Urpo Lamminmäki

Homogeneous Detection of Avidin Based on Switchable Lanthanide Luminescence

We have developed switchable lanthanide luminescence-based binary probe technology for homogeneous detection of avidin, which is a tetrameric protein. Two different nonluminescent label moieties--a light-absorbing antenna ligand and a lanthanide ion carrier chelate--were conjugated to separate biotins, which is known as avidins natural ligand. The assay was based on binding of the two differently labeled biotins on separate binding sites on the target protein and consequent self-assembly of a luminescent complex from the two label moieties. Specific luminescence signal was observed only at the presence of the target protein. The characteristics of the switchable lanthanide luminescence assay were compared to the reference assay, based on lanthanide resonance energy transfer. Both assays had a limit of detection in the low-picomolar concentration range; however, the lanthanide chelate complementation-based assay had wider dynamic range and its optimization was more straightforward. The switchable lanthanide luminescence technology could be further applied to generic protein detection, using reagents that are analogous to the proximity ligation assay principle.

Luminescence switching by hybridization-directed mixed lanthanide complex formation.

We have developed a homogeneous assay method in which the lanthanide ion carrier and light absorbing components of a luminescent lanthanide chelate are separated in two distinct molecules that can together form a luminescent mixed chelate complex. The separated label moieties were conjugated to oligonucleotides which were used as probes to detect a complementary target DNA. The background signal of the assay was very low, indicating the signal was highly dependent on the hybridization of the two probes on adjacent positions on the target oligonucleotide.

Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling

Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkels mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.

Study on nonspecificity of an immuoassay using Eu-doped polystyrene nanoparticle labels.

Nanoparticle labels have been shown to improve the sensitivity of a sandwich immunoassay significantly. Further improvement in sensitivity is limited by nonspecific binding of the nanoparticle labels. Here, an experimental characterization of assay performance was carried out using clinically important analytes thyroid stimulating hormone and prostate-specific antigen. Particular attention was paid to characterization of nonspecific binding properties of nanoparticle labels. Therefore, different particle sizes and high affinity monoclonal antibodies (Mab) and their Fab and scFv recombinant antibody fragments were investigated. Combination of Fab fragment as a capture antibody and Mab as a detector antibody on a nanoparticle label resulted in high signal-to-background ratio consistently. Against the expectations no significant difference in nonspecific binding was found using fragmented antibodies compared to Mabs. The results also suggested that nonspecific binding was independent of the particle size. The particle size had a significant effect on the specific signal favouring the use of small particles giving a high specific signal. This study indicated that nonspecific binding is not readily affected by the physical size of the nanoparticle label or antibodies used in the assay.

Engineering of a broad-specificity antibody: Detection of eight fluoroquinolone antibiotics simultaneously

Recombinant sarafloxacin-recognizing antibody was engineered with the use of novel fluoroquinolone (FQ) derivatives. A monoclonal FQ antibody, 6H7, was targeted to random mutagenesis to broaden the specificity of the antibody in development of a generic assay for FQ antibiotics. Engineering involved the synthesis of different small-sized FQ molecules to immobilize and detect the mutant antibodies. Selections with labeled FQs resulted in several mutant antibodies with increased affinity or wider specificity toward different FQs. The best characterized mutant antibody was capable of recognizing seven of eight targeted FQs below maximum residue limits set by the European Union. The results are promising in regard to the development of a multiresidue immunoassay for FQs based on a single antibody.

Resonance Energy Transfer from Lanthanide Chelates to Overlapping and Nonoverlapping Fluorescent Protein Acceptors

Forster resonance energy transfer (FRET) is a powerful tool in studying biomolecular interactions. Intrinsically fluorescent lanthanide chelates are increasingly used as FRET donors due to their long emission lifetime that enables the use of time resolution. Fluorescent proteins, on the other hand, owe their popularity to the intrinsic luminescent properties, facilitating their use as fusion proteins. In this investigation, two energy transfer pairs, terbium(III) chelate with green fluorescent protein (GFP) and europium(III) chelate with yellow fluorescent protein (YFP), were studied by expressing the fluorescent protein acceptor as a fusion protein together with Rab21 GTPase. GTP-conjugated lanthanide chelates were used as donor conjugates. In contrast to conventional FRET observed with the Tb(3+)-GFP pair, a phenomenon called nonoverlapping FRET (nFRET) was observed with the Eu(3+)-YFP pair. In nFRET, the sensitized emission of the acceptor was measured at shorter wavelength than where the emission of the donor was observed. Regardless of the lower signal levels, nFRET resulted in a substantially higher signal-to-background ratio. Conventional FRET from sensitized acceptor yielded a single apparent fluorescence emission lifetime, while with nFRET two lifetimes were observed. The lanthanide chelates together with fluorescent proteins enable a straightforward and sensitive assay technology in nFRET applications.

Two ScFv antibody libraries derived from identical VL–VH framework with different binding site designs display distinct binding profiles

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

Role of lectin microarrays in cancer diagnosis

The majority of cell differentiation associated tumor markers reported to date are either glycoproteins or glycolipids. Despite there being a large number of glycoproteins reported as candidate markers for various cancers, only a handful are approved by the US Food and Drug Administration. Lectins, which bind to the glycan part of the glycoproteins, can be exploited to identify aberrant glycosylation patterns, which in turn would help in enhancing the specificity of cancer diagnosis. Although conventional techniques such as HPLC and MS have been instrumental in performing the glycomic analyses, these techniques lack multiplexity. Lectin microarrays have proved to be useful in studying multiple lectin–glycan interactions in a single experiment and, with the advances made in the field, hold a promise of enabling glycomic profiling of cancers in a fast and efficient manner.

Genetically encoded protease substrate based on lanthanide-binding peptide for time-gated fluorescence detection.

The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb(3+)-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb(3+)-LBP-GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases.

Modulating the binding properties of an anti‐17β‐estradiol antibody by systematic mutation combinations

The anti‐17β‐estradiol antibody 57‐2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57‐2 variants by systematically combining mutations previously identified from phage display–derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light‐chain and a heavy‐chain variant containing a four‐residue random insertion in complementarity determining region CDR‐H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross‐reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time‐resolved fluorescence based immunoassays. The double‐mutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57‐2 crystal structures. These analyses provided information about the steroid‐binding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time‐resolved fluorescence based immunoassay.