Note: Descriptions are shown in the official language in which they were submitted.
2~ ~227
The present invention relates generally to an
immunological detection technique and more particularly,
to the specific dPtection of authentic delta-bilirubin
using a monoclonal antibody.
Bilirubin, the breakdown product of
hemoglobin, is comprised of four fractions.
Unconjugated bilirubin is a lipophilic, neuro-toxic
fraction. Sugar-conjugated bilirubin, which may be
either mono- or di-conjugated, is hydrophilic and non-
toxic. The fourth fraction, known as delta-bilirubin,
was first identified in 1981. It is naturally covalently
bonded to albumin protein.
Delta-bilirubin has been found to be of
clinical importance. It is useful in diagnosing a
number of hepatobiliary disorders, especially
obstructive jaundice. Recently, it was determined that
delta bilirubin is of value in the early diagnosis of
liver allograft rejection (Clinical Chemistry, Vol. 36:
9-14, 1990~.
Liver transplantation is an accepted form of therapy
for patients suffering liver disease; however, non-
invasive tests indicative of rejection are reguired.
Post-operative quantification of enzymes, such as
aspartate transaminase and alkaline phosphatase, does
not adequately distinguish ~etween rejection and
infection in liver patients. Determination of delta-
bilirubin, either alone or in concert with conjugated
bilirubin, has been shown to be an unusually sensitive
marker of liver function or dysfunction. A decline in
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the delta-bilirubin fraction with a concomitan~ increase
in the amount of conjugate bilirubin, or a consistently
low delta bilirubin value (less than 30~ total
bilirubin) is a specific indication of liver rejection.
The function of delta-bilirubin as an accurate
prognosticator in liver patients and, further, as an
index of the resolution of jaundice has lead to the
requirement for an accurate, specifi- technique for its
detection.
Methods have previously been devised to det~ct
and quantify delta-bilirubin; however, use of these
methods is limited with respect to efficiency, accuracy
and especially with respect to specificity. A method
utilizing high pressure liquid chromatography (HPLC) has
been established (Lauff et al., CRC Series on Liquid
Chromatography (1984) Vol.ll pp. 32-39) but has proven
to be a laborious detection technique. In addition, the
HPLC method is not sensitive enough for incisive
diagnostic use. Thin film slide methods, wherein delta-
bilirubin is calculated indirectly, are analytically
less precise than the HPLC method. Other assaying
technique, such as diazo-based methods, utilized in the
past have been unsuccessful in detecting delta-bilirubin
which is lost during deproteinization of the sample due
to the covalent attachment of bilirubin to albumin.
Albumin is a very large molecule having molecular weight
of 69,000 daltons whereas the molecular weight of
bilirubin is only 584.7 daltons. As a result, bilirubin
tends to be hidden or masked from reagents within the
"folds" of the albumin protein which is often
precipitated out of the sample during bilirubin analysis
using diazo-based methods.
In order to overcome the disadvantages of
methods currently existing for the determination of
delta-bilirubin, provision of an accurate, specific
means for dElta-bilirubin detection is required.
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- 2~2~7
A monoclonal antibody specific against
bilirubin has previously been prepared (Chemical
Abstracts, Volume 104, 1986). Such an antibody is not,
however, effective against the delta-bilirubin fraction
of bilirubin. Delta-bilirubin is structurally different
from other forms of bilirubin due to its specific
covalent linkage to albumin.
Other anti-bilirubin monoclonal antibodies
have been prepared which are specific against haptenic
bilirubin of a bilirubin-albumin complex (Shimizu, S. et
al, Biochimica et Bio~hvsica Acta, 967, 1988). However,
the bilirubin-albumin complex contains an average of 8
to 11 bilirubins per molecule of serum albumin. In
contrast, authentic delta-bilirubin, to which the
monoclonal antibody of this invention is directed,
comprises a one-to-one molecular complex of bilirubin
and albumin wherein the bonding is covalent and is in
the vicinity of residues 95-297 from the N-terminus of
the albumin protein.
The present invention provides a monoclonal
antibody, produced using hybridoma technology, that is
specific against authentic delta-bilirubin. The two
components of delta-bilirubin, albumin and bilirubin,
appear to be covalently bound by an amide linkage
between a free propionic acid side chain of bilirubin
and an epsilon amino group of a lysine moiety within the
albumin protein. Extensive research by the present
inventor has lead to identi~ication o~ the specific
3 --
.
covalent linkage region on the albumin molecule. The
highly specific nature of the covalent bond between
albumin and bilirubin is characteristic of delta-
bilirubin. This bond is believed to be the antigenic
determinant recognized by the monoclonal antibody.
Other forms of bilirubin, both conjugated and
unconjugated, do not exhibit this specific bond to
albumin and further, do not react appreciably with the
monoclonal antibody of the present invention.
Accordingly, the invention provides a
monoclonal antibody or a fragment thereof, comprising an
antigen binding site specific against delta-bilirubin.
Another aspect of the present invention
provides an immunoassay method for detecting and
quantifying delta-bilirubin in a biological sample
comprising contacting the sample with the monoclonal
antibody or fragment thereof comprising an antigen
binding site specific against delta-bilirubin and
quantifying ~he amount of said antibody that
subsequently binds to the delta-bilirubin of the
biological sample to be analyzed.
A further aspect of the present invention
provides a kit for quantifying delta-bilirubin in a
biological sample comprising an immobilized enzyme-
labelled monoclonal antibody or fragment thereof
comprising an antigen binding site specifically directed
against delta-bilirubin, substrate specific to said
enzyme label, a wash solution, means for administering
said biological sample to the immobilized monoclonal
antibody and means for administering said substrate to
the antibody.
Yet another aspect of the present invention
provides a kit for quantifying delta-bilirubin in a
biological sample comprising immobilized monoclonal
antibody or fragment thereof, comprising an anttgen
binding site specifically directed against delta-
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bilirubin, a wash solution, a solutlon containing a
label, means for administering said biological sample to
the immobilized monoclonal antibody and means for
administering the label to the antibody.
A further aspect of the invention provides an
antibody-producing hybridoma cell line characterized by
its ability to produce monoclonal antibodies specific
against delta-bilirubin.
Still a further aspect provides a hybrid
myeloma cell line characterized by its ability to
produce monoclonal antibodies specific against delta-
bilirubin, wherein said cell line is prepared through
the fusion of an immunocyte cell from an animal
sensitized to delta-bilirubin and a myeloma cell line.
Yet another aspect of the invention provides a
monoclonal antibody or a fragment thereof, comprising an
antigen binding site which is directed specifically
against delta-bilirubin, wherein the hybrid cell line
producing said antibody is a hybrid of an immunocyte
cell from an animal sensitized to delta-bilirubin and a
myeloma cell line.
According to the preferred embodiment of the
present invention, hybridoma technology is utilized to
produce the desired monoclonal antibody. Immunization
of experimental animals with the antigen, human delta
bilirubin, elicits an immune response wherein antibodies
directed specifically against delta-bilirubin are
synthesized. Lymphocytes, the blood cells that secrete
antibodies, are stimulated to divide upon exposure to
the antigen and the result is clones of lymphocytes
producing a single type of antibody, specifically an
antibody directed against delta-bilirubin.
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~ ~ ~ L/~ 2, ~
Lymphocytes produced as a result of the
immunization process are isolated from the immunized
animal and combined with myeloma cells. Fusion of
myeloma cells to lymphocytes to form hybridoma cells may
be stimulated by a fusogen, preferably a chemical
fusogen. Especially preferred as a chemical fusogen is
polyethylene glycol. Selection for fused hybridoma
cells by suspension of the cells in appropriate
selection media allows for the growth of immortal fused
cells only. The selection medium prefer~bly utilized
contains hypoxanthine, aminopterin and thymidine and is
commonly known in the art as HAT medium. Further
screening for hybridomas capable of producing the
monoclonal antibody of the invention may be performed by
employing enzyme-linked immunosorbent assay (ELISA~
techniques.
Upon characterization and isolation of a
hybridoma cell-line capable of producing monoclonal
antibodies specific to delta-bilirubin, the monoclonal
antibody produced can itself be isolated and used to
detect and quantify delta-bilirubin in biological
samples. Several immunoassay methods are known and
described in the art (Talwar, G.P., Non-IsotopiC
Immunoassays and their Applications). These immunoassay
methods may be adapted to be useful in the detection and
guantification of delta-bilirubin in a biological sample
using the monoclonal antibody of the present invention.
One such method comprises labelling the
monoclonal antibody or fragment thereof having an
antigen binding site specific to delta-bilirubin with a
detectable label compound, reacting the labelled
antibody with the biological sample to be analyzed, and
detecting the resulting amount of labelled
antibody/delta-bilirubin complex in the sample. The
isolated monoclonal antibody is appropriately labelled
such that it may be detected by colorimetric,
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fluorometric, or radio-chemical techniques. Further, it
may be labelled with an enzyme for detection by a
subsequent substrate rea~tion or it may be labelled wlth
an additional antibody for detection by any of the
above-mentioned techniques.
A displacement method for the detection of
delta-bilirubin in a biological sample may also be used.
This includes complexation of the monoclonal antibody to
labelled exogenous delta-bilirubin to form an
antibody/delta-bilirubin complex prior to its addition
to the sample to be analyzed for delta-bilirubin
content. Upon addition of the complex to the sample,
any endogenous delta-bilirubin present will displace the
labelled exogenous delta-bilirubin which can
subsequently be quantified. Thus, the amount of
displaced labelled exogenous delta-bilirubin is equal to
the amount of endogenous delta-bilirubin in the
biological sample. The method of quantification will
vary depending on the label employed. The previously
described labels are applicable to this method also.
The preferred embodiment of the invention is
further described in the following specific, non-
limiting examples.
MATERIALS
(a) Immunizing Antigen: Human delta bilirubin isolated
as described in Clin. Chemist~y, 28: 629-637 (1982).
(b) Screening Antigen: Uncon~ugated and conjugated
bilirubin isolated and dissolved in solution as per Wu
et al., Clin. Chem., 26: 1323-1335 (1980).
(c) Tissue Culture Media
2~ 7~
Complete Medium: RPMI 1640 (Flow
Laboratories, Mississauga, Ontario) was supplemented
with 10% V/V fetal calf serum (Gibco, Burlington,
Ontario), 10 mM Hepes buffer and 5 x 10-5 M 2-
mercaptoethanol.
Serum-free Medium: Complete medium prepared
without the fetal calf serum.
HAT Medium: Complete medium supplemented with
5 x lO- 3 M hypoxanthine, 2 x 10-5 M aminopterin and 8 x
0- 4 M thymidine.
HT Medium: Complete supplemented with 5 x lO-
5 M hypoxanthine and 8 x 10-4 M thymidine.
(d) ELISA Solutions and Reagents
Phosphate Buffered Saline (PBS): 8 g NaCl,
0.2 g ~Cl, 1.15 g Na2HPO4 and 0.2 g KH2PO4 in one litre
H20, pH 7.4 to 7.6.
Coating Buffer: 17.2 g NaHCO3 and 8.6 g
Na2 C3 in one litre of distilled H20, pH within 002 pH
units of 9.6 without ad~ustment. This buffer must be
made fresh on day of use.
Washing Buffer: PBS containing 0.05~ Tween-
20.
Peroxidase Substrate: A single 0-
phenylenediamine tablet (J.D. Biologicals) dissolved in
2.5 ml of distilled H20 containing 2.5 ,ul of 30% H2 2 .
2 ~ ~
EXAMPLE 1 - Il~MUNIZATION
Female Balb/c mice, 6 to 8 weeks of aye, were
immunized with an intraperitoneal (ip) injection of 50
~1 (100 ~g) of antlgen, human delta-bilirubin in
Freund's complete adjuvant. The animals were further
boosted with intraperitoneal injections, having the same
amount of antigen but prepared in Fraund's incomplete
adjuvant, in week 3, week 6 and week 9 following the
initial injection. Subsequent t~ these injections, the
animals were given a minimum rest period of 4 weeks.
Three days prior to preparation of the hybridoma cell
line, the animal received a final ip boost of 50 ,ul (100
,ug) of antigen suspended in 0.5 ml of PBS solution.
EXAMPLE 2 - FUSION
The spleens were asept~cally removed from the
immunized animals and pressed through a wire mesh screen
to achieve a single cell suæpension. Splenocytes were
mixed with Sp2/0 myeloma at a spleen/myeloma ratio of
5:1 and washed three times with serum free medium.
Following the final wash, the supernatant was completely
removed and the cell pellet was broken by gentle
agitation. Polyethylene glycol (PEG) 1450 tAmerican
Type Culture Collection, Rockville, Maryland) was
diluted to 50~ with 37C serum free medium and added
dropwise to the pellet over 60 seconds. The PEG/cell
suspension was incubated at 37C for a further 90
seconds before being diluted with 10 ml of serum free
medium over a 5 minute period. Following a further 10
minute incubation at 37C, the cells were resuspended in
HAT (hypoxanthine, aminopterin and thymidine) medium (5
x 106 cells/ml) and plated into 96 well tissue culture
plates (5 x 105/well). The plates were then incubated
_ g _
.
2 ~
at 37C in a humidified CO2 incubator. Every three
days, the wells were refilled with 100 ,ul of HAT medium
until vigorous hybridoma growth was evident.
EXAMPLE 3 - SCREENING ~SSAY
96 well ELISA plates (Nunc, Burlington,
Ontario) were coated overnight with bilirubin antigen
dissolved in coating buffer t10 ,ug/ml). The plates are
washed twice with wash buffer and the wells refilled
wlth 50 ,ul aliquots of wash buffer. Supernatants (50
,ul/well) were transferred aseptically from the tissue
culture plates and the ELISA plates incubated at 37C
for two hours. The wells were emptled, washed four
times and refilled wlth wash buffer containing goat
anti-mouse IgG/IgM conjugated to 1:1000 dilution of
horseradish peroxidase (J.D. Biologicals, Toronto,
Ontario). Following a final two hour incubation, the
wells were emptied, washed five times with wash buffer
and refilled with peroxidase substrate solution (100
~l/well). The reaction was allowed to proceed for 30
minutes before being terminated by the addition of 8M
H2 S4 ( 25 ,ul/well). Absorbance of the wells was read at
495 nm in a BioTek ELISA plate reader. Wells giving
absorbance readings greater than the mean plus three
times the standard deviation of the negative control
wells (no primary antibody) were regarded as positive.
EXAMPLE 4 - CELL FREE~ING
Those wells determined to be positive twice by
the screening ELISA were transferred into 24 well tissue
culture plates (Nunc) and allowed to grow to confluency.
The supernatants of each well were removed and frozen at
-70C. The cells were detached and suspended in fetal
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~ 7
calf serum containing 10% dimethyl sulfoxide as a
cryoprotectant. Cells were frozen overnight at -70C
and subsequently transferred to liquid nitrogen.
EXAMPLE 5 - CLONING
Two parental hybridomas were selected for
cloning by limiting dilution. Cells were resuspended at
1 x 104/ml in HT (hyposanthine and thymidine) medium and
added in 200 ~l aliquots to 8 wells of a 96 well tissue
culture plate. The remaining wells were filled with 100
,ul of HT medium and using a multichannel pipette, a
serial two-fold dilution series was created. Following
incubation and cell growth, the supernatants were tested
for bilirubin reactivity by ELISA. The highest dilutlon
well positive for bilirubin reactive antibody was
recloned by limiting dilution.
EXAMPLE 6 - ASCITES PRODUCTION
Male Balb/C mice 6 to 8 weeks of age (Jackson
Laboratories) were given a single ip injection of 0.5 ml
Pristane (Sigma Chemicals, St. Louis, Missouri). Ten
days later, the mice received 5 x 106 hybridoma cells by
ip injection. Ascites appeared 10 to 14 days later and
was collected. Cells and fibrin were removed by
allowing the ascites to clot. The resulting ascites was
stored at -70C.
EXAMPLE 7-- DETECTION OF DELTA-BILIRUBIN
The following data illustrates the specificity
4 ~
of a monoclonal antibody of the present invention,
specifically monoclonal #108.
The method used to detect the response of the
monoclonal antibody to three different samples of
bilirubin, namely authentic human serum-isolated delta
bilirubin, human bile-isolated conjugated bilirubin
(typically 70-75% di- and 20-25% mono-conjugate) and
unconjugated bilirubin, involved an enzyme-substrate
reaction.
The monoclonal antibody was bound to reaction
wells into which bilirubin samples were added.
Commercially available albumin-specific antibody (IgG)
pre-linked to peroxidase (POD) was added to the reaction
wells which were subsequently washed of extraneous non-
bound matter after a suitable reaction time. A
substrate for peroxidase, such as phenylenediamine, was
added and the color of the dye product formed after 1
hour was measured at 630 nm for each sample.
It should be noted that the response of the
monoclonal antibody to both conjugated and unconjugated
bilirubin was negligible.
TABLE 1. Response of Monoclonal Antibody Measured as
Absorbance at 630 nm to Delta-bilirubin (B~ ),
Conjugated bilirubin ~Bc) and Unconjugated
bilirubin (Bu).
Srp~ o~ Cono~ntr~210n ol IJlllrubln
ElLllrubln (1 y/100 Pl~
0.5 0.25 0.125 0.063 0.03 0.16 O.OB
~1 0.65 0.75 1.06 0.32 0.06 0.025 0.02
~C . 10 - O . 0~5 0 . 05 0 . 015 0 . 01
~u S O . 0~ 0 . 01 0 . 01 0 . OOS O . 002
^ Negllyl~le
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