A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877
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A monoclonal antibody and an enzyme immunoassay
for human Ala-IL-877
Natalia N. Nashkevicha, Svetlana Akalovicha, Natalia Lounevab,George A. Heavnerc, Nikolai N. Voitenoka,d,*
aLaboratory of Cellular and Molecular Immunology, Institute of Hematology and Blood Transfusion, Dolginovsky Tract 160, Minsk, BelarusbByelorussian Institute for Hereditary Diseases, Minsk, Belarus
cCentocor Inc., Malvern, PA, USAdFund for Molecular Hematology and Immunology, Post Box 338, Moscow 125493, Russia
Received 4 February 2002; received in revised form 30 April 2002; accepted 21 June 2002
Interleukin-8 (IL-8) plays a central role in neutrophil chemotaxis and exerts a wide range of effects on various cells, ranging
from tumor angiogenesis to impairment of neuronal signaling. Two main forms of IL-8 exist, one containing 77 amino acids
(Ala-IL-877) and a second containing 72 amino acids (Ser-IL-872), which comprise more than 90% of IL-8 protein in cell
cultures. IL-877 was reported to be produced predominantly by endothelial cells and is known as endothelial IL-8. IL-872predominates in monocyte cultures and is known as leukocyte IL-8. While both forms have equal chemotactic activity in
vivo, recent data suggest that their biological activities might be different. Here we describe the generation of a mouse
monoclonal antibody (mAb) specific for IL-877 and the development of a corresponding immunoassay. Various immunization
protocols were investigated. Immunization with conjugates of a peptide from the N-terminus of IL-877 (NTP77) resulted in the
production of an IgG1 mAb (N11) that recognizes human IL-877 and neutralizes its chemotactic activity. A sensitive ELISA
specific for IL-877 was developed using N11 for capture and a biotinylated mAb to IL-872 for detection. Using this
immunoassay it was shown that the only form of IL-8 secreted in cell culture was IL-877 and that the IL-872 present was the
result of proteolysis of IL-877. IL-877 was detected in plasma and cerebrospinal fluid (CSF) from patients with sepsis and
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Ala-interleukin-8; Monoclonal antibody; ELISA; Culture supernatants; Septic plasma
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0022 -1759 (02 )00279 -X
Abbreviations: IL-8, interleukin-8; mAb, monoclonal antibody; Ig, immunoglobulin; NTP77, N-terminal peptide of IL-877; CFA/IFA,
complete/incomplete Freunds adjuvant; i.p., intraperitoneally; BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; Ova,
ovalbumin; Sulfo-MBS, m-maleimidobenzoyl sulfosuccinimide ester; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; TNF-a, tumornecrosis factor-a; IL-1-h, interleukin-1-h; LPS, lipopolysaccharide; PI, protease inhibitors cocktail; CSF, cerebrospinal fluid.
* Corresponding author. Laboratory of Cellular and Molecular Immunology, Institute of Hematology and Blood Transfusion, Dolginovsky
Tract 160, 223059 Minsk, Belarus. Tel.: +7-375-172-344-483; fax: +7-375-172-310-472.
E-mail address: firstname.lastname@example.org (N.N. Voitenok).
Journal of Immunological Methods 270 (2002) 3751
Interleukin-8 (IL-8) belongs to the CXC family of
chemokines (Baggiolini, 2001) and plays a central
role in neutrophil chemotaxis and activation (Baggio-
lini et al., 1994). In addition, IL-8 exerts pleiotropic
actions (Mukaida, 2000) including angiogenesis (Bel-
perio et al., 2000), alteration of neuronal signaling
(Giovannelli et al., 1998) and leukemic cell apoptosis
(Terui et al., 1998). IL-8 is produced by multiple cell
types under the influence of bacterial products, in
response to inflammatory cytokines such as tumor
necrosis factor and interleukin-1, or after malignant
transformation (Matsushima and Oppenheim, 1989;
Baggiolini et al., 1994; Mukaida, 2000).
IL-8 secreted by cultured monocytes showed het-
erogeneity in the N-terminal sequence and the amino
acid length of the IL-8 protein ranged from 79 amino
acids through 77, 72, 71, 70 to a 69-amino-acid
variant (Lindley et al., 1988; Van Damme et al.,
1988; Yoshimura et al., 1989; Schroder et al., 1990).
Two main forms of IL-8 containing 77 or 72 amino
acids were termed Ala-IL-877 (IL-877) and Ser-IL-872(IL-872), respectively, and account for more than 90%
of the IL-8 in monocyte culture (Yoshimura et al.,
1989). The various forms of IL-8 are generated via
proteolytic cleavage of the primary 99-amino-acid
precursor following cleavage of the 20-amino-acid
signal sequence (Yoshimura et al., 1989; Baggiolini
et al., 1994). IL-877 was reported to be produced
predominantly by endothelial cells and is known as
endothelial IL-8 (Gimbrone et al., 1989; Hebert et
al., 1990; Huber et al., 1991) while IL-872 is known as
leukocyte-derived IL-8 (Hebert et al., 1990; Bag-
giolini et al., 1994). While IL-872 is a more potent
neutrophil chemoattractant when tested by in vitro
assays (Hebert et al., 1990; Nourshargh et al., 1992),
both forms have equal in vivo neutrophil chemotactic
activity (Nourshargh et al., 1992) attributed to the
GluLeuArg (ELR) motif (amino acids 6967)
(Baggiolini et al., 1994).
Recent data suggest that the biological activity of
IL-877 may differ from that of IL-872 in terms of its
capacity to induce apoptosis of leukemic cell lines
(Terui et al., 1998, 1999).
The production and role of IL-877 and IL-872 in
inflammatory responses and pathology in vivo are
still obscure, since existing immunoassays are unable
to distinguish the two molecules. N-terminal amino
acid sequencing and molecular size estimation by
polyacrylamide gel electrophoresis (PAGE) were
used for the identification of IL-8 forms in cell
cultures (Lindley et al., 1988; Van Damme et al.,
1988; Yoshimura et al., 1989) and in vivo (Renne-
kampff et al., 2000).
In this report we describe the production of a
monoclonal antibody (mAb) to IL-877 and the devel-
opment of a specific enzyme immunoassay. This
ELISA was able to quantify IL-877 in cell cultures,
blood plasma and cerebrospinal fluid (CSF) obtained
from patients with sepsis and meningitis.
2. Materials and methods
2.1. Animals and reagents
Female BALB/c mice (BALB/cJCitMoise) of 10
12 weeks of age were obtained from the breeding
nucleus of the Shemyakin & Ovchinnikov Institute of
Bioorganic Chemistry, Moscow. Mice were housed in
accordance with institutional guidelines.
Recombinant human IL-877 and IL-872 (r-IL-8)
were obtained from Peprotech, Rocky Hill, NJ, or
kindly donated by Dr. Ji Ming Wang (Lab. Molecular
Immunoregulation, NCI at Frederick, Frederick, MD).
Synthetic IL-877 was kindly donated by Prof. Marco
Baggiolini, Kocher Inst., University Bern. The peptide
AVLPRSAKELRC-NH2 (NTP77) corresponding to
the N-terminal part of human IL-877 was synthesized
by the Protein Design group at Centocor, Malvern, PA
on Rink resin using an ABI 431A Synthesizer with the
standard single coupling FMOC protocol. After cleav-
age using a cocktail of trifluoroacetic acid (TFA),
phenol, dithiothreitol, thioanisole and water, the pep-
tide was purified by reverse phase (C-18) HPLC using
a linear gradient of 3080% acetonitrile in 0.1% TFA.
The purified peptide had the correct amino acid
analysis and mass spectra. The N-terminal pentapep-
tide of IL-877, AVLPR, was synthesized by PerSeptive
Biosystems, Department of Microbiology and Immu-
nology, University of Maryland, School of Medicine,
Baltimore, MD. Recombinant human IL-877 fused to
human FcIgG (FcIgG IL-877) and recombinant
human tumor necrosis factor-a (TNF-a) receptor p-55 fused to human FcIgG (FcIgGp-55) (Scallon et
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375138
al., 1995) were kindly provided by Dr. Bernard
Scallon, Centocor. The N-terminal peptide corre-
sponding to the N-terminal 22 amino acids of the
human IL-8 receptor CXCR2 was kindly donated by
Dr. Ernst Brandt (Forschungszentrum, Borstel, Ger-
many). Rabbit polyclonal antibody to human IL-8 was
kindly donated by Prof. Sergei Ketlinsky (Inst. Ultra-
pure Biopreparates, St. Petersburg, Russia). Complete
(CFA) and incomplete (IFA) Freunds adjuvants were
from Sigma, St. Louis, MO. RIBI adjuvant MPL-
TDM system was from RIBI ImmunoChem Research,
2.2. Conjugation protocol
Bovine serum albumin (BSA) (Pierce, Rockford,
IL), ovalbumin (Ova) and keyhole limpet hemocyanin
(KLH) (both from Sigma) were used for peptide
conjugation. The peptide AVLPRSAKELRC-NH2,
corresponding to the N-terminal sequence of IL-877,
was conjugated to carrier proteins in two ways: (1)
adding sulfhydryl groups to the peptide with 2-imi-
nothiolane (Jue et al., 1978; Aithal et al., 1988)
followed by conjugation to BSA, Ova, or KLH using
the heterobifunctional agent m-maleimidobenzoyl sul-
fosuccinimide ester (sulfo-MBS) (Kitagawa et al.,
1982); or, (2) by water-soluble carbodiimide in a
single-step coupling reaction (Catty and Raykundalia,
BALB/c mice were immunized intraperitoneally
(i.p.) on the splenic side with 25 or 50 Ag ofrecombinant human FcIgGIL-877 fusion protein or
NTP77 conjugated to BSA, Ova or KLH in 100 Al ofphosphate-buffered saline (PBS) emulsified in 100 Alof CFA. Mice were boosted i.p. on the contralateral
side at monthly intervals with 2550 Ag of recombi-nant human FcIgGIL-877 fusion protein, 2550 Agof NTP77 conjugate or 2030 Ag of synthetic IL-877in 100 Al of saline solution emulsified at 1:1 with IFA.Blood samples were collected from the tail vein of
anaesthetized mice on day 20 after each immuniza-
tion. Each group consisted of three or more mice.
Serial dilutions of mouse sera in PBS with 1.0% BSA
were assayed in triplicate in 96-well polystyrene
plates (Corning Inc., Corning, NY) coated with
NTP77, IL-877, IL-872 or NTP77 conjugates at 1 Ag/ml and developed with goat anti-mouse horseradish
peroxidase conjugate (Bio-Rad, Richmond, CA). Titer
was defined as the last dilution, which yielded a value
above the negative control (normal mouse sera). The
levels of IL-8 specific antibodies in mouse sera were
expressed as relative units (RU) against the standard
mAb WS-4 at 1 Ag/ml.
2.4. Monoclonal antibodies: generation and purifica-
Spleen cells were fused to the myeloma cells
P3X63-Ag8.653 and cloned as previously described
(Lane et al., 1984; Panyutich et al., 1991). Hybridoma
clones producing antibodies were identified by ELISA
using microtiter plates coated with free NTP77, NTP77conjugates, corresponding carriers, IL-877, IL-872 or
FcIgGIL-877 fusion protein as indicated in Table 1
and were developed with peroxidase-labeled goat
anti-mouse antibody. Selected hybridoma wells were
subcloned twice by limiting dilution over a BALB/c
peritoneal macrophage feeder layer. The IgG subclass
of the mAbs was determined using an isotyping kit
from Fisher Biotech (Pittsburgh, PA). The resulting
mAbs were produced by injecting hybridoma cells i.p.
into BALB/c mice primed with pristane (Sigma). For
in vitro analysis, mAbs were purified from hybridoma
supernatants or ascites by Protein A or Protein G
chromatography (Pharmacia Fine Chemicals, Piscat-
away, NJ). Protein concentrations were determined by
the Bradford dye binding assay (Bio-Rad).
2.5. Characterization of monoclonal antibodies to
2.5.1. Hybridomas origin and isotyping data
Hybridomas specific for IL-872 (4C, 3A, N6) and
anti-IgG clone C12 were derived from mice immu-
nized with recombinant proteins FcIgGIL-877 and IL-
8. The previously described anti-IL-872 hybridoma
clonesWS-4 and BS-1 (Ko et al., 1992) and hybridoma
H6 were from our internal panel. Hybridoma H9,
specific for the sulfo-MBS-link, was derived from
sequential immunization with BSANTP77 and
OvaNTP77 (sulfo-MBS coupled). N11 and D5 clones
were derived from immunization with KLHNTP77(sulfo-MBS coupled) followed by BSANTP77 (car-
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 39
bodiimide conjugate) and synthetic IL-877. All these
hybridoma clones produced IgG1, n isotype antibod-ies, except the clone D5 that produced an IgM mAb.
2.5.2. Chemotaxis assay
Neutrophil chemotaxis was performed against
recombinant IL-877 and IL-872 using 48-well micro
Boydens chambers as previously described (Falk et
al., 1980). Briefly, binding buffer containing 1% BSA
in RPMI 1640 with or without mAb (at 0.550 Ag/ml)and IL-877 or IL-872 (at 10 ng/ml) was placed in the
lower compartment of the chamber. A 5-Am polycar-bonate filter (Neuroprobe, Cabin John, MD) was
placed at the intersection of the upper and lower
compartments. Freshly isolated neutrophils (106
cells/ml) in binding medium were added to the upper
compartment of the chamber. After incubation for 2 h
at 37 jC, the upper surface of the filter was scrapedand the resulting cells fixed with methanol and stained
with Leukostat (Fisher Scientific). Data were analyzed
using the BIOQUANT program (R & M Biomatrics,
Nashville, TN) and the results were expressed as the
mean number of migrated cells per 10 fields at 10magnification.
SDS-PAGE was performed by the Laemmli proce-
dure using 20% acrylamide separation gels. Samples
were electrotransferred from gels to nitrocellulose
membranes (Hybond C) and incubated with mAb
N11, 4C or WS-4 at 10 Ag/ml, followed by reactionwith the alkaline phosphatase-labeled goat anti-mouse
IgG antibody (Bio-Rad) and developed using NBT/
BCIP as a substrate.
Mononuclear leukocytes isolated from the fresh
heparinized blood of healthy donors using standard
Ficoll/Hypaque protocol were incubated for 3.5 h in
60 mm plastic dishes (Corning Glass Works, Corning,
NY) at 37 jC in RPMI5% FCS (Sigma) in thepresence of 100 ng/ml lipopolysaccharide (LPS) from
E. coli and brefeldin at 10 Ag/ml (both from Sigma).Cell smears were fixed in ice-cold acetone for 10 min
or in neutral buffered 10% formalin solution for 10
min and stained with mAb N11, WS-4 or an isotype
control mouse IgG1 mAb at 10 Ag/ml overnight at 4jC, followed by treatment with horseradish peroxi-dase-conjugated rabbit anti-mouse antibody (Dako) or
an LSAB kit (Dako) according to the manufacturers
protocol. All steps of staining of formalin-fixed
smears were performed in the presence of 0.01%
2.6. ELISA detecting IL-8
The selection of mAb pairs for the detection of IL-
877 and optimization of an IL-877 ELISA was per-
formed using the basic protocol described below.
Microtiter plates were coated with 5 Ag/ml of thecapture mAb in 100 Al of 0.1 M sodium carbonatebuffer, pH 9.6 or PBS, pH 7.4 overnight at 4 jC.After washing the plates three times with 0.3 M NaCl
in PBS, pH 7.4 and 0.05% Tween-20 (PBS0.3 M
NaClTw), unbound sites were blocked by adding
Specificity of mAbs generated after immunization with FcIgG
IL-877 or NTP77 conjugates
Antigen Absorbance (492 nm)
4C C12 H9 N11 D5
IL-872 2.717 0.045 0.186 0.015 0.237
IL-877 2.717 0.038 0.221 2.481 2.367
NTP77 0.030 0.046 0.216 2.153 0.025
BSANTP77a 0.040 0.051 2.207 2.419 0.051
BSANTP77b 0.039 n.t.c 0.166 2.239 0.105
BSA 0.020 0.050 0.160 0.012 0.023
OvaNTP77a 0.028 0.080 2.328 2.246 0.021
Ova 0.019 0.063 0.221 0.012 0.025
FcIgGIL-877 2.595 1.624 0.196 1.797 1.451
Human IgG 0.036 1.000 0.267 0.019 0.028
FcIgGp55d 0.052 1.210 0.268 0.020 0.035
KLHNTP77a 0.015 n.t. 2.223 1.232 0.028
KLH 0.021 n.t. 0.152 0.010 0.025
KLHCXCR2 NTPa,e n.t. n.t. 1.927 0.015 n.t.
BSAAVLPRb n.t. n.t. n.t. 0.025 n.t.
Microtiter plates were coated with 0.1 Ag of each antigen per welland tested against hybridoma clone supernatants diluted 1/5 with
PBS0.5% BSA0.05% Tween-20, followed by development with
peroxidase-labeled goat anti-mouse antibody as described in the
Materials and Methods. Means of duplicates are shown. Absorb-
ency data considered as positive recognition are printed in bold.a Conjugation with sulfo-MBS.b Conjugation with carbodiimide.c n.t.non-tested.d Recombinant protein consisting of human TNF-R p55 fused to
FcIgG.e Hemocyanin conjugated via sulfo-MBS with N-terminal
peptide (22 amino acids) of CXCR2 receptor of IL-8.
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375140
0.5% BSA in PBS0.3 M NaClTw at room temper-
ature (RT) for 1 h. IL-872, IL-877 (stored at 10 Ag/mlin PBS1% BSA at 70 jC) and samples wereserially diluted in PBS0.3 M NaCl0.5% BSATw,
added to the wells (in triplicate) and incubated at RT
for 1 h. Plates were washed three times with PBS0.3
M NaClTw after each subsequent step. Antibodies
used in the liquid phase were labeled with biotin
using Sulfo-NHS-LC-Biotinylation Kit (Pierce, Rock-
ford, IL) and added at 0.51 Ag/ml for 1 h at RT.After washing, the plates were incubated with horse-
radish peroxidase-conjugated avidin. Plates were
washed and developed with 0.4 mg/ml o-phenylene-
diamine (Sigma) in 20 mM citrate buffer, pH 4.7
containing 0.01% H2O2. After 10 min, the reaction
was quenched with 5% H2SO4 and absorbance values
were read at 492 nm. When a rabbit antibody to IL-8
was used for the liquid phase, plates were developed
with an alkaline phosphatase-labeled goat anti-rabbit
IgG (Boehringer Mannheim, Germany) using p-nitro-
phenylphosphate (Labsystems, Helsinki, Finland)
and read at 405 nm.
2.7. Induction of IL-8 in cell cultures
Mononuclear leukocytes (MNL) in RPMI10%
FCS, isolated from fresh heparinized blood using the
standard Ficoll/Hypaque protocol, were incubated in
24-well plastic plates (Corning Glass Works) for 1 h at
37 jC as previously described (Chaly et al., 2000).Adherent MNL were incubated in RPMI2% FCS in
the presence of 100 ng/ml LPS. At different time
points, supernatant was aspirated and stored frozen or
at 4 jC with or without a protease inhibitor cocktail(PI) (Protease inhibitor cocktail tablets Complete,
Mini, Roche Diagnostics, Mannheim, Germany). Bre-
feldin (10 Ag/ml) was added to some of the cultures 15min before the addition of LPS. Supernatants from
human breast adenocarcinoma cell lines, cultured in
either serum-free or serum-containing media were
kindly provided by Dr. Rosalba Salsedo, Lab. Molec-
ular Immunoregulation, FCDRC, Frederick, MD.
Neuroblastoma cell lines SK-N-MC (ATCC HTB
10) and SK-N-SH (ATCC HTB 11) were obtained
from ATCC (Rockville, MD) and cultured in IMDM
10% FCS. Human umbilical vein endothelial cells
(HUVEC) were purchased from Clonetics (Walker-
ville, MD) and subcultured according to the manu-
facturers instructions in Endothelial Cell Growth
Medium (EGM, Clonetics). Neuroblastoma cells and
HUVEC were stimulated for 20 h with either human
TNF-a at 510 ng/ml or interleukin-1-h (IL-1-h)(both Peprotech) at 5 ng/ml.
2.8. Detection of IL-877 in clinical samples
Blood and CSF samples were obtained from the
Dept. of Intensive Care Medicine of the Pediatric
Infections City Hospital, Minsk, and Urgent Medicine
Hospital, Minsk. Blood samples were anticoagulated
with EDTA (5 mM final concentration) and mixed
with the protease inhibitor cocktail at the recommen-
ded concentration. Samples were immediately centri-
fuged and the plasma separated and stored at 40 or 70 jC. Cerebrospinal fluid samples were preservedwith PI and stored frozen at 40 jC.
3.1. Serum antibody responses to FcIgGIL-877 and
conjugates of NTP77
Mouse sera were screened for their ability to bind
NTP77 or r-IL-872. Since the fusions of spleens from
more than 20 selected animals immunized with
FcIgGIL-877 or BSANTP77 failed to produce a
single mAb specific for IL-877, we briefly present our
observations on antibody responses and an immuni-
zation protocol that resulted in the generation of mAbs
specific for IL-877.
The use of 50 Ag FcIgGIL-877 fusion protein inCFA for priming followed by boosting with 25 Ag ofFcIgGIL-877 in IFA resulted in an approximately
three-fold greater antibody response to IL-8 as
compared to the same sensitization using free IL-8
(Fig. 1). Freunds adjuvant with FcIgGIL-877yielded about 2.5-fold higher anti-IL-8 antibody
levels when compared with RIBI adjuvant (not
shown). We were unable to boost mice with 50 AgFcIgGIL-877 in IFA since gross lesions and mor-
bidity were observed in mice primed with 50 Ag ofFcIgGIL-877 in CFA and boosted with the same
dose of FcIgGIL-877 in IFA. No morbidity or
significant lesions were present in mice boosted with
25 Ag of FcIgGIL-877 in IFA.
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 41
Surprisingly, the second boost with FcIgGIL-877in IFA failed to stimulate an anti-IL-8 response (Fig.
1), while antibody titers to human IgG increased (not
shown). In contrast, the use of r-IL-8 in IFA for the
second boost instead of FcIgGIL-877 resulted in
more than a three-fold increase of anti-IL-8 antibody
levels (Fig. 1). Interestingly, when r-IL-8 was used for
the first boost, antibody levels were two-fold less
Mice immunized using the mixed FcIgGIL-877/r-
IL-8 immunization protocol (Fig. 1) showed higher
antibody titers to IL-872 compared to NTP77 (about
20:1, data not shown). More than 10 fusions of
spleens from these mice failed to produce NTP77specific mAbs, while numerous hybridomas to IL-872or human IgG were generated (4C, C12, Table 1).
We primed a group of mice with 50 Ag of BSANTP77 conjugate in CFA followed by two boosts with
the same antigen in IFA at monthly intervals. As in the
case of sensitization with FcIgGIL-877, the second
boost with the BSANTP77 conjugate gave no higher
antibody titers to NTP77 (not shown). However, when
OvaNTP77 conjugate was used for the second boost,
an anti-NTP77 response that was about 3.5-fold
greater was observed (not shown). Nevertheless,
fusions of spleens from selected mice from this
protocol failed to produce a single anti-NTP77 hybrid-
oma, while mAbs reacting with irrelevant conjugates
coupled through a sulfo-MBS-link were identified
(H9, Table 1), indicating that the sulfo-MBS bond
region itself induced a specific antibody response after
the priming and two boosts. Immunization with sulfo-
MBS coupled KLHNTP77 followed by sequential
boosting with carbodiimide conjugated BSANTP77and synthetic IL-877 gave two NTP77-specific hybrid-
omas in the first spleen fusion (N11 and D5) as shown
in Table 1.
3.2. Specificity of monoclonal antibodies derived from
mice immunized with FcIgGIL-877 and NTP77conjugates
Multiple mAbs to IL-872 (4C, Table 1, 3A and N6,
Fig. 6) or human IgG (C12, Table 1) were produced
by fusions of splenocytes from mice immunized with
FcIgGIL-877. Antibody 4C did not react with NTP77(Table 1) which contained the chemotactic ELR motif
but competed with N11 mAb for IL-877 binding (data
not shown). It was the most efficient mAb in neutral-
izing IL-8 among those available for comparison (data
not shown), including mAb WS-4 (Ko et al., 1992).
Similar to WS-4 (Harada et al., 1993), 4C cross-
reacted with rabbit IL-8 (data not shown). Each
mAb capable of binding to the 72-amino-acid form
of IL-8 also recognized IL-877 (Table 1).
Hybridoma H9 was derived from mice immunized
with sulfo-MBS coupled conjugates of NTP77 with
BSA and Ova. This mAb recognized sulfo-MBS
coupled BSANTP77 and an irrelevant conjugate of
KLH and the N-terminal peptide of human CXCR2
but did not react with carbodiimide conjugated BSA
NTP77, indicating sulfo-MBS-link specificity.
Monoclonals D5 and N11 showed specificity for
the 77-amino-acid form of IL-8 and were derived
from the mouse immunized with sulfo-MBS coupled
KLHNTP77 followed by carbodiimide conjugated
BSANTP77 and synthetic IL-877. D5 reacted with
IL-877 and FcIgGIL-877 but did not react with
NTP77 and showed weak recognition of IL-872. In
contrast, mAb N11 reacted with IL-877, FcIgGIL-877
Fig. 1. Comparison of the antibody response to human IL-8 after the
first and the second boost with FcIgGIL-877 or recombinant IL-8.
On day 0, groups of three mice were primed with FcIgGIL-877 in
CFA and boosted on day 30 and on day 60 with FcIgGIL-877 (n)or r-IL-8 (o) in IFA. Blood for titration was taken on day 20 aftereach immunization. Sera were assayed for IL-8 specific antibodies
by serial dilutions in an ELISA and titers expressed as relative units
against the standard mAb WS-4 at 1 Ag/ml. Values are shown forthe meanF S.E. (n= 3). Mice primed with r-IL-8 showed less than1.5 RU after the first boost with r-IL-8 in Freunds adjuvant (not
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375142
and free or conjugated NTP77 but did not recognize
IL-872. This mAb was selected for further study.
3.3. Characterization of antibody N11
3.3.1. Specificity of mAb N11 in ELISA
The specificity of N11 adsorbed on polystyrene
plates was characterized using a liquid phase poly-
clonal antibody to IL-8 in an ELISA format. IL-877showed concentration-dependent binding to N11 (Fig.
2A), while no binding of IL-872 was seen. Both forms
of IL-8 bound to the BS-1 mAb that recognized IL-872(Fig. 2B).
IL-877 is converted to IL-872 by cleavage of the
AVLPR peptide with serine proteases at the ArgUSerbond (Yoshimura et al., 1989). We explored the
possibility that the peptide AVLPR could inhibit bind-
ing of IL-877 to N11. Fig. 3 shows that the AVLPR
peptide was capable of inhibiting the binding of N11
to IL-877 and NTP77 but a 103 molar excess of
AVLPR over IL-877 was needed to obtain a significant
inhibition. The binding of N11 to NTP77 was more
sensitive to inhibition by AVLPR (Fig. 3) suggesting
that the affinity of N11 to NTP77 was much lower than
to IL-877. Interestingly, N11 mAb was unable to bind
a solid phase BSAAVLPR conjugate (Table 1) and
free AVLPR peptide (not shown).
3.3.2. Characterization of N11 using immunoblotting
and cell immunostaining
The ability to recognize antigens after denaturing
PAGE and in fixed cell and tissue preparations repre-
sents an important characteristic of mAbs to cytokines.
Antibody N11 recognized IL-877 and conjugates of
NTP77 on immunoblots after Laemmli denaturing
PAGE, while IL-872 was not recognized (data not
shown). As expected, mAbs 4C and WS-4 recognized
both forms of IL-8. When tested in immunohistolog-
ical staining of human mononuclear leukocytes acti-
vated with LPS for 3.5 h in the presence of brefeldin,
N11 showed efficient immunostaining of cell smears
fixed with ice-cold acetone or formalin/0.01% saponin
(data not shown).
Fig. 2. Comparison of the anti-IL-877 mAb N11 (A) and the anti-IL-
872 mAb BS-1 (B) used as capture antibodies in the ELISA.
Microtiter plates were coated with mAbs (5 Ag/ml) and incubatedwith IL-877 or IL-872 as described in the Materials and Methods. A
rabbit anti-IL-8 antibody was used for the liquid phase, followed by
an alkaline phosphatase-labeled goat antibody to rabbit IgG. Means
of triplicates are shown.
Fig. 3. Inhibition of binding of the anti-IL-877 mAb N11 or the anti-
IL-872 mAb H6 to plastic adsorbed IL-877 or NTP77 in the presence
of increasing concentrations of the AVLPR peptide or NTP77.
Microtiter plates were coated with 0.1 Ag/ml of r-IL-877 (.) orNTP77 (5) and incubated with mAb N11 or the anti-IL-872 mAb H6in the presence of increasing concentrations of AVLPR ( +AVLPR)
or NTP77 ( +NTP77) and were developed with peroxidase-labeled
goat anti-mouse antibody.
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 43
3.3.3. N11 mAb is efficient in homologous ELISA
Dimeric and oligomeric forms of proteins in sol-
ution can be recognized by so-called oligomeric or
homologous (hm) ELISA, which uses the same
mAb specific for a single epitope on a monomeric
protein for both the solid and the liquid phase (Corti et
al., 1992; Petyovka et al., 1995). In a homologous
ELISA, N11 recognized IL-877 in PBS0.5% BSA at
concentrations ranging from 2 to 16 ng/ml (Fig. 4A),
demonstrating that IL-877 is a dimer (or oligomer) at
physiologically relevant concentrations. The non-
ionic detergent Tween-20 dissociated IL-877 to mono-
mers that were not recognized by N11/N11 hm-
ELISA (Fig. 4A), but were recognized in a heterol-
ogous ELISA consisting of captured N11 and bio-
tinylated H6 (Fig. 4B). These results are similar to
those obtained for the oligomeric forms of IL-872(Petyovka et al., 1995) and TNF-a (Corti et al.,1992; De Groote et al., 1993; Petyovka et al., 1995).
3.3.4. Antibody N11 neutralizes chemotactic activity
of IL-877Antibody N11 completely inhibited chemotactic
activity of IL-877, while chemotactic activity of IL-872was not affected in the presence of increasing concen-
trations of N11 (Fig. 5A). As expected, the mAb 4C to
IL-872 inhibited chemotactic activity of both forms of
IL-8 (Fig. 5B).
Fig. 4. Titration of IL-877 in a homologous ELISA (A) using N11
mAb for capture and biotinylated N11 for detection (N11/N11*),
compared to a heterologous ELISA (B) for IL-877 consisting of N11
for capture and biotinylated H6 (N11/H6*) for detection in the
presence (.) or in the absence (o) of 0.05% Tween-20. * Representsbiotinylated mAb, the same as in Figs. 6 and 7.
Fig. 5. The effect of the anti-IL-877 mAb N11 (A) and the anti-IL-872mAb 4C (B) on the human neutrophil chemotactic activity of IL-877(.) and IL-872 (o). Spontaneous cell migration differed, but wastypically in the range of 1530 cells per high power field (No./HPF).
The mean and F S.E. of three experiments are shown.
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375144
3.4. ELISA for human IL-877
3.4.1. Selection of a partner antibody for N11 mAb in
To develop a sandwich immunoassay specific for
IL-877, we used a combination of N11 with a second
mAb recognizing both IL-872 and IL-877. A panel of
mAbs to IL-872 that were previously described (BS-1
and WS-4, Ko et al., 1992) or produced in the current
study (3A, 4C, H6 and N6), was tested to select the best
partner for N11. Fig. 6 shows the titration of IL-877 in
an ELISA using immobilized N11 (A) with different
biotinylated anti-IL-8 mAbs for detection and the
reverse configurations using biotinylated N11 (B).
When N11 was used for capture, BS-1, H6 and N6
were the best for detection, although WS-4 showed
lower sensitivity. In the reverse configuration using
biotinylated N11 for detection, the same mAbs showed
reversed sensitivity with the WS-4/N11 combination
being the most sensitive (Fig. 6B). Antibody 4C was
unable to detect IL-877 when paired with N11 (data not
3.4.2. Comparison of ELISAs using the N11 mAb for
either capture or detection
Since both forms of IL-8 may be produced simulta-
neously (Yoshimura et al., 1989; Schroder et al., 1990),
an ELISA for IL-877 must be able to detect IL-877 in the
presence of various concentrations of IL-872. We
compared the efficiency of N11 for either capture or
detection of IL-877 in the presence of increasing con-
centrations of IL-872. Fig. 7A shows that the recog-
nition of IL-877 was inhibited by the presence of
increasing concentrations of IL-872, when mAb H6
that recognizes both forms of IL-8 was used as the
capturing antibody. In contrast, recognition of IL-877 in
an ELISA using N11 to capture IL-877 was not affected
in the presence of IL-872. The combination of N11 as
the capturing antibody and biotinylated BS-1 or H6
for detection was selected for further study. The detec-
tion limit of this ELISA for IL-877 was between 25 and
50 pg/ml (Fig. 7B).
The ELISA using mAbs BS-1, WS-4 or 4C (rec-
ognizing IL-872) for capture and biotinylated N6, H6
or BS-1 for detection recognized both forms of IL-8
and was called the total IL-8 ELISA. The total
ELISA using BS-1 and N6 and the ELISA for IL-877showed similar IL-8 titration curves both in PBS0.3
M NaCl0.5% BSATw and normal donor plasma
(Fig. 7B and C). Since the total IL-8 determined by
this assay is the sum of IL-872 and IL-877, the
concentration of IL-872 in the samples was estimated
by subtracting the concentration of IL-877 from the
3.5. Production of IL-877 by cultured cells
The ELISA for IL-877 was used to study the
production of IL-877 in various cell cultures. We
determined the concentration of IL-877 in supernatants
from the cultures of endothelial cells, epithelial tumor
cell lines, neuroblastoma cell lines SH and MC,
myelo-monocytic cells THP-1, neutrophils and mono-
cytes. Cells were cultured in media either with or
Fig. 6. Comparison of ELISAs for IL-877 using different anti-IL-872mAbs paired with the anti-IL-877 mAb N11 used as the capture
antibody (A) or as the detection antibody (B).
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 45
without serum and resting or stimulated with LPS,
TNF-a or IL-1-h. Both forms of IL-8 were detected inthe supernatants from all cell cultures. The levels of
IL-877 ranged from 5% to 70% of the total IL-8
protein and were more dependent on the duration of
cell culture and the presence of serum in the culture
medium than on cell type (data not shown). To better
understand the mechanisms of the generation of IL-
877 in cell cultures, we studied the production of IL-
877 and IL-872 by human monocytes. In monocytes
stimulated with LPS for 3.5 h in the presence of the
Fig. 8. The effect of protease inhibitors on the accumulation of IL-
877 and total IL-8 in supernatants of LPS-stimulated monocytes. The
plastic adherent fraction of human blood mononuclear leukocytes
(adherence to 24-well plastic plates at 106/ml in 1 ml for 1 h) was
stimulated with LPS (100 ng/ml) and incubated in the absence (A)
or in the presence (B) of a protease inhibitor cocktail in 0.5 ml
RPMI2% FCS at 37 jC for the indicated times. Supernatants werestored frozen at 40 jC after the addition of the protease inhibitorcocktail to all samples.
Fig. 7. (A) Detection of IL-877 in the presence of IL-872 in an
ELISA using mAb N11 as a capture antibody (E), compared to anELISA using mAb N11 as the detection antibody (D) paired withanti-IL-872 mAb H6. IL-877 was added to the capture antibody at 0.8
ng/ml simultaneously with increasing concentrations of IL-872 (at
0.62540 ng/ml). (B) Standard titration curves of IL-877 and IL-872in an ELISA using mAb N11 for capture and the biotinylated anti-
IL-872 mAb H6 for detection. Open symbols: titration of IL-872 (5)and IL-877 (o) in PBS0.3 M NaCl0.5% BSA0.05% Tween-20; closed symbols: IL-872 (n) and IL-877 (.) were added tonormal donor plasma at 4 ng/ml and further diluted in PBS0.3 M
NaCl0.5%BSA0.05%Tween-20. (C) The same titration of IL-872and IL-877 as in B using total ELISA consisting of anti-IL-872mAb
BS-1 for capture and biotinylated mAb N6 for detection.
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375146
secretion inhibitor brefeldin, followed by lysis with
Triton X-100 buffer containing protease inhibitors, no
IL-872 was detected but levels of 100250 ng of IL-
877 per 106 monocytes were found (data not shown).
When the levels of both forms of IL-8 in the culture
medium of monocytes stimulated with LPS were
determined over the course of incubation, no IL-872was detected in the culture medium in the first 6 h
(Fig. 8A). IL-872 was detected after 6 h with levels
increasing with time with a corresponding decrease in
levels of IL-877. By 12 h IL-877 comprised about 80%
of the total IL-8. This decreased to less than 30% at 24
h. When a cocktail of protease inhibitors was added at
the beginning of incubation, only IL-877 was detected
during the first 18 h (Fig. 8B). When cell-free super-
natants from both 6 and 12 h monocyte cultures were
incubated at 37 jC for 6 h, IL-877 was converted toIL-872. This could be prevented by adding a cocktail
of protease inhibitors (data not shown). The conver-
sion of the IL-877 to IL-872 was significant in serum
containing media without any cells (about 75% of
exogenous IL-877 was converted to the 72-amino-acid
form during 20 h at 37 jC in RPMI10% FCS) andno conversion was detected in serum-free medium
(data not shown).
3.6. Detection of IL-877 in samples of plasma and
Using the ELISA for IL-877 described above, we
examined samples of human plasma and cerebrospinal
fluid taken from patients with various forms of
meningococcal infection and sepsis. To prevent pro-
teolytic conversion of IL-877, blood and CSF were
collected in plastic tubes containing both EDTA and a
cocktail of protease inhibitors and were centrifuged
not later than 1 h after blood collection to prevent
possible IL-8 release from platelet or leukocyte depots
(Su et al., 1996). The titration of IL-877 in normal
donor plasma containing a PI cocktail was identical to
that in PBS0.3 M NaCl0.5% BSATw (Fig. 7B).
The content of IL-877 in 20 samples of normal
volunteer donor blood was below the detection limit
(data not shown); however, both IL-877 and IL-872were detected at various concentrations in the samples
of plasma and CSF from patients with meningitis and
sepsis (Table 2). It was surprising to find substantial
levels of IL-877 in some samples of blood plasma that
contained little or no total IL-8, as detected by a
total IL-8 ELISA. This paradoxical combination
was never observed in CSF samples from the same
Detection of IL-877 and total IL-8 in human blood plasma and CSF from patients with meningitis and sepsis
Patient Diagnosis Blood plasma CSF
Total IL-8 a
1 Meningitis of unidentified etiology 0.172 0.216 0.150 0.300
2 Meningococcal meningitis, Meningococcemia 0.840 0.216 0.220 1.466
3 Meningococcal meningitis, Meningococcemia < 0.060 < 0.015 1.808 7.375
4 Purulent meningitis of unidentified etiology, fifth day 1.124 0.358 0.146 0.620
4a Ibid, 11th day of disease 0.310 6.050 0.140 0.196
5 Meningitis of unidentified etiology 1.562 < 0.015 n.t.b n.t.
6 Hydrocephalia of unidentified etiology n.t. n.t. 3.804 5.544
7 Meningococcemia 2.018 < 0.015 n.t. n.t.
8 Meningococcemia 1.292 < 0.015 n.t. n.t.
9 Meningoencephalitis 1.724 0.250 1.760 2.010
10 Meningoencephalitis 0.361 0.604 16.150 42.150
11 Encephalitis 2.036 0.225 0.654 2.250
12c Major surgery, sepsis 1.432 0.132 n.t. n.t.
a The data on total IL-8 were obtained using the BS-1-N6* ELISA; similar results were obtained using total IL-8 ELISAs 4C-H6*,
WS-4-N6* and H6-4C* (* indicates biotinylated mAb).b n.t.non-tested.c Patient No.12 was an adult; all other patients were from the pediatric clinic, 9 months15 years old.
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 47
patients (Table 2), which showed elevated levels of
IL-877 together with higher total IL-8.
Because of the potential utility of measuring IL-877in culture supernatants, body fluids and tissue sec-
tions, we produced a mAb specific for this form of IL-
8 and developed a corresponding ELISA.
To generate antibodies to the N-terminus of IL-877,
mice were immunized with a recombinant antigen of
IL-877 fused to the Fc portion of human IgG or with
conjugates of the synthetic peptide AVLPRSA-
KELRC-NH2, which corresponds to the N-terminal
12 amino acids of IL-877.
The use of FcIgGIL-877 in Freunds adjuvant for
the priming and the first boost showed the immune
response augmenting function of FcIgG, which may
be relevant to the enhanced immunogenicity of fusion
proteins or of antigens coupled to molecules that
interact with immunocompetent cells such as C3b
(Dempsey et al., 1996) and thyroglobulin (Mnich et
However, the use of FcIgGIL-877 for the second
boost showed no immunoenhancing effect and the
antibody response to the Fc domain prevailed over
the response to IL-8. While recombinant IL-8 was
approximately three-fold weaker as an immunogen
for priming and the first boost compared to FcIgG
IL-877, it induced a three-fold higher antibody
response to IL-8 when used for the second boost
instead of FcIgGIL-877. We observed a similar loss
of immunoenhancing function of a carrier protein
after the second boost in the course of immunization
against BSANTP77. The substitution of BSA for
Ova as the carrier protein for the second boost
markedly enhanced antibody responses to NTP77.
This observation might be relevant to the so-called
antigen competition phenomenon (Landsteiner,
1945). It seems that the FcIgG fragment in recombi-
nant FcIgGIL-877 and carrier proteins in conju-
gates of NTP77 were necessary for efficient priming
and the first boost, while they inhibited the antibody
response toward the weaker antigens, IL-8 (Fig. 1)
and NTP77, after the second boost. The N-terminus
of IL-877 seems to be a weaker antigen compared to
the FcIgG fusion partner, the 72-amino-acid body
of IL-877 itself and even to the sulfo-MBS chemical
bond between NTP77 and the carrier protein. Anti-
IL-877 mAbs were produced by substituting both the
carrier protein and the chemical bond in the NTP77conjugate in the course of immunization followed
by a final pre-fusion immunization with synthetic
The mAb N11 that resulted from this protocol
reacted with IL-877, FcIgGIL-877 and both the free
and conjugated AVLPRSAKELRC-NH2 peptide of
IL-877 but did not recognize IL-872. IL-877 may be
converted to IL-872 by cleavage with serine proteases
at the ArgUSer bond (Yoshimura et al., 1989; Hebertet al., 1990; Padrines et al., 1994) thus releasing the
N-terminal AVLPR pentapeptide of IL-877. This pep-
tide was shown to induce apoptosis in leukemic cells
(Terui et al., 1999). N11 was unable to bind immobi-
lized AVLPR peptide although the binding of N11
with IL-877 and NTP77 was inhibited by the presence
of an approximately 103 molar excess of AVLPR.
These data indicate that the epitope recognized by
N11 overlaps but does not coincide with the AVLPR
sequence. In addition, these data demonstrate that the
chance of inhibiting the recognition of IL-877 in an IL-
877 ELISA by naturally occurring AVLPR peptide is
The combination of N11 with H6 or BS-1 that
recognize IL-872 was used to develop an IL-877-
specific ELISA. It was shown that recognition of
IL-877 was not affected by an excess of IL-872 added
to the samples when N11, specific for IL-877, was
used as the capture antibody. This ELISA showed
specificity for IL-877 with a detection limit of 2550
pg/ml, sufficient for measuring IL-877 in cell culture
and human body fluids.
The study of the production of IL-877 by mono-
cytes and other cells in vitro has revealed that cultured
cells secreted only the IL-877 form, while IL-872appeared at later time points of the cell culture due
to post-secretion proteolytic degradation of IL-877 in
the culture medium. This is in agreement with pre-
vious observations using N-terminal sequencing and
estimation of the molecular weight of IL-8 by SDS-
PAGE (Yoshimura et al., 1989).
To contact circulating blood leukocytes and induce
their attachment and transendothelial emigration, IL-8
must be exposed on the blood surface of endothelial
cells (Rot, 1992; Tanaka et al., 1993; Rot et al., 1996;
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375148
Middleton et al., 1997). The latest data indicate that
IL-8 exposed on the circulation side of endothelium in
response to LPS is produced by endothelial cells
themselves, rather than in extravascular tissue
(Dumont et al., 2000).
Since it was shown here that IL-877 secreted by
cultured cells persisted in vitro for a few hours and
since leukocyte adherence and transendothelial emi-
gration occur within a few minutes (Girard and
Springer, 1995; Baggiolini, 1998), we suggest that
the only form of IL-8 involved in neutrophilendo-
thelium interactions in vivo is IL-877. The chance of
fast intravascular proteolytic conversion of endothe-
lium generated IL-877 to IL-872 is rather small since
proteases in blood circulate with corresponding pro-
tease inhibitors. We have detected no conversion of r-
IL-877 to IL-872 after 20 h of incubation at 37 jC infresh normal donor plasma that was anticoagulated
with EDTA and separated 30 min after venepuncture
(data not shown). IL-877 was recently isolated from a
wound healing area and was suggested to stimulate
keratinocyte proliferation (Rennekampff et al., 2000).
IL-872, on the other hand, is extremely resistant to
further proteolytic degradation and might accumulate
in inflammatory tissue reservoirs. In such places, IL-
872 has been suggested to play a detrimental role in
chronic inflammation (Yoshimura et al., 1989). The
study of IL-877 and IL-872 localization in human
tissue sections is currently performed using N11 and
other mAbs. Since N11 is capable of neutralizing IL-
877, it might be used in monkey models of inflamma-
tion in vivo to explore the role of this form of IL-8 in
leukocyte transendothelial emigration.
To test which form of IL-8 is produced in vivo, we
used samples of human plasma and cerebrospinal
fluid taken from patients with sepsis and meningitis.
IL-877 was clearly detected in these samples, indicat-
ing that it is produced during inflammatory responses
in vivo. It was surprising to find that high plasma
levels of IL-877 in some of the blood samples were not
accompanied by adequate levels of total IL-8 (i.e.
the sum of the 72- and 77-amino-acid forms). These
data imply that the total IL-8 ELISA was unable to
detect IL-8 in some plasma samples, while the IL-877specific ELISA detected IL-877. This situation was
never observed in the samples of CSF, where high
levels of IL-877 were accompanied by the same or
higher levels of total IL-8.
Blockade of the recognition of IL-8 in plasma
samples in a total IL-8 ELISA has long been
known. Sylvester et al. (1992) have demonstrated
that in human sera, IL-8 is bound with high affinity
to IgG, which interferes with the ELISA and was
suggested to serve as an IL-8 scavenger auto-
antibody (Sylvester et al., 1992; Leonard, 1996;
Kurdowska et al., 2001). We suggest that the total
IL-8 ELISA in our experiments was blocked via the
same mechanism. In favor of this suggestion is the
absence of this situation in the samples of CSF that
contain little or no IgG compared to plasma. Alpha
2-macroglobulin has also been shown to form com-
plexes with IL-8 (Kurdowska et al., 2000). Since the
ELISA for IL-877 was not blocked in the same
plasma samples, the N-terminal region of IL-877 is
probably available for binding with N11 in hypo-
thetical complexes. Interestingly, this phenomenon
was observed in 21 plasma samples from 36 chil-
dren, while in adult sepsis, it was found in 1 plasma
from 12 patients. In a preliminary study using
captured N11 and liquid phase antibody to human
IgG (Sylvester et al., 1992), we detected IL-877/IgG
complexes in the samples of human blood plasma.
The investigation of this phenomenon is beyond the
scope of this report.
This observation addresses once more the often
discussed problem: Immunoassays for detecting
cytokines: What they are really measuring? (Mire-
Sluis et al., 1995). While the use of an IL-877 ELISA
as a diagnostic tool in diseases in which IL-8 may be
implicated requires further investigation, the antibod-
ies and assays described in this report should be useful
in experimental studies of IL-8 in host defense and
We thank Dr. Bernard Scallon, Centocor Inc.,
Malvern, PA, for the production of FcIgGIL-877 and
the donation of FcIgGp-55; Prof. Marco Baggiolini,
Kocher Inst., University Bern, Switzerland, for the
gift of synthetic IL-877; Dr. Ernst Brandt, Forschungs-
zentrum, Borstel, Germany, for providing the N-
terminal peptide of human CXCR2; Anna Portyanko,
Medical University, Minsk, Belarus, for help with
immunohistology; Dr. Joost Oppenheim, LMI, NCI at
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 49
Frederick, MD, for providing the BIOQUANT
software for chemotaxis assays; Dr. Ilia Tichonov,
Inst. Hematology, Minsk, Belarus, for help with
mouse serum assays and production of mAb H6; Dr.
Yuri Chaly, Inst. Hematology, Minsk, for his expert
assistance in the manuscript preparation; Dr.
Alexander Kudin, Medical University, Minsk, Bela-
rus, for help in obtaining clinical samples of plasma
and CSF; Dr. Ji Ming Wang, LMI, NCI at Frederick,
MD, for the gift of recombinant human IL-877; and
Prof. Sergei Ketlinsky, Institute of Highly Pure
Biopreparations, St. Petersburg, Russia, for the gift
of a rabbit antibody to human IL-8.
The work was supported by Research Grant from
Centocor Inc., Malvern, PA, a Grant from INTAS,
Brussels, research funding from the Office of Interna-
tional Affairs, Department of Health & Human
Services, National Cancer Institute, NIH, Bethesda,
MD, and from the Ministry of Health, Republic of
Aithal, H., Knigge, K., Kartha, S., Czyzewski, E., Toback, G.,
1988. An alternative method utilizing small quantities of ligand
for affinity purification of monospecific antibodies. J. Immunol.
Methods 112, 633.
Baggiolini, M., 1998. Chemokines and leukocyte traffic. Nature
Baggiolini, M., 2001. Chemokines in pathology and medicine. J.
Intern. Med. 250, 91.
Baggiolini, M., Dewald, B., Moser, B., 1994. Interleukin-8 and
related chemotactic cytokinesCXC and CC chemokines.
Adv. Immunol. 55, 97.
Belperio, J.A., Keane, M.P., Arenberg, D.A., Addison, C.L., Ehlert,
M.D., Burdick, M.D., Strieter, R.M., 2000. CXC chemokines in
angiogenesis. J. Leukoc. Biol. 68, 1.
Catty, D., Raykundalia, C., 1988. Production of polyclonal anti-
bodies. In: Catty, D. (Ed.), AntibodiesA Practical Approach
(Vol. vol. 1 IRL Press, Oxford.
Chaly, Y.V., Selvan, R.S., Fegeding, K.V., Kolesnikova, T.S., Voi-
tenok, N.N., 2000. Expression of IL-8 gene in human mono-
cytes and lymphocytes: differential regulation by TNF and IL-1.
Cytokine 12, 636.
Corti, A., Fassina, G., Marcucci, F., Barbanti, E., Cassani, G.,
1992. Oligomeric tumour necrosis factor alpha slowly con-
verts into inactive forms at bioactive levels. Biochem. J. 284,
De Groote, D., Grau, G., Dehart, I., Frannchimont, P., 1993. Stabi-
lisation of functional tumor necrosis factor-a by its soluble TNFreceptors. Eur. Cytokine Netw. 4, 359.
Dempsey, P., Allison, M., Akkaraju, S., Goodnow, C., Fearon, D.,
1996. C3d of complement as a molecular adjuvant: bridging
innate and acquired immunity. Science 271, 348.
Dumont, R.A., Car, B.D., Voitenok, N.N., Junker, U., Moser, B.,
Zak, O., OReilly, T., 2000. Systemic neutralization of interleu-
kin-8 markedly reduces neutrophilic pleocytosis during experi-
mental lipopolysaccharide-induced meningitis in rabbits. Infect.
Immun. 68, 5756.
Falk, W., Goodwin Jr., R.H., Leonard, E.J. 1980. A 48-well micro
chemotaxis assembly for rapid and accurate measurement of
leukocyte migration. J. Immunol. Methods 33, 239.
Gimbrone, M.A., Obin, M.S., Brock, A.F., Luis, E.A., Hass, P.E.,
Hebert, C.A., Yip, Y.K., Leung, D.W., Lowe, D.G., Kohr, W.J.,
Darbonne, W.C., Bechtol, K.B., Baker, J.B., 1989. Endothelial
interleukin-8: a novel inhibitor of leukocyte-endothelial interac-
tions. Science 246, 1601.
Giovannelli, A., Limatola, C., Raggozzino, D., Mileo, A.M., Rug-
gieri, A., Ciotti, M.T., Mercanti, D., Santoni, A., Eusebi, F.,
1998. CXC chemokines interleukin-8 (IL-8) and growth-related
gene product a (GROa) modulate Purkinje neuron activity inmouse cerebellum. J. Neuroimmunol. 92, 122.
Girard, J., Springer, T., 1995. High endothelial venules (HEV):
specialized endothelium for lymphocyte migration. Immunol.
Today 16, 449.
Harada, A., Sekido, N., Kuno, K., Akiyama, M., Kasahara, T.,
Nakanishi, I., Mukaida, N., Matsushima, K., 1993. Expression
of recombinant rabbit IL-8 in Escherichia coli and establishment
of the essential involvement of IL-8 in recruiting neutrophils
into lipopolysaccharide-induced inflammatory site of rabbit
skin. Int. Immunol. 5, 681.
Hebert, C.A., Luscinskas, F.W., Kiely, J.-M., Luis, E.A., Darbonne,
G.L., Bennett, G.L., Liu, C.C., Obin, M.S., Gimbrone, M.A.,
Baker, J.B., 1990. Endothelial and leukocyte forms of IL-8.
Conversion by thrombin and interactions with neutrophils. J.
Immunol. 145, 3033.
Huber, A.R., Kunkel, S.L., Todd, R.F., Weiss, S.J., 1991. Regula-
tion of transendothelial neutrophil migration by endogenous
interleukin-8. Science 254, 99.
Jue, R., Lambert, J.M., Pierce, L.R., Traut, R.R., 1978. Addition of
sulfhydryl groups to Escherichia coli ribosomes by protein
modification with 2-iminothiolane (methyl 4-mercaptobutyrimi-
date). Biochemistry 17, 5399.
Kitagawa, T., Kawasaki, T., Munechika, H., 1982. Enzyme immu-
noassay of Blascidin S with high sensitivity: a new and conven-
ient method for a preparation of immunogenic (haptenprotein)
conjugates. J. Biochem. 92, 585.
Ko, Y., Mukaida, N., Panyutich, A., Voitenok, N.N., Matsushima,
T., Kawai, T., Kasahara, T., 1992. A sensitive enzyme-linked
immunosorbent assay for human interleukin-8. J. Immunol.
Methods 149, 227.
Kurdowska, A., Alden, S.M., Noble, J.M., Stevens, M.D., Carr,
F.K., 2000. Involvement of alpha-2-macroglobulin receptor in
clearance of interleukin 8-alpha-2-macroglobulin complexes by
human alveolar macrophages. Cytokine 12, 1046.
Kurdowska, A., Noble, J.M., Steinberg, K.P., Ruzinski, J.T., Hud-
son, L.D., Martin, T.R., 2001. Anti-interleukin 8 autoantibody:
interleukin 8 complexes in the acute respiratory distress syn-
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375150
drome. Relationship between the complexes and clinical disease
activity. Am. J. Respir. Crit. Care Med. 163, 463.
Landsteiner, K., 1945. The Specificity of Serological Reactions
Harvard Univ. Press, Cambridge 2nd rev.
Lane, R.D., Crissman, R.S., Lachman, M.F., 1984. Comparison of
polyethylene glycols as fusogens for producing lymphocyte
myeloma hybrids. J. Immunol. Methods 72, 71.
Leonard, E.J., 1996. Plasma chemokine and chemokineautoanti-
body complexes in health and disease. Methods 10, 150.
Lindley, I., Aschauer, H., Seifert, J., 1988. Synthesis and expression
in E. coli of the gene encoding monocyte-derived neutrophil-
activating factor: biological equivalence between natural and
recombinant neutrophil-activating factor. Proc. Natl. Acad.
Sci. U. S. A. 85, 9199.
Matsushima, K., Oppenheim, J.J., 1989. Interleukin 8 and MCAF:
novel inflammatory cytokines inducible by IL 1 and TNF. Cy-
tokine 1, 2.
Middleton, J., Neil, S., Wintle, J., Clark-Lewis, I., Moore, H., Auer,
E., Hub, E., Rot, A., 1997. Transcytosis and surface presentation
of IL-8 by venular endothelial cells. Cell 91, 385.
Mire-Sluis, A.R., Gaines-Das, R., Thorpe, R., 1995. Immunoassays
for detecting cytokines: what are they really measuring? J. Im-
munol. Methods 186, 157.
Mnich, S.J., Veenhuizen, A.W., Monahan, J.B., Sheehan, K.C.,
Lynch, K.R., Isakson, P.C., Portanova, J.P., 1995. Characteriza-
tion of a monoclonal antibody that neutralizes the activity of
prostaglandin E2. J. Immunol. 155, 4437.
Mukaida, N., 2000. Interleukin-8: an expanding universe beyond
neutrophil chemotaxis and activation. Int. J. Hematol. 72, 391.
Nourshargh, S., Perkins, J.A., Showell, H.J., Matsushima, K., Wil-
liams, T.J., Collins, P.D., 1992. A comparative study of the
neutrophil stimulatory activity in vitro and in vivo pro-inflam-
matory properties in vivo of 72 amino acid and 77 amino acid
IL-8. J. Immunol. 148, 106.
Padrines, M., Wolf, M., Walz, A., Baggiolini, M., 1994. Interleukin-
8 processing by neutrophil elastase, cathepsin G and proteinase-
3. FEBS Lett. 352, 231.
Panyutich, A., Voitenok, N., Lehrer, R., Ganz, T., 1991. An enzyme
immunoassay for human defensins. J. Immunol. Methods 141,
Petyovka, N., Lyakh, L., Voitenok, N.N., 1995. Homologous ELI-
SA for detection of oligomeric human TNF: properties of the
assay. J. Immunol. Methods 186, 161.
Rennekampff, H.O., Hansbrough, J.F., Kiessig, V., Dore, C., Sti-
cherling, M., Schroder, J.M., 2000. Bioactive interleukin-8 is
expressed in wounds and enhances wound healing. J. Surg.
Res. 93, 41.
Rot, A., 1992. Endothelial cell binding of NAP-1/IL-8: role in
neutrophil emigration. Immunol. Today 13, 291.
Rot, A., Hub, E., Middleton, J., Pons, F., Rabeck, C., Thierer, K.,
Wintle, J., Wollf, B., Zsak, M., Dukor, P., 1996. Some aspects of
IL-8 pathophysiology: chemokine interaction with endothelial
cells. J. Leukoc. Biol. 59, 39.
Scallon, B.J., Trinh, H., Nedelman, M., Brennan, F.M., Feldmann,
J., Ghrayeb, J., 1995. Functional comparisons of different tu-
mour necrosis factor receptor/IgG fusion proteins. Cytokine 7,
Schroder, J.-M., Sticherling, M., Henneicke, H.H., Preissner, W.C.,
Christophers, E., 1990. IL-1 alpha or tumor necrosis factor-al-
pha stimulate release of three NAP-1/IL-8-related neutrophil
chemotactic proteins in human dermal fibroblasts. J. Immunol.
Su, S.B., Mukaida, N., Matsushima, K., 1996. Rapid secretion of
intracellularly pre-stored interleukin-8 from rabbit platelets upon
activation. J. Leukoc. Biol. 59, 420.
Sylvester, I., Yoshimura, T., Sticherling, M., Schroder, J.M., Ceska,
P., Peichl, P., Leonard, E.J., 1992. Neutrophil attractant protein-
1-immunoglobulin G immune and free anti-NAP-1 antibody in
normal human serum. J. Clin. Invest. 90, 471.
Tanaka, Y., Adams, D.H., Shaw, S., 1993. Proteoglycans on endo-
thelial cells present adhesion-inducing molecules to leukocytes.
Immunol. Today 14, 111.
Terui, Y., Ikeda, M., Tomizuka, H., Kasahara, T., Ohtsuki, T., Uwai,
M., Mori, M., Itoh, T., Tanaka, M., Yamada, M., Shimamura, S.,
Ishizaka, Y., Ikeda, K., Ozawa, K., Miura, Y., Hatake, K., 1998.
Activated endothelial cells induce apoptosis in leukemic cells by
endothelial interleukin-8. Blood 92, 2672.
Terui, Y., Tomizuka, H., Mishima, Y., Ikeda, M., Kasahara, T.,
Uwai, M., Mori, M., Itoh, T., Tanaka, M., Yamada, M., Shima-
mura, S., Ishizaka, Y., Ozawa, K., Hatake, K., 1999. NH2
terminal pentapeptide of endothelial interleukin 8 is responsible
for the induction of apoptosis in leukemic cells and has an
antitumor effect in vivo. Cancer Res. 59, 5651.
Van Damme, J., Van Beeumen, J., Opdenakker, G., Billiau, A.A.,
1988. Novel, NH-terminal sequence-characterized human
monokine possessing neutrophil chemotactic, skin-reactive,
and granulocytosis-promoting activity. J. Exp. Med. 167, 1364.
Yoshimura, T., Robinson, E.A., Appella, E., Matsushima, K.,
Showalter, S.D., Skeel, A., Leonard, E.J., 1989. Three forms
of monocyte-derived neutrophil chemotactic factor (MDNCF)
distinguished by different lengths of the amino-terminal se-
quence. Mol. Immunol. 26, 87.
N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 51
IntroductionMaterials and methodsAnimals and reagentsConjugation protocolImmunizationMonoclonal antibodies: generation and purificationCharacterization of monoclonal antibodies to IL-8Hybridomas origin and isotyping dataChemotaxis assayImmunoblotsImmunohistochemistry
ELISA detecting IL-8Induction of IL-8 in cell culturesDetection of IL-877 in clinical samples
ResultsSerum antibody responses to FcIgG-IL-877 and conjugates of NTP77Specificity of monoclonal antibodies derived from mice immunized with FcIgG-IL-877 and NTP77 conjugatesCharacterization of antibody N11Specificity of mAb N11 in ELISACharacterization of N11 using immunoblotting and cell immunostainingN11 mAb is efficient in homologous ELISAAntibody N11 neutralizes chemotactic activity of IL-877
ELISA for human IL-877Selection of a partner antibody for N11 mAb in ELISAComparison of ELISAs using the N11 mAb for either capture or detection
Production of IL-877 by cultured cellsDetection of IL-877 in samples of plasma and cerebrospinal fluid