Virchows Archiv B Cell Pathol (1992) 62:259-262
VirchowsArchivB CellPathology
lncludingMalecularPatholo~
9 Springer-Verlag 1992
Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64 Evidence that IND.64 reacts with the cell proliferation associated nuclear antigen previously defined by Ki-67 Giiran Key, Fabienne Meggetto*, Michael H.G. Becker, Talal AI Saati*, Carsten Schliiter, Michael Duchrow, Georges Delsol*, and Johannes Gerdes ForschungsinstitutBorstel, DivisionMolecularImmunology,Parkallee22, W-2061 Borstel, Federal Republic of Germany * Institute of Pathology,Universityof Toulouse, F-31059 Toulouse, France Received March 3, AcceptedMay 22, 1992
Summary. The immunohistochemical characteristics of the monoclonal antibody IND.64 are very similar to those of the monoclonal antibody Ki-67. The aim of this study was to further characterize this new antibody and to compare it with Ki-67 using immunobiochemical methods. Our results demonstrate that the similarity between the antibodies holds true even at the molecular level. Immunoblot analysis of IM-9-cell lysates with both antibodies showed a double band with apparent molecular weights of 395 kD and 345 kD, respectively. Competition ELISAs using a synthetic peptide derived from the thus far determined Ki-67 eDNA sequence as competitor, indicate that IND.64 may recognize the same epitope as Ki-67. The IND.64 epitope resides at least within a 20 amino acid sequence which also contains the Ki-67 epitope. Since IND.64 is of the IgG2b subclass, while Ki-67 is of the IgG1 subclass, the two antibodies may be useful for double immunostaining. In addition, IND.64 may help in determining the still unknown function of the anigen it recognizes.
Key words: IND.64 - Monoclonal antibody - Cell proliferation - Ki-67
Introduction Determination of the growth fraction of a given cell population provides important information for tumor diagnosis and prognosis. In recent years the use of histochemistry in connection with specific antibodies against proliferation-associated antigens has received increasing interest. One of the most widely used antibodies in this field is the monoclonal antibody Ki-67 (Gerdes et al. 1983) which reacts with a nuclear antigen expressed in G1, S, G2 and M, but not in Go (Gerdes et al. 1984). Correspondence to: J. Gerdes
Ki-67 has turned out to be a good marker for determining the growth fraction in a wide variety of normal and malignant human tissues (reviewed by Gerdes 1990), and there is increasing evidence that the Ki-67 index is of prognostic value even in individual cases of a variety of human cancers (Gerdes etal. 1987; Grogan etal. 1988; Hall et al. 1988; Wintzer et al. 1991). Recently, Delsol's group have generated a monoclonal antibody, the immunohistochemical staining pattern of which was identical to that of Ki-67 in frozen sections of several human tissues. Thus, it was of interest to determine whether this antibody recognizes the same antigen as Ki-67 or another molecule also associated with cell proliferation. In Western blots, Ki-67 reacts with two polypeptide chains with apparent molecular weights of 345 kD and 395 kD, that were found only in proliferating cells and not in quiescent cells (Gerdes et al. 1991). By immunoscreening of lambda gtl 1 expression libraries with Ki-67, several cDNA clones could be detected that all contained a 62 bp element with high homology (65-100%). This element was presumed to encode for the epitope recognized by the antibody Ki-67. In ELISA tests and Western blots the antibody Ki-67 reacted with bacterially expressed fusion proteins corresponding to parts of the so far determined eDNA sequence. The aim of this study was to compare the monoclonal antibodies IND.64 and Ki-67 at the molecular level.
Material and methods Cells and specimen. IM-9 (multiple myelomacell line, ATCC No. CCL 159, courtesyof Prof. H. Stein, Berlin, FRG) and the mouse hybridoma cell lines were cultured under standard conditions in RPMI 1640medium(Gibco, Berlin,FRG) supplementedwith antibiotics and 10% fetal calf serum. Antibodies. The monoclonal antibodies Ki-67 and IND.64 were prepared as described previously (Gerdes et al. 1983; Meggetto et al. in press). Alkaline phosphatase-conjugatedgoat anti-mouse IgG was obtained from Dianova (Hamburg, FRG).
260
Peptide synthesis. The synthetic peptide used in this study was kindly synthesized by Dr. Kullmann, Institut fiir Neurobiochemie, Hamburg, FRG. This peptide represents the amino acid sequence deduced from a 62 bp DNA region presumed to encode for the Ki-67 epitope (Gerdes et al. 1991).
targeted with goat anti-mouse alkaline phosphatase conjugate (Dianova, Hamburg, FRG), diluted 1 : 10000 in blocking solution. The phosphatase activity was visualized using nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates (Leary et al. 1983).
Preparation of recombinant parts of the Ki-67 antigen
Competition ELISA
Construction ofplasmids. Nucleotides 1-1002 of the recently published Ki-67 antigen cDNA sequence (Gerdes et al. 1991) were cloned between the Eco R1 and Pstl sites of the pAX4a+ vector (Markmeyer et al. 1990) (Medac, Hamburg, FRG). The resulting plasmid was designated pAX X2/1 and was transformed into E. coli JM 109 (Yanisch-Perron et al. 1985). A 100 bp fragment including the 62 bp repetitive element which presumably encodes for the Ki-67 epitope was ligated between the Eco R1 and Bam H1 sites of the pEV vectors (Crowl et al. 1985) in all reading frames. The resulting plasmids were designated pEVvrf l/B3, pEV-vrf 2/B3, pEV-vrf 3/B3 and transformed into E. coli RR1 (Bolivar and Backmann 1979).
Polystyrene microtiter plates were incubated overnight at 4~ C with 0.5 Ixg/well of lysates of induced E. coli JM109 pAX X2/1 expressing the 1002 bp insert in a total volume of 50 p.l PBS. After 30 min incubation at room temperature with the synthetic peptides at different concentrations, the monoclonal antibodies were subjected to precoated ELISA plates. Nonspecific binding was blocked by 2% gelatin in TBS (TBS-Gel). The plates were incubated 1 h at 37~ C. After three washes with TBS all wells were incubated for 1 h at 37~ C with 50 lal of a goat anti-mouse AP conjugate (Dianova, Hamburg, FRG), diluted 1 : 10000 in TBS-Gel followed by three washes with TBS and an incubation with 50 Ixl/well phosphatase substrate (Sigma 104-0, 1 mg/ml in TBS, pH 9,5) at 37~ C. Optical densities were evaluated at 405 nm on a Flow immunoplate reader (FLOW Laboratories, Meckenheim, FRG).
Preparation of bacterial cell lysates. An overnight culture of E. coli JMI09 containing pAX X2/1 was diluted to an O.D.55o of 0.3, grown to an O.D.55o of 1.0 and induced with 1 mM isopropylthio-fl-galactoside (IPTG) according to Markmeyer et al. (1990). After an additional incubation for 2.5 h at 30~ C, bacteria were harvested by centrifugation, washed once in phosphate-buffered saline (PBS), resuspended in 1/20 volume of PBS and stored at - 2 0 ~ C until further used. E. coli RR1 harboring pEV-vrf 1-3/B3 plasmids were cultured at 37~ C in M9 medium (Sambrook et al. 1989) supplemented with 40 Ixg/ml ampicillin, 0.5% glucose and casamino acids, respectively, until they reached on O.D.sso of 0.8. Subsequently bacteria were harvested and lysates were prepared for SDS-PAGE and Western blots as described below.
Preparation of native Ki-67 antigen IM-9 cells (6 • 107) were pelleted by centrifugation at 400 • g for 10 min and resuspended in 500 ~tl PBS containing 1 mM phenylmethylsulfonyl-fluoride (PMSF). The cell suspension was snap-frozen in liquid nitrogen and homogenized in a mortar cooled with liquid nitrogen. The cell debris was solubilized by boiling for 5 min in 0.5 ml double concentrated SDS-PAGE sample buffer (Laemmli 1970) with 100 mM DTT (see below). The insoluble DNA was destroyed by short sonication.
Antibody subclass determination The subclass determination was performed with the dip-stick method (Holland Biotechnology BV, Leiden, The Netherlands) according to the manufacturer's instructions.
Affinity determination of monoclonal antibodies Ki-67 and IND.64 The relative affinities of both antibodies were determined by use of the method described by Rath et al. (1988). Briefly, in the competition ELISA (see above) the synthetic peptide was used in different concentrations to compete for the antibody binding. The affinity values were calculated from the resulting optical densities and the corresponding peptide molarity.
Results Isotype analysis A c c o r d i n g to the d i p - s t i c k a s s a y Ki-67 p r o v e d to be o f IgG1 k a p p a a n d I N D . 6 4 o f I g G 2 b k a p p a s u b t y p e .
SDS-PAGE SDS gel electrophoresis using 5% or 7.5% separation gels, respectively, and 3.5% stacking gels was performed according to Laemmli (1970). Sample buffer contained 50mM Tris/HC1, pH 6.8, 4% SDS, 20% glycerin and 100 mM DTT. Samples were boiled for 5 min before electrophoresis. Molecular weight markers were obtained from Pharmacia/LKB (Freiburg, FRG) or Sigma (M/inchen, FRG).
Immunoreactivity of Ki-67 and IND.64 with the native nuclear antigen I M - 9 cells were lysed, s u b j e c t e d to S D S - P A G E , a n d W e s t e r n b l o t s were i m m u n o s t a i n e d with Ki-67 a n d I N D . 6 4 , respectively. As d e m o n s t r a t e d in Fig. 1, b o t h a n t i b o d i e s r e a c t e d with two p o l y p e p t i d e c h a i n s w i t h a n a p p a r e n t m o l e c u l a r w e i g h t o f 345 k D a n d 395 k D , respectively.
Immunoblotting Proteins were transferred to nitrocellulose membranes (BA 85, Schleicher & Schuell, Dassel, FRG) by the Western blot technique (Towbin et al. 1979). The transfer was performed at 50 mA for 16h in a water-cooled device. Unoccupied binding sites were blocked by incubation for 1 h with 1% BSA or gelatine in Trisbuffered saline (TBS). After 1 h incubation with the primary antibody and thorough washing with TBS, the bound antibody was
Immunoreactivity of Ki-67 and IND.64 with two bacterially expressed proteins deduced from Ki-67-cDNA 1002 b p a n d 100 b p f r a g m e n t s o f the Ki-67 c D N A were b a c t e r i a l l y expressed, a n d the lysates were s u b j e c t e d to
261
Fig. 1. Immunoblot analysis of lysates obtained from IM-9 cell line cells. Western blots were immunostained with the monoclonal antibodies Ki-67 and IND.64. Both antibodies react with a double band with molecular weights of 345 kD and 395 kD
S D S - P A G E . Subsequently, Western blots were immunostained with Ki-67 and IND.64. As shown in Fig. 2, the antibodies reacted with the fusion proteins in similar patterns, while the controls (no insert for the p A X X2/1 derived fusion protein, wrong reading frame for the pEV-vrf/B3 derived fusion protein) were negative for both antibodies.
Competition ELISA For further comparison of the antigen binding characteristics o f the two antibodies a synthetic peptide containing the putative epitope recognized by Ki-67 was tested in a competition ELISA. As shown in Fig. 3, the binding o f both antibodies to the recombinant parts of the Ki-67 antigen could be inhibited with the peptide in a dose dependent manner. Thus, the peptide was able to compete for the paratopes of both Ki-67 and IND.64.
Relative affinities of Ki-67 and IND.64 Using the synthetic peptide in a molar range from 1.6 • 10 -4 M to 4 • -8 M, the relative affinity constants could be calculated from a similar competition assay: K i - 6 7 : 2 • 10 -6 M; I N D . 6 4 : 1 3 x 10 -6 M. Thus, the rel-
Fig. 2. Immunoblot analysis of recombinant parts of the Ki-67 antigen. A Bacteria were transformed with a vector containing a 1002 bp cDNA fragment (lanes 1 and 3) or, as a control, with the same vector containing no specific insert (lanes 2 and 4). Ki-67 and IND.64 react with identical bands representing the expression
product of the DNA insert. B A 100 bp fragment, containing the 62 bp element presumed to contain the epitope recognized by antibody Ki-67 was expressed in bacteria in the correct (lanes 1 and 3) and in a wrong (lanes 2 and 4) reading frame. The antibodies Ki-67 and IND.64 only react with the lysates from bacteria containing the cDNA insert in the correct reading frame
262 Acknowledgements. The authors wish to thank Margrit Hahn, Bet-
tina Baron and Claudia Wohlenberg for their skillful technical assistance. The work was partly supported by the Dr. Mildred Scheel Stiftung ffir Krebsforschung (project W49/90/Ge2) and L'Association pour la Recherche sur le Cancer (ARC).
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References
IND.84
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.E
40
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o 0
2
4
6
8
10
12
14
Peptlde concentration [IJg/welll
Fig. 3. The synthetic peptide deduced from the 62 bp eDNA element presumed to encode for the Ki-67 epitope was tested in a competition ELISA for its ability to compete for the binding of the monoclonal antibodies Ki-67 and IND.64 to the solid phase bound pAX X2/1-derived fusion protein. Both antibodies are displaced by the peptide in a dose-dependent manner
ative affinity of I N D . 6 4 was found to be 6.5 times lower than that of Ki-67.
Discussion
Because of the similar immunohistochemical staining pattern o f Ki-67 and IND.64, the aim of this study was to compare the immunobiochemical characteristics o f these antibodies. Recently, the group from Borstel (Gerdes et al. 1991) found that the antibody Ki-67 reacts with two polypeptides of 345 k D and 395 kD, which are detected exclusively in proliferating cells. The results of the present study show that the antibody I N D . 6 4 detects two polypeptides with identical apparent molecular weights. Furthermore, both antibodies similarly stained Western blots of two different recombinant parts of the Ki-67 antigen. It was noteworthy that I N D . 6 4 even recognized the pEV-vrf/B3-derived fusion protein, representing the translation product of the 62 bp Ki-67 c D N A fragment which was recently assumed to encode for the epitope recognized by Ki-67. In addition, the synthetic peptide which was deduced from the 62 bp c D N A was able to compete for the binding of both antibodies. Thus, we conclude that Ki-67 and I N D . 6 4 react with the same antigen, and in the light of these findings it is very likely that both antibodies recognize the same epitope. At least, the epitopes of I N D . 6 4 and Ki-67 must be located close to each other on a domain of only twenty amino acids in length. Considering that preparations of tissue or nuclei were used as immunogens for the generation o f both monoclonal antibodies, it is tempting to assume that this area of the antigen is imm u n o d o m i n a n t when the whole molecule is used for immunization. In conclusion IND.64 can be regarded as a true Ki-67 equivalent, exhibiting a different affinity and immunoglobulin subclass. This antibody m a y be useful for double immunostaining and m a y contribute to understanding the presently unknown function of the antigen it recognizes.
Bolivar F, Backmann K (1979) Plasmids of Escherichia coli as cloning vectors. Meth Enzymol 68: 245-267 Crowl R, Seamans C, Lomedico P, McAndrew S (1985) Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene 38:31-38 Gerdes J, Lemke H, Baisch H, Wacher H-H, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715 Gerdes J, Li L, Schliiter C, Duchrow M, Wohlenberg C, Gerlach C, Stahmer I, Kloth S, Brandt E, Flad H-D (1991) Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol 138: 867-873 Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31:13-20 Gerdes J, Stein H, Pileri S, Rivano MT, Gobbi M, Ralfkiaer E, Nielsen KM, Pallesen G, Bartels H, Palestro G, Delsol G (1987) Prognostic relevance of tumour-cell growth fraction in malignant non-Hodgkin's lymphomas. Lancet ii:448-449 Gerdes J (1990) Ki-67 and other proliferation markers useful for immunohistological diagnostic and prognostic evaluations in human malignancies. In: Osborn M (ed) Seminars in cancer biology, vol 1. Saunders Scientific Publications, London New York, pp 99-206 Grogan TM, Lippman SM, Spier CM, Slymen DJ, Rybski JA, Rangel CS, Richter LC, Miller TP (1988) Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki67. Blood 71:1157-1160 Hall PA, Richards MA, Gregory WM, d'Ardenne A J, Lister TA, Stansfeld AG (1988) The prognostic value of Ki-67 immunostaining in Non-Hodgkin's Lymphoma. J Pathol 154:223-235 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Leary J J, Brigati D J, Ward DC (1983) Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA of RNA immobilized on nitrocellulose: Bioblots. Proc Natl Acad Sci USA 80:4045-4049 Markmeyer P, Riihlmann A, Englisch U, Cramer F (1990) The pAX plasmids: new gene-fusion vectors for sequencing and expression of proteins in Escherichia coli. Gene 93:129-134 Meggetto F, A1 Saati T, Cohen-Knafo E, Roubinet F, Selves J, Bouche G, Key G, Gerdes J, Delsol G (1992) Production of monoclonal antibody (IND.64) identifying cell cycle-associated antigen using spleen cells from nude mice bearing Ichikawa tumour. J Pathol (in press) Rath S, Stanley CM, Steward MW (1988) An inhibition immunoassay for estimating relative antibody affinity and affinity heterogeneity. J Immunol Meth 106:245-249 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbour Laboratory, New York Towbin H, Staehelin T, Gordon J (1979) Electrophoretical transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350~354 Wintzer H-O, Zipfel I, Schulte-Mrnting J, Hellerich U, von Kleist S (1991) Ki-67 immunostaining in human breast tumors and its relationship to prognosis. Cancer 67:421-428 Yanisch-Perron C, Vieira J, Messing J (1985) Improved M 13 phage cloning vectors and host strains: nucleotide sequences of the M 13mp18 and pUC 19 vectors. Gene 33 : 103-119
VirchowsArchivB CellPathology
lncludingMalecularPatholo~
9 Springer-Verlag 1992
Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64 Evidence that IND.64 reacts with the cell proliferation associated nuclear antigen previously defined by Ki-67 Giiran Key, Fabienne Meggetto*, Michael H.G. Becker, Talal AI Saati*, Carsten Schliiter, Michael Duchrow, Georges Delsol*, and Johannes Gerdes ForschungsinstitutBorstel, DivisionMolecularImmunology,Parkallee22, W-2061 Borstel, Federal Republic of Germany * Institute of Pathology,Universityof Toulouse, F-31059 Toulouse, France Received March 3, AcceptedMay 22, 1992
Summary. The immunohistochemical characteristics of the monoclonal antibody IND.64 are very similar to those of the monoclonal antibody Ki-67. The aim of this study was to further characterize this new antibody and to compare it with Ki-67 using immunobiochemical methods. Our results demonstrate that the similarity between the antibodies holds true even at the molecular level. Immunoblot analysis of IM-9-cell lysates with both antibodies showed a double band with apparent molecular weights of 395 kD and 345 kD, respectively. Competition ELISAs using a synthetic peptide derived from the thus far determined Ki-67 eDNA sequence as competitor, indicate that IND.64 may recognize the same epitope as Ki-67. The IND.64 epitope resides at least within a 20 amino acid sequence which also contains the Ki-67 epitope. Since IND.64 is of the IgG2b subclass, while Ki-67 is of the IgG1 subclass, the two antibodies may be useful for double immunostaining. In addition, IND.64 may help in determining the still unknown function of the anigen it recognizes.
Key words: IND.64 - Monoclonal antibody - Cell proliferation - Ki-67
Introduction Determination of the growth fraction of a given cell population provides important information for tumor diagnosis and prognosis. In recent years the use of histochemistry in connection with specific antibodies against proliferation-associated antigens has received increasing interest. One of the most widely used antibodies in this field is the monoclonal antibody Ki-67 (Gerdes et al. 1983) which reacts with a nuclear antigen expressed in G1, S, G2 and M, but not in Go (Gerdes et al. 1984). Correspondence to: J. Gerdes
Ki-67 has turned out to be a good marker for determining the growth fraction in a wide variety of normal and malignant human tissues (reviewed by Gerdes 1990), and there is increasing evidence that the Ki-67 index is of prognostic value even in individual cases of a variety of human cancers (Gerdes etal. 1987; Grogan etal. 1988; Hall et al. 1988; Wintzer et al. 1991). Recently, Delsol's group have generated a monoclonal antibody, the immunohistochemical staining pattern of which was identical to that of Ki-67 in frozen sections of several human tissues. Thus, it was of interest to determine whether this antibody recognizes the same antigen as Ki-67 or another molecule also associated with cell proliferation. In Western blots, Ki-67 reacts with two polypeptide chains with apparent molecular weights of 345 kD and 395 kD, that were found only in proliferating cells and not in quiescent cells (Gerdes et al. 1991). By immunoscreening of lambda gtl 1 expression libraries with Ki-67, several cDNA clones could be detected that all contained a 62 bp element with high homology (65-100%). This element was presumed to encode for the epitope recognized by the antibody Ki-67. In ELISA tests and Western blots the antibody Ki-67 reacted with bacterially expressed fusion proteins corresponding to parts of the so far determined eDNA sequence. The aim of this study was to compare the monoclonal antibodies IND.64 and Ki-67 at the molecular level.
Material and methods Cells and specimen. IM-9 (multiple myelomacell line, ATCC No. CCL 159, courtesyof Prof. H. Stein, Berlin, FRG) and the mouse hybridoma cell lines were cultured under standard conditions in RPMI 1640medium(Gibco, Berlin,FRG) supplementedwith antibiotics and 10% fetal calf serum. Antibodies. The monoclonal antibodies Ki-67 and IND.64 were prepared as described previously (Gerdes et al. 1983; Meggetto et al. in press). Alkaline phosphatase-conjugatedgoat anti-mouse IgG was obtained from Dianova (Hamburg, FRG).
260
Peptide synthesis. The synthetic peptide used in this study was kindly synthesized by Dr. Kullmann, Institut fiir Neurobiochemie, Hamburg, FRG. This peptide represents the amino acid sequence deduced from a 62 bp DNA region presumed to encode for the Ki-67 epitope (Gerdes et al. 1991).
targeted with goat anti-mouse alkaline phosphatase conjugate (Dianova, Hamburg, FRG), diluted 1 : 10000 in blocking solution. The phosphatase activity was visualized using nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates (Leary et al. 1983).
Preparation of recombinant parts of the Ki-67 antigen
Competition ELISA
Construction ofplasmids. Nucleotides 1-1002 of the recently published Ki-67 antigen cDNA sequence (Gerdes et al. 1991) were cloned between the Eco R1 and Pstl sites of the pAX4a+ vector (Markmeyer et al. 1990) (Medac, Hamburg, FRG). The resulting plasmid was designated pAX X2/1 and was transformed into E. coli JM 109 (Yanisch-Perron et al. 1985). A 100 bp fragment including the 62 bp repetitive element which presumably encodes for the Ki-67 epitope was ligated between the Eco R1 and Bam H1 sites of the pEV vectors (Crowl et al. 1985) in all reading frames. The resulting plasmids were designated pEVvrf l/B3, pEV-vrf 2/B3, pEV-vrf 3/B3 and transformed into E. coli RR1 (Bolivar and Backmann 1979).
Polystyrene microtiter plates were incubated overnight at 4~ C with 0.5 Ixg/well of lysates of induced E. coli JM109 pAX X2/1 expressing the 1002 bp insert in a total volume of 50 p.l PBS. After 30 min incubation at room temperature with the synthetic peptides at different concentrations, the monoclonal antibodies were subjected to precoated ELISA plates. Nonspecific binding was blocked by 2% gelatin in TBS (TBS-Gel). The plates were incubated 1 h at 37~ C. After three washes with TBS all wells were incubated for 1 h at 37~ C with 50 lal of a goat anti-mouse AP conjugate (Dianova, Hamburg, FRG), diluted 1 : 10000 in TBS-Gel followed by three washes with TBS and an incubation with 50 Ixl/well phosphatase substrate (Sigma 104-0, 1 mg/ml in TBS, pH 9,5) at 37~ C. Optical densities were evaluated at 405 nm on a Flow immunoplate reader (FLOW Laboratories, Meckenheim, FRG).
Preparation of bacterial cell lysates. An overnight culture of E. coli JMI09 containing pAX X2/1 was diluted to an O.D.55o of 0.3, grown to an O.D.55o of 1.0 and induced with 1 mM isopropylthio-fl-galactoside (IPTG) according to Markmeyer et al. (1990). After an additional incubation for 2.5 h at 30~ C, bacteria were harvested by centrifugation, washed once in phosphate-buffered saline (PBS), resuspended in 1/20 volume of PBS and stored at - 2 0 ~ C until further used. E. coli RR1 harboring pEV-vrf 1-3/B3 plasmids were cultured at 37~ C in M9 medium (Sambrook et al. 1989) supplemented with 40 Ixg/ml ampicillin, 0.5% glucose and casamino acids, respectively, until they reached on O.D.sso of 0.8. Subsequently bacteria were harvested and lysates were prepared for SDS-PAGE and Western blots as described below.
Preparation of native Ki-67 antigen IM-9 cells (6 • 107) were pelleted by centrifugation at 400 • g for 10 min and resuspended in 500 ~tl PBS containing 1 mM phenylmethylsulfonyl-fluoride (PMSF). The cell suspension was snap-frozen in liquid nitrogen and homogenized in a mortar cooled with liquid nitrogen. The cell debris was solubilized by boiling for 5 min in 0.5 ml double concentrated SDS-PAGE sample buffer (Laemmli 1970) with 100 mM DTT (see below). The insoluble DNA was destroyed by short sonication.
Antibody subclass determination The subclass determination was performed with the dip-stick method (Holland Biotechnology BV, Leiden, The Netherlands) according to the manufacturer's instructions.
Affinity determination of monoclonal antibodies Ki-67 and IND.64 The relative affinities of both antibodies were determined by use of the method described by Rath et al. (1988). Briefly, in the competition ELISA (see above) the synthetic peptide was used in different concentrations to compete for the antibody binding. The affinity values were calculated from the resulting optical densities and the corresponding peptide molarity.
Results Isotype analysis A c c o r d i n g to the d i p - s t i c k a s s a y Ki-67 p r o v e d to be o f IgG1 k a p p a a n d I N D . 6 4 o f I g G 2 b k a p p a s u b t y p e .
SDS-PAGE SDS gel electrophoresis using 5% or 7.5% separation gels, respectively, and 3.5% stacking gels was performed according to Laemmli (1970). Sample buffer contained 50mM Tris/HC1, pH 6.8, 4% SDS, 20% glycerin and 100 mM DTT. Samples were boiled for 5 min before electrophoresis. Molecular weight markers were obtained from Pharmacia/LKB (Freiburg, FRG) or Sigma (M/inchen, FRG).
Immunoreactivity of Ki-67 and IND.64 with the native nuclear antigen I M - 9 cells were lysed, s u b j e c t e d to S D S - P A G E , a n d W e s t e r n b l o t s were i m m u n o s t a i n e d with Ki-67 a n d I N D . 6 4 , respectively. As d e m o n s t r a t e d in Fig. 1, b o t h a n t i b o d i e s r e a c t e d with two p o l y p e p t i d e c h a i n s w i t h a n a p p a r e n t m o l e c u l a r w e i g h t o f 345 k D a n d 395 k D , respectively.
Immunoblotting Proteins were transferred to nitrocellulose membranes (BA 85, Schleicher & Schuell, Dassel, FRG) by the Western blot technique (Towbin et al. 1979). The transfer was performed at 50 mA for 16h in a water-cooled device. Unoccupied binding sites were blocked by incubation for 1 h with 1% BSA or gelatine in Trisbuffered saline (TBS). After 1 h incubation with the primary antibody and thorough washing with TBS, the bound antibody was
Immunoreactivity of Ki-67 and IND.64 with two bacterially expressed proteins deduced from Ki-67-cDNA 1002 b p a n d 100 b p f r a g m e n t s o f the Ki-67 c D N A were b a c t e r i a l l y expressed, a n d the lysates were s u b j e c t e d to
261
Fig. 1. Immunoblot analysis of lysates obtained from IM-9 cell line cells. Western blots were immunostained with the monoclonal antibodies Ki-67 and IND.64. Both antibodies react with a double band with molecular weights of 345 kD and 395 kD
S D S - P A G E . Subsequently, Western blots were immunostained with Ki-67 and IND.64. As shown in Fig. 2, the antibodies reacted with the fusion proteins in similar patterns, while the controls (no insert for the p A X X2/1 derived fusion protein, wrong reading frame for the pEV-vrf/B3 derived fusion protein) were negative for both antibodies.
Competition ELISA For further comparison of the antigen binding characteristics o f the two antibodies a synthetic peptide containing the putative epitope recognized by Ki-67 was tested in a competition ELISA. As shown in Fig. 3, the binding o f both antibodies to the recombinant parts of the Ki-67 antigen could be inhibited with the peptide in a dose dependent manner. Thus, the peptide was able to compete for the paratopes of both Ki-67 and IND.64.
Relative affinities of Ki-67 and IND.64 Using the synthetic peptide in a molar range from 1.6 • 10 -4 M to 4 • -8 M, the relative affinity constants could be calculated from a similar competition assay: K i - 6 7 : 2 • 10 -6 M; I N D . 6 4 : 1 3 x 10 -6 M. Thus, the rel-
Fig. 2. Immunoblot analysis of recombinant parts of the Ki-67 antigen. A Bacteria were transformed with a vector containing a 1002 bp cDNA fragment (lanes 1 and 3) or, as a control, with the same vector containing no specific insert (lanes 2 and 4). Ki-67 and IND.64 react with identical bands representing the expression
product of the DNA insert. B A 100 bp fragment, containing the 62 bp element presumed to contain the epitope recognized by antibody Ki-67 was expressed in bacteria in the correct (lanes 1 and 3) and in a wrong (lanes 2 and 4) reading frame. The antibodies Ki-67 and IND.64 only react with the lysates from bacteria containing the cDNA insert in the correct reading frame
262 Acknowledgements. The authors wish to thank Margrit Hahn, Bet-
tina Baron and Claudia Wohlenberg for their skillful technical assistance. The work was partly supported by the Dr. Mildred Scheel Stiftung ffir Krebsforschung (project W49/90/Ge2) and L'Association pour la Recherche sur le Cancer (ARC).
Z,ooI =P ;,~
80
~
eo
g
--X-- KI-e7 -e-
References
IND.84
E
.E
40
~
2o
0
A
o 0
2
4
6
8
10
12
14
Peptlde concentration [IJg/welll
Fig. 3. The synthetic peptide deduced from the 62 bp eDNA element presumed to encode for the Ki-67 epitope was tested in a competition ELISA for its ability to compete for the binding of the monoclonal antibodies Ki-67 and IND.64 to the solid phase bound pAX X2/1-derived fusion protein. Both antibodies are displaced by the peptide in a dose-dependent manner
ative affinity of I N D . 6 4 was found to be 6.5 times lower than that of Ki-67.
Discussion
Because of the similar immunohistochemical staining pattern o f Ki-67 and IND.64, the aim of this study was to compare the immunobiochemical characteristics o f these antibodies. Recently, the group from Borstel (Gerdes et al. 1991) found that the antibody Ki-67 reacts with two polypeptides of 345 k D and 395 kD, which are detected exclusively in proliferating cells. The results of the present study show that the antibody I N D . 6 4 detects two polypeptides with identical apparent molecular weights. Furthermore, both antibodies similarly stained Western blots of two different recombinant parts of the Ki-67 antigen. It was noteworthy that I N D . 6 4 even recognized the pEV-vrf/B3-derived fusion protein, representing the translation product of the 62 bp Ki-67 c D N A fragment which was recently assumed to encode for the epitope recognized by Ki-67. In addition, the synthetic peptide which was deduced from the 62 bp c D N A was able to compete for the binding of both antibodies. Thus, we conclude that Ki-67 and I N D . 6 4 react with the same antigen, and in the light of these findings it is very likely that both antibodies recognize the same epitope. At least, the epitopes of I N D . 6 4 and Ki-67 must be located close to each other on a domain of only twenty amino acids in length. Considering that preparations of tissue or nuclei were used as immunogens for the generation o f both monoclonal antibodies, it is tempting to assume that this area of the antigen is imm u n o d o m i n a n t when the whole molecule is used for immunization. In conclusion IND.64 can be regarded as a true Ki-67 equivalent, exhibiting a different affinity and immunoglobulin subclass. This antibody m a y be useful for double immunostaining and m a y contribute to understanding the presently unknown function of the antigen it recognizes.
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