LAAKEL MOHAMED (CA)
PEPE JEFFREY C (US)
| 1. | An immortalized cell, comprising SV40 T Antigen, infected with hepatitis C virus (HCV), wherein said cell replicates HCV. |
| 2. | An immortalized cell according claim 1 selected from ST E4, STE8, STD5, VeroB9, VeroD7, VeroE9, BartD9, BartG3, BartE9, and MDBKB10. |
| 3. | A method of screening for an agent that inhibits HCV replication, comprising contacting the immortalized cell according to claim 1 or claim 2 with a candidate agent and determining an inhibition of HCV replication. |
BACKGROUND OF THE INVENTION Hepatitis C virus (HCV) is a major cause of post transfusion hepatitis, which often leads to liver cirrhosis and cancer. HCV is a worldwide health problem. Development of vaccines and other antiviral therapies is awaited. To develop this, cell culture systems for propagating HCV are needed, but the levels of virus replication reported up to now have been too low. Primary human and chimpanzee hepatocytes are susceptible to HCV, and do replicate the virus (see U. S. Patent No. 6,096, 541), but primary hepatocytes are difficult to obtain, and usually survive less than two weeks in culture. Full length HCV RNA has been successfully transferred into a number of cell lines, but replicative levels are not stable and become undetectable within a few weeks. More recently, sub-genomic regions of HCV have been cloned and expressed at high levels in minireplicons, but none of these minireplicons supports virus replication.
Hence, a need exists for the development of one or more systems capable of stably supporting HCV replication. Furthermore, there is a need for a stable and efficient cell culture system permissive for HCV infection to allow the identification of novel antiviral agents to treat or prevent HCV infection. The present invention meets such needs, and further provides other related advantages.
SUMMARY OF THE INVENTION This invention provides methods for growing cultured human cells under conditions that permit biochemical and genetic manipulations. In one particular embodiment, the lifespan of these cultured human cell lines is prolonged by immortalization. One way in which this is achieved is by using the SV40 large T-antigen but other methods can also be used. In another embodiment, the cells lines are transformed so that they carry a chemical marker such as the dominant selection marker neo. This invention also provides methods for infecting human cell lines with HCV under conditions and for a time sufficient to allow propagation of the infected cell lines, stable retention of infection, and production of HVC particles over many generations.
In one embodiment, provided is an immortalized cell, comprising SV40 T Antigen, infected with hepatitis C virus (HCV), wherein said cell replicates HCV.
In another preferred embodiment, the invention provides an immortalized cell selected from ST-E4, ST-E8, ST-D5, Vero-B9, Vero-D7, Vero-E9, Bart-D9, Bart-G3, Bart-E9, and MDBK-B10.
In another aspect, the invention also provides methods for using these stably transformed and infected human cell lines for identifying antiviral agents, such as anti-HCV agents (with or without other antiviral compounds), useful for the treatment or prevention of HCV infections. In one embodiment, the invention provides a method of screening for an agent that inhibits HCV replication, comprising contacting the immortalized cell according to claim 1 with a candidate agent and determining an inhibition of HCV replication.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic of exemplary embodiments of the methods of using cell culture to replicate HCV.
Figure 2 shows results of RT-PCR of RNA samples isolated from HCV infected cells.
Figure 3 shows results of RT-PCR with RNA isolated from HCV infected cells and the effect of the amount of RNA used in the PCR.
Figure 4 shows the RT-PCR results using 200 ng RNA isolated from control (non-infected) cells, infected cells, and supernatants.
Figure 5 shows primer selection can affect detection of HCV with RT-PCR.
Figure 6 shows alignment of candidate HCV genome sequenced from PCR product from infected cells with other known HCV genomes.
Figure 7 shows result of plaque assay to examine permissiveness of transformed cell lines to infection by HCV.
Figure 8 shows the result of RT-PCR and nested PCR to detect HCV in infected transformed cells.
Figure 9 shows alignment of candidate HCV genome sequenced from PCR product from infected cells with other known HCV genomes.
Figure 10 shows the result of Southern blot analysis to detect HCV in infected transformed cells.
Figure 11 shows the result of a plaque assay to quantify HCV from transformed cell lines infected with HCV.
Figure 12 shows the result of a plaque assay to quantify HCV from transformed cell lines infected with HCV (no plaques detected).
Figure 13 shows the result of nested PCR on the RT-PCR sample of Vero-B9 infected with S1 supernatant using primers H6 and R3.
Figure 14 shows the result of RT-PCR and nested PCR samples from infected ST-E4, ST-E8 and ST-D5 probed with DIG-labeled nested PCR product from Figure 13.
Figure 15 shows result of HPLC to quantify HCV-specific PCR products generated from transformed cell lines infected with HCV.
Figure 16 shows the detection by immunofluorescense of SV Large T Ag in H2.35 cells transformed with pSV3-neo and serially passaged.
Figure 17 shows the detection by immunofluorescense of SV Large T Ag in ST-E8 cells transformed with pSV3-neo and serially passaged.
Figure 18 shows the detection by immunofluorescense of SV Large T Ag in Vero-B9 cells transformed with pSV3-neo and serially passaged.
Figure 19 shows the detection by immunofluorescense of SV Large T Ag in MDBK-B10 cells transformed with pSV3-neo and serially passaged.
Figure 20 shows the result of RT-PCR and nested PCR to detect HCV in transformed cells that had been serially passaged.
Figure 21 shows the result of RT-PCR and nested PCR to detect HCV negative strand in transformed cells that had been passaged five times.
Figure 22 shows the result of RT-PCR to detect HCV negative strand in transformed cells that had been serially passaged 8,9, and 11 times, and one week post-infection (p. i.).
Figure 23 shows the result of a kinetic experiment to detect the negative strand in the cell clones MDBK-B10 and ST-E4.
Figure 24 shows the result of nested PCR to detect HCV positive strand in H2.35 cell line infected with HCV.
Figure 25 shows the results of the cytopathic effect of HCV supernatant S2 infecting ST-D5, ST-E4 and ST-E8 cell lines.
Figure 26 shows the results of immunofluorescence detection of HCV core protein in HCV infected transformed cell lines.
Figure 27 shows the results of immunofluorescence detection of HCV protein NS5 in HCV infected transformed cell lines.
Figures 28A and 28B show the results of western blot analysis of HCV infected cells to detect the expression of the HCV NS5B (A) and core (B) proteins.
DETAILED DESCRIPTION OF THE INVENTION As set forth above, the present invention provides compositions and methods for making and using cell lines that stably replicate HCV. The
instant invention, therefore, relates generally to the surprising discovery that certain immortalized cell lines are useful for the stable propagation of hepatitis C virus (HCV). Accordingly, such cell lines are further useful for screening for and identifying candidate antiviral agents that inhibit HCV replication.
Discussed in more detail below are methods of replicating HCV in cell culture and suitable uses thereof, as well as representative compositions and therapeutic uses.
In the present description, any concentration range, percentage range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. As used herein, "about"or"comprising essentially of'mean 15%. The use of the alternative (e. g.,"or") should be understood to mean either one, both or any combination thereof of the alternatives. In addition, it should be understood that the individual compounds, or groups of compounds, derived from the various combinations of the sequences, structures, and substituents described herein, are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular sequences, structures, or substituents is within the scope of the present invention.
Transformation and Immortalization of Cell Lines As set forth above, the instant invention generally provides a method of stably replicating HCV in cells permissive to infection by HCV. In one preferred embodiment, cells permissive to infection by HCV and for stable replication of HCV are transformed cells, more preferably the transformed cells are immortalized, and even more preferably the immortalized cells express SV40 large T Ag. In certain embodiments, the cells suitable for transformation include H2.35, ST, Vero, Bart, and MDBK. Techniques for transformation and immortalization of a variety of cell types (such as primary culture cells,
secondary culture cells, cell lines, etc. ) is known in art, as described in Salmon et al. (Mol. Therapy 2: 404,2000, and references cited therein).
In another embodiment, the immortalized cells are generated by transfection with a nucleic acid molecule that encodes SV40 large T Ag. In a preferred embodiment, the T Ag nucleic acid is carried on a vector, such as a plasmid, YAC, shuttle vector, and the like. Optionally, the vector can further comprise a nucleic acid that encodes a selectable marker, such as neomycin.
In a preferred embodiment, the vector is a plasmid and more preferably is pSV3-neo.
A suitable transformed cell useful in the instant invention can be detected, for example, by indirect immunofluorescence antibody (IFA) analysis.
For example, the expression of SV40 large T Ag can be evaluated by IFA as follows : cells are grown on glass coverslips, gently washed with prewarmed phosphate-buffered saline (PBS), fixed with immunofluorescent buffer (IF buffer, Bio-Rad) containing 3% formaldehyde for at least 1 h at room temperature, and then treated with 3% Triton X-100 in IF buffer for another 1h.
After washing with PBS, the cells are incubated with mouse anti-SV40 Large T Ag monoclonal antibody (alternatively, a human monoclonal antibody against SV40 large T antigen (Ag), clone 101, (Research Diagnostic, Inc.) can be used) for 1h, washed with PBS, and incubated under the same conditions with fluorescein labeled anti-mouse IgG (Sigma) and blue Evans. UV light microscopy can be used to examine the labeled cells. Exemplary transformed cells suitable for use with the instant invention include ST-E4, ST-E8, ST-D5, Vero-B9, Vero-D7, Vero-E9, Bart-D9, Bart-G3, Bart-E9 and MDBK-B10.
Preferred transformants should be stable over many passages. For example, the expression of the protein SV40 Large T Ag in transformants H2.35, ST-E8, Vero-B9 and MDBK-B10 remains stable after 40 passages (see, e. g. , Figures 16-19).
Infection of Transformed Cells Infection of transformed cells suitable for use in the instant invention can be accomplished using procedures known in the art (see, e. g., U. S. Patent No. 6,096, 541). The different transformed cells can be infected with HCV and subsequently sequentially passaged, wherein the supernatant of the previous passage is used to infect fresh cells. The presence of HCV will typically be monitored during the sequential infection and passage of the cells to verify the continued permissiveness to HCV infection. The summary of the third and fourth passages is as follows : 3rd Passage (plates 24/well) 1 ST-E4 Vero-B9 Bart-D9 ST-E8 Vero-D7 Bart-G3 ST-DS VeroE9 Bart-E9 Transformed Cell Lines 4th Passage (Flasks 12. 5 cm~) Infection with supernatant from Sl S2 Incubation of infected cells ST-E4 Vero-B9 Bart-D9 ST-E8 Vero-D7 Bart-G3 MDBK-B10 ST-D5 VeroE9 Bart-E9 Transformed Cell Lines (Flasks 12. 5 cm2) .. Infection with patient sera Sa/5 S6/1 S6/10 S7/1 I. Passage only with supernatant from S/a5 and S 1/6 ' Incubation of infected cells
After three passages, a cytopathic effect may be observed in some cell clones. For example, ST-transformed cells, MDBK-B10 and Vero-B9 showed some cytopathic effects, and in particular, the ST clones showed subsequent lysis at about seven days post infection (p. i. ). The cells are at the 5th and the 6th passage.
Detection of HCV in Infected Cells PCR Replication of HCV can be monitored directly (e. g., plaque assay) or indirectly (e. g. , RT-PCR, nested PCR, HPLC of HCV nucleic acids), and qualitatively (e. g. , Southern blots, western blots) or quantitatively (e. g. , PCR, plaque assay), and preferably a combination of methods are used to verify the presence of HCV in infected cells.
For example, for RT-PCR, RNA from infected transformed cells can be extracted using Trizol LS reagent (GIBCO BRL). Total RNA isolated from cells can be used in Titanium One-Step RT-PCR (Clontech) to confirm the presence of HCV (see, e. g. , Figure 2). In comparison with the positive control (RNA transcript from full-length infectious clone H77C), the PCR product is of the expected size (185 base pairs (bp) ). Quantification of the concentration of RNA from the three cell lines showed that 2. 3 pg was used per reaction for Vero-B9,1. 7 pg for MDBK-B10 and 3. 6 pg for Bart-D9. Figure 3 shows that when the RNA concentration varies from 1 to 500 ng, a proportional increase in the RT-PCR product is observed, particularly for MDBK-B10 and Vero-B9 cells. However, for Bart-D10 cells, there is no observable RT-PCR product. For MDBK-B10 and Vero-B9, lane 4 shows the negative RT control reaction (RT was heat inactivated). Figure 4 shows the RT-PCR results using 200 ng RNA isolated from control (non-infected) cells, infected cells, and supernatants. For Vero-B9, only the infected cells show the PCR product at 185 bp.
In certain preferred RT-PCR embodiments, primers are selected from HCV genome internal sequences, 5'UTR, or 3'UTR. To increase specificity of the RT-PCR product, combination primers were designed (see Figure 5, top). Conditions were used to obtain a PCR product in one step RT- PCR reaction. As used herein, primers useful in the instant invention include H6 (SEQ ID NO: 1, sense primer), R3 (SEQ ID NO: 3, anti-sense primer), R7 (SEQ ID NO: 2), H3 (SEQ ID NO: 4), and R8 (SEQ ID NO : 5). For example, the H6-R3 combination gave a product at the correct size (185 bp). Similarly, the
H6-R7 gave a product of the right size, although less product was produced (239 bp). Using samples from the H3-R7 and H3-R8 combination, a second round PCR (nested PCR) using different combinations of primers was performed. The results are summarized at the bottom of Figure 5 showing a strong band of the right size with the H6-R7 combination.
In certain embodiments, transformed cells are passaged further (e. g. , 7th and 8th passage) and then analyzed again for the presence of HCV.
For example, as described herein, the replication of HCV can be followed by RT-PCR and nested PCR. For example, first round RT-PCR can be performed using primers H3 and R8, wherein the expected product size is 290 bp. The results from the first round PCR can be seen in Figure 8A. Lanes 3,5, 6,9 and 10 had faint bands approximately 290 bp in size when compared to the 100 bp DNA ladder in lanes 1 and 8. A second round of PCR was done using internal primers H6 and R3. This primer combination had an expected product size of 185 bp, which was detectable in the samples from the infected cells (Figure 8B).
In certain other embodiments, transformed cells are passaged further (e. g., 9th, 10th and the 11th passage), and the replication of HCV was followed by RT-PCR and nested PCR. Figure 20 shows only some of the cell clones that have been infected with different supernatants of HCV virus. The virus is still able to be detected in these cells clones that have been infected with supernatants from passages 8,9, and 11. The band at position 185 bp shows that there is the continual presence of the virus in these later passages.
The band is from the positive strand of the RNA.
Figure 21 shows the detection of the negative strand in one cell clone, MDBK-B10, infected with supernatant Sa/5. The band at position 185 bp. confirms the replication of the virus. The virus must first produce the negative strand in order to make the positive strand. The negative strand is the intermediary step for replication before it passes to the positive strand. The band at position 185 bp. for the negative strand (Figure 21) was excised, purified and sent for sequencing. The sequence matches the known HCV
sequences from the Genebank, confirming that the negative strand is from HCV.
The detection of HCV negative strand in later passages is shown in this gel (Figure 22). This experiment was done with RNA extracted from the different cell clones infected with supernatants from passages 8,9 and 11, one week p. i. A band at position 185 bp is apparent in lanes 9 and 10, it is MDBK- B10 infected with two supernatants (S2, and Sa/5). In theory, the reason that a band at position 185 bp for the other clones is not seen may be due to the time when the virus is replicating. One week p. i. may not be the exact moment when the virus is replicating ; hence, the negative strand in the other clones is not detected.
The gel in Figure 23 is the result of a kinetic experiment to detect the negative strand in the cell clones MDBK-B10 and ST-E4. In lanes 2 and 6 there is the appearance of a band at position 185 bp in Day 1 p. i. for clone MDBK-B10 infected with either S2 or Sa/5. The clone ST-E4 infected with Sa/5 shows a strong band at position 185 bp starting from Day 0 (6 hrs. after the virus was absorbed on the cells) and the intensity slowly decreases by day 3 but the band is always present. The cell clone ST-E4 infected with S2 also shows a band at 185 bp ay Day 0 but it is less intense. The band slowing decreases in intensity by Day 3. The virus is able to replicate early on in these cell clones.
Figure 24 shows the replication of HCV in the H2.35 cell line.
H2.35 cells are mouse hepatocytes transformed by SV40. A band at position 185 bp is apparent in cells infected with either S1 or S2 supernatant at passage 3, done under two different experimental procedures. The band at 185 bp is the result of a nested PCR using the positive strand of DNA. The band in lane 5 was excised, purified and sent for sequencing. The sequencing results of the band at 185 bp, (lane 5, Figure 22) of H2.35 cells infected with S2 supernatant line up with the known sequences of HCV from the Genebank, showing that HCV is replicating in H2.35 cell line.
To confirm the presence of the HCV genome and to validate the PCR products, the band containing the candidate nucleic acid was excised from the gel, purified, quantified by spectrophotometry (Beckman DU 520 General Purpose Spectrophotometer), and then sequenced using methods known in the art. Sequence analysis and alignment was done using Lasergenee software (DNASTAR Inc., Madison, WI). This alignment was compared to other HCV genotypes (e. g., 1a and 1b in Figure 6, and see Figure 9). The nucleic acid sequence of the purified PCR product shows almost perfect identity with other known HCV genome sequences.
Southern Blot Hybridization Another way to confirm the presence of HCV is to perform the Southern blot hybridization using an HCV genome specific labeled probe. In instant invention, the anti-sense primer R3 labeled with DIG was used (see, e. g. , Example 12). Briefly, the RT-PCR and nested PCR products were transferred to a nylon membrane and subsequently detected by DIG-labeled R3 probe (Figure 10). The three nested PCR sample bands were detected by autoradiography. The specificity of the probe is evident since only the amplified HCV products were detected and not the p-actin despite the fact that ß-actin had a very intense band on the gel. No bands were detected in the control lane showing there was no contamination during amplification by PCR or during subsequent steps.
Immunofluorescence Detection of HCV Protein Indirect immunofluorescence antibody (IFA) analysis was carried out with an anti-Human SV40 large T ag, clone 101, monoclonal antibody (Research Diagnostic, Inc.). This expression of SV40 large T ag was evaluated by IFA as follows : cell grown on glass coverslips were gently washed with prewarmed phosphate buffered saline (PBS) and fixed with immunofluorescent buffer (IF buffer, Bio-Rad) containing 3% formaldehyde for at least 1h at room temperature, then treated with 3% Triton X-100 in IF buffer for another 1h.
After 1 wash with conventional PBS, the cells were incubated with the mouse anti-SV40 Large ag monoclonal antibody for 1 h, washed with PBS and incubated under the same conditions the fluorescein-labeled anti-mouse IgG (Sigma) and blue Evans. The cells were then examined under UV light microscopy.
The slides in Figure 26 confirm the presence of the HCV virus at the protein level. The anti-core antibody to HCV was used in this experiment.
The fluorescence is evident in all the infected clones, ST-E4, ST-D5, ST-E8, H2.35, Vero-B9 and MDBK-B10. There is no evidence of fluorescence in the non-infected cells.
The slides in Figure 27 confirm the presence of the HCV virus at the protein level. The anti-NS5 antibody to HCV was used in this experiment.
The fluorescence is evident in all the infected clones, ST-E4, ST-D5, ST-E8, H2.35, Vero-B9 and MDBK-B10. There is no evidence of fluorescence in the non-infected cells.
Western Immunoblot These are the results of Western blotting (Figure 28A and 28B) using protein extracted from different cell clones infected with different supernatants. Figure 28A shows the results of probing with the monoclonal mouse-anti NS5B antibody. The control is purified NS5B protein (Fract. ici).
There is a band at position 68 kD in all the infected cell clones. There is no band at this position in the uninfected sample (ST-D5 NI). Figure 28B shows the results of probing with the monoclonal antibody mouse anti-core antibody.
There is a band at position 21 kD in all the infected samples. There is no band at this position in the uninfected samples (Vero NI and MDBK NI). These blots confirm that at the there is expression of the HCV NS5B and core proteins in the infected cell clones.
Quantification of the HCV Plaque Assay In this kind of experiment, the ability of transformed cell lines to replicate HCV and produce plaques is used to quantify the amount of virus produced. For example, ST clones (ST-D5, ST-E8, ST-E4) and a Vero clone (Vero-D10) were placed (transformed cell lines) in 6-wells plates (1x106cells/well) and infected with serial dilutions of supernatants from the 4th passage described herein to determine the potential infectivity of these clones with HCV. Inoculum was removed after 2h, and cells were overlaid with EMEM supplemented with 5% HS, and 0.5% SeaKem LE agarose. After 4 days, plaques were quantified by manual counting after the cells were fixed with 7% formaldehyde and stained with 0.5% crystal violet. For example, both the ST-T and the clone ST-D5 produced high levels of HCV replication (see, e. g. , Figure 7). The results of the plaque assay are from the supernatants of cells of the invention in the 4th passage. Figure 11 shows that the infectivity of HCV varied from clone to clone and from transformed cell line to another. Both ST : r and the clone ST-E4 presented high level of HCV replication. Figure 12 also shows that in the MDBK-B10 and Vero-B9 tested there is no production of the plaques, which may or may not mean that there is no replication of HCV in these transformed cell lines.
Thus, in one preferred embodiment, replication of HCV is directly detected in a transformed cell line. In one aspect, the method of direct detection is the plaque assay.
Dot Blot Hybridization In certain embodiments, HCV infection of transformed cells is detected by dot blot hybridization. For example, the PCR Digoxigenin (DIG) Probe Synthesis kit (Roche) can be used to put a DIG label on PCR products.
Briefly, a nested PCR on the RT-PCR sample of Vero-B9 infected with S1 supernatant using H6 and R3 as the primers was used and the PCR DIG labeling mix. The controls are an unlabeled Vero-B9 S1 PCR product and the
control template supplied with the kit. The cycling conditions are described previously in the materials and methods for the nested PCR. The samples were run on a 2% agarose gel at 100V for 1 hour. The kit control gave a band at the expected 500 bp size (data not shown). In Figure 13, the nested PCR product for the unlabeled sample gave the expected band at 185 bp. The DIG labeled nested PCR product gave a band at a higher position than 185 bp. The increase in the band size is due to the presence of the DIG label. This confirms that the nested PCR has a DIG label on it. This band was excised from the gel and purified using the Qiagen gel purification kit. The purified DIG labeled PCR product was used as a probe in DOT blot and Northern blot experiments.
Samples were prepared and deposited in a dot blot apparatus for subsequent probing with the DIG-labeled nested PCR product. The samples that were deposited on the nylon membrane are as follow. As a standard, tenfold serial dilutions of unlabeled Vero-B9 infected with S1 supernatant were deposited in duplicata in 10-fold decreasing concentrations from 25 ng/pL, 2.5 ng/uL, etc. to 2.5 fg/uL. As a control, a DIG labeled DNA marker was deposited in a single lane at tenfold serial dilutions from 25 ng/pL to 2.5 fg/pL. The RT- PCR and the nested PCR samples from infected ST-E4, ST-E8 and ST-D5 were deposited in duplicata, each undiluted and diluted 1/10. A scheme of the deposit layout is below. 1 2 3 4 5 6 7 8 9 10 11 12 Vero B9 DIG ST E4 Vero B9 ST E8 ST D5 A 25 ng 25ng loue X 10ul 10ul (15ul) (15ul) RT-RT-RT- PCR PCR PCR B 2.5ng 2.5ng 10ul 2.5ng 10ul 10ul (15ul) (15ul) RT- (15ul) RT-RT- PCR PCR PCR C 0. 25ng 0. 25ng 15ul 0. 25ng 15ul 15ul (15ul) (15ul) (1/10) (15ul) (1/10) (1/10) D 25pg 25pg 15u 25pg 15ul 15ul 15ul) (15ul) (1/10) (15ul) (1/10) (1/10 E 2.5pg 2.5pg 15ul 2.5pg 15ul 15ul (15ul) (15ul Nested (15ul) Nested Nested F 0.25pg 0.25pg 15ul 0.25pg 15ul 15ul (15ul) (15ul Nested (15u)) NestedNested 25fg 25fg 15ul 25fg 15ul 15ul 15ul) (15ul) (1/10) (15ul) (1/10) (1/10) H 2.5fg 2.5fg 15ul 2.5fg 15ul 15ul (15ul) (15ul) (1/10) (15ul) (1/10) (1/10)
The nylon membrane was UV-cross-linked for 8 min. to fix the samples on the membrane and then prehybridized in DIG hybridization solution for 2 hours at 55°C. The membrane was sealed in a pouch with a DIG-labeled PCR probe and further incubated overnight at 55°C. The membrane was then washed in the same manner as for a Southern blot (see Example 12).
The hybridized membrane was exposed for a minimum of 3 hours to Kodak BioMax film. As shown in Figure 14, the unlabeled PCR product gave a signal that was proportional to the concentration deposited on the membrane. In contrast, the three samples showed an intense signal, and even the RT-PCR samples showed a good signal. In one preferred embodiment, the aforementioned method is useful for the detection and the quantification of HCV genome according to the instant invention.
HPLC A new method for the detection and the quantification oh HCV genome using the HPLC was established as follows. The first step is to set up
the optimal conditions for the separation of the different reagents of RT-PCR reactions. (see Figure 15).
Identification of Inhibitors of HCV Replication In another aspect, the instant invention provides methods for screening agents that inhibit the replication of HCV by contacting candidate agents with transformed cells infected with HCV and detecting a decrease or abolishment of HCV replication. One such agent tested was suramin. The cytopathic effect of HCV supernatant S2 upon infection of ST-D5, ST-E4 and ST-E8 cell lines is shown in Figure 25. The top row shows the effect of different dilutions of the virus 10-2, 104, 10-6 ten days post-infection. The cytopathic effect is evident because the virus has killed of all the cells. The control (NI, untreated cells) cells were not affected. When the cells were treated with different candidate agents to inhibit HCV replication, such as suramin, there was an effect on the cytopathcity of the virus. First, a concentration of 500 pg/mL suramin is toxic to the cells because at this concentration the uninfected cell control were killed. At a concentration of 250 pg/mL suramin the cytopathic effect of the virus is reduced by 4-5 logs. The uninfected control cells were not affected at this concentration.
The present invention also pertains to methods for treating or preventing HCV infection, comprising administering to a subject in need thereof a composition comprising at least one agent that inhibits HCV replication as identified in the methods of the instant invention, and a pharmaceutically acceptable carrier, diluent, or excipient, at a dose sufficient to inhibit HCV replication. In certain embodiments, an infection is due known group or subgroup of HCV. A subject suitable for treatment with a HCV replication inhibitor formulation may be identified by well-established indicators of risk for developing a disease or well-established hallmarks of an existing disease. For example, indicators of an infection include fever, pus, microorganism positive cultures, inflammation, and the like. Infections that may be treated with a HCV replication inhibitor of the subject invention include those caused by or due to
HCV, whether the infection is primary, secondary, opportunistic, and the like.
Examples of HCV include any antigenic variant of these viruses.
The pharmaceutical compositions that contain one or more HCV replication inhibitor of the invention may be in any form that allows for the composition to be administered to a subject, such as a human or animal. For example, compositions of the present invention may be prepared and administered as a liquid solution or prepared as a solid form (e. g., lyophilized), which may be administered in solid form, or resuspended in a solution in conjunction with administration. An HCV replication inhibitor composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject or patient or bioavailable via slow release. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units. In certain preferred embodiments, any of the aforementioned pharmaceutical compositions comprising a HCV replication inhibitor or cocktail of inhibitors of the invention are in a container, preferably in a sterile container.
In still another embodiment, the HCV replication inhibitor pharmaceutical composition is preferably sterile.
All of the U. S. patents, U. S. patent application publications, U. S. patent applications, non-U. S. patents, non-U. S. patent applications and non- patent publications referred to in this specification, are incorporated herein by reference, in their entirety. The invention having been described, the following examples are intended to illustrate, and not limit, the invention.
EXAMPLES EXAMPLE 1 CELL CULTURES The cells used are MDBK (ATCC), Vero (ATCC), and ST (ATCC).
The two first cell lines were maintained in EMEM and ST were maintained in DMEM media supplemented with glutamin 2 mM, 1 % of Penicillin/Streptomycin and 10% of fetal bovine serum under conditions of 5% C02 in air at 37°C.
EXAMPLE 2 PLASMID PREPARATION pSV3 neo was obtained from ATCC. It was propagated in E. coli and supercoiled DNA was isolated according to QIAGEN plasmid purification handbook (QlAfilter plasmid Maxi Protocol). The yield and purity of the DNA were very good. The DNA concentration was about 1.8 pg/pL for about 600 uL of total volume.
EXAMPLE 3 TRANSFORMATION OF CELLS The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome in this invention by transfecting the cells with the pSV3 neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers "neo". The"neo"sequence codes for the bacterial phosphotransferase APH (39//that inactivates the aminoglycoside G418.
ST, Vero and MDBK cells were trasfected the pSV3neo plasmid using the Lipofectamine Plus Reagent (Gibco BRL) with some modifications.
Supercoiled DNA was introduced into tissue culture cells (~ 10 g for approximately 1-5x106 cells in six well plates). About 72 h after exposure to
DNA, the cells were trypsinized, replated and G418 was added to the medium at the concentration as indicated in Table 1. At this step, the transformed cells were passeged four time in the selection medium and with Vero, ST and MDBK transformed cells the fourth passage was successful.
The next step in our planning is to do the sub-cloning of the already transformed cells by limiting dilution method. Cultures were diluted and plated at 60,50, 40,30, 20,10, 5, and I cells per 200 1 in 96 well plates (12 wells per each dilution). The cultures were observed microscopically for identification of wells containing single cells, and subsequently for their proliferation.
For ST transformed cells, they were at the 39th passages and obtained 4 potential clones which were growing in 12.5 cm2 flasks.
For Vero transformed cells, they were at the 33th passages and obtained 8 potential clones which were growing in 12.5 cm2 flasks.
For MDBK transformed cells, they were at the 33th passages and obtained 3 potential clones which were growing in 12.5 cm2 flasks.
Summary of the passages of clones Transformed cell lines Number of potential Number of passages clones Vero-T 8 33th MDBK-T 3 33th ST-T 4 39th EXAMPLE 4 RNA EXTRACTION RNA extraction were done using Trizol LS from GibcoBRL. RNA was extracted from cells and supernatants. 0.75 ml of Trizol LS was added for every 0.25 ml of supernatant and mixed. After 5 minutes of incubation at room
temperature, 0.2 ml of chloroform was added to each sample followed by a further 15 minutes of incubation at room temperature. Samples were centrifuged for 15 minutes at 4°C at 12 000 rpm to separate the phases. The upper aqueous phase containing the RNA was removed and placed in a clean Eppendorf tube to which was added 0.5 ml of isopropanol. The samples were left at room temperature for 10 minutes then centrifuged for 10 minutes at 4°C at 12 000 rpm to precipitate the RNA. The supernatant was removed, the RNA washed in 1 ml of 75% ethanol and centrifuged for 5 minutes at 4°C at 7500 rpm. The ethanol was removed, the pellets air-dried and the RNA was resuspended in 50: 1 of DEPC-treated water. RNA samples were quantified by spectrophotometry.
EXAMPLE 5 ONE STEP RT-PCR RT-PCR is a very sensitive and versatile technique that is used to measure gene expression in cultured cells. The RT-PCR was made using the Titanium One-Step RT-PCR kit (Clontech Cat# K1403-1). It is a unique method since the RT-PCR can be performed in a single step, in a single tube. There is an advantage using this Titanium kit since traditional RT-PCR reactions are usually performed in two consecutive steps, in two different reactions, the first a first-strand cDNA synthsis step using reverse transcriptase followed by a PCR step using a thermostable DNA polymerase, Taq Polymerase. The two-step procedures require either multiple tubes or the sequential addition of enzymes and reagents. Another advantage of the Titanium kit is that since no additional reagents are needed after the reaction is initiated, the possibility of cross- contamination is reduced.
EXAMPLE 6 PREPARING RT-PCR REACTIONS For one reaction use 5pL of 10x One-Step, Buffer, 1, uL of 50X dNTP mix, 0.5 pL of recombinant Rnase Inhibitor (40 units/pL) 25 pL of
thermostabilizing reagent, 10 pL of GC-Melt, 1 pL of Oligo (dT) primer 1 uL of 50X RT-Titanium Taq Enzyme mix.
Aliquot out the 43.5 pL mix into PCR thin walled tubes (Ultident).
To each tube add: 1-5.5 uL of RNA sample (200 ng/pL), 1 uL of PCR Primer mix (50 pmol/µL) and DEPC H2O to complete the volume to 50 pL.
The primers (Invitrogen Life Technologies) that we used for the RT-PCR at the 3'UTR region are: H6: GTG CAG CCT CCA GGA CC (SEQ ID NO: 1) R3: GTA CCA CAA GGC CTT TC (SEQ ID NO: 3) EXAMPLE 7 CYCLING CONDITIONS The RT-PCR reactions were run in hot-lid Biometra thermocycling machines using the following program: 50°C for 1 hr., 94°C for 2 min. and 35 cycles of 94°C for 30 secs, 60°C for 30 secsl and 72 °C for 1 min. the 72° C for 8 min.
EXAMPLE 8 NESTED PCR In the conditions where we did not observe a PCR product in the RT-PCR reactions a nested PCR was made. Nested PCR consists of using the RT-PCR product (1St round) in a second PCR reaction.
EXAMPLE 9 PREPARING NESTED PCR REACTIONS There were two ways to prepare these reactions. The first consists of preparing a 1x reaction mix using 1 µL dNTP mix, 3.75 µL 20 mM MgCI2, 5 pL 10x Buffer, 1 pL of forward primer, 2 pL of reverse primer, 0.36 pL of Taq polymerase enzyme, 2 pL of the RNA sample and H20 to complete the volume to 50 pL.
The second method consists of using the Platinum Super Mix (Canadian Life Technologies). For one reaction prepare a reaction mix with 45 pL of the Platinum Super Mix, 1, uL of forward primer (50 pmol/µL), 2 pL of reverse primer (50 pmol/pL), and 2 pL of the RNA sample (200ng/pL).
The primers (Invitrogen Life Technologies) that were used at the 3'UTR region are H6: GTG CAG CCT CCA GGA CC (SEQ ID NO: 1) R7: ATG GTG CAC GGT CTA CGA GAC (SEQ ID NO: 2) R3: GTA CCA CAA GGC CTT TC (SEQ ID NO: 3) H3: GAA AGC GTC TAG CCA TGG CGT (SEQ ID NO : 4) R8: GGT TTA GGA TTC GTG CTC ATG G (SEQ ID NO : 5) EXAMPLE 10 CYCLING CONDITIONS The PCR reactions were run in hot-lid Biometra thermocycling machines using the following program: 94°C for 2 min. , and 35 cycles of 94°C for 30 secs, 60 °C for 30 secs. and 72°C for 1 min. the 72° C for 8 min.
EXAMPLE 11 AGAROSE GEL ANALYSIS An agarose was gel was prepared using 1.5% agarose and 4% ethidium bromide diluted in 1x TAE (0.04M Tris-acetate, 0. 001M EDTA Buffer). The samples were mixed with 6x loading buffer (0.25% bromophenol blue and 30% glycerol in water) and loaded onto the gel. The gel was run at 100V for 1 hr. in 1x TAE as the running buffer. The gel was observed under UV light (Herolab) and photographed using a Biometra software.
EXAMPLE 12 ELECTROPHORESIS AND SOUTHERN BLOT HYBRIDIZATION RT-PCR and nested PCR samples from cells and/or supernatants were deposited as well as RT-PCR samples of ß-actin and H20
as controls. 10 pl of each sample was loaded with 2 pl of 6x loading buffer and 2 pl of ethidium bromide. 5 pi of DIG DNA marker (Roche) was also deposited.
A 1.5% agarose gel was prepared and run at 60V for 30 minutes then at 80V for 3 hours. Southern hybridization was carried out according to Sambrook et aL using DIG detection as opposed to radioactivity. After gel electrophoresis, the gel was denatured for 45 minutes in 1.5 M NaCI, 0.5 N NaOH with gentle agitation. The gel was then rinsed briefly with ddH20 followed by 30 minutes of soaking in neutralizing solution (0.5 M Tris, 1.5 M NaCI) with gentle agitation.
The neutralizing solution was changed and the gel left to soak for a further 15 minutes. The wick (long piece of 3MM Whatman paper) was wet in 10x SSC and placed over the glass support with the ends of the wick in the glass dish filled with 10x SSC (liquid should reach almost to the level of the glass support).
The nylon membrane (Nytran Supercharge, Schleicher & Schuell) was soaked briefly in ddH20 until it was completely wet then soaked for 5 minutes in 10x SSC. Three pieces of 3MM Whatman paper were cut to the size of the gel and soaked in 2x SSC until wet. 10x SSC was used as the transfer buffer. The apparatus was assembled and left to transfer for 2 days. Once the transfer was complete the membrane was fixed with UV light for 7 minutes. The gel was stained with ethidium bromide for twenty minutes to ensure that the transfer was successful. The DIG Luminescence Detection Kit for Nucleic Acids (Roche) was used for detection. The membrane was prehybridized at 55°C for 2 hours. Hybridization with the DIG-labeled oligo probe complimentary to the DNA samples was carried out overnight at 55°C. Following hybridization, the membrane was washed twice in 2x SSC, 0. 1% SDS for 20 minutes followed by two 30 minute washes in 0. 1x SSC, 0.1% SDS at 55°C and a third at room temperature. The membrane was then blocked with 1x blocking solution (Roche blocking reagent in maleic acid) for 2 hours with gentle agitation. The membrane was incubated with the DIG-alkaline phosphatase antibody for 4 hours at room temperature, and then was equilibrated for 5 minutes in detection buffer. The detection substrate (CSPD, an alkaline phosphatase substrate) was added and left on the membrane for 10 minutes in the dark then removed
and the membrane incubated at 37°C for 10 minutes to enhance luminescence.
The membrane was exposed to Biomax film (Kodak) overnight.
EXAMPLE 13 QUANTIFICATION BY DOT BLOT HYBRIDIZATION Samples were deposited on a nylon membrane (Nytran Supercharge, Schleicher & Schering) using the dot blot vacuum apparatus.
After aspirating the samples, the DNA was fixed to the membrane by UV crosslinking for 6 minutes. The membrane was placed in a hydridization cylinder with 50ml of DIG Easy Hyb solution (Roche) and placed in the oven at 55°C for 4 hours. After prehybridization, the membrane was placed in a Kapak plastic pouch with the diluted probe. The probe was a DIG-labeled second PCR product. A first round sample of D supernatant was amplified using the DIG Probe PCR Labeling Kit (Roche). Hybridization was allowed to proceed overnight. The membrane was washed twice for 20 minutes in 2x SSC, 0. 1% SDS at room temperature, twice for 30 minutes at 55°C in 0. 1x SSC, 0.1% SDS, and once for 30 minutes in 0. 1x SSC, 0. 1% SDS with the oven off. The membrane was blocked in 1x blocking solution for 2 hours at room temperature.
The membrane was placed in a plastic Kapak pouch with DIG-alkaline phosphatase antibody for 4 hours at room temperature. The membrane was rinsed briefly in washing buffer then washed overnight in washing buffer at room temperature. The following morning, the membrane was washed twice for 30 minutes at room temperature. The membrane was equilibrated in detection buffer for 5 minutes then placed in a plastic pouch with 5 ml of diluted CSPD (alkaline phosphatase substrate) for 10 minutes in the dark. The CSPD was removed, the pouch resealed and placed at 37°C for 10 minutes to enhance luminescence. The membrane was taped in a cassette and exposed to a film for 1 hour.
EXAMPLE 14 IMMUNOFLUORESCENCE MICROSCOPY ANALYSIS Indirect immunofluorescence antibody (IFA) analysis was carried out with an anti-Human SV40 large T ag, clone 101, monoclonal antibody (Research Diagnostic, Inc.). This expression of SV40 large T ag was evaluated by IFA as follows : cell grown on glass coverslips were gently washed with prewarmed phosphate-buffered saline (PBS) and fixed with immunofluorescent buffer (IF buffer, Bio-Rad) containing 3% formaldehyde for at least 1h at room temperature, then treated with 3% Triton X-100 in IF buffer for another 1h.
After 1 wash with conventional PBS, the cells were incubated with the mouse anti-SV40 Large T ag monoclonal antibody for 1h, washed with PBS and incubated under the same conditions the fluorescein-labeled anti-mouse IgG (Sigma), and blue Evans. The cells were then examined under UV light microscopy.
EXAMPLE 15 TREATMENT OF CELL CLONES WITH SURAMIN The cell clones ST-D5, ST-E4 and ST-E8 were plated at a concentration of 1 x 105 cells/well in 12-well plates. Each cell clone was infected with S2 supernatant from P12 of the respective clone in the following dilutions 10-2, 10-4, 10-6. The cell clones were infected first and then treated with Suramin sodium salt (Sigma S-2671) at two concentrations (0.5 g/mL and 0.25 g/mL). The controls for each cell clone include non-infected cells treated with the same concentrations of Suramin, infected cells not treated with Suramin and non-infected cells not treated with Suramin. The virus was incubated on the cells overnight.
EXAMPLE 16 NEGATIVE STRAND DETECTION The first step is to prepare the cDNA from RNA extracted from the infected cell clones. Only the forward primer H6 (5'-GTG CAG CCT CCA GGA CC-3') (SEQ ID NO: 1) was used here.
A 1x reaction mix consisted of 400 ng of RNA, 2 pL of H6 primer (50 pmol/pL), I pL RNAsin RNAase Inhibitor (40 U/pL) (Promega), 1 pL of 0.1 M DTT, 1 pL of 2.5 mM dNTP mix (Invitrogen), 4 uL of 5X RT Buffer and DEPC ddH20 to complete the volume to 20 pL. Aliquot the samples. Heat them for 15 minutes at 65°C. Put the samples on ice and add MMLV-RT enzyme (1U/rxn) (Pharmacia). Incubate at 42°C for 45 minutes. Add more MMLV-RT enzyme (0.5U/rxn) and incubate 30-45 min longer at 42°C.
The next step is the first round PCR using the cDNA and the external primers H3 (5'GAA AGC GTC TAG CCA TGG CGT-3') (SEQ ID NO: 4) and R8 (5'-GGT TTA GGA TTC GTG CTC ATG G-3') (SEQ ID NO: 5). A typical reaction mix consisted of 5 uL of 10x Taq Buffer, 1 uL of 2.5 mM dNTP mix, 2 pL of both H3 and R8 (50 pmol/L). 0. 5 uL of Taq enzyme, 10 uL of cDNA and ddH20 to complete the reaction volume to 50 pL. The Biometra PCR Thermocycler was used with the cycling conditions for the nested-2 protocol (40 cycles).
The last step is a nested PCR using the PCR product from the first round and the inner primers H6 and R3 (5'-GTA CCA CAA GGC CTT TC- 3') (SEQ ID NO: 3). A typical reaction mix consisted of 5 uL of 10x Taq Buffer, 1 uL of 2.5 mM dNTP mix, 2 pl of both H6 and R3 (50 pmol/pL) 0.5 pL of Taq enzyme, 10 pL of cDNA and ddH20 to complete the reaction volume to 50 pL.
The Biometra PCR Thermocycler was used with the cycling conditions for the nested-2 protocol (40 cycles).
EXAMPLE 17 WESTERN BLOT HYBRIDIZATION Protein Extraction The proteins were extracted from the infected cell clones using the following procedure. A volume of 750 uL of Trizol (Invitrogen) was added to disrupt the cells. Then a volume of 200 L of chloroform was added. The aqueous phase was removed after centrifugation at 10000 g for 15 min. at 4°C.
The cellular DNA was precipitated with 0.3 mL of 100% ethanol/750 uL of Trizol. The tubes were vortexed and centrifuged at 2000 g for 5 min. at 4°C.
The supernatant is now used for protein precipitation by adding 1.5 mL/0.75 ml Trizol with isopropyl alcohol. Store the samples at 15-30°C for 10 min.
Centrifuge the protein precipiate at 12 000 g for 10 min. at 4°C.
Remove the supernatant and wash the pellet 3x with 0.3M Guanadine hydrochloide (Sigma) dissolved in 95% ethanol. Add 2.0 mL of wash solution/0. 75 mL of Trizol. During each cycle store the protein pellet in the wash for 20 min. at room temperature and centrifuge at 7500 g for 5 min. at 4°C. After the final wash, vortex the pellet in 2.0 mL of 100% ethanol. Store the pellet in ethanol 20 min. at room temp. Centrifuge at 7500 g for 5 min. at 4°C. Let the pellet dry for 5-10 min. Dissolve it in 1% SDS (Sigma) by pipetting. Store the proteins at-20°C.
Protein Quantification The proteins were quantified by using the DC Protein assay (Bio- Rad). Dilutions of BSA standard were prepared (0.2 mg/mL to 1.5 mg/mL).
Add 20 uL of reagent S to reagent A that will be needed for the experiment.
Pipet 5 uL of standard and protein samples into a clean dry microplate. Add 25 pL of reagent A into each well. Add 200 uL of reagent B into each well. Put the plate into a microplate reader (Spectra Max 190), use the mixing function. After 15 min. read the absorbance at 750 nm:
SDS-PAGE Prepare a 12% polyacrylamide gel as follows : Assemble the glass plates according to the Bio-Rad instruction. Prepare the resolving gel by mixing 3.3 mL of ddH20,4. 0 mL of 30% acrylamide mix (Bio-Rad), 2.5 mL of 1.5 M Tris (pH 8.8) 0.1 mL of 10 % SDS, 0.1 mL of 10% ammonium persulfate and 6 L TEMED. Pour the acylamide solution into the gap between the glass plates.
Leave 5 cm free for the stacking gel. Overlay the acylamide solution with ddH20. Once the gel has polymerized (30 min. ) pour off the overlay. Prepare the 5% stacking gel by mixing 2.7 mL of ddH20,0. 67 mL of 30 % acrylamide mix, 0.5 mL of 1.0 M Tris (pH 6.8) 0.04 mL of 10% SDS, 0.04 ml, of 10 % ammonium persulfate and 4 L of TEMED. Pour in the stacking gel solution and carefully insert the comb. Wait 30 min. until the gel polymerizes.
Prepare the samples by boiling them for 5 min. in 2X SDS-gel- loading buffer (100 mM Tris-HCI (pH 6.8), 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol) to denature the proteins. Mount the gel in the electrophoresis apparatus with 1X Tris-Glycine electrophoresis buffer (25 mM Tris, 250 mM glycine (pH 8.3), 0. 1% SDS) Load 15 L of the samples and include a rainbow molecular weigh marker (Bio-Rad). Run the gel at 60 V for 15 min. then increase the voltage to 100 V until the dye front runs down to the bottom (2hrs.).
Blotting Cut six pieces of Whatman 3MM paper and one piece of PVDF membrane (Bio-Rad) to the exact size of the polyacyramide gel. Soak the PVDF membrane in 100% methanol for 5 min. then equilibrate it in Transfer Buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS and 20% methanol.
Soak the Whatman paper in the transfer buffer. Soak the gel in 10% methanol dissolved in transfer buffer and then equilibrate the gel in transfer buffer. Set up the transfer apparatus as follows : On the anode (bottom electrode) place a wet cushion, three pieces of the wet Whatman paper, the gel, the PVDF membrane, three pieces of filter paper and another wet cushion. Make sure that there are no air bubbles trapped in between the gel and the membrane.
Place the apparatus in the electrophorsis apparatus with the transfer buffer. Apply 100 V of current for two hours. After the transfer, the gel may be stained with Coomasie Brilliant Blue to check the efficiency of transfer.
The membrane in now reading for overnight blocking in 5% milk dissolved in PBS and 0. 1% Tween 20. The primary antibodies (Biodesign) used were monoclonal mouse anti-HCV core antibody (1/50), and monoclonal mouse anti- HCV NS5 antibody (1/100). All the antibodies are dissolved in the blocking buffer. The membranes were incubated for 2 hrs. in the primary antibody.
Then the membranes were washed 3x 10 min. each in PBS. Then the membranes were incubated in the secondary antibody for 2 hrs. The secondary antibody was anti-mouse peroxidase (1/2000) for all the primary monoclonal antibodies. The membranes were washed 3x 10 min. each in PBS.
Detection The ECL Plus detection system (Amersham Biosciences) was used to reveal the proteins on the membrane. Mix the detection solutions A and B in a ratio of 40: 1. A volume of 2mL of solution A and 50 pL of solution B is used for one membrane. Place the membrane protein side, up on a sheet of Saran wrap. Apply the detection reagent on the membrane and incubate for 5 min. at room temperature. Drain off excess detection reagent and place the membrane protein side down on a clean sheet of Saran wrap. Place the blot protein side up in a X-ray film cassette. Expose it to a sheet of autoradiography film.
(Hyperfilm ECL, Amersham-Biosciences) for 15 secs. Develop the film and repeat exposure with a new film based on the results of the first.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.