A sensitive TSH assay in spot-coated microwells utilizing recombinant antibody fragments.

We have developed a novel TSH immunoassay based on a simplified test protocol suitable for point-of-care testing yet providing 3rd generation TSH assay sensitivity. The antibody density and the functional solid phase capacity were improved up to six-fold by capturing the site-specifically biotinylated recombinant Fab fragment or single chain antibody fragment onto the surface of immobilized streptavidin. An important mechanism for obtaining a low limit of detection (0.003 mIU/l) was the reduction of the coated area to a size ("spot") more closely coinciding with the excitation beam. The reporter technology was based on time-resolved fluorometric detection of inherently fluorescent europium chelates of high quantum yield. The ready-to-use assay concept employed the all-in-one (Aio!) principle--holding all assay components in a dry form in the microtitration well--to provide a simple assay protocol. Direct signal measurement from the surface was done after the washing step without a separate development step. It is concluded that size reduction and site-specific labeling of the antibodies to create a surface with high functional capacity provides a rapid, highly sensitive immunoassay.

Innovations in serum and urine markers in prostate cancer current European research in the P-Mark project.

An overview is given of serum and urine prostate cancer markers that are currently under investigation and subsequently the P-Mark project is introduced. There are many markers showing promise to overcome the limitations of prostate specific antigen (PSA). Eventually, these markers should be able to increase the specificity in diagnosis, differentiate between harmless and aggressive disease and identify progression towards androgen independence at an early stage. In the P-Mark project, several recently developed, promising markers will be evaluated using clinically well-defined biorepositories. Following successful evaluation, these markers will be validated on a sample set derived from two large, European, prostate cancer studies and used for the identification of special risk groups in the general population. In addition, novel markers will be identified in the same biorepositories by different mass spectrometry techniques.

Syntheses and properties of luminescent lanthanide chelate labels and labeled haptenic antigens for homogeneous immunoassays.

Lanthanide chelate labels containing substituted 4-(arylethynyl)pyridine as the chromogenic moiety and iminobis(acetic acid) groups as the chelating part were synthesized. N-Succinimidyl esters of the carboxy derivatives of thyroxine and progesterone were prepared and coupled to the aliphatic amino groups of the synthesized lanthanide chelates. The luminescence properties of the chelates and labeled haptenic antigens were measured in ethanol and in an aqueous buffer containing either albumin or detergents as luminescence-modulating compounds. The energy transfer enhanced ion luminescence of the derivatives containing a para-amino-substituted phenyl ring showed particularly strong dependence on environmental changes, which makes these derivatives well suited for homogeneous time-resolved fluoroimmunoassay based on the use of external luminescence modulators.

Effect of C-H bonds on the quenching of luminescent lanthanide chelates.

The quenching of europium(III) and terbium(III) chelate luminescence by high-energy C-H vibrational manifolds was studied with two types of stable chelates, i.e., a seven-dentate phenylethynylpyridine derivative and a nine-dentate terpyridine derivative. The replacement of C-H bonds by C-D bonds in the chelating parts of the ligands had a clear positive effect on Eu(3+) luminescence but a negligible effect on Tb(2+) luminescence. In aqueous solution, however, the positive effect was undetectable, if the chelating ligand did not create complete shielding of the ion against aqueous quenching. In chelates, where the coordination of water molecules to the inner sphere is prevented, the residual quenching through C-H vibrational quanta can be avoided by replacement of all C-H bonds in the vicinity of the emitting ion by C-D bonds.

A time-resolved study of the mechanism of the energy transfer from a ligand to the lanthanide(III) ion in solutions and solid films.

The photochemical properties of some lanthanide chelates developed for immunohistochemistry have been studied in water and in solid Langmuir-Blodgett films. The fluorescence and triplet-state lifetimes of 4-(phenylethynyl)pyridine (PET), di[(phenylethynyl)pyridine] (D-PET), phenylterpyridine (PTP) and their tetra- or penta-acid derivatives (-TA or -PA) were measured in the presence and absence of Gd(III)-, Tb(III)- and Eu(III)-ions. The mechanism for the total process and the rate constants and quantum yields for the individual reaction steps and for the total process were determined in water solution. Time-resolved absorption and luminescence methods were also used to study the energy transfer between an amphiphilic 4-[4-[(C(10)H(12))(2)NCO]phenylethynyl]-pyridine tetra acid (A-PET-TA) and the Tb(III)- and Eu(III)-ions in solid Langmuir-Blodgett films. Luminescence and transient absorption rate constants were determined.

Lanthanide chelates as a tool in nucleic Acid chemistry.

The potentiality of lanthanide chelates as photoluminescent markers and cleaving agents of nucleic acids is discussed, the main emphasis being on the chelates derived from aromatic nitrogen bases.

Synthesis of europium(III) chelates suitable for labeling of bioactive molecules.

Two different kinds of europium(III) chelates, luminescent and nonluminescent, were prepared. The chelates were coupled to bioanalytical reagents, such as antibodies, after activations of the amino group on the chelates with thiophosgene,2,4,6-trichloro-1,3,5-triazine, or iodoacetic anhydride. The reactivities of the activated luminescent chelates in the labeling of antibodies as well as the effects of both the coupling ratio and the linkage group to the luminescence quantum yield of the antibody-bound chelate were studied in aqueous buffer solution.

Influence of coupling method on the luminescence properties, coupling efficiency, and binding affinity of antibodies labeled with europium (III) chelates.

A series of chelating 4-(phenylethynyl)pyridines having various 1,3,5-triazin-2-ylamino groups at the para position of the phenyl ring was synthesized. Their europium chelates were coupled to antibodies and the properties of antibody conjugates analyzed by fluorometry and in time-resolved fluorometric immunoassay. The substituents in the triazine ring were observed to have various effects on the chelate luminescence, the labeling properties of the chelates, and the immunoreactivity of labeled antibodies. The series of substituted triazinyl derivatives serves as a model of bioreactive groups that can be applied when certain properties are searched for, such as improved chelate solubilities, minimized internal quenching, different effects on the ligand triplet state, and stipulated coupling reactivities.

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits.

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1). The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.