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Methods for simultaneously detecting both members of a binding pair

a technology of binding pair and detection method, which is applied in the field of simultaneous detection of both members of a binding pair, can solve the problems of reducing the detection efficiency of antibodies, so as to enhance the ability to detect infections and improve the detection efficiency. the effect of rapid and sensitiv

Inactive Publication Date: 2009-01-08
BIOE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004]The invention is based on a rapid and sensitive method for simultaneously detecting both members of a binding pair, such as a ligand and receptor or an antigen and host antibody, from a biological sample. Methods of the invention can, for example, enhance the ability to detect infections at an early stage, leading to earlier treatment of the infection.

Problems solved by technology

Immunoassay techniques are limited in their ability to detect the presence of viral contaminants in early stages of infection, with the window period between infection with a virus and detection by immunoassay techniques varying from two to four weeks for HIV and up to about 10 weeks for hepatitis C virus (HCV).
Techniques such as reverse-transcriptase polymerase chain reaction (RT-PCR) or branched chain DNA analysis can shorten the time period between infection and detection, but are cost prohibitive for use on an individual donor basis and do not eliminate the window period.

Method used

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  • Methods for simultaneously detecting both members of a binding pair
  • Methods for simultaneously detecting both members of a binding pair
  • Methods for simultaneously detecting both members of a binding pair

Examples

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example 1

[0036]Biotinylation of Proteins: Antibodies were conjugated at a concentration of 5 mg / ml; viral antigens were conjugated at a concentration of 1 mg / ml. To biotin label anti-viral protein antibodies and viral antigens, the proteins were exchanged into 100 mM KH2CO3 buffer (pH 8.3) using an appropriate size Centricon (Amicon) filter.

[0037]Biotin N-hydroxysuccinimidyl ester (Molecular Probes, Eugene, Oreg.) in DMSO (10 mg / ml, Sigma Chemical Co., St. Louis, Mo., Cat. #D8779) was prepared immediately prior to use, and added to the protein to be biotinylated in a 5:1 or 10:1 molar ratio. Reactions were performed by vortexing the protein solution lightly, and adding the biotin / DMSO to the protein solution and mixing thoroughly. Protein and biotin ester were reacted for one hour at room temperature in the dark. Conjugated protein was separated from free biotin by separation on a 10 ml Sephadex-25 gel column or spin column using 1×PBS to elute. Individual 1 ml fractions were collected and a...

example 2

[0039]Production of Analyte Capture Beads: Analyte capture beads were prepared by completely resuspending avidin-coated paramagnetic beads (7 μm, Spherotech, VM-60-100) by mixing well. Beads (typically 3.8×106 beads) were placed in a 50 ml centrifuge tube and mixed with 30 ml of 1×PBS. After retaining beads on the side of the tube with magnets, all PBS was removed. The beads were washed two more times with PBS.

[0040]After the final PBS wash, the required volume of biotinylated antibody (typically 40 μg) and 2 ml of 1×PBS were added to the beads. The beads were resuspended by vortexing continuously for a minimum of 3 hours, or by vortexing for one hour and then storing overnight at 4° C. Beads stored overnight were vortexed for an additional 2 hours the next morning. Approximately 30 ml of buffer containing 1% FBS and 0.1% NaN3 in PBS were used to wash the conjugated beads three times. Beads were resuspended in 19.25 ml of the same buffer and stored at 4° C. until use.

[0041]To label ...

example 3

[0042]Detection of Viral Antigens and Host Antibody: Plasma samples from normal individuals and from individuals positive for HCV, HIV, or HBV were obtained from New York Biologicals (Southampton, N.Y.), Scantibodies Laboratory (Santee, Calif.), or Intergen BioDiagnostics (Purchase, N.Y.). Plasma samples were treated with Triton-X 100 detergent to a final concentration of 0.5% to lyse viral membranes and expose core particles prior to testing. E. coli derived recombinant viral antigens, including surface and core antigens, were obtained from BiosPacific (Emeryville, Calif.) or Intergen BioDiagnostics. Antigens were added to normal non-pathologic serum samples for development of a reference standard curve and for use in spike and recovery analysis.

[0043]Flow cytometric analysis was performed on a Coulter EPICS Profile TI, a Coulter XL, or a Partec PAS flow cytometer using linear forward vs. side light scatter to gate the bead population. Fluorescence signal was amplified logarithmica...

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Abstract

Methods and kits for simultaneously measuring both members of a binding pair are described.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 138,536, filed May 3, 2002, which is a continuation of U.S. application Ser. No. 09 / 365,065, filed Jul. 30, 1999 (now U.S. Pat. No. 6,383,740). The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.TECHNICAL FIELD[0002]The invention relates to methods for simultaneously detecting both members of a binding pair in a biological sample.BACKGROUND OF THE INVENTION[0003]Blood products used for transfusion and transfer of blood components must be routinely screened for the presence of infectious agents such as human immunodeficiency virus (HIV), hepatitis viruses, human T-lymphocytotropic virus, and cytomegalovirus. Such agents typically are detected by either identification of viral antigens or by detection of an immune response to the virus (i.e., host-derived anti-viral antibodies) using enzy...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/543G01N21/78G01N33/52G01N33/53G01N33/569G01N33/573G01N33/576G01N33/577
CPCG01N33/54306Y10S435/974Y10S435/975Y10S435/96Y10S435/973Y10S435/971Y10S435/968G01N2474/20
Inventor COLLINS, DANIEL P.
Owner BIOE
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