Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2002-02-21
2004-09-14
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007500, C435S007940, C435S975000, C436S518000, C436S547000, C436S548000, C530S380000, C530S388100, C530S388150
Reexamination Certificate
active
06790625
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to antibodies raised against a placental protein.
BACKGROUND OF THE INVENTION
References referred to in the text by a number enclosed by parenthesis are listed at the end of the specification.
The goal of pregnancy management is the delivery of a mature, healthy infant, without encountering complications which can adversely affect the well being of both the mother and the newborn. A significant percentage of pregnancies are affected by various disorders. Among them are preterm delivery, intrauterine growth retardation and preeclampsia. These complications negatively impact the outcome of affected pregnancies, at enormous cost both to the patients as well as to the health system.
Placental Protein 13 (PP13) is a protein which was previously isolated from human placental tissue (U.S. Pat. No. 4,500,451 to Bohn, et al., the contents of which are incorporated herein by reference). The protein was characterized by the following parameters: electrophoretic mobility, isoelectric point, sedimentation coefficient, molecular weight determined by ultracentrifugation molecular weight determined by SDS-PAGE electrophoresis, extinction coefficient and carbohydrate content. The amino acid composition (residues per 100 residues) was determined but not the amino acid sequence.
PP13 was used to develop an assay for the early stage detection of three specific pregnancy-related disorders: intrauterine growth retardation, preeclampsia and preterm deliver (U.S. Pat. No. 5,198,366 to Silberman, the contents of which are incorporated herein by reference). Both a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed using labeled PP13 and anti PP13 polyclonal antiserum, respectively. However, experimental results were given only for the RIA, and not for the ELISA. No further properties of PP13 are disclosed in the Silberman patent. There have also been reports in the literature regarding the determination of other placental proteins and their relationship to pregnancy disorders (1-3).
The ELISA fulfils requirements of objectivity, simplicity, sensitivity and specificity previously only attained by radioimmunoassay (4). A methodological comparison of ELISA and RIA reveals several advantages of the former method:
1. ELISA is absolutely safe and does not require a specially designed laboratory and trained personnel for working with radioactive material.
2. Two-antibody sandwich ELISA is a more sensitive, rapid and easily quantitatable method.
3. Enzymes are rather stable as compared with radioactive tracers and cause a high level of result reproducibility.
4. The enzymatic activity may be measured easily using the spectrophotometric principle of an ELISA-reader, which is much cheaper and simpler in handling than a gamma-counter.
5. ELISA is more suitable for automation.
It is therefore desirable to develop an improved ELISA for the determination of PP13 levels.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide monoclonal antibodies (Mab) capable of binding PP13.
It is a further object of the invention to provide an immunoassay which measures the level of PP13 in biological fluids.
In one aspect of the invention, there is provided a Mab capable of binding PP13. In particular, the invention provides hybridoma clones selected from the group consisting of clones #26-2, 27-2-3, 215-28-3, 534-16 and 606-8-11-67, as well as the Mab produced by these clones. These clones have been deposited in accordance with the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes of the Pasteur Institute of 25, Rue du Docteur Roux, Paris, France. The following are the depository details of the clones:
Clone #
Accession No.
Deposit Date
26-2
I-2134
Mar. 4, 1999
27-2-3
I-2135
Mar. 4, 1999
215-28-3
I-2136
Mar. 4, 1999
534-16
I-2137
Mar. 4, 1999
606-8-11-67
I-2138
Mar. 4, 1999
In another aspect of the invention, there is provided an immunoassay for measuring the level of PP-13 in a biological fluid comprising the steps of: (a) bringing the fluid into contact with a Mab according to the invention, thereby forming Mab-PP-13 complexes; (b) exposing the complexes to a second antibody linked to a signal-generating molecule, the second antibody being capable of binding the complexes; and (c) providing conditions conducive to the production of a signal generated by the signal-generating molecule.
In the present specification, the term “signal-generating molecule” relates to a molecule capable of generating, either directly or indirectly, a detectable signal. The signal may be, e.g. a radioactive emission or a spectrophotometric absorbance at a specific wavelength. Preferably, the signal will be a color which can be detected by a spectrophotometric reader. The signal-generating molecule may generate the signal directly, e.g. by reacting itself with a chromogenic substrate, or indirectly, e.g. by binding to another molecule which is able to generate a signal. In a preferred embodiment, the signal-generating molecule is a ligand which generates a signal indirectly by binding to a ligand-binding molecule which is linked to an enzyme, which in turn catalyzes a reaction resulting in color formation.
The biological fluid may be any fluid which may contain PP13, such as placental extract or blood serum. Preferably, the fluid is blood serum. In one embodiment of this aspect of the invention, the Mab, which binds one site on PP13, is bound to a solid phase such as a microtiter well or a bead. The second antibody will be capable of binding another site on PP13, and may be polyclonal or monoclonal. In a preferred embodiment, the second antibody is also a Mab according to the invention.
In a further aspect of the invention, there is provided a kit for measuring the level of PP-13 in a biological fluid comprising: (a) a Mab according to the invention; (b) a second antibody linked to a signal-generating molecule; and (c) PP-13 standard solutions. In a preferred embodiment, the second antibody is also a Mab, as described above. The kit may be used to carry out an immunoassay as described above.
REFERENCES:
patent: 4500451 (1985-02-01), Bohn et al.
patent: 5198366 (1993-03-01), Silberman
patent: 6548306 (2003-04-01), Admon et al.
patent: 0 283 606 (1988-09-01), None
patent: WO 99/38970 (1999-08-01), None
XP-000915077: Than et al., “Isolation and Sequence Analysis of a cDNA Encoding Human Placental Tissue Protein 13 (PP13), a New Lysophospholipase, Homologue of Human Eosinophil Charcot-Leyden Crystal Protein”,Placente, (1999), 20(8), pp. 703-710.
Paltieli Yoav
Rabinovitch Lev
Browdy and Neimark PLLC
Diagnostic Technologies Ltd.
Nguyen Bao-Thuy L.
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