Rat Antikeratin Monoclonal Antibodies are Specific for
Stratified Squamous Epithelia
NENA W. CHIN, M.D., PH.D., BAI-TANG LAI, M.D., AND KARL W. LANKS, M.D., PH.D.
Laboratory Service, Veterans Administration Medical Center
and Department of Pathology, State University of New YorkHealth Science Center at Brooklyn, Brooklyn, New York;
and Beijing Lung Tumor Research Institute, Beijing,
Peoples Republic of China
Seven monoclonal antibodies of rat origin were generated
against human epidermal keratins and were assayed by immunoblot and by immunoperoxidase staining of frozen sections
from various human epithelial tissues. By immunoblotting, the
antibodies recognized two different subsets of high molecular
weight cytokeratin polypeptides. Unlike the majority of the
reported mouse monoclonal antibodies obtained to similar immunogens, these rat monoclonal antibodies showed an unexpected degree of specificity, i.e., they stained only stratified
squamous epithelia and not simple epithelia nor urothelia. The
potential of using rats and the appropriate immunogen to generate more tissue-specific anticytokeratin monoclonal antibodies is discussed. (Key words: Rat; Antikeratin; Monoclonal
antibodies) Am J Clin Pathol 1988; 89: 315-320
THE CYTOKERATINS (CKs) of epithelial tissues
comprise a most intriguing class of intermediate filaments that are related biochemically and immunologically to epidermal keratins. By two-dimensional polyacrylamide gel electrophoresis, a total of 19 polypeptides
have been resolved in various human epithelia and distinct subsets are reported to be characteristic for each
epithelial type.16 For diagnostic purposes, it would be
desirable if a panel of monoclonal antibodies to specific
CKs could be used to identify the tissue of origin of
poorly differentiated epithelial tumors. Various groups
of investigators have pursued this goal by generating
murine monoclonal antibodies to CK extracts from
human epidermal tissues,7"9'25"26 bovine muzzle epidermis,6 and cultured epithelial cells such as HeLa
cells,4'20 PtKj cells,15 human hepatoma cells,7"9 and pig
kidney epithelial cells." Extended studies have been
performed using these monoclonal antibodies but, so
far, it has not been possible to establish tissue specificity
based on the type of epithelium.3101719'21'24 Nevertheless, some degree of specificity albeit limited, has been
reported. For example, those monoclonal antibodies
generated using CK-enriched preparations from HeLa
Received March 24, 1987; received revised manuscript and accepted
for publication August 31, 1987.
Supported in part by a Veterans Administration Research Advisory
Group Award to Dr. Chin.
Address reprint requests to Dr. Chin: Laboratory Service (113)
Brooklyn Veterans Administration Medical Center, 800 Poly Place,
Brooklyn, New York 11209.
cells and other cultured epithelial cells as immunogens
stain only simple epithelia and not stratified epithejj a 4,7,1 !,i5,2o j n con t r ast, those obtained using epidermal
keratins as immunogens stain both stratified and simple
epithelia.6"9'25"26 For diagnostic purposes, the latter
group of monoclonal antibodies may be useful as a universal probe for all types of carcinomas.
From a biochemical standpoint, the reactivity of the
monoclonal antibodies to more than one CK polypeptides may explain the lack of tissue specificity. Indeed,
the results of the immunoblot analysis indicate that
common antigenic determinants exist in the various CK
species (see review2), thus making it difficult to obtain
monospecific antibodies. A possible approach to overcome this problem may have been found by Roop and
co-workers22 who reported the production of monospecific antisera by injecting synthetic peptides bearing the
unique carboxyl terminal amino acid sequences of two
mouse keratin subunits. It remains to be seen whether
this approach can provide monospecific antibodies with
the desired tissue specificity.
At present, all the reported monoclonal antibodies to
cytokeratin are of murine origin. This study reports a
group of rat monoclonal antibodies to human epidermal
keratins that specifically recognize stratified squamous
epithelia with the exception of esophagus. Unlike the
conventional murine monoclonal antibodies to the
same immunogen, i.e., epidermal keratins, the antibodies reported herein do not stain simple epithelia nor
transitional cell epithelia. Bearing in mind the potential
usefulness of tissue-specific antikeratin monoclonal antibodies in the diagnosis of epithelial tumors, this study
suggests the use of rats and an appropriately selected
immunogen, for example, a CK preparation from a
solid carcinoma, to generate monoclonal antibodies
with the desired specificity.
315
CHIN, LAI, AND LANKS
316
Materials and Methods
Preparation of Human Epidermal Keratin
The extraction procedure was performed as described
by Fuchs and Green.5 Human plantar callus was filed
into a fine powder and then Dounce-homogenized in 5
mL of 10 mmol/L TRIS-HC1 (pH 7.6) containing 10
mmol/L ethylenediaminetetraacetate (EDTA). The homogenate was then centrifuged at 8000 g for 5 minutes,
and the pellet washed several times in TRIS-EDTA
buffer. Extraction was achieved byfirstresuspending the
pellet in a solution containing 2% sodium dodecylsulfate
(SDS) and 10 mmol/L 2-mercaptoethanol at 37 °C for
15 minutes followed by sonication (4X15 s). The suspension was then heated at 100 °C for 2 minutes and
centrifuged to remove any insoluble residue. The final
preparation was stored at -20 °C after electrophoretic
analysis of the constituent polypeptides.
SDS-Polyacrylamide Gel Electrophoresis
Electrophoresis was performed in a one-dimensional
5 to 15% gradient slab gel according to the discontinuous TRIS-glycine system of Laemmli,13 and modified
as previously described.'
Immunization Procedure
Six male Wistar rats were immunized by intraperitoneal inoculation with 100 jug of human epidermal keratin emulsified with Freund's complete adjuvant. Two or
more such injections were administered at one- to twoweek intervals, with a final booster given 72 hours before the fusion.
Hybridoma Production
Monoclonal antibodies were generated according to
the basic outline of Kohler and Milstein,12 and modified
as previously described.14 P3U-1 myeloma cells (kindly
provided by Dr. J. Lewis) and rat spleen cells were fused
in the presence of 30% (v/v) polyethylene glycol 1000,
and the fused cells grown in Costar 96-well microculture
plates (105 cells per well). After culture for 2 weeks in
Dulbecco-modified Eagle minimal essential medium
with 10% fetal calf serum containing hypoxanthine and
thymidine, the culture supernatants were screened by
RIA against purified epidermal keratin. Hybridomas
that were positive by RIA were further screened by immunoblot analysis. Those hybridomas that were specific
for keratin subunits were subcloned by limiting dilution
in soft agar.
A.J.C.P. • March 1988
Solid-Phase RIA
The solid-phase RIA involved drying 1 ng of extracted
human epidermal keratin onto each well of a 96 well
U-bottomed polyvinyl plate, followed by rinsing twice
with 1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) to minimize nonspecific adsorption. Then each well was incubated sequentially at room
temperature for 30 minutes with 50 uL each of the following: (1) hybridoma culture supernatant; and (2) l25Ilabeled rabbit antirat immunoglobulin G (107 cpm/
mL). The wells were rinsed with PBS, then with 1% BSA
in PBS after each incubation. Individual wells were cut
from the plates and the radioactivity was determined in
a gamma counter.
Immunoblot Analysis
To identify the keratin subunits recognized by the
hybridoma antibodies, the separated epidermal keratin
polypeptides were subsequently electrophoresed onto
nitrocellulose paper according to the procedure of Towbin and co-workers.23 After a brief rinse in 10 mmol/L
TRIS-HCl/saline (pH7.4) the whole sheet was incubated
in 50% newborn calf serum in the same buffer for 1 hour
at 37 °C. One-centimeter vertical strips were then cut
from the sheet and incubated for 30 minutes at room
temperature with the various undiluted hybridoma culture supernatants, followed by 125I-labeled rabbit antirat
IgG (5 X 106 cpm/mL) for 1 hour. The strips were
washed extensively with TRIS-saline buffer between incubations and exposed for autoradiography after air
drying.
Indirect Immunoperoxidase Staining
Fresh surgical specimens were obtained from the Laboratory Service of the Brooklyn Veterans Administration Medical Center except cervix tissue which was
kindly provided by Dr. I. Hitti of Wyckoff Heights Hospital of Brooklyn. Cryostat sections were air-dried for 1
to 2 hours, thenfixedin methanol for 10 minutes at -20
°C. Following inactivation of endogenous peroxidase
activity by a 5-minute incubation with H202-methanol
solution (1:5), the sections were washed in PBS and serially incubated with the following reagents with intervening PBS washes: (1) hybridoma supernatant for 30
minutes; (2) biotinylated rabbit antirat IgG for 30 minutes; (3) avidin-biotinylated horseradish peroxidase
complex for 45 minutes; and (4) solution of 3-amino-9ethyl carbazole (AEC) and 1% H 2 0 2 for 20 minutes.
Sections were counterstained with hematoxylin and
mounted in Geltol.
ANTIKERATIN MONOCLONAL ANTIBODIES
Vol. 89 • No. 3
317
Results
Hybridoma Production and Selection
Two fusion experiments were performed. Of 1,920
wells plated, 161 contained hybridomas and were
screened by solid-phase RIA followed by immunoblot
analysis. There was no absolute correlation between the
RIA and the immunoblot reactivity, i.e., some supernatants were strongly positive by RIA but negative by immunoblot whereas others were weakly positive by RIA
but clearly positive by immunoblot. The monoclonal
antibodies were of the IgG type since both screening
methods were specific for that immunoglobulin class.
Immunoblot Analysis
Reactivity of the hybridoma supernatants that were
weakly to strongly positive by RIA was tested against the
human keratin preparation originally used as antigen
and the results are shown in Figure 1. Lane A is the
Coomassie-blue stained gel of the extracted human epidermal keratins showing several major bands with apparent molecular weights ranging from approximately
46 to 67 kD. Lanes B, C, D, and E are the immunoblots
of the same keratin extracts incubated with the polyclonal antibody, monoclonal antibodies 12H6, 1F6, and
a nonreactive hybridoma, respectively. As expected, the
antikeratin antiserum recognizes most, if not all of the
keratin subunits, whereas each monoclonal antibody
recognizes a major subset of polypeptides, namely those
either in the 66 to 67 kD (lane C) or 66 kD (lane D)
molecular weight range and a few minor bands. Based
on these two immunoblot staining patterns, the monoclonal antibodies can be divided into two main groups:
(1) 12H6, 5F5; and (2) 1F6, 5B3, 12F11, 12H2, 18B1.
The nonreactive hybridoma shows little antibody reactivity (lane E).
Immunoperoxidase Staining
The seven monoclonal antibodies that showed intense
staining on the immunoblots were tested on a series of
methanol-fixed frozen sections and the results are summarized in Table 1. As can be seen, all the monoclonal
antibodies (except 12F11 and rarely 1F6) stained all
stratified squamous epithelia except esophagus. Surprisingly, 12F11 was nonreactive on tissue sections even
though it clearly stained some of the keratin bands by
immunoblot analysis; 1F6 did not stain oral mucosa
and tongue. Transitional and simple epithelia were negative with the monoclonal antibodies as were the nonepithelial tissues and HeLa cell monolayer. The polyclonal rat antibody was always included as a positive
<^K
52K46K-
B
D
FIG. 1. Coomassie-blue stained gel of human epidermal keratins (A)
and the corresponding immunoblots after reaction with the rat antikeratin antiserum (B), with monoclonal antibodies 12H6 (C), 1F6 (D)
and with nonreactive hybridoma (£). (K = kDa)
control and indeed, it stained all types of epithelial tissues examined including esophagus and transitional cell
epithelium. Figure 2 shows the avidin-biotin immunoperoxidase staining patterns of normal stratified squamous epithelia (skin, oral mucosa, tongue, tonsil and
exocervix) using the monoclonal antibody 18B1. Of the
sections used in this study, exocervix is the only tissue
without a keratinized layer. There was no apparent difference in the immunostaining pattern by the two
groups of monoclonal antibodies, and all the tissues
showed cytoplasmic staining in the suprabasal layers
only. The basal layer was consistently nonreactive with
the monoclonal antibodies. The staining pattern was essentially diffuse, but occasionally individual or small
groups of cells stained heavily as in the tongue and tonsil
sections. In contrast, the antiserum showed both basal
and suprabasal staining of the different stratified squamous epithelia (not shown).
Preliminary results on the staining of neoplastic tissues are also included in Table 1. Well-differentiated
squamous cell carcinomas of the skin, tongue, and lung
were stained with the monoclonal antibodies and the
reaction product was seen in the keratin whorls as well
as the superficial, more mature-looking malignant epithelium (not shown). The staining with 1F6 in the carci-
CHIN, LAI, A N D L A N K S
318
AJ.C.P. • March 1988
Table 1. Reactivity of Rat Monoclonal Antibodies to Frozen Sections of Human Tissues
1F6
5B3
5F5
Stratified epithelia
Skin
Oral mucosa
Tongue
Tonsil
Exocervix
Esophagus
Urinary bladder
+
+
+
—
+
+
+
+
+
—
+
+
+
+
+
—
Simple epithelia
Liver
Lung
Thyroid
Kidney
Prostate
Synovial tissue
Endocervix
Breast
-
*
*
-
*
*
—
+
±
*
*
+
*
*
+
*
•
*
—
*
*
*
*
—
*
*
*
*
—
Nonepithelial tissues
Muscle
Connective tissue
Lymphocytes
—
—
—
—
Cultured cells
HeLa cells
-
-
-
Neoplastic epithelia
Skin: SCC
BCC
Tongue
Lung
SCC
Adenoc
Stomach
Colon
Urinary bladder
12F11
12H2
12H6
18B1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Polyclonal
Antibody
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
SCC: squamous cell carcinoma; BCC: basal cell carcinoma; Adeno: adenocarcinoma.
* Not done.
noma of the tongue was essentially negative since only
patchy stain was present in the external keratinized
layer. As expected, carcinomas of the basal layer of the
skin, of glandular and of urothelial epithelia were all
negative with the monoclonal antibodies.
Discussion
This report has described the generation and characterization of a panel of seven rat monoclonal antibodies
raised against human epidermal keratins. This batch of
antibodies is unique because rat spleen was used, instead
of the conventional mouse spleen, to generate the antibodies. Rats were used because a single spleen could
provide enough cells for one or more fusions and because a sufficient amount of immune serum could be
obtained in the same experiment to serve as a positive
control. Unexpectedly, a total of seven rat monoclonal
antibodies was easily obtained which showed a tissue
specificity that is different from the majority of the con-
ventional murine monoclonal antibodies directed to the
same immunogen, i.e., epidermal keratins. The murine
antikeratin monoclonal antibodies generally stain skin
as well as simple epithelia and other stratified epithelia
including urothelium.3,8,9'20'25 In contrast, the rat monoclonal antibodies stain in this study stratified squamous
epithelia only, with the exception of esophagus and
there is no reactivity with simple epithelia and transitional cell epithelia.
Two murine monoclonal antibodies to epidermal
keratins deserve mention because their tissue specificity
is similar in some respect to that of the rat monoclonal
antibodies presented herein. Like the rat monoclonal
antibodies, the AE2 murine monoclonal antibody of
Sun and associates24,26 stains the suprabasal cells of the
epidermis (keratinized) and not the esophagus and
bladder (nonkeratinized). However, the rat antibodies in
this study, in addition, also stain other tissues that are
not keratinized but which have the potential to keratinize (e.g., oral mucosa, tongue, tonsil and exocervix). The
Vol. 89 • No. 3
ANTIKERATIN MONOCLONAL ANTIBODIES
319
FIG. 2. Biotin-avidin immunoperoxidase staining of methanol-fixed cryostat sections of the skin (A), oral mucosa {B), tongue (C), tonsil (Z>), and
exocervix (E) using rat antikeratin monoclonal antibodies. Positive immunoreactivity is indicated by the presence of dark gray to black areas. (F)
High-power magnification displaying the granular positive stain in the cytoplasm. Hematoxylin (A-E, XI20; F, X630).
K-6 antibody of Osborn and co-workers'8 is reported to
stain squamous epithelia but specific details are not
given.
The lack of tissue staining in esophagus, simple epithelia and bladder using these rat monoclonal antibodies can be explained by the fact that these epithelia contain CKs that are different from those found in foot sole
epidermis, the source of the immunogen in this study.16
An exception to this observation is CK #5 (58 kD)
which is present in both esophagus and skin. From this
immunoblot analysis, however, it is apparent that there
was no reactivity against any polypeptide in the 58 kD
region; thus, this is consistent with the absence of tissue
staining in esophagus.
Common antigenic determinants in the higher molecular weight CK subunits were noted on the immunoblots using the various rat monoclonal antibodies. Even
though these antibodies reacted with two different subsets of CK polypeptides, the overlapping activity in the
66 kd band probably accounted for the lack of differential tissue staining by the two sets of antibodies. One of
the monoclonal antibodies, 12F11, did not stain any of
the tissues examined despite a positive immunoblot. A
similar observation has been reported by Lane15 who
CHIN, LAI, AND LANKS
320
found that three of his six monoclonal antibodies have
not reacted with any frozen tissue sections. It is conceivable that the antibodies do not recognize the "native"
(non-SDS denatured) antigen. Another of our antibodies, 1F6, did not stain oral mucosa and tongue; it would
be of interest to determine if this antibody might be
useful in excluding carcinomas of the oral cavity. Preliminary data using malignant epithelia concur with the
staining reactivity of their normal counterparts.
The tissues that were stained by the rat monoclonal
antibodies have the same histologic phenotype as that of
the immunogen, that is, both are related to stratified
squamous epithelia. This type of relationship also exists
in other studies in which murine monoclonal antibodies
to CKs of simple epithelial cell lines4,7"915 stained only
simple epithelia. This is the only study currently that has
used the rat system to generate monoclonal antibodies
to keratin, and because of the tissue specificity obtained,
albeit limited, it may be possible to immunize rats with
an appropriately selected immunogen and produce
monoclonal antibodies which may have the desired tissue specificity.
Acknowledgements. The authors thank Mr. V. Shah for technical
assistance, Dr. R. Libbin for photomicrography, Dr. I. Hitti for providing fresh tissue from cervix, and Mrs. A. Sellitti for clerical assistance.
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