[go: up one dir, main page]

WO1996040790A1 - Presentation d'antigene natif sur des billes ou autres phases solides - Google Patents

Presentation d'antigene natif sur des billes ou autres phases solides Download PDF

Info

Publication number
WO1996040790A1
WO1996040790A1 PCT/US1996/009381 US9609381W WO9640790A1 WO 1996040790 A1 WO1996040790 A1 WO 1996040790A1 US 9609381 W US9609381 W US 9609381W WO 9640790 A1 WO9640790 A1 WO 9640790A1
Authority
WO
WIPO (PCT)
Prior art keywords
solid phase
functionalities
substituents
beads
streptavidin
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.)
Ceased
Application number
PCT/US1996/009381
Other languages
English (en)
Inventor
Kenneth A. Davis
Barnaby Abrams
James E. Bishop
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Becton Dickinson and Co
Original Assignee
Becton Dickinson and Co
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 Becton Dickinson and Co filed Critical Becton Dickinson and Co
Publication of WO1996040790A1 publication Critical patent/WO1996040790A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Definitions

  • the critical parameter is the recognition of the antigen. This is strongly affected by the conformation of the target, and, in general, it is essential to have the target in a corifirmation resembling its native conformation. As conformation is strongly influenced by the environment of a molecule, it becomes extremely difficult to attach a target to a bead or other structure to facilitate its use in the desired application, yet retain a corifirmation similar to its native conformation.
  • the instant invention presents a rapid simple method for preparing solid phases, preferably beads, with antigens or other substituents presented on the surface in such a manner that the antigens/substituents retain their original functionality and conformation, as well as much of their native structure, to permit their use in a wide array of applications.
  • the substituent is isolated from the hydrophobic surface of the bead or other solid phase by coating the surface with a milk protein, alone or in combination with linker molecules, thereby preventing interaction of the substituents with the bead surface.
  • the complexes of the instant invention are formed by attaching the substituent(s) to the solid phase, either directly via surface functionalities or via a linking group, or both. Regardless of the method of attachment used, the bead, other than the substituent binding sites, is coated with a milk protein or a milk protein derivative such as dephosphocasein (as used herein the term "milk” includes whole milk, powdered milk, and modified milk, milk proteins, and milk protein derivatives).
  • This coating serves a two-fold basis preventing binding of the substituents to other sites on the solid phase (i.e., as a blocking agent) and preventing interaction of the bound substituents with the hydrophobic surfaces of the beads. In this way, the substituents retain their natural structure and properties.
  • the coating is obtained by immersing the solid phase, prior to attaching substituents, in solutions or mixtures containing milk for a period of approximately thirty minutes; however, it can also be stored for extended periods of time in such solutions.
  • the solid phase is a bead which can be any of those known to be useful in flow cytometry applications or other applications for antigen presentation.
  • the beads must be capable of forming a stable complex with the substituents.
  • the bead is comprised of polymethylmethacrylate possessing arnino functionalities on the surface. These beads can often be directly attached to the substituents via the amino functionality.
  • activated biotin is attached to the amino groups and the beads are then coated with milk protein.
  • the beads are then admixed with streptavidin creating a streptavidin layer on the bead, which is capable of binding biotinylated substituents. It is to be understood, however, that other beads and/or surface functionalities can also be used.
  • the substituents can then be attached to the beads by the appropriate chemical reaction.
  • the substituent is reacted with biotin to form a biotinylated complex which readily binds to the streptavidin layer on the beads.
  • biotinylated complex which readily binds to the streptavidin layer on the beads.
  • substituents will be chosen based on the ultimate desired use of the solid phase and/or the problem desired to be solved.
  • substituents will include, but are not limited to, antigens, antibodies, other biologically active molecules, and fluorophores.
  • Fluorescent proteins are particularly useful with beads which are prepared in accordance with this invention. They produce a specific brightness and emission spectrum which is unaffected by any interactions between the bead surface and the fluorophores. Further, the beads exhibit a high utility as standards for flow cytometry, as the fluorophore coated beads produce the same fluorescence as stained cells.
  • Polymethacrylate amino beads (6.1 microns PMMA-RNH2, Bangs Laboratories, Inc., Carmel, IN) were washed once with 10 volumes PBS + 0.1% sodium azide, twice with three volumes PBS +0.1% SDS, and twice with PBS. The washed beads were then suspended (3% w/v) in PPBS containing 1.5mM NHS-XX-Biotin, and incubated, with mixing, a room temperature for one hour. The beads were then washed twice with three volumes of PBS, and subsequently, suspended and stored overnight in a PBS +0.1% sodium azide + 2% non ⁇ fat dry milk.
  • Example 3 Attachment of biotinylated protein to streptavidin beads Streptavidin beads (Example 1), at a suspension of 2.5% in PBS, were admixed with biotinylated hCG or hLH (Example 2) and incubated at room temperature for two hours.
  • the beads were then washed twice in 5 volumes PBS +0.1% sodium azide +0.5% BSA, and suspended at 2.5% w/v in the same buffer.
  • the beads present the protein in an unhindered conformation and can be used to bind and/or sort antibodies for hCG or hLH.
  • Biotinylated beads were reacted with excess streptavidin conjugates of fluorescein isotheocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll - a protein (Per CP), PE/Cy5 reactive dye (Biological Detection Systems), or allophycyanin (APC).
  • FITC fluorescein isotheocyanate
  • PE phycoerythrin
  • Per CP peridinin chlorophyll - a protein
  • PE/Cy5 reactive dye Biological Detection Systems
  • allophycyanin APC
  • SA-PE beads were prepared as in Example 4, except that BSA, Tween 20, gelatin, and Tetronic 908 were used in place of the milk protein. These compounds were much less efficient in blocking the non-specific abso ⁇ tion, resulting in a much higher interfering signal on a flow cytometer, then when milk was used.
  • the milk protein derivative, dephosphocasein was found to be at least as efficient a blocker as whole milk protein.
  • biotinylated CD4 prepared as described in Example 2.
  • the beads were titrated and found to have approximately 10 ⁇ molecules.
  • a control lot of streptavidin coated polystyrene beads prepared by coating the bead directly with streptavidin, without any blocking) were similarly reacted with biotinylated CD4.

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)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (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)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Cette invention concerne un procédé simple et rapide de préparation de phases solides présentant des antigènes ou d'autres substituants sur leur surface, et d'une manière telle que ces antigènes ou substituants conservent leur valence fonctionnelle et leur conformation originelles ainsi qu'une grande partie de leur structure native, ce qui permet de les utiliser dans une gamme d'applications étendue. Le substituant est isolé de la surface hydrophobe d'une bille, ou d'une autre phase solide, par enduction de ladite surface de lait ou d'une protéine du lait, seul ou en combinaison avec des molécules de liaison, ce qui permet d'empêcher une interaction entre les substituants et la surface de la bille.
PCT/US1996/009381 1995-06-07 1996-06-07 Presentation d'antigene natif sur des billes ou autres phases solides Ceased WO1996040790A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US43843795 1995-06-07
US08/438,437 1995-06-07

Publications (1)

Publication Number Publication Date
WO1996040790A1 true WO1996040790A1 (fr) 1996-12-19

Family

ID=23740666

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/009381 Ceased WO1996040790A1 (fr) 1995-06-07 1996-06-07 Presentation d'antigene natif sur des billes ou autres phases solides

Country Status (1)

Country Link
WO (1) WO1996040790A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190039041A1 (en) * 2013-05-14 2019-02-07 Genomics Usa, Inc. Compositions and methods for entrapping protein on a surface

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0282192A1 (fr) * 1987-03-09 1988-09-14 Quidel Immuno-essai compétitif pour les haptènes utilisant une phase solide de protéine-haptène couplés
EP0643305A2 (fr) * 1993-09-13 1995-03-15 Ciba Corning Diagnostics Corp. Sous-revêtement de surfaces de phases solides et méthode de revêtement direct

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0282192A1 (fr) * 1987-03-09 1988-09-14 Quidel Immuno-essai compétitif pour les haptènes utilisant une phase solide de protéine-haptène couplés
EP0643305A2 (fr) * 1993-09-13 1995-03-15 Ciba Corning Diagnostics Corp. Sous-revêtement de surfaces de phases solides et méthode de revêtement direct

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SIPE, JEAN D. ET AL: "Direct binding enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA)", J. IMMUNOL. METHODS (1989), 125(1-2), 125-35 CODEN: JIMMBG;ISSN: 0022-1759, 1989, XP002018648 *
UEBEL, R. ET AL: "Improved immunoassay for perphenazine", CLIN. CHIM. ACTA (1992), 213(1-3), 111-13 CODEN: CCATAR;ISSN: 0009-8981, 1992, XP002018649 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190039041A1 (en) * 2013-05-14 2019-02-07 Genomics Usa, Inc. Compositions and methods for entrapping protein on a surface
US10556218B2 (en) * 2013-05-14 2020-02-11 Pure Transplant Solutions L.L.C. Compositions and methods for entrapping protein on a surface
US11260363B2 (en) 2013-05-14 2022-03-01 Pure Transplant Solutions L.L.C. Compositions and methods for entrapping protein on a surface

Similar Documents

Publication Publication Date Title
US4228237A (en) Methods for the detection and determination of ligands
US5384073A (en) Ligand gold bonding
US5424193A (en) Assays employing dyed microorganism labels
EP0077671B1 (fr) Réactifs immunologiques employant des supports polymères
US5571726A (en) Kit containing glutaraldehyde coated colloidal metal particles of a preselected size
US5616467A (en) Method and kit for analyte detection employing gold-sol bound antibodies
JP2757965B2 (ja) 発光アリールスルホネートシアニン染料を用いた物質の標識ないし検出方法
US4743542A (en) Method for forestalling the hook effect in a multi-ligand immunoassay system
CN103097895B (zh) 核酸或免疫色谱法用试剂组合物、核酸或免疫色谱法测定方法及核酸或免疫色谱法测定用试剂盒
EP2518193A2 (fr) Procedes pour lier de facon reversible un compose de Biotine a un support
US4689310A (en) Methods for attaching ligands or anti-ligands to a solid phase
JP6333181B2 (ja) バイオアッセイのシグナル増幅のための方法およびシステム
EP0394398B1 (fr) Nanoparticule comportant une enzyme liee et un ligand pour utilisation dans des analyses
NO152955B (no) Reagens for bestemmelse av et immunologisk aktivt materiale
JP2002517728A (ja) 分析対象物質を検出する方法
WO1999017120A1 (fr) Preparation de conjugues au moyen de groupes de liaison polyethyleneglycol
US4791069A (en) Methods for attaching ligands or anti-ligands to a solid phase
JPH07301632A (ja) イムノアッセイ試薬およびそれを用いたイムノアッセイ法
EP0276165B1 (fr) Liposomes susceptibles de localiser certains objets et leur utilisation dans un immuno-essai
WO1996040790A1 (fr) Presentation d'antigene natif sur des billes ou autres phases solides
CA1289876C (fr) Systeme en phase solide incorporant des sels de tetrazolium, destines a etre utilise pour des essais ligand-recepteur
Ngo et al. New approach to heterogeneous enzyme immunoassays using tagged enzyme-ligand conjugates
EP0136347A1 (fr) Surfaces insolubles traitees pour inhiber la liaison d'une proteine non-specifique
EP1702076B1 (fr) Procede et composition de stabilisation de reactifs liquides
US20080108147A1 (en) Reduction of non-specific binding in immunoassays

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase