Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-11-14
2003-10-14
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007210, C435S007930, C435S007950, C436S172000, C436S800000, C436S536000, C436S546000
Reexamination Certificate
active
06632624
ABSTRACT:
DESCRIPTION
1. Technical Field
The subject of the present invention is a method for the immunoassay of human chromagranin A, with which it is possible in particular to assay not only chromogranin A in intact form, but also the major fragments of this chromogranin A.
Such assay may be used in particular for the diagnosis and follow-up of pathologies such as, for example, pheochromocytoma and intestinal carcinoids.
2. Prior Art
Chromogranin A (CgA) is a protein having a molecular weight of 48 kDa, a pI of 4.9, whose human form contains 439 amino acids as described by Konecki et al., 1987, in reference [1]. It belongs to the family of granins with which it shares structural and physiological similarities. CgA, like chromogranin B, shows marked inter-species preservation which presupposes a major function. CgA is largely represented in the secretagogues of endocrine and neuroendocrlne cells of which, along with the other granins, it forms one of the main components. It also acts at this level as a regulatory element of the cosecretion of other entities, such as the catecholamines in the adrenal gland.
CgA also plays an essential prohormone role via the release of active peptides, subsequent to intra-granular and extra-matricial proteolytic degradation.
In appended
FIG. 1
, SEQ ID NO:1 is shown of the 439 amino acids described by Konecki et al., corresponding to human chromogranin-A.
FIG. 2
shows this sequence in simplified fashion, identifying in this figure some of the peptides able to be released in man, i.e. the peptides: vasostatin/&bgr; granin, chromostatin, pancreastatin, parastatin and prochromacin. Protelysis affects the dibasic sites numbered and denoted by an asterisk in
FIG. 2
, which are distributed along the CgA sequence and total 10 in number in human CgA. This proteolysis has been described as a recurrent phenomenon either side of the protein ends, and it has been shown that it is specific to tissues and that it leads to substantial differences in the tissue distribution of the peptides produced. The degree of intensity and the type of proteolysis may therefore be responsible for the great variability of the fragments found in the tissues, blood circulation and urine.
Recent work by Corti et al., reference [2], has confirmed this diversity by showing that in patients suffering from pheochromocytoma there exist circulating forms of different conformation and in varying proportions from one person to another. In the same way, it has been shown, through research on the WE-14 peptide, that CgA is proteolysed in different manner in normal tissues and neoplasic tissues of the pancreatic gastro-enteral tract.
In addition to its detection in normal tissues and in corresponding neoplasias, numerous studies see a diagnostic advantage in the assay of CgA. The levels of circulating CgA are significantly high in cases of pheochromocytoma, carcinoid and endocrine pancreatic tumour. This data has been confirmed and extended to other pathologies: neuroblastoma, tumours of the gastro-intestinal tract, essential hypertension. The presence of CgA has been shown in a great number of neurodegenerative pathologies including Alzheimer's (see reference Munoz, Lab. Invest., 1991, vol. 64, pages 826-832 [15]). Some authors have also shown that the presence of CgA in cancers of the prostate could be the sign of an unfavourable development, as in the case of kidney cancer. The assay of Cga therefore offers a major advantage.
Document U.S. Pat. No. 4,758,522 [3] describes an immunoassay of CgA in which the CgA is measured by competitive assay with radiolabelled CgA for the sites of an anti-human CgA antibody.
Syversen et al. in reference [4], have described an assay of chromogranin A with the ELISA technique using an antibody directed against a C-terminal fragment of CgA corresponding to SEQ ID NO:1 amino acids 210 to 439 and radioimmunoassay of pancreastatin.
Corti et al., in reference [2], have also described two assays of CgA using monoclonal antibodies directed against SEQ ID NO:1 amino acids 81 to 90 and SEQ ID NO:1 amino acids 68 to 70 of human CgA.
These assays therefore take into consideration some CgA fragments, but they do not permit differentiation of pathological type on the results of the assay.
Indeed, proteolysis of the molecule and the multiplicity of the circulating fragments require an assay configuration which is able to detect the majority of these entities.
The purpose of the present invention is precisely a method of CgA immunoassay which measures the levels of intact CgA and the levels of major fragments found in circulating blood.
DISCLOSURE OF THE INVENTION
According to the invention, the immunoassay method for human chromogranin A (CgA) present in a sample comprises the use of at least one monoclonal or polyclonal antibody which binds specifically to an epitope positioned extending from (SEQ ID NO:1) amino acids 145 to 284 from the N-terminal end of human CgA.
According to the invention, the imunoassy method for human chromograinin A (CgA) present in a sample comprises the use of at least one monoclonal or polyclonal antibody which binds specifically to an epitope positioned extending from (SEQ ID NO:1) amino acids 145 to 284 from the N-terminal end of human CgA.
To implement this assay, to the sample to be assayed is added a quantity of labelled human CgA and the antibody which binds specifically to an epitope positioned in SEQ ID NO:1 amino acids 145 to 234 of CgA, and it is then left to incubate. In this manner, competitive conditions are set up between the sample CgA and the labelled CgA for the sites of this antibody. When the assay is conducted in heterogeneous phase, using a radioelement as label, it is possible to separate the antibody-hCgA complexes formed by immunoprecipitation using an appropriate antibody. By then determining the radioactivity of the complexes formed, it is possible to determine the CgA concentration of the sample with reference to a standard curve obtained from standard samples with known CgA concentrations.
It is also possible to conduct the assay in homogeneous phase, using labelled hCgA for example and an antibody able to modify the signal emitted by the labelled hCgA.
In this assay, advantageously the antibody used is specific to an epitope positioned in SEQ ID NO:1 amino acids 219 to 234 of human CgA, or the antibody specific to an epitope positioned in SEQ ID NO:1 amino acids 145 to 197 of human CgA. These antibodies are preferably monoclonal antibodies.
To carry out this assay, the preparation of the standard samples and labelled CgA may use purified human CgA from endocrine or neuroendocrine cells of human origin, or preferably recombinant human CgA.
According to a second embodiment of the invention, the immunoassay is a sandwich-type assay using a first monoclonal or polyclonal antibody which binds specifically to a first epitope positioned in SEQ ID NO:1 amino acids 145 to 234 of human CgA, and a second monoclonal or polyclonal antibody which binds specifically to a second epitope different from the first and also positioned in SEQ ID NO:1 amino acids 145 to 234 of human CgA, one of the two antibodies being labelled.
In this second embodiment of the invention, two antibodies are used that are specific to different epitopes, positioned in SEQ ID NO:1 amino acids 145 to 234 of CgA. This assay may be conducted in heterogeneous phase by immobilizing the first antibody on a solid phase and using a second, labelled antibody.
This assay may also be conducted in homogeneous phase, using a first labelled antibody and a second antibody able to modify the signal emitted by the first labelled antibody.
By way of example, the first antibody may be specific to an epitope positioned in the sequence of SEQ ID NO:1 amino acids 145 to 197 of CgA and the second antibody may be specific to an epitope positioned in SEQ ID NO:1 amino acids 219 to 234 of CgA.
It is also possible to use in this assay a first antibody specific to an epitope positioned in SEQ ID NO:1 amino acids 219 to 2
Aunis Dominique
Bellanger Laurent
Degorce François
Cheu Changhwa Jacob
CIS bio International
Le Long V.
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