WO2001057532A1 - Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents - Google Patents
Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents Download PDFInfo
- Publication number
- WO2001057532A1 WO2001057532A1 PCT/EP2001/000829 EP0100829W WO0157532A1 WO 2001057532 A1 WO2001057532 A1 WO 2001057532A1 EP 0100829 W EP0100829 W EP 0100829W WO 0157532 A1 WO0157532 A1 WO 0157532A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- microorganisms
- chaotropic
- concentration
- filter
- membrane
- Prior art date
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Classifications
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- 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
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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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
Definitions
- the present invention is m the field of diagnostic and is related to a method and kit for the rapid detection of whole microorganisms on retaining membranes by the use of chaotropic agents .
- etiological agents of malaria especially Plasmodi um falciparum, leishma iasis , gonorrhea, syphilis, tuberculosis, malaria, tick-borne Lyme disease, as well as meningitis due to a variety of vectors such as Nei sseria , Haemophi lus , Streptococcus , Li s te ⁇ a and Mycobacteri u , a rapid diagnostic method putting the whole pathogen m evidence does not exist and there is a need for such rapid method.
- etiological agents of malaria especially Plasmodi um falciparum, leishma iasis , gonorrhea, syphilis, tuberculosis, malaria, tick-borne Lyme disease, as well as meningitis due to a variety of vectors such as Nei sseria , Haemophi lus , Streptococcus , Li s t
- the document EP-0306206 describes a diagnostic device comprising a funnel that channels the fluid under analysis onto a defined part of a filter membrane that retains the analyte.
- the funnel is thereafter removed and the membrane is further treated by signal - generating materials and other reagents as washing fluids, applied simultaneously to the test area and adjacent area of the membrane
- the simultaneous treatment of adjacent area supposedly provides a negative control to assist m detecting non-specific binding and thereby false-positive results
- the sample is confined to a discrete area of the filter and the whole of the filter is then treated with reagent, thereby giving a true negative control .
- Such an improved device relying on direct determination of the presence of pathogens on the membrane is indeed applicable only with water. More charged fluids such as serum, accumulated into a single discrete area of the filter tend to clog this filter up, prevent the easy flow-through of subsequently added fluids (such as a wash medium) and cause the succeeding washing fluids, applied to the whole of the membrane, to flow around that area without penetrating it. Further, even with water, the device is specifically developed and meant to visualise false positive cases, thereby indicating that the occurrence of false positive cases is possible, even with water. No such device applied to detect whole microorganisms by direct labelling is m use for body fluids such as serum, cerebrospmal fluid, pericardial fluid, urine or other body fluid.
- the chaotropic agent is contacted together with different surfactants with the sample under analysis, not as a washing solution, and no attempt is made to detect m a direct way the pathogenic entity condensed on the surface of a membrane, but a lateral flow lmmunoassay is used m the detection of the analyte present m the treated biological fluid.
- the document US-5714343 describes a device consisting m absorbing pads surmounted by a retaining membrane.
- the fluid is passed through the retaining membrane and the microorganisms potentially retained on the membrane are visualised by a chromogenic agent having an oxidation potential such that the reagent can be reduced by microbial dehydrogenase, yielding a visibly coloured product indicative of the presence of microorganisms m the sample.
- the retaining membrane having pores (0.75 to 1.2 ⁇ m) larger than the dehydrogenase-active microorganisms it is supposed to retain, the capture of the bacteria on such filters is achieved by mechanical retention.
- the document EP 0 234 941 indicates that treatment of viral subunits with denaturing concentrations of guanidme (from 5 to 8 molar) (after the purification of the virus and its disruption into subunits with detergents) improves the purity of these subunits for the adsorption of antibodies m an Elisa. It is known that denaturing concentrations of chaotropic agents destroy protein assemblies into subunits, as is the case with the alpha and beta subunits of human cho ⁇ onic gonadotropm. Sometimes, as is the case with the subunits of HCG, the immunogenicity of the subunits is not affected by this hypertonic treatment.
- Tuberculoprotems are isolated by treating tubercular material with chaotropic agents (guanidme, urea and phenol) which reduce the tubercle pathogen to lmmunogenic tuberculoprotems (FR-2082226) .
- chaotropic agents guanidme, urea and phenol
- FR-2082226 tubercle pathogen to lmmunogenic tuberculoprotems
- the present invention is related to a method and kit for the rapid detection of whole microorganisms, which do not present or reduce the possible false positive or false negative results affecting the method of the state of the art . Summary of the invention
- a simple detection system able to demonstrate a direct way, m a sensitive way and a specific way the presence of whole pathogens belonging to the bacterial kingdom or else eukaryotic parasites as Plasmodium falciparum or toxoplasma m various body fluids as serum, cerebrosp al fluid, pericardial fluid etc. condensed on a retaining membrane, is not yet available, although such a detection would greatly improve the rapid diagnostic of several pathogens.
- Such a system must, per force, exploit the specific binding capacities existing among specific binding partners for the microorganisms one wishes to detect, as specific antibodies, protein A, protein G.
- the present invention is related to a method for the detection of one or more microorganisms present m a liquid sample, preferably a biological liquid sample, said method comprising at least the following steps:
- the chaotropic agent solution comprises urea, guanidme, thiourea, isothiocyanate or a mixture thereof and the concentration of the chaotropic agent m the solution is higher than 2M, preferably between 4 and 8M (denaturat g concentration) .
- the microorganisms could be present m any biological sample, preferably m a charged biological sample such as sputum, blood, serum, plasma, cephalo- rachidian fluid or hop obtained from any animal patient, including the human and are preferably pathogenic bacteria or eukaryotic parasites detected either simultaneously or successively.
- Contaminants of beverages and foods, as listeria, yeasts and molds are also detectable.
- the type of filter used to retain the analytes under study is usually cellulose, paper, nitrocellulose or nylon, or other types selected by the man skilled m the art.
- the specific labelled reagents used for the detection of microorganisms are preferably antibodies, possibly coupled directly or indirectly to a marker, such as gold micellae, biotme, enzymes, chromophores, stained latex beads, enzymes, fluorophores , radioactive compounds or a mixture thereof .
- a marker such as gold micellae, biotme, enzymes, chromophores, stained latex beads, enzymes, fluorophores , radioactive compounds or a mixture thereof .
- the present invention is related also to a kit comprising means and media for performing the detection method according to the invention, said kit comprising filtering membranes of predetermined pore size to retain specific microorganisms, one or several chaotropic agents, one or several labelled reagents (signal-generating reagents) , one of them being specific for the monitored microorganisms .
- the method and kit could be adapted to allow the rapid detection of microorganisms present a sample by using an automate and means for performing automatically the specific detection according to the invention.
- binding partner being itself coupled to a marker (i.e. an agent susceptible to amplify the labelling, as colloidal gold or end enzyme) .
- a marker i.e. an agent susceptible to amplify the labelling, as colloidal gold or end enzyme
- microorganisms having the size of bacteria or bigger (spirochetes, trypanosomes and worms) present m body fluids as serum andcoat obtained by means of specific binding partners as antibodies, protein A or protein G is not easy.
- microorganisms m solution or condensed on the surface of a membrane resist the disruptive action of high molar concentrations of chaotropic agents as guanidme, urea, thiocyanate, thiourea, isothiocyanate, perchlorate etc. while becoming readily accessible therewith to recognition by specific binding partners .
- the same microorganisms condensed from the same sera on the same retaining membranes and treated with the same chaotropic agents used at a mola ⁇ ty too low to exploit their disruptive potential on proteins (as for example 0.5 molar guanidme used to destroy the activity of gra ⁇ .4- bacterial dehydrogenase) are not recognised by their specific binding partners . Therefore a tenfold higher concentration of the chaotropic agents is unexpectedly beneficial m the conduct of an lmmunoassay.
- nucleic acid is obtained following treatment of cells and viruses with chaotropic agents indicates that the cell membranes, the nuclear membranes and tertiary structures of proteins viruses are destroyed by this treatment. This effect precludes any evident application of chaotropic agents at high molar concentrations for the purpose of the present invention.
- Chaotropic agents as urea and guanidme applied at concentrations ranging between 4 to 8 molar, useful and necessary for the exploitation of their protein denaturing properties and their ability to suppress hydrogen bonds, are currently used either to eliminate proteins from nucleic acid preparations or else to separate protein subunits, thereafter isolated by separation methods based on the different physico-chemical properties of the separated products. For example, the alpha and beta subunits of cho ⁇ onic gonadotropm are separated m the presence of 8 molar urea at 40 °C and the two subunits are subsequently isolated by passage on a DEAE-cellulose chromatography column.
- Purified tuberculoprotems are isolated by submitting tubercular material to a precipitation stage at an acid pH, carried out m the presence of a substance capable of rupturing hydrogen bonds, as urea, guanidme, formamide, phenol, etc.
- the precipitated material is thereafter contacted with a modified cellulose bearing a basic group such as DEAE (see document FR-2082226) .
- DEAE modified cellulose bearing a basic group
- Example 2 An analysis identical to that described Example 1 was performed with a solution that was 7 molar m urea. The same positive result as m example 1 was obtained, whereas concentrations lower than about 4.5 molar were ineffective m revealing a satisfactory signal. A concentration of urea superior to about 8 molar reduced the signal .
- Example 4 [0038] The same experiment as m example 1 was conducted with the chaotropic agents 4 molar potassium thiocyanate, 2 molar thiourea, 6 molar guanidme containing 2 molar thiourea, 3 molar urea mixed with 3 molar guanidme. Solubilisation difficulties due to the presence of various surfactants currently used m lmmunoassays and known from the man of the art did not allow higher mola ⁇ ties ' concentrations for thiocyanate and thiourea than those here applied. These chaotropic agents were not superior to urea or guanidme used alone.
- Example 5 Example 5
- the sputum underwent regular analysis and was found to contain TB bacilli by the bacilloscopy technique based on the Ziehl-Nielsen Stain.
- the sputum was fluidised by addition of 0.25% N-acetyl- cysteine and 1% NaOH (final concentrations) .
- This fluidising method is standard in mycobacteriology for the treatment of sputum before its use in cultures.
- the NaOH is added as a fluidisation medium and also in order to decontaminate the sample and kill all other bacteria present, before culture for tubercle bacilli is initiated.
- the NaOH is not indispensable for the liquefaction and N- acetyl-cysteine, at concentrations between 0.5% and 2% and alkaline pH, is able to liquefy sputum in two minutes.
- Other methods of fluidisation based on hypochlorite or sodium dodecyl sulphate, were found to work just as well. In particular, bleach may be appropriate for low income countries because it is cheap, available locally and it better respects the integrity of the retaining membrane than NaOH.
- a neutralisation of the fluid with HCl was needed before passing it on a nitro-cellulose retaining membrane.
- Other means of fluidisation proved compatible with the retaining membranes used. Any fluidismg agent that respects the antigemcity of the bacteria and does not alter the retaining membrane is satisfactory.
- a fiber-glass prefilter pad was placed on top of the nitrocellulose retaining membrane, so as to retain the unsolubilised material remaining m the fluidised sputum.
- 200 ⁇ l of fluidised sputum were passed through the prefilter and the 0.8 ⁇ m nitrocellulose membrane, before the fiberglass prefilter was removed.
- the retaining membrane was washed with 2 times 100 ⁇ l of 0.2M phosphate pH 7.2 containing 0.2% Tween 20. After the wash, the membrane was treated with 6 M guanidme, and further contacted with a rabbit antiserum against M. tuberculosis appropriately diluted 1:160 m phosphate-buffered saline pH 7.2, containing 0.2% Tween 20.
- the signal present on the membrane amplified with a reagent consisting in 30 nm gold-sensitised rabbit anti-goat IgG.
- the sensitivity of the system detects about 3000 mycobacte ⁇ al entities/ ml.
- Example 6 The detection of mycobacteria in sputum was done as m example 5, with a membrane whose pore size was 0.45 ⁇ m. This small pore size was found acceptable although it retarded the flow rate, but a slower flow of the reagents enhanced the sensitivity of the method by allowing a longer contact and prolonged reaction time of the reagents with the antigens.
- Rabbit anti-mycobacterial serum was used to label the antigen retained on the membrane.
- the secondary partner of reaction was composed of antibodies against rabbit IgG raised m donkeys.
- the enhancer was 30 nm-gold-labelled Protein G., which reacts well with equine antibodies.
- the sensitivity reached was about 3000 entities/ml .
- a membrane with a pore-size of 0.45 ⁇ m was used to collect the mycobacteria suspended m sputum m an experiment applied as m example 5.
- a secondary binding partner consisting of Protein A-20 nm colloidal gold, was applied (Sigma Chemical Co, St Louis, MO) .
- the results were similar to those obtained m example 6, when the secondary antibody was gold-sensitised goat anti-rabbit IgG, i.e. 3000 particles/ml .
- specimens for assay are urethral and cervical swabs, and contutica, with the aim to detect the etiological agent .
- Human cava was spiked with decreasing amounts of the pathogen Chlamydia trachomati s Elementary bodies. Antibodies raised rabbits were used to label the pathogen. One and a half millilitres of the spiked ur e were passed on a filter with mean more size 0.2 ⁇ m, that was able to retain the pathogen on its surface. No prefilter was useful .
- Toxoplasmosis is caused by Toxoplasma gondii , a sporozoan whose individual cells are 4 to 7 ⁇ m long. Diagnosis relies essentially on detection of specific antibodies m serum but the sites most commonly attacked are the lymph nodes, brain, eyes and lungs. Direct examination of sputum, vaginal exudates, spinal, pleural and peritoneal fluids are possible, but rarely practised. No diagnostic kits exist for direct examination of sputum and body fluids. [0047] Toxoplasma gondn organisms were mixed with sputum, the sputum was liquefied and 1 ml passed on an 0.8 ⁇ m membrane fitted with a prefilter. The same procedure as m example 7 was followed. The results were excellent, with the detection of about 200 pathogenic entities m the sample .
- Sputum was spiked with known concentrations of Nei sse ⁇ a gonorrhoeae antigen, as example 5.
- the pore-size of the retaining membrane was 0.45 ⁇ m, compatible with the size of the organisms (0.6 to 1. O ⁇ m m diameter) .
- the solubilisation of the sputum was done with 0.2% Sodium dodecyl sulphate at pH 9.5, treatment that was found not to alter the immunoreactivity of the pathogen.
- Example 11 The microscopic observation of the adult schizonts of Plasmodiu falciparum m blood smears is the laboratory diagnostic of malaria.
- Human fresh whole blood was spiked with formalin-inactivated schizonts. After the spiking, the blood was hemolysed with 1% Nonidet P 40 (final concentration) and 500 ⁇ l of the fluid was passed on a glass prefilter and collected on a retaining membrane with 0.8 ⁇ m. Guanidme at a 4 molar concentration at pH 8,0 was found satisfactory in this analysis. A high concentration of 8 molar urea was also found adequate but this remarkable resistance of the schizonts may have been obtained by their fixation with formalin.
- the mouse antibodies used to label the antigen were revealed with Gold-labelled Protein G. Distinct red spots on the membrane signalled the presence of individual organisms.
- a great number of possibilities were investigated, using either antibodies labelled with peroxidase or with gold, using protein A and protein G labelled with gold or with peroxidase, and the usefulness of secondary amplification steps was also investigated.
- the most reliable and easy method was found to be the one described m example 7: 500 ⁇ l to 1.5 ml (depending on the proteinic load of the processed sample) of sample is passed through a retaining membrane. The organisms presumably concentrated on the surface of the membrane are then treated with a solution 6 molar m guanidme hydrochloride at pH 8. After a wash, the membrane is treated with rabbit antibodies against the analyte and the presence of the antibodies potentially attached to the antigenic determinants of the analyte are put m evidence with gold- labelled protein A.
- solubilising solution consisting of 5% N-acetylcysteine and 0.5% mercaptoethanol in 1% NaOH brought at pH 12.
- solubilising solution is standard procedure but other solubilising methods (e.g. sodium hyposulphite) known by the person skilled in the art are equally applicable.
- Example 13 100 ⁇ l of sputum found positive for TB by a microscopic analysis was mixed with 100 ⁇ l of a solution that was 7 molar in guanidine at pH 12.00. After solubilisation of the sputum, 100 ⁇ l of NaClO at 12° was added to the mixture and digestion of the sputum was pursued during 15 minutes at Room Temperature. The sample was thereafter mixed with 1.3 ml of a solution that was 7 molar in guanidine at pH 8.5 and processed as per example
- a red central spot is observed after completion of the test, not observable when negative samples are processed in an identical manner.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01905689A EP1252518B1 (en) | 2000-02-01 | 2001-01-26 | Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents |
DE60110369T DE60110369T2 (en) | 2000-02-01 | 2001-01-26 | PROCESS FOR FAST DETECTION OF ALL MICRO-ORGANISMS OF RESTRAINT MEMBRANES USING CAOTROPIC MEANS |
AU2001233710A AU2001233710A1 (en) | 2000-02-01 | 2001-01-26 | Method for the rapid detection of whole microorganisms on retaining membranes byuse of chaotropic agents |
US10/182,793 US6846648B2 (en) | 2000-02-01 | 2001-01-26 | Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents |
AT01905689T ATE294393T1 (en) | 2000-02-01 | 2001-01-26 | METHOD FOR THE RAPID DETECTION OF ENTIRE MICROORGANISMS VIA RETENTION MEMBRANES USING CAOTROPIC AGENTS |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00870013.0 | 2000-02-01 | ||
EP00870013A EP1122542A1 (en) | 2000-02-01 | 2000-02-01 | Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents |
Publications (1)
Publication Number | Publication Date |
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WO2001057532A1 true WO2001057532A1 (en) | 2001-08-09 |
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ID=8175698
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/000829 WO2001057532A1 (en) | 2000-02-01 | 2001-01-26 | Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents |
Country Status (6)
Country | Link |
---|---|
US (1) | US6846648B2 (en) |
EP (2) | EP1122542A1 (en) |
AT (1) | ATE294393T1 (en) |
AU (1) | AU2001233710A1 (en) |
DE (1) | DE60110369T2 (en) |
WO (1) | WO2001057532A1 (en) |
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GB0304832D0 (en) * | 2003-03-04 | 2003-04-09 | Secr Defence | Assay method |
US20060046305A1 (en) * | 2003-10-15 | 2006-03-02 | National University Of Singapore | Method and apparatus for detecting analyte with filter |
GB2409519B (en) * | 2003-12-22 | 2005-11-09 | Prail Price Richardson Diagnos | Microorganism detector |
US20090148870A1 (en) * | 2006-05-18 | 2009-06-11 | Ericson Daniel G | Rapid Detection of Mycobacterium Tuberculosis and Antimicrobial Drug Resistance |
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US9555099B2 (en) | 2012-05-16 | 2017-01-31 | Immune Design Corp. | Vaccines for HSV-2 |
JP6091158B2 (en) * | 2012-10-23 | 2017-03-08 | デンカ生研株式会社 | Method to increase sensitivity of immunoassay system by pretreatment of urine with denaturant |
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CN105717310A (en) * | 2016-04-15 | 2016-06-29 | 肖乐义 | Immuno-fluorescent staining method for detecting mycobacterium tuberculosis in leukocytes and kit |
CN105759038A (en) * | 2016-04-15 | 2016-07-13 | 肖乐义 | Immunoassay method and kit for assaying mycobacterium tuberculosis from biological samples |
US11173126B2 (en) | 2016-06-01 | 2021-11-16 | Infectious Disease Research Institute | Nanoalum particles comprising a PAA sizing agent |
GB201703383D0 (en) | 2017-03-02 | 2017-04-19 | Gargle Tech Ltd | Testing for particulates |
WO2020049569A2 (en) | 2018-09-05 | 2020-03-12 | Hero Scientific Ltd. | Testing for particulates |
KR20220125149A (en) | 2019-05-25 | 2022-09-14 | 액세스 투 어드밴스드 헬스 인스티튜트 | Compositions and methods for spray drying adjuvant vaccine emulsions |
WO2022149135A2 (en) | 2021-01-06 | 2022-07-14 | Hero Scientific Ltd. | Filtration sampling devices |
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EP0234941A2 (en) * | 1986-02-26 | 1987-09-02 | THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce | Competitive elisa for the detection of antibodies |
US4695537A (en) * | 1982-05-21 | 1987-09-22 | The University Of Tennessee Research Corp. | Particles sensitized with detergent-treated antigen for agglutination immunoassay |
US4808518A (en) * | 1985-02-11 | 1989-02-28 | University Of Tennessee Research Corporation | Recovery of cytomegalovirus antigen and use thereof in an assay |
EP0325045A2 (en) * | 1987-12-21 | 1989-07-26 | Syntex (U.S.A.) Inc. | Immunoassay method for detecting antigens |
US5155023A (en) * | 1990-02-09 | 1992-10-13 | Synbiotics Corporation | Enzyme immunoassay procedure for amphipathic analytes |
US5714343A (en) * | 1991-01-24 | 1998-02-03 | Orion Corporation Ltd. | Method and kit for the detection of microorganisms |
Family Cites Families (2)
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US5482834A (en) * | 1982-05-17 | 1996-01-09 | Hahnemann University | Evaluation of nucleic acids in a biological sample hybridization in a solution of chaotrophic salt solubilized cells |
US4978613A (en) * | 1989-01-17 | 1990-12-18 | Abbott Laboratories | Beta-lactamase assay employing chromogenic precipitating substrates |
-
2000
- 2000-02-01 EP EP00870013A patent/EP1122542A1/en not_active Withdrawn
-
2001
- 2001-01-26 WO PCT/EP2001/000829 patent/WO2001057532A1/en active IP Right Grant
- 2001-01-26 AU AU2001233710A patent/AU2001233710A1/en not_active Abandoned
- 2001-01-26 DE DE60110369T patent/DE60110369T2/en not_active Expired - Fee Related
- 2001-01-26 EP EP01905689A patent/EP1252518B1/en not_active Expired - Lifetime
- 2001-01-26 US US10/182,793 patent/US6846648B2/en not_active Expired - Fee Related
- 2001-01-26 AT AT01905689T patent/ATE294393T1/en not_active IP Right Cessation
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4695537A (en) * | 1982-05-21 | 1987-09-22 | The University Of Tennessee Research Corp. | Particles sensitized with detergent-treated antigen for agglutination immunoassay |
US4808518A (en) * | 1985-02-11 | 1989-02-28 | University Of Tennessee Research Corporation | Recovery of cytomegalovirus antigen and use thereof in an assay |
EP0234941A2 (en) * | 1986-02-26 | 1987-09-02 | THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce | Competitive elisa for the detection of antibodies |
EP0325045A2 (en) * | 1987-12-21 | 1989-07-26 | Syntex (U.S.A.) Inc. | Immunoassay method for detecting antigens |
US5155023A (en) * | 1990-02-09 | 1992-10-13 | Synbiotics Corporation | Enzyme immunoassay procedure for amphipathic analytes |
US5714343A (en) * | 1991-01-24 | 1998-02-03 | Orion Corporation Ltd. | Method and kit for the detection of microorganisms |
Also Published As
Publication number | Publication date |
---|---|
EP1122542A1 (en) | 2001-08-08 |
DE60110369T2 (en) | 2006-03-09 |
ATE294393T1 (en) | 2005-05-15 |
DE60110369D1 (en) | 2005-06-02 |
EP1252518A1 (en) | 2002-10-30 |
AU2001233710A1 (en) | 2001-08-14 |
EP1252518B1 (en) | 2005-04-27 |
US20030022160A1 (en) | 2003-01-30 |
US6846648B2 (en) | 2005-01-25 |
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