Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-03-11
2001-08-21
Ceperley, Mary E. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S007500, C435S007930, C435S346000, C530S808000, C530S809000
Reexamination Certificate
active
06277964
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to monoclonal antibodies having a high affinity for polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene and benzo [a]pyrene, hybridoma cell lines producing said monoclonal antibodies, and immunoassays for monitoring and quantifying the amount of PAHs present in the environment with a high sensitivity using said monoclonal antibodies.
The present invention also relates to PAH conjugates useful as an imunogen in preparing said antibodies and as a standard substance in a competitive assay.
DESCRIPTION OF THE PRIOR ART
In the measurement of PAHs existing in the environment, sixteen bicyclic to hexacyclic standard compounds such as naphthalene, phenanthrene and benzo [a]pyrene are generally analyzed, which are defined by the U.S. Environmental Protection Agency (EPA), although PAHs include a great number of compounds.
Methods for measuring PAHs in a sample include Soxlet extraction with toluene, gas chromatography, high performance liquid chromatography, thin layer chromatography, immunoassay, and the like.
However, Soxlet extraction with toluene measures the total amount of oil components including asphaltenes, resins, etc. in addition to all PAls contained in a sample, and therefore, it is impossible to analyze each individual PAR component.
Gas chromatography and high performance liquid chromatography can analyze and quantify all the sixteen PAH components defined by the EPA, contained in a sample. However, these methods can not monitor a number of samples concurrently, and therefore, they are mainly used for a precise analysis in a laboratory.
On the other hand, thin layer chromatography and immunoassaycan rapidly analyze a number of samples concurrently. However, these methods are inferior to gas chromatography and high performance liquid chromatography in terms of their resolving powers, and therefore, it is difficult to distinguish all the sixteen PAH components defined by the EPA. Accordingly, these methods are used, when the purpose of the measurement is merely the distinguishment of the groups of bi-, tri-, tetra-, penta- and hexa-cyclic components of PAHs or the grasp of the relative concentrations of the groups of the above components.
As described above, an analytic method used varies depending on the purpose of the measurement, and an immunoassay is suitable for a field monitoring where the rapid measurement of many samples is required.
In monitoring PAHs in an exhaust gas or a drained water or PAHs in.a contaminated soil by an immunoassay, the following technical problems may arise:
(1) In general, it is difficult to prepare an antibody against a low molecular antigen such as PAHs;
(2) The structures of the sixteen standard PAHs greatly resemble to each other, and. therefore, it is difficult to obtain an antibody which distinguishes each individual structure of them; and
(3) PAHs are slightly soluble in water and it is necessary to carry out an antigen-antibody reaction in an organic solvent, but the antibody is mainly comprised of a high molecular weight protein, and therefore, it is likely to lose its activity in the organic solvent.
U.S. Pat. No. 5,358,851 discloses a polyclonal antibody prepared by conjugating p-tolylacetic acid with bovine &ggr;-globulin and using the resulting conjugate as an immunogen. The patent specification also discloses a method for analyzing the presence or the amount of aromatic ring-containing compounds, such as benzene, toluene and xylene, in a sample using the polyclonal antibody.
U.S. Pat. No. 5,449,611 discloses a monoclonal antibody prepared by conjugating &bgr;-methylnaphthalene with bovine serum albumin and using the resulting conjugate as an immunogen. The patent specification also discloses a method for analyzing the presence of PAHs in a sample using the monoclonal antibody.
In addition, M. Gomes and R. M. Santella [Chem. Res. Toxicol. 3, pp.307-310 (1990)] discloses a monoclonal antibody prepared by using 6-aminobenzo [a]pyrene bound to bovine serum albumin as an immunogen. The document describes that the monoclonal antibody not only reacts with benzo [a]pyrene, but also cross-reacts with metabolic products thereof and some of other PAHs.
However, the polyclonal and monoclonal antibodies disclosed in these~documentsare not satisfactory in terms of their specificity and affinity for the antigen, when used for monitoring PAHs.
SUMMARY OF THE INVENTION
One object of the present invention is to solve the technical problems as described above and to provide a monoclonal antibody more suitable for monitoring PARs by an immunoassay.
Another object of the present invention is to provide a hybridoma cell line producing said antibody, and an immunoassay for analyzing PAHs in a sample using said monoclonal antibody.
A further object of the present invention is to provide a PAH conjugate useful as an immunogen in preparing said antibody and as a standard substance in a competitive assay.
According to the present invention, these and other objects are accomplished by a monoclonal antibody specifically recognizing polycyclic aromatic compounds selected from the group consisting of monoclonal antibodies PAH-1, PAH-3, PAH-6 and PAH-7.
Also, the present invention provides a hybridoma cell line producing the above monoclonal antibody, which is selected from the group consisting of hybridomas PAH-1, PAH-3, PAH-6 and PAH-7.
Furthermore, the present invention provides an immunoassay for analyzing polycyclic aromatic compounds in a sample, which comprises:
(a) carrying out an antigen-antibody reaction of a sample containing polycyclic aromatic compounds with the above monoclonal antibody in an aqueous solution containing an organic solvent; and
(b) detecting the polycyclic aromatic compounds bound to the antibody.
In addition, the present invention provides a conjugate of the following formula (1):
Ar—CO—(CH
2
)
n
—CONH—Z (1)
wherein Ar is a tricyclic, tetracyclic or pentacyclic aromatic compound, n is an integer of 1 to 3, and Z is a carrier protein or a labeling substance.
The present inventors established hybridoma cell lines producing anti-PAH monoclonal antibodies suitable for the measurement of PAHs, by binding PAHs to carrier proteins through a spacer to prepare novel PAH conjugates, immunizing an animal using the conjugates asan immunogen, fusing antibody-producing cells of the immunized animal with myeloma cells to prepare hybridoma cell lines, and selecting the hybridoma cell lines suitably. Also, we could construct an immunoassay for the measurement of PAHs using the monoclonal antibodies.
REFERENCES:
patent: 5015586 (1991-05-01), Severn et al.
patent: 5358851 (1994-10-01), Peck
patent: 5449611 (1995-09-01), Friedman et al.
patent: 2-501351 (1990-05-01), None
patent: WO88/09798 (1988-12-01), None
patent: WO96/13605 (1996-05-01), None
K. Li et al, Anal. Chem., 71, 302-309, Jan. 1999.*
J. Chagnaud et al, Polycyclic Arom. Compd., 3 (Suppl), 663-672, 1993.*
E. Quelven et al, Polycyclic Aromatic Compounds, 13, 93-103, 1999.*
R. Niessner, Analytical Methods and Instrumentation, 1 (3), 134-144, 1993.*
M. Liu et al, Analytical Letters, 31 (12), 2025-2038, 1998.*
H. Wallin et al, Cancer Letters, 22, 163-170, Jan. 1999.*
Gomes, M., et al., Chem. Res. Toxicol. 3, 307-310 (1990).
Ichimura Naoya
Kataoka Chiwa
Kitagawa Hirohisa
Miura Kazunobu
Ohtani Yutaka
Ceperley Mary E.
Osaka Gas Company Limited
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