A rapid enzyme-linked immunosorbent assay for human factor XI

THROMBOSIS RESEARCH 50; 329-334, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

BRIEF COMMUNICATION A RAPID ENZYME-LINKED

IMMUNOSORBENT

ASSAY FOR HUMAN FACTOR XI

Yutaka Komiyama, Hiroyuki Nishikado, Midori Masuda, Hiroshi Egawa, Norifumi Kobayashi", Shinzo Kobatake", Shuji Matsuura" and Kenjiro Murata Department of Clinico-laboratory Medicine, Kansai Medical University, Moriguchi, Osaka 570 and * Osaka Laboratory of Wako Pure Chemicals Co., Amagasaki, Hyogo 661, JAPAN

Accepted in revised form 3.2.1988 by Editor S. Okamoto)

(Received 27.11.1987;

INTRODUCTION Factor XI (F.XI) participates in an early phase of the intrinsic blood coagulation system (1). F.XI levels are measured generally by coagulant Two radioimmunoassays assay, but the precision falls in a high range (2). have been reported by Saito (3) and Scott (4). but they are not applicable to routine examinations.

F.XI

We report a rapid enzyme-linked immunosorbent assay (ELISA) for human using two anti-F.XI monoclonal antibodies (MCAs) (5). MATERIALS AND METHODS

Human F.XI was purified by immunoaffinity Purification of human F.XI. Fifty milligrams of anti-F.XI MCA (Z-l) chromatography of anti-F.XI MCA. (5) was coupled to 3 g of CNBr-activated Sepharose 48 (Pharmacia Fine ACD Chemicals) and equilibrated with sodium phosphate buffer (pH 7.3). plasma (400 ml) was applied on anti-F.XI MCA column (1.2 x 9 cm) and washed with 0.02X ammonia water containing 0.15 M NaCl and 0.1 M sodium acetate Then F.XI was eluted buffer (pH 4.5) containing 1.5 M NaCl, successively. with 4 M guanidine HCl in the acetate buffer and immediately deionized with This guanidine eluate was further purified with Blue Sephadex G-25. Sepharose CL-6B equilibrated with 40 mM Tris, 10 mM succinate buffer (pH 8.34) containing 1 mM EDTA, 1 mM benzamidine, 50 ug/ml polybrene and 0.02x The Purified F.XI (600 ug) was obtained from the void fraction. NaN3. purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6) and the specific coagulant activity was 196 unit/mg protein. Key words: Factor XI, enzyme-linked antibody.

immunosorbent

329

assay, monoclonal

RAPID ELISA FOR FACTOR XI

330

One

unit

F.XI

of

is defined

as Protein

normal pooled plasma. of Lowry et al. (7), Method

of

which

ELISA

for

recognize

All

described according

before to the

(5). method

peroxidase Bekelite)

(POD) coated

(9). with

procedures

were

carried

Anti-F.XI

in plasma.

of Parham The solid

et al. (8). and the phase was a 96-well

of

IgG

assayed

the

antigen

level

the F.XI molecule, (2-l) was digested

fraction

of

in a cold MCAs

of MCA

(100

(4-l) and blocked with 300 ul of 1% bovine coating buffer was 50 mM sodium bicarbonate We

out

epitopes Anti-F.XI

50 pl

50,

No.

that amount of activity present in 1.0 ml of concentration was determined by the method

F.XI

human

different

Vol.

F.XI

(2-l

were with

room. and

4-l),

produced pepsin

as

Fab' was labeled with microplate (Sumitomo

pg/ml)

of

anti-F.XI MCA The

serum albumin (BSA). buffer (pH 9.5). in plasma

(F.XI:Ag)

by

one-step

Fifty microliters of POD-labeled anti-F.XI MCA (2-l) ELISA, as follows. Fab' and 150 ul of sample plasma (1 in 20 dilution) or standard F.XI were aoolied to the well of a 96-well microolate and incubated at 37°C for one

h&r.

After

washing

sodium phosphate 7.3) containing POD activity on

with

0.1

M

buffer (pH 0.5% Tween 20, the plate was

assayed with orthophenylenediamine-H,O,. The dilution buffer was 0.1 M sodium phosphatebuffer containing 0.15 0.1%

BSA.

(pH 7.3) M NaCl and

Results

expressed

as

duplicated

1.5 E

mean

g z 1.0 I.0 aJ

were value

of

assay.

a

Plasma was Sample plasma. obtained from patients with liver cirrhosis (age range: 52-73, mean age=61), essential hvDertension 418-76,

mean

asthma age=43)

(age and

(age range: age=46) and (age range: female=59),

(age

=; v) n z

6

range:

age=63), bronchial range: 17-71, mean allergic rhinitis 12-65 healthy 15-79,

purified

F.X

1eve1 (Fg/ml )

, mean volunteers male=223,

FIG.

frozen at Healthy -90°C until use. volunteers of the aging study were selected by standard blood and urine tests which included blood count, glucose, liver and kidney function

and

Effect

of

F.XIa-a1AT

on

ELISA

for

Purified F.XI (2-6 pg/O.5 ml) mixed with F.XIa-cllAT (O:O, =:0.1, mzo.2, mr0.4 0.5

ml)

and

assayed

as

F.XI

was us/

described

in method.

test.

RESULTS Standard

0

curve

and

precision

of

AND ELISA

DISCUSSION for

human

F.XI.

Our

ELISA

for

F.XI

2

Vol. 50, No. 2

RAPID ELISA FOR FACTOR XI

in plasma can be completed within about one hour. As previously described, antiF.XI MCAs (2-l and 4-l) recognize both F.XI and its derivative, F.XIa-alantitrypsin complex (F.XIa-a,AT) Then we examined the (5). effect of F.XIa-oIAT on our assay system. As shown in Fig. 1, added F.XIa-o1AT (0.1-0.4 ug/ml plasma), did not increased the color development. Since F.XIaalAT level was not reached such high level (0.4 pg/ml plasma) even in the patients with DIC (10, 11). effect of F.XIa-aIAT on our ELISA system was negligible in routine sample.

331

F.XI:Ag level (pg/ml)

Fig. 2 shows the typical standard curve obtained using FIG. 2 purified F.XI, diluted with buffer. The same curve Standard curve of ELISA for human F.XI could also be obtained when purified F.XI was diluted Purified F.XI was diluted with buffer with buffer containing 5% alone (0) or 5% F.XI-deficient plasma F.XI-deficient plasma. in buffer (a) and assayed as described The recovery test of purified in method. F.XI (l-4 pg/ml plasma in the patient plasma (1.75-4.16 ug/ml plasma) was satisfactory Correlation (90- 110%). between this ELISA and routine coagulant assay was determined with use of the plasma of 8 patients with liver cirrhosis, 20 normal volunteers and 10 patients with essential hypertension (range of F.XI:Ag, 1.2-6.8 ug/ml Correlation coefficient was 0.94, and correlation equation of plasma). this ELISA to coagulant assay was Y=O.91Xt2.4 (n=38). Many investigators have Age variation of F.XI:Ag level in normal adults. The levels of reported age variations in coagulation factors (2, 12). fibrinogen and factor VIII vary with age, while those of factor X and As shown in Table 1, there was no significant difprothrombin do not. The normal level of F.XI:Ag was 4.lsO.54 ference among each age groups. ug/ml plasma. Plasma levels of contact factors are F.XI:Ag level in various diseases. known to differ among clinical conditions (DIC, liver cirrhosis) (13). Using our rapid ELISA, we screened the F.XI:Ag levels of patients with The F.XI:Ag level in patients with various diseases, as shown in Table 2. liver cirrhosis was lower than normal, but that in essential hypertension was higher.

Vol. 50, No. 2

RAPID ELISA FOR FACTOR XI

TABLE 1

F.XI:Ag

Level and Age of Healthy Adults

F.XI:Ag (ug/ml plasma)

age 15-19 20-29 30-39 40-49 50-59 60-69 70-79

(n=21) (n=59) (n=64) (n=39) (n=24) (n=67) (n=8)

Data are expressed evaluated

4.08kO.47 4.1520.59 4.1420.50 4.18kO.56 4.1220.43 4.1720.61 4.2420.43 as mean+S.D. by Student's

and statistically t-test.

TABLE 2 F.XI:Ag Levels of the Patients with Various Diseases

F.XI:Ag (ug/ml plasma) Normal level Liver cirrhosis Essential hypertension Bronchial asthma Allergic rhinitis

4.1520.54 2.29i0.58 5.1150.77 5.44+0.99 4.02k1.10

(n=282) (n==l6)** (n=20)** (n=37)= (n=34)

Data are expressed as mean&S.D. and statistically evaluated by Student's t-test. +M.. Significantly differ-ant from normal level (~~0.01)

Recently, Freyria et al. (14) reported that the kallikrein-kinin system potentiates contact system activity in the plasma of asthmatics. Therefore, we screened the F.XI:Ag lava1 in the patients with two allergic As shovn in Table 2, the F.XI:Ag level in the patients with diseases. bronchial asthma vas significantly higher than normal, whereas that in allergic rhinitis vas similar to the normal level. We developed a new rapid ELISA for human F.XI using two anti-F.XI MCAs, which rtcognixed different epitopes. In the clinical study, we demonstrated that F.XI:A is elevated in the patients with bronchial asthma. This rapid ELI sA for F.XI vi11 be a useful tool for screening of F.XI:Ag in various diseases, ACKNOWLEDGMENT This work was supported by Grant-in-Aid

for Encouragement

of Young

Vol. 50, No. 2

Scientists Culture.

RAPID ELISA FOR FACTOR XI

333

(61771957) from the Japanese Ministry of Education,

Science and

REFERENCES 1.

KURACHI, K. and DAVIE, E.R. Human factor XI. Methods Enzymol. 80, 211220, 1981.

2.

DODDS, W. J ., MOYNIHAN, A.C., BENSON, R.E. and HALL, C.A. The value of age- and sex-matched controls for coagulation studies. Br. J. Haemato1. 29, 305-317, 1975.

3.

SAITO, H. and GOLDSMITH, Jr. G.H. Plasma thromboplastin antecedent (PTA, factor XI): A specific and sensitive radioimmunoassay. Blood 50, 377-385, 1977.

4.

SCOTT, C.F.. SINHA, D., SEAMAN, F.S.. WALSH, P.N. and COLMAN, R.W. Amidolytic assay for human factor XI in plasma: Comparison with a coagulant assay and a new rapid radioimmunoassay. Blood 63. 42-50, 1984.

5.

NISHIKADO, H., KOMIYAMA, Y., MASUDA, M., EGAWA. H. and MURATA, K. Murine monoclonal antibodies to human factor XI. Thromb. Res. 42, 225-234. 1986.

6.

LAEMMLI, V. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, 1970.

7.

FARR, A.L. and RANDALL, R.J. Protein LOWRY, O.N., ROSENBROUGH. N.J., measurement with the folin phenol reagent. J. Biol. Chem. 193, 265276, 1951.

8.

PARHAM, P. On the fragmentation of monoclonal from BALB/c mice. J. Immunol. 131, 2895-2902,

9.

ISHIKAWA, E., YOSHITAKE, S., IMAGAWA, M. and SUMIYOSHI, A. Preparation of monomeric Fab'-horseradish peroxidase conjugate using thiol groups in the hinge and its evaluation in enzyme immunoassay and immunohistochemical staining. Ann. NY Acad. Sci. 240, 74-89, 1983.

10.

NISHIKADO, H., KOMIYAMA, Y., MASUDA, M., EGAWA, H. and MURATA, K. Factor XIa-olantitrypsin complex - Elevation in the patients with DIC -. Thromb. Res. 44, 489-501, 1986.

11.

NISHIKADO, H., KOMIYAMA, Y., MASUDA, M., EGAWA, H. and MURATA, K. Determination of main inhibitor of activated factor XI. Thromb. Res. 48, 145-151, 1987.

12.

MATSUDA T. Aging, blood coagulation, fibrinolysis and blood viscosity. Jap. J. Clin. Hematol. 28, 1085-1092, 1987 (in Japanese).

13.

SAITO, H. Contact factors in health and disease. Seminar Thromb. Hemostas. 13, 36-49, 1987.

14.

FREYRIA, A.M., LASSER, E.C., LYON, S.G. and

IgGl, IgG2a. and IgG2b 1983.

SIMON, R.A. Alpha 2-macro-

334

RAPID ELISA FOR FACTOR XI

Vol. 50, No. 2

globulin-kallikrein potentiates contact system activity: Possible effect in asthma. Int. Arch. Allergy Appl. Immunol. 83, 341-347, 1987.