WO1993001272A2 - Bioassay method - Google Patents

Bioassay method Download PDF

Info

Publication number
WO1993001272A2
WO1993001272A2 PCT/GB1992/001210 GB9201210W WO9301272A2 WO 1993001272 A2 WO1993001272 A2 WO 1993001272A2 GB 9201210 W GB9201210 W GB 9201210W WO 9301272 A2 WO9301272 A2 WO 9301272A2
Authority
WO
WIPO (PCT)
Prior art keywords
embryo
test
circulation
yolk sac
test substance
Prior art date
Application number
PCT/GB1992/001210
Other languages
French (fr)
Other versions
WO1993001272A3 (en
Inventor
Margaret Kathryn Pratten
Peter Frederick Thomas Cumberland
Original Assignee
University Of Leicester
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.)
Filing date
Publication date
Priority claimed from GB919114638A external-priority patent/GB9114638D0/en
Priority claimed from GB919114955A external-priority patent/GB9114955D0/en
Application filed by University Of Leicester filed Critical University Of Leicester
Priority to EP92914060A priority Critical patent/EP0594660A1/en
Priority to JP5502083A priority patent/JPH06509469A/en
Publication of WO1993001272A2 publication Critical patent/WO1993001272A2/en
Publication of WO1993001272A3 publication Critical patent/WO1993001272A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to bioassays for potentially biologically harmful substances.
  • One method used in carrying out a bioassay is to extract rat embryos and incubate them floating in a nutrient medium containing the test substance.
  • the yolk sac contains enzymes which may metabolise the test substances so that the embryo may receive derivatives of some substances rather than the test substances per se.
  • test substances may combine with metabolites in the yolk sac, forming addition compounds with properties different to the pure test substances.
  • a further complication is the subsequent metabolism of the test substance-metabolite intermediates. These and other reactions can lead to either metabolic activation or deactivation of the test substance.
  • assays generally involving isolated cell populations grown in tissue culture or extracts of tissue homogenates, can yield valuable information about the effects of potentially hazardous chemicals on various systems within such cell populations or homogenates, assays for potentially mutagenic, carcinogenic and angiogenic substances are preferably carried out in multicellular or whole animal systems.
  • the present invention provides an in vitro method for bioassay which has advantages over existing in_ vitro methods and which avoids the need for the use of multi ⁇ cellular or whole animal systems as conventionally used; although such systems may also be desired as back-up or for a complete assay, in general, if the method of the present invention indicates that the substance is harmful, the requirement for further testing can in general be eliminated.
  • the invention comprises a method for bioassay comprising passing test reagents into the circulation of an embryo in such manner that the reagents are not modified by any intermediate metabolism, culturing the embryo in culture medium and assessing embryonic development.
  • the method may comprise injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism.
  • the yolk sac may be bypassed by cannulation of the vitelline circulation.
  • the embryo may be placed in a-well on a microscope stage.
  • the well may be formed from agar and may be dyed to provide contrast when viewing the blood vessels beneath the yolk sac surface.
  • the well may be heated.
  • the vitelline circulation may be cannulated by a microcannula, which may have an internal tip diameter of between 1 and 2 ⁇ .
  • the microcannula may be coated with a silicon- ising agent.
  • the microcannula may be connected to a micro- injection pump and may be used to inject a test substance which may comprise a fluorescent dye, such as fluorescein or rhodamine, so that a check may be made as to whether the test substance has been satisfactorily injected.
  • a test substance which may comprise a fluorescent dye, such as fluorescein or rhodamine
  • the test substance may be injected into the embryo in a total volume of less than or equal to one microlitre.
  • the embryo may be a Wistar rat conceptus which may be between 10 and 11 days old.
  • the test substance may be injected into the embryo over the course of four minutes.
  • the rat embryos may be cultured in heat-inactivated rat serum containing antibiotics such as gentamycin, penicillin and streptomycin, for example.
  • the embryo may be cultured in heat-inactivated homologous serum which may contain antibiotics.
  • the embryo whether a rat conceptus or not, may be cultured in synthetic or semi-synthetic medium.
  • Embryos may be used which are not surrounded by a visceral yolk sac, for example chicken embryos in fertilised eggs.
  • the test reagent may be administered as before by micro-cannulation directly into the extra- embryonic circulation, possibly the vitelline circulation or chlorioallantoic blood vessels, and the egg contents may be released from the shell into a plastic container and incubated therein, or a window may be cut into the shell to access the embryo.
  • test reagent may be necessary to test sample embryos whic have been treated otherwise than by direct cannulatio to ensure that the test reagent has survived th transfer into the embryo in unmodified form, but this would in general be an easier task than performin multiple micro-cannulations.
  • test substances may b determined by quantitation means which may compris immunological, biochemical and morphometric assays.
  • the biochemical assays may comprise protein, DNA, DNA synthesis, protein synthesis and enzym synthesis and the morphometric assays may compris crown-rump measurements, somite number measurements, yolk sac diameter measurements and vitelline circulatio assessment.
  • the morphometric assays may additionall comprise established morphometric scoring assays.
  • the method may be used to assess factors whic are toxic specifically to the developing embryo, or t assess angiogenic and anti-angiogenic factors. Th visceral yolk sac may also be bypassed by cannulation o the embryo heart at an early stage of development with a microcannula. Angiogenic activity of the test substances may be assessed by morphometry of the developing blood vascular system.
  • test substance may be added with a radio- label, such as tritiated thymidine, and injected into the embryo heart.
  • a radio- label such as tritiated thymidine
  • the embryo may then be homogenised and an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess in vitro the entire mass response to the test substance.
  • test kits for carrying out the method.
  • test kits may include microcannulae, dyed agar wells, culture media, a work station comprising a micro-injection pump, a micro- manipulator, a heating stage and dissection head for a microscope, together, if desired, with a microscope.
  • the method comprises injecting test substances and appropriate control substances into a post- implantation Wi ⁇ tar rat embryo between 9 and 13 days old, bypassing the visceral yolk sac of the embryo so that the substances are not modified by visceral yolk sac metabolism, culturing the injected embryo in culture medium and quantitating subsequent embryonic development.
  • the yolk sac is bypassed by micro-cannulation of the vitelline circulation.
  • the cannula is inserted so that the flow of the cannula contents is with the direction of blood flow.
  • the microcannula used in the cannulation has an internal tip diameter of between 1 and 2 urn, and preferably of 1.2 ⁇ m.
  • the microcannula is made of borosilicate and may be given an aqueous-repellent coating with trimethylchlorosilane to prevent the test substances as well as the embryonic tissue adhering to the wall of the cannula.
  • the microcannula is connected to a micro- injection pump set to deliver nano- or even pico-litre volumes over the course of one second to four minutes.
  • the test substances comprise a fluorescent dye such as fluorescein or rhodamine and the embryo checked to see if the test substance has been satisfactorily injected:
  • test substance is injected into the embryo in a total volume of less than or equal to one microlitre. Volumes substantially greater than this lead to artefacts of the method and can lead to an embryo dying before the bioassay is complete. At 10.5 days the rat conceptus has a negligible blood vascular system in the yolk sac, which can be exploited in the assessment of potentially angiogenic substances.
  • the embryo After cannulation, the embryo is cultured in roller bottles in heat-inactivated rat serum containing antibiotics such as gentamycin, penicillin and streptomycin. It will be appreciated, however, that the embryo may be cultured in any suitable medium such as a synthetic or semi-synthetic medium.
  • the effects of the test substances are determined in immunological, biochemical and morphometric assays.
  • the biochemical assays can for example be for total protein and total DNA content and for protein and DNA synthesis, thus assessing the entire mass response of the embryo to the test substances. It will be appreciated, however, that specific enzymes or proteins can be assayed and an assessment made of the induction, or otherwise, of biochemical systems which are involved in toxification/detoxification reactions.
  • One such system is the P450 Cytochrome system.
  • DNA content may be assayed with 4'-6-diamidino-2- phenylindole (DAPI).
  • Morphometric assays comprise crown-rump measurements, somite number measurements, yolk sac diameter measurements and visual inspection of the vitelline circulation.
  • test substance can be added with a radio- label such as tritiated thymidine and injected into the embryo heart.
  • a radio- label such as tritiated thymidine
  • the embryo is then homogenised and an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess the entire mass response in. vitro to the test substance.
  • a test kit for carrying out the bioassay comprises microcannulae, sterile dyed black agar blocks for holding the embryos for the micro-cannulation procedure, culture medium sachets, a work station comprising a micro-injection pump, a micro-manipulator and a microscope comprising a dissecting head and a heated stage, the heating being desirable for maintaining viability of the embryo during the injection procedure and being preferable to maintaining the laboratory at a sufficient temperature for this.
  • One method used in carrying out a bioassay is to extract rat embryos and incubate them floating in a nutrient medium containing the test substance.
  • the method may comprise injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism.
  • Embryos may be used which are not surrounded by a visceral yolk sac, for example chicken embryos in fertilised eggs.
  • the test reagent may be administered as before by micro-cannulation directly into the vitelline circulation, and the egg contents may be released from the shell into a plastic container and incubated therein, or a window may be cut into the shell to access the embryo.
  • test reagent may be introduced into the albumen close to the embryo.
  • test reagent may be necessary to test sample embryos which have been treated otherwise than by direct cannulation to ensure that the test reagent has survived the transfer into the embryo in unmodified form, but this would in general be an easier task than performing multiple micro-cannulations.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Toxicology (AREA)
  • Pathology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Endocrinology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Rheumatology (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

A bioassay method comprises passing test reagents into the circulation of an embryo in such manner that the reagents are not modified by any intermediate metabolism. This can be effected in e.g. rat embryos by microcannulation directly into the vitelline circulation by passing the visceral yolk sac. In chick embryos the test reagent can be administered directly into the extra-embryonic circulation. The embryo is cultured and assessed as to its embryonic development.

Description

BIOASSAYMETHOD
This invention relates to bioassays for potentially biologically harmful substances.
One method used in carrying out a bioassay is to extract rat embryos and incubate them floating in a nutrient medium containing the test substance.
Under normal circumstances it is difficult to administer directly substances to living post- implantation embryo such as a rat embryo as currently used, due to the presence of the embryonic visceral yolk sac. The yolk sac contains enzymes which may metabolise the test substances so that the embryo may receive derivatives of some substances rather than the test substances per se.
Moreover, the test substances may combine with metabolites in the yolk sac, forming addition compounds with properties different to the pure test substances. A further complication is the subsequent metabolism of the test substance-metabolite intermediates. These and other reactions can lead to either metabolic activation or deactivation of the test substance.
Although in vitro assays, generally involving isolated cell populations grown in tissue culture or extracts of tissue homogenates, can yield valuable information about the effects of potentially hazardous chemicals on various systems within such cell populations or homogenates, assays for potentially mutagenic, carcinogenic and angiogenic substances are preferably carried out in multicellular or whole animal systems.
The present invention provides an in vitro method for bioassay which has advantages over existing in_ vitro methods and which avoids the need for the use of multi¬ cellular or whole animal systems as conventionally used; although such systems may also be desired as back-up or for a complete assay, in general, if the method of the present invention indicates that the substance is harmful, the requirement for further testing can in general be eliminated.
The invention comprises a method for bioassay comprising passing test reagents into the circulation of an embryo in such manner that the reagents are not modified by any intermediate metabolism, culturing the embryo in culture medium and assessing embryonic development.
When using mammalian, e.g. rat embryos, the method may comprise injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism.
The yolk sac may be bypassed by cannulation of the vitelline circulation. During cannulation the embryo may be placed in a-well on a microscope stage. The well may be formed from agar and may be dyed to provide contrast when viewing the blood vessels beneath the yolk sac surface. The well may be heated.
The vitelline circulation may be cannulated by a microcannula, which may have an internal tip diameter of between 1 and 2 μ .
The microcannula may be coated with a silicon- ising agent.
The microcannula may be connected to a micro- injection pump and may be used to inject a test substance which may comprise a fluorescent dye, such as fluorescein or rhodamine, so that a check may be made as to whether the test substance has been satisfactorily injected.
The test substance may be injected into the embryo in a total volume of less than or equal to one microlitre. The embryo may be a Wistar rat conceptus which may be between 10 and 11 days old.
The test substance may be injected into the embryo over the course of four minutes.
After injection of the test substances, the rat embryos may be cultured in heat-inactivated rat serum containing antibiotics such as gentamycin, penicillin and streptomycin, for example.
Where the embryo is not a rat conceptus, the embryo may be cultured in heat-inactivated homologous serum which may contain antibiotics.
Otherwise, the embryo, whether a rat conceptus or not, may be cultured in synthetic or semi-synthetic medium.
Embryos may be used which are not surrounded by a visceral yolk sac, for example chicken embryos in fertilised eggs. The test reagent may be administered as before by micro-cannulation directly into the extra- embryonic circulation, possibly the vitelline circulation or chlorioallantoic blood vessels, and the egg contents may be released from the shell into a plastic container and incubated therein, or a window may be cut into the shell to access the embryo. However, it may be possible to avoid the nee for micro-cannulation by introducing the test reagen into the albumen close to the embryo.
It may be necessary to test sample embryos whic have been treated otherwise than by direct cannulatio to ensure that the test reagent has survived th transfer into the embryo in unmodified form, but this would in general be an easier task than performin multiple micro-cannulations.
The effects of the test substances may b determined by quantitation means which may compris immunological, biochemical and morphometric assays.
The biochemical assays may comprise protein, DNA, DNA synthesis, protein synthesis and enzym synthesis and the morphometric assays may compris crown-rump measurements, somite number measurements, yolk sac diameter measurements and vitelline circulatio assessment. The morphometric assays may additionall comprise established morphometric scoring assays.
The method may be used to assess factors whic are toxic specifically to the developing embryo, or t assess angiogenic and anti-angiogenic factors. Th visceral yolk sac may also be bypassed by cannulation o the embryo heart at an early stage of development with a microcannula. Angiogenic activity of the test substances may be assessed by morphometry of the developing blood vascular system.
The test substance may be added with a radio- label, such as tritiated thymidine, and injected into the embryo heart. The embryo may then be homogenised and an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess in vitro the entire mass response to the test substance.
The invention also comprises test kits for carrying out the method. Such test kits may include microcannulae, dyed agar wells, culture media, a work station comprising a micro-injection pump, a micro- manipulator, a heating stage and dissection head for a microscope, together, if desired, with a microscope.
One typical method for a bioassay according to the invention will now be described.
The method comprises injecting test substances and appropriate control substances into a post- implantation Wiεtar rat embryo between 9 and 13 days old, bypassing the visceral yolk sac of the embryo so that the substances are not modified by visceral yolk sac metabolism, culturing the injected embryo in culture medium and quantitating subsequent embryonic development.
The yolk sac is bypassed by micro-cannulation of the vitelline circulation. The cannula is inserted so that the flow of the cannula contents is with the direction of blood flow. The microcannula used in the cannulation has an internal tip diameter of between 1 and 2 urn, and preferably of 1.2 μm. Typically, the microcannula is made of borosilicate and may be given an aqueous-repellent coating with trimethylchlorosilane to prevent the test substances as well as the embryonic tissue adhering to the wall of the cannula.
The microcannula is connected to a micro- injection pump set to deliver nano- or even pico-litre volumes over the course of one second to four minutes. The test substances comprise a fluorescent dye such as fluorescein or rhodamine and the embryo checked to see if the test substance has been satisfactorily injected:
The test substance is injected into the embryo in a total volume of less than or equal to one microlitre. Volumes substantially greater than this lead to artefacts of the method and can lead to an embryo dying before the bioassay is complete. At 10.5 days the rat conceptus has a negligible blood vascular system in the yolk sac, which can be exploited in the assessment of potentially angiogenic substances.
After cannulation, the embryo is cultured in roller bottles in heat-inactivated rat serum containing antibiotics such as gentamycin, penicillin and streptomycin. It will be appreciated, however, that the embryo may be cultured in any suitable medium such as a synthetic or semi-synthetic medium.
The effects of the test substances are determined in immunological, biochemical and morphometric assays. The biochemical assays can for example be for total protein and total DNA content and for protein and DNA synthesis, thus assessing the entire mass response of the embryo to the test substances. It will be appreciated, however, that specific enzymes or proteins can be assayed and an assessment made of the induction, or otherwise, of biochemical systems which are involved in toxification/detoxification reactions. One such system is the P450 Cytochrome system.
DNA content may be assayed with 4'-6-diamidino-2- phenylindole (DAPI). Morphometric assays comprise crown-rump measurements, somite number measurements, yolk sac diameter measurements and visual inspection of the vitelline circulation.
The test substance can be added with a radio- label such as tritiated thymidine and injected into the embryo heart. The embryo is then homogenised and an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess the entire mass response in. vitro to the test substance.
A test kit for carrying out the bioassay comprises microcannulae, sterile dyed black agar blocks for holding the embryos for the micro-cannulation procedure, culture medium sachets, a work station comprising a micro-injection pump, a micro-manipulator and a microscope comprising a dissecting head and a heated stage, the heating being desirable for maintaining viability of the embryo during the injection procedure and being preferable to maintaining the laboratory at a sufficient temperature for this.
One method used in carrying out a bioassay is to extract rat embryos and incubate them floating in a nutrient medium containing the test substance. When using mammalian, e.g. rat embryos, the method may comprise injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism.
Embryos may be used which are not surrounded by a visceral yolk sac, for example chicken embryos in fertilised eggs. The test reagent may be administered as before by micro-cannulation directly into the vitelline circulation, and the egg contents may be released from the shell into a plastic container and incubated therein, or a window may be cut into the shell to access the embryo.
However, it may be possible to avoid the need for micro-cannulation by introducing the test reagent into the albumen close to the embryo.
It may be necessary to test sample embryos which have been treated otherwise than by direct cannulation to ensure that the test reagent has survived the transfer into the embryo in unmodified form, but this would in general be an easier task than performing multiple micro-cannulations.

Claims

1. A method for bioassay comprising passing test reagents into the circulation of an embryo in such manner that the reagents are not modified by any intermediate metabolism, culturing the embryo in culture medium and assessing embryonic development.
2. A method according to claim 1, in which the embryo is a mammalian embryo.
3. A method according to claim 2, in which the mammalian embryo is a rat embryo.
4. A method according to claim 3, in which the rat embryo is a Wistar rat conceptus.
5. A method according to claim 3 or claim 4, in which the embryo is between 10 and 11 days old.
6. A method according to any one of claims 1 to 5, in which the embryo has a visceral yolk sac and comprising injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism.
7. A method according to claim 6, in which the yol sac is bypassed by cannulation of the vitellin circulation.
8. A method according to claim 7, in which, durin cannulation, the embryo is placed in a well on microscope stage.
9. A method according to claim 8, in which the wel is formed from agar.
10. A method according to claim 9, in which the wel is dyed to provide contrast when viewing the bloo vessels beneath the yolk sac surface.
11. A method according to any one of claims 8 to 10, in which the well is heated.
12. A method according to any one of claims 1 to 11, in which the vitelline circulation is cannulated with a microcannula.
13. A method according to claim 12, in which the microcannula has an internal tip diameter of between 1 and 2 urn.
14. A method according to claim 12 or claim 13, in which the microcannula is coated with a siliconising agent.
15. A method according to any one of claims 12 to
14, in which the microcannula is connected to an injection pump.
16. A method according to any one of claims 12 to
15, in which the microcannula is used to inject a test substance containing a fluorescent dye.
17. A method according to any one of claims 1 to 16, comprising injecting a total volume of less than or equal to one microlitre.
18. A method according to any one of claims 1 to 17, in which a test substance is injected into the embryo over the course of four minutes.
19. A method according to any one of claims 1 to 18, in which, where the embryo is a rat embryo, it is cultured in heat-inactivated rat serum containing an antibiotic.
20. A method according to any one of claims 1 to 18, in which the embryo is cultured in synthetic or semi- synthetic medium.
21. A method according to claim 1, utilising embryos which are not surrounded by a visceral yolk sac.
22. A method according to claim 21, utilising chicken embryos in fertilised eggs.
23. A method according to claim 21 or claim 22, in which the test reagent is administered by micro¬ cannulation directly into the extra-embryonic circulation.
24. A method according to claim 22, in which the egg contents are released from the shell into a plastic container and incubated therein.
25. A method according to claim 22, in which a window is cut into the shed to access the embryo.
26. A method according to claim 21, in which the test reagent is introduced into the albumen.
27. A method according to any one of claims 1 to 26, in which the test substance is added with a radiolabel.
28. A method according to claim 28, in which the test substance is injected into the embryo heart.
29. A method according to claim 28, in which the embryo is homogenised for assessment of the effects of the test substance, an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess in-vitro the entire mass response to the test substance.
30. A test kit for carrying out a bioassay comprising microcannulae, dyed agar wells, culture media, a work station comprising a micro-injection pump, a micromanipulator, a heating stage and a dissection head for a microscope.
PCT/GB1992/001210 1991-07-06 1992-07-03 Bioassay method WO1993001272A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP92914060A EP0594660A1 (en) 1991-07-06 1992-07-03 Bioassay method
JP5502083A JPH06509469A (en) 1991-07-06 1992-07-03 bioassay method

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB919114638A GB9114638D0 (en) 1991-07-06 1991-07-06 Bioassay method
GB9114638.1 1991-07-06
GB919114955A GB9114955D0 (en) 1991-07-11 1991-07-11 Bioassay method
GB9114955.9 1991-07-11

Publications (2)

Publication Number Publication Date
WO1993001272A2 true WO1993001272A2 (en) 1993-01-21
WO1993001272A3 WO1993001272A3 (en) 1993-03-18

Family

ID=26299196

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1992/001210 WO1993001272A2 (en) 1991-07-06 1992-07-03 Bioassay method

Country Status (6)

Country Link
EP (1) EP0594660A1 (en)
JP (1) JPH06509469A (en)
AU (1) AU2230992A (en)
CA (1) CA2112897A1 (en)
IE (1) IE922172A1 (en)
WO (1) WO1993001272A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006716A1 (en) * 1993-08-30 1995-03-09 Northwestern University Rat pluripotent embryonic stem cells and method of obtaining and using same
WO1997031266A1 (en) * 1996-02-21 1997-08-28 Neumann Norbert J Process for determining the phototoxicity and/or photosensitivity of substances or mixtures thereof, and use thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006262863A (en) * 2005-03-25 2006-10-05 Gifu Univ Method for evaluating external stimulation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU461120A1 (en) * 1972-10-30 1975-02-25 Харьковский Научно-Исследовательский Институт Микробиовакцин И Сывороток The method of determining the biological activity of substances
DE3530336A1 (en) * 1985-08-24 1987-02-26 Wittmann J Dr Process for culturing the poultry embryo

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU461120A1 (en) * 1972-10-30 1975-02-25 Харьковский Научно-Исследовательский Институт Микробиовакцин И Сывороток The method of determining the biological activity of substances
DE3530336A1 (en) * 1985-08-24 1987-02-26 Wittmann J Dr Process for culturing the poultry embryo

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL SOCIETY TRANSACTIONS vol. 15, no. 5, October 1987, LONDON pages 922 - 923 ERIC P.K. MENSAH-BROWN ET AL. 'THE FATE OF I125-LABELLED PROTEINS AFTER INJECTION INTO 11.5 DAY RAT EMBRYONIC CIRCULATION.' *
DATABASE WPI Section Ch, Week 7603, Derwent Publications Ltd., London, GB; Class B04, AN 76-04952X/03 & SU,A,461 120 (KHARK MICROB VACCIN) 9 July 1975 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006716A1 (en) * 1993-08-30 1995-03-09 Northwestern University Rat pluripotent embryonic stem cells and method of obtaining and using same
US6171858B1 (en) 1996-02-12 2001-01-09 Norbert J. Neumann Proscess for determining the phototoxicity and/or photosensitivity of substances or mixtures thereof, and uses thereof
WO1997031266A1 (en) * 1996-02-21 1997-08-28 Neumann Norbert J Process for determining the phototoxicity and/or photosensitivity of substances or mixtures thereof, and use thereof

Also Published As

Publication number Publication date
EP0594660A1 (en) 1994-05-04
CA2112897A1 (en) 1993-01-21
AU2230992A (en) 1993-02-11
JPH06509469A (en) 1994-10-27
IE922172A1 (en) 1993-01-13
WO1993001272A3 (en) 1993-03-18

Similar Documents

Publication Publication Date Title
AU633029B2 (en) Stabilized lyophilized mammalian cells and method of making same
CN104215756B (en) The mensuration system of bio-related substance
US8586294B2 (en) Detection of protein translocation by beta-galactosidase reporter fragment complementation
Lakhotia et al. Damaging agitation intensities increase DNA synthesis rate and alter cell‐cycle phase distributions of CHO cells
EP0777861B1 (en) Test kit and method for detection of target cells and molecules
Frampton et al. Aqueous two‐phase system‐mediated antibody micropatterning enables multiplexed immunostaining of cell monolayers and tissues
Holter et al. Micromethods and their application in the study of enzyme distribution in tissues and cells
Krishnan Histochemical studies on reproductive and nutritional cycles of the holothurian, Holothuria scabra
WO1993001272A2 (en) Bioassay method
US20050084914A1 (en) Assays with primary cells
CN110887967A (en) Method for detecting antibody binding capacity and application thereof
Stirewalt et al. Schistosoma mansoni: histochemical analysis of the postacetabular gland secretion of cercariae
Head et al. Identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling
Braasch et al. Cell cycle components and their potential impact on the development of continuous in vitro penaeid cell replication
US20030044766A1 (en) Methods and devices for detecting cell-cell interactions
CN110887961A (en) Solid phase carrier and kit for antibody screening
EP2516620B1 (en) Apparatus and method for cell exploitation
DE69032663T2 (en) ENZYME TEST AND TEST SET FOR MEASURING CELLULAR ACTIVATION
US5472846A (en) Test kit and method for amplification and detection of antigen cells
Clark Modeling the Complexity of the Metastatic Niche Ex Vivo
JPH03505669A (en) Improvements in bioassays
KR20220094191A (en) Methods for Independent Analysis of Multiple Biological Processes in Encapsulated 3D Cell Co-culture
Godman et al. A description of echo-9 virus infection in cultured cells: II. Cytochemical observations
Kiyomoto et al. Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo
Ardiyan A REVIEW OF QUALITY CONTROL IN HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY STAINING

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AT AU BB BG BR CA CH CS DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO PL RO RU SD SE US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE BF BJ CF CG CI CM GA GN ML MR SN TD TG

AK Designated states

Kind code of ref document: A3

Designated state(s): AT AU BB BG BR CA CH CS DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO PL RO RU SD SE US

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE BF BJ CF CG CI CM GA GN ML MR SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1992914060

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2112897

Country of ref document: CA

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1992914060

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 1994 170345

Country of ref document: US

Date of ref document: 19940524

Kind code of ref document: A

WWW Wipo information: withdrawn in national office

Ref document number: 1992914060

Country of ref document: EP