Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1996-09-23
2000-12-12
Chin, Christopher L.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
435 71, 435 79, 435 792, 435 795, 436532, 436815, 530810, 530811, 530816, 530839, G01N 33533
Patent
active
061597465
DESCRIPTION:
BRIEF SUMMARY
The present invention refers to a solid phase immunoassay for detecting specific inhibitors of proteolytic enzymes in biological fluids or in any kind of solution containing them, as well as for detecting proteolytic activities in any solution containing them.
At present, a number of methods for assay of protease and anti-proteolytic activity on protein substrate are available. Among the known methods, the assays in which proteases and their inhibitors are determined by using mono- or polyclonal antibodies are the most specific and sensitive. However, no one of such procedures is universally suitable given the diversity and specificity of the proteases, thus requiring different methods for different classes of proteases.
The assay of the invention comprises contacting a tubulin protein or a tubulin-like peptide covalently linked to a suitable support with a solution containing a proteases together with a related inhibitor and determining said inhibitor by using a monoclonal antibody which specifically recognizes the free end of the linked tubulin. Within the meaning of the term "tubulin-like peptide" is comprised any peptide on which the more common classes of proteases are active and the free end of which is identical to the free end of tubulin. With the term "free end", it is intended the end of the tubulin protein or of the peptide which is not linked to the support; as said protein or peptide are linked to the support preferably via their N-terminus, in general the "free end" corresponds to the C-terminus end of the amino acid sequence.
With the present assay it is possible to determine the inhibitors of the more common classes of proteases at the same time, using the same peptidic substrate and the same detection antibody.
The method of the present invention may also be employed to detect the presence of the above proteases in the tested solution, by detecting them with specific known inhibitors.
With the present assay it is also possible to compare the inhibition potency of a given inhibitor which is normally obtained in admixture with unknown substances, thus permitting to follow the purification of said inhibitor.
The method of the invention is accurate, precise, rapid and easy to practice. The intra- and inter- assay precision are well within the range of values currently accepted for analytical purposes.
With the assay of the present invention, the activity of the inhibitors can also be determined when any impurity is present in the tested solution, as after the enzymatic reaction the peptidic substrate is thoroughly washed before detecting the activity of the inhibitor.
Another object of the present invention is a kit for use in the above method for determining anti-proteolytic activities, which comprises a support to which a tubulin protein or a tubulin-like peptide is covalently linked and a solution containing a monoclonal antibody which specifically recognizes the free end of the linked tubulin.
Preferred proteases for which the method of the present invention is particularly useful are the proteases of the serine, aspartic and cysteine classes.
As mentioned above, the peptidic substrate which is employed in the assay of the present invention for determining the inhibition of anti-proteolytic substances against the above proteases is tubulin or a tubulin-like peptide. Preferably, animal tubulin is employed, particularly preferred being bovine brain tubulin.
Such a protein is obtained by means of purification and separation procedures known in the art. For instance, bovine brain tubulin may be purified from bovine brain extract according to Islam K. and R. G. Burns, FEBS Lett., 123, 1981, pp. 181-185 and R. G. Burns and Islam K., Europ. Jour. Biochem., 117, 1981, 515-519; separation of tubulin from microtubule-associated proteins (MAPs) may be performed by known chromatographic procedures, preferably by ion-exchange chromatography according to Vallee et al. in "Methods in Enzymology", 134, 1986, pp. 89-116.
In general, tubulin is obtained, and employed in the present assay, as an equimolar mi
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Carrano Lucia
Denaro Maurizio
Islam Khalid
Chin Christopher L.
Gruppo Lepetit S.p.A.
Homan Ruth E.
Nguyen Bao-Thuy L.
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