EP1042672A1 - Solid phase test for endotoxin - Google Patents
Solid phase test for endotoxinInfo
- Publication number
- EP1042672A1 EP1042672A1 EP99915905A EP99915905A EP1042672A1 EP 1042672 A1 EP1042672 A1 EP 1042672A1 EP 99915905 A EP99915905 A EP 99915905A EP 99915905 A EP99915905 A EP 99915905A EP 1042672 A1 EP1042672 A1 EP 1042672A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- endotoxin
- solid phase
- phase apparatus
- indicator
- buffer components
- 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- Withdrawn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
-
- 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
-
- 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
- G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
- G01N2333/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
Definitions
- This invention relates to a solid phase test for endotoxin, in ⁇ particular a test such as in a flat sheet or in capillary form for the detection of endotoxin in aqueous solutions. More particularly the invention relates to a direct solid phase chromogenic assay, which permits a quick biochemical identification of endotoxin and is sensitive to picogram endotoxin amounts.
- Horseshoe crab (limulus) amoebocyte lysate provokes together with endotoxin from gram-negative bacteria a gelatination induced by a stepwise activation of several coagulation factors contained in the lysate.
- An assay comprising the extract (lysate) of horseshoe crab blood cell (amoebocyte) to induce this gelatination in a reagent glass as a liquid phase system is well known as a method and it is called the limulus test.
- the application of the LAL to measure endotoxin involves the gelatination of the sample (gelation method, turbidimetry assay) in the reagent glass or a more sensitive method based on a chromogenic substrate and measurement by optical absorption.
- gelation method turbidimetry assay
- -Today different LAL from horseshoe crab 2 are - 2 - commercialized through the world: Limulus Polyphemus, Tchypleus tridendatus, Tachypleus glgas and Carcinoscorpius rotundicauda.
- All commercialized LAL testing methods based on a chromogenic substrate are liquid-phase assays.
- the principle is that in a reagent glass containing a lysate and sample, bacterial endotoxin contained in the sample initiates activation of a cascade of serine protease (Factor B, Factor C, Proclotting enzyme, Coagulin) enzymes in LAL that cleave the end product coagulogen into a peptide coagulin which produces clotting.
- a cascade of serine protease Factor C
- Proclotting enzyme Proclotting enzyme
- a synthetic peptide having an amino acid sequence in common with the hydrolysis sites of coagulogen, namely the chromogenic substrate Boc-Leu-Gly-Arg-p-nitroanilide (pNA) or the fluoro-genic substrate Boc-Leu-Gly-Arg-4-methylcoumaryl-7-amide.
- the colourless substrate is rapidly cleaved to form the chromophore p-nitroaniline or the fluorogenic 4-methylcoumaryl-7-amide.
- Tanaka disclosed a quantitative kit for endotoxin determination which comprises an insoluble carrier on which is immobilized an endotoxin-sensitive factor derived from a limulus amoebocyte which specifically reacts with endotoxin without reacting with ⁇ -glucan.
- the test system involves the indication of endotoxin when colouring occurs in the liquid phase.
- the present invention provides an analytical test strip for the detection of endotoxin in aqueous solutions in which the strip is wetted at the bottom with the sample. After a certain period of time, until the sample flows from the bottom to the top, an indicator is added at the top, to develop a red colour which is indicative for the presence or the absence of endotoxin in the sample.
- the present invention involves an improvement to a quick test strip for the detection of endotoxin.
- the test strip provides a dry analytical element for endotoxin which comprises an absorbent carrier divided in four portions impregnated with: 1 ) a reagent system of limulus factors; 2) synthetic chromogenic substrate derivatized with p-nitroaniline; 3) a buffer system containing chloride acid and sodium nitrite; 4) a buffer system containing chloride acid and ammonium.
- the first portion contains a solid support and a limulus " amoebocyte lysate or substances containing endotoxin sensitive factors which react with endotoxin to produce coagulin.
- the method of the immobilization of the lysate or of the endotoxin sensitive factor to the solid support are: 1 ) absorption; 2) adsorption by ionic forces or/and hydrophobic interaction, 3) covalent binding.
- the procedures for binding the lysate in the solid support are generally known the art.
- the solid support which is employed in this test strip is one which is capable of absorbing endotoxin from the sample.
- the sample containing endotoxin reacts with reagents in the first portion and the reaction products are transported by flow to the dry region of the first support and thus to the second portion of the test strip.
- the solid support is one which is capable of absorbing both sample and binder.
- an hydrophillic polymers solid support examples are chromatographic papers, nitrocellulose, dextran and glass fibres.
- chromatographic hydrophobic polymers are used though if using chromatography papers, it is important to use reagents which do not react with ⁇ -glucan, as described for example in US patent 5,550,030.
- the solid support is typically made with the same material as the first support and it is one which is capable of absorbing the liquid and reactants flowing from the first support, and which when wetted is this way, provides for flow and analyte by capillary attraction from the first portion, and through the second portion into the third portion.
- solution should react with immobilized chromogenic substrate.
- suitable supports are chromatographic papers, nitrocellulose, dextran and glass fibres. Preferably, chromatographic papers are used.
- the solid support absorbed with the chromogenic substrate is one which is capable otabsorbing reactants flowing from the first support.
- the solid support When dried, the solid support is also one which is capable of absorbing the liquid and the chromophore p-nitroaniline flowing from the second support, and which when wetted is this way, provides for flow from the second portion, and through the third portion into the fourth portion.
- the fourth portion contains a solid support and a buffer containing ⁇ N HCI containing 0.5% ammonium.
- a solid support Preferably, to make the support, it is impregnated with the buffer system, and dried.
- the solid support When absorbed with the buffer system and dried, the solid support is also one which is capable of absorbing the liquid and the chromophore p-nitroaniline flowing from the third support, and which when wetted is this way, provides for flow from the third portion, and through the dried region.
- the third and fourth portions are those through which when the flowing free p- nitroaniline released by the action of enzymes is derivatized to its diazanium salt, typically formed in the fourth portion.
- Fig. 5 shows a quantitative test strip.
- Portion 1 hydrophillic polymeric powder was incubated under pyrogen free conditions in 0.5 lU/ml (1 g/ml) of limulus amoebocyte lysate (Charles River- Sulzfel Germany) until saturated absorption of the liquid was reached. After drying, the module has been filled to reach a height of 1 cm. 0.1 cm has then been filled with neutral polymeric powder.
- Tris-HCI is mixed with polymeric powder to obtain a concentration of 0.025 mmol/l and inserted between portions 2 and 3 with a height of 0.5 cm. 0.1 cm has been filled with neutral polymeric powder.
- Portion 3 Polymeric powder was submerged in a solution of 1 N HCI containing 0.1 % (w/v) sodium nitrite, and dried. A quantity sufficient for 0.5 cm has been inserted into the module. 0.1 cm has been filled with neutral polymeric powder.
- Portion 4 1 cm of polymeric powder was submerged with in solution of 1 N HCI containing 0.5 % (W/v) ammonium sulfate, dried and has been filled into the module. 0.1 cm has been filled with neutral polymeric powder.
- Portion 5 2.5 mg of N 1 -naphthlethylenediamine in mixed with 0.5 cm polymeric material.
- Portion 2 consisted of 0.5cm x 0.5 cm ultra high molecular weight polyethylene - 10 - sheet material (Porex Technologies) which was spotted with 1 .5 /mol/ml of chromogenic substrate (t-butoxycarbonyl-leucyl-glycyl-arginine-pa-ranitroanilide) for ⁇ ndoxin (Pefachrome LAL Code NR 1 1 1 79-01 ISO 9001 -1 N29001 Pentapharm AG BASEL CH) . The strip was applied just above the neutral sephadex portion.
- Portion 4 consisted of a 0.5 cmx 0.5 cm strip of filter paper (Schleicher & Shuel 23 SL) which was submerged with in solution of 1 N HCI containing 0.5 % (W/v) ammonium sulfate, dried and applied above the third portion.
- Portion 5 consisted of a 0.5 cmx 0.5 cm strip of filter paper (Schleicher & Shuel 23 SL) applied above the fourth portion.
- sample 1 add the sample to the first portion of the test strip or immerse the first portion in the sample (example 2) or fill the reservoir with the sample (example 1 ) .
- clotting enzyme is produced through the cascade reaction by the action of activated Factor C or proclotting enzymes.
- portion 3 after the .capillary flow through the chromogenic substrate and in the presence of Tris-HCI, the activated clotting enzyme and/or factor B flowing from the lower portion cleaves p-nitroaniline. The cleaved p-nitroaniline being transported to the third and fourth portion and derivatized to its diazonium salt.
- the diazonium salt is coupled to N1 -napthylethylenediamine to form a diazoamino of a red colour.
- the assay can be performed with different sensitivities, according to the grade of contamination to be analyzed.
- several test strips have been elaborated to read the minimal acceptable concentration of endotoxin in aqueous solution (Table 1 ).
- Figure 4 illustrates the resulting absorbance due to the - 12 - variation of the LAL which allow 4 classes of test-strips. Applied to the test-strip of example 2, it is possible to prepare a test-strip especially for the detection of endotoxin concentration in dialysate-solution, for which the acceptable limit is prescribed to be below 0.5 EU/ml. Detection spots for this prepared test strip are illustrated in Figure 5.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9807814.0A GB9807814D0 (en) | 1998-04-09 | 1998-04-09 | Solid phase test for endoxin |
GB9807814 | 1998-04-09 | ||
PCT/GB1999/001099 WO1999053322A1 (en) | 1998-04-09 | 1999-04-09 | Solid phase test for endotoxin |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1042672A1 true EP1042672A1 (en) | 2000-10-11 |
Family
ID=10830245
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99915905A Withdrawn EP1042672A1 (en) | 1998-04-09 | 1999-04-09 | Solid phase test for endotoxin |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1042672A1 (en) |
AU (1) | AU3432899A (en) |
CA (1) | CA2327169A1 (en) |
GB (1) | GB9807814D0 (en) |
WO (1) | WO1999053322A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003038433A1 (en) * | 2001-10-29 | 2003-05-08 | Arkray, Inc. | Test apparatus |
ATE452986T1 (en) | 2003-03-17 | 2010-01-15 | Charles River Lab Inc | METHODS AND COMPOSITIONS FOR DETECTING MICROBIAL CONTAMINANTS |
JP2008522640A (en) | 2004-12-02 | 2008-07-03 | チャールズ リバー ラボラトリーズ, インコーポレイテッド | Methods and compositions for detection and / or quantification of Gram positive bacteria contaminants |
EP1836492B1 (en) | 2005-01-13 | 2008-12-31 | Charles River Laboratories, Inc. | Method for classifying a microorganism in a biological sample |
US20130078665A1 (en) * | 2012-07-18 | 2013-03-28 | Deepika Bodapati | Test strip, a test kit and a method for detection of endotoxin in food |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4717658A (en) * | 1985-12-03 | 1988-01-05 | Miles Inc. | Gram negative bacteria screening method with horseshoe crab amebocyte lysate (LAL) |
GB8624253D0 (en) * | 1986-10-09 | 1986-11-12 | Harvey W | Endotoxin assay |
CA1303983C (en) * | 1987-03-27 | 1992-06-23 | Robert W. Rosenstein | Solid phase assay |
DK255887D0 (en) * | 1987-05-20 | 1987-05-20 | Claus Koch | IMMUNOASSAY |
US5198339A (en) * | 1990-07-13 | 1993-03-30 | Board Of Regents, The University Of Texas System | Method for detection of gram-negative bacterial lipopolysaccharides in biological fluids |
JP3322700B2 (en) * | 1992-09-14 | 2002-09-09 | 生化学工業株式会社 | Endotoxin-specific measuring agent for solid-phase reaction |
US5773306A (en) * | 1996-05-24 | 1998-06-30 | Trustees Of The University Of Pennsylvania | Methods and kits for the detection of endotoxin |
-
1998
- 1998-04-09 GB GBGB9807814.0A patent/GB9807814D0/en not_active Ceased
-
1999
- 1999-04-09 EP EP99915905A patent/EP1042672A1/en not_active Withdrawn
- 1999-04-09 WO PCT/GB1999/001099 patent/WO1999053322A1/en not_active Application Discontinuation
- 1999-04-09 CA CA002327169A patent/CA2327169A1/en not_active Abandoned
- 1999-04-09 AU AU34328/99A patent/AU3432899A/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO9953322A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1999053322A1 (en) | 1999-10-21 |
GB9807814D0 (en) | 1998-06-10 |
AU3432899A (en) | 1999-11-01 |
CA2327169A1 (en) | 1999-10-21 |
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