SE423456B - Method of producing antiserum for histological identification of endocrine-active cells, especially cells producing substance P - Google Patents

Method of producing antiserum for histological identification of endocrine-active cells, especially cells producing substance P

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Publication number
SE423456B
SE423456B SE8006439A SE8006439A SE423456B SE 423456 B SE423456 B SE 423456B SE 8006439 A SE8006439 A SE 8006439A SE 8006439 A SE8006439 A SE 8006439A SE 423456 B SE423456 B SE 423456B
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Sweden
Prior art keywords
antiserum
cells
producing
granule fraction
peptide
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Application number
SE8006439A
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Swedish (sv)
Other versions
SE8006439L (en
Inventor
R Hakansson
J Thorell
Original Assignee
Milab Malmo Immunalaboratorium
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Milab Malmo Immunalaboratorium filed Critical Milab Malmo Immunalaboratorium
Priority to SE8006439A priority Critical patent/SE423456B/en
Publication of SE8006439L publication Critical patent/SE8006439L/xx
Publication of SE423456B publication Critical patent/SE423456B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Method of producing an antiserum to a peptide or protein component from endocrine-active cells for identification of such cells by applying the antiserum to a microscopic section. Tissue with said endocrinally active cells is disintegrated, and from the disintegrated product one separates in familiar fashion by centrifuging and filtering steps an insoluble granule fraction, which contains said peptide or protein component. The insoluble granule fraction is suspended in aqueous solution and the antiserum is prepared by using the aqueous suspension. Especially intended are antisera to a granule fraction from tumours, especially carcinoids producing substance P.

Description

10 20 ' gzs 30 35 40 n' 5 sooe4à9-7 ledningsvis angivna sättet enligt uppfinningen erhallit de ckännetecken, som framgår av patentkravet 1. 10 20 'gzs 30 35 40 n ' 5 sooe4à9-7 the method according to the invention according to the invention characteristics as claimed in claim 1.

",Särskilt avses att genom sättet enligt uppfinningen isolera en peptid- eller proteinkomponent från sådana endo- -krint aktiva celler, som tidigare inte har kunnat identifie- ras genom tillämpning av-gängse teknik, och att.framställa antisera mot denna komponent, vilka sedan kan appliceras på mikroskopiska histologiska snitt för pâvisande av dessa cel- lers förekomst i snittet. I , '_ Uppfinningen ekemplifieras i det följande genom beskriv- ning av sättets tillämpning på vissa endokrint aktiva celler i mag-tarmkanalen tillhörande det s k enterokromaffina cell- systemet. W e 0 0 _ Eftersom peptidhormon-producerande celler i mag-tarmkana~ len ej förekommer samlade utan är diffust utspridda, möter det stora svårigheter att isolera enstaka peptider från sådana celler, Vid den som exempel valda tillämpningen har därför i stället valts möjligheten att isolera de ifrågavarande cell- komponenterna ur en tumör, utgången från celler av den tumör- ggrupp, som betecknas som substans P-producerande carcinoid.In particular, it is intended that by the method according to the invention isolate a peptide or protein component from such endocytes -cryptally active cells, which have not previously been able to identify race through the application of current technology, and to produce antisera against this component, which can then be applied to microscopic histological sections to detect these cells occur in the section. I, '_ The invention is exemplified in the following by describing application of the method to certain endocrine active cells in the gastrointestinal tract belonging to the so-called enterochromaffin cell the system. W e 0 0 _ Because peptide hormone-producing cells in the gastrointestinal tract ~ len does not appear collected but is diffusely scattered, meets the great difficulty in isolating individual peptides from such cells, In the example selected application has therefore in instead, the possibility of isolating the cells in question components of a tumor, the output of cells of the tumor group, which is designated as substance P-producing carcinoid.

, Tumörcellerna disintegreradesgenom homogenisering i 0,3 M csukroslösning, och det erhållna homogenisatet rensades från celler, cellkärnor och andra större fragment genom centrifu- gering vid 1000 xlg i 20 minuter vid 0°C. Supernatanten fick passera en serie MHLIPOR-filter'med avtagande porstorlek: 8000, 5000, 3000, l200 och 800 nm. Filtratet, en olöslig frak- tion med partikelstorleken mindre än 800 nm centrifugerades därefter vid 40000 x g i 30 minuter. Det därigenom erhållna sedimentet eller pelleten slammades upp i destillerat vatten, g dialyserades mot vatten under något dygn och lyofiliserades.The tumor cells were disintegrated by homogenization in 0.3 M sucrose solution, and the resulting homogenate was purified cells, cell nuclei and other larger fragments by centrifugation fermentation at 1000 xlg for 20 minutes at 0 ° C. The supernatant received pass a series of MHLIPOR filters with decreasing pore size: 8000, 5000, 3000, l200 and 800 nm. The filtrate, an insoluble fraction particle size less than 800 nm was centrifuged then at 40,000 x g for 30 minutes. It thereby obtained the sediment or pellet was slurried in distilled water, g was dialyzed against water for a few days and lyophilized.

Det lyofiliserade materialet suspenderades därefter i en vattenlösning och homogeniserades, varigenom erhölls en sus- pension, bestående av såväl lösliga som olösliga komponenter i granulas väggar och innehåll. Ett antal kaniner immunisera- des genom intrakutan injektion av suspensionen tillsamman med s k Freunds adjuvans. Efter sex veckor kunde antikroppar mot någon komponent i suspensionen påvisas genom att tumörceller från substans P-producerande carcinoider reagerade vid kon- ventionell immunohistokemisk färgning., ' pDet antigen, som vid det beskrivna tillvägagângssättet 10 8006439-7 ger upphov ti11 antikropparna i erhâiïet antiserum, har ännu inte kunnat bestämt identifieras.The lyophilized material was then suspended in one aqueous solution and homogenized to give a suspension pension, consisting of both soluble and insoluble components in granule walls and contents. A number of rabbits immunize by intracutaneous injection of the suspension together with s k Freund's adjuvant. After six weeks, antibodies against any component of the suspension is detected by tumor cells from substance P-producing carcinoids reacted at con- conventional immunohistochemical staining. The antigen, as in the described procedure 10 8006439-7 gives rise to the antibodies in the obtained antiserum, has yet could not be definitively identified.

Vid appïicering av antiserumet på histoïogiska snitt från normal mag-tarmkanai har man funnit, att antikropparna kan bindas tiil de endokrint aktiva ceiïer, från viïka substans P-producerande carcinoider anses utgå. En serie carcinoidtumö- rer har undersökts med PAP-färgat antiserum, framstäïit eniigt ovan, och man har då funnit, att sådana tumörer visar en po- sitiv reaktion med antiserumet. Däremot reagerar detta inte med ce11er från tumörer av annat ursprung.When applying the antiserum to histological sections from normal gastrointestinal tract it has been found, that the antibodies can be bound to the endocrine active cells, from viika substance P-producing carcinoids are considered deleted. A series of carcinoid tumors have been tested with PAP-stained antiserum, presented unanimously above, and it has then been found that such tumors show a positive positive reaction with the antiserum. However, this does not react with ce11s from tumors of other origin.

Claims (4)

gsoo6459-7 10 l5 2.0 25 PATENTKRAVgsoo6459-7 10 l5 2.0 25 PATENT CLAIMS l. Sätt att framställa ett antiserum mot en peptid- eller proteinkomponent-från endokrint aktiva celler för identifiering av sådana celler genom applicering av anti- serumet på mikroskopiska snitt, k ä n n«e t e c k n a t av att vävnad med de nämnda endokrint aktiva cellerna desintegreras, att från den desintegrerade produkten av- skiljes påfi och för sig känt sätt en olöslig granulafrak- tion, innefattande den nämnda peptid- eller proteinkompo- nenten härrörande från cytoplasman och med en partikel- storlek < 800 mm, att den olösliga granulafraktionen sus- penderas i vattenlösning och att antiserumet framställes med användning av den sålunda erhållna vattensuspensionen.A method of preparing an antiserum to a peptide or protein component from endocrine active cells for identifying such cells by applying the antiserum to microscopic sections, characterized in that tissue with said endocrine active cells disintegrates, from the disintegrated product is separated in a manner known per se an insoluble granule fraction, comprising the said peptide or protein component originating from the cytoplasm and having a particle size <800 mm, that the insoluble granule fraction is suspended in aqueous solution and that the antiserum is prepared using the aqueous suspension thus obtained. 2. Sätt enligt krav l, k ä n n e t e c k n a t av att den vävnad som desintegreras utgöres av tumörer från de nämnda cellerna. e s2. A method according to claim 1, characterized in that the tissue that is disintegrated consists of tumors from said cells. e s 3. Sätt enligt något av krav l eller 2, k ä n n e - t e c k n a t av att sedimentet slammas i vatten och lyofi- liseras före suspenderingen i vattenlösning.3. A method according to any one of claims 1 or 2, characterized in that the sediment is slurried in water and lyophilized before the suspension in aqueous solution. 4. Sätt enligt något av krav l - 3, k ä n n e - t e c k n a t av att granulafraktionen framställes från tumörer av typ substans P-producerande carcinoid. 10 l5 8006439-7 SAMMANDRAG Sätt att framställa ett antiserum mot en peptid- eller proteinkomponent från endokrint aktiva celler för identifiering av sådana celler genom applicering av anti- serumet på mikroskopiska snitt. Vävnad med de nämnda endokrint aktiva cellerna desintegreras, och från den desintegrerade produkten avskiljes på i och för sig känt sätt genom centrifugerings- och filtreringssteg en olöslig granulafraktion, som innefattar den nämnda peptid- eller proteinkomponenten. Den olösliga granul- afraktionen suspenderas i vattenlösning och med använd- ning av vattensuspensionen framställes antiserumet. Särskilt avses antisera mot en granulafraktion från tumörer, speciellt substans P-producerande carcinoid.4. A method according to any one of claims 1 - 3, characterized in that the granule fraction is prepared from tumors of the substance substance P-producing carcinoid. SUMMARY Methods of preparing an antiserum to a peptide or protein component from endocrine active cells for identification of such cells by applying the antiserum to microscopic sections. Tissue with said endocrine active cells is disintegrated, and from the disintegrated product an insoluble granule fraction comprising the said peptide or protein component is separated in a manner known per se by centrifugation and filtration steps. The insoluble granule fraction is suspended in aqueous solution and the antiserum is prepared using the aqueous suspension. In particular, antisera are intended against a granule fraction from tumors, especially substance P-producing carcinoid.
SE8006439A 1980-09-15 1980-09-15 Method of producing antiserum for histological identification of endocrine-active cells, especially cells producing substance P SE423456B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
SE8006439A SE423456B (en) 1980-09-15 1980-09-15 Method of producing antiserum for histological identification of endocrine-active cells, especially cells producing substance P

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
SE8006439A SE423456B (en) 1980-09-15 1980-09-15 Method of producing antiserum for histological identification of endocrine-active cells, especially cells producing substance P

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SE8006439L SE8006439L (en) 1982-03-16
SE423456B true SE423456B (en) 1982-05-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003307A1 (en) * 1982-03-15 1983-09-29 Hakansson, Rolf Method for production of antiserum against granula fraction from endocrinologically active cells

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003307A1 (en) * 1982-03-15 1983-09-29 Hakansson, Rolf Method for production of antiserum against granula fraction from endocrinologically active cells

Also Published As

Publication number Publication date
SE8006439L (en) 1982-03-16

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