Method for producing reagent for antibody detection and use thereof
Inventors
Futami, Junichiro • Kakimi, Kazuhiro • Maekawa, Ryuji • Shiraki, Masato
Assignees
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Publication Number
US-10822384-B2
Publication Date
2020-11-03
Expiration Date
Abstract
The present invention provides the following: a method for efficiently producing a reagent for detecting an antibody that specifically binds with an insoluble antigen protein present in a liquid sample; a reagent for antibody detection produced by the production method; and a use of the antibody. In a step for solubilizing an antigen protein, it is possible to efficiently solubilize and recover the antigen protein by using a cationizing agent; therefore, when compared to conventional methods, it is possible to efficiently produce a reagent for detecting an antibody that has bound to multiple antigen protein molecules in a carrier.
Core Innovation
A reagent for detection of antibody binding to an epitope of an antigenic protein is formed by cationizing an antigenic protein and immobilizing the cationized, denatured antigenic protein on a carrier. The carrier includes a suspension of magnetic beads, with the cationized, denatured antigenic protein irreversibly immobilized on the magnetic beads.
The cationization forms cationized SH groups on the denatured antigenic protein. The cationized SH groups are formed from reaction with a (3-bromopropyl)trimethylammonium (TAP-Br) cationizing agent or with an alkyl halide cationizing agent.
Cationization is described as improving solubilization of poorly soluble full-length, membrane, and cancer antigenic proteins, while preserving and exposing epitopes for antibody detection. The approach inhibits time-dependent disulfide formation that can cause aggregation, and produces a more storage-stable antibody-detection reagent.
Claims Coverage
Two independent claims are present. They cover bead-based antibody-detection reagents having cationized, denatured antigenic proteins irreversibly immobilized on magnetic beads, where the proteins include cationized SH groups formed by reaction with specific cationizing agents.
Bead-based antibody detection reagent with cationized, denatured antigenic protein immobilized on magnetic beads
A suspension of magnetic beads and a cationized, denatured antigenic protein irreversibly immobilized on the magnetic beads for detection of antibody binding to an epitope of an antigenic protein.
TAP-Br formed cationized SH groups on cationized, denatured antigenic protein
The cationized, denatured antigenic protein has cationized SH groups formed from reaction with a (3-bromopropyl)trimethylammonium (TAP-Br) cationizing agent.
Magnetic-bead antibody detection reagent with cationized, denatured antigenic protein immobilized on magnetic beads
A suspension of magnetic beads and a cationized, denatured antigenic protein immobilized on the magnetic beads for detection of antibody binding to an epitope of an antigenic protein.
Alkyl halide cationizing agent formed cationized SH groups
The cationized, denatured antigenic protein has cationized SH groups formed from reaction with an alkyl halide cationizing agent.
The independent claims define a magnetic-bead reagent for antibody epitope detection using a cationized, denatured antigenic protein immobilized on the beads, with cationized SH groups formed by reaction with TAP-Br or an alkyl halide cationizing agent.
Stated Advantages
Improves solubilization of poorly soluble full-length, membrane, and cancer antigenic proteins.
Preserves and exposes epitopes for antibody detection.
Inhibits time-dependent disulfide formation that causes aggregation.
Provides a more storage-stable antibody-detection reagent.
Documented Applications
Multiplex detection of patient serum antibodies against multiple cancer antigens regardless of HLA type using labeled secondary antibodies and a bead-based readout.
Detection of antibody binding to epitopes of antigenic proteins using an antibody-detection workflow that includes labeled secondary antibodies and a magnetic-bead reagent readout (e.g., Bio-Plex).
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