Ca2+-regulated photoproteins: effective immunoassay reporters

Abstract

Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are "self-contained", triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element--bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects.

Keywords: Ca2+-regulated photoprotein; PCR-ELISA; bioluminescence; immunoassay; multiplex assay; re-engineered photoproteins.

Figure 1.

Bioluminescence of Ca²⁺-regulated photoprotein. (A) Scheme for the reaction: photoprotein is a complex of single-chain polypeptide, containing Ca²⁺-binding sites (I, II, III) and pre-oxidized coelenterazine (CE-O₂). The binding of Ca²⁺ results in coelenterazine decarboxylation yielding a stable complex of polypeptide, three Ca²⁺ and coelenteramide (CM), carbon dioxide and a quantum of light. (B) Kinetics of aequorin (-○-) and obelin (-Δ-) bioluminescence signals. (C) Bioluminescence spectra of aequorin (solid line) and obelin (dashed line). (D) Obelin amount versus bioluminescence.

Figure 2.

Generalized scheme for dual bioluminescent immunoassay using aequorin in tandem with firefly luciferase, according to [31]. The surface was activated by antibodies of two types (1) against two antigens (2). Secondary antibodies (3) were tagged with digoxigenin and biotin. The sandwiches, formed on the surface were detected using conjugate aequorin-antidigoxigenin Fab fragment and biotinylated luciferase-streptavidin complex (4). Ca²⁺ injection triggered flash-type aequorin bioluminescence and then the mixture of luciferin, ATP and Mg²⁺ was placed into the wells to initiate luciferase bioluminescence.

Figure 3.

Scheme for PCR-ELISA method developed by Vlieger et al. [30]. 1. amplicon labeled strands immobilization on the surface; 2. hybridization with tagged probe; 3. immuno-enzymatic complex formation; 4. amplicon detection through enzyme reaction.

Figure 4.

Color photoprotein obelin mutants as reporters in dual immunoassay according to [56]. (A) Bioluminescence spectrum of “violet” (W92F, H22E) and “greenish” (Y138F) obelin mutants (thick lines); band-pass optical filters I and II transmission (thin lines). (B) Simultaneous dual-color bioluminescence immunoassay of LH and FSH: the surface was activated with anti-α-FSH (the same for both hormones) IgG. Sandwich immunocomplex formed at incubation of hormones and conjugates of obelin mutants with corresponding hormones anti-β-subunits (specific for every hormone) IgGs mixture. Reporters’ bioluminescence was simultaneously triggered by single injection of Ca²⁺ solution, divided using band-pass optical filters and measured with a two-channel photometer.