WO2002048672A2 - Reactifs et procedes a utiliser dans la detection d'analytes - Google Patents

Reactifs et procedes a utiliser dans la detection d'analytes Download PDF

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Publication number
WO2002048672A2
WO2002048672A2 PCT/GB2001/005437 GB0105437W WO0248672A2 WO 2002048672 A2 WO2002048672 A2 WO 2002048672A2 GB 0105437 W GB0105437 W GB 0105437W WO 0248672 A2 WO0248672 A2 WO 0248672A2
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WO
WIPO (PCT)
Prior art keywords
reagent
immunoassay
pbs
immunoreactant
tween
Prior art date
Application number
PCT/GB2001/005437
Other languages
English (en)
Other versions
WO2002048672A3 (fr
Inventor
Rudolf A. Kruithof
Original Assignee
Ra Laboratories Limited
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
Application filed by Ra Laboratories Limited filed Critical Ra Laboratories Limited
Priority to AU2002222146A priority Critical patent/AU2002222146A1/en
Publication of WO2002048672A2 publication Critical patent/WO2002048672A2/fr
Publication of WO2002048672A3 publication Critical patent/WO2002048672A3/fr

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the invention relates to reagents and methods for use in the detection of analytes and in particular for use in immunoassays such as sandwich and competition immunoassays but not limited thereto.
  • analytes concentration of one or more compounds (generally referred to as analytes) in a liquid sample.
  • use is made of antibodies or fragments thereof which bind specifically to the analyte of interest.
  • antibodies bound to a solid support are employed in a variety of sandwich and competitive type assays.
  • Sandwich assays are considered to be highly sensitive and are particularly appropriate for use with samples having low concentrations of analytes. However, they are also useful in determining the concentration of large molecules such as proteins. On the other hand, competition assays are particularly useful for detecting the presence of smaller analytes (e.g organic molecules) or multiple analytes in a sample (e.g. drug analysis of a patient fluid sample) .
  • analytes e.g organic molecules
  • multiple analytes in a sample e.g. drug analysis of a patient fluid sample
  • Prior art assays generally involve a washing step after the liquid sample has been incubated with each new reactant. It is therefore the aim of the present invention to provide an assay in which washing steps were not required but the assay still performed as well or better than those in which washing steps were essential.
  • an immunoassay characterised in that the method excludes washing steps and comprises initially diluting a sample containing an analyte to be tested, with a first reagent having a haemolysing property upon blood cells in the test sample thus preventing aggregation thereof, introducing a solid phase to the test sample mixture on which there is coupled a first immunoreactant, introducing a second immunoreactant, said second immunoreactant being a labelled immunoreactant, and further introducing a substrate /visualisation complex capable of binding the labelled immunoreactant to from a conjugate; wherein said substrate /visualisation complex is diluted by a second reagent, the second reagent comprising a mixture of phosphate buffer, protein and detergent, said reagent adapted to improve interaction between the solid phase and the substrate whilst also optimising visualisation of the analyte of interest.
  • the first reagent comprises a haemolysin although it can simply comprise haemolytic properties such as by hypotonic shock.
  • haemolysin can simply comprise haemolytic properties such as by hypotonic shock.
  • This is achieved by way of a mixture of purified H2O which generally has a pH of 5.5 and has added thereto sodium hydroxide which increases the pH up towards pH 7 thereby creating hypotonic shock to the cells in the sample.
  • the first reagent comprises a haemolysin it is Saponin but other haemolysins may be used.
  • the first reagent comprises phosphate buffer such as PBS.
  • the first reagent generally comprises PBS (pH 7.4, 10 mM) and 0.66% Saponin.
  • the second reagent comprises an mixture of phosphate buffer, protein and detergent.
  • the phosphate buffer is PBS and the protein is BSA, also the detergent is Tween, such as Tween-20, but need not be limited thereto.
  • the reagent comprises 3 parts PBS A + 1 part PBS- Tween in the form of 3 parts PBSA (PBS pH 7.4, 10 mM + 1% BSA) and 1 part PBS pH 7.4, 10 mM)-Tween (0.05% Tween 20). It is envisaged that the assays described above may also be used in respect of urine samples or spinal fluid samples.
  • a first immunoreactant which in this case is an amount of the analyte of interest, namely T4, is bound to a solid phase such as polystyrene beads.
  • the binding is carried out in the usual manner which will be well known to a person skilled in the art but an example of which is reproduced below (See Procedure A).
  • the polystyrene beads are preferably Luminex® beads but any suitable solid phase can be used.
  • the sample to be tested which may or may not contain the analyte or analytes of interest is diluted with a first reagent usually in the amount of about 2.5 ⁇ l sample to 75 ⁇ l of first reagent.
  • the first reagent comprises phosphate buffer namely PBS (pH 7.4, 10 mM) and 0.66% of a haemolysin such as Saponin.
  • PBS pH 7.4, 10 mM
  • a haemolysin such as Saponin
  • Saponin is used in this example it is envisaged that other haemolysins or mixtures thereof would also be suitable.
  • hypotonic shock has also been used successfully to lyse the cells. This is achieved by way of a mixture of purified H2O which generally has a pH of 5.5 has added thereto sodium hydroxide which increases the pH up towards pH 7 thereby creating hypotonic shock.
  • a second immunoreactant is introduced If as in this case the first immunoreactant is a T4 antigen the second immunoreactant to be used would usually be an anti-T4 antibody. Further, the second immunoreactant is usually labelled with an enzyme or other suitable label such as biotin etc. In this case the anti-T4 was biotinylated i.e. labelled with biotin. The assay mixture is then incubated for about half an hour at 37°C.
  • a substrate /visualisation complex is then prepared for adding to the assay. This allows visualisation of the amount/ concentration of the analyte (T4) in the sample.
  • the substrate has to be capable of specifically binding the label to form a conjugate.
  • Streptavidin is therefore used to bind to the biotin. Visualisation of the conjugate occurs through the fact that the streptavidin is coupled with phycoerythrin.
  • substrate /visualisation complexes can be utilised depending on the label used for example Horse radish Peroxidase conjugate and substrate or Alkaline Phosphatase conjugate or substrate.
  • the substrate /visualisation complex is diluted with a second reagent.
  • the second reagent comprises an admixture of phosphate buffer, protein and detergent.
  • PBS is the phosphate buffer whilst BSA is used as the protein with Tween 20 as the detergent.
  • the second reagent comprises 3 parts PBSA + 1 part PBS-Tween 20 generally in the form of 3 parts PBSA (PBS pH 7.4, 10 mM + 1% BSA) and 1 part PBS (pH 7.4, 10 mM)-Tween (0.05% Tween 20).
  • test sample which may contain the analyte or analytes of interest (in this case hepatitis B or C, or HIV antigen) is diluted with the first reagent generally in the region of 2.5 ⁇ l sample to 75 ⁇ l of first reagent.
  • the composition of the first reagent and its action has already been described above in Example 1.
  • a first immunoreactant which is can be an antibody or antigen, but for example is HIV-1 P24, is bound to a solid phase.
  • Polystyrene Luminex® beads are again used as the solid phase and bound and activated as described Procedure A. However, any suitable solid phase can be used The solid phase with the antibody bound thereto is then added to the assay. The mixture is then incubated for half an hour at 37°C.
  • Other first immunoreactants can include glycoproteins, GP41 or GP120.
  • a second immunoreactant which again can be an antibody or antigen, for example Anti Human IgG is introduced.
  • the second immunoreactant is usually labelled by an enzyme or other suitable label. In this case it is labelled with biotin as in Example 1 above.
  • a substrate /visualisation complex is then prepared for adding to the assay. This allows visualisation of the amount/ concentration of the analyte (Hepatitis or HIV) in the sample.
  • the substrate has to be capable of specifically binding the label to form a conjugate.
  • Streptavidin is therefore used to bind to the biotin. Visualisation of the conjugate occurs through the fact that the streptavidin is coupled with phycoerythrin.
  • substrate/visualisation complexes can be utilised depending on the label used for example Horse radish Peroxidase conjugate and substrate or Alkaline Phosphatase conjugate or substrate.
  • the substrate /visualisation complex is diluted with a second reagent the action and composition of which is described in Example 2 above.
  • Reagents should be allowed to come to room temperature before use. The supernatant should be kept to determine the amount of beads lost during procedure.
  • Method for BEAD COUPLING - Prepare protein to be coupled in PBS (500 ⁇ l of 40 - 100 ⁇ g/ml is required) .
  • Beads should not be vortexed from stages 3 of bead activation to step 2 of bead coupling. Beads may stick to the side of the microcentrifuge tubes during activation. This is normal. It should be noted that only the final blocking buffer (PBS BSA azide) contains azide. Azide should not be added to any other reagents as the azide will couple to the beads rather than the protein.
  • the advantage of the present invention is that it allows for automation of immunoassays. This is because the washing steps are no longer required due to the use of the first and second reagents described above. Further the lack of washing steps in the immunoassays carried out according to the present invention does not adversely affect the quality of the results produced thereby.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un dosage immunologique caractérisé en ce que le procédé exclut des étapes de lavage et consiste initialement à diluer un échantillon contenant un analyte à tester, un premier réactif présentant une propriété hémolysante sur les globules de l'échantillon de test, empêchant ainsi l'agrégation desdits globules, à introduire une phase solide dans le mélange de l'échantillon de test au niveau duquel est couplé un premier immunoréactif, à introduire un second immunoréactif, ledit second immunoréactif étant un immunoréactif marqué, et également à introduire un complexe de substrat/ visualisation pouvant se lier à l'immunoréactif marqué afin de former un conjugué; ledit complexe de substrat/ visualisation étant dilué par un second réactif, le second réactif comprenant un mélange de tampon de phosphate, de protéines et de détergent, ledit réactif étant adapté pour améliorer l'interaction entre la phase solide et le substrat tout en optimisant également la visualisation de l'analyte à examiner.
PCT/GB2001/005437 2000-12-12 2001-12-11 Reactifs et procedes a utiliser dans la detection d'analytes WO2002048672A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002222146A AU2002222146A1 (en) 2000-12-12 2001-12-11 Reagents and methods for use inn the detection of analytes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0030360A GB2372319A (en) 2000-12-12 2000-12-12 A solid phase immunoassay involving blood cells which assay excludes washing steps
GB0030360.2 2000-12-12

Publications (2)

Publication Number Publication Date
WO2002048672A2 true WO2002048672A2 (fr) 2002-06-20
WO2002048672A3 WO2002048672A3 (fr) 2003-01-16

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AU (1) AU2002222146A1 (fr)
GB (1) GB2372319A (fr)
WO (1) WO2002048672A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009055382A2 (fr) * 2007-10-21 2009-04-30 Yasger, Paul Dosages immunologiques en une étape présentant une sensibilité et une spécificité accrues
WO2009086343A3 (fr) * 2007-12-31 2010-05-06 3M Innovative Properties Company Compositions et procédés de capture de micro-organismes
CN109187121A (zh) * 2018-08-06 2019-01-11 中国人民解放军第三0二医院 联合免疫效果早期快速评价方法中的试剂及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5935825A (en) * 1994-11-18 1999-08-10 Shimadzu Corporation Process and reagent for amplifying nucleic acid sequences

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6047962A (ja) * 1983-08-26 1985-03-15 Terumo Corp 血中の抗原または抗体量の測定方法およびそれに使用する試験液
JPS60244861A (ja) * 1984-05-19 1985-12-04 Masakatsu Hashimoto 溶血式測定方法及びそれに使用する試薬キツト
JPH0255955A (ja) * 1988-08-19 1990-02-26 Green Cross Corp:The Ｃｓｆ測定用ｅｉａ試薬キットおよび測定方法
US5650288A (en) * 1995-07-14 1997-07-22 Macfarlane; Gordon D. Immunophilin-bound immunosuppressant assay

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5935825A (en) * 1994-11-18 1999-08-10 Shimadzu Corporation Process and reagent for amplifying nucleic acid sequences

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Mc Cubbin V. and Frank M.B. :'Detection of recombinant proteins by ELISA.' In: Frank M.B. ed., Molecular Biology Protocols. & http://omrf.ouhsc.edu/frank/elisa.html October 2, 1997 XP002217404 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009055382A2 (fr) * 2007-10-21 2009-04-30 Yasger, Paul Dosages immunologiques en une étape présentant une sensibilité et une spécificité accrues
WO2009055382A3 (fr) * 2007-10-21 2009-09-24 Yasger, Paul Dosages immunologiques en une étape présentant une sensibilité et une spécificité accrues
WO2009086343A3 (fr) * 2007-12-31 2010-05-06 3M Innovative Properties Company Compositions et procédés de capture de micro-organismes
CN101952727A (zh) * 2007-12-31 2011-01-19 3M创新有限公司 微生物捕获用组合物和方法
JP2011509083A (ja) * 2007-12-31 2011-03-24 スリーエム イノベイティブ プロパティズ カンパニー 微生物捕捉用組成物及び微生物を捕捉する方法
CN109187121A (zh) * 2018-08-06 2019-01-11 中国人民解放军第三0二医院 联合免疫效果早期快速评价方法中的试剂及其应用

Also Published As

Publication number Publication date
WO2002048672A3 (fr) 2003-01-16
GB2372319A (en) 2002-08-21
GB0030360D0 (en) 2001-01-24
AU2002222146A1 (en) 2002-06-24

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