Biochimica et Biophysica Acta, 1080(1991)34-39 © 1991ElsevierSciencePublishersB.V. All rightsreserved0167-4838/91/$03.50 ADONIS 016748389100300D

34

BBAPRO 33996

Presence of human immunoglobulin G anti serum pancreatic elastase 1 autoantibodies and their influence on elastase 1 radioimmunoassay H. Asada t, K. Shibata

l,

K. Uchida ~, A. Yamauchi 2, M. Kovo 2, y . Takeyama H. Ohyanagi 3 and Y. Saitoh 3

3,

t Shionogi Diagnostics Research Laboratories, Diagnostics Division, Shionogi & Co., Ltd., Mishima, Settsu-shi, Osaka (Japan), 2 Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka (Japan) and 3 The 1st Department of Surgery, Kobe UniL~rsity School of Medicine, Chuo-loA Kobe (Japan)

(Received 15 March 1991)

Keywords: Humanpancreaticelastase 1; Autoantibody;Radioimmunoassay;Affinityconstant Human immunoglobulin G (IgG) anti human pancreatic elastase 1 autoantibodies were detected in sera of patients with pancreatic disorders. The characteristics of these anti elastase 1 autoantibodies and their influence on radioimmunoassRy (RIA) for elastase 1 were investigated. They were placed in the IgG class by the double antibody method, and most were assumed to be of a monoclonal type from their elution profiles in gel filtration analysis. The presence of autoantibodies in serum caused an increase in apparent elastase I values and a decrease in the recovery of elastase 1 exogenously added to the serum. These results suggest that elastase 1 immunoassay data for autoantibody positive sera can cause misjudgement of clinical stages of patients.

Introduction Some enzymes are bound to T-globulin in human sera. In 1964, Wilding et al. [1] reported for the first time that amylase was bound to serum IgA in macroamylasemia. In 1967, Ganrot also reported that serum lactate debydrogenase (LDH) became linked to immunoglobulin in lupoid cirrhosis [2]. Subsequently, several investigators demonstrated that the immunoglobulins involved in LDH-immunoglobulin complexes mostly belong to IgA [2-5] and in a few cases to IgG [6,7]. In 1970, Hirata et al. [8] reported t.he first case of a patient with spontaneous hypoglycemia who had insulin-binding antibodies with no history of insulin injection. Serum elastase 1 increases in patients with pancre-

Abbreviaftons: BocTACK, t-butoxycarbonyltri-alanyl-chloromethyl ketone; LDH, lactate dehydrogenase;BSA, bovineserum albumin; PEG, pol,/(ethyleneglycol);a I AT-E1, at-antitrypsin-elastase1; a 2 MG-EI, a2-macrosIobulin-elastase 1; IgG, immunoglobulin;RIA, radioinununoa~say;DEAE, diethylaminoethyl. Correspondence:H. Asada, ShionogiDiagnosticsResearch Laboratories, DiagnosticsDivis;.on,Shionogi& Co., Ltd., 2-5-1, Mishima, Sensu-shi,Osaka 566, Japan.

atic tumors or pancreatitis; paiticularly, in the case of acute pancreatitis. It rapidly increases to abnormal levels, and this has been used to develop a radioimmunoassay for it to diagnose pancreatic diseases. However, we found autoantibodies to human pancreatic elastase 1 in the sera of some patients who suffered from acute pancreatitis or had ever been attacked by pancreatic diseases. In the present paper, we describe the characteristics of the autoantibodies and their influence on the elastase 1 radioimmunoassay. Materials and Methods Serum specimen

Serum specimens used in the present experiments were obtained from patients and healthy ¢olunteers. All of the sera were stored at - 2 0 ° C until use. H u m a n pancreatic elastase 1 a n d chemicals

Human pancreatic elastase 1 was kindly offered by Dr. N. Yoshida, Shionogi Research Laboratories (Osaka, Japan). Its purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis. All chemicals were of analytical grade, unless otherwise noted.

35

t-Butoxycarbonyl tri-alanyl-chloromethyl ketone (BocTACK) A peptide chloromethyl ketone serine proteinase inhibitor [9-11] was synthesized by Dr. K. Inouye, Shionogi Research Laboratories (Osaka, Japan) and was used as an inactivator for t2Sl-labelled elastase 1 or intact elastase 1. The influence of serum inhibitors, a~ antitrypsin (Sigma St. Louis, MO) and a 2 macroglobulin (Sigma), on the assay was avoided by using this inhibitor.

125I-lahelled human pancreatic elastase 1 Human pancreatic elastase 1 was isotope-labelled with nsI by the chloramine T method [12] with some modifications. To a solution of 10/~g of astase 1 in 50/zl of 0.5 M phosphate buffer (PB; pH 7.5) placed in a polypropylene tube were added 55.5 MBq of Na~eSI (Amersham, Buckinghamshire, U.K.; 37 M Bq/10 ~l) and 10 tti of chloramine T (2 m g / m l in 0.5 M PB). The mixture was shaken for 30 s at room temperature and the reaction was stopped by addition of 50 t~l of Na2S206 (2.5 m g / m l in 0.1 M PB) and 50/~1 of KI (50 m g / m l in distilled water). 10 t*l of 1% (w/v) bovine serum albumin (BSA; Sigma) in 0.1 M PB was then added to the reaction mixture and applied to a Sephadex G-25 (10 x 50 mm, Pharmacia, Uppsala, Sweden) column. The iodinated elastase 1 was eluted with 0.1 M PB pH 7.5 and collected by a minicollector. The enzyme activity of m2Sl-elastase 1 (3.7 MBq//~g) was then inactivated with BocTACK; ~2Sl-elastase 1 was diluted with 0.05 M phosphate-buffered saline (PBS) containing 0.2% (w/v) BSA, 0.05% (w/v) NaN 3 and 0.01% (w/v) BocTACK. After being left standing for 12 h at room temperature, the resulting solution of 12Sl-elastase 1 (1.5 n g / m l ) was used for radioimmunoassay.

Other 1251-labelled compounds al-Microglobulin (Toray Research Center, Kanagawa, Japan) and phospholipase A 2 (Shionogi Research Laboratories) were also labelled with 12sI by the chloramine T method as described above. The respective iodinated compounds were purified by gel filtration method (Sephadex G-25, 10 x 150 ram, Pharmaeia), followed by dilution with PBS containing 0.2% (w/v) BSA and 0.05% (w/v) NaN 3.

Anti-elastase 1 antibody The antibody for human pancreatic elastase 1 was prepared using rabbits. Intact elastase 1 (1.37 m g / m l , 100 /zl/rabbit) was emulsified in Freund's complete adjuvant (Sigma) and injected intra-dermaily to three adult male rabbits at multiple sites. Each rabbit was given an equal amount of elastase 1 in booster injections 3 times. Blood was collected from the central ear

artery and the sera were stored at - 8 0 o C. Antibody titers were determined by radioimmunoassay. The cross-reactivity of the antiserum (F-137) thus obtained was tested by immunoelectrophoresis according to the method of Yagi et al. [13]. Electrophoresis and precipitation were carried out on a 0.8-mm thin agar gel (Ciba Coming Diagnostics, Palo Alto, CA, U.S.A.). A single precipitin line was formed with intact elastase 1 in the ~2 ~ Y region and another with an unidentified component of normal human serum in the /3t ~/32 region. To find the effect of the unknowri component on tbt; rndioimmunoassay, the F-137 antiserum was preincubated with an equal volume of normal human serum at 4 ° C for 24 h and applied to radioimmunoassay; the component did not interfere with the radioimmunoassay.

Goat anti-rabbit immunoglobulin Goat anti rabbit immunoglobulins f't,:ed on i m munobeads were obtained from Bio-Rad (CA, U.S.A., Control No. 70718).

Radioimmunoassay for pancreatic elastase 1 The reaction was carried out in disposable polypropylene tubes (Shionogi, Osaka Japan). To obtain tile standard curve of elastase 1, BocTACK-treated elastase 1 was dissolved in 80% (v/v) fetal calf serum (Flow Laboratories, North Ryde, Australia) solution (0.05 M PBS, pH 7.4, 0.05% (w/v) NaN 3) at concentrations of 0.5 to 50 ng/ml. Antiserum and t2Sl-labelled elastase 1 were adequately diluted with 0.2% (w/v) BSA solution (0.05 M PBS, pH 7.4, 0.05% (w/v) NAN3). 100 t~l of the serum sample or standard elastase 1 and the ~zSI-labelled elastase 1 were placed in a tube and to which was added the antiserum (1 × 10S-fold diluted, 100/Ll). After being shaken for a while, the tube was warmed at 37 °C for 3 h and the goat anti rabbit IgG antibody-fixed immunobeads were added (100/zg/500 /~i). The mixture was warmed at 37 ° C for 30 rain and then centrifuged at 3000 rpm for 10 rain. After removal of the supernatant, the radioactivity of the precipitates was counted with an Aloka ARC-600 gamma counter. For comparison, a commercially available radioimmunoassay kit for serum elastase 1 (Dainabot Co., Tokyo, Japan) was used.

Recovery of elastase 1 To demonstrate the presence of the autoantibodies in st'urn, the recovery of elastase 1 was examined. Standard elastase 1 was dissolved in the test serum at two different concentrations and the elastase 1 concentrations were determined by the procedures described above.

High performance liquid chromatography (HPLC) HPLC was performed with a Shimadzu HPLC system; Rheodyne Model 7162 loop injector (Rheodyne,

36 CA, U.S.A.), two model LC-9A pumps, gradient programmer SCL-6B and FRAC-100 fraction collector (Pharmacia). An adequate volume of the sample was injected onto a Superose 12 column (10 × 300 mm, Pharmacia) and eluted with a 0.05 M PBS (pH 7.5) at a flow rate of 1 ml/min. The radioactivity of each eluate (0.25 ml) was counted by an Aloka ARC-600 gamma counter.

Detection of autoantibodies in serum The autoantibodies in serum were detected by HPLC. A mixture of 100 /~1 of the serum and l ~ l elastase 1 solution (0.74 KBq/0.2 ng per 100/~1) was incubated at 3 7 " C for 1 h, then loaded on a Superose 12 column (Pharmacia) and eluted with 0.05 M PBS (pH 7.5). The radioactivity of the eluates (each, 0.25 ml) was counted.

Binding specificity of autoantibodies The binding specificity of the autoantibodies was examined by the inhibition analysis as described in the above section. Briefly, a serially diluted testing inhibitor ( a I microglobulin, pancreatic secretory trypsin inhibitor (Shionogi Research Laboratories), phospholipase A2, trypsin (Sigma) or elastase 1 itself) was added to the mixture of t~I-elastase 1 and the adequately diluted scram. The mixture was incubated at 37 °C for 12 h and the B / F separation was performed by the PEG method as described above. Results

The presence of the autoantibodies in serum was tentatively defined by low recoveries of the elastase 1 added to the test serum. Some typical cases are given

Purification of 7-globulin in serum

TABLE 1

The autoantibody positive serum (0.5 ml) was diluted 3-fold with distilled water and subjected to DEAE-Sepharose anion exchange chromatography (10 × 30 mm, Pharmacia) according to the procedure of Nadkarni et al. [14]. The purity of the ,,,-globulin thus obtained was confirmed by Titan agarose gel electrophoresis (Helena Laboratories, Beaumont, TX, U.S.A.).

Recovery o f elastase 1 added to the sera tested

Determination of y-globulin class The immunoglobulin class of the autoantibodies was determined by the double antibody method [15]. A mixture of 100 ~.! of the serum and m~l-elastase 1 (0.74 KBq/100/zl) was incubated at 4 ° C for 12 h, to which was then added 2.5 mg of anti-human IgG specific mouse monoelonal antibody 2-25 (5 m g / m l in 0.05 M PBS, pH 7.5) [16] and further incubated at 37 ° C for 1 hr. The binding of the mouse monoclonal antibody to the elastase 1-autoantibody complex was examined by gel filtration analysis.

Cases 1-6 were autoantibody negative and 7-10 were positive. No.

1

2

3

4

5

6

Characterization of anti-elastase 1 autoantibodies The binding affinity and the serum level of the autoantibodies were determined by Scatchard analysis [17]. To a mixture of 50/zl of adequately diluted serum and tZSl-elastase 1 (0.74 KBq/0.144 ng/100 /zl) was added a serially diluted BoeTACK-treated elastase 1. The mixture was incubated at 3 7 " C for 12 h, to which was then added 1000 /zl of 0.05 M PBS (pH 7.4) containing 14% (w/v) poly(ethylene glycol) (PEG) 6000 (Sigma). The bound ~zSI-elastase 1 was separated by centrifugation (3000 x g, 20 min). After removal of the supernatant the remaining radioactivity of the precipitates was counted with an Aloka ARC-600 type gamma counter.

Elastase 1 added (ng/ml)

7

8

9

10

Recovery found (ng/ml)

(%)

0 2.5 7.5

0.98 3.74 9.75

107 115

0 2.5 7.5

1.63 3.95 9.59

96 105

0 2.5 7.5

1.19 4.01 9.72

109 112

0 2.5 7.5

1.22 3.77 8.20

1Ol 94

0 2.5 7.5

2.56 5.36 11.24

106 112

0 2.:; 7.5

2.09 4.66 9.71

102 ;~2

0 2.5 7.5

3.75 4.79 7.65

42 52

0 2.5 7.5

6.01 6.45 6.87

18 11

0 2,5 7.5

9.26 9.99 11.19

29 26

0 2.5 7.5

9.17 10.44 13.17

51 53

37 alAT-E1 T BocTACK-E 1

a2MG-E1

1

5

!:

7 o

i°

1 ,~-:~:- , 5

5

7,5

10

12.5

15

17.5

l~'lufion volume (rul)

Fig. i. Gel filtration analysis of serum preincubated with I~I-EI. (e) autoantibody positive serum; ( • ) negative serum. Markers ( ~) in the figure indicate the fractions where the standard elastase I or its conjugates should be eluted. El; elastase 1.

in Table I. Cases 1-6 were autoantibody negative, but cases 7 - 1 0 positive. Actually, 34 out of 320 cases were positive. Fig. 1 shows Superose 12 column elutton profiles of ~:SI-elastase 1 preincubated with the autoantibody positive and negative human sera. ~25I-elastase 1 in the autoantibody negative serum was mainly eluted in the same fractions as the standard elastase 1 (BocTACK-treated elastase 1), but the radioactivity in the positive serum was mainly found in larger molecular fractions, between the az-macroglobulin-elastase 1 complex ( a 2 MG-E1) and *.he at-antitrypsin-elastase 1 complex ( a 1 AT-E1) fractio~,~. When the autoantibody-elastase 1 complex was incubated at p H 3.0 (0.2 M citrate buffer) for 10 rain at

=1.6×109M

,

10

12.5

15 17.5 Elutton volume (rot)

room temperature, the radioactivity of the complex completely disappeared and was recovered in the standard elastase 1 fractions (data not shown). The immunoglobulin class of the autoantibodies was identified for three samples of antibody positive sera by means of the double antibody method. As shown in Fig. 2, most of the radioactivity of ~zSI-elastase 1 incubated with the mouse monoclonal antibody 2-25 was eluted in the void volume. Since this monoclonal antibody specifically binds to human immunoglobulin G and shows no cross reaction with the other class of immunoglobulins, we concluded that the autoantibodB

°'t

0.5. : Ka

J•

Fig. 2. Determination of IgG class by a double antibody method. Reactivityof 12Sl-elastase 1-autoantibody immune complex to murine antibody against human IgG was confirmed by gel filtration analysis. Autoantibody positive serum was preincubated with ~ZSl-elastase 1 and further incubated with routine anti human lgG antibody. The symbol ( • ) means 'before incubation with the murine antibody' and (e), 'after'.

A

.

.

7,5

-f

Ka=54X10sM

0.4-

0.3-

¢.. 4p

~ 0.3-

c @

4) O U.

.o 0.~-

~c 0.2::1

\

0 m

,s

0.1. 0.t-

0 1

2

3

4

5

6 7 Bound antigen ( x 10- ~0M)

,

\ ,

,

\

i

+

3 4 5 6 7 Bound antigen ( x l 0 tOM)

Fig. 3. Scatchard plots for autoantibodies occurring in two serum samples (A, serum No. 2; B, serum No. 4).

38 TABLE II

Discussion

Affinity constants and serum let'els of six species of autoantibodies

Serum No. 1 2 3 4 5

AffinityConst. (I/mol) 2.0" l0 s 1.6"109 4.5" l0 s 2.1 • 10'~ 5.4" 10s 3.3" 10I°

Amount(ng/ml) I 500 1000 2300 7800 1900 120

ies belonged to the IgG class. The binding affinity and the serum content of the autoantibodies were determined by Scatchard analysis (Fig. 3). The plots of three other autoantibodies were nearly the same as shown in Fig. 3B. The range of the affinity constant of each autoantibody to elastase 1 was 2.0" 108 to 3.3" 10 I° ( l / m o l ) from which the serum content of each autoantibody was calculated to be 120 to 7800 n g / m l assuming that one molecule of the autoantibody might bind to one molecule of elastase 1 (Table II). Serum No. 2 showed two binding affinities, high (1.6.109) and tow (4.5 • 108), suggesting that two different autoantibodies had been formed (Fig. 3A). The inhibition analysis showed that the binding of the autoantibodies to elastase 1 was highly specific, it was hardly inhibited by other serum components other than elastase 1 (data not shown). This was also confirmed by HPLC. When the autoantibody positive serum was incubated with I~I-labelled pancreatic phospholipase A 2 or aj-microglobulin, no shift of the radio-labelled materials occurred in the gel filtration pattern, suggesting that the autoantibodies were specific to pancreatic elastase 1 (data not shown). Subsequently, the influence of the autoantibody on radioimmunoassay of serum elastase 1 was investigated. When y-globulin fraction after D E A E - S e p h arose anion-exchange chromatography (purified autoantibodies) was added to autoantibody-negative sera, the apparent values were markedly elevated (Table III). A similar elevation was noted when the commercially available kit was used.

Several papers have been reported that the presence of autoantibody in serum will result in elevation of the actual values of antigens in immunoassays [18,19]. Recently, we developed a new radioimmunoassay method for human pancreatic elastase 1, basically according to the method established by Ooyama et ai. [20] and found the prcsence of pancreatic elastase 1 autoantibodies in the serum. The autoantibody was dominant in sera showing high elastase 1 values; it was detected in 27 out of 142 sera with values over 384 n g / d i [21], but in only 7 out of 178 sera with values under this. No autoantibody was detected in 69 healthy volunteers with no history of pancreatic diseases. The occurrence of anti-elastase 1 autoantibody seems to be more frequent than that of other kinds of human enzymes; the occurrence of the anti-creatme l,:ina~e antibody being from 0.23 to 0.61%, that of anti-LDH from 0.32% to 0.15%, that of anti-alkaline phosphatase from 0.26% to 0.03% and that of amylase from 0.18% to 0.04% [22]. Autoantibodies of five different origins were used to form the immune complex with 125I-elastase 1, but there was no remarkable difference in the elution profile from a gel filtration column. The peak of the immune complex was a sharp singlet (Fig. 1), while the immune complex formed with a polyelonal rabbit antibody was broad and had shifted to a higher molecular area. This result suggests that the autoantibodies for elastase 1 are of the monoclonal type like that for insulin [23,24]. The binding affinities of these autoantibodies from five origins were evaluated in a liquid phase and all, except one, had single affinity constants. The exception, serum No. 2 in Table II indicated the presence of two different types of autoantibody. Compared to the affinity (4.5.109 l / m o l ) and working concentration (5 n g / m l ) of polyclonal rabbit antl-elastase I antibody all the autoantibodies in Table II seemed to have similar or higher affinities to elastase 1 and that the serum levels were quite high. These results show that interfer-

TABLE Ill Influence of anti-elastase 1 autoantibodies on RIAs for elastase 1

100 ~g of partially purified autoantibody (anti El) was added to five sera (autoantibody-negative) and changes of the elastase t values were summarized Serum No.

RIA in our laboratory

1 2 3 4 5

anti E1 ( (ng/ml) 4.36 4.49 5.30 3.86 2.48

)

anti El ( + (ng/ml) 8.53 9.38 11.05 8.99 7.07

Dainabot kit )

%

change

196 209 208 233 285

anti E1 ( (ng/ml) 3.76 4.27 5.08 4.25 2.79

)

anti El ( + (ng/ml) 6.45 6.89 7.43 6.78 5.36

)

%

change

172 161 146 160 192

39 e n c e from t h e a u t o a n t i b o d y p r e s e n t in t h e s e r u m can elevate t h e e l a s t a s e 1 values o b t a i n e d by radioimm u n o a s s a y a n d d e c r e a s e t h e recovery o f elastase 1 a d d e d to t h e s e r u m b e c a u s e o f c o m p e t i t i v e b i n d i n g o f the autoantibody. In this r e p o r t , we have d e s c r i b e d t h e c h a r a c t e r i s t i c s o f a u t o a n t i b o d i e s a n d t h e i r influence o n t h e radioimm u n o a s s a y for elastase 1. All five a u t o a n t i b o d i e s t e s t e d h e r e b e l o n g to t h e l g G class a n d have q u i t e high affinity a n d specificity for e l a s t a s e 1. Since t h e elastase 1 a u t o a n t i b o d y i n t e r f e r e s with t h e r a d i o i m m u n o a s s a y s y s t e m for e l a s t a s e 1, d e t e r m i n a t i o n s s h o u l d b e p e r f o r m e d carefully, especially with a u t o a n t i b o d y - p o s i t i v e sera.

Acknowledgements W e w o u l d like to t h a n k Dr. K. l n o u y e for t h e synthesis o f B o c T A C K , Dr. N. Y o s h i d a for t h e g e n e r ous gift o f h u m a n e l a s t a s e 1 a n d Mr, K. E n d o h for his precious information.

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5 Biewenga, J. and F~itkamp, T.E.W. (1975) Clin. Chim. Acta. 58, 239-249. 6 Biewenga. J. (1973'~ Clin. Chim. Acta. 47, 139-147. 7 Kindmark, C.O. (1960) Scand. J. clin. Lab. Invest. 24, 49-53. 8 Hirata, Y.. lshizu, H,, Ouchi, N., Motomura, S., Abe, M., Hara, Y., Wakasugi. H., Takahashi. I., Sakano, H., Tanaka, M., Kawano, H. and Kanesaki, T. (1970)J. Jap. Diabetic Soc. 13, 312-320. 9 Ardelt, W., Koj, A., Chudzik, J. and Dubin, A. (1976) FEBS Left. 67, 156-160. 10 Tuhy, P.M. and Powers, J.C. (1975) FEBS Left. 50, 359-361. 11 Dimicoli, J.-L., Renaud, A., Lestienne. P. and Bieth, J.G. (1979) J. Biol. Chem. 25, 5208-5218. 12 Hunter, W.M. and Greenwood, F.C. (1962) Nature 194, 495-4%. 13 Yagi, Y., Maier. P.K. and Pressman, D. (1%2) J. lmmunol. 89, 736-744. 14 Nadkarni, G.D. and Jeejeebhoy, K.N. (1%8) Indian J. Biochem. 5, 16-18. 15 Kajita, Y., Majima, T., Yoshimura, M., Hachiya, T.. Miyazaki, T., ljichi, H. and Oehi, Y. (1978)Clin. Chim. Acta 89, 485-492. 16 Hosoi, S., Shinomiya, K., Nakano, H., Mikawa, H. and Harada, S. (1985) J. Allergy Clin. Immunol. 75, 320-327. 17 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660-672. 18 Armitage, M., Wilkin, T., Wood, P., Casey, C. and Loveless, R. (1988) Diabetes 37, 1392-13%. 19 Paganelli, R., Quinti, !., D'Offizi, G.P., Papetti, C., Nisini, R. and Aiuti, F. (1988) J. Clin. Lab. lmmunol. 26, 153-157. 20 Ooyama. T., Kawamura, K., Orimo, H. and Murakami, M. (1978) lgaku-no-ayumi 105, 91-97. 21 Buchler, M., Malfertheiner, P., Uhl, W. and Beget, H.G. (1986) Klin Wochenschr 64, 1186-1191. 22 Tozawa, T. (1989) Electrophoresis 10. 640-644. 23 Hirata, Y.. Tominaga, M., Itoh, J. and Noguchi, A. (1974) Ann. intern. Med. 81,214-218. 24 Uchigata, Y., Yap, K., Takayama-Hasumi, S. and Hirata, Y. (1989) Diabetes 38. 663-666.