Characteristics of the epitope of leukotriene B4 recognized by a highly specific mouse monoclonal antibody

Vol. 123, No. 3, 1984

September 28, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 944-950

CHARACTERISTICS OF THE EPITOPE OF LEUKOTRIENE B 4 RECOGNIZED BY A HIGHLY SPECIFIC MOUSE MONOCLONAL ANTIBODY

J.Y. Lee, T. Chernov, and E.J. Goetzl

Howard Hughes Medical Institute and the Department of Medicine, University of California Medical Center, San Francisco, California 94143

Received August 20, 1984

Mouse monoclonal IgG2b antibodies to leukotriene B 4 bind [3H]leukotriene B 4 with an affinity one-thirtieth to one-third that of different rabbit antibodies to leukotriene B 4. The concentrations of related ligands required to inhibit by 50% the binding of [3H]leukotriene B 4 define cross- reactivities of approximately 100% for carboxyl-derivatives of leukotriene B4, 10% for 12(S)-leukotriene B 4 and 8 __cis-leuk°triene B4, which were not distinguished from leukotriene B 4 by polyclonal antibodies, 3-5% for the two isomers of 6 trans-leukotriene B4, 5% for 20-OH-leukotriene B 4 and 20-COOH- leukotriene B4, and less than i% for other leukotrienes, mono-hydroxy-eicosatetraenoic acids, and the two leukotriene B4-1ike isomers of 8,15-di-hydroxy- eicosatetraenoic acid. Thus the monoclonal combining site is highly specific for the di-hydroxy-triene portion of leukotriene B 4. ©1984 Acad~icPress, Inc.

The most potent leukocyte chemotactic factor generated by the oxygenation

of arachidonic acid is 5(S),12(R)-di-hydroxy-eicosa-6,14 cis-8,10 trans-

tetraenoic acid, which is designated leukotriene B 4 (LTB4) (1,2). The recog-

nition of nM concentrations of LTB 4 by stereospecific receptors on human

polymorphonuclear (PMN) leukocytes (3,4) and eosinophils elicits chemotaxis,

increased adherence to surfaces, lysosomal degranulation, enhanced expression

of complement receptors, and greater cytotoxicity (1,5-7). LTB 4 suppresses

human T-lymphocyte functions specifically by inhibiting helper cell and

augmenting suppressor cell activities (8), and evokes increased human lymphocyte

cytotoxicity (9). The introduction of LTB 4 into tissues induces an influx

Abbreviation s used are: LTB4, leukotriene B4, 5(S),12(R)-di-hydroxy-eicosa- 6,14 ci__ss-8,10 trans-tetraenoic acid; PMN, polymo'rphonuclear; BGG, bovine gamma globulin; HSA, human serum albumin; EDC, l-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride; HPLC, high performance liquid chromatography; 8-cis-LTB4, 5(S),12(S)-dihydroxy 6,10-trans-8,14-cis-eicosatetraenoic acid; 8(R),I5(S)-LT, 8(R),15(S)-dihydroxy-5 cis-9,11,13 trans-eicosatetraenoic acid; 8(S),15(S)-LT, 8(S),15(S)-dihydroxy-5 cis-9,11,13 trans- eicosatetraenoic acid; TETA, triethylenetetramine tetrahydrochloride; HAT, hypoxanthine, aminopterin, and thymidine; LTB4-MCI , mouse monoclonal anti- LTB4; PEG, polyethylene glycol; and LTB4-APA , N-(3-aminopropyl) amide-LTB 4.

0006-291X/84 $1.50 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. 944

Vol. 123, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of PMN leukocytes (i0) and, secondarily, increased microvascular permeability

(11) and hyperalgesia (12).

Rabbit polyclonal antibodies to LTB4, which had been raised with immuno-

gens consisting of LTB 4 coupled to serum albumin through the 12-oxy or 1-

carboxyl substituents, provided sensitive radioimmunoassays for quantities

of LTB 4 as small as 0.03 pmol (13-15). However, such antibodies cross-

reacted extensively with biologically important isomers of LTB4, such as the

double lipoxygenation product 8 cis-LTB 4 (16). A mouse monoclonal antibody

to LTB 4 now is described, that is sufficiently specific to distinguish LTB 4

from its naturally-occurring isomers.

MATERIALS AND METHODS

Dulbecco's modified Eagle's medium with 4.5 g of glucose/L (Cell Culture Facility, UCSF), fetal bovine serum (Sterile Systems, Inc., Logan, Utah), polyethylene glycol (PEG) (m.w. 3,350), recrystallized bovine gam~na globulin (BGG), human serum albumin (HSA) (Sigma Chemical Co., St. Louis, Mo.), complete and incomplete Freund's adjuvant (Grand Island Biological Co.~ Grand Island, N.Y.), arachidonic acid (Supelco, Inc., Bellefonte, Pa.), [ H]LTB 4 (180-221Ci/mmol)(Amersham Corp., Arlington Heights, Ill.) triethylenetetramine tetrahydrochloride (TETA) (Aldrich Chemical Co., Inc., Milwaukee, Wisc.), [5,6,8,9,11,12,14,15-3H(N)] arachidonic acid (specific activity 80- 120 Ci/mmol), [14,15-3H (N)]-LTC 4 (34 Ci/mmol), [14,15-OH(N)]-LTD4 (36 Ci/ mmol) (New England Nuclear, Inc., Boston, Mass.), l-ethyl-3-(3-dimethyl amino-propyl)-carbodiimide hydrochloride (EDC) (Pierce Chemical Co., Rockford, Ill.), polyethylene glycol (PEG) (m.w. 3000-3700) (MCB Manufacturing Chemists, Inc., Cincinnati, Ohio), affinity-purified monospecific rabbit antibodies to mouse IgGl, IgG2a , IgG2b , IgG3, IgA, and IgM (Zymed Laboratories, Inc., South San Francisco, Calif.), and high-performance liquid chromatography (HPLC)-grade organic solvents that had been redistilled from glass (Burdick and Jackson Laboratories, Inc., Muskegon, Mich.) were obtained as noted. [3H]-I2-HETE (specific activity = 21-39 Ci/mmol), [3H]-5-HETE (specific activity = 34-47 Ci/mmol), [3H]-labeled (specific activity = 23-36 Ci/mmol) and non-radioactive 6-trans isomers of LTB 4 were prepared, purified by HPLC, and characterized as described (1,3). The 5(S),12(S)-dihydroxy 6,10-trans- 8,14-cis-eicosatetraenoic acid isomer of LTB 4 (8-cis-LTB 4) was prepared and purified by published methods (16) and was supplied in synthetic form by Prof. E. J. Corey (Harvard University, Cambridge, Mass.). Synthetic LTB4, 5(S),12(S)-di-hydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid (12(S)- LTB4) , N-(3-aminopropyl) amide-LTB 4 (LTB4-APA) , 20-OH-LTB4, 20-COOH-LTB4, LTC4, LTD4, LTE4, and 8(R),15(S)- and 8(S),15(S)-dihydroxy-5 cis-9,11,13 trans-eicosatetraenoic acid (8(R),I5(S)-LT and 8(S),I5(S)-LT, respectively) were supplied generously by Dr. J. Rokach (Merck Frosst Canada, Inc., Quebec, Canada).

LTB 4 was coupled through the C 1-carboxyl group to amino-groups on triethylenetetramine tetrahydrochloride (TETA)-modified HSA as described (17); the preparations of LTB4-HSA contained 5.9-11.6 LTB 4 per HSA, as quantified by the incorporation of [3H]LTB4. Seven six-week old female Balb/c mice were injected intradermally, intramuscularly, and intraperitoneally with 150 ~g of LTB4-HSA in complete Freund's adjuvant on Day 0, 21, and 35, and then intraperitoneally three times with 100 ~g of

945


LTB4-HSA in incomplete Freund's adjuvant at 7-14 day intervals. Three days after a last intraperitoneal and intrasplenic injection with 100 ~g of aqueous LTB4-HSA , the spleen was removed and splenic cells were fused by a polyethylene glycol (PEG) technique (I g/ml) in serum-free medium (18) with a 1:1 ratio of mouse myeloma P3X63AG8 cells (American Type Culture Collection, Rockville, Md.) that are resistant to 20 ~g/ml of 8-azaguanine and do not grow in hypoxanthine, aminopterin, and thymidine (HAT)-containing selective medium (19). Fused cells were distributed into 500-600 wells of 96-well plastic plates (Falcon Plastics, Oxnard, Calif.) in Dulbecco's modified Eagle's medium with L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (I00 U/ml), streptomycin (i00 ~g/ml), and 20% (v:v) fetal bovine serum and exposed to HAT selective medium after 24 hr.

[3H]LTB4 binding activity in supernates of hybridoma colonies that developed at 2-3 weeks, clones that were established subsequently by a limiting-dilution technique, and larger scale cultures of the clones of hybridoma cells was quantified by precipitation of mouse immunoglobulins with PEG in the presence of BGG as described (17), except that the final concentration of PEG was raised to 13.0 g per 100 ml. In order to determine the Ig subclass of antibodies to LTB4, the Ig-bound [3H]LTB4 in replicate samples was precipitated with 10-50 ~I of rabbit antibodies to different mouse Igs and 5-25 ~i of goat anti-rabbit IgG (Chemicon International, Inc., Los Angeles, Calif.). Statistical significance was determined with a standard paired t-test.

RESULTS AND DISCUSSION

The incubation of replicate aliquots of 6 nCi of [3H]LTB4 with 2, 5,

10, 20, and 40 ~I of medium containing mouse monoclonal anti-LTB 4 (LTB 4-

MCI) without and with 10 pmol of non-radioactive LTB 4 resulted in respective

binding of a mean of 5 and 0%, 7 and 2%, 13 and 7%, 17 and 11%, and 25 and

18% of the [3H]LTB4 (n = 3). The dilution of monoclonal anti-LTB 4 was

adjusted to achieve binding of 8-11% of the [3H]LTB4 in five consecutive

studies of the concentration-dependence of competitive inhibition of binding

by LTB 4 (Fig. I). Three pmol of LTB 4 inhibited by 51% ± 18% (mean ± S.D.)

the bidding of [3H]LTB4 by monoclonal anti-LTB 4 and one pmol of LTB 4 was the

smallest quantity which inhibited binding significantly. The linear

Scatchard plot of the binding data revealed a K a of 0.5 x 109 M -I for the

monoclonal combining site, as compared to an average K a of 3-9 x 109 M -I for

different populations of rabbit antibodies to LTB 4. Thus a radioim~unoassay

for LTB 4 utilizing the mouse monoclonal antibody would have a sensitivity

approximately one-thirtieth that of an assay of similar technique (15) with

rabbit polyclonal anti-LTB 4 (Fig. i) and one-third that of a double antibody

precipitation assay with rabbit polyclonal anti-LTB 4 (14).

946


110

lOO v 90 O 80 z 70 W ~ 6o < 50

~ 4 0

a 3 0

0 2O W o 10

~ 0 - - - - - - Z m -1C

W

I t 0.03 0'.1 0'.3 1 :3 10 3'0 100

QUANTITY OF COMPETING LIGAND (p moles)

Fig. I: Structural determinants of the binding of LTB 4 by monoclonal ( ) and rabbit polyclonal ( .... ) antibodies. Each point and bracket depicts the mean and standard deviation of the results of five (LTB 4, O) or three (12(S)-LTB4, O ; 12(R)-6 trans-LTB4, ~ ; 8 cis-LTB4, D ; LTB4-APA , • ; 20-OH-LTB4, x ) experiments. The di---fference in the displacement of [3H]LTB4 from the monoclonal antibody by LTB 4 and by 3 pmol of 12(S)-LTB 4 was significant with a p value less than 0.05, and by other concentrations of 12(S)- LTB4, 12(R)-6 trans-LTB4, 8 cis-LTB4, and 20-OH-LTB 4 was significant with a p value less than 0.01. Similarly, the differences in displacement of [3H]LTB4 from rabbit antibodies by LTB 4 and by LTB4-APA and 20-OH-LTB 4 were significant with a p value less than O . O l .

The specificity of the monoclonal anti-LTB 4 was assessed both by direct

quantification of the binding of other radiolabeled ligands and by competitive

inhibition by non-radioactive ligands of the binding of [3H]LTB4. The incuba-

tion of replicate portions of 20, 40, and 60 nCi of other lipoxygenase

products of arachidonic acid with 50 ~I of anti-LTB 4 (n = 3), resulted in

binding of a mean of only 0.4% or less of [3H]LTD4 and less than 0.2% of

[3H]-12(S)-6 trans-LTB4, [3H]LTC4, [3H]5-HETE, and [3H]12-HETE. The profile

of competitive inhibition of the binding of [3H]LTB4 by monoclonal antibody

was distinctly different from that of binding by polyclonal antibodies (Fig.

i). The monoclonal antibody distinguished LTB 4 from isomers differing only

in hydroxyl-group position, such as 12(S)-LTB4, or in the position of the

cis-double bond in the triene between the hydroxyl-groups, such as 8 cis-

LTB4, which the polyclonal antibodies bound with an affinity equal to that

for LTB4o The omega-oxidation products of LTB4, 20-OH-LTB 4 and 20-COOH-

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Table I - Specificity of Mouse Monoclonal Antibody to LTB 4

Relative Inhibition of Binding Cross-Reactive Ligand of [3H]LTB4 (3 pmol, mean, n=3)

LTB 4 100 12(S)-LTB4 56 12(R)-6 trans-LTB 4 14 12(S)-6 trans-LTB 4 8 8 cis-LTB 4 16 LTB4-APA 74 20-OH-LTB 4 < 0. 20-COOH-LTB 4 < O. 8(R),15(S)-LT 7 8(S),15(S)-LT 4 LTC 4 < 0. LTD 4 < 0. LTE 4 < O. 5-HETE < I 12-HETE < 1

LTB4, and the 6 trans-isomers of LTB 4 bound with less affinity than LTB 4

using either type of antibody (Fig. 1, Table I). The cross-reactivity of

the different double bond isomers of LTB 4 and the two isomers of 8,15(S)-LT

appeared to be 4-16%, when calculated from the relative inhibition of

binding of [3H]LTB4 by 3 pmol of each of the ligands (Table I). However,

the slopes of the other curves of competitive inhibition were different than

that for LTB 4. When calculated in terms of the quantity of ligand required

to inhibit by 50% the binding of [3H]LTB4, the extent of cross-reaction was

approximately 10% for 12(S)-LTB 4 and 8 cis-LTB4, 5% for 20-OH-LTB 4 and 20-

COOH-LTB4, 3-5% for the 6 trans-isomers of LTB4, and less than i% for the

isomers of 8,15(S)-LT. LTB4-APA , which has an aliphatic addition to the C-I

carboxyl-group, is bound by the monoclonal antibody with an apparent affinity

no different than that for LTB 4 free ~cid, while the polyclonal antibodies

discriminate significantly between LTB4-APA and the free acid of LTB 4 (Fig.

I). The more restricted epitope of the monoclonal activity was not a

function of the immunogen, which was the same in both cases. The specifi-

city of the monoclonal antibody was similar to that of the polyclonal anti-

bodies when examined only with sufficiently heterologous non-binding ligands

(Table I). The extent of competition with [3H]LTB4 was less than i% for 5-

HETE and 12-HETE and less than 0.1% for LTC4, LTD4, and LTE 4.

948


The reaction of complexes of [3H]LTB4 and mouse monoclonal antibody

with mono-specific rabbit antibodies to different isotypes of mouse Ig and

then goat anti-rabbit IgG to increase the extent of precipitation, indicated

that the LTB4-binding mouse monoclonal antibody LTB4-MCI is of the IgG2b

class.

The greater specificity of the monoclonal antibody than polyclonal

antibodies for the di-hydroxy-triene position of the LTB 4 molecule, suggest

that it will be more useful for identification of LTB 4 in biological mixtures

of related compounds. However, at least 1 pmol of LTB 4 will be required for

precise quantification due to the lower affinity of the monoclonal antibody.

The essentially unlimited supplies of highly specific monoclonal anti-LTB 4

that now are available also may permit analyses of the role of LTB 4 in a

variety of experimental models, as anti-LTB 4 neutralizes the leukocyte-

directed activities of LTB 4 in vitro.

ACKNOWLEDGEMENTS

This work was supported in part by NIH grants HL-31809 and AI-31896.

REFERENCES

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14. Lewis, R.A., Mencia-Huerta, J.M., Soberman, R.J., Hoover, D., Marfat, A., Corey, E.J., and Austen, K.F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7904-7908.

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19. Littlefield, J.W. (1964) Science 145, 709-710.

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Characteristics of the epitope of leukotriene B4 recognized by a highly specific mouse monoclonal antibody