EP1352241A1 - Technique multiplex par absorbance - Google Patents

Technique multiplex par absorbance

Info

Publication number
EP1352241A1
EP1352241A1 EP01992259A EP01992259A EP1352241A1 EP 1352241 A1 EP1352241 A1 EP 1352241A1 EP 01992259 A EP01992259 A EP 01992259A EP 01992259 A EP01992259 A EP 01992259A EP 1352241 A1 EP1352241 A1 EP 1352241A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
antibody
conjugated
substrate
marker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01992259A
Other languages
German (de)
English (en)
Inventor
Bassam Damaj
Ali-Samer Al-Assaad
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.)
Biosignature Diagnostics Inc
Original Assignee
Biosignature Diagnostics Inc
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 Biosignature Diagnostics Inc filed Critical Biosignature Diagnostics Inc
Publication of EP1352241A1 publication Critical patent/EP1352241A1/fr
Withdrawn legal-status Critical Current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
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • ELISA solid-phase heterogeneous enzyme-linked immunosorbent assay
  • the enzyme-labeled ligand is an enzyme-labeled antibody.
  • Commonly used enzymes include alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, ⁇ - glucuronidase, luciferase, and urease.
  • the enzyme-labeled ligand binds the target molecule/bound antibody complex.
  • a substrate specific for the enzyme, is added to the solid phase support.
  • Target molecules include, but are not limited to, antigens, or fragments thereof, including antigens derived from microorganisms and other pathogens, antibodies or fragments thereof, including antibodies produced in response to antigens derived from microorganisms and other pathogens, tumor markers, oligonucleotides, nucleic acids, carbohydrates, proteins, chemicals, drugs, receptors, haptens, hormones, gamma globulins, allergens, viruses, virus subunits, bacteria, toxins such as those associated with tetanus and with animal venoms, enzymes, nucleic acid molecules including DNA fragments, RNA fragments, and artificial nucleic acid fragments, and self-antibodies generated in autoimmune disease, or any mix thereof.
  • antigens When using antigens to detect target molecules, several antigens, each specific for a different target molecule of interest, are bound to a reaction surface.
  • the antigen may be bound to any reaction surface, solid or semisolid phase support, including, but not limited to, polystyrene wells of microtitre plates, glass or plastic strips, glass or plastic beads, membranes, microplates, magnetic particles, latex particles, nitrocellulose particles, polyacrylamide beads, magnetic beads, polystyrene, polyurethane, agarose, collagen, gelatin, SEPHAROSE, SEPHADEX, SEPHARON, nylon, rayon, and test tubes.
  • the antigen is bound to a single reaction well of the reaction surface.
  • attachment to the reaction surface may be non-covalent wherein the molecule binds to the reaction surface through adsorption.
  • the attachment may be covalent wherein the molecule is chemically coupled to the reaction surface with a linker molecule.
  • a wash step may be performed after the reaction well is coated.
  • a sample suspected of containing at least one marker may be added to the reaction well.
  • sample can be any sample of interest, including, but not limited to, a biological sample, including, but not limited to, serum, blood, urine or saliva, a soil sample, or any liquid sample, including water.
  • Biological samples may be of either human or non-human origin.
  • the markers bind different, specific bound molecules.
  • a wash step may be performed after the bound molecule/marker complexes are formed to remove unbound molecules.
  • the ligand may be any antagonist having a binding affinity for the reactant, including, but not limited to, single chain antibody fragments, antibody, antibody fragments, artificial antibodies, peptides, chemical entities, lipids, carbohydrates, artificial nucleic acid sequences, DNA, RNA, antigen or fragments thereof.
  • the sample and solution are incubated for a sufficient time to allow the first marker to bind with the first conjugated ligand, the second marker to bind with the second conjugated ligand and the third marker to bind with the third conjugated ligand.
  • 0.5 mg of dialyzed antibody was added to 1.5 mg of the enzyme in 10 ml of 10 mM PBS.
  • 80 ⁇ l 25% glutaraldehyde was added and mixed gently.
  • the solution was let stand at room temperature for 2 hrs.
  • the reaction was stopped by adding an equivalent volume (10 ml) of PBSLE (10 mMPBS containing 100 mMlysine and 100 mM ethanolamine).
  • PBSLE 10 mMPBS containing 100 mMlysine and 100 mM ethanolamine.
  • the solution was desalted with a SEPHADEX G25 column in PBSN (10 mMPBS with 0.05MNaN 3 ).
  • the antibody-coated plate was rinsed by flooding with washing buffer (20 mM TrisHCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) a minimum of three times. Each well was filled with blocking buffer (0.2% BSA, 0.01% Gelatin in TBS, pH 7.5) dispensed using multichannel pipettes and incubated 15 min at 37° C. The plate was rinsed three times with washing buffer and any residual liquid was removed by gently flicking it face down onto paper towels.
  • washing buffer (20 mM TrisHCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5
  • blocking buffer (0.2% BSA, 0.01% Gelatin in TBS, pH 7.5
  • Step I 200 ⁇ l aliquots of 1/50 serum dilution in PBS of the test antigen sample solutions (patient serum or plasma enzyme) or the standard antigen dilutions were added to the antibody-coated wells and incubated for 30 minutes at 37° C. The plates were rinsed three times in wash buffer and residual liquid was removed by blotting.
  • ONGP's OD was quenched almost 10 fold from 1.025 to 0.174 due to interference from previous enzymes or their buffers.
  • series 2 (Table 2b), ABTS was added first. OD reading at 405nm was followed by a washing step. PnPP was added second and followed by a washing step after reading the OD at 405nm. ONGP was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.039, 1.800 and 0.175 for ABTS, PnPP and ONGP respectively as compared with 0.992, 1.816 and 1.025 obtained from reading the OD of the same marker plated individually in single wells. ONGP's OD was quenched almost 10 fold from 1.025 to 0.175 due to interference from previous enzymes or their buffers.
  • ONGP was added first. OD reading at 405nm was followed by a washing step. PnPP was added second and followed by a washing step after reading the OD at 405nm. ABTS was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.089, 1.962 and 0.266 for ONGP, PnPP and ABTS respectively as compared with 1.025, 1.816 and 0.992 obtained from reading the OD of the same marker plated individually in single wells. ABTS's OD was quenched almost 4 fold from 0.992 to 0.266.
  • ONGP was added first. OD reading at 405nm was followed by a washing step. ABTS was added second and followed by a washing step after reading the OD at 405nm. PnPP was added third and the OD was read at 405nm. This sequential addition of substrates worked, as the OD values obtained at 405nm were 1.069, 1.282 and 1.727 for ONGP, ABTS and PnPP respectively as compared with 1.025, 0.992 and 1.816. This series showed OD levels of absorbance of all the three markers compatible with the OD readings seen in Table 1. Table 1 : Single marker data from single wells.
  • Table 2b Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
  • Table 2c Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
  • Table 2d Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
  • Table 2e Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
  • the assay involves the simultaneous measurement of three cytokine markers (IL-1)
  • the assay detects and quantitates at least three markers from the same sample, in the same well and at the same time on a solid phase support following the specific sequential addition of substrates as illustrated in Example I.
  • Table 4 Triple marker data from single wells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (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)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant de détecter et de quantifier simultanément des molécules cibles multiples dans un seul puits de réaction d'une surface de réaction. Des anticorps de différentes molécules cibles sont liés à une surface de réaction. Une solution échantillon supposée contenir les molécules cibles est ajoutée à la surface de réaction et incubée. Au cours de l'incubation, les molécules cibles forment des complexes avec les anticorps liés. Après une étape de lavage, un mélange d'anticorps liés à une enzyme est ajouté à la surface de réaction. Ces anticorps liés à une enzyme ont une affinité pour les complexes molécules cibles/anticorps liés. L'addition séquentielle de substrats enzymatiques spécifiques permet de détecter des molécules cibles multiples sans empoisonner les enzymes. L'invention concerne également un nécessaire de détection simultanée de molécules cibles multiples.
EP01992259A 2000-12-21 2001-12-21 Technique multiplex par absorbance Withdrawn EP1352241A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US25828600P 2000-12-21 2000-12-21
US258286P 2000-12-21
US94782301A 2001-09-06 2001-09-06
US947823 2001-09-06
PCT/US2001/049745 WO2002050537A1 (fr) 2000-12-21 2001-12-21 Technique multiplex par absorbance

Publications (1)

Publication Number Publication Date
EP1352241A1 true EP1352241A1 (fr) 2003-10-15

Family

ID=26946549

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01992259A Withdrawn EP1352241A1 (fr) 2000-12-21 2001-12-21 Technique multiplex par absorbance

Country Status (4)

Country Link
EP (1) EP1352241A1 (fr)
AU (1) AU2002232722A1 (fr)
CA (1) CA2432398A1 (fr)
WO (1) WO2002050537A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3217176B1 (fr) * 2016-03-11 2019-08-07 Scienion AG Procede immunoenzymatique permettant la detection et l'identification sequentielles d'analytes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4948726A (en) * 1986-06-02 1990-08-14 Longoria Claude C Enzyme immunoassay based on membrane separation of antigen-antibody complexes
US5180806A (en) * 1988-05-16 1993-01-19 The Scripps Research Institute Polypeptides and compositions of human papillomavirus latent proteins, diagnostic systems and methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0250537A1 *

Also Published As

Publication number Publication date
AU2002232722A1 (en) 2002-07-01
WO2002050537A1 (fr) 2002-06-27
CA2432398A1 (fr) 2002-06-27

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