New monoclonal antibodies against bilitranslocase 177
IzvIrNI zNaNstveNI člaNek
New monoclonal 
antibodies against 
bilitranslocase
1 Department for 
the Production of 
Diagnostic Reagents 
and for Research, Blood 
Transfusion Center of 
Slovenia, Ljubljana
2 Department of 
Pathology, Izola General 
Hospital, Izola
3 Department of 
Systems Biology and 
Biotechnology, National 
Institute of Biology, 
Ljubljana
* Authors contributed 
equally to this work, 
Ljubljana
Korespondenca/
Correspondence:
Uroš rajčević,  
uros.rajcevic@ztm.si
Ključne besede:
svetlocelični karcinom 
ledvičnih celic; 
bilitranslokaza; 
monoklonska protitelesa; 
UOk171
id 1548zdrav vestn | maj – junij 2017 | letnik 86 OnkologijaIzvirni znanstveni članek
New monoclonal antibodies against 
bilitranslocase as a diagnostic tool in 
determining the progress of clear cell renal 
cell carcinoma
Novi monoklonski protitelesi proti bilitranslokazi kot 
diagnostično orodje pri ugotavljanju napredovanja 
svetloceličnega karcinoma ledvičnih celic
alexandra Bogožalec košir,*1,3 tjaša lukan,*1, 3 Mateja kukovec,2 vivijana snoj,2  
sendi Montanič,1 vladka čurin Šerbec,1 Uroš rajčević1
Abstract
Background: Monoclonal antibodies (mAbs) are an important tool in diagnostics and research, es-
pecially when we are dealing with a protein marker of unknown primary structure as in the case 
of bilitranslocase (BTL). BTL is also expressed on kidney cells, where it acts as an organic anion 
transporter. We have shown earlier that there are differences in bilitranslocase expression in normal 
kidney cells versus early grade kidney cancer.
Methods: We developed monoclonal antibodies against extra- and intra-cellular domains of bil-
itranslocase protein model. To also gain a deeper insight in bilitranslocase expression in clinical 
samples, we assessed BTL expression in different grades of clear cell kidney cell carcinoma (ccRCC).
Results: Both new monoclonal antibodies bind to a protein in UOK171 cells but not in the negative 
control. Binding of mAb is specific. mAb produced by cell line 2A9/2E9 (peptide 298–310; intracel-
lular domain) is more suitable for immunohistochemical analyses as it gives stronger intensity of 
binding than mAb produced by cell line 11C9/2G9 (peptide 235–246; extracellular domain). Anti-
body 2A9/2E9 stains bilitranslocase in proximal renal tubules of normal kidneys but not in the sur-
rounding stroma. Staining decreases in grade I compared to normal kidney, gradually increases in 
grades II and III, and decreases again in grade IV of ccRCC tissue.
Conclusions: Our results show that these antibodies can be used in different immunoassays. Fur-
thermore, specificity and affinity of our mAbs allowed us to use them in the analysis of progressive 
grades of clear cell renal cell carcinoma in a limited number of patients. Thus, mAbs developed here 
can be used as a diagnostic tool that could help distinguish between early and late grades of clear cell 
renal cell carcinoma.
Izvleček
Uvod: Monoklonska protitelesa (mAbs) so pomembno orodje v diagnostiki in raziskovanju, še po-
sebej kadar obravnavamo proteinski označevalec neznane primarne strukture, kot je v primeru bili-
translokaze (BTL). BTL se izraža tudi na celicah ledvic, kjer deluje kot prenašalec organskih anionov. 
V prejšnjih študijah smo ugotovili, da se bilitranslokaza izraža različno v normalnih ledvičnih celi-
cah v primerjavi z zgodnjimi fazami ledvičnega raka.
178 zdrav vestn | maj – junij 2017 | letnik 86
ONkOlOgIja
Key words:
clear cell renal 
cell carcinoma; 
bilitranslocase; 
monoclonal antibodies; 
UOk171
Citirajte kot/Cite as:
zdrav vestn. 2017;  
86:177–84.
Prispelo: 17. 5. 2016 
sprejeto: 17. 3. 2017
Metode: Razvili smo monoklonska protitelesa proti zunaj- in znotrajceličnim domenam proteinske-
ga modela bilitranslokaze. Da bi pridobili globlji vpogled v izražanje bilitranslokaze tudi v kliničnih 
vzorcih, smo ugotavljali izražanje bilitranslokaze tudi pri različnih gradusih svetloceličnega karcino-
ma ledvičnih celic (ccRCC).
Izsledki: Obe novi monoklonski protitelesi se vežeta na protein v celicah UOK171, ne pa tudi v ne-
gativnih kontrolah. mAb, ki ga proizvaja celična linija 2A9/2E9 (peptid 298–310; znotrajcelična do-
mena), je bolj primeren za imunohistokemijske preiskave, ker daje večjo intenziteto vezave kot mAb, 
ki ga proizvaja linija 11C9/2G9 (peptid 235–246; zunajcelična domena). Protitelo 2A9/2E9 obarva 
bilitranslokazo v proksimalnih ledvičnih tubulih, ne pa v okoliški stromi. Obarvanje se zmanjšuje v 
gradusu I v primerjavi z normalno ledvico, postopno naraste v gradusih II in III ter ponovno pade 
v gradusu IV ccRCC.
Sklepi: Naši rezultati kažejo na to, da se lahko ta protitelesa uporabijo pri različnih imunskih testih. 
Poleg tega sta nam specifičnost in afiniteta naših mAb omogočila njihovo uporabo pri analizi na-
predovalih gradusov svetloceličnega karcinoma ledvičnih celic pri omejenem številu bolnikov. mAb, 
razvita v tej študiji, se lahko uporabijo kot diagnostično orodje za razlikovanje med zgodnjimi in 
poznimi gradusi svetloceličnega carcinoma ledvičnih celic.
1 Introduction
In this work, we describe the use of 
anti-peptide monoclonal antibodies aga-
inst bilitranslocase as a diagnostic tool. 
Bilitranslocase is a membrane protein 
localized in hepatocyte plasma mem-
brane, basolateral domain of proximal 
tubules in the kidney, gastric epithelia 
and endothelial cells. Its main known 
biological purpose is to transfer organic 
anions from blood to hepatocytes (1,2,3).
Although the primary structure of bi-
litranslocase was predicted over a deca-
de ago (4), a model of three-dimensional 
structure has been proposed only recen-
tly (5), due to the low ability of membra-
ne proteins to form a crystal structure 
and inability of isotopic reorientation 
in solution that might prevent structure 
solving using X-ray crystallography or 
NMR (5).
Until recently, the most widely used 
antibody targeting bilitranslocase was 
an anti-peptide polyclonal antibody pro-
duced by immunization of rabbit with a 
multi-antigen peptide, corresponding to 
segment 65–75 of the primary bilitran-
slocase structure (4). In one of our recent 
studies monoclonal antibodies against 
segment 65–75 were described  (6) and 
in the present research monoclonal an-
tibodies against segments 235–246 and 
298–310 are characterized. According to 
the three-dimensional structure, both 
segments 65–75 and 235–246 belong to 
extracellular domains of bilitranslocase, 
crucial for its transport function, while 
segment 289–310 is an intracellular one 
and its function is yet unknown.
In our previous research we were able 
to show that expression of bilitranslo-
case is down-regulated in grade I clear 
cell renal cell carcinoma (ccRCC), using 
monoclonal antibodies against peptide 
65–75 (6).
Renal cell carcinoma (RCC) repre-
sents 2 % of all cancers and is the seventh 
most commonly diagnosed cancer in 
men and the ninth most commonly di-
agnosed cancer in women (7). It usually 
occurs in people between the ages of 50 
and 70, where men are more exposed 
than women (8). Its incidence worldwi-
de is about 300 000 new cases per year, 
a quarter of them present with advanced 
disease. RCC arises from the proximal 
renal tubular epithelium (9) and is a he-
terogeneous disease made up of a num-
ber of different histological types of can-
New monoclonal 
antibodies against 
bilitranslocase
New monoclonal antibodies against bilitranslocase 179
IzvIrNI zNaNstveNI člaNek
cer  (10) that occur as an inherited and 
as a sporadic, non-inherited cancer (11). 
The most common histological diagnosis 
is a clear cell renal cell carcinoma (ccR-
CC) which represents 85 % of kidney tu-
mors  (12,13). The most studied form of 
inherited ccRCC is the one associated 
with von Hippel-Lindau (VHL) syndro-
me  (11). Affected individuals are at risk 
for developing tumors in many organs, 
including the kidney. In VHL syndrome, 
one VHL allele, located on chromosome 
3p25 and encoding for tumor suppres-
sor protein is inherited with a mutation, 
when the other, wild-type VHL allele, is 
inactivated or silenced, and ccRCC ari-
ses. Defects in the VHL gene also appear 
to be responsible for a high percentage 
of tumors in patients with sporadic ccR-
CC (14,15).
Recent high throughput sequencing 
projects have identified driver genes in 
ccRCC beyond VHL  (16,17,18). These 
studies identified frequently mutated 
tumor suppressors, including PBRM1, 
BAP1 and SETD2, all of which function 
as chromatin and/or histone modifiers 
and intriguingly map to the frequently 
lost 3p21 locus. New reports also suggest 
that additional pathogenic mechanisms 
are at play and suspect that ccRCC is a 
metabolic disease  (19), with the results 
potentially yielding novel therapeutic 
insights for the care of kidney cancer.
Prognosis is closely related to the 
stage of disease  (9). The average life 
expectancy for patients whose disease 
is already advanced (locally invasive or 
metastatic renal-cell carcinoma) is about 
13 months (9). Mortality rates of RCC 
are rising with its incidence worldwi-
de of about 102 000 deaths per year (7). 
Therefore it is crucial to detect disease 
before it spreads, which is a major pro-
blem, since in the early development the 
disease is characterized only by nonspe-
cific symptoms (9,12,20). More specific 
symptoms such as flank pain, hematuria, 
and a palpable abdominal mass are signs 
of advanced disease (9).
2 Materials and methods
2.1 Cell culture
Human clear cell renal carcinoma 
UOK 171 cell line was obtained at NIH-
-NCI, Bethesda MD, USA. Cells were 
grown in Dulbecco modified Eagle’s me-
dium (MP Biomedicals), supplemented 
with 10 % (v/v) fetal bovine serum, 1 % 
(v/v) glutamine, 0.34 % (v/v) penicillin 
(Sigma) and 0.34 % (v/v) streptomycin 
(Sigma) in a humidified incubator at + 
37 °C and 5 % CO2. Cells were seeded 
to 6-well plates (1 × 105–4 × 105 cells per 
well) with round coverslips (2r = 13 mm), 
treated with Poly-L-Lysine (Sigma). 
Next day cells were ready for the immu-
nocytochemistry assay.
2.2 Immunocytochemistry
Cells were rinsed with PBS and fixed 
with ice-cold 70 % methanol (Sigma) in 
PBS for 20 min at +4°C. They were then 
permeabilized with 0.2 % Triton (Tech-
nicon) in PBS for 10 min at room tem-
perature (RT) and blocked with 4 % FCS 
in PBS for 15 min at RT. Samples were 
incubated with primary antibodies for 
2h at RT. Cells were then incubated with 
appropriate fluorescent secondary anti-
bodies Alexa Flour 555 Goat anti-Mouse 
IgG (Life Technologies), at concentrati-
on 2 μg/ml in 4 % FCS and 0.1 % Triton 
in PBS for 1h at RT. Cell nuclei were stai-
ned by Hoechst 33258, pentahydrate (bis-
-benzimide) FluoroPure™ grade stain 
(Invitrogen), diluted 1:1000 in the same 
buffer as antibodies and incubated for 1 
h at RT. Cells were washed three times 
with PBS between all incubation steps. 
After the final three washes with 4 % 
New monoclonal 
antibodies against 
bilitranslocase
180 zdrav vestn | maj – junij 2017 | letnik 86
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Fig. 1: Detection of bilitranslocase in UOk171 cells by monoclonal antibodies against peptides B 
(extracellular) and C (intracellular). specificity of binding of mabs 2a9/2e9 (peptide C) and 11C9/2g9 
(peptide B) to bilitranslocase is confirmed by using negative control. Magnification bars: 10 μm.
FCS and 0.1 % Triton in PBS, slides were 
mounted in Fluoromount (Sigma). Ne-
gative controls were treated identically 
with omission of the primary antibody. 
The cells were visualized by fluorescent 
microscopy (Nikon, Eclipse Ti-E).
2.3 Immunohistochemistry
Immunohistochemistry (IHC) was 
performed on paraffin embedded, archi-
ved tissue sections of ccRCC and normal 
human kidney tissue sections. Immuno-
histochemical analysis was performed 
on five samples (n=5) in triplicates for 
each grade  (21) of carcinoma, except 
grade 1, where only three samples (n=3) 
in triplicates were used. We also tested 
normal kidney samples. Two samples 
in five parallels were used. Briefly, tissue 
sections were deparaffinized in xylene, 
rehydrated in graded ethanol and trea-
ted with sodium citrate buffer (20 mi-
nutes at 95–100°C) for antigen retrieval. 
After washing in water and PBS, incuba-
tion with undiluted primary antibodies 
in supernatant was performed at RT for 
2h. Tissue slides were washed with PBS 
and treated with 2 % hydrogen peroxide 
(Sigma) in PBS for 8 min to block en-
dogenous peroxidases, followed by incu-
bation with secondary goat anti-mouse 
IgM antibodies (Sigma), conjugated with 
HRP (diluted 1:1000) for 2h at RT. After 
washing with PBS, reaction products 
were visualized by diaminobenzidine 
(DAB) as chromogen. Slides were rinsed 
with water. Mayer’s hematoxylin (Sigma) 
and Scott’s buffer (0.2 % (w/v) sodium 
bicarbonate, 2 % (w/v) magnesium sulfa-
te) were used as a counterstain. Sections 
were dehydrated in graded ethanol fol-
lowed by xylene before they were moun-
ted with Eukitt mounting agent (Sigma) 
and inspected by pathologist for staining 
intensity, staining area, background and 
ccRCC grading. Negative controls were 
treated identically with omission of the 
New monoclonal 
antibodies against 
bilitranslocase
New monoclonal antibodies against bilitranslocase 181
IzvIrNI zNaNstveNI člaNek
Fig. 2: staining of bilitranslocase in four grades of human clear cell renal cell carcinoma (ccrCC) and 
normal human kidneys using mab 2a9/2e9. Magnification bar: 100 μm.
primary antibody. Isotype controls were 
carried out by using monoclonal antibo-
dies of the same antibody class against 
blood group antigen A.
2.4 IHC Statistics
All diagnostic stainings were per-
formed in triplicate. Negative controls 
were repeated five times. Pathologist 
inspected the IHC preparations for sta-
ining intensity, staining area, backgro-
und and ccRCC grading all by grading 
from 0–3. Mean and standard deviation 
of intensity values were calculated. Null 
hypothesis that there were no differences 
in mean values of staining intensities be-
tween ccRCC grades, and ccRCC grades 
and normal kidney was tested by unpai-
red (homoscedastic) two-tailed student’s 
t-test. The null hypothesis was rejected 
with p < 0.05.
3 Results
3.1 Binding of mAbs to BTL
Antibodies were tested for their abili-
ty to bind bilitranslocase in UOK171 cell 
line by fluorescent immunocytochemi-
stry (Fig. 1). The results show that both 
monoclonal antibodies bind to a protein 
New monoclonal 
antibodies against 
bilitranslocase
182 zdrav vestn | maj – junij 2017 | letnik 86
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Fig. 3: staining intensities of the specimens of all four grades of ccrCC(gr.I-
Iv) and normal human kidney tissue (gr. 0).  Black lines above the histogram 
connect normal kidney with significantly differential grades in ccrCC. Blue 
lines above the histogram connect grade I with II-Iv ccrCC. asterisks above the 
connecting lines indicate the level of significance between the tested pairs.
in UOK171 cells but not in the negative 
control. The binding of monoclonal an-
tibodies is specific.
3.2 Applications of mAbs
Based on the results of a pilot IHC 
test we concluded that mAb produced 
by cell line 2A9/2E9 (peptide 298–310; 
intracellular domain) is more suitable 
for immunohistochemical analyses as it 
gives stronger intensity of binding than 
mAb produced by cell line 11C9/2G9 
(peptide 235–246; extracellular domain) 
and no background staining. Hence, this 
antibody was tested by immunohisto-
chemical staining of bilitranslocase in 
all four grades of human clear cell renal 
cell carcinoma (ccRCC) and normal hu-
man kidneys (Fig. 2). The results showed 
that our monoclonal antibody 2A9/2E9 
stains bilitranslocase in proximal renal 
tubules of normal kidneys but not in 
the surrounding stroma. Figure 2 also 
shows that staining decreases in grade I, 
gradually increases in grades II and III, 
but then decreases again in grade IV of 
ccRCC tissue.
To confirm the above-mentioned re-
sults, we performed a statistical analysis 
of staining intensities in different gra-
des of ccRCC and normal kidney (Fig. 
3), n=5 for each grade, except grade 1 
(n=3). Compared to normal kidney, bili-
translocase staining intensity decreased 
in grade I ccRCC tissue as we reported 
before  (6). The difference however was 
not found to be significant (p = 0.086). 
Staining increases from grade I on and 
there is a significant difference between 
grades III and IV and normal kidney tis-
sue. There is also a significant difference 
between grades II, III and IV and grade 
I of ccRCC.
4 Discussion
For potential diagnostic purposes 
we selected two anti-peptide monoclo-
nal antibodies against bilitranslocase 
(2A9/2E9 and 11C9/2G9, respectively). 
Antibodies are specific for extracellular 
domain crucial for BTL transport func-
tion and C-terminal intracellular doma-
in, respectively. They specifically bind 
both domains in the protein (Fig. 1, 2), 
enabling us to follow the expression of 
bilitranslocase in different tissues as well 
as in different cell lines according to the 
proposed model of BTL  (5). Specificity 
of antigen-antibody binding was charac-
terized by different immunoassays (Fig. 
1, 2). Moreover, to make sure our antibo-
dies are specific for the targeted antigen, 
we introduced a set of isotype monoclo-
nal antibodies (monoclonal antibodies 
of the same class, against another anti-
gen) as a control, which confirmed our 
results as specific (Fig 1, 2).
BTL expression in kidney cells was 
previously described in animal mo-
dels (23) where it acts as a transporter of 
New monoclonal 
antibodies against 
bilitranslocase
New monoclonal antibodies against bilitranslocase 183
IzvIrNI zNaNstveNI člaNek
flavonoids from the diet. BTL expression 
was also demonstrated in normal human 
kidney and kidney cancer (6,23), where it 
was localized in distal tubules in normal 
kidney (6) and proximal and distal renal 
tubules while it was absent in renal corti-
cal tumors (23). ccRCC originates in the 
epithelium of the proximal convoluted 
tubule, which transports glomerular fil-
trate from the glomerulus to the descen-
ding part of the nephron. In our study, 
we detected BTL expression in the epi-
thelium of proximal tubules in normal 
kidneys but not in the surrounding stro-
ma (Fig 2). We have shown earlier that 
BTL expression decreases in early grade 
ccRCC compared to normal kidneys (6) 
in a limited number of patients. To get 
a better insight in BTL expression in 
ccRCC and to demonstrate our novel 
monoclonal antibodies against bilitran-
slocase as a tool in research and possibly 
in the diagnostics of this potential new 
biomarker, we performed this study on a 
larger number of kidney tumor samples 
from patients with progressive grades of 
ccRCC (stages I-IV) and normal kidney 
controls. We are able to confirm the dec-
rease in BTL expression from normal 
kidney where it stained the proximal 
tubules to grade I ccRCC (Fig. 2, 3). The 
observed difference in BTL expression 
was statistically insignificant (Fig. 3). 
However, we were able to demonstrate 
that significant differences in bilitranslo-
case exist as the cancer progresses from 
stage I towards stage IV (Fig. 3). There is 
also a significant difference between bili-
translocase expression in normal kidney 
cells and grades III and IV of ccRCC. 
This way, based on BTL expression, we 
were able to distinguish between nor-
mal kidneys and high grades (III-IV) 
and low- versus high-grade tumors (I 
vs. II-IV), while it was impossible to dis-
tinguish between grades II-IV based on 
BTL expression. Why it appears that the 
BTL expression decreases in low-grade 
ccRCC compared to high-grade ccRCC 
remains elusive. The fact that ccRCC is 
suspected to be a metabolic disease (19) 
and that different genetic changes seem 
to be directed toward metabolic path-
ways in our opinion this may be related 
to the disrupted transport and perhaps 
metabolism of antioxidants, where bili-
translocase plays a role  (22). The exact 
role of bilitranslocase in the progression 
of ccRCC will have to be revealed by ad-
ditional testing.
5 Conclusions
In this work, specific monoclonal 
antibodies 2A9/2E9 and 11C9/2G9, of 
extracellular and intracellular C-termi-
nal domains, respectively, of predicted 
primary structure of rat bilitranslocase 
were produced and characterized. De-
scribed monoclonal antibodies could in 
future be used as a diagnostic tool that 
could help distinguish between early and 
late stages of clear cell renal cell carcino-
ma.
6 Acknowledgements
The authors gratefully acknowledge 
the help of Marjana Šprohar and Maja 
Černilec, BTCS in design and help in 
performance of immunoassays. Sabina 
Passamonti and Michela Terdoslavich 
are acknowledged for their help. This 
work was partially funded by European 
Regional Development Fund (Crossbor-
der Cooperation Program Italy-Slove-
nia 2007–2013, Trans2care project). The 
authors gratefully acknowledge the gift 
of UOK 171 cell line from dr. W. Marston 
Linehan, Urologic Oncology Branch, 
National Cancer Institute, Bethesda, Ma-
ryland.
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antibodies against 
bilitranslocase
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