/ . Biochem. 86, 943-949 (1979)

Enzyme Immunoassay of Pancreatic Glucagon at the Picogram Level Using /3-D-Galactosidase as a Label Susumu IWASA,* Hayao UENO,* Tsutomu MIYA,* Mitsuhiro WAKIMASU,* Koichi KONDO,* and Akira OHNEDA** •Chemical Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., Jusohonmachi, Yodogawa-ku, Osaka 532, and "the Third Medical Department, Tohoku University School of Medicine, Sendai, Miyagi 980 Received for publication, March 19, 1979

An enzyme immunoassay of pancreatic glucagon was established by using E. coli fi-Dgalactosidase [EC 3.2.1.23] as a marker. In order to increase the sensitivity of the immunoassay, different peptides obtained from glucagon fragments were used to produce the enzyme conjugate and the immunogen. Antiserum N6E raised against C-terminal fragment peptide (15-29) could be diluted to more than 1 : 100,000 in the assay and was highly specific for pancreatic glucagon. The antiserum reacted well with the C-terminal fragment peptide (21-29) as well as another fragment peptide (15-29) and pancreatic glucagon. The enzyme immunoassay using antiserum N6E and fragment peptide (21-29)-enzyme conjugate could detect as little as 1 to 2 pg of glucagon. The mean recovery of glucagon added to serum specimens was 104% and the coefficients of variation were 3.7-14.5% (within assay) and 9.0-18.5% (between assay).

Many reports have appeared in recent years on the radio-immunological determination of glucagon in plasma (1-6), but few papers have dealt with the enzyme immunoassay (EIA) of glucagon (7). The EIA method was inferior to the established radioimmunoassay (R1A) method as regards both sensitivity and precision. Van Weemen and Schuurs reported that the Abbreviations: EIA, enzyme immunoassay; RIA, radioimmunoassay; PG, pancreatic glucagon; GLI, glucagon-like immunoreactive substances; BSA, bovine serum albumin; MBS, /ne/a-maleimidobenzoyl Nhydroxysuccinimide ester; RIgG, rabbit immunoglobulin G. Vol. 86/. No. 4, 1979

943

sensitivity of estrogen EIA could be increased when the estrogen derivatives used to produce the enzyme conjugate and the immunogen differed slightly (8, 9). The present work was undertaken to develop a sensitive method for determining pancreatic glucagon (PG) by EIA employing /3-r>galactosidase as a label. In these studies, different peptides obtained from glucagon C-terminal fragments were used to produce the enzyme conjugate and the immunogen in order to increase the sensitivity of PG EIA. Such a sensitive EIA method was expected to be as useful as the RIA technique for studies of the secretory mechanism of this hormone.

944

S. IWASA, H. UENO, T. MIYA, M. WAKIMASU, K. KONDO, and A. OHNEDA

II to VII were prepared by the conventional solution method. The synthetic methodology and MATERIALS AND METHODS purification procedures have been described preMaterials—Seven different peptides were used viously (10). The analytical data for the products are shown in Table I together with their structures. in the present study. Gut glucagon extract (gut glucagon-like I: Peptide pancreatic glucagon (1-29) Peptide II: synthetic C-terminal glucagon immunoreactive substances: gut GLI) from canine jejunum and antiserum R517 reacting with gut fragment (15-29) Peptide HI: synthetic C-terminal glucagon GLI as well as PG were gifts from Dr. H. Sakurai, fragment (15-29) containing Kyoto University (11). Antiserum 30K specific norleucine instead of methio- for PG was obtained from the Diabetes Research nine at the third position from Fund, South Western Medical School at Dallas, Texas (12). Antisera N2C and N6E were prepared the C-terminus Peptide IV: synthetic C-terminal glucagon by immunizing rabbits with peptide II conjugated with bovine serum albumin (BSA) by means of fragment (21-29) Peptide V: synthetic C-terminal glucagon glutaraldehyde (13). Antiserum N14B was prepared by using peptide VII instead of peptide IL fragment (22-29) These peptide-BSA conjugates were emulsified with Peptide VI: synthetic C-terminal glucagon an equal volume of Freund's complete adjuvant fragment (23-29) Peptide VU: synthetic N-terminal glucagon and then injected at multiple subcutaneous sites along the dorsal surface. fragment (1-10) Purified porcine glucagon (peptide 1) was purchased Preparation of Peptide-Enzyme Conjugate— from Sigma Chemical Co., St. Louis, Mo. Peptides PG and its fragment peptides were labeled with TABLE I. Analytical data for glucagon fragment peptides. Pancreatic glucagon (peptide I, 1-29) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-TrpLeu-Met-Asn-Thr Peptide H (15-29): la]o = -30.0° (c~0.3 in 50% AcOH); Rf 0.59" amino acid analysis: Arg 2.15; Trp 0.91; Asp 3.13; Thr 0.99; Ser 0.87; Glu 2.20; Ala 1.05; Val 0.96; Met 1.00; Leu 1.07; Phe 1.07 (79%)" Peptide m (15-29): [ a ]g=-3O.9° (c=0.3 in 50% AcOH); Rf 0.60 amino acid analysis: Arg 2.11; Trp 0.97; Asp 3.40; Thr 0.93; Ser 0.83; Gin 2.10; Ala 1.00; Val 1.04; Leu 0.91; Phe 0.84; Nle 1.06 (74%) Peptide IV (21-29): [a]§=-26.0° (c=0.2 in 80% AcOH); Rf 0.76 amino acid analysis: Trp 1.07; Asp 2.06; Thr 0.98; Glu 1.06; Val 0.97; Met 1.00; Leu 1.04; Phe 1.11 (73%) Peptide V (22-29): [a]^=-23.5° (c=0.5 in 80% AcOH); Rf 0.83 amino acid analysis: Trp 0.93; Asp 1.09; Thr 0.94; Glu 1.00; Val 0.90; Met 1.01; Leu 1.00; Phe 0.96 (81%) Peptide VI (23-29): [a]^=-15.9° (c=0.5 in 80% AcOH); R, 0.70 amino acid analysis: Trp 1.04; Asp 1.00; Thr 0.91; Glu 0.99; Val 1.07; Met 0.99; Leu 1.00 (78%) Peptide VH (1-10): [a]g=-35.0° (c=0.5 in 1% AcOH); Rf 0.54 amino acid analysis: His 0.92; Asp 0.93; Thr ZOO; Ser 1.80; Glu 1.04; Gly 1.00; Tyr 0.96; Phe 0.99 (84%) • Thin layer (cellulose) chromatography with n-butanol : pyridine : acetic acid : water (15 : 10 : 3 : 12 by volume). b Average recovery. / . Biochcm.

945

GLUCAGON ENZYME IMMUNOASSAY jS-D-galactosidase [EC 3.2.1.23] (Boehringer Mannheim, Ltd., West Germany) using mem-maleimidobenzoyl TV-hydroxysuccinimide ester (MBS) as a coupling reagent, as described elsewhere (14). Briefly, PG or the fragment peptide was mixed with MBS in tetrahydrofuran, then the MBSacylated peptide obtained was incubated with the enzyme. Next, the reaction mixture was directly chromatographed on a Sepharose 6B column using 0.02 M phosphate buffer (pH 7.0) containing 0.1 M NaCl, 1 DIM MgCl,, 0.1% BSA, and 0.1% NaN, '(buffer A) as an eluent. The fractions of eluate containing the peak of enzyme activity were used for glucagon assay. Preparation of Insolubilized Goat Anti-Rabbit Immunoglobulin G (RIgG)—IgG fractions of antiRlgG serum (Miles Lab. Inc., Kankakee, El.) were prepared by a combination of Na,SO4 precipitation and DEAE-cellulose column chromatography, and coupled to microcrystalline cellulose activated with CNBr (IS). The immunoadsorbents were suspended at a concentration of 5 % and used for assays. Assay Procedures—Enzyme immunoassays (EIA) of PG were performed by the doubleantibody solid phase method (16, 17). As an assay buffer, 0.02 M phosphate saline (pH 7.3) containing 0.5 % human serum albumin and 0.5 % EDTA (buffer B) was used. A 50 pt\ sample was allowed to stand at 4°C for 3 days with 200 p\ of buffer B, 50 fi\ (500 U) of Aprotinin (Antagosan®, Hoechst Co., West Germany), 100 fi\ of antiserum, and 100 //I of the peptide-enzyme conjugate containing about 0.2 fiU of /9-r>galactosidase activity (25°C; lactose as a substrate) (IS). The antiserum was diluted until it could bind approximately 30% of the added enzyme activity. Next, 100 /il of the anti-RIgG antibody-coupled cellulose suspension was added and the mixture was shaken at 30°C for another 3 h. After three washings with buffer A, the solid phase was resuspended in 500^1 of the substrate reagent containing 10 fig of 4-methyl-umbelliferyl-/3-D-galactoside (P-L Biochemicals Inc., Milwaukee, Wis.) as a substrate (19) and incubated overnight at room temperature. The reaction was terminated by adding 3 ml of 0.1 M carbonate buffer (pH 10.5) and the suspension was centrifuged. Thefluorescenceintensity of the supernatant was measured using a fluorometer with excitation at 365 nm and emission at 450 nm.

Vol. 86, No. 4, 1979

RESULTS Specificity of Antisera against Glucagon Fragment—Two types of antiserum were used in EIA for the determination of PG: (1) Antisera 30K and R517 raised against peptide I (2) Antisera N2C and N6E raised against peptide II The specificities of antisera N2C and N6E were studied in EIA employing peptide IV-enzyme conjugate. Figure 1 shows the reactivity of N6E with various C-terminal peptide fragments and gut GLI. This antiserum bound to peptides I, II, EH, and IV much more strongly than to peptides V and VL It did not cross-react with gut GLI and it was thus considered to be as PG-specific as Unger's 30K antiserum. Antiserum N2C gave similar results, although they are not shown in Fig. 1. Effect of Combinations of Antiserum and Enzyme-Labeled Peptide on the Sensitivity of PG

PG

GF

0.03

0.13 OS 10 GLI (ug/tub*)

Fig. 1. Inhibition-binding curves of the enzyme-labeled peptide IV with standard pancreatic glucagon (PG), C-terminal glucagon fragments (GF; described in Table I) and gut glucagon-like immunoreactive substances (GLI) in the enzyme immunoassay using antiserum N6E. PG (1-29, peptide I) (O), GF (15-29, peptide II) (•), GF (15-29, peptide III) (•), GF (21-29, peptide IV) (x), GF (22-29, peptide V) (A), GF (23-29, peptide VI) (A), and gut GLI (3). B,: Enzyme activity bound to the solid phase without added PG, GF, or gut GLL B: Enzyme activity bound to the solid phase with added PG, GF, or gut GLI.

946

S. IWASA, H. UENO, T. M1YA, M. WAKIMASU, K. K O N D O , and A. OHNEDA

TABLE n . Midpoints for pancreatic glucagon (in ng/ml) in enzyme immunoassays using various combinations of antiserum and peptide-enzyme conjugates. Peptide in immunogen'

Antissrurri

30K

R517

N2C

N6E

Final dilution factor of antiserum

Peptide in enzyme conjugate*

40,000

I

12,000 1,600

n m

1,600

IV

32,000

i

6,000 1,600

n m

1,600

IV

60,000

i

200,000 60,000

n m

200,000

IV

40,000

i

200,000 100,000

n m

200,000

IV

Sensitivity midpoint (ng/ml)

b

1.1 0.20

1.8 0.35

0.88 9.2 2.9 0.85 0.60 2.2 3.4 0.53

» Described in " MATERIALS AND METHODS." » Described in " RESULTS." « The binding of the peptideenzyme conjugate to antibody was too weak for the detection of enzyme activity bound to the solid phase. EIA—Peptides I to IV were each used as a peptide in the enzyme conjugate. Table n shows the effect of each combination of antiserum and enzyme-labeled peptide on the sensitivity of PG EIA. Sensitivity is expressed as the midpoint, i.e., the concentration of PG which reduces the binding of the peptide-enzyme conjugate to antibody to half the value obtained in the absence of free PG, and the results presented here represent average values obtained from assays carried out in duplicate. Antisera 30K and R517 gave acceptable sensitivity in two combinations, but the use of the antisera with the peptide Il-enzyme conjugate resulted in about six times higher sensitivity than the conventional use of the antisera with peptide I-enzyme conjugate, despite the fact that more antibody was required to obtain the same degree of binding of the peptide-enzyme conjugate. Antisera 30K and R517 had such weak immune reactivity against peptide HI- or rV-enzyme conjugate that the enzyme activity bound to the antisera was undetectable. The assays using N2C and N6E show that peptide I

or IV is preferable as a peptide in the enzyme conjugate. In the N2C assay system, the peptide I- or IV-enzyme conjugate gave ten times more sensitivity than the peptide Il-enzyme conjugate. Curves obtained using 30K with peptide Il-enzyme conjugate and N6E with peptide IV-enzyme conjugate were steeper than those obtained using 30K with peptide I-enzyme conjugate and N6E with peptide Il-enzyme conjugate, particularly in the low concentration region of PG, where steeper curves result in increased sensitivity and precision (Fig. 2). The detection limit of the assays using 30K with the peptide Il-enzyme conjugate and N6E with the peptide IV-enzyme conjugate was 20 pg/ml and 40 pg/ml, respectively. Sample Dilution Curves and Measurement of Glucagon Added to Human Sera—Plasma specimens from rats which received intravenous arginine infusion at a dose of 1 g/kg body weight for 30 min were serially diluted 1 :1 to 1 : 16 with buffer B and assayed for PG by EIA using the combination of antiserum N6E and peptide IV-enzyme conjugate. As shown in Fig. 3, the curves for standard J.

Biochem.

GLUCAGON ENZYME IMMUNOASSAY

947 TABLE HI. Recovery of pancreatic glucagon added to human sera.»

'••••. '"""^rxx ••. •-. X *

80

60

• • • - .

Recovered

' * • •

• .

\ N

\

no.

(Pg/ml)

1

40 x

^



20

0.06

Q25

PG

1.0

4.0

(ng/ml)

2

Fig. 2. Comparison of various combinations of antiserum and peptide-enzyme conjugates. In the 30K assay system, peptide I (O) or II ( • ) was used as a peptide in the enzyme conjugate, and in the N6E assay system, peptide II ( A ) or IV ( A ) was used. Peptides I, n , and IV are described in Table I. B, and B are described in Fig. 1.

"X

90 60

i/i

1/16

1/4

0

Sampla dilut on factor

0.06

0.25 P G

1.0





58 139 268 508 1,218

92 111 107 102 122



95 168 251 330 570 1,100





73 156 235 475 1,005

116 125 94 95 101





92 130 190 488 768

146 104 76 98 77



102 194 232 292 590 870

•4.0

(ng/ml )

Fig. 3. Standard curve for pancreatic glucagon (PG) (O) and curves for serially diluted plasma specimens ( • ) measured by the enzyme immunoassay using antiserum N6E and peptide IV-cnzyme conjugate (described in " RESULTS ").

PG and for the serially diluted plasma were parallel in specimens containing different levels of PG. Various amounts of PG were added to human sera to give final concentrations of 63-1,000 pg/ml and the mixtures were assayed for PG by EIA using the combination of antiserum N6E and peptide IV-enzyme conjugate. The recovery of PG added ranged from 76 to 146% with an average of about 104% for 15 determinations (Table HI). Vol. 86, No. 4, 1979

62 120 201 330 570 1,280

(%)

» Mean±S.D.=104±17%. •> Each sample was assayed for glucagon by EIA using antiserum N6E at a final dilution of 1:200,000 with peptide IV-enzyme conjugate. Assay procedures were as described in " MATERIALS AND METHODS."

Ov

30

1/4

— 63 125 250 500 1,000

63 125 250 500 1,000

\

1/W

(pg/ml)

63 125 250 500 1,000 3

20

(pg/ml)

Coefficients of Variation in the EIA—In order to investigate the reproducibility and precision of EIA using the combination of antiserum N6E and peptide IV-enzyme conjugate, the coefficients of variation were measured at six different PG levels. They were 3.7-14.5 % (within assay) and 9.0-18.5 % (between assay). These results show that the immunoassay is as reproducible and precise as RIA. DISCUSSION The development of a highly sensitive EIA method for measuring peptide hormones such as glucagon at the femtomole level requires an enzyme with a high specific activity, and thus ^-D-galactosidase

948

S. IWASA, H. UENO, T. MIYA, M. WAKIMASU, K. KONDO, and A. OHNEDA

from E. coli has been used to label glucagon or its fragment peptides (20). However, even when this enzyme was used in glucagon EIA, the conventional method was not as sensitive as RIA (7). We used various combinations of antibody and peptide-enzyme conjugates, and found that the sensitivity could be increased by using peptide El-enzyme conjugate in an anti-PG system and peptide IV-enzyme conjugate in an anti-peptide II system. These combinations improved the sensitivity of glucagon EIA to such an extent that the technique is now comparable to RIA. Such combinations may reduce the affinity of the antibody for the peptide-enzyme conjugate and thus give free glucagon at very low concentrations a better chance to compete. The antisera obtained by immunizing peptide II were specific for PG, probably because the N-terminal and middle portions of the PG molecule contain the antigenic sites shared by gut GLI, as suggested by Assan and Slusher (21), and Jacobsen et al. (22). PG-specific antisera raised against C-terminal glucagon fragments were also reported by Nishino et al. (23, 24) and Ohneda et al. (25). In fact, antisera N2C and N6E raised against C-terminal glucagon fragment (peptide II) did not cross-react with gut GLI, although antiserum N14B raised against an N-terminal glucagon fragment (peptide VII) did react with gut GLI. Such PG-specific antisera are useful in immunoassays for PG determination, since plasma usually contains GLI of intestinal origin. Antisera N2C and N6E used here bound peptide II-enzyme conjugate more strongly than free PG as they were prepared by immunizing peptide II, and the assays using the antisera with peptide II-enzyme conjugate proved to be relatively insensitive. The assays using these antisera and peptide IV-enzyme conjugate, however, showed high sensitivity and were comparable to the assays using 30K or R517 antiserum against PG. PG and its fragment peptides were conjugated very easily with £-D-galactosidase using MBS as a coupling reagent (14), but the immunoreactivity of the PG-enzyme conjugate obtained here was much more markedly affected by the coupling conditions than the immunoreactivity of the fragment peptide-enzyme conjugates. That is probably because PG has a lysine residue at the 12th position from its N-terminus, the e-amino

group of which can be coupled to an SH group of the enzyme by MBS, while the C-terminal fragment peptides II to IV have no free amino group other than the N-terminal amino group. These enzyme-labeled fragment peptides could be used in the EIA for more than 3 months without losing their enzymatic and immunological activities, and their high stability resulted in good reproducibility and precision of the EIA method. Such an EIA procedure using a sensitive combination of antiserum and peptide-enzyme conjugate may be extended to other peptide hormones by linking a suitable fragment peptide to an enzyme in order to increase the sensitivity and specificity. The authors wish to acknowledge the valuable advice and suggestions offered by Prof. T. Nambara of Tohoku University. They are grateful to Drs. M. Nishikawa and M. Hori of this laboratory for their encouragement and support. They also thank Dr. H. Sakurai of Kyoto University for his generous gift of antiserum R517 and gut extract, and Dr. M. Fujino and his coworkers of this laboratory for their kind gift of some synthetic peptides. REFERENCES 1. Unger, R.H., Eisentraut, A.M., McCall, M.S., & Madison, L.L. (1961) / . Clin. Invest. 40, 1280-1289 2. Lawrence, A.M. (1966) Proc. Natl. Acad. Sci. U.S. 55, 316-320 3. Hazzard, W.R., Crockford, P.M., Buchanan, K.D., Vance, J.E., Chen, R., & Williams, R.H. (1968) Diabetes 17, 179-186 4. Young, J.D. & Kraegen, E.W. (1968) Aust. J. Exp. Biol. Med. Scl. 46, 697-705 5. Nonaka, K. & Foa, P.P. (1969) Proc. Soc. Exp. Biol. Med. 130, 330-336 6. Heding, L.G. (1971) Diabetobgia 7, 10-19 7. Asano, T., Kato, K., Ishikawa, E., & Okumura, M. (1977) Igaku no Ayumi (in Japanese) 103, 25-26 8. Van Weemen, B.K. & Schuurs, A.H.W.M. (1972) FEBS Lett. 24, 77-81 9. Van Weemen, B.K. & Schuurs, A.H.W.M. (1975) Immunochemistry 12, 667-670 10. Fujino, M., Wakimasu, M., Shinagawa, S., Kitada, C , & Yajima, H. (1978) Chem. Pharm. Bull. 16, 539-548 11. Sakurai, H., Kuzuya, H., Kurahachi, H., Fukase, M., & Imura, H. (1973) Jap. J. Nud. Med. 10, 135-136 12. Maier, V. & Pfeiffer, E.F. (1978) Horm. Metab. Res. 10, 177-182 / . Biochem.

GLUCAGON ENZYME IMMUNOASSAY 13. Garaud, J.C., Moody, A.J., Eloy, R., & Grenier, J.F. (1976) Horm. Metab. Res. 8, 241-243 14. KJtagawa, T. & Aikawa, T. (1976) J. Biochem. 79, 233-236 15. Iwasa, S., Kondo, K., Miya, T., & Takeda, K. (1978) Immunopharmacology 1,3-12 16. Van Wecmen, B.K. & Schuurs, A.H.W.M. (1971) FEBS Lett. 15, 232-236 17. Comoglio, S. & Cclada, F. (1976) / . Immunol. Methods 10, 161-170 18. Wallenfels, K., Zarnitz, MX., Laule, G., Bender, H., & Keser, M. (1959) Biochem. Z. 331, 459-463 19. Woollen, J.W. & Walker, P.G. (1965) Clin. Chim. Ada 12, 647-658

Vol. 86, No. 4, 1979

949 20. Exley, D. & Abukncsha, R. (1978) FEBS Lett. 91, 162-165 21. Assan, R. & Slusher, N. (1972) Diabetesll, 843-855 22. Jacobsen, H., Demandt, A., Moody, AJ..& Sundby, F. (1977) Biochim. Biophys. Ada 493, 452-459 23. Nishino, T., Imagawa, K., Shin, S., Uehata, S., Hashimura, E., & Yanaihara, N. (1977) Folia Endocrinologica Japonica 53, 551 24. Nishino, T., Kohira, T., Shin, S., Uehata, S., Hashimura, E., Imagawa, K., & Yanaihara, N. (1978) Folia Endocrinologica Japonica 54, 454 25. Ohneda, A., Watanabe, K., Wakimasu, M., & Fujino, M. (1979) Horm. Metab. Res. 11, 463-467

Enzyme immunoassay of pancreatic glucagon at the picogram level using beta-D-galactosidase as a label.

/ . Biochem. 86, 943-949 (1979) Enzyme Immunoassay of Pancreatic Glucagon at the Picogram Level Using /3-D-Galactosidase as a Label Susumu IWASA,* Ha...
376KB Sizes 0 Downloads 0 Views