Lumit™ Immunoassay Labeling Kit Technical Manual
Instructions for Use of Product(s)
VB2500
Literature # TM602
The Lumit™ Immunoassay is based on NanoLuc® Binary Technology (NanoBiT). NanoBiT is a structural complementation system designed for biomolecular interaction studies. The NanoBiT® system is composed of two subunits, Large BiT (LgBiT; 18kDa) and Small BiT (SmBiT; 11 amino acid peptide), that have been optimized for stability and minimal self-association due to weak affinity (190μM). In Lumit™ Immunoassays, antibodies are chemically labeled with SmBiT and LgBiT subunits. Upon binding of the labeled antibodies to an analyte, the subunits come into close proximity, reassemble into a functional luciferase enzyme and generate a luminescent signal in the presence of substrate. Other immunoassay formats, including competition and indirect immunoassays, can also be developed using this technology.
HaloTag is a fusion protein that covalently binds its ligand (HaloTag® Ligand) under physiological conditions and has been used in variety of applications, including antibody labeling. Labeling is a two-step process in which amine reactive HaloTag® Succinimidyl Ester (O4) ligand (509kDa) reacts with primary amines of lysine amino acids on the antibodies. For this reaction, antibodies should be in an amine-free buffer without any protein preservative. Antibodies labeled with HaloTag® Ligand are then incubated with HaloTag®-LgBiT (50kDa) or HaloTag®-SmBiT (31kDa) fusion protein to make a covalent conjugate of antibody-HaloTag®-LgBiT or antibody-HaloTag®-SmBiT.
Summary of Changes
The following changes were made to the 4/22 revision of this document:
- In Section 3.A, the use of two Zeba™ columns of the same size, in series, was clarified.
- Miscellaneous text edits were made to Sections 1 and 2.
- The cover image was updated.
Revised 4/22.