ULTRASENSITIVE DETECTION OF VIRUS PARTICLES AND VIRUS-LIKE PARTICLES

Abstract

The present invention relates to a method for quantitatively and/or qualitatively determining virus particles containing at least one binding site for a capture molecule and at least one binding site for a probe, to a kit for carrying out said method, and to various uses.

Claims

1.-15. (canceled)

16. A method for quantitatively and/or qualitatively determining virus particles containing at least one binding site for capture molecules and at least one binding site for probes, wherein the method comprises: (a) immobilizing capture molecules on a substrate, (b) contacting the virus particles with the capture molecules, (c) immobilizing the virus particles on a substrate by binding to capture molecules, (d) contacting the virus particles with the probes and (e) binding the probes to the virus particles, and wherein the probes are capable of emitting a specific signal and (b) and (d) can be carried out simultaneously or (d) can be carried out before (b).

17. The method of claim 16, wherein a spatially resolved determination of a probe signal is carried out.

18. The method of claim 16, wherein the virus particles are selected from virus, virion, bacteriophage, parts or fragments of the former.

19. The method of claim 16, wherein the substrate is composed of a material selected from plastic, silicon, silicon dioxide.

20. The method of claim 16, wherein the substrate is composed of glass.

21. The method of claim 16, wherein the substrate has a hydrophilic surface prior to (a).

22. The method of claim 21, wherein a hydrophilic layer is applied to the substrate prior to (a).

23. The method of claim 22, wherein the hydrophilic layer is selected from PEG, poly-lysine, dextran, derivatives thereof.

24. The method of claim 22, wherein prior to application of the hydrophilic layer the substrate is hydroxylated and functionalized with reactive groups (amino groups).

25. The method of claim 24, wherein functionalization with amino groups is achieved by contacting the substrate with APTES (3-aminopropyltriethoxysilane).

26. The method of claim 25, wherein the substrate is contacted with APTES in the gas phase.

27. The method of claim 24, wherein functionalization with amino groups is achieved by contacting the substrate with ethanolamine.

28. The method of claim 16, wherein the capture molecules are covalently bonded to the substrate or a coating thereof.

29. The method of claim 16, wherein the binding sites of the virus particles are epitopes and the capture molecules and probes are antibodies or aptamers or combinations thereof.

30. The method of claim 16, wherein the probes are labeled with fluorescent dyes.

31. The method of claim 16, wherein detection is carried out by spatial-resolution fluorescence microscopy.

32. A kit for carrying out the method of claim 16, wherein the kit comprises one or more of a substrate, optionally with hydrophilic surface, capture molecules, probes, substrate with capture molecules, solutions, buffers.

Description

EXAMPLES

Example 1

[0114] The experiment was carried out in commercially available 3D NHS microtiter plates (PolyAn GmbH) containing 384 reaction chambers (RCs). The RCs of the microtiter plates were coated with antibodies: clone anti-gp8-E1 (prod. #ABIN793840, lot #77410, antibodies-online.com) as capture molecule (15 μl; μg/ml in 100 mM MES, pH=4.7; incubation overnight). Thereafter, the RC was subjected to a wash program consisting of washing and aspiration three times, in each case with phosphate-buffered saline (PBS) containing 0.1% Tween 20 and PBS. In the next step, the RCs were coated with 50 μl of Smartblock (Candor Bioscience GmbH) at room temperature (RT) for 1 h and were subjected to the above-described wash program again after this time had passed. Thereafter, 15 μl of the sample, in sequential dilution in human EDTA blood plasma in quadruplicate, were in each case loaded in RCs and incubated at RT. After incubation overnight, the RCs were washed using the wash program, the RCs were sucked dry and detection antibodies were loaded. The detection antibodies were in each case labeled with one type of fluorescent dye. The antibody RL-ph1 (prod. #LS-C146750, LifeSpan BioScience) was labeled with the fluorescent dye CF488 and the antibody LRL-ph2 (prod. #LS-C146751, lot #76955, LifeSpan BioScience) was labeled with the fluorescent dye CF633. The detection antibodies were diluted together in PBS to give a final concentration of 1.25 ng/ml for each antibody. 15 μl of antibody solution were loaded per RC and incubated at room temperature for 1 h. After this time had passed, the plate was washed 5 times with PBS containing 0.1% Tween 20 and 5 times with PBS. After complete suction, the RCs were filled with 20 μl of water and the plate was sealed with a film.

[0115] The measurement was carried out in a TIRF microscope (Leica) with a 100× oil immersion objective. For this purpose, the glass base of the microtiter plate was generously coated with immersion oil and the plate was introduced into an automated stage of the microscope. Thereafter, what was consecutively recorded per RC at 5×5 positions was in each case two images in two fluorescence channels (excitation/emission=635/705 nm and 488/525 nm). For both channels, what was selected was the maximum laser output (100%), an exposure time of 500 ms and a gain value of 1300. The image data were then evaluated.

[0116] For the analysis, a quadratic ROI of 800 was first used. This means that the outermost 100 pixels on each side of each image were in each case not included in the evaluation, meaning that a 1000×1000 pixels image gives rise to an 800×800 pixels image. In the next step, intensity thresholds for each channel were ascertained on the basis of the negative control. For said threshold, all images of the negative control were averaged for each channel and what was ascertained was that intensity value above which only 0.1% of the total pixels (ergo 640 pixels) are present. In the evaluation step, the intensity threshold was first applied for each image in each channel and images of the same position were then compared with one another in both values. What were counted per image were only those pixels in which, in both channels, the pixel at the exact same position is above the intensity threshold of the channel. Lastly, the number of pixels was averaged over all images in each RC and, afterwards, the mean values of the average pixel numbers of the replicate values were ascertained and the standard deviation was specified.

[0117] The results are summarized in FIG. 1.

[0118] FIG. 1 shows a concentration series of serially diluted M13 phages (Ph.D.-7 Phage Display Library; prod. #E8102L, lot #0061005, New England BioLabs) in negative human EDTA blood plasma. The figure shows a linear relationship between the measurement signal and the concentration of M13 phages over 5 log dilution steps and a distinguishability of the lowest concentration in relation to the background.

Example 2

[0119] The experiment, the result of which is summarized in FIG. 2, was carried out analogously to Example 1, with the difference that the phages were diluted in TRIS-buffered saline instead of in human blood plasma. This also gave rise to different intensity thresholds, which however were ascertained using the same rule (0.1% of the negative control). The evaluation was carried out analogously to Example 1.

Example 3

[0120] shows the phages after measurement using Hoechst stain. For the experiment, commercial microtiter plates (Greiner Bio-one; Sensoplate Plus) containing 384 reaction chambers (RCs) were used. First of all, the surface of the microtiter plate was constructed. For this purpose, the plate was placed into a desiccator in which a bowl containing 5% APTES in toluene was situated. The desiccator was flooded with argon and incubated for one hour. Thereafter, the bowl was removed and the plate was dried under vacuum for 2 hours. 20 μl of a 2 mM solution of SC-PEG-CM (MW 3400; Laysan Bio) in deionized H.sub.2O were filled into the reaction chambers of the dry plate and incubated for 4 hours. After the incubation, the RC was washed three times with water and then incubated with in each case 20 μl of an aqueous 200 mM EDC solution (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Sigma) and with 50 mM NHS (N-hydroxysuccinimide, Sigma) for 30 minutes. The plate was washed again with three times with deionized water. Thereafter, the RCs were coated with clone anti-gp8-E1 (prod. #ABIN793840, lot #77410, antibodies-online.com) antibodies as capture molecule (20 μl; μg/ml in PBS; 1 hour). Thereafter, the RC was treated with the wash program consisting of, in each case, washing and complete suction three times with TBS containing 0.1% Tween 20 and TBS. In the next step, the RCs were coated overnight with 50 μl of Smartblock (Candor Bioscience GmbH) at room temperature (RT) and, after this time had passed, were again subjected to washing and complete suction three times using tris(hydroxymethyl)aminomethane-buffered saline (TBS; pH=7.4). The samples were diluted sequentially in tris(hydroxymethyl)aminomethane (TRIS) buffer containing the dye Hoechst stain (1 μg ml-1) and incubated for one hour. Thereafter, 15 μl of sample were in each case loaded in RCs in triplicate and incubated at RT for 1 hour. After the incubation, the RCs were washed three times with TBS and, after this time had passed, the plate was washed 3 times with TBS. After complete suction, the RCs were filled with 20 μl of TBS and the plate was sealed a film.

[0121] The measurement was carried out in a TIRF microscope (Leica) with a 100× oil immersion objective. For this purpose, the glass base of the microtiter plate was generously coated with immersion oil and the plate was introduced into the automated stage of the microscope. Thereafter, what was consecutively recorded per RC at 5×5 positions was one image in the fluorescence channel (excitation/emission=405/450 nm). What was selected was the maximum laser output (100%), an exposure time of 500 ms and a gain value of 800. The image data were then evaluated. For the channel, an intensity threshold was set at 4000 grayscales. In the evaluation step, the intensity threshold was first applied for each image in each channel and images of the same position were then compared with one another in both values. What were counted per image were only those pixels in which, in both channels, the pixel at the exact same position is above the intensity threshold of the channel. Lastly, the number of pixels is averaged over all images in each RC and, afterwards, the mean values of the average pixel numbers of the replicate values are ascertained and the standard deviation is specified.

[0122] The results are summarized in FIG. 3.

[0123] FIG. 3 shows a concentration series of serially diluted M13 phages (Ph.D.-7 Phage Display Library; prod. #E8102L, lot #0061005, New England BioLabs) in TBS containing Hoechst stain. The figure shows a concentration-dependent relationship of M13 phages over log dilution steps (10{circumflex over ( )}9-10{circumflex over ( )}5), and the last two concentration steps could no longer be distinguished from the background.

[0124] What is clearly shown is the suitability of Hoechst stain as dye adhering to DNA and RNA in the virus assay for staining of the virus particles. It would thus be possible to detect intact viruses, or to distinguish between empty coats and coats containing DNA and/or RNA.

Example 4

[0125] FIG. 4 shows the phages from FIG. 3 after the measurement using Höchst stain. The RCs of the microplate from Example 3 were washed three times with TBS and were incubated for 1 hour with the detection antibodies, 1.25 μg/ml RL-ph1 (prod. #LS-C146750, LifeSpan BioScience) labeled with CF488 fluorescent dye and 1.25 μg/ml LRL-ph2 (prod. #LS-C146751, lot #76955, LifeSpan BioScience) labeled with CF633 fluorescent dye. After washing three times with TBS, the samples were measured TIRF microscope (Leica) with a 100× oil immersion objective. For this purpose, the glass base of the microtiter plate was generously coated with immersion oil and the plate was introduced into the automated stage of the microscope. Thereafter, what was consecutively recorded per RC at 5×5 positions was in each case two images in two fluorescence channels (excitation/emission=635/705 nm and 488/525 nm). For both channels, what was selected was the maximum laser output (100%), an exposure time of 500 ms and a gain value of 800. The image data were then evaluated. For this purpose, intensity thresholds for each channel were ascertained on the basis of the negative control. For said threshold, all images of the negative control were averaged for each channel and what was ascertained was that intensity value above which only 0.1% of the total pixels (ergo 1000 pixels) are present. In the evaluation step, the intensity threshold was first applied for each image in each channel and images of the same position were then compared with one another in both values. What were counted per image were only those pixels in which, in both channels, the pixels at the exact same position were above the intensity threshold of the channel. Lastly, the number of pixels was averaged over all images in each RC and, afterwards, the mean values of the average pixel numbers of the replicate values were ascertained and the standard deviation was specified.

[0126] The experiment shows both the concentration-dependent relationship of M13 phages over 4 log dilution steps (10{circumflex over ( )}6-10{circumflex over ( )}3). It shows that a subsequent staining with fluorescently labeled antibodies is possible. Furthermore, the experiment shows that microtiter plates with PEG coating achieve approximately the same sensitivity as in the case of 3D NHS plates with a shorter incubation time (cf. FIG. 2).

Example 5

[0127] The experiment was carried out in commercially available 3D NHS microtiter plates (PolyAn GmbH) containing 384 reaction chambers (RCs). The RCs of the microtiter plates were coated with anti-VSV-G antibody P5D4 (Sigma) as capture molecule (15 μl; 10 μg/ml in 100 mM MES, pH=4.7; overnight). Thereafter, the RC was treated with the wash program consisting of, in each case, washing and complete suction three times using phosphate-buffered saline (PBS) containing 0.1% Tween 20 and PBS. In the next step, the RCs were coated with 50 μl of Smartblock (Candor Bioscience GmbH) at room temperature (RT) for 1 h and, after this time had passed, were again subjected to washing and complete suction three times using tris(hydroxymethyl)aminomethane-buffered saline (TBS; pH=7.4). The samples were sequentially in tris(hydroxymethyl)aminomethane-buffered saline (TBS) incubated for one hour. Thereafter, 15 μl of sample were in each case loaded in RCs in triplicate and incubated at RT for 1 hour. After the incubation, the RCs were subjected to washing and complete suction three times using TBS and were admixed with 15 μl of detection antibodies. The detection antibodies were in each case labeled with one type of fluorescent dye. Anti-VSV-G antibodies P5D4 (Sigma) were in each case labeled with CF488 and with CF633. The detection antibodies were diluted together in TBS to give a final concentration of 1.25 ng/ml for each antibody. 15 μl of antibody solution were loaded per RC and incubated at RT for 1 h. After this time had passed, the plate was washed 3 times with TBS. After complete suction, the RCs were filled with 20 μl of TBS and the plate was sealed a film.

[0128] The measurement was carried out in a TIRF microscope (Leica) with a 100× oil immersion objective. For this purpose, the glass base of the microtiter plate was generously coated with immersion oil and the plate was introduced into the automated stage of the microscope. Thereafter, what was consecutively recorded per RC at 5×5 positions was in each case two images in two fluorescence channels (excitation/emission=635/705 nm and 488/525 nm). For both channels, what was selected was the maximum laser output (100%), an exposure time of 500 ms and a gain value of 800. The image data were then evaluated. For this purpose, intensity thresholds for each channel were ascertained on the basis of the negative control. For said threshold, all images of the negative control were averaged for each channel and what was ascertained was that intensity value above which only 0.1% of the total pixels (ergo 1000 pixels) are present. In the evaluation step, the intensity threshold was first applied for each image in each channel and images of the same position were then compared with one another in both values. What were counted per image were only those pixels in which, in both channels, the pixels at the exact same position were above the intensity threshold of the channel. Lastly, the number of pixels was averaged over all images in each RC and, afterwards, the mean values of the average pixel numbers of the replicate values were ascertained and the standard deviation was specified.

[0129] The results are summarized in FIG. 5.

[0130] FIG. 5 shows a concentration series of serially diluted virus particles of an HIV safety strain (for the biosynthesis, see J Mol Biol. 2017 April 21; 429(8): 1171-1191. doi: 10.1016/j.jmb.2017.03.015., which contains a VSV glycoprotein in the lipid membrane) diluted in TBS from cell culture medium. The figure shows a linear relationship between the measurement signal and the concentration of virus particles over 3 log dilution steps and a distinguishability of the lowest concentration in relation to the background. Since a virus particle with lipid coat is concerned here, the method according to the invention can be used on for the analysis of this taxonomic group.

Example 6

[0131] FIG. 6 shows the number of virus particles per μm.sup.2 in the individual dilution steps of the data from FIG. 5. This alternative evaluation of the data allows absolute counting of virus particles and is thus independent of a standard. For the evaluation, what were multiplied for each recording were the grayscale values of each corresponding pixel from both images in which fluorescence color channels were obtained. The resultant image, or the grayscale value matrix, was smoothed on the basis of a 2D Gaussian function (“imgaussfilt”) with a standard deviation of 4 pixels. This was followed by the determination of the local maxima on the basis of the function “imregionalmax”. The number of local maxima above a threshold corresponds here to the number of particles, the threshold corresponding to the threshold ascertained in FIG. 5. In this way, the number of particles was determined for each recording and, finally, the mean value was formed over RCs of the same concentration.