EP1999457A1

EP1999457A1 - Method for determining molecules or molecule parts in biological samples

Info

Publication number: EP1999457A1
Application number: EP07723151A
Authority: EP
Inventors: Lars Philipsen; Ansgar J. Pommer; Raik BÖCKELMANN; Bernd Bonnekoh; Harald Gollnick
Original assignee: MPB Meltec Patent und Beteiligungs GmbH
Current assignee: MPB Meltec Patent und Beteiligungs GmbH
Priority date: 2006-03-09
Filing date: 2007-03-09
Publication date: 2008-12-10
Also published as: DE102006010907A1; US20100120060A1; WO2007101706A1

Abstract

The invention relates to a method for determining molecules, molecule groups, and/or molecule parts in biological samples. According to said method, the sample is spiked at least once with at least one light emitting marker, and the light emitted by the marker is measured. The emission of light that is inherent in the probe is reduced or canceled by means of a bleaching process before the light emitted by the marker is measured.

Description

Method for the determination of molecules or molecular parts in biological samples

The invention relates to a method for the determination of molecules, groups of molecules and / or parts of molecules in biological samples, d. H. Samples of human, animal, plant or microbial origin.

It has long been common to use fiororophore-labeled antibodies for the histo- and cytochemical examination of cells. The fluorophore-labeled antibodies react specifically with antigens that are expressed on the surface of certain cells. Cells carrying such antigens are thus labeled with the fluorophore via the antibody. When the fluorophores are exposed to adequate excitation light, the fluorophores are excited to emit photons, which can be measured by a photodetector.

Following this basic binding principle, fluorescent labels coupled not only to antibodies but also to other detector molecules such as lectins, nucleic acids, inorganic or organic molecules, probes and other ligands are used to analyze biological samples. Accordingly, the following statements apply mutatis mutandis not only for antibodies, but also for analogous applications with other detector molecules such as lectins, nucleic acids, inorganic or organic molecules or probes and other ligands.

However, biological cell and tissue structures already genuinely contain a variety of fluorophores whose fluorescence may adversely affect the measurement results. This is particularly the case when the spectrum of light emitted by the genuinely existing fluorophores overlays the light emission spectrum of the fluorophores of the labeling substances, for example the fluorophore-labeled antibodies.

The genuine fluorescence of tissue or cell samples, for example, proves to be particularly disadvantageous in the study of skin tissue samples. It is known that collagen is one of the endogenous skin fluorophores (Sandby-Moller J, Thieden E, Philipsen PA, Heydenreich J, Wulf HC: Skin auto-fluorescence as a biological UVR dosimeter, Photodermal Photoimmunol Photomed 2004, 20: 33-40). , In addition, skin ingredients carry such as reduced nicotinamide adenine dinucleotide phosphate, flavins, elastin and melanin contribute to genuine skin fluorescence (Konig K, Rieman I: High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution, J biomed Opt 2003, 8, 432-439) Also, a phenomenon is known that - unlike normal skin - psoriasis plaques show red autofluorescence due to protoporphyrin IX accumulation in the keratinized layer (Bissonnette R, Zeng H, McLean DI, Schreiber WE, Roscoe DL, Lui H: Psoriatic plaque exhibit red autofluorescence that is due to protoporphyrin IX J Invest Dermatol 1998, 11 1: 586-591).

Image analysis methods are known from the prior art with which the disturbing autofluorescence can be largely or completely removed from multispectral images. With strong autofluorescence of the samples or unfavorable superimposition of the autofluorescence with the fluorescence of the added markers, however, this can lead to significant signal information being lost.

Because of the genuine fluorescence of skin tissue samples, it was thus not possible or insufficiently possible to examine skin tissue samples in this regard without interference using fluorophores.

The object of the invention is to eliminate the disadvantages of the prior art. In particular, a method for the determination of molecules, groups of molecules and / or parts of molecules in biological samples is to be specified, which is particularly suitable for the investigation of skin tissue of human or animal origin and blood preparations.

This object is solved by the features of claim 1. Advantageous embodiments of the invention will become apparent from the features of claims 2 to 18. In accordance with the invention, a method for the determination of molecules, groups of molecules and / or parts of molecules is provided in biological samples, comprising at least one time displacement of the sample with at least one light emitting marker and measuring the light emission of the marker, wherein prior to measuring the light emission of the marker, the light emission inherent in the sample is reduced or eliminated by bleaching becomes. The bleaching of the sample should be done before applying the light-emitting label.

The invention is based on the finding that by means of a bleaching step, the light emission inherent in the sample can be reduced or eliminated. The bleaching step is carried out before the first application of a light-emitting marker. Due to the reduction or elimination of the inherent light emission, the light spectrum emitted by the marker after application thereof is not affected by light emissions originating from sources other than the markers. It has surprisingly been found in the experiments carried out according to the invention that the minimization or elimination of autofluorescence and nonspecific fluorescence in cell and tissue samples including tissue sections can advantageously be achieved by repeated soft bleaching, and in particular useful in the analysis of skin tissue samples; see also the following examples. Thus, in the method of minimizing autofluorescence, the biological sample is subjected to repeated soft bleaching, i. subjected to at least two bleaching steps before the sample is first mixed with a light-emitting marker. Furthermore, it has been found in the experiments carried out according to the invention that the bleaching is preferably carried out with light whose wavelength range corresponds to the excitation wavelength of the marker (s) used, such as the wavelength of 488 nm in the case of FITC; see also the following examples. As shown in the examples, by the method of the present invention, autofluorescence and specific fluorescence in cell and tissue samples can be achieved within a relatively short time by means of three bleaching steps with light of two wavelengths, i. within about two to three hours so that the sample basically has no inherent fluorescence emitting at the excitation wavelength of the light-emitting marker intended for use, and on the other hand the sample is intact so far that it can be analyzed with light-emitting markers ; see also FIGS. 1 and 2.

The term "inherent light emission" is understood to mean the emission of light by constituents of the sample without the addition of light-emitting substances to the sample have been. The sample's inherent light emission includes autofluorescence as well as nonspecific fluorescence of the sample, but also any other perturbing light emissions such as fluorescence originating from the coating material of a slide or from substances used for sample fixation.

The term "inherent fluorescence" used hereinafter is understood to mean the emission of light by constituents of the sample without fluorescence-emitting substances having been added to the sample. The fluorescence inherent in the sample includes autofluorescence as well as nonspecific fluorescence of the sample, but also any other interfering light emissions such as fluorescence originating from the coating material of a slide or from substances used for sample fixation.

By the term "bleaching" (also referred to as "bleaching") is meant the destruction of the fluorophores contained in the sample. In particular, destroying the fluorophores involves destroying the fluorophore groups of molecules of the sample which cause the inherent light emission or inherent fluorescence.

A biological sample is understood to mean samples of human, animal, plant or microbial origin. The term "microbial" derived from the term

"Microbes" includes the entire spectrum of microorganisms including bacteria, viruses, fungi, mono- and multicellulars, algae, blue-green algae as well as

Prions (see Pschyrembel, KHn. Wörterbuch, 259th edition, de Gruyter Berlin - New York

2002, S. 1063 + 1064): "Microbes: see (see) microorganisms - microorganisms: also microbes, microorganisms, bacteria, viruses, protozoa, fungi (small mushrooms, so-called Funguli, s.

Fungi) ".). The sample can be, for example, tissue or liquids, such as

Blood, lymph or secretions act as well as preparations made from them.

For example, blood preparations, such as cell preparations of mononuclear cells,

Lymphocytes, monocytes, granulocytes, platelets and erythrocytes from the blood to be examined by the method according to the invention. The invention thus enables the examination of samples which have a strong autofluorescence. The method is therefore particularly suitable for the fluorescence analysis of autofluorescent tissue samples or autofluorescent cell cultures of human or animal origin. Fluorescence analysis of autofluorescent cell cultures involves fluorescence analysis of adherent cells as well as suspension cells. The method according to the invention is particularly suitable for fluorescence analysis of human or animal skin tissue.

The method according to the invention will be explained in more detail below with reference to the fluorescence analysis. However, this is not intended to be a limitation of the method according to the invention to the examples of fluorescence analysis given here.

The inherent fluorescence of a sample is eliminated or reduced according to the invention by bleaching the sample.

The bleaching is carried out so that the binding sites for the fluorophore-labeled markers which are applied to the biological sample after bleaching according to the invention are not destroyed.

The bleaching of the sample should be done before applying the fluorophore-labeled label.

In a preferred embodiment, the bleaching of the samples is performed by irradiating the sample with light. The wavelength of the light is chosen so as to destroy the functional groups of molecules of the sample which cause their inherent fluorescence.

In the case of fluorescence analysis, a light-emitting marker is to be understood as meaning a fluorophore-labeled marker. Fluorophore-labeled markers include, for example, fluorophore-labeled antibodies, lectins, nucleic acids, probes and other binding molecules. The fluorophore-labeled label can be any type of fluorophore, but preferably the fluorophore is selected from the group consisting of fluorescent nanoparticles (quantum dots) and fluorochromes. In a preferred embodiment, the bleaching is carried out until the signal intensity of the inherent fluorescence has been reduced to a predetermined level. For this example, the sample can be exposed to the light of a specific wavelength for a given period of time.

The bleaching according to the invention can be repeated several times. This is particularly advantageous if it is not known what time of exposure of the light is required to reduce the signal intensity of the inherent fluorescence to a predetermined level. In this case, it is preferred that several bleaching cycles be performed, each bleaching cycle comprising bleaching the sample for a predetermined period of time and measuring the signal strength of the residual inherent fluorescence.

Conveniently, upon completion of bleaching, measuring the signal strength of the residual inherent fluorescence is performed.

The light used to bleach the sample is preferably generated by the excitation light source of an inverted or upright fluorescence microscope. Accordingly, the light source may in particular be a mercury-vapor lamp, halogen lamp, xenon lamp, a laser, a light-emitting diode, in each case in single or multiple or combinations thereof.

The wavelength of the light used for bleaching, in one embodiment of the invention, is chosen to be similar to the excitation wavelength of a fluorophore to be used as a marker. Alternatively, light having a wavelength range including the excitation wavelength of the fluorophore may also be selected. By bleaching the biological sample with light corresponding to or including the wavelength of the fluorophore, it is achieved that the inherent fluorescence of the sample excited by that wavelength is destroyed. If, after bleaching according to the invention, the fluorophore-labeled marker is applied to the sample which is excited at this wavelength, the measurement of the fluorescence is thus no longer disturbed by the inherent fluorescence of the sample. When two or more markers with different excitation wavelengths are used, the bleaching according to the invention preferably comprises bleaching with light whose wavelength range comprises all excitation wavelengths of the markers used. Of course, the bleaching can also take place in n substeps by the action of light with the excitation wavelength of a first marker, then by the action of light with the excitation wavelength of an ith marker and finally by the action of light with the excitation wave of the nth marker.

The duration of bleaching depends on the time required to lower the inherent fluorescence of the biological sample below a predetermined level. Conveniently, several bleaching cycles are provided, wherein the duration of the individual bleaching step is preferably between 1 and 60 minutes, more preferably between 5 and 45 minutes, particularly preferably between 10 and 30 minutes.

The number of bleaching cycles should be chosen such that the fluorescence inherent in the biological sample which emits at a given excitation wavelength is preferably at most 10%, more preferably at most 3%, most preferably at most 0.3%, based in each case on fluorescence intensity. This also applies if only one bleaching step is carried out.

To determine the inherent fluorescence of a sample, prior to carrying out the method according to the invention, fluorescence measurements should first be carried out in order to determine an indication of the number of bleaching cycles required and the duration of a bleaching step.

The method is suitable for the preparation of samples to be analyzed by luminometry, microscopy, fluorescence microscopy, confocal microscopy, multi-epitope-ligand mapping (MELK), flow cytometry or fluorescence activated cell sorting (FACS). The multi-epitope ligand cartography (MELK) is described inter alia in DE 197 09 348 A1 and EP 0 810 428 A1. The term "fluorophore-labeled marker" used here is a synonym for the term "fluorescence-labeled marker".

The method according to the invention can be used in the practical or theoretical disciplines of medicine for diagnostic purposes or for the identification of new pathogenetic or therapeutic target structures or for therapy / medication monitoring.

The invention is explained in more detail below by means of examples with reference to the drawings. Show

Fig. Ia is a fluorescence micrograph of skin tissue (63-fold

Enlargement);

FIG. 1b is a graph of the variation of autofluorescence. FIG

Areas of the sample shown in Fig. Ia as a function of the number of bleaching cycles; and

Fig. 2 fluorescence microscopic images of lymphocytes. A) cells incubated with PBS before the bleaching step according to the invention; B) the same cells after the bleaching step according to the invention. C) Fluorescence signal of the subsequent reaction with fluorophore-labeled antibody.

The fluorescence micrograph of a skin tissue sample shown in Fig. Ia was obtained prior to carrying out the method according to the invention. It is a strong autofluorescence to detect. For two regions of the image (circled in FIG. 1 a), the fluorescence intensity in FIG. 1 b is shown as a function of the number of bleaching cycles. In FIG. 1b, the dashed curve corresponds to the region of a hair follicle (hair follicle) of the epidermis bounded by dashed lines in FIG. 1 a, while the line dotted in FIG. 1 a corresponds to the region of the dermis bounded by a dotted line in FIG. = Dermis) corresponds. The dashed-dotted line in Fig. Ia bounds the epidermis of the skin tissue sample. In Fig. Ib, the ordinate shows the fluorescence intensity, while the abscissa represents the number of bleaching cycles, and the letters b and f used therein for image pick-up after bleaching are before bleaching. To recognize is that the signal strength of autofluorescence has been significantly reduced after five bleach cycles.

sample preparation

The sample was prepared as follows: Biopsies of the skin tissue of patients suffering from psoriasis as well as healthy skin tissue were taken under local anesthesia. The biopsies had a diameter of 6 mm. After removal, the biopsies were snap frozen. Of the frozen biopsies, 5-μm thick sections were made by means of a cryotome. The sections were then air-dried at room temperature for 10 minutes, then immersed in acetone at room temperature for 10 seconds, dried again and finally stored at -20 ° C. Immediately prior to performing the method of the invention, the sections were dipped for 10 minutes for rehydration in PBS, pH 7.4.

Bleaching and fluorescence measurements

To perform the bleaching cycles, the section to be examined (ie, skin), hereinafter referred to as a sample, was applied to a slide and applied to the stage of an inverse wide field fluorescence microscope (Leica DM IRE2 with a xenon fluorescent excitation lamp) equipped with fluorescence filters for fluorescein isothiocyanate (FITC) and phycoerythrin (PE). For fluorescence measurements, a CCD camera (Apogee KX4) was used.

The sample should be examined using fluorophore-labeled antibodies. As fluorophores FITC and PE were chosen. The sample was examined as follows:

A. Eliminating the inherent fluorescence of the sample emitted at the excitation wavelengths of FITC and PE. Three bleach cycles were performed.

1.) Measuring the inherent fluorescence, in each case a picture was taken with fluorescence replacements for FITC and PE. The exposure time was 5 seconds (see Fig. 2A for FITC); 2.) a) bleaching the sample with light of a wavelength of 488 nm excitation wavelength from FITC) for 15 min; b) bleaching the sample with light of a wavelength of 546 nm for 15 minutes;

3.) measuring the inherent fluorescence as in 1.); 4.) wait 10 minutes;

5.) measuring the inherent fluorescence as in 1.);

6.) a) bleaching the sample with light of a wavelength of 488 nm

(Excitation wavelength of FITC) for 15 min; b) bleaching the sample with light

Wavelength of 546 nm for 15 min; 7.) measuring the inherent fluorescence as in 1.); 8.) 10 min wait; 9.) measuring the inherent fluorescence; 10.) a) bleaching the sample with light of a wavelength of 488 nm

(Excitation wavelength of FITC) for 15 min; b) bleaching the sample with light of a wavelength of 546 nm for 15 minutes;

11.) Measure the inherent fluorescence as in 1.).

The result is shown in Fig. 2B for FITC. It can be seen that the sample has no inherent fluorescence emitted at an excitation wavelength of 488 nm.

B. Determining molecules or moieties of the sample using FITC-labeled antibodies and PE-labeled antibodies.

The sample obtained according to section A. was admixed in a known manner with a solution containing FITC-labeled antibodies, incubated and the solution containing FITC-labeled antibodies not bound to the sample was removed. Subsequently, the FITC-labeled antibodies bound to binding sites (antigens) on the sample were excited at 488 nm by the action of light and the emitted fluorescence radiation was measured (see FIG. 2C for FITC-labeled antiCDδ antibodies). The obtained photograph shows for the first time the light emitted by added FITC without being influenced by the autofluorescence of the sample). After measurement, the antibody-bound FITC molecules were bleached. After bleaching, the sample may be spiked with other FITC-labeled antibodies in several cycles as described above to determine additional antigens on the sample.

After the cycle of FITC-labeled antibodies and bleaching of the FITC molecules bound to the sample, the sample was spiked with a solution containing PE-labeled antibodies, incubated, and the solution, the PE-labeled antibodies not bound to the sample contained, removed. Subsequently, the PE-labeled antibodies bound to binding sites (antigens) on the sample were excited at 546 nm by the action of light and the emitted fluorescence radiation was measured. Again, several cycles with different PE-labeled antibodies can be performed.

Claims

claims

1. A method for the determination of molecules, groups of molecules and / or parts of molecules in biological samples, comprising at least one time displacement of the sample with at least one light-emitting marker and measuring the light emission of the marker, characterized in that before measuring the light emission of the marker of the Sample inherent light emission is reduced or eliminated by bleaching, wherein the bleaching is carried out prior to the displacement of the sample with the at least one light-emitting marker.

2. The method according to claim 1, characterized in that the sample inherent light emission comprises autofluorescence, non-specific fluorescence or both.

3. The method according to any one of the preceding claims, characterized in that the light-emitting marker emits fluorescent light.

4. The method according to any one of the preceding claims, characterized in that the bleaching of the samples is carried out by the action of light on the sample.

5. The method according to claim 4, characterized in that the wavelength of the light is selected so that the functional groups of the sample, which cause their inherent light emission, are destroyed.

A method according to any one of the preceding claims, characterized in that the sample is selected from the group comprising human, animal, plant and microbial tissue.

7. The method according to any one of claims 1 to 6, characterized in that the sample is a blood sample.

8. The method according to any one of claims 1 to 6, characterized in that the human or animal tissue is skin tissue.

9. The method according to any one of claims 1 to 6, characterized in that the sample is cell or tissue culture material.

10. The method according to any one of claims 3 to 9, characterized in that the light-emitting marker has a light-emitting unit selected from the group comprising fluorescence nanoparticles (quantum dots) and fluorochromes.

11. The method according to any one of the preceding claims, characterized in that the light-emitting unit with an antibody, a lectin, a probe and / or another ligand is conjugated.

12. The method according to any one of the preceding claims, characterized in that the bleaching is repeated several times.

13. The method according to any one of the preceding claims, characterized in that the sample inherent light emission has been reduced to a predetermined signal strength.

14. The method according to any one of the preceding claims, characterized in that the bleaching is carried out until the sample inherent light emission has been reduced to a predetermined signal strength.

15. The method according to any one of claims 4 to 14, characterized in that the light used for bleaching the sample by means of one or more mercury vapor lamps, halogen lamps, xenon lamps, lasers, light-emitting diodes or combinations thereof is generated.

16. The method according to any one of the preceding claims, characterized in that the biological sample is a blood preparation of human or animal origin.

17. The method according to any one of the preceding claims, characterized in that the biological sample is a skin sample of human origin.

18. The method according to any one of the preceding claims, characterized in that the biological sample is cell or tissue culture material of human origin.