of interleukin-8. Immunol. Invest. 20, 89.
Deuel, T.F., Keim, P.S., Farmer, M. and Heinrikson, R.L. (1977) Amino acid sequence of human platelet factor 4. Proc. Natl. Acad. Sci. USA 74, 2256. Gregory, H., Young, J., Schr6der, J.-M., Mrowietz, U. and Christophers, E. (1988) Structure determination of a human lymphocyte derived neutrophil activating peptide (LYNAP). Biochem. Biophys. Res. Commun. 151,883. Holmes, W.E., Lee, J., Kuang, W.J., Rice, G.C. and Wood, W.I. (1991) Structure and functional expression of a human interleukin-8 receptor. Science 253, 1278. Larsen, C.G., Anderson, A.O., Appella, E., Oppenheim, J.J. and Matsushima, K. (1989) The neutrophil activating protein (NAP-I) is also chemotactic for T lymphocytes. Science 243, 1464. Luster, A.D., Unkeless, J.C. and Ravetch, J.V. (1985) y-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315, 672. Martich, G.D., Danner, R.L., Ceska, M. and Suffredini, F. (1991) Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of antiinflammatory agents. J. Exp. Med. 173, 1021. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, H.F., Leonard, E.J. and Oppenheim, J.J. (1988) Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp. Med. 167, 1883. Murphy, P.M. and Tiffany, H.L. (1991) Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 253, 1280. Richmond, A., Balentien, E., Thomas, H.G., Flaggs, G., Barton, D.E., Spiess, J., Bordoni, R., Francke, U. and Derynck, R. (1988) Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to/3-thromboglobulin. EMBO J. 7, 2025. Schr6der, J.-M., Mrowietz, U., Morita, E. and Christophers, E. (1987) Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity. J. Immunol. 139, 3474. Sticherling, M., Schr6der, J.-M. and Christophers, E. (1989) Production and characterization of monoclonal antibodies against the novel neutrophil activating peptide NAP/IL-8. J. Immunol. 143, 1628. Sticherling, M., Bornscheuer, E., Schr6der, J.-M. and Christophers, E. (1991) Localization of neutrophil-activating peptide-1/interleukin-8-immunoreactivity in normal and psoriatic skin. J. Invest. Dermatol. 96, 26. Strieter, R.M., Kunkel, S.L., Showell, H.J., Remick, D.G., Phan, S.H., Ward. P.A. and Marks, R.M. (1989) Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-a, LPS, and IL-1/3. Science 243, 1467. Strieter, R.M., Phan, S.H., Showell, H.J., Remick, D.G., Lynch, J.P., Genord, M., Raiford, C., Eskandari, M., Marks, R.M. and Kunkel, S.L. (1989) Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts. J. Biol. Chem. 264, 10621.
38 Sylvester, I., Rankin, J.A., Yoshimura, T., Tanaka, S. and Leonard, E.J. (1990) Secretion of neutrophil attractant/ activation protein by lipopolysaccharide-stimulated lung macrophages determined by both enzyme-linked immunosorbent assay and N-terminal sequence analysis. Am. Rev. Respir. Dis. 141, 683. Thronton, A.J., Strieter, R.M., Lindleyl I., Baggiolini, M. and Kunkel, S.L. (1990) Cytokine-induced gene expression of a neutrophil chemotactic factor/IL-8 in human hepatocytes. J. Immunol. 144, 2609. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. Van Zee, K.J., DeForge, L.E., Fischer, E., Marano, M.A., Kenney, J.S., Remick, D.G. Lowry, S.F. and Moldawer, L.L. (1991) IL-8 in septic shock, endotoxemia, and after IL-1 administration. J. Immunol. 146, 3478. Waits, A., Peveri, P., Aschauer, H. and Baggiolini, M. (1987) Purification and amino acid sequencing of NAF, a novel
neutrophil-activating factor produced by monocytes. Biochem. Biophys. Res. Commun. 149, 755. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E.A., Appella, E., Oppenheim, J.J. and Leonard, E.J. (1987) Purification of a human monocyte-derived neutrophil chemotactic factor that shares sequence homology with other host defence cytokines. Proc. Natl. Acad. Sci. USA 84, 9233. Yoshimura, T., Robinson, E.A., Appella, E., Matsushima, K., Showalter, S.D., Skeel, A. and Leonard, E.J. (1989) Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amimo-terminal sequence. Mol. Immunol. 26, 87. Yoshitake, S., Imagawa, M., Ishikawa, E., Niitsu, Y., Urushizaki, I., Nishiura, M., Kanazawa, R., Kurosaki, H., Tachibana, S., Nakazawa, N. and Ogawa, H. (1982) Mild and efficient conjugation of rabbit Fab' and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay. J. Biochem. 92, 1413.
27
JIM 06485
A highly sensitive enzyme-linked immunosorbent assay for the measurement of interleukin-8 in biological fluids N o b u o Ida a, Shingou Sakurai b, Kazuo Hosoi b and Tetsunosuke Kunitomo a a Medical Devices and Diagnostics Research Laboratories, Toray Industries, Inc., ! 1! 1 Tebiro, Kamakura, Kanagawa 248, Japan, and b Mishima Plants, Toray Industries, Inc., 4845 Mishirna, Shizuoka 411, Japan (Received 3 March 1992, revised received 5 May 1992, accepted 29 May 1992)
Interleukin-8 (IL-8) is a chemotactic and activating cytokine for neutrophils, which plays an important role in acute inflammatory responses. We aimed to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for IL-8 and established 18 clones of anti-IL-8 monoclonal antibodies (mAbs). These mAbs were evaluated in terms of their antigen-binding affinities, and five clones were selected and used for the comparative study of various combinations of antibodies in sandwich ELISA. Affinity purified rabbit polyclonal antibody was also used in this study. One antibody pair, which showed relatively high sensitivity and which was not severely interfered with blood components, was selected and the assay conditions were optimized by choosing the appropriate buffer for sample dilution and by directly labeling the second antibody with enzyme. The finalized ELISA, using polyclonal antibody as first (coated) antibody and horseradish peroxidase-labeled mAb (clone EL139) Fab' fragment as second antibody, could detect as low as 2.5 pg/ml (0.125 pg/well) of IL-8 by in total 2 h incubation, without being affected by body fluid components. The ELISA was specific to IL-8, showing no cross-reactivity with other cytokines or various IL-8 family proteins which share some amino acid sequence homology with IL-8. As an example of its application to clinical specimens, plasma samples from patients with septic shock were measured. The results showed that sepsis patients contain significantly higher levels of plasma IL-8 compared to normal controls. When analyzed by gel-filtration chromatography, IL-8 in sepsis plasma was eluted in a molecular weight (M r) region corresponding to the monomer form. The ELISA established here is expected to be effectively used for further investigations on the relationship between IL-8 and various diseases. Key words: Interleukin-8; ELISA; Sepsis
Introduction Correspondence to: N. Ida, Medical Devices and Diagnostics Research Laboratories, Toray Industries, Inc., 1111 Tebiro, Kamakura, Kanagawa 248, Japan. Tel.: 81-467-322111. Fax: 81-467-32-4791. Abbreviations: Ab, antibody; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; IL-8, interleukin-8; mAb, monoclonal antibody; Mr, molecular weight; PBS, phosphate-buffered saline.
Interleukin-8 (IL-8), which is also known as monocyte-derived neutrophil chemotactic factor (MDNCF) (Yoshimura et al., 1987), neutrophil activating factor (NAF) (Waits et al, 1987), neutrophil activating peptide-1 (NAP-l) (Schr6der et al., 1987), or T cell chemotactic factor (TCF)
28 (Larsen et al., 1989), is a potent chemotactic and activating cytokine for neutrophils. It is produced by various types of cells such as monocytes (Yoshimura et al., 1987; Larsen et al., 1989), lymphocytes (Gregory et al., 1988), endothelial cells (Strieter et al., 1989a), hepatocytes (Thronton et al., 1990), and fibroblasts (Strieter et al., 1989b), and believed to play an important role as a mediator of acute inflammatory reactions. It belongs to a family of low molecular weight (8-10 kDa) proteins including connective tissue activating peptide III (CTAP-III) (Castor et al., 1983), /3-thromboglobulin (/3-TG) (Begg et al., 1978), platelet factor 4 (PF4) (Deuel et al., 1977), interferon-y inducible protein (IP-10) (Luster et al., 1985) and growth related oncogene (GRO) (Richmond et al., 1988), with 29-46% sequence homology at the protein level. Structural and functional analyses of IL-8 have been advancing rapidly, since its cDNA was cloned and sequenced (Matsushima et al., 1988). For example, three-dimensional structure was determined by NMR (Clore et al., 1990) and X ray crystallographic analysis (Baldwin et al., 1991), and two types of its receptor gene were isolated recently (Holmes et al., 1991; Murphy et al., 1991). As expected from its important role in acute inflammatory responses, IL-8 is also reported to be involved in the pathogenesis of several diseases including rheumatoid arthritis (Brennan et al., 1990), psoriasis (Sticherling et al., 1991), and sepsis (Martich et al., 1991; Van Zee et al., 1991). In order to further clarify the relationship between IL-8 and these various disorders, accurate determination of IL-8 levels in body fluids is an important technique. Widely used bioassays, which measure the chemotactic activity or the release of lysosomal enzymes from activated neutrophils (Yoshimura et al., 1987; Schr6der et al., 1987), are not easily applied to clinical samples, because these assays are not sensitive enough, time-consuming, and most importantly, results are affected by other bioactive substances contained in the body fluids. Several immunological assays have also been developed and successfully used for the determination of IL-8 levels in culture supernatants or in plasma samples (Ceska et al., 1989; Sticherling et al., 1989; Sylvester et al., 1990; DeForge et al., 1991). In these assays, how-
ever, interference of body fluid components have not been fully examined. In this paper, we describe the establishment of anti-IL-8 mAbs with high antigen-binding affinity, and the development of a highly sensitive ELISA for IL-8 which is directly applicable to clinical samples.
Materials and methods
IL-8 preparation IL-8, used as the immunogen and the standard sample for ELISA, was produced from poly I : Cstimulated human fibroblast cell, DIP-2, and purified to homogeneity by silica column chromatography, heparin column chromatography and reverse-phase HPLC. This preparation was a mixture of three isoforms of IL-8 with 72, 77, and 79 amino acid residues, which are known to be caused by differential processings from the same precursor (Yoshimura et al., 1989), and the contents of these three isoforms were about 50, 40 and 10%, respectively. Concentration of IL-8 in the purified preparation was determined by amino acid analysis.
Polyclonal antibody Two New Zealand White rabbits were immunized s.c. with 350 /.Lg each of IL-8 in Freund's complete adjuvant. The rabbits were boosted twice with the same amount of IL-8 in Freund's incomplete adjuvant at intervals of 2 and 3 weeks. 10 days after the final immunization, they were bled and the antiserum (110 ml total) was separated. The antiserum obtained was diluted twice with PBS and applied to IL-8 immobilized affinity column to which 10 mg of IL-8 was covalently coupled, and the bound antibody was eluted with 0.1 M glycine-HCl buffer (pH 2.4). By this purification, 7.5 mg of antibody was obtained from 30 ml of antiserum.
Establishment bridoma
of anti-IL-8 mAb-producing hy-
Ten female B A L B / c mice (8 weeks of age) were immunized i.p. with 10-20 ~g of IL-8 in an adjuvant of aluminum potassium sulfate with inactivated Bordetella pertussis (Wako Pure Chemical, Osaka, Japan). They were boosted twice or
29 three times with 10 ~g of antigen (first with aluminum potassium sulfate adjuvant, second and third in phosphate buffered saline (PBS)) at the intervals of 10-40 days. 3 days after the last immunization, the spleen cells were isolated, mixed with P3U1 myeloma cells at a ratio of 10: 1, fused in 50% polyethylene glycol 1500 (Boehringer Mannheim, Germany) and seeded into 96-well microplate in HAT medium (RPMI 1640) containing 15% fetal calf serum and 2 ng/ml human IL-6. The anti-IL-8 mAb secreting hybridomas were screened by ELISA method. Microtiter plates (Nunc, Denmark) were coated with IL-8 (0.5 /zg/ml in PBS 100/xl/well) at 4°C overnight, and blocked with 0.5% BSA in PBS. Hybridoma culture supernatant (100 ~1) was dispensed into each well and incubated for 1 h at room temperature, followed by sequential incubations with biotinylated goat anti-mouse IgG (Zymed, San Francisco, CA), streptavidin-horseradish peroxidase (HRP) conjugate (Zymed, San Francisco, CA), and the enzyme substrate (0.1 M sodium acetate-citrate buffer (pH 5.5) containing 0.006% H 2 0 z and 0.2 mg/ml 3,3',5,5'-tetramethyl benzidine (TMB)). The plate was washed with the washing solution (2-fold diluted PBS containing 0.025% Tween 20) three times between each incubation step. Finally, 1 N H 2 S O 4 was added to stop the reaction, and the color development was measured at 450 nm. Cells in positive wells were further cultured in HT medium overnight, and on the next day, the supernatants were withdrawn again, serially diluted in PBS containing 0.25% BSA and 0.05% Tween 20, and tested for the reactivity with solid phase IL-8 as described above. Positive wells which showed the reactivity at relatively higher dilution rate (with lower concentration of mAb) were selected and cloned twice by limiting dilution.
m,4b production and purification Hybridomas were cultured in serum-free medium (Celgrosser-H, Sumitomo Pharmaceuticals, Osaka, Japan) and the supernatants were used for the characterization of mAbs. Purified mAbs, used in IL-8 ELISA, were purified from mouse ascites by ammonium sulfate precipitation and protein A column chromatography.
Isotyping of mAb The subclasses of mAbs were determined using commercially available ELISA kit (Zymed, San Francisco, CA).
Western blotting Western blotting was performed according to the method of Towbin et al. (1979). Briefly, IL-8 sample (75/xg/ml, 100/zl) was separated by 20% gel SDS-PAGE, and electrically blotted to a membrane (Clear Blot Membrane-P, Atto, Tokyo, Japan). After the sheet was blocked with 5% skim milk, 2 p.g/ml of anti-IL-8 mAbs, biotinylated goat anti-mouse IgG or biotinylated goat anti-mouse IgM (Cappel, West Chester, PA), and streptavidin-HRP conjugate were sequentially added and incubated. Finally, the enzyme activity was detected with color developing kit (Immunostaining H R P kit, Konica, Tokyo, Japan).
Epitope analysis Epitopes of MAbs were examined using eight synthetic peptides (15 amino acid length each), which cover the whole sequence of IL-8 with overlapping of six or seven amino acid residues between each peptide. The peptides, Serl-Lys15 (SAKELRCQCIKTYSK), Cys9-Lys23 (CIKTYSKPFHPKFIK), Phe17-Gly31 (FHPKFIKELRVIESG), Leu25-Ile39 (LRVIESGPHCANTEI), His33-Arg47 ( H C A N T E I I V K L S G D R), V a 1 4 1 - G l u 5 5 (VKLSDGRELCLDPKE), Leu49-Glu63 ( L C L D P K E N W V Q R V V E ) and Val58-Ser72 (VQRVVEKFLKRAENS), were synthesized by solid phase method and covalently attached to microplate wells ('Co-Bind' Micromenbranes, Newark, N J) according to the manufacturer's instruction. After blocking, serially diluted MAbs (2-0.016 /xg/ml) were dispensed into each well and incubated for 1 h. The bound mAbs were detected by the sequencial reaction of biotinylated goat anti-mouse IgG or biotinylated goat anti-mouse IgM, streptavidin-HRP, and the enzyme substrate.
Evaluation of the affinity of mAb The affinity of each mAb to the antigen IL-8 was estimated by two different sandwich ELISA methods as follows. In evaluation (1), the plate
30 was c o a t e d with r a b b i t p o l y c l o n a l a n t i - I L - 8 antib o d y a n d b l o c k e d . Serially d i l u t e d IL-8 was a d d e d a n d i n c u b a t e d at 25°C for 1 h. T h e n , 2 / . ~ g / m l o f each mAb, biotinylated rabbit anti-mouse IgG (Tago, B u r l i n g a m e , CA), s t r e p t a v i d i n - H R P conjugate, a n d t h e e n z y m e s u b s t r a t e w e r e s e q u e n t i a l l y d i s p e n s e d a n d i n c u b a t e d . A f t e r t h e r e a c t i o n was s t o p p e d with 1 N H 2 8 0 4 , t h e a b s o r b a n c e at 450 n m was m e a s u r e d . A O D v a l u e ( d i f f e r e n c e b e t w e e n t h e a b s o r b a n c e o f 1 n g / m l IL-8 s a m p l e a n d o f 0 n g / m l IL-8) was c a l c u l a t e d f r o m t h e s l o p e o f the s t a n d a r d curve, a n d u s e d as an index o f t h e affinity o f m A b . I n e v a l u a t i o n (2), t h e p l a t e was c o a t e d with e a c h m A b (2 / ~ g / m l ) o v e r n i g h t at 4°C, b l o c k e d , a n d t h e n serially d i l u t e d IL-8 was a d d e d a n d i n c u b a t e d at 25°C for 1 h. B i o t i n y l a t e d rabbit anti-IL-8 polyclonal antibody, streptavidinHRP conjugate, and the enzyme substrate were s e q u e n t i a l l y d i s p e n s e d a n d i n c u b a t e d f o l l o w e d by a d d i t i o n o f 1 N H 2 5 0 4 , a n d t h e a b s o r b a n c e at 450 m was m e a s u r e d . A O D was c a l c u l a t e d as d e s c r i b e d in e v a l u a t i o n (1). B i o t i n y l a t i o n o f t h e a n t i b o d y was p e r f o r m e d by using N H S - L S - b i o t i n (Pierce, R o c k f o r d , IL).
Pepsin digestion and HRP labeling of rnAb 5 m g o f E L 1 3 9 m A b , in 5 ml o f 0.1 M s o d i u m c i t r a t e b u f f e r ( p H 3.5), was d i g e s t e d with 50 /zg o f p e p s i n (Sigma, St. Louis, M O ) at 37°C for 1 h. G e n e r a t e d F ( a b ' ) 2 f r a g m e n t was l a b e l e d with H R P ( B o e h r i n g e r M a n n h e i m , G e r m a n y ) by male i m i d e - h i n g e m e t h o d ( Y o s h i t a k e et al., 1982). P u r i f i c a t i o n o f H R P - E L 1 3 9 F a b ' c o n j u g a t e was p e r f o r m e d with h y d r o x y a p a t i t e c o l u m n H P L C .
ELISA for IL-8 A m i c r o t i t e r p l a t e was c o a t e d with r a b b i t anti° IL-8 p o l y c l o n a l a n t i b o d y (0.5 / x g / m l in PBS) at 4°C overnight, a n d b l o c k e d with 0.5% B S A in PBS at 25°C for 2 h. A f t e r e a c h well was w a s h e d with 400 ~1 o f w a s h i n g s o l u t i o n (2-fold d i l u t e d PBS c o n t a i n i n g 0.025% T w e e n 20), 100 ~1 o f t h e r e a c t i o n b u f f e r (0.1 M T r i s - H C l p H 8.0 c o n t a i n ing 0.25% B S A , 0.05% T w e e n 20, 0.5% e a c h o f n o r m a l m o u s e a n d r a b b i t s e r u m ) was d i s p e n s e d , a n d 5 0 / z l o f s a m p l e s o r s t a n d a r d IL-8 was a d d e d a n d i n c u b a t e d at 25°C for 1 h. T h e p l a t e was s h a k e n on a m i c r o p l a t e m i x e r t h r o u g h o u t the i n c u b a t i o n time. H R P - l a b e l e d EL139 F a b ' m A b
TABLE I CHARACTERIZATION OF mAbs AOD represents the difference of the absorbance values of 1 ng/ml IL-8 standard and 0 ng/ml IL-8 standard, when measured by sandwich ELISA in combination with each mAb and rabbit polyclonal antibody. Clone
EC81 EC226 EF79 EI7 EIl80 EK21 EK35 EK50 EK56 EK57 EK60 EL138 EL139 EL140 EL152 EM12 EM24 EM30
Subclass
IgG1,K IgM,K IgG1,K IgG1,K IgG1,K IgG1,A IgG1,K IgG1,K IgG1,K IgG1,K IgG1,K IgG2a,x IgG1,K IgG1,K IgG1,K IgG1,K IgG1,A IgG1,K
a Not determined.
Affinities Evaluation (1) AOD
Evaluation (2) AOD
1.76 n.d. a 0 1.75 0.70 0.01 2.78 0.53 1.48 0.37 0.13 0.02 2.65 0.03 1.59 0.59 0 0.75
6.29 0 0.01 0.42 0.22 0.01 2.74 1.29 1.68 1.21 0.02 0.32 1.48 0 1.96 2.54 0.03 3.78
Western blot
Reactivity with synthetic peptide
+ + + + + + + + + + + + + -
Cys9-Lys23 Leu49-Glu63 Va158-Ser72 --
31 (0.5 /~g/ml in 100 tzl of PBS containing 0.25% BSA, 0.05% Tween 20 and 0.3 M NaCI) and the substrate solution (100 tzl of 0.1 M sodium acetate-citrate (pH 5.5) containing 0.006% H 2 0 2 and 0.2 mg/ml TMB) were sequentially added and incubated at 25°C for 30 min each, with three washing steps between each incubation. The colorimetric reaction was stopped by adding 100/xl of 1 N H2SO 4 and the absorbance at 450 nm (reference at 595 nm) was measured. The assay was usually performed in duplicate.
Results
Characterization of mAb Based on the results of ELISA screening, 18 hybridomas were selected and established. Table I summarizes the results of the characterization of mAbs. The most abundant isotype was IgG1,K (14 among 18 clones). The other four clones were IgGl,h (two clones), IgG2a,K (one clone) and IgM,K (one clone). By Western blotting analysis, 13 mAbs showed reactivity with membrane blot-
B
C
2.0
1.5
r -u-
1.0
.fl
0.5
<
o "-'q" . . . . . 20
40
"q 80
20
40
80 0
I 20
I 40
l
80
[m-s] (pe/ma) Fig. 1. Measurement of IL-8 in buffer and serum by various combinations of antibodies. 96 well microplate was coated with 100 ~1 of EC81 (A), EM30 (B) or rabbit polyclonal antibody (C), 1 / z g / m l each at 4°C overnight, and blocked with 0.5% BSA. Various concentrations of IL-8, diluted in either buffer (e, PBS containing 0.25% BSA and 0.05% Tween 20) or serum ( A , pool of two healthy subjects), 100/zl, were reacted at 25°C for 1 h. Biotinylated EL139 (1 /xg/ml), streptavidin-HRP conjugate and the enzyme substrate were serially reacted at 25°C for 30, 15 and 30 min, respectively, with a washing step between each incubation. Finally, the color development was stopped by the addition of 1 N HzSO4, and the absorbance at 450 nm (reference at 595 nm) was measured.
ted IL-8, while five clones were negative in the assay. The 13 positive mAbs all stained the adjacent two bands, which were thought to be two isoforms of IL-8 (72 amino acid residues and 77 amino acid residues), with nearly equal densities. The epitope of each mAb was analyzed by using synthetic peptides, and EK50, EK56 and EK60 showed reactivities against the peptides corresponding to Cys9-Lys23 (CIKTYSKPFHPKFIK), Leu49-Glu63 (LCLDPKENWVQRVVE) and Va158-Ser72 (VQRVVEKFLKRAENS), respectively. The other 15 clones, however, reacted with none of the eight peptides. Results of the estimation of mAb affinities are also shown in Table I. In these evaluations, each MAb was used either as capture (coated) antibody (evaluation (2)) or reporter (second) antibody (evaluation (1)) in combination with rabbit polyclonal antibody, and serially diluted standard IL-8 was measured by sandwich ELISA. The slope of the standard curves was compared and the promising five clones, EC81, EK35, EL139, EM12 and EM30, were selected for the further studies. Comparison of antibody combinations and the examinations on the inhibitory effects of serum components The five selected mAbs (see above) and the affinity purified rabbit polyclonal antibody were used as coated (first) and biotinylated (second) antibody, and the possible 36 antibody combinations were compared. In each combinations, IL-8 samples diluted in buffer (PBS containing 0.25% BSA and 0.05% Tween 20) and in normal human serum (pool of two healthy subjects) were assayed and the standard curve was constructed for each sample. Figs. 1A and 1B indicate the results of two combinations (EC81/biotin-EL139 and EM30/biotin-EL139) which gave especially good performance (high sensitivity) for a buffer sample. As seen in these systems, however, serum samples generally gave lower color developments compared to buffer samples, which suggest the inhibitory effects of serum components. In case of EC81/biotin-EL139, inhibition turned out to be caused by some cross-reactive material to coated mAb (EC81) (data not shown), so we purified the inhibitory substance by Sephacryl S-200 gel-filtration column chromatography and
32 the affinity chromatography using EC81 mAb-immobilized column, and analyzed its N terminal sequence. The sequence obtained, N L A K G K E E S L D S - , exactly corresponded with that of C T A P - I I I (Castor et al., 1983). In case of E M 3 0 / E L 1 3 9 , we could not clarify the inhibitory mechanism, although the inhibition seemed to be caused by some rather heat-unstable (destroyed by 30 rain incubation at 56°C) substance. A m o n g the antibody combinations tested, polyclonal antibody/biotin-EL139 gave relatively good performance in terms of sensitivity and also the serum inhibition (Fig. IC), so we focused on this antibody pair and tried to improve the assay system.
2.0
/ 1.5
Recovery from various biological samples In order to further examine the interference of body fluid components, recovery tests from nor-
]
H ~r
1.0
.o
<
0.05 0.5
Optimization of the ELISA conditions and the evaluations of minimun detection limit and intraassay variation By diluting the samples three-fold with 0.1 M Tris-HC1 buffer (pH 8.0) containing 0.25% BSA and 0.05% Tween 20 (mixing 100/~1 of the buffer with 50 ~1 of sample in the assay well), we could avoid serum inhibition, and by labeling EL139 mAb (after pepsin digestion) directly with H R P instead of biotin, a simple and rapid (2 h total) protocol was established. Fig. 2 shows the standard curve and the result of the estimation of minimum detection limit by this finalized protocol. The standard curve was linear in the range from 0 to 300 p g / m l IL-8. Absorbance of 600 p g / m l standard was above the measurable range (0-3.0) of the microplate reader (data not shown). A similar standard curve was obtained when standards diluted in normal pool plasma was measured. The minimum detection limit was estimated to be 2.5 p g / m l , since mean + 2 SD value of 0 p g / m l standard was lower than the mean - 2 SD value of 2.5 p g / m l standard, both for the buffer and plasma samples. Table II shows the intra-assay variations of three plasma specimens each containing various concentrations of exogeneously added IL-8. Coefficient of variation (CV) value of 16 wells were all less than 6%.
5
In 0
0
O0 2.5 5 25 50
100
200
12.5 300
[IL-8] (pg/ml)
Fig. 2. Standard curve and the estimation of minimum detection limit. Standard IL-8, diluted either in buffer (e, PBS containing 0.25% BSA and 0.05% Tween 20) or plasma (A, pool of four citrated plasmas from healthy subjects) were measured accordingto the ELISAprocedure described in the materials and methods section. Each point represents mean+_ 2 SD value of n = 6 (1.25-300 pg/ml 1L-8)or n = 12 (0 pg/ml 1L-8) measurements.
mal serum, plasma, urine and culture medium (RPMI 1640 containing 10% FCS) samples were performed. As shown in Table III, mean recovery rates were 91.5, 93.1, 105.0 and 108.0%, respectively, which indicated that these specimens do
TABLE II INTRA-ASSAYVARIATIONSOF PLASMASAMPLES Samples are a pool of four plasma specimens (containing citrate as anticoagulant) obtained from healthy volunteers. IL-8 was added to give final concentrations of 20 (L), 60 (M) and 180 pg/ml (H), respectively. Sample
n
Mean (pg/ml)
SD
CV (%)
L M H
16 16 16
19.3 54.6 157.8
1.13 2.91 6.83
5.8 5.3 4.5
Mean
5.2
33 not i n t e r f e r e with t h e e s t a b l i s h e d E L I S A . Since t h e r e existed s o m e positive s a m p le s ( e x c e e d 2.5 p g / m l ) in s e r u m a n d urine, we p e r f o r m e d t h e a n t i b o d y - a d s o r p t i o n e x p e r i m e n t s . W h e n t h e samples w e r e p r e - i n c u b a t e d with a n t i- I L - 8 polyclonal antibody, all s am p l e s s h o w e d a l m o s t n e g a t i v e resuits, while t h e p r e - i n c u b a t i o n with t h e s a m e c o n c e n t r a t i o n o f c o n t r o l ( n o n - i m m u n e ) rabbit I g G did not affect t h e assay. T h e s e results i n d i c a t e that the positive s a m p l e s f r o m h e a l t h y subjects i n d e e d c o n t a i n e n d o g e n e o u s IL-8, or at least s o m e s u b s t an ce r e a c t i v e with a n ti - I L - 8 rabbit
polyclonal a n t i b o d y (and m A b c l o n e EL139), b u t not with rabbit IgG.
Cross-reacticity with other cytokines V a r i o u s cytokines, h u m a n IFN-o~, IFN-/3, I F N 3', IL-2, IL-6, G - C S F an d G M - C S F , w e r e d i l u t e d in P BS c o n t a i n i n g 0.25% B S A an d 0.05% T w e e n 20 at the c o n c e n t r a t i o n s o f 1 n g / m l , an d m e a s u r e d by this E L I S A . A s shown in T a b l e IV, results w e r e all b e l o w the d e t e c t i o n limit, 2.5 p g / m l . T a b l e I V also shows the cross-reactivity and t h e inhibitory effects o f v ar i o u s IL-8 family
TABLE III RECOVERY OF EXOGENEOUSLY ADDED IL-8 FROM VARIOUS BIOLOGICAL SAMPLES IL-8 was added to serum, plasma, urine and culture medium (RPMI 1640 containing 10% FCS) sample at the final concentrations of 100 pg/ml, left for 1 h at room temperature, and assayed. For antibody adsorption test, affinity purified rabbit anti-lL-8 polyclonal antibody or control (non-immune) rabbit IgG, final concentrations 20 ~g/ml each, were added to each sample, left for 1 h at room temperature, and assayed. Values below detection limit (2.5 pg/ml) were assumed to be 0 pg/ml for the calculation of recovery rate. Sample
[IL-8] (pg/ml) Without addition
Serum 1 2 3 4 5 6 7 8 9 10 11
15.5 61.0 5.0 4.0 10.0 11.5 8.0 17.0 3.5 6.5 9.5
% recovery 20/zg/ml Anti-IL-8 added
20/~g/ml Rabbit IgG added
100 pg/ml IL-8 added
< 2.5 < 2.5 < 2.5 < 2.5 < 2.5 < 2.5 < 2.5 3.0 < 2.5 < 2.5 < 2.5
19.0 55.0 4.5 < 2.5 11.0 10.0 11.5 15.0 3.5 9.0 8.5
106.5 152.0 96.5 86.5 101.1 108.5 105.5 94.5 103.0 115.0 89.0
Mean
91.0 91.0 91.5 82.5 91.0 97.0 97.5 77.5 99.5 108.5 79.5 91.5
Plasma 1 2 3 4 5
< 2.5 4.5 3.0 < 2.5 4.5
87.0 100.5 93.0 94.5 102.5
Mean
87.0 96.0 90.0 94.5 98.0 93.1
Urine 1 2 3 4
< 2.5 < 2.5 16.0 4.0
< 2.5 < 2.5 < 2.5 < 2.5
< 2.5 < 2.5 17.5 3.5
104.0 104.0 118.5 113.5
Mean
Culture medium
104.0 104.0 102.5 109.5 105.0
< 2.5
< 2.5
< 2.5
108.0
108.0
34 T A B L E IV C R O S S R E A C T I V I T Y W I T H V A R I O U S C Y T O K I N E S A N D IL-8 F A M I L Y P R O T E I N S Sample
[IL-8] ( p g / m l )
% recovery
Without IL-8 addition IFN-a IFN-/3 IFN-y IL-2 IL-6 G-CSF GM-CSF /3-TG CTAP-III PF-4 IP-10 GRO
(1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (1 n g / m l ) (45 n g / m l ) (100 n g / m l ) (100 n g / m l ) (100 n g / m l ) (100 n g / m l )
< < < < < < < < < < <
50 p g / m l IL-8 added
2.5 2.5 2.5 2.5 2.5 2.5 2.5 3.0 2.5 2.5 2.5 2.5
proteins,/3-TG, CTAP-III, PF-4, IP-10 and GRO. Neither protein, 45-100 ng/ml, showed cross-reactivity, or interfered with the assay of 50 pg/ml IL-8.
Comparison between serum and plasma specimens Results of Table III and some other preliminary investigations indicated that serum (but not plasma) samples from normal subjects often show positive results. Furthermore, the serum levels were collectively high or low, depending on the source of the sample, namely the places and periods they were collected, suggesting the possibility that the results were varied by the conditions of blood separation. To check this possibility, we prepared plasma and serum samples from the same blood specimens at the same time, and compared their IL-8 levels by ELISA. As indicated in Table V, serum samples showed obviously higher values than corresponding plasma samples (containing EDTA as anticoagulant). The values of serum samples declined to undetectable levels by pre-incubation with anti-IL-8 polyclonal antibody but not with the same concentrations of non-immune rabbit IgG (data not shown). These results suggest that IL-8 is produced from blood cells during the separation of serum samples. On the other hand, the recovery rate of exogenously spiked IL-8 was reasonable both for serum samples (89-103%) and plasma samples (92-109%).
46.0 44.5 50.5 52.5 50.0
86.0 89.0 101.0 105.0 100.0
TABLE V COMPARISON OF SIMULTANEOUSLY PREPARED SERUM AND PLASMA SAMPLES FROM HEALTHY SUBJECTS Blood samples of seven healthy volunteers were collected each into two commercially available sterile glass tubes (Nipro, Osaka, Japan), one of which contains E D T A - 2 K as anticoagulant. The tubes were left at room temperature for about 1 h (plasma samples) or 2 h (serum samples) and centrifuged at 4°C for 15 min. Separated samples were stored frozen until assayed. Sample
[IL-8] ( p g / m l ) Without IL-8 addition
Serum 1 2 3 4 5 6 7
23.0 31.0 9.0 57.0 7.5 12.5 7.0
% recovery 100 p g / m l IL-8 added 124.5 131.5 107.5 146.5 98.0 115.5 100.5
Mean
Plasma 1 2 3 4 5 6 7 Mean
101.5 100.5 98.5 89.5 91.5 103.0 93.5 96.9
< < < < < < <
2.5 2.5 2.5 2.5 2.5 2.5 2.5
98.5 94.0 108.5 92.0 92.0 95.0 93.5
98.5 94.0 108.5 92.0 92.0 95.0 93.5 96.2
35 In order to examine the effects of various anticoagulants, we also examined the citrated-plasma (Table III) and heparinized plasma samples (data not shown), besides EDTA-plasma indicated in this table. In all cases, most of the samples contained undetectable levels of IL-8, and no serious problems were observed as to the recovery rates. (Mean recovery rate was 86.6% for heparinized plasma, 96.2% for EDTA plasma, and 93.1% for citrated plasma, respectively.)
Application to clinical samples Plasma samples from patients with septic shock were collected and assayed. Although differences between individuals were prominent, the mean IL-8 concentration of 16 sepsis patients (157.6 pg/ml) was significantly higher than that of 29 healthy subjects (0.8 pg/ml), as shown in Fig. 3. In order to obtain a clue about how IL-8 exists in the blood circulation, we separated IL-8 in sepsis plasma by Sephacryl S-200 column chromatography and measured each fraction by the ELISA. As shown in Fig. 4, the ELISA reactive material was eluted in a M r region of 5-10 kDa as a single peak, which suggested that IL-8 in the plasma exists as monomer, free from any carrier proteins.
Discussion Cytokines play key roles in many types of host defence reactions through their diverse biological activities. Recently, they are also attracting much attention as pathogenic or marker substances in various diseases. This is also the case for IL-8, an important mediator of proinflammatory responses, which is reported to have relationship with rheumatoid arthritis (Brennan et al., 1990), psoriasis (Sticherling et al., 1991) and sepsis (Martich et al., 1991; Van Zee et al., 1991). For further investigation of its relationship to various diseases, accurate quantitation of IL-8 levels in body fluid samples is a basic and indispensable technique. Since the reported IL-8 assays, based on biological or immunological methods, are not ideal for this purpose, we aimed to develop a sensitive and simple ELISA, which is directly applicable to clinical specimens.
1000
1
500
'-"
100
--,
50
o0 I
10
.
2.5
.
.
.
.
.
L
.
.
.
.
00 0000 • O O • • • 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Normal
.
•
Sepsis
Fig. 3. Plasma IL-8 levels in healthy volunteers and sepsis patients. T h e horizontal dotted line indicates the detection limit (2.5 p g / m l ) . At the time of blood sampling, most of the sepsis patients had fever of higher than 38°C, systolic blood pressure of less than 100 m m H g and blood endotoxin levels of higher than 10 p g / m l . Septic shock was due to the co-infection of gram-positive and gram-negative bacteria in two patients (out of 16), gram-positive bacteria in four cases, gramnegative bacteria in four cases and fungus in one case. In five patients, the etiological agent was not identified.
The key factor determining the sensitivity of ELISA is the antigen-binding affinity of antibodies. For the purpose of obtaining high affinity clones, we roughly checked the mAb affinities in the first screening of hybridomas by measuring the reactivity of serially diluted mAbs with the coated antigen. 18 established clones of mAbs were further analyzed precisely in terms of their affinity to IL-8 by another evaluation method, sandwich ELISA in combination with polyclonal antibody. Although this evaluation seems to be better than the first screening method, which would be affected by the conformational change of IL-8 induced by the adsorption to the solid phase, it has another drawback. Namely, it could be affected by the overlapping of the epitope of
36
$
$
$
$
$
¢-1
•
20
<
10
10
15
2O
Fraction number
25
30
(2 ml/tube)
Fig. 4. Gel-filtration column chromatographyof IL-8 in sepsis plasma. 200 /xl of plasma sample from sepsis patient, which contained 790 pg/ml of IL-8, was subjected to gel filtration on a column of Sephacryl S-200 (15 x420 mm) equilibrated with PBS containing 0.1% BSA. Fractions of 2 ml were collected and the IL-8 level in each fraction was measured by ELISA. Arrows at the top indicate the elution position of the following Mr markers: 670 (thyroglobulin, 670 kDa), 158 (gamma globulin, 158 kDa), 44 (ovalbumin, 44 kDa), 17 (myoglobin, 17 kDa), 1.35 (vitamin B-12, 1.35 kDa).
each mAb and the dominant epitope of the polyclonal antibodies. In fact, our preliminary analysis using synthetic peptides indicated that the epitopes of polyclonal antibodies are not uniformly distributed along the IL-8 sequence but rather limited in the C terminal peptide (Va158-Ser72), which would be in accordance with the results that a combination of polyclonal antibody and EK60 (which recognizes Va158-Ser72 peptide) showed poor performance. We think there remains a possibility that combination of mAbs other than the selected five clones give good results, when used as m A b / m A b sandwich ELISA. In some mAb combinations examined, such as E C 8 1 / E L 1 3 9 or E M 3 0 / E L 1 3 9 (Figs. 1A and 1B), extremely high sensitivity was obtained for the IL-8 sample in the buffer, while most of these ELISAs were severely interfered with serum components, resulting in low sensitivity for IL-8 in serum. We tried to clarify the inhibitory mechanism and identified the interfering substance in E C 8 1 / E L 1 3 9 ELISA as CTAP-III, one of the IL-8 related peptides existing in serum, which cross-react with coated EC81. Similar cross-reactivity of anti-IL-8 mAbs with this protein or/3-TG, which is the N terminally processed form of
CTAP-III, was also reported by other groups (Sticherling et al., 1898; Sylvester et al. 1990). We must pay careful attention to these cross-reactivities in the immunological assay for IL-8. As indicated in Table V, normal serum samples contained higher levels of IL-8 compared to plasma samples prepared from the same blood. Since the value was lowered to a undetectable level by preincubation with anti-IL-8 polyclonal antibody, the presence of internal IL-8 in the serum samples, possibly produced by blood cells during the separation of serum, was suggested. When plasma samples were measured, levels in normal subjects were generally low and the recovery of exogenously added IL-8 was good (mean 96.2%), so we decided to use plasma for the IL-8 determination. It would be necessary to interpret the results carefully, when measuring the blood cell-derived substances in serum samples. Very recently, Darbonne et al. (1991) reported that IL-8 is effectively trapped onto the surface of red blood cells in physiological conditions. Existence of internal IL-8 after serum separation could be explained by this phenomenon. As an example of the ELISA application to clinical samples, we measured IL-8 in the plasma of patients with septic shock, and detected much higher levels compared to healthy subjects. This result is in accordance with the former informations that IL-8 is induced in vitro and in vivo by the stimulation with LPS or bacterial infusion (Yoshimura et al., 1987; Martich et al. 1991; Van Zee et al., 1991). We further analyzed one of the IL-8 positive sepsis plasmas by gel-filtration column chromatography and found out that ELISA-detectable IL-8 in the plasma exists entirely as monomer form. Although the result is rather preliminary, it provided the first information as to how IL-8 exists in the human blood circulation. The minimum detection limit of the established ELISA, 2.5 p g / m l , is much lower than the previously reported assays, and the results could be obtained in totally 2 h incubation without being affected by body fluid components. Through this high sensitivity and specificity, the ELISA is expected to be effectively used for further investigation on the involvement of IL-8 in various diseases.
37
Acknowledgements We would like to thank Dr. K. Matsushima, Kanazawa University, for supplying recombinant 1P-10, Dr. S. Kanesaka, Showa University Fujigaoka Hospital, and Dr. Y. Yamamoto, Nippon Medical School, for supplying plasma samples from sepsis patients, and Dr. O. Ishida, SMI Bristol Ltd., for collecting serum and plasma samples from healthy subjects. We also thank Dr. T. Kanno for preparing rabbit antiserum to IL-8, Mr. T. Maruyama for peptide synthesis, Dr. T. Sasagawa for amino acid sequencing, Ms. T. Hosaka for helping in IL-8 ELISA, and Dr. P.B. Sehgal, New York Medical College, for valuable discussions.
References Baldwin, E.T., Weber, I.T., St. Charles, R., Xuan, J.C., Appella, E., Yamada, M., Matsushima, K., Edwards, B.F.P., Clore, G.M., Gronenborn, A.M. and Wlodawer, A. (1991) Crystal structure of interleukin 8: symbiosis of NMR and crystallography. Proc. Natl. Acad. Sci. USA 88, 502. Begg, G.S., Pepper, D.S., Chestermann, C.N. and Morgan, F.G. (1978) Complete covalent structure of human /3thromboglobulin. Biochemistry 17, 1739. Brennan, F.M., Zachariae, C.O.C., Chantry, D., Larsen, C.G., Turner, M., Maini, R.N., Matsushima, K. and Feldmann, M. (1990) Detection of intrleukin 8 biological activity in synovial fluids from patients with rheumatoid arthritis and production of interleukin 8 mRNA by isolated synovial cells. Eur. J. Immunol. 20, 2141. Castor, C.W., Miller, J.W. and Walz, D.A. (1983) Structural and biological characteristics of connective tissue activating peptide (CTAP-III), a major human platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 80, 765. Ceska, M., Effenberger, F., Peichl, P. and Pursch, E. (1989) Purification and characterization of monoclonal and polyclonal antibodies to neutrophil activation peptide (NAP-I). The development of highly sensitive ELISA methods for the determination of NAP-1 and anti-NAP-1 antibodies. Cytokine 1, 136. Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, M. (1990) Three-dimensional structure of interleukin 8 in solution. Biochemistry 29, 1689. Darbonne0 W.C., Rice, G.C., Mohler, M.A., Apple, T., H6bert, C.A., Valente, A.J. and Baker, J.B. (1991) Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin. J. Clin. Invest. 88, 1362. DeForge, L.E. and Remick D.G. (1991) Sandwich ELISA for detection of picogram quantities of interleukin-8. Immunol. Invest. 20, 89.
Deuel, T.F., Keim, P.S., Farmer, M. and Heinrikson, R.L. (1977) Amino acid sequence of human platelet factor 4. Proc. Natl. Acad. Sci. USA 74, 2256. Gregory, H., Young, J., Schr6der, J.-M., Mrowietz, U. and Christophers, E. (1988) Structure determination of a human lymphocyte derived neutrophil activating peptide (LYNAP). Biochem. Biophys. Res. Commun. 151,883. Holmes, W.E., Lee, J., Kuang, W.J., Rice, G.C. and Wood, W.I. (1991) Structure and functional expression of a human interleukin-8 receptor. Science 253, 1278. Larsen, C.G., Anderson, A.O., Appella, E., Oppenheim, J.J. and Matsushima, K. (1989) The neutrophil activating protein (NAP-I) is also chemotactic for T lymphocytes. Science 243, 1464. Luster, A.D., Unkeless, J.C. and Ravetch, J.V. (1985) y-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315, 672. Martich, G.D., Danner, R.L., Ceska, M. and Suffredini, F. (1991) Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of antiinflammatory agents. J. Exp. Med. 173, 1021. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, H.F., Leonard, E.J. and Oppenheim, J.J. (1988) Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp. Med. 167, 1883. Murphy, P.M. and Tiffany, H.L. (1991) Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 253, 1280. Richmond, A., Balentien, E., Thomas, H.G., Flaggs, G., Barton, D.E., Spiess, J., Bordoni, R., Francke, U. and Derynck, R. (1988) Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to/3-thromboglobulin. EMBO J. 7, 2025. Schr6der, J.-M., Mrowietz, U., Morita, E. and Christophers, E. (1987) Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity. J. Immunol. 139, 3474. Sticherling, M., Schr6der, J.-M. and Christophers, E. (1989) Production and characterization of monoclonal antibodies against the novel neutrophil activating peptide NAP/IL-8. J. Immunol. 143, 1628. Sticherling, M., Bornscheuer, E., Schr6der, J.-M. and Christophers, E. (1991) Localization of neutrophil-activating peptide-1/interleukin-8-immunoreactivity in normal and psoriatic skin. J. Invest. Dermatol. 96, 26. Strieter, R.M., Kunkel, S.L., Showell, H.J., Remick, D.G., Phan, S.H., Ward. P.A. and Marks, R.M. (1989) Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-a, LPS, and IL-1/3. Science 243, 1467. Strieter, R.M., Phan, S.H., Showell, H.J., Remick, D.G., Lynch, J.P., Genord, M., Raiford, C., Eskandari, M., Marks, R.M. and Kunkel, S.L. (1989) Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts. J. Biol. Chem. 264, 10621.
38 Sylvester, I., Rankin, J.A., Yoshimura, T., Tanaka, S. and Leonard, E.J. (1990) Secretion of neutrophil attractant/ activation protein by lipopolysaccharide-stimulated lung macrophages determined by both enzyme-linked immunosorbent assay and N-terminal sequence analysis. Am. Rev. Respir. Dis. 141, 683. Thronton, A.J., Strieter, R.M., Lindleyl I., Baggiolini, M. and Kunkel, S.L. (1990) Cytokine-induced gene expression of a neutrophil chemotactic factor/IL-8 in human hepatocytes. J. Immunol. 144, 2609. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. Van Zee, K.J., DeForge, L.E., Fischer, E., Marano, M.A., Kenney, J.S., Remick, D.G. Lowry, S.F. and Moldawer, L.L. (1991) IL-8 in septic shock, endotoxemia, and after IL-1 administration. J. Immunol. 146, 3478. Waits, A., Peveri, P., Aschauer, H. and Baggiolini, M. (1987) Purification and amino acid sequencing of NAF, a novel
neutrophil-activating factor produced by monocytes. Biochem. Biophys. Res. Commun. 149, 755. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E.A., Appella, E., Oppenheim, J.J. and Leonard, E.J. (1987) Purification of a human monocyte-derived neutrophil chemotactic factor that shares sequence homology with other host defence cytokines. Proc. Natl. Acad. Sci. USA 84, 9233. Yoshimura, T., Robinson, E.A., Appella, E., Matsushima, K., Showalter, S.D., Skeel, A. and Leonard, E.J. (1989) Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amimo-terminal sequence. Mol. Immunol. 26, 87. Yoshitake, S., Imagawa, M., Ishikawa, E., Niitsu, Y., Urushizaki, I., Nishiura, M., Kanazawa, R., Kurosaki, H., Tachibana, S., Nakazawa, N. and Ogawa, H. (1982) Mild and efficient conjugation of rabbit Fab' and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay. J. Biochem. 92, 1413.