DE19616321A1 - Production of glycolipid mixture from porcine spleen - Google Patents
Production of glycolipid mixture from porcine spleenInfo
- Publication number
- DE19616321A1 DE19616321A1 DE1996116321 DE19616321A DE19616321A1 DE 19616321 A1 DE19616321 A1 DE 19616321A1 DE 1996116321 DE1996116321 DE 1996116321 DE 19616321 A DE19616321 A DE 19616321A DE 19616321 A1 DE19616321 A1 DE 19616321A1
- Authority
- DE
- Germany
- Prior art keywords
- verotoxin
- glycolipid
- test kit
- verotoxins
- receptor mixture
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
Abstract
Description
Im Rahmen der klinisch-mikrobiologischen Labordiagnostik ist der schnelle und direkte Nachweis von Verotoxinen im Stuhl ein wichtiges Verfahren zur Aufklärung von Gastroenteritiden und den postinfektiösen lebensbedrohlichen Symptomen (HUS, TTP, HC). Der bisherige Stand der Technik zum Nachweis von Verotoxinen im Stuhl beschränkt sich auf sehr kosten- und zeitaufwendige Verfahren, die im wesentlichen auf der Detektion der Verotoxine mit Hilfe von Antikörpern basieren (Sandwich-ELISA). Die Durchführung der PCR zum Nachweis verotoxinogener Bakterien direkt mit Stuhlextrakten ist bisher nicht standardisierbar.In the context of clinical microbiological laboratory diagnostics, the fast and Direct detection of verotoxins in stool is an important clarification procedure of gastroenteritis and the post-infectious life-threatening symptoms (HUS, TTP, HC). The previous state of the art for the detection of verotoxins in Chair is limited to very costly and time-consuming procedures that in the are essentially based on the detection of verotoxins with the help of antibodies (Sandwich ELISA). Carrying out the PCR for the detection of verotoxinogenic Bacteria directly with stool extracts cannot yet be standardized.
Zur Verbesserung der Sandwich-ELISA wurde von einigen Autoren (z. B. Basta et al.) die Rezeptor-ELISA (RELISA) eingeführt, bei der ein Antikörper durch einen toxinspezifischen Zellrezeptor (z. B. Gb 3) ersetzt wurde. Derartige Rezeptoren (Gb 2/3/4) sind allerdings nur aus mehreren exotischen Quellen (Fuchsbandwurm, Meeresschnecken etc.) bzw. mit einem hohem Aufwand aus Blut-, Hela-, Verozellen oder aus menschlichen Nieren zu isolieren und anschließend zu mischen. Es wurde nun gefunden, daß in der Schweinemilz die Glykolipide Gb 2/3/4 ausreichend und in günstiger Mischung vorhanden sind. Darüber hinaus fällt dieses Organ in großem Umfang als Schlachtnebenprodukt mit geringen Kosten an. Daher betrifft die vorliegende Erfindung ein Verfahren zur Herstellung eines Glykolipidrezeptorengemisches mit den Merkmalen im Anspruch 1 sowie das danach hergestellte Glykolipidrezeptorengemisch. Zur Isolierung von Glykolipiden aus Schweinemilz wurde das Verfahren nach FOLCH modifiziert angewendet. So konnte Gb 2/3/4 in ausreichender Menge isoliert und im verseiften Zustand für den Nachweis von Verotoxinen angewendet werden. Dabei ist ökonomisch interessant, daß die Gb 2/3/4 in ausreichender Mischung gleichzeitig für den Nachweis aller Verotoxine 1, 2 und Varianten in einem Untersuchungsschrift verwendet werden können.To improve the sandwich ELISA, some authors (e.g. Basta et al.) introduced the receptor ELISA (RELISA), in which an antibody by a toxin-specific cell receptor (e.g. Gb 3) was replaced. Such receptors (Gb 2/3/4) are, however, only from several exotic sources (fox tapeworm, Marine gastropods etc.) or with a lot of effort from blood, hela, vero cells or isolate from human kidneys and then mix. It has now been found that the glycolipids Gb 2/3/4 are sufficient and available in a favorable mixture. In addition, this falls Organ on a large scale as a slaughter by-product at low cost. Therefore The present invention relates to a method for producing a Glycolipid receptor mixture with the features in claim 1 and that after prepared glycolipid receptor mixture. For the isolation of glycolipids Pork spleen was modified using the FOLCH method. So could Gb 2/3/4 isolated in sufficient quantity and in the saponified state for detection of verotoxins. It is economically interesting that the Gb 2/3/4 in sufficient mixture simultaneously for the detection of all verotoxins 1, 2 and variants can be used in an examination document.
Mit der Verwendung von Glykolipidrezeptoren aus Schweinemilz liegt ein billiger und sehr spezifischer sowie ubiquitär anwendbarer Test für alle bekannten Verotoxine vor. Die Erfindung betrifft daher auch ein Verfahren zur Detektion von Verotoxinen mit den Merkmalen im Anspruch 4 sowie ein Testkit mit den Merkmalen im Anspruch 5. Vorteilhafte Ausgestaltungen ergeben sich aus den Ansprüchen 6-10. Der leicht an Mikrotiterplatten anzuheftende Rezeptor ermöglicht die Testung von gleichzeitig vielen Proben (45 Doppelbestimmungen in einem Arbeitsgang) auf Anwesenheit von Verotoxinen aus Patientenmaterialien.With the use of glycolipid receptors from pig spleen lies a cheaper and very specific and ubiquitous test for all known verotoxins in front. The invention therefore also relates to a method for the detection of verotoxins with the features in claim 4 and a test kit with the features in claim 5. Advantageous refinements result from claims 6-10. The easy one The receptor to be attached to microtiter plates enables testing at the same time many samples (45 double determinations in one operation) for the presence of Verotoxins from patient materials.
Das Verfahren zur Herstellung von Glykolipidrezeptoren sowie der Einsatz und die Durchführung der RELISA ergibt den Aufbau eines Testkits. The process for the preparation of glycolipid receptors as well as the use and the Implementation of the RELISA results in the construction of a test kit.
- 1. 50 g Trockenmasse (lyophilisiert) ergeben nach Extraktion, Reinigung und Verseifung nach FOLCH eine Ausbeute von ca. 20 mg Gb 2/3/4.1. 50 g of dry matter (lyophilized) give after extraction, cleaning and Saponification according to FOLCH a yield of approx. 20 mg Gb 2/3/4.
-
2. Reinigung mit den Modifikationen
- - Verwendung eines Chloroform/Methanol-Gemisches anstelle einer Chloroform/Methanol-Phasentrennung
- - Temperatur der Verseifung 37°C anstelle 100°C
- - Use of a chloroform / methanol mixture instead of a chloroform / methanol phase separation
- - Saponification temperature 37 ° C instead of 100 ° C
-
3. Herstellung einer Stammlösung:
10 mg/ml in Methanol gelöst (Stammlösung) sind mindestens 2 Jahre bei 4°C haltbar.3. Preparation of a stock solution:
10 mg / ml dissolved in methanol (stock solution) can be stored at 4 ° C for at least 2 years. - 4. Bindung des Glykolipidgemisches an mit Nitrozellulose beschichteten Mikrotiterplatten (1,2 µg/well; d. h. 15 µl der Stammlösung auf 10 ml Kopplungspuffer). Dadurch bessere Bindung des Rezeptors an die Mikrotiterplatte. Die mit Rezeptoren beladenen Mikrotiterplatten sind bei -20°C mindestens 2 Jahre haltbar.4. Binding of the glycolipid mixture to coated with nitrocellulose Microtiter plates (1.2 µg / well; i.e. 15 µl of the stock solution to 10 ml Coupling buffer). This improves the binding of the receptor to the microtiter plate. The microtiter plates loaded with receptors are at least 2 at -20 ° C Lasts for years.
- 5. Verwendung für den Verotoxinnachweistest: 100 µl/well einer Stuhlaufschwemmung (1 g/l ml PBS), d. h. ca. 45 Stuhlproben pro Mikrotiterplatte als Doppelbestimmung.5. Use for the verotoxin detection test: 100 µl / well of a stool suspension (1 g / l ml PBS), d. H. approx. 45 stool samples per microtiter plate as a double determination.
- 6. Inkubation: 2 h bei 37°C6. Incubation: 2 h at 37 ° C
- 7. Weiterverarbeitung nach dem ELISA-Verfahren (Tschäpe et al.)7. Further processing according to the ELISA method (Tschäpe et al.)
- 8. Als Positivkontrolle dient gereinigtes Verotoxin 1, 2 bzw. der internationale Teststamm E. coli CDC933 (slt1/2).8. Purified verotoxin 1, 2 or international serves as positive control Test strain E. coli CDC933 (slt1 / 2).
- - Beschichtung von Mikrotiterplatten mit einem Glykolipidgemisch Gb 2/3/4 ent sprechend Anlage 4.1. (mindestens 2 Jahre Haltbarkeit der Mikrotiterplatten bei -20°C).- Coating microtiter plates with a glycolipid mixture Gb 2/3/4 ent speaking Appendix 4.1. (At least 2 years shelf life of the microtiter plates -20 ° C).
- - Bindung der Positiv- bzw. Negativkontrolle von Verotoxin 1, 2 und Varianten an die Kontroll-wells.- Binding of the positive or negative control of verotoxin 1, 2 and variants the control wells.
- - Herstellung einer ready for use Anti-Verotoxin 1- bzw. Anti-Verotoxin 2-Antikörper verdünnung für Mikrotiterplatten- Production of a ready for use anti-verotoxin 1 or anti-verotoxin 2 antibody dilution for microtiter plates
- - Herstellung eines markierten Anti-Kaninchen-Antikörpers zur Detektion (z. B. Enzymmarkierung)- Preparation of a labeled anti-rabbit antibody for detection (e.g. Enzyme labeling)
Basta, M., Karmali, M., Lingwood, C. (1989). RELISA und Verseifung. J. of Clinical Microbiology Z, 1617-1622.Basta, M., Karmali, M., Lingwood, C. (1989). RELISA and saponification. J. of Clinical Microbiology Z, 1617-1622.
Folch, J., Lees, M., Stanley, G.H.S. (1957). Glycolipid-Extraktion. J. Biol. Chem. 226, 497-509.Folch, J., Lees, M., Stanley, G.H.S. (1957). Glycolipid extraction. J. Biol. Chem. 226, 497-509.
Tschäpe, H., Prager, R., Streckel, W., Fruth, A., Tietze, E., Böhme, G. (1995). Vertoxinogenic Citrobacter freundii associated with severe gastroenteritis and cases of haemolytic uraemic syndrome in a nursery school: green butter as the infection source. Epidemiol. Infect. 114, 441-450.Tschäpe, H., Prager, R., Streckel, W., Fruth, A., Tietze, E., Böhme, G. (1995). Vertoxinogenic Citrobacter freundii associated with severe gastroenteritis and cases of haemolytic uraemic syndrome in a nursery school: green butter as the infection source. Epidemiol. Infect. 114, 441-450.
Claims (10)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE1996116321 DE19616321A1 (en) | 1996-04-24 | 1996-04-24 | Production of glycolipid mixture from porcine spleen |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE1996116321 DE19616321A1 (en) | 1996-04-24 | 1996-04-24 | Production of glycolipid mixture from porcine spleen |
Publications (1)
Publication Number | Publication Date |
---|---|
DE19616321A1 true DE19616321A1 (en) | 1997-10-30 |
Family
ID=7792278
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
DE1996116321 Withdrawn DE19616321A1 (en) | 1996-04-24 | 1996-04-24 | Production of glycolipid mixture from porcine spleen |
Country Status (1)
Country | Link |
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DE (1) | DE19616321A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0896223A1 (en) * | 1997-08-07 | 1999-02-10 | Nitto Denko Corporation | Immunoassay method and immunoassay kit |
-
1996
- 1996-04-24 DE DE1996116321 patent/DE19616321A1/en not_active Withdrawn
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0896223A1 (en) * | 1997-08-07 | 1999-02-10 | Nitto Denko Corporation | Immunoassay method and immunoassay kit |
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Legal Events
Date | Code | Title | Description |
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8139 | Disposal/non-payment of the annual fee |