LU86652A1 - METHOD FOR DETECTING TISSUE LESIONS - Google Patents

Description

I “T BL-3963

_ Q A lf C 0 GRAND-DUCHÉ DE LUXEMBOURG

Brevet N ° Q 0 0 v t

Monsieur le Ministre 4 du 10 novembre 1986 de l’Économie et des Classes Moyennes, = Î £ La | Service de la Propriété Intellectuelle

__Z_ LUXEMBOURG

^

Demande de Brevet d'invention /. ---------------------------------—................ .................................................. .—------------------------------------------------ - (i) I. Requête

La soc, -dite PROGEN. BIOTECHNIK GmbH, in Neuenheimer Feld. 519, __________ ". (2) -U ...-...... 6900 ......... Heidelberg ------------------------ -------------------------------------------------- ----------------------------------..._______________ représentée par E.Megers & ET Freilinger, Ing.conseils en propreind., ^ ^ 46 rue du Cimetière. Luxembourg, ______ agissant en qualité de mandataires________ déposent) ce —dix novembxe. mil-Jieuf cent, quatre vingt six ___ (4) à 10_______ heures, au Ministère de l'Économie et des Classes Moyennes, à Luxembourg: 1. la présente requête pour l'obtention d'un brevet d'invention concernant: nVerfahrerL .. .to find ..... of ...... tissue lesions "__ (5) 2. la description en langue______allemande_________________________________de l'invention en trois exemplaires; 3. ............- .............................................. planches de dessin, en trois exemplaires; '4th la quittance des taxes versées au Bureau de l'Enregistrement à Luxembourg, le 10 novembre 1986_______; 5th la délégation de pouvoir, datée de _____ Heidelb.org .________________ le 5 novembre. 1986 ____;! 6.le document d'ayant cause (authorization); 1 déclare (nt) en assumant la responsabilité de cette déclaration, que l '(es) inventeur (s) est (sont): (6) 1 ... 2 -.... .Dr.Garda ..... BROTHER ...... Bergheimer .... S ± rasse 11 OA, ..... 1 .....-.... 6,900.Heidelberg ___________________________!., 2 ., ....... Prof ........ Dr We rner Wilhelm FRANKE, ..... Landfriedstrasse 6, D - 6900 Heidelberg revendique (nt) pour la susdite demande de brevet la priorité d'une (des) demande (s) de (7) ........ .................................................. .................................................. ......................................... déposée (s) en (8) __________________________________________________________________ le (9) .............................................. ... ”.............................................. .................................................. ...----------------------------------------------- -------------------------------------------------- ----- sous le N ° (10) ....................... ".............. .................................................. .................................................. .................................................. ..............................................__... ........

au nom de (11) __________________________ “_______________________________________________________________________________________________________________________________________________________________ élitfélisent) domicile pour lui (elle) et, si désigné, pour son mandataire, à Luxembourg________________ 46 rue du ..... Cimetière.f ........... Luxembourg .____________________________________________________________________________________________ (12) solliciteQnt) la délivrance d'un brevet d'invention pour l'objet décrit et représenté dans les annexes susmentionnées, avec ajournement de cette délivrance à ............... .............. dix-huit _ ................................ .......................................... mois. (13)

Le d ^ ô / ^ iji / mandataire: ...... Ernest ..... Meyers ....................-...... ....................-............................. .-........... — .................. — ................. ..........., (14) Π. Procès-verbal de Dépôt

La susditéaemande de brevet d’invention a été déposée au Ministère de l’Économie et des Classes Moyennes, Service de la Propriété Intellectuelle · «iLuxembourg, en date du: 10 novembre 1986

/ ** '· **' \, I

/ Z 'f. \ Pr. Le Ministre de l'Economie et des Classes Moyennes, à ......... IQ ................... heures f *> \ pjd .

U \ P j Le chef du service de Impropriété intellectuelle, ™ -: - y / _. w / EXPLICATIONS RELATIVES AU FOR Wed XVTJirt / _ / (1) s'il ya lieu "Demande de certificat d'addilion au brevei principal, à la demande de brevet principal No ..........: THu \ ../.......**- (2) inscrire les nom, prénom, profession, adresse du demandeur, lorsque celui-ci est un particulier ou les dénomination sociale, formme juridique, adresse du siège social Jusque le demandeur est une personne morale - (3) inscrire * *

BL-396 3 / BM

PATENT APPLICATION Procedure for finding tissue lesions PROGEN BIOTECHNIK GmbH Im Neuenheimer Feld 519 D. 6900 Heidelberg 1 A 57 019 5.11.86 * »* g

METHOD FOR FINDING VIALS

The invention relates to a method for locating tissue lesions, in which body fluid is drawn off and examined.

It is known to find hepatitis by detecting liver-specific enzymes in the blood serum. Myocardial lesions can also be found by reading myocardial-specific enzymes and other soluble proteins. Enzymes have only a limited activity and many proteins or cell-specific proteins are not soluble in serum and other body fluids. This limits the application of this method.

The object of the invention is to design a method of the type mentioned at the outset so that it can be used as widely as possible.

The invention is characterized in that soluble fragments of insoluble, antigen-bearing, intracellular and non-secreted structures and proteins, immunologically cytoskeletal proteins which can be assigned to the damaged tissue, are immunologically determined and quantified.

A further development of the invention is characterized in that the antigen-carrying proteins are intermediate filament proteins.

The invention makes use of the following findings from cell biology:

Intermediate filament (IF) proteins, which, as components of the cytoskeleton, belong to the insoluble cell components, jr v 2 A 57 019 30.10.86.

^ are proteins of high cell type specificity distinguished by their high stability ♦ The assignment is shown in Table I below.

TABLE 1

Tissue intermediate filament protein

Muscle desmin

Neuronal neurofilament proteins (NF-L, NF-M, NFH)

Mesenchymal vimentin

Astrocyte glia filament

Epithelial cytokératine

After cell lesions, insoluble proteins and structures are often proteolytically broken down. After degradation reactions, for example, IF proteins are used to obtain the relatively stable alpha-helical centerpieces, which can now be detected and determined in body fluids using an immunological process.

The detection procedure is carried out with the help of a sandwich ELISA, which is made up of two specific antibodies for each filament type.

Serum from blood, cerebrospinal fluid, urine, amniotic fluid and Punktat e.

Intermediate filament proteins can be mixed in insoluble form in the dots. So that these are also taken into account in the serological examination, - * »3 A 57 019 30.10.86.

ayf the stripped punctate has a degrading effect until at least a large part of the intermediate filament proteins that may be present in the punctate is broken down into the alpha-helical center piece. Then the soluble fraction is separated and then the alpha-helical middle pieces contained in the soluble fraction are identified immunologically in the soluble phase.

In order to ensure that the alpha-helical centerpieces are not destroyed to the extent that they hinder identification, it is recommended that they only be degraded until they are 60 to 100 percent by weight at a temperature of 0 to 40 ° C (degrees Celsius) are soluble in physiological saline. This is done by stopping the activity of the enzyme used for the breakdown by means of suitable inhibitors. It is expedient here to break down the intermediate filament proteins only to such an extent that the alpha-helical center piece remains intact at at least 80 percent by weight.

There are various cytokératins (No. 1 - 19), whose expression patterns are assigned to different epithelial tissues. In the case of cytokeratins, the type of epithelial tissue assigned is therefore preferably also determined and quantified. Also for this assignment has sufficient characteristics in the alpha-helical centerpieces.

The standardization required for the quantification is expediently carried out on the basis of a defined standard, the standard being formed from purified alpha-helical middle pieces of the respectively assigned intermediate filament proteins.

The invention allows the results of the determination and quantification to be taken immediately after the serum has been withdrawn. 4 a 57 019 30.10.86.

determine. That is a particular advantage.

4th

The invention further makes it possible to control the course of pathological or therapeutic processes - for example radiation, chemotherapy - in which tissue is destroyed by the removal of the body fluid and the determination and quantification several times in succession during the course of a geue-contaminating treatment injurious treatment and / or afterwards.

The invention will now be explained in more detail with reference to the accompanying examples.

1% 5 A 57 019 10/30/86.

* EXAMPLE 1

Detection of lesions in epithelial tissue 2ur differential diagnosis ik

The cytokératins present are immunologically determined in 1 ml (milliliter) of patient blood serum by a sandwich ELISA. A first monoclonal antibody PAN-CI is used for this. the so-called catcher antibody, which is directed against a first epitope, which occurs in all cytokratin fragment complexes present in the serum. Further monoclonal antibodies C-401 to C-419 are provided, so-called detector antibodies, which are directed individually against second epitopes, which are specifically assigned to the individual known 19 cytokeratins. The second epitope is independent and different from the first epitope.

The detector antibodies are enzymatically labeled with peroxidase.

For standardization, the purified alpha-helical middle piece of the 19 different cytokératins is subjected to the same sandwich ELISA with the same antibodies. The 19 different cytokeratin values thus obtained then serve as reference values for the quantitative evaluation of the measurement results. _

In this way the relative proportion of the individual cytokératins is determined. This allows an exact specification of the damaged epithelial tissue and conclusions about the extent of the injury.

If only cytokératins 8 and 18 are to be re-examined, this suggests a lesion of hepatocytes, renal epithelium or acini, the pancreas and the tumors derived from these cell types and makes others' * 6 A 57 019 30.10.86.

Tissue unlikely to be the site of the lesion.

EXAMPLE 2

Detection of lesions in the inner epithelial area

See Example 1, but with the limitation to the capture antibody PAN-CI and those Detektoi antibodies C-408, D-418 and C-419, which are assigned to the cytokeratins No. 8. No. 18 and No. 19 . If cytokératins No. 8, No. 18 and No. 19 are detected with sufficient proportions, this indicates a lesion in the epithelial area of the intestinal tract and makes other organs seem unlikely as the site of the lesion.

EXAMPLE 3

Follow-up of chemotherapy for liver hepatoma

The examination and determination of cytokératins 8 and 18 according to Example 1 is repeated on blood samples taken monthly from the same patient.

EXAMPLE 4

Early diagnosis if suspected neural tube defects during pregnancy are centrifuged 10 ml amniotic fluid punctate. The sediment is crushed in a 3-fold volume, based on the wet weight, of a phosphate-buffered saline solution <10 rnM (millimolar) sodium phosphate pH 7.4, 150 mH sodium chloride) with the help of a knife homogenizer to a pulp-like consistency. Chymotrypsin is added to the homogenate in a ratio of 1: 1000, calculated on the wet weight of the tissue. This is followed by an incubation step at 30 ° C in the Uasser bath, which is stopped after 60 minutes. The enzyme activity is stopped by adding 5 mM 2-7 A 57 019 30.10.86.

Nitro-4-carboxyphenyl-N-N-diphenyl carbamate (NCDC). The homogenate is then centrifuged for 30 minutes at 10g (.g = gravitational constant).

Under these conditions, 80 to 95 percent by weight of the alpha-helical middle pieces are released from the IF proteins of the homogenate in a still identifiable state.

The two centrifuged supernatants obtained are separated, each individually, immunologically examined for their content of neurofilament and gliafilament protein with a sandwich ELISA. For this purpose, a first monoclonal antibody NF-8 or GL-8, the so-called catcher antibody, is used for the two filament types, which is directed against a first epitope of the alpha-helical middle piece of the filament proteins. In addition, a second monoclonal antibody NF-100 or GL-1Û0, the so-called detector antibody, is used, which is directed against a second epitope of the alphahelical middle part of the associated filament. The second epitope is independent and different from the first epitope.

The detector antibodies NF-100 and GL-100 are coupled with peroxidase and are detected in the binding to the alpha-helical center via the enzyme activity of the peroxidase. The standardization is carried out in accordance with example 1.

The occurrence of glia or neurofilament fragments in amniotic fluid samples indicates neural tube defects and thus provides a much more reliable and faster prenatal diagnostic test than previously known methods used.

k 4 <· »8 A 57 019 30.10.86.

EXAMPLE 5-7

See Example 1-4, but instead of the individual capture antibodies assigned to the individual cytokinatins, capture antibodies are used which recognize the individual cytokératins in combination. For example, the combination C-501 and C-504 recognizes the cytokeratin number X, the combination C-501 and C-503 recognizes the cytokeratin (X + 1).

EXAMPLE 8

Obtaining the antibodies

Vimentin is cleaned from bovine eye lenses, reconstituted as filament, cleaved with chymotrypsin, and the alpha-helical agent is separated chromatographically and used to produce the monoclonal antibodies using hybridoma technology. The specificity of the antibodies is determined with the aid of the purified alpha-helical fragments and cross-reactions with the other IF proteins by immune reactions in the native (or renatured) and denatured state of these proteins are excluded.

Claims (12)

1. A method for finding tissue lesions, in which body fluid is drawn off and examined, characterized in that soluble fragments of insoluble, antigen-bearing, intracellular and non-secreted structures and proteins, immunologically assignable to the damaged tissue, cytoskeleton proteins are determined and quantified immunologically will. 2. The method according to claim 1, characterized in that the antigen-carrying proteins are intermediate filament proteins. 3. The method according to claim 1 or 2, characterized in that the body fluid withdrawn is blood and the determination is made in blood serum. 4. The method according to claim 1 or 2, characterized in that the body fluid withdrawn is cerebrospinal 1iquor. 5. The method according to claim 1 or 2, characterized in that the removed body fluid is urine. 6. The method according to claim 1 or 2, characterized in that the removed body fluid is amniotic fluid. 7. The method according to claim 1 or 2, characterized in that the removed body fluid is obtained by puncturing. 10 a 57 019 5.11.86. , * * · » 8. The method according to claim 7, characterized in that s has a degrading effect on the withdrawn punctate until soluble fragments have formed from at least a large part of the structural proteins which may be present in the punctate, that the soluble fraction is then separated and that then in the fragments contained in the soluble fraction can be identified immunologically. 9. The method according to any one of the preceding claims, characterized in that, in the case of cytokeratin in the body fluid, the type of epithelial tissue assigned in each case is determined and guaranteed on the basis of the relative proportion of the identified cytokératins No. 1 to 19. 10. The method according to any one of the preceding claims, characterized in that the quantification takes place on the basis of a defined standard, the standard being formed from purified biochemically defined fragments of the cytoskeleton. 11. The method according to any one of the preceding claims, characterized in that the quantification is carried out on the basis of a defined standard, the standard being formed from purified alpha-helical elements of the respectively assigned intermediate filament proteins. 1i A 57 019 5.11.86. \ * "k 12. The method according to any one of the preceding claims, characterized in that for monitoring the course of medical treatment, the removal of the body fluid and the determination and quantification take place several times in succession during the treatment and / or afterwards.