Molecular and Cellular Probes (1991) 5, 261-269
Direct measurement of calpastatin subtypes by sandwich enzyme immunoassay using monoclonal antibodies Hiroko Yokota, Masahiko Katayama, Fumitsugu Hino,* Ikunoshin Kato, Emiko 2 Takano,' Masatoshi Maki, Masakazu Hatanaka 2 and Takashi Murachi' Biotechnology Research Laboratories, Takara Shuzo Co ., Ltd., Otsu, Shiga 520-21, Japan, 'Institute for Virus Research and 2 Department of Clinical Science and Laboratory Medicine, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan (Received 8 September 1990, Accepted 1 November 1990)
Six stable hybridoma cell lines secreting monoclonal antibodies to human calpastatin were established . All monoclonal antibodies belong to the IgG1 subclass and recognized different epitopes on calpastatin . At least two groups were distinguished ; the first group was specific for muscle-type (M-) calpastatin and the second group recognized not only M-calpastatin but also erythrocyte-type (E-) calpastatin . The inhibitory effect of all monoclonal antibodies on calpastatin activity was relatively low even at high concentrations of antibodies . Enzyme immunoassay systems were developed for direct determination of calpastatin subtypes in human cells requiring no other sample treatment than the disruption of the cells . The assay methods were, in principle, based on the sandwich enzyme immunoassay using epitope-specific monoclonal antibodies . The enzyme immunoassay system for M-calpastatin was specific for M-calpastatin and could not detect E-calpastatin . The enzyme immunoassay system for total calpastatin detected not only M-calpastatin but also E-calpastatin . The sensitivity of these assay systems was 10 pmol I -1 of calpastatins . Antigenicity of calpastatins was found to be unchanged in the presence of EDTA and haemoglobin . Good reproducibilities of withinand between-assay series and excellent recovery of exogenous calpastatins from cell lysates were observed . From these results, it seems that our newly developed subtype-specific enzyme immunoassay systems for calpastatins are useful in biochemical studies and clinical testing for determination of calpastatin subtypes .
KEYWORDS : proteinase inhibitor, calpastatin, enzyme immunoassay, monoclonal antibodies,
INTRODUCTION Calpastatin is an endogenous protein inhibitor acting specifically on calpain (EC 3 .4.22 .17 ; Ca 21 -dependent cysteine endopeptidase) .' Both calpain and calpastatin are very widely distributed in animal cells .2' The physiological roles of calpastatin have not been clarified, but the calpain-calpastatin system may be an important factor in the in vivo regulation of Ca2 dependent proteolysis ." The primary structure of human calpastatin was determined by nucleotide sequencing of cloned
cDNA from human liver mRNA ." Human calpastatin consisted of 673 amino-acid residues and had a domain structure with four internally repetitive sequences (domain I-IV) and one N-terminal nonhomologous sequence (domain L) . Previously, we reported the purification of two different molecular species of calpastatin from pig heart muscle (107 kDa in SDS-PAGE ; Muscle-type) and pig erythrocytes (68 kDa in SDS-PAGE ; erythrocyte-type) ." Muscle-type calpastatin (M-calpastatin)
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© 1991 Academic Press Limited
H . Yokota et al.
consisted of domains L and I-IV . 12 Erythrocyte-type calpastatin (E-calpastatin) consisted of domain II-IV and lacked the N-terminal region of M-calpastatin .' 3 A quantitative distribution study was carried out earlier on calpastatin in various tissues and cells by an enzymological method . Takano et ai . 14 reported an enzyme immunoassay (EIA) system for measuring calpastatin in erythrocyte by using polyclonal antibody, but they had to prepare a haemoglobin-free sample to avoid the interference by the inherent peroxidase activity of haemoglobin . Their method could not distinguish M- and E-calpastatins . In order to investigate the biological and physiological roles of calpastatin, it was necessary to develop a rapid, simple, sensitive and subtype-specific assay method for calpastatin . In this paper, we describe direct EIA systems for human total calpastatin (M-calpastatin plus E-calpastatin) and M-calpastatin, which are based on sandwiching the antigen using epitopespecific monoclonal antibodies (MAbs). This is the first report on the subtype specific MAbs against calpastatins and their application for sandwich EIA .
MATERIALS AND METHODS Materials Bovine serum albumin (BSA : RIA grade) was purchased from Sigma Chemical Co . (USA) ; Peroxidase (EC 1 .11 .1 .7), from Boehringer Mannheim (Germany) ; Sephadex G-25 and G-200 were obtained from Pharmacia Fine Chemicals (Sweden) ; 96-well multititre plate and EIA plate from Nunc (Denmark) . Other chemicals came from Nacalai Tesque Ltd (Japan) . Human M-calpastatin and its domain I and domain III, produced by expression of the respectively cloned genes in Escherichia coil, were purified by heat treatment and DEAF-cellulose chromatography as described previously ." Human E-calpastatin was purified from the cytosol fraction of human erythrocytes." M-calpastatin and E-calpastatin thus prepared were used as both the immunogen and the EIA standard .
Production of MAbs Female BALB/c mice were immunized with human Mcalpastatin and cell fusion was performed by the standard procedures by using mouse myeloma P3U1 cell ." Hybridoma colonies were screened for specific antibody production by enzyme-linked immunosorbent assay (ELISA) and were cloned by the limiting dilution method . Anti-calpastatin antibody secreting
clones were grown intraperitoneally in pristanetreated BALB/c mice for ascites fluid production .
Detection of anti-calpastatin antibodies Anti-calpastatin antibodies were detected using the ELISA according to the standard procedure ." For this, 50 pl of sample was incubated in calpastatin- or its related antigens-coated microtitre EIA plate for 1 h at 25 ° C . After that the plate was washed three times with 10 mm phosphate-buffered saline (pH 7 . 4; PBS), and then 50 pl of peroxidase (POD) conjugated goat anti-mouse IgG (Cappel) was added to each well . The plate was left for 1 h at 25 ° C and then washed four times with PBS . The POD activity was determined as described below .
Purification of MAbs Monoclonal antibodies were purified from ascites fluids by precipitation with ammonium sulphate, followed by Mono Q FPLC (Pharmacia) .' 9 The protein concentration of purified IgG was calculated from A280 =1 . 4 for 1 mg,ml - ' . The purified MAb was labelled with POD by the periodate method . 20
Sandwich EIA Microtitre EIA plates were coated with MAb solution (200 pl well - ') at 10 gg ml - ' in coating buffer (0. 1 nit sodium carbonate, pH 8. 5) and incubated for 16 h at 4 ° C . The plates were washed three times with PBS and blocked with PBS containing 1 % BSA (200 gl well - ') for 3 h at 25° C. Following blocking the plates were washed three times with PBS . A volume of 100 gl of the standard or sample was added to each well, and the plate was left for 1 h at 25 ° C without shaking . After that, the plate was washed four times with PBS, and 100 pl of POD-labelled MAb, prepared by the periodate method 20, in PBS-containing 1 % BSA, corresponding to 200 ng of MAb was added to each well . The plate was left for 1 h at 25 ° C without shaking . Following incubation the plate was washed four times with PBS. The plate was then incubated for 15 min at 25 °C with a solution (100 pl well - ') of 0 . 1 M citrate-0-2m phosphate buffer, pH 5 . 0, containing 1 g I - ' o-phenylenediamine and 0 . 01 % (v/v) hydrogen peroxide . The reaction was stopped by the addition of 1 N sulphuric acid (100 pl well - '). The POD activity was determined by measuring absorbance value at 492 nm with a microplate reader (Titertek Multiskan, Model MCC, Flow Lab .) . The
Enzyme immunoassay for calpastatin concentration of calpastatins were expressed in pmol I - ', which can be converted into ng ml - ' using molecular weight 73,000 for M-calpastatin 10 and 46,000 for E-calpastatin . 13
Effect of MAbs on the interaction of calpastatin and calpain
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting SDS-PAGE was performed as described by Laemmli . 23 Electrophoretic transfer of the protein from gel to nitrocellulose paper was done by the procedure of Towbin et a! ., 24 and the nitrocellulose paper was treated for antibody binding using the procedure of Kittler . 25
The inhibitory effect of MAbs on calpastatin activity was determined using the method for assay of calpastatin activity . For determination of calpastatin activity, the calpastatin sample was preincubated with a fixed amount of calpain I, purified from human erythrocyte," at 30° C for 30 min, then the decrease in proteolytic activity was measured using casein as the substrate. One unit of calpastatin is defined as the amount of protein which inhibits one unit of calpain under the standard assay conditions, as described previously ." For determination of inhibitory effect of MAbs on calpastatin activity, the antibody solution was first added to the calpastatin sample and the mixture was allowed to stand for 1 h at 30 °C prior to the addition of calpain . As a control experiment, 50 mm Tris-buffered saline (pH 7 . 5 ; TBS) was used in place of the antibody solution . The calpastatin activity remaining was measured and the inhibitory effect of MAbs was expressed as a percentage of the control value .
Protein determination The protein concentration of calpastatin was determined by the method of Lowry et a! . 22 with BSA as standard .
Preparation of cell lysate The haemolysate was prepared by haemolyzing the erythrocytes with 10 vol of 20 mm Tris-HCI (pH 7 . 5), containing 1 mm ethylenediaminetetraacetic acid (EDTA), 1 mm ethylene glycol bis((3-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA), and 5 mm -mercaptoethanol . Cell lines were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum . All cultured cells were harvested at their confluent stage . The cultured cells were washed with PBS and then lysed at 4 ° C in phosphate buffer containing 1 % (v/v) Triton X-100, 5 mm EDTA and 1 mm phenylmethylsulphonyl fluoride . The solubilized cells were cleared of debris by centrifugation at 10,000 rpm for 5 min .
RESULTS Characterization of MAbs Fifty-two of 904 hybridoma cultures were shown to produce antibodies to M-calpastatin as determined by ELISA . Of the 52 wells containing antibodies, 10 wells having relatively higher titre in ELISA were selected for cell cloning by the limiting dilution method . From 10 wells, six hybridoma cells showed good growth, and were taken out and cultured in bulk. They were then injected into the peritoneal cavity of BALB/c mice and grown as ascitic tumours, and the ascites containing MAbs were obtained . The titre of antibodies in the ascitic fluid was determined by ELISA . Table 1 summarizes the titre of each ascitic fluid, subclass, specificity and the inhibitory potency on calpastatin activity of each purified MAb . Ouchterlony test revealed that all six MAbs belong to IgG1 subclass . All antibodies inhibited the calpastatin activity to calpain, depending on the concentration of MAb, but the inhibitory potency was relatively low even in the presence of excess antibody . Of the six MAbs, one (CSL1-5) recognizes domain III, three (CSL4-7, CSF1-2 and CSF2-2) recognize domain I, and two (CSL5-10 and CSF3-3) recognize neither domain I nor domain Ill . The epitope of MAbs CSL5-10 and CSF3-3 should be localized either domain II or IV, which must await further experimental verification . The specificity of MAbs were further investigated by immunoblotting . MAb CSF1-2, which recognized domain I (Table 1), specifically reacted with purified M-calpastatin but did not react with E-calpastatin . Moreover, this MAb reacted with a protein of similar size with M-calpastatin in the lysate of cultured human lung cell, while no reactive protein band was observed with lysate of human erythrocyte (Fig . 1b) . MAb CSL1-5, which recognized domain III, reacted not only with M-calpastatin but also with E-calpastatin . CSF3-3 also reacted with both . These two MAbs reacted with the proteins of similar size respectively with M-calpastatin and E-calpastatin in the lysate of cultured human lung cell and human erythrocyte (Figs 1c and 1d) .
H. Yokota et al.
264 Table 1 .
Reactivity of MAbs with human calpastatins
CSL1-5 CSL4-7 CSL5-10 CSF1-2 CSF2-2 CSF3-3
G, G, C, G, G, G,
1 2 5 2 1 1
x x x x x x
10 , 10 5 10° 10 5 10 4 105
Inhibition of M-calpastatint
+ + + + + +
+ + +
5 4 5 10 1 2
32 28 57 55 30 34
• Dilution of the ascitic fluid required to reduce the binding of POD-labelled goat anti-mouse IgG to 50% of the maximum absorbance in ELISA. t The inhibitory activity of purified MAbs as assayed as described in Materials and methods . T Molar ratio of M-calpastatin to MAb .
Fig . 1 . Immunoblot analysis for the specificity of MAbs to purified calpastatins and the lysate of erythrocyte or A549 cell . Human erythrocyte haemolysate (100 µg protein; lane 1), A549 cell lysate (50 µg protein ; lane 2), 5 jig of E-calpastatin (lane 3) and 5 µg of M-calpastatin (lane 4) were electrophoresed by using 12 . 5% polyacrylamide gel, and blotted to nitrocellulose membrane . The nitrocellulose membrane was stained with amido black (a), and immunostained with CSF1-2 (b), CSF3-3 (c) and CSL1-5 (d) . The positions of molecular size markers in kDA are shown .
Enzyme immunoassay for calpastatin
Table 2 .
Sandwich enzyme immunoassay for M-calpastatin (a) and E-calpastatin (b)
POD-labelled MAb Immobilized MAb
+ + + +
+ + +
+++ ++ ++ ++
++ + ++ -
+++ ++ ++ -
(a) CSL1-5 CSL4-7 CSL5-10 CSF1-2 CSF2-2 CSF3-3 (b) CSL1-5 CSL4-7 CSL5-10 CSF1-2 CSF2-2
The results of EIA were graded in accordance with the obtained absorbance (A) at 492 nm ; -, A < 0 .2; +, 0 .2 < A < 0. 5 ; + +, 0'5