Note: Descriptions are shown in the official language in which they were submitted.
1315220
This invention relates to monoclonal antibodies
capable of reacting with human carcinoma cells and to a
process of producing these monoclonal antibodies. __
1315220
The advent of the hydridoma technology(l) for the
- production of large amounts of high titered monoclonal anti-
bodies ~MCAs) with a unique specificity for the antigen
has greatly improved the possibility of probing the cell-
surface antigenic complex of tumor cells and spurred efforts
to generate tumor-specific antibodies. Investigators have
used a variety of approaches for producing MCAs that recognize
various antigenic structures on malignant and/or normal breast
tissues.( 1 ) In the present specification, the Applicant
describes the use of hybridoma technology to generate a panel
of hybridoma-secreting monoclonal antibodies that react with
a high degree of selectivity to human breast cancer cells.
The potential of these MCAs to probe surface protein differ-
ences between normal and malignant breast cancer cells will
also be decribed. BT-20 breast carcinoma cell line was used
as a source of antigens because it was derived from a poorly
differentiated tumor(l9) and has been well characterized and
- -~ ~ extensively studied.(20 26) More importantly for the present
invention, these cells were very tumorigenic when implanted in
the nude athymic mice, producing nodules with the same mor-
phologic characteristics as the original tumor.(21) These
cells seem to have kept the properties of breast carcinoma.
In U.S. Patent No. 4,522,918 there are described
monoclonal antibodies which demonstrate a reactivity with
human breast cancer. However, these antibodies do not react
with 100~ certainty with the result that tests that can be
made with these antibodies cannot be totally reliable.
It is the object of the present invention to provide
methods of production, selection and characterization of
monoclonal antibodies capable of reacting with a large panel r
of cultured human breast carcinoma cells~and biopsies.
- l~r
'~''''~"''`''".
- 1 3 1 5220
This invention relates to a process for producing
monoclonal antibodies capable of reacting with human breast
carcinoma cells, which comprises:
a) providing a breast cancer cell line of known
aggressiveness;
b) culturing confluent monolayers of the breast
cancer cell line in serum-free culture medlum, overnight;
c) harvesting the cultured cell line mechanically,
such as with a rubber policeman and in the absence of trypsin;
d) immunizing mice with the harvested cell line
obtained in (c);
- e) fusing spleenocytes of mice immunized as in (d)
with myeloma cell line to give hybridoma cultures;
f) selecting hybridoma culture supernatants that
contain antibodies reacting in an ELISA assay with BT-20
versus HsL-100 used here as a discriminatory cell line of
. normal human breast origin; and
g) isolating stable hybridoma producing monoclonal
antibodies by cloning.
In accordance with a preferred embodiment of the
invention, the breast cancer cell line has been selected from
a group of breast cancer cell lines consisting of BT-20,
BT-483, BT-549, CaMa, ZR-75-30, ZR-75-1, MCF-7, SKBR 3 and
T-47D, the most preferred being BT-20.
- In accordance with another preferred embodiment
step (d) is carried out by hyper immunizing sALB/c mice.
In accordance with another preferred embodiment of
the invention, spleenocytes of mice are fused with the myeloma
cell line, NS-l.
In accordance with yet another embodiment of the
invention isolated stable hybridoma producing monoclonal
1 3 1 5220
antibodies are separated into different groups based on several
criteria. The following grouping are evident monoclonal anti-
bodies which:
: 1. recognize a protein moiety;
2. correspond to an antigen which is localized
in the cytoplasm;
3. react with substantially 100% of breast
cancer biopsies of primary and metastatic
origin in cryostat sections,
4. do not react or react very weakly with
normal breast tissue;
and/or
1. recognize a glycoprotein moiety,
2. correspond to an antigen which is localized
at cell surface;
3. react with a limited number of primary
and metastatic breast cancer biopsies; and ,
- 4. also recognize normal tissues of breast and
non-breast origin.
The invention is illustrated by means of the
-- , annexed drawings, in which:
i~ FIGURE l represents the SDS-PAGE electrophoresis
of purified monoclonal antibodies;
FIGURE 2 represents curves showing the quantitative
binding of lF10B4, lFlOG2 and 3C6F9 to BT-20 and HBL-100
cell lines; and
FIGURE 3 is a photomicrograph of four immuno-
fluorescence staining of acetone-fixed cultured cell lines
and primary breast carcinomas.
MATERIAL AND METHODS
CULTURED HUMAN CELL LINES: Nine well established
human ~ ~ry tumor cell lines (26 to 32) were maintained as -~
k
.
1 3 1 5220
,,~
continuous cultured and repeatedly tested to ensure the
absence of any mycoplasma infection. Most of these cell lines
were either purchased from the American Type Culture Collection or
kindly obtained from EG & G Mason Research Institute (Rockville,
Maryland). Characteristics of each of these cell lines is
given in Table 1. Additional cell lines used included cultures
of apparently normal m~mm~ry cells, HBL-100, an epitheiial
cell line derived from human milk sample (33), and two human
fetal foreskin cells lines, SW-480 and CC-95. All cell lines
were routinely maintained in RPMI-1640 medium (Grand Island
Biological Co.; Burlingtony Ontario, Canada) supplemented with
0.2 mM L-glutamine, 20 mM hepes buffer, lOYo heat-inactivated
fetal calf serum and antibiotics (penicillin 100 IU/ml,
streptomycin 10 ~ug/ml and fungizone 10 ,ug/ml). Single cell
suspensions of each monolayer culture were obtained by
treatment with trypsin-EDTA in Hank's balanced salt solution.
Cells were maintained at 37C in a humidified atmosphere of
95% air and 5% CO2. Tests for mycoplasma contamination
- using agar culture were performed and only negative cell
lines were further used.
HYBRIDOMA METHODOLOGY:
(a) Hyperimmunization and Fusion: Female BALB/c mice
were hyperimmunized by 3 subcutaneous injections of 10 BT-20
cells emulsified in complete Freund's adjuvant. For all the
immunizations BT-20 cells were cultured in serum-fr-ee RPMI
medium for 24 hours before harvesting and were detached from
the culture vessel by means of a rubber policeman. Rise in
serum antibody titers in immunized mice was monitored weekly
and animals showing high response to BT-20 by the ELISA
(Enzyme-Linked Immunosorbant Assay) technlque were selected
; for fusion. At least 1 month after the last immunization,
.
1 ~ 1 5220
mice were boosted i.p. with 107 BT-20 cells. Three days
later spleenocytes were fused with the non-immunoglobulin
producing myeloma cell line NS-l (P3-NSl/l-Ag4-1) (34),
using 50% polyethylene glycol as fusing agent.
(b) Antibody-screening assay: As already mentioned
an ELISA technique was used for antibody screening in either
culture supernatants, purified ascitic fluid or in sera of
immunized mice. In preparation for this test, cells (BT-20
and HBL-100) were grown until confluence in sterile tissue
culture multiwell plates (96 flat bottom wells, Linbro) using
complete RPMI medium. Confluent monolayers were then
incubated overnight at 37C in serum-free RPMI-medium and
fixed with a solution of 0.1% glutaraldehyde in PBS (5 min.
at room temperature). This was followed by 3 washes in PBS
pH 7.4 and blocking of non-specific sites by an incubation at
- 37C (200 yl/well) with a blocking solution. This solution
TM
contained 20 ml of citrated milk (100 g of Crino milk powder
~ with 12.3 g of citric acid), 1 g of polyvinyl-pyrolidone (PVP-
- ~ 40) and O.S g of BSA ~bovine serum albumin). To this was
added 75 ml of Tris-HCl buffer, the volume was completed to
100 ml and the pH adjusted to 7.5. The blocking solution
was always filtered on nylon membrane paper before its use.
After blocking the non-specific sites, cells were
rinced twice in PBS then 100 ~ul of antibodies (at different
dilutions) were added to each well and incubated at 37C for
90 min. Cells were then rinced 3 times in PBS and 100 ~1 of
f __ horseradish peroxidase-labeled goat anti-mouse IgG (Miles
~~~ Lab.) diluted 1/75 in dilution buffer (0.5 g of PVP-40 and
0.5 g of BSA, pH 7.5) was added and incubated for 90 min. at
37C. ~ ;
5 --
`~
1 3 1 5220
After 3 washes in PBS, lOO,ul of the substrate (2,2'
azinodi-(3-ethylbenzthiazoline sulfonate and oxygen peroxide,
v:v) was added and incubated for 10 min at 37C and for 20 min
at room temperature, in the dark. The reaction was stopped by
the addition of 50 ~uliwell of a solution of lOYo sodium dodecyl-
sulfate. The optical density was read in a spectrophotometer
at 405 nm wavelength (Titerteck Multiskan photometer, Flow
Lab.).
(c) Selection and cloninq of hybridomas: Following
fusion with ~S-l cells approximately 96% (311) of the seeded
wells showed rapid growth within two weeks. Only 128 (40%)
secreted antibodies that bound to BT-20 cells in the ELISA
assay, of these 21 (16%) reacted weakly or failed to react
with the discriminatory cell line B L-100. After 3 to 5
primary screens, hybridoma cultured were cloned and recloned
twice using the limiting dilution technique in the presence of
irradiated 3T3 mouse fibroblasts as a feeder layer. Six of
these clones were further characterized (Table 2) and injected
into pristane-primed BALB/c mice (2,6,10,14-tetramethyl-
pentadecane, 96%) for the production of ascitic fluid.
(d) Characterization and purification of M~A~: The
heavy and light chain composition of each MCAs was determined
using Ouchterlony gels first against affinity-purified goat-
antiserum specific for each of the murine subclass (IgGl,
IgG2a, IgG2b, IgG3, IgM and IgA) and then to monos~ecific
goat-antimouse immunoglobulin isotypes (Cappel Lab.,
Cochranville, PA). Quantitation of MCAs in culture super-
natants of the cloned hybridomas was performed by a standard
radioimmunoassay using goat anti-mouse IgG (Miles Lab.).
~ 30 Mouse ascitic fluids were clarified by centrifugation (2000
-- rpm for 30 min at 4C) and the immunoglobulin fraction purified
;
1 3 t 5220
by three sequential Am~onium sulfate precipitation. In this
technique, the ~onium sulfate (50/O) solution was added drop
by drop to the ascitic fluid and the mixture kept under
constant agitation. The mixture was then transferred to 50 ~1
conical tubes and placed for at least 16 hours at 4C. The
precipitate was recovered by centrifugation for 20 min at 4C.
This procedure was repeated twice with the precipitate, but
in the second and third precipitation incubation was for only
- 2 hours at 4C. The final precipitate was dissolved in a
O.Ol M Carbonate buffer pH 8.3 and dialysed for 16 hours at
, 4C against the same buffer~ After dialysis the precipi-
tate was diluted in Carbonate buffer so as to dissolve
completely the precipitate. The solution was filtered first
on a Watman no 1 (Canlab) filter paper then on a millipore
membrane 0.2 ~m pores under vacuum (filter unit, type LS
Nalgene, 0.2 ym, Nalge), aliquoted into sterile tubes
(1 mg/ml) and fro~en at -70C until use. The purity of
each MCAs was confirmed by SDS-PAGE electrophoresis (Fig. 1)
under reduced conditions (2-mercaptoethanol). Lanes 1 and 2
show lFlOB4 and lFlOG2(yl) while lane 3 is 3C6F9(y2a).
IMMUNOFLUORESCENCE STUDIES ON CULTURED CELL LINES:
In preparation for indirect immunofluorescence, freshly
trypsinized cells obtained during the logarithymic growth
phase of a culture, were seeded onto pretreated tissue culture
slides and incubated overnight in complete medium. Cells
- were first washed, then either fixed with cold acetone at 4C
for 10 min, or immiediately stored at -70C until used. For
these studies, slides were incubated with hybridoma super-
natants for 20 min at room temperature, then for 30 min with
fluoresceinated goat anti-mouse Ig (Cappel Lab., Cockranville,
PA) diluted 1/30 in PBS. After two washes for 10 min in
_ 7 _
1 3 1 5220
PBS, slides were mounted with 50% glycerol in PBS and were
examined with an epifluorescent microscope. The appropriate
working dilutions of these three MCAs, as tested on acetone-
fixed BT-20 and HBL-100 cells, were established at 1/50
for lFlOB4 and lFlOG2 (20 ~g/ml PBS) and 1/20 for 3C6F9
(50 ~g/ml).
These dilutions were selected because they gave a strong
reactivity on BT-20 cells in the presence of a clear back-
ground.
IMMUNOHISTOCHEMICAL REACTIVITY ON HUMAN TISSUES:
Specimens obtained either directly from the operating room or
from fresh autopsies were snap frozen in liquid
nitrogen and transferred at -70C until used. The specimens
included, (a) 30 primary breast tumors, (b) 7 metastatic
m~m~ry cancers (5 lymph nodes and 2 liver metastasis), (c)
7 benign breast tissues including 5 normal m~mm~ry tissues,
one fibrodenoma and one sclerosing adenosis; and (d) 6
normal tissues of non breast origin. Acetone-fixed cryostat
sections of each specimen were studied using an indirect
immunofluorescence staining. Tissue sections were incubated
for 30 min at room temperature with 250,ul of the diluted
MCAs (same concentrations as determined on BT-20 cells); after
extensive washing with PBS, they were incubated for 30 min
with 100 ~ul of fluoresceinated rabbit anti-mouse IgG (Cappel
Lab., Cockranville, PA) diluted 1/30 in Pss. Fixed sT-20
and HBL-100 monolayers were always used as control slides.
These sections were examined with an epi-illumination
fluorescent microscope. In all instances, an adjacent
section of the frozen tissue was stained with hematoxylin-
eosin and microscopic findings were correlated both with theimmunofluorescence studies and the pathoIogical diagnosis
1 3 1 5220
~,
,,,
performed on paraffin-embedded sections of these cases.
RESULTS
- QUANTITATIVE COMPARISON OF ANTIBODY REACTIVITY ON
BT-20 AND HBL-100: Isotype analysis of these MCAs showed that
antibodies lFlOB4 and lFlOG2 were both of the yl subclass,
while 3C6F9 represented a y2a immunoglobulin (Table 2). All
three MCAs produced high titers of IgG in hybridoma super-
natants fluid. To reach a more quantitative comparison of
their affinity to the target antigens, purified ascitic fluid
10 were used at a concentration of 1 mg/ml of proteins and the
effect of antibody dilution on the differential reactivity
:
to BT-20 versus HBL-100 was determined (Fig. 2). Using
an ELISA assay, we could demonstrate two different patterns
of reactivity; lFlOB4 and lFlOG2 showing a high degree of
selectivity bound strongly to BT-20 (up to 10 2 dilution for
lFlOB4 and 10 3 dilution for lFlOG2 and weakly (10 1 dilution)
or not at all with HBL-100 (Fig. 2). On the other hand at low
dilutions, 3C6F9 reacted with both HBL-100 and BT-20, however,
at 10 3 dilution reactivity was still very strong with BT-20
- 20 and was completely lost with HBL-100. We could also note that
even at 10 dilution 3C6F9 reacted very strongly with BT-20
(Fig. 2). These results were further confirmed on acetone-
fixed cell monolayers by a standard avidin-biotin peroxydase
technique using commercially available reagents (Vectastain,
Trade Mark of Cederlane Lab., Ontario Canada) and the three
- MCAs at the same dilutions.
.
1 31 5220
IMMUNOFLUORESCENCE STUDIES ON CULTURED NORMAL AND
- MALIGNANT CELL LINES. With reference to Fig. 3, there is
illustrated the immunofluorescence staining of acetone-fixed
cultured cell lines and primary breast carcinomas (a) HBL-100
incubated with 3C6F9, (b) BT-20 cells incubated with lFlOG2,
(c) primary c-arcinoma incubated with 3C6F9 and (d) primary
breast carcinoma incubated with lFlOG2. The different pattern
of reactivity demonstrated by these two MCAs will be noted;
IFlOG2 shows a granular cytoplasmic reactivity often associated
with a perimuclear accentuation of staining while 3C6F9 shows
a fine linear positivity at the periphery of the cells. The
enlargement for (a), (b), (c) and (d) is as follows: (a), (b)
and (d) x 1000, c x 400. To localize the antigenic structures
defined by lFlOB4, lFlOG2 and 3C6F9, indirect immunocyto-
~ chemical staining of acetone-fixed and unfixed m~mm~ry
~~ epithelial cells was performed (Table 3). For both lFlOB4
and lFlOG2, a strong cytoplasmic staining was observed on
acetone-fixed BT-20 cells, while unfixed preparations of the
same breast cancer cell line were negative. The appearance
of the cytoplasmic staining was coarsely granular and was
uniformly distributed throughout the cytoplasm with some
accentuation around the perinuclear membrane (Fig. 3b). A
similar pattern was noted for the nine human breast cancer
cell lines studied. However, as illustrated in Table 3,
the intensity of the staining varied from one cell line to
another and there was no correlation between the intensity
of the fluorescence staining and any of the different
,, ,~
characteristics of these cell lines i.e. tumor origin,
tumorigenicity in aminals, hormonal receptors content and
growth in soft agar (Table 1). In contrast to the positivity
-- 10 --
1 3 1 5220
of malignant breast cancer cell lines, lFlOB4 and lFlOG2
showed no staining with the three normal cell lines examined
(Table 3).
Results obtained with 3C6F9 were substantially
different: this monoclonal antibody recognized both acetone-
fixed and unfixed preparations of normal and malignant cell
lines. A positive reaction was detected with all the cell
lines examined (Table 3) regardless of their origin, the
intensity of the reaction varied however, from one cell line
to another and was weaker with the normal cell lines. This
fluorescence was localized, in all instances, at the periphery
of the cells and was uniformly distributed among the cells of
a given preparation (Fig. 3a). These data indicate that the
antigenic determinant recognized by 3C6F9 is associated with
the plasma membrane of the cells and is expressed on both
normal and malignant cultured epithelial cell lines.
REACTIVITY WITH HUMAN MAMMARY TUMORS: To determine
if antigens recognized by these 3 MCAs are also present in
breast tumors of human origin, tissue sections of mammary
tumors were stained by indirect immunofluorescence. Two
groups of m~mm~ry tumors were examined, the first consisted
of 30 primary infiltrating ductal or lobular carcinomas, the
second included 7 metastatic breast cancers (lymph nodes or
liver metastases). As reported in Table 4, l.F10B4 and lFlOG2
exhibited the capacity to react with a wide spectrum of breast
carcinomas of primary and metastatic origin. lFlOs4 recognized
97% of the primary tumors tested while lFlOG2 recognized 100%
of both infiltrating ductal or lobular m~mm~ry carcinomas.
Similar results were obtained for metastatic breast tumors
(Table 4) i.e.; whatever the site of the metastasis, both MCAs
~ ~;
; :
1 31 5220
recognized lO~/o of all the metastatic breast cancers
examined. In 67% ( 20 out of 30) of the primary tumors
studied, a clearly cytoplasmic pattern of reactivity could
be identified (Fig. 3d). This type of staining was identical
to that seen with m~mm~ry epithelial cell lines (~ig. 3b). In
the r~m~in;ng tumors (10 out of 30) the reactivity was mainly
localized at the periphery of the cells usually in a homo-
geneous, discontinuous pattern.
On the other hand, 3C6F9 reacted strongly with
2~/o of the primary breast cancers examined and weakly with 4~/0
of them, illustrating a more limited spectrum of recognition
of primary brea~t cancers. This feature was further accen-
tuated when metastatic tumors were studied, only 4~/0 of these
metastatic tumors stained weakly with 3C6F9 (Table 4). In all
the positive cases, a linear reactivity localized at the cell
.
membrane was observed with 3C6F9 (Fig. 3c). This pattern
of staining was also similar to that noted on cultured
m~ ry epithelial cell lines (Fig. 3a).
- REACTIVITY ON NORMAL BREAST AND NON BREAST TISSUES:
The reactivity of these 3 MCAs was also evaluated on cryostat
sections of 7 benign breast tissues ( 5 normal - - ry tissues,
one fibroadenoma and one sclerosing adenosis), and 6 normal
- . tissues of non breast origin. With lFlOB4 and lFlOG2,
immunofluorescence was negative in glanaular structures in
- -ry lobules but few focal reactivities were sometimes
found in ducts. In these areas a weak staining of the luminal
pole of the epithelial cells lining the ducts was observed.
With 3C6F9 however, a peripherally located reactivity of
variable intensity was noted in mammarY glands and ducts.
On normal tissues of other origin, lFlOB4 and
lFlOG2 showed by fluorescence a very weak reactivity-in some
~l 2_- ,
r~~
1315220
renal tubular epithelial cells, in few keratinocytes and
epithelial cells of sebaceous glands ln the skin and a moderate
staining in biliary ducts of the liver. All mesenchymal
structures, including smooth and striated muscle tissues,
lymph nodes and connective tissues were negative. These
data suggest that the antigenic detel ;n~nts recognized by
lFlOB4 and lFlOG2 are not restricted to malignant - -ry
cells but are probably present in very small amounts in few
normal epithelial cells of other tissues. On the other hand,
with 3C6F9 a strong reactivity was detected on all medial
smooth muscle cells in blood vessels, whereas a moderate
reactivity was found in biliary ducts. Moreover, a weak
positivity was found focally on the basement membrane of the
skin and kidneys specimens studies. Therefore, 3C6F9
recognizes an epitope that is present in the cell membrane of
a large spectrum of normal epithelial cells of different
origin.
An overall ~x ination of the data presented herein
suggests that MCAs described can be divided into two different
groups on the basis of their differential reactivities to human
tumor specimens and cultured cell lines of either malignant or
normal origin. Two of the MCAs namely lFlOB4 and lFlOG2,
showing a wide spectrum of reactivity to human breast
carcinomas, recognize cytoplasmic structures in primary and
metastatic - -ry tumors but fail to react with normal
cultured cell lines and react weakly or locally with
biopsies of benign breast tumors and normal tissues of breast
and non breast origin. On the other hand, 3C6F9 recognizes a
surface membrane det~ inAnt expressed on both normal and
neoplastic mammary epithelial cells as well as on medial
smooth muscle cells in blood vessels.
.
i ::
; :::~
-- 1315220
- ~ It is interesting to note that all three MCAs
reacted with - ry tumor cell lines without discrimination.
On the other hand, while lFlOB4 and lFlOG2 show (even at low
dilutions) little reactivity to ~BL-100, 3C6F9 demonstrate a
comparatively stronger reactivity to the three normal cell
lines studied (Table 3).
Cells in culture may change their behavior and
become unrepresentative of their in vivo counterparts in
expression of certain tumor-associated antigens, particularly
those associated with differentiation. In order to have a
more valid assessment of the antigens recognized by those
three MCAs, their binding to frozen sections of mammary tumors
has been examined using indirect immunofluorescence techniques.
~~ It could be demonstrated that 3C6F9 react with 630/o (19 out
of 30) of the primary breast cancers examined and with only
43/0 (3 out of 7) of the metastatic breast tumors. This
monoclonal antibody identifies an epitope localized at the
periphery of the cells and, as revealed by examination of
unfixed and acetone-fixed cultured cell lines, the antigenic
determinant seems to be associated with the plasma membrane
of both normal and malignant cells.
On the other hand, lFlOB4 and lFlOG2 recognize
a wide spectrum of m~mm~ry tumors staining almost all of the
30 primary and 7 metastatic breast cancers examined, except
for one primary tumor which was negative with lFlOB4. These
two MCAs recognize structures localized in the cytoplasm of
these neoplastic cells, they do not recognize (or very weakly)
epithelial structures of normal breast tissues (7 biopsies)
and non m~mm~ry tissues (6 biopsies).
This wide range of reactivity of lFlOB4 and lFlOG2
for carcinoma of the breast, their different pattern of
- 14 -
Z@ .~
1 31 5220
staining within a single tumor, between different tumors of
the same histological type and between the various types is
very interesting. The fact that they recognize cytoplasmic
determinants, which are usually better preserved in conven-
tional formalin-fixed paraffin-embedded tissues, suggested,
that these two MCAs may have diagnostic, prognostic and
therapeutic applications. Thus MCAs used singly or in
combination could be used in:
1. Radioimmunoassay for the development of
diagnostic kits for the detection of cir-
culating antigens, aiding in the diagnosis,
follow up and the prognosis of breast cancer
patients.
2. In vivo localization of micrometastasis at
the draining lymph nodes or at distant location
(bone, lung, brain) when labeled with radioactive
isotopes.
3. Treatment of primary and/or metastatic breast
lesions using MCAs either alone or coupled to
toxins, drugs or radioactive isotopes.
, -~'~.
: : ~
~ 31 ~2~
a) ~ o 0 0 ~ ,~
~D N ~ t~l ~ ~ ~ ~
~ P~
'~ !
,-'h+ + ~ D
In ~ U
n ~
H
n C
D O D ~D ~D a) ~ ~
' ~ r~ ~ ~ r r ~ r r r
~; ~ r~
' ~ r~ r
~ , r
r~ U U U U r
H f
~ ~ O U~ r
E~ ,3 , ,4~ 3 tr~
_ ., .
In r,
-I ~ C U U ~.) U t.) UU U C) ID . ~
~~ f~ ' C
t~ ~ H H H H H H H ~,~ . t,
C I
., ~r ,~
O ID O ID
r~ rc 'C C~ O U ~r
~ r~ r ~r t ~ O r
~D 't ~ I~ q~ ~ ~ 4 ~ ~ ~ en
2 >~ t~ J '~ 3 en
,, J I ~ _ _ _ L~ 'C ~
, 1 rrJ
f ~-r ~ ~ ~ S- ~, jt~
~ ~ S-e e ,: ~
f~ fl a. a a~
ID r ~
O., ~ C
~ I O
~1 r ~ ~1 ~1-~
- ,,-1 ~ ~ ~ I I r l S- ~ U ~ -I
O ~ fl C ~ U~ a
e I ~: I I ~ m~
~ a E~ o x I
u ~ m q q c~ N N X t~ E~ ta ,R U
l6
TABLE 2
INITIAL SCREENING AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES
IN THE SUPERNATANTS OF CLONED HYBRIDOMAS
Antibody Immunoglobulin ELISAC
Designation
Class and Concentration BT-20 HBL-100
subclass (ug/ml)
3C6F9 IgG2ak 81.2 +++ +
3C6H6 IgGlk 88.0 + o
3C6D7 IgGlk 19.6 + o
lFlOB4 IgGlk 126.0 ++ 0
~1 lFlOHl IgGlk 108.9 +++ +
lFlOG2 IgGlk 135.7 ++ +
__
a. Initial screenings and characterization of cloned hybridomas was performed
using culture supernatant diluted 1:10 in PBS. r~
b. Subclass determination was performed using Ouchterlony gels while quantitation
of MAs in culture supernatant was determined using a standard radioimmunoassay.
c. Optical density (405 nm) was graded a~ follows: (O) = 0.2 or lower,
(+) = 0.4 - 02: (++) = 0.6 - 0.4, and (+++) = 0.8 or higher.
1 31 5220
r~ ' + + + R +
,~ o o o oo o o o o o o o
~, o ~
r o
, -~ ~ o o o oo o o o o o o o
-
H ~ ~ Uq U~ uq u7 uq v~
~ O +
+ + + + + + + +
E~ O ~j ~ rl H H H H H H H H H O O O
~ ~1'
o
O r~l + +
> ~--1 H H H H H H H H H O O O
-
U~
r~
r~
~0 ,~ ,~ O
I I ~ 00
~a~ . N ~ 1` ~ 1` r~
a E~ I o p3 3 o
m q q c~ N N ~, uq E~ :d uq t~
m
--18
:~
1 3t 5220
a Fluorescence of intracellular (I) and surface (S) structures
was graded as follows: (O) negative; (+) weakly positive,
(++) moderate; and (+++) strongly positive staining in
the maiority of the cells examined (more than 80%) .
b nt = not tested.
:, ,
.
~q
: ~
- 1 31 5220
C)
~ O 1~ 0 0 1~ rl ~
l_ ___ ~_~ O ~
-1 o -1 0 0 d~
C r~
r ~rl
V
~ _ ~ _ _ _ +
U~ r rl O ~e O
O u~) O
~ Q~
'~ O ~ U
O r-
F ~v- a
O ~ ~ 3
~2 z --O ~o~ ~o ^ a~
~ 7 o- ,5::
E~ ~ zo ~ O
u~ ~ 1 ~,
7~ --~ ~o
o t` ~ ,-
rl C~
~ _ ~ 3 ~ ~
~, Q, h
' a
H J O Co~) 14 . O 0 ~
~ ~ o m ~
m ~ '+ ~,
r ~ _ , + ~
- h
~ O
-- 20 --
.
~ ; :~
:
: : ~ :
1315220
i .
REFERENCES
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