a monoclonal antibody and an enzyme immunoassay for human ala-il-877

15
A monoclonal antibody and an enzyme immunoassay for human Ala-IL-8 77 Natalia N. Nashkevich a , Svetlana Akalovich a , Natalia Louneva b , George A. Heavner c , Nikolai N. Voitenok a,d, * a Laboratory of Cellular and Molecular Immunology, Institute of Hematology and Blood Transfusion, Dolginovsky Tract 160, Minsk, Belarus b Byelorussian Institute for Hereditary Diseases, Minsk, Belarus c Centocor Inc., Malvern, PA, USA d Fund 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 Abstract 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-8 77 ) and a second containing 72 amino acids (Ser-IL-8 72 ), which comprise more than 90% of IL-8 protein in cell cultures. IL-8 77 was reported to be produced predominantly by endothelial cells and is known as ‘‘endothelial’’ IL-8. IL-8 72 predominates 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-8 77 and the development of a corresponding immunoassay. Various immunization protocols were investigated. Immunization with conjugates of a peptide from the N-terminus of IL-8 77 (NTP 77 ) resulted in the production of an IgG1 mAb (N11) that recognizes human IL-8 77 and neutralizes its chemotactic activity. A sensitive ELISA specific for IL-8 77 was developed using N11 for capture and a biotinylated mAb to IL-8 72 for detection. Using this immunoassay it was shown that the only form of IL-8 secreted in cell culture was IL-8 77 and that the IL-8 72 present was the result of proteolysis of IL-8 77 . IL-8 77 was detected in plasma and cerebrospinal fluid (CSF) from patients with sepsis and meningitis. 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; NTP 77 , N-terminal peptide of IL-8 77 ; CFA/IFA, complete/incomplete Freund’s 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, tumor necrosis 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: [email protected] (N.N. Voitenok). www.elsevier.com/locate/jim Journal of Immunological Methods 270 (2002) 37 – 51

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Page 1: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

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

Abstract

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

meningitis.

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 Freund’s 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, tumor

necrosis 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: [email protected] (N.N. Voitenok).

www.elsevier.com/locate/jim

Journal of Immunological Methods 270 (2002) 37–51

Page 2: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

1. Introduction

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

Glu–Leu–Arg (ELR) motif (amino acids 69–67)

(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 30–80% 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 (FcIgG–p-55) (Scallon et

N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 37–5138

Page 3: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

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) Freund’s adjuvants were

from Sigma, St. Louis, MO. RIBI adjuvant MPL-

TDM system was from RIBI ImmunoChem Research,

Hamilton, MT.

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,

1988).

2.3. Immunization

BALB/c mice were immunized intraperitoneally

(i.p.) on the splenic side with 25 or 50 Ag of

recombinant human FcIgG–IL-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 25–50 Ag of recombi-

nant human FcIgG–IL-877 fusion protein, 25–50 Agof NTP77 conjugate or 20–30 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-

tion

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

FcIgG–IL-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

IL-8

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 FcIgG–IL-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 BSA–NTP77 and

Ova–NTP77 (sulfo-MBS coupled). N11 and D5 clones

were derived from immunization with KLH–NTP77(sulfo-MBS coupled) followed by BSA–NTP77 (car-

N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 37–51 39

Page 4: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

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

Boyden’s chambers as previously described (Falk et

al., 1980). Briefly, binding buffer containing 1% BSA

in RPMI 1640 with or without mAb (at 0.5–50 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 scraped

and 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 � 10

magnification.

2.5.3. Immunoblots

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 reaction

with the alkaline phosphatase-labeled goat anti-mouse

IgG antibody (Bio-Rad) and developed using NBT/

BCIP as a substrate.

2.5.4. Immunohistochemistry

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 RPMI–5% FCS (Sigma) in the

presence 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 4

jC, followed by treatment with horseradish peroxi-

dase-conjugated rabbit anti-mouse antibody (Dako) or

an LSAB kit (Dako) according to the manufacturer’s

protocol. All steps of staining of formalin-fixed

smears were performed in the presence of 0.01%

saponin (Sigma).

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 the

capture mAb in 100 Al of 0.1 M sodium carbonate

buffer, 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 (PBS–0.3 M

NaCl–Tw), unbound sites were blocked by adding

Table 1

Specificity of mAbs generated after immunization with FcIgG–

IL-877 or NTP77 conjugates

Antigen Absorbance (492 nm)

Antibodies

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

BSA–NTP77a 0.040 0.051 2.207 2.419 0.051

BSA–NTP77b 0.039 n.t.c 0.166 2.239 0.105

BSA 0.020 0.050 0.160 0.012 0.023

Ova–NTP77a 0.028 0.080 2.328 2.246 0.021

Ova 0.019 0.063 0.221 0.012 0.025

FcIgG–IL-877 2.595 1.624 0.196 1.797 1.451

Human IgG 0.036 1.000 0.267 0.019 0.028

FcIgG–p55d 0.052 1.210 0.268 0.020 0.035

KLH–NTP77a 0.015 n.t. 2.223 1.232 0.028

KLH 0.021 n.t. 0.152 0.010 0.025

KLH–CXCR2 NTPa,e n.t. n.t. 1.927 0.015 n.t.

BSA–AVLPRb n.t. n.t. n.t. 0.025 n.t.

Microtiter plates were coated with 0.1 Ag of each antigen per well

and tested against hybridoma clone supernatants diluted 1/5 with

PBS–0.5% BSA–0.05% 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) 37–5140

Page 5: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

0.5% BSA in PBS–0.3 M NaCl–Tw at room temper-

ature (RT) for 1 h. IL-872, IL-877 (stored at 10 Ag/ml

in PBS–1% BSA at � 70 jC) and samples were

serially diluted in PBS–0.3 M NaCl–0.5% BSA–Tw,

added to the wells (in triplicate) and incubated at RT

for 1 h. Plates were washed three times with PBS–0.3

M NaCl–Tw 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 0.5–1 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 RPMI–10%

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 RPMI–2% 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 15

min 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-

facturer’s instructions in Endothelial Cell Growth

Medium (EGM, Clonetics). Neuroblastoma cells and

HUVEC were stimulated for 20 h with either human

TNF-a at 5–10 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 preserved

with PI and stored frozen at � 40 jC.

3. Results

3.1. Serum antibody responses to FcIgG–IL-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

FcIgG–IL-877 or BSA–NTP77 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 FcIgG–IL-877 fusion protein in

CFA for priming followed by boosting with 25 Ag of

FcIgG–IL-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). Freund’s adjuvant with FcIgG–IL-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 AgFcIgG–IL-877 in IFA since gross lesions and mor-

bidity were observed in mice primed with 50 Ag of

FcIgG–IL-877 in CFA and boosted with the same

dose of FcIgG–IL-877 in IFA. No morbidity or

significant lesions were present in mice boosted with

25 Ag of FcIgG–IL-877 in IFA.

N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 37–51 41

Page 6: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

Surprisingly, the second boost with FcIgG–IL-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 FcIgG–IL-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

(Fig. 1).

Mice immunized using the mixed FcIgG–IL-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 BSA–

NTP77 conjugate in CFA followed by two boosts with

the same antigen in IFA at monthly intervals. As in the

case of sensitization with FcIgG–IL-877, the second

boost with the BSA–NTP77 conjugate gave no higher

antibody titers to NTP77 (not shown). However, when

Ova–NTP77 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 KLH–NTP77 followed by sequential

boosting with carbodiimide conjugated BSA–NTP77and 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 FcIgG–IL-877 and NTP77

conjugates

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

FcIgG–IL-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 BSA–NTP77 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

KLH–NTP77 followed by carbodiimide conjugated

BSA–NTP77 and synthetic IL-877. D5 reacted with

IL-877 and FcIgG–IL-877 but did not react with

NTP77 and showed weak recognition of IL-872. In

contrast, mAb N11 reacted with IL-877, FcIgG–IL-877

Fig. 1. Comparison of the antibody response to human IL-8 after the

first and the second boost with FcIgG–IL-877 or recombinant IL-8.

On day 0, groups of three mice were primed with FcIgG–IL-877 in

CFA and boosted on day 30 and on day 60 with FcIgG–IL-877 (n)

or r-IL-8 (o) in IFA. Blood for titration was taken on day 20 after

each 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 for

the meanF S.E. (n= 3). Mice primed with r-IL-8 showed less than

1.5 RU after the first boost with r-IL-8 in Freund’s adjuvant (not

shown).

N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 37–5142

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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 ArgUSer

bond (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 BSA–AVLPR 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 incubated

with 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 (.) or

NTP77 (5) and incubated with mAb N11 or the anti-IL-872 mAb H6

in 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) 37–51 43

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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 PBS–0.5% 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. * Represents

biotinylated 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 was

typically in the range of 15–30 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) 37–5144

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3.4. ELISA for human IL-877

3.4.1. Selection of a partner antibody for N11 mAb in

ELISA

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

shown).

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 PBS–0.3

M NaCl–0.5% BSA–Tw 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

‘‘total’’ IL-8.

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) 37–51 45

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without serum and resting or stimulated with LPS,

TNF-a or IL-1-h. Both forms of IL-8 were detected in

the 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

RPMI–2% FCS at 37 jC for the indicated times. Supernatants were

stored frozen at � 40 jC after the addition of the protease inhibitor

cocktail 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 an

ELISA using mAb N11 as the detection antibody (D) paired with

anti-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

0.625–40 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 PBS–0.3 M NaCl–0.5% BSA–0.05% Tween-

20; closed symbols: IL-872 (n) and IL-877 (.) were added to

normal donor plasma at 4 ng/ml and further diluted in PBS–0.3 M

NaCl–0.5%BSA–0.05%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) 37–5146

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secretion inhibitor brefeldin, followed by lysis with

Triton X-100 buffer containing protease inhibitors, no

IL-872 was detected but levels of 100–250 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 to

IL-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 RPMI–10% FCS) and

no conversion was detected in serum-free medium

(data not shown).

3.6. Detection of IL-877 in samples of plasma and

cerebrospinal fluid

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 PBS–0.3 M NaCl–0.5% BSA–Tw (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

Table 2

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

IL-877(ng/ml)

Total IL-8 a

(ng/ml)

IL-877(ng/ml)

Total IL-8

(ng/ml)

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 months–15 years old.

N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 37–51 47

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patients (Table 2), which showed elevated levels of

IL-877 together with higher ‘‘total’’ IL-8.

4. Discussion

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 FcIgG–IL-877 in Freund’s 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

al., 1995).

However, the use of FcIgG–IL-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 FcIgG–IL-877. We observed a similar loss

of immunoenhancing function of a carrier protein

after the second boost in the course of immunization

against BSA–NTP77. 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 FcIgG–IL-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

IL-877.

The mAb N11 that resulted from this protocol

reacted with IL-877, FcIgG–IL-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; Hebert

et 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

rather small.

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 25–50

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) 37–5148

Page 13: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

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 neutrophil–endo-

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 in

fresh 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

pathology.

Acknowledgements

We thank Dr. Bernard Scallon, Centocor Inc.,

Malvern, PA, for the production of FcIgG–IL-877 and

the donation of FcIgG–p-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) 37–51 49

Page 14: A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

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

Belarus.

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