Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1989-12-05
1994-08-30
Kepplinger, Esther M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 71, 435 72, 435 792, 435 793, 435 794, 435 795, 436501, 436524, 436811, 436578, 436540, 5303882, C12Q 100, G01N 3353, G01N 33566, G01N 33551, G01N 33543, G01N 33541, A61K 3514, A61K 3704, C07K 300, C07K 1300, C07K 1500, C07K 1700
Patent
active
053427564
DESCRIPTION:
BRIEF SUMMARY
This invention relates to measuring type III collagen degradation.
Type III collagen is found in several types of connective tissue throughout the human body. Its proportion is characteristically high in young tissue, e.g. in the early phases of wound healing or during the initial phase of development of a fibrosis.
It has become evident in recent years that the development of a fibrotic condition in a parenchymal organ, e.g. cirrhosis of the liver, is dependent on both the synthesis and the degradation of connective tissue. Many diseases are associated with an increase in the amount of collagen synthesized, but as long as the degradative mechanisms can compensate for the increased synthesis, no accumulation of collagen takes place.
Several methods have been described in recent years that allow an estimate to be made of the amount of type III collagen synthesized. The degradation of collagen has been assessed by determining urinary hydroxyproline excretion. However, this method is tedious, needs 24 hour urine collection, and is not specific for a particular collagen type. There is a need for a specific method for determining type III collagen degradation.
The present invention provides such a method, which is quick and simple to practise.
It has been found that the aminoterminal non-helical end of the type III collagen molecule, the so-called telopeptide region, which can be assayed by immunological methods, e.g. by a radioimmunoassay, provides significant information on the degradation of type III collagen.
This discovery can be used in any known method of immunoassay using an antibody specific to type III collagen amino-terminal telopeptide. The invention accordingly provides a method for assaying type III collagen degradation products, which comprises contacting a sample which may contain such products with an antibody specific to type III collagen amino-terminal telopeptide and a label under conditions such that the label becomes bound in an amount which depends on the amount of type III collagen degradation products present in said sample and then assaying the bound and/or unbound label as a measure of the type III collagen degradation products present in said sample. Any method of immunoassay can be used, but preferably a heterogenous method involving a phase separation step, such as those known by the initials RIA, ELISA, FIA, TR-FIA, and IRMA.
Preferably the new method is operated as a radioimmunoassay using isolated human aminoterminal type III collagen telopeptide, and an antibody specific to it. The telopeptide is labelled with a radionuclide, preferably iodine 125, using for example the chloramine-T method, the free iodine being separated by a disposable reverse phase cartridge. Other methods of labelling using enzymatic or fluorescent labels, e.g. europium, can also be used.
Type III collagen has usually been isolated from suitable tissues containing it after pepsin treatment of the tissue. Pepsin digests away the cross-linked telopeptide regions and thus brings helical atelo type III collagen into solution. This material cannot be used for the preparation of the telopeptide antibody required for the present invention or for the telopeptide itself. In accordance with a feature of the invention, therefore, highly purified type III collagen, suitable for us in the method of the invention, is made by extracting suitable human tissue, preferably uterine leiomyoma, with a salt (sodium chloride) solution of suitable molarity and pH to produce a solution of type III collagen in its untruncated form, still containing the telopeptide regions. The type III collagen solution is further purified by salt precipitations and chromatographical methods using techniques known to those skilled in the art. The aminoterminal telopeptide is finally isolated after bacterial collagenase degradation of the highly purified type III collagen. The liberated telopeptide is then purified by chromatographic methods preferably using high performance liquid chromatography (HPLC).
The purified human type III collagen thus prep
REFERENCES:
patent: 4628027 (1986-12-01), Gay
Chemical Abstracts vol. 106, (1987) Abstract No. 106:115529d, Anal. Biochem. 1986, 158(2), 334-45.
Chemical Abstracts vol. 105, (1986) Abstract No. 95762y, J. Histochem. Cytochem. 1986, 34(8), 1003-11.
Rennard et al. (1980) Enzyme Linked Immunoassay for Connective Tissue Components Anal. Biochem 104:205-214.
Risteli Juha
Risteli Leila
Kepplinger Esther M.
Orion-yhtyma Oy
Preston D. R.
LandOfFree
Type III collagen degradation assay does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Type III collagen degradation assay, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Type III collagen degradation assay will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-29413