CA2044227A1 - Detection of delta-bilirubin - Google Patents
Detection of delta-bilirubinInfo
- Publication number
- CA2044227A1 CA2044227A1 CA 2044227 CA2044227A CA2044227A1 CA 2044227 A1 CA2044227 A1 CA 2044227A1 CA 2044227 CA2044227 CA 2044227 CA 2044227 A CA2044227 A CA 2044227A CA 2044227 A1 CA2044227 A1 CA 2044227A1
- Authority
- CA
- Canada
- Prior art keywords
- bilirubin
- delta
- antibody
- monoclonal antibody
- biological sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT
A novel method for the determination of authentic delta-bilirubin is provided. This method utilizes a novel monoclonal antibody produced using hybridoma technology and directed specifically against delta-bilirubin rather than against bilirubin that is synthetically or chemically linked to albumin. The antibody is able to distinguish between delta-bilirubin and other forms of bilirubin by recognition of the specific covalent linkage present between bilirubin and albumin in delta-bilirubin. Detection of delta-bilirubin is important in the diagnosis of various hepatobiliary disorders and in the early prognosis of liver transplant patients. As a result, an immunoassay kit for delta-bilirubin detection is also provided.
A novel method for the determination of authentic delta-bilirubin is provided. This method utilizes a novel monoclonal antibody produced using hybridoma technology and directed specifically against delta-bilirubin rather than against bilirubin that is synthetically or chemically linked to albumin. The antibody is able to distinguish between delta-bilirubin and other forms of bilirubin by recognition of the specific covalent linkage present between bilirubin and albumin in delta-bilirubin. Detection of delta-bilirubin is important in the diagnosis of various hepatobiliary disorders and in the early prognosis of liver transplant patients. As a result, an immunoassay kit for delta-bilirubin detection is also provided.
Description
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 2 ~ 2 ~
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.
- 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 .
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-2 ~ , 2 ~ ~
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.
~ ~ ~ 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, 2 ~ '~
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.
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 ~ 7 calf serum containing 10% dimethyl sulfoxide as a cryoprotectant. Cells were frozen overnight at -70C
and subsequently transferred to liquid nitrogen.
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.
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.
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
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 2 ~ 2 ~
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.
- 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 .
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-2 ~ , 2 ~ ~
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.
~ ~ ~ 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, 2 ~ '~
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.
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 ~ 7 calf serum containing 10% dimethyl sulfoxide as a cryoprotectant. Cells were frozen overnight at -70C
and subsequently transferred to liquid nitrogen.
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.
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.
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
Claims (14)
1. A monoclonal antibody or a fragment thereof, comprising an antigen binding site specific against delta-bilirubin.
2. A monoclonal antibody as defined in claim 1, wherein said antibody is directed against the unique conformation of delta-bilirubin.
3. A monoclonal antibody as defined in claim 1, wherein said antibody is directed against the covalent bond specific to delta-bilirubin which links bilirubin to albumin.
4. A monoclonal antibody as defined in claim 3, wherein said bond is in the vicinity of amino acid residues 95-297 from the N-terminus of the albumin protein.
5. A monoclonal antibody as defined in claim 1, wherein said antibody is labelled.
6. An immunoassay method for detecting and quantifying delta-bilirubin in a biological sample comprising contacting the biological sample with the monoclonal antibody as defined in claim 1 and quantifying the amount of said antibody bound to delta-bilirubin.
7. A method as defined in claim 6, wherein the monoclonal antibody is labelled.
8. A method as defined in claim 7, wherein the label comprises the enzyme peroxidase and the amount of antibody bound to delta-bilirubin is quantified by addition of a substrate for peroxidase.
9. A kit for quantifying delta-bilirubin in a biological sample comprising 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.
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.
10. A kit for quantifying delta-bilirubin in a biological sample comprising immobilized monoclonal antibody or fragment thereof, comprising an antigen binding site specifically directed against delta-bilirubin;
a wash solution;
a solution containing a label;
means for administering said biological sample to the immobilized monoclonal antibody; and means for administering the label to the antibody.
a wash solution;
a solution containing a label;
means for administering said biological sample to the immobilized monoclonal antibody; and means for administering the label to the antibody.
11. A kit as defined in claim 10, wherein the label is selected from the group comprising an enzyme, a fluorescent group, a chromophore, an immunolabel or delta-bilirubin labelled with an enzyme, a fluorescent group, a chromophore, or an immunolabel.
12. An antibody-producing hybridoma cell line characterized by its ability to produce monoclonal antibodies specific against delta-bilirubin.
13. 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.
14. 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US53644890A | 1990-06-12 | 1990-06-12 | |
| US536,448 | 1990-06-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2044227A1 true CA2044227A1 (en) | 1991-12-13 |
Family
ID=24138543
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2044227 Abandoned CA2044227A1 (en) | 1990-06-12 | 1991-06-10 | Detection of delta-bilirubin |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2044227A1 (en) |
| DE (1) | DE4119395C2 (en) |
-
1991
- 1991-06-10 CA CA 2044227 patent/CA2044227A1/en not_active Abandoned
- 1991-06-12 DE DE19914119395 patent/DE4119395C2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE4119395A1 (en) | 1991-12-19 |
| DE4119395C2 (en) | 1996-09-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0783104A1 (en) | Method for assaying soluble amyloid precursor protein | |
| EP0158973A2 (en) | Multisite immunometric assay | |
| US5620856A (en) | Monoclonal antibody assay and kit for detecting metal cations in body fluids | |
| JP2750423B2 (en) | Method for measuring vitamin B12 and reagent for measuring vitamin B12 | |
| JP7013051B2 (en) | Antibodies that specifically bind to bovine pregnancy-related glycoprotein 1 and their uses | |
| US20100093006A1 (en) | High affinity anti-n1,n12-diacetylspermine monoclonal antibody | |
| JP5798679B2 (en) | Monoclonal antibody specifically recognizing human liver-carboxyl esterase 1, hybridoma cell line producing said antibody and use thereof | |
| EP0479929B1 (en) | Vitamin b12 assay | |
| FI119990B (en) | Sandwich immunoassay for N-peptide | |
| EP0401370B1 (en) | Enzyme immunoassay according to sandwich method of human iv-type collagen | |
| JPWO2009044561A1 (en) | Anti-proNT / NMN monoclonal antibody | |
| JP4663831B2 (en) | Monoclonal antibodies, cell lines, and methods for measuring N1, N12-diacetylspermine | |
| CA2044227A1 (en) | Detection of delta-bilirubin | |
| KR20200099421A (en) | An antibody specifically binding symmetric dimethylarginine and uses thereof | |
| JP4664340B2 (en) | Monoclonal antibodies, cell lines, and methods for measuring N1, N12-diacetylspermine | |
| KR100493932B1 (en) | Monoclonal antibody recognizing resistin, production method and use thereof | |
| CA2355893C (en) | Antiuracil monoclonal antibody | |
| JPH0677017B2 (en) | A method for the immunoassay of human dehydrogenase type IV collagen | |
| AU634158B2 (en) | Anti-endothelin antibody | |
| EP0938678A1 (en) | Method and kit for the diagnosis of troponin i | |
| JP2002209579A (en) | Anti-human hemoglobin monoclonal antibody, cell line producing the same and kit containing the same for detecting human hemoglobin | |
| JP2558956B2 (en) | Immunological assay method for human osteocalcin, reagent and kit therefor, antibody against human osteocalcin, hybridoma producing the same, and method for producing the same | |
| JP2001000181A (en) | Cell line producing human anti-c-reactive protein monoclonal antibody, its preparation and monoclonal antibody | |
| US6291198B1 (en) | Antibody that recognizes pyrazine derivative and method for measuring 1,2-dicarbonyl derivative using said antibody | |
| JP2004215649A (en) | Anti-c-reactive protein monoclonal antibody, cell strain producing the same, method for producing the anti-c-reactive protein monoclonal antibody, and kit for detecting anti-c-reactive protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |