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Biomedical subjects

Pekka Hänninen

Publications and source records attributed to Pekka Hänninen.

6 recordsLinked to original sources
PubMed2006

Theoretical assessments of errors in rapid immunoassays-how critical is the exact timing and reagent concentrations?

The reliability of rapid immunoassay is a concern due to an incomplete incubation to a non-equilibrium state and is susceptible to different error factors causing variance. The most critical point in the process should be found in order to improve the accuracy, and reproducibility of immunoassays, and enhance the system robustness. In this paper, the behavior of rapid assays is predicted by simulations using mechanistic assay model, based on antibody-analyte binding reaction kinetics. This antibody-analyte binding reaction kinetics model was constructed for a generic three-component (immunometric) assay and the parameters were chosen to be those of a known surface binding assay. The effects of the exact incubation timing and the initial reagent concentrations were studied focusing on the early phase of incubation, the non-equilibrium state. The magnitudes of errors in the input parameters were estimated using knowledge from practical immunoassays. According to simulations, inaccurate incubation timing adds error in the results at very short incubation times, especially in low analyte concentrations. The inaccurate reagent concentrations increase variance at short incubation times, as well. The error decreases rapidly after the first few minutes of incubation.

Antigen-Antibody Complex↗
PubMed2006

Two-photon excited fluorescence energy transfer: a study based on oligonucleotide rulers.

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor-acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.

DNA, Single-Stranded↗
PubMed2006

Calibration of bioaffinity assays using kinetic data.

Bioaffinity assays are usually calibrated by using a set of standard measurements fitted to a simple empirical model. In this paper, a new calibration approach based on mechanistic model of reaction kinetics is presented. When the calibration assay is known in terms of reaction mechanism, incubation time, initial concentration, and rate constants, one can back-calculate concentrations of unknown samples measured in a nonequilibrium time point. This paper describes a calculation method of unknown sample concentrations based on kinetically measured single calibration assay point. The theoretical results are verified by two common in-vitro diagnostic assays.

Biological Assay↗
PubMed2004

New separation-free assay technique for SNPs using two-photon excitation fluorometry.

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia trade mark TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot trade mark assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.

DNA Primers↗
PubMed2003

Reaction kinetics of a two-photon excitation microparticle based immunoassay--from modelling to practice.

Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations.

Antibodies↗