Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-05
2002-03-26
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S172000, C436S501000, C436S513000, C436S518000, C436S524000, C436S536000, C422S068100, C422S073000, C422S082050, C250S298000, C250S39600R, C250S397000, C250S458100, C250S459100, C250S461100, C250S461200, C356S004010, C356S004050, C356S073000, C356S213000, C356S317000, C356S318000, C356S336000, C356S337000, C356S341000, C356S342000, C356S441000, C356S442000, C356S945000
Reexamination Certificate
active
06361956
ABSTRACT:
BACKGROUND OF THE INVENTION
Immunoassay is an established biospecific assay method, widely used in routine diagnostics and research laboratories. Another group of biospecific assays is DNA and RNA hybridisation assays. Two biospecific probes, a primary probe and a secondary probe (i.e. an antibody, DNA or RNA probe) are usually used in biospecific assays. They are both connected to the specific determinants of the analyte molecules and form a complex of three molecules (sandwich structure). Normally, one of these two reagents is labelled. Nowadays, commonly used labels are radioisotopes, enzymes, luminescent and fluorescent labels. The most common sample material, which is assayed, is blood serum.
The method according to this invention refers to those fluorometric biospecific assays where one biospecific reagent is attached to microparticles serving as a solid matrix. The solid matrix with the attached components is later referred to as the solid phase. The second biospecific reagent is labelled with a fluorescent label and is later referred simply as the label. In regard to their functional principles, assays can be divided in two main groups: 1) assays which have excess reagent bound to a solid phase, which in immunology are called immunometric assays, and 2) assays which have a limited amount of reagent bound to a solid phase. These are referred to as competitive assays.
The first type is applicable for the analyte molecule Ag which can provide at least two specific determinants. In this type of assay, the initial concentration of the biospecific reagent Ab bound to the solid phase exceeds the amount of the analyte Ag. The other reagent in the reaction is the labelled reagent Ab*. Complexes AbAgAb* are bound to the solid phase, and the signal response of the assay is linear. DNA- and RNA-assays are also performed according to the same principle.
The second type is applicable to small analyte molecules Ag. In this assay, the biospecific reagent Ab bound to the solid phase A, is in a limited concentration in relation to the analyte. The other reagent in the reaction is the labelled analyte molecule Ag*, i.e. the labelled analyte molecule is used as the reagent. The components Ag and Ag* bind to the solid phase reagent Ab in proportion to their relative concentrations. This reaction is known as a competitive binding reaction, and the signal response of this type of assay is non-linear.
In addition to these two main groups, the method of this invention is applicable to study the reaction kinetics between different biomolecules Ab and Ab*, i.e. to monitor the formation of reaction products as a function of time. In these studies, we simply monitor how the labelled molecule Ab* binds to the solid phase molecule Ab.
The previously used symbols Ab, Ag and Ab* used here refer to biospecific molecules in general, and are not restricted to immunological antibodies and antigens.
The problem with conventional assays and research methods lies in their complexity. For example, the determination of hormones from blood with a fluorometric immunometric assay method often requires several steps: separation of cells from the serum, dispension and dilution of the serum sample, incubation with the solid phase, separation of the free fraction by washing, incubation with the label, separation of the free label by washing, addition of a measurement solution and measurement of the signal.
Separation of cells from serum is necessary because in conventional fluorometric assays the strong light absorption of haemoglobin affects measurements. In the first type of assays referred to above, the reagents must be added in two separate phases, including separation of the free fraction from the bound fraction between these steps, otherwise the assay response, (i.e. the concentration of the product AbAgAb*) is decreased if the analyte concentration exceeds the binding capacity of the solid phase. This harmful phenomenon appears only when all assay components are added simultaneously, and is called the “hook effect” in the literature. The free label must also be separated; otherwise the signal from the bound label can not be measured because of the high background signal contributed by the free label.
There is a constant need for simpler and more cost-effective analyses within routine diagnostics. The new biospecific assay method of the present invention using fluorescent labels involves only one step, and does not require separation of the label, and is particularly suitable for assays in whole blood or in other biological suspensions. In addition, this invention makes it possible to perform real-time measurements of bioaffinity reaction kinetics.
Two-photon Excitation
Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be absorbed into the same chromophore. In this case, the absorbed energy is the sum of the energies of the two photons. According to the concept of probability, the absorption of a single photon in a dye, is an independent event, and the absorption of several photons is a series of single, independent events. The probability of absorption of a single photon can be described as a linear function as long as the energy states that are to be excited are not saturated. The absorption of two photons is a non-linear process. In two-photon excitation, dye molecules are excited only when both photons are absorbed simultaneously. The probability of absorption of two photons is equal to the product of probability distributions of absorption of the single photons. The emission of two photons is a thus a quadratic process.
The properties of the optical system used for fluorescence excitation can be described with the response of the system to a point-like light source. A point-like light source forms, due to diffraction, an intensity distribution in the focal plane characteristic to the optical system (point spread function). When normalised, this point spread function is the probability distribution of how the photons from the light source reach the focal area. In two-photon excitation, the probability distribution of excitation equals the normalised product of intensity distributions of the two photons. The probability distribution thus derived, is 3-dimensional, especially in the vertical direction, and is clearly more restricted than for a single photon. Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted 3-dimensional vicinity of the focal point is excited.
When a dye is two-photon excited, the scattering of light in the vicinity of the focal point and from the optical components, is reduced remarkably compared to normal excitation. Furthermore, two-photon excitation decreases the background fluorescence outside the focal point, in the surroundings of the sample and in the optics. Since the exciting light beam must be focused onto as a small point as possible, two-photon excitation is most suitable for the observation of small sample volumes and structures, which is also the case in the method according to this invention.
The advantage of two-photon excitation is also based on the fact that visible or near-infrared (NIR) light can, for example, be used for excitation in the ultraviolet or blue region. Similarly, excitation in the visible region can be achieved by NIR light. Because the wavelength of the light source is considerably longer than the emission wavelength of the dye, the scattering at a wavelength of the light source and the possible autofluorescence can be effectively attenuated by using low-pass filters (attenuation of at least 10 orders of magnitude) to prevent them from reaching the detector.
Since in two-photon excitation the density of photons per unit volume and per unit time must be very high in order to make two photons to be absorbed into the same chromophore, it is useful to use lasers which generate short pulses with high repetition rate. A practical laser for two-photon excitation is for example a passively Q-switch
Hänninen Pekka
Soini Erkki
Soini Juhani
Le Long V.
Lydon James C.
Padmanabhan Kartic
Soini Erkki
LandOfFree
Biospecific, two photon excitation, fluorescence detection... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Biospecific, two photon excitation, fluorescence detection..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Biospecific, two photon excitation, fluorescence detection... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2834592