Bioluminescent aptamer-based microassay for detection of melanoma inhibitory activity protein (MIA).

Melanoma inhibitory activity protein (MIA) does obviously offer the potential to reveal clinical manifestations of melanoma. Despite a pressing need for effective diagnosis of this highly fatal disease, there are no clinically approved MIA detection ELISA kits available. A recommended MIA threshold has not yet been defined, mostly by reason of variability in immunoglobulins' affinity and stability, the difference in sample preparation and assay conditions. Here we present a pair of high-affinity DNA aptamers developed as an alternative recognition and binding element for MIA detection. Their stability and reproducible synthesis are expected to ensure this analysis under standard conditions. The devised aptamer-based solid-phase microassay of model standard and control human sera involves luciferase NLuc as a highly sensitive reporter. Bioluminescence dependence on MIA concentration ranges in a linear manner from 2.5 to 250 ng mL-1, providing a MIA detection limit of 1.67 ± 0.57 ng mL-1.

Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features.

Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering "character".

Starch-Coated Magnetic Iron Oxide Nanoparticles for Affinity Purification of Recombinant Proteins.

Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins (cardiac troponin I, survivin, and melanoma inhibitory activity protein) bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100-500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated.

N-extended photoprotein obelin to competitively detect small protein tumor markers.

Two variants of Ca2+-regulated photoprotein obelin, extended from the N-terminus with small tumor markers - melanoma inhibitory activity protein (MIA) and survivin, one of the protein inhibitors of apoptosis, were designed, obtained and studied. Both domains in the obtained hybrid proteins exhibit the properties of the initial molecules: the main features of Ca2+-triggered bioluminescence are close to those of obelin, and the tumor markers' domains are recognized and bound by the corresponding antibodies. The obtained hybrids compete with the corresponding tumor markers for binding with antibodies, immobilized on the surface and their use has been shown to be promising as bioluminescent labels in a one-stage solid-phase competitive immunoassay.

Hybrid Minimal Core Streptavidin-Obelin as a Versatile Reporter for Bioluminescence-based Bioassay.

Ca2+ -regulated photoprotein obelin was genetically fused with a minimum-sized core streptavidin. Hybrid protein (SAV-OL) was produced by bacterial expression and applied as a specific bioluminescent probe in diverse solid-phase assays. The obtained results clearly demonstrate specific activity of each domain indicating its proper folding with favorable space orientation. SAV-OL has been shown to be a much more sensitive label than the chemical conjugate of a full-length streptavidin with obelin.

Bioluminescent detection probe for tick-borne encephalitis virus immunoassay.

To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity. Hybrid protein was applied as a label for solid-phase immunoassay of the antigens, associated with the tick-borne encephalitis virus (native glycoprotein E or extracts of the infected strain of lab ticks). The assay demonstrates high sensitivity (0.056 ng of glycoprotein E; 10(4)-10(5) virus particles or 0.1 pg virions) and simplicity and is competitive with conventional methods for detection of TBEV.

Hydrogen-bond networks between the C-terminus and Arg from the first α-helix stabilize photoprotein molecules.

Previous studies have stated that aequorin loses most of its bioluminescence activity upon modification of the C-terminus, thus limiting the production of photoprotein fusion proteins at its N-terminus. In the present work, we investigate the importance of the C-terminal proline and the hydrogen bonds it forms for photoprotein active complex formation, stability and functional activity. According to the crystal structures of obelin and aequorin, two Ca(2+)-regulated photoproteins, the carboxyl group of the C-terminal Pro forms two hydrogen bonds with the side chain of Arg21 (Arg15 in aequorin case) situated in the first α-helix. Whereas, deletion or substitution of the C-terminal proline could noticeably change the bioluminescence activity, stability or the yield of an active photoprotein complex. Therefore, modifications of the first α-helix Arg has a clear destructive effect on the main photoprotein properties. A C-terminal hydrogen-bond network is proposed to be important for the stability of photoprotein molecules towards external disturbances, when taking part in the formation of locked protein conformations and isolation of coelenterazine-binding cavities.

Simultaneous bioluminescent immunoassay of serum total and IgG-bound prolactins.

Novel dual-analyte single-well bioluminescence immunoassay (BLIA) for total and IgG-bound prolactins was developed on the base of Ca(2+)-regulated photoprotein obelin mutants with altered color and kinetics of bioluminescence signal as reporters. The mutants W92F-H22E and Y138F were chemically conjugated with monoclonal mouse anti-hPRL and anti-hIgG immunoglobulins and thus displayed signals from total prolactin and IgG-bounded prolactin (macroprolactin) correspondingly. Bioluminescence of the reporters was simultaneously triggered by a single injection of Ca(2+) solution and discriminated via bioluminescent signal spectral and time resolution. The developed microplate-based immunoassay allows detection of two prolactin forms in crude serum without additional manipulations (e.g., gel chromatography or PEG-precipitation). Total prolactin bioluminescence immunoassay in standard, control, and clinical sera offers high sensitivity and reproducibility. The BLIA results show good correlation with those obtained by RIA and immunoassay after gel chromatography.