Patent Application
20150275295
The invention relates to a novel method for detecting at least one target molecule(s) in a sample, in particular for detecting peptides, proteins, lipids or carbohydrate in a sample; at least one probe for use in the said method; a plurality, or library, of said probes for use in said method; and a kit of parts for carrying out said method.
Molecular interactions involving, for example, peptides or proteins (e.g., antibody-antigen interactions, hormone-receptor interactions, virus-receptor interactions, enzyme-substrate interactions, to name but a few) represent the most complex and important processes in any biological system. Their detection can provide valuable information concerning the status of the system and so can provide important information of diagnostic, therapeutic or commercial value. For example, the following is a non-exhaustive list of the sorts of information that can be derived by monitoring peptide or protein interactions.
However, unlike nucleic acid-based detection, there is currently no effective methodology for the amplification of peptides, proteins, lipids or carbohydrate-based interactions in a biological system. This means, especially where quantity is low, many interactions go unrecognised or undetected. Specific amplification of such interactions or signals is needed to provide a suitably sensitive method. Such a method would have significant impact in all areas of biological and medical research. The latter includes, but is not limited to: characterization of antibody-mediated immune responses for diagnostic and vaccine related use; screening for protein-protein interactions in biological processes or cellular signalling; screening of drug-protein binding or interaction, such as off-target or non-specific binding that could lead to side effects; screening for protein-glycoprotein binding, such as identification of virus-receptor binding for cellular entry; the characterization of post-translational glycan and oligosaccharide modifications on proteins for characterization and development of biologic drugs; and screening for protein-phospholipid interaction in biological processes such as determining how blood clotting proteins bind to cellular membranes.
In the area of monitoring antibody-mediated immune responses, being able to monitor peptide or protein-based signals in a biological system would be of great value. For example, any disease involving an infectious agent will very likely produce a specific host response against that agent. This includes conditions such as encephalitis resulting from infectious agents that are presently difficult to diagnose. Additionally, antibody-mediated immune responses from non-infectious diseases such as cancer, autoimmune diseases and chronic fatigue syndrome also represent areas likely to benefit from monitoring a peptide or protein-based signal in a biological system.
Whilst it is currently possible to monitor peptides or proteins in response to an antibody-mediated immune response, most of the current technologies for peptide or protein-based detection only allow for examination of immune responses against a single or small number of targeted agents. This makes the interrogation of a system repetitive, laborious, time consuming and expensive. These drawbacks could be overcome if it was possible to provide a high-throughput peptide or protein-based screening method, or microarray, for antibody monitoring. Whilst high-throughput peptide-based microarrays for antibody monitoring have been reported, these are not widely used due to their low sensitivity and lack of reproducibility (H. Andresen and C. Grötzinger (2009) Deciphering the antibodyome-peptide arrays for serum antibody biomarker diagnostics. Current Proteomics, 2009, 6, 1-12).
The invention described herein thus aims to overcome the disadvantages associated with the prior art.
Statements of Invention
According to a first aspect of the invention there is provided a probe for detecting and/or quantifying at least one target molecule(s) in a sample comprising:
Reference herein to a binding partner that is specific for said target molecule(s) means the binding partner is able to bind to said target molecule(s) to the exclusion of binding with other target molecule(s) of either a different or similar nature and, indeed, in some instances is unable to bind with any other target molecule(s).
In a preferred embodiment of the invention a plurality of probes may be provided as a probe library, once new probes are developed this library may be expanded and, additionally, or alternatively, said library may also be customized for a particular purpose such as, without limitation, hospital-based diagnosis such as for example, the diagnosis of acute respiratory infections, where approximately 100 probes may be needed. The expanded library may, however, comprise 105 or 106 probes and when of this size it is expected to pick up mimitopes (epitopes mimicking the original native epitopes), this will make the library extremely powerful and useful for certain applications, such as the investigation of cross-reactive antigens for autoimmune diseases and biomarker discovery.
In a preferred embodiment of the invention said binding partner has at least one epitope that is specific for said target molecule(s) but, ideally, it has a plurality of epitopes that are specific for said target molecule(s).
Most preferably, said binding partner comprises at least one and, ideally, a plurality of peptides and/or proteins which, individually or collectively, comprise at least one and, preferably, a plurality of epitopes that are specific for said peptide or protein to be detected.
In a further preferred embodiment of the invention said probe is further provided with a tag or label that facilitates the identification of same in a multiplex assay. Tags or labels of this sort are characterised by being amplifiable by PCR and so, ideally, comprises a further short DNA sequence of a distinctive nature that is, preferably, easy to read.
When working the invention a group of probes for detecting a specific type or class of target molecule(s) may be provided with a common tag whereby the presence or amount of this type or class of target molecule(s) can be determined using said tag prior to, or possibly after, detecting individual members of the class using the distinctive barcode. Alternatively, a specific type of sample may be provided with a common tag whereby the detection of a particular target molecule(s) in the assay can be linked to a particular sample, for example, and without limitation, a particular tag may be used to designate a particular patient sample and the barcodes associated with the different probes may be used to detect different target molecule(s) found in or associated with that patient sample.
In some respects this tag or label can be viewed as a secondary barcoding system. The first identification sequence of nucleotides or barcoding region (typically between 18-5 nucleotides) is used to identify specific target molecule(s) whilst the secondary barcoding system is used to identify specific samples or groups/types of target molecule(s). For example, where specific samples are to be monitored, if 10 different serum samples are investigated in one study, we can combine all the PCR products into a single next generation sequencing run (greatly reducing cost), and the secondary barcoding will allow us, during sequence analysis, to identify the particular sample from which each specific target molecule(s) came.
In a further preferred embodiment of the invention said tag or label is attached to said probe at a site remote from said binding partner so as not to interfere with the binding function of same. Ideally said tag or label is incorporated into at least one of the primer sequences i) or ii) of the oligonucleotide b) of the probe of the invention. More preferably said tag or label is incorporated into both primer sequences i) or ii) of the oligonucleotide b) of the probe of the invention.
In a further preferred embodiment of the invention said first sequence is positioned nearest to said binding partner and said second sequence is positioned furthest away from said binding partner. Alternatively, said second sequence is positioned nearest to said binding partner and said first sequence is positioned furthest away from said binding partner.
In a further preferred embodiment of the invention said identification sequence of nucleotides or Barcode comprises, or consists of the following group of nucleotides, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 nucleotides and, in any event, a number of nucleotides sufficient to provide the number of sequence combinations required to work the assay. For example 10 nucleotides provide for 1,048,576 combinations whereas 15 nucleotides provides for 1,073,741,824 combinations. Our current preferred design contains 16 nucleotides for 4,294,967,296 combinations.
In a further preferred embodiment of the invention for low-throughput applications, said barcode region may include or comprise at least one restriction enzyme site for the enzymatic cleavage of same, such as a BamH1 or HindIII site, although any other suitable restriction enzyme site known to those skilled in the art may be used.
In a further preferred embodiment of the invention said probe comprises single-stranded DNA, although double-stranded DNA may be used to provide stability, reduce non-specific interactions and alleviate potential steric hindrance.
According to a second aspect of the invention there is provided a plurality of probes for use in a multiplex assay to detect at least one target molecule(s) in at least one sample wherein each probe comprises:
In a preferred embodiment of the invention said first and second sequences are common to a number, and ideally all, of the probes to facilitate the amplification of said oligonucleotide in the herein described method of the invention.
In yet another preferred embodiment of the invention at least two of said probes, and ideally more, are provided with different binding partners whereby a number of different target molecules(s) in at least one sample can be identified.
More preferably still, probes used to detect a specific type, or group of target molecules(s) are provided with a first common tag or label whereas probes used to detect another specific type or group of target molecules(s) are provided with a second common tag or label. Additionally or alternatively, probes used to identify a specific sample are provided with another common tag or label. Ideally, these tags or labels are short nucleotide sequences provided in at least one, or both, of the primer regions i) or ii) of the oligonucleotide b) of the probe of the invention. In this context short means 3-15 nucleotides long, ideally 9-11 nucleotides long including any one of 9, 10 or 11 nucleotides. The current preferred tag/label in our design is 10 nucleotides long.
According to a third aspect of the invention there is provided a method for detecting at least one target molecule(s) in a sample comprising:
According to a fourth aspect of the invention there is provided a multiplex method for detecting at least one target molecule(s) in at least one sample comprising:
In a preferred method of the invention separating said conjugates can be undertaken using any preferred laboratory technique such as washing, filtration, migration, precipitation, immuno-precipitation or centrifugation.
Ideally, immuno-precipitation is practiced where antibodies to the binding partner of the probe, or the peptide or protein to be detected, are used to selectively remove the conjugate(s) from the sample, ideally the antibodies are monoclonal, although polyclonal antibodies may also be used.
In a further preferred method of the invention detecting said target molecule(s) in said sample can be undertaken by sequencing said identification sequence of nucleotides or barcode; moreover, in the fourth aspect of the invention this can additionally or alternatively be undertaken by sequencing said tag.
In a further preferred method of the invention said sample is selected from the group comprising a sample of: blood; serum; semen; lymph fluid; cerebrospinal fluid; tears; saliva; urine; feces; tissue; and sweat. Alternatively, the sample may be an environmental sample such as water, soil or oil.
Those skilled in the art will know how to conduct polymerase chain reaction (PCR) to amplify said oligonucleotide.
Those skilled in the art will also appreciate that the specificity of the binding partner for its counterpart ensures the specificity of the assay and so eliminates non-specific binding or background noise, moreover, it also ensures specific binding at low concentrations and so where the size of the molecular signal is small. This feature, coupled with the PCR amplification step, ensures the small signal is detectable and so significantly increases the sensitivity of the assay. More advantageously still, the coupling of each probe with a tag ensures the results of the assay can be rapidly realized, thus increasing the efficiency of the system and lending it to high through-put screening. Additionally, the use of multiple probes within an assay method enables multiplex investigations and so enables one to determine whether a particular signal is present in multiple samples and/or whether a number of signals are present in either a single sample or multiple samples.
According to yet a further aspect of the invention there is provided a kit for detecting at least one target molecule(s) in at least one sample comprising: at least one probe or a library of probes in accordance with the invention, optionally, at least one primer pair for polymerase chain reaction (PCR) amplifying said probe and/or sequencing said probe and/or reagents or instructions pertaining thereto.
Those skilled in the art will appreciate that in so far as the probe is concerned the invention involves:
2) Specific incorporation of a unique identifier or bar-code (BC) region in O so that a large number of P-O probes can be used in a single assay/tube; and ideally
Those skilled in the art will also appreciate that the invention, advantageously, can be worked by creating probe libraries that are specific for particular lines of enquiry or investigation. Thus, for example, libraries can be created that include probes designed to detect selected pathogens, such as bacteria and viruses and, more advantageously probes that are designed to detect the immunodominant epitopes of said pathogens. Yet more particularly, libraries of probes can be created to detect pathogens known to cause specific diseases, such as, but not limited to, human encephalitis or respiratory diseases. Indeed, a library of probes may be created, containing, e.g. 100-150 P-O probes covering the major respiratory diseases. Further, libraries of probes can be created to undertake serological testing to determine, for example, the presence of enteroviruses.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.
Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.
Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.
Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
The invention will now be described by way of example only with reference to the following figures:
Although the invention can be applied to all areas of biological research and development, we will use the monitoring of immune responses (antibodies) to illustrate the mode of practice.
As shown in
Specific steps of practice:
Although different methods can be used to capture specific peptide or protein and binding partner-P binding, we prefer to do this in liquid phase to increase the specificity (i.e., reduce background binding). Magnetic beads coated with, e.g. specific antibodies (e.g., anti-human IgG or anti-human IgM) are incubated with human serum first, followed by extensive washing. The P-O probe library will then be added to the antibody-bead mixture in a suitable buffer system. After incubation, the beads will be washed extensively to remove any unbound P-O probes.
We herein demonstrate the working of the invention using two example probes/P-O and positive antibodies specific for each. The target protein. in this case was epitopes specific for influenza virus and denuge virus.
Antibodies Used in this Study
We have also designed the oligo portion of the P-O conjugate so that the products of the PCR enrichment step are immediately ready for quantification by deep sequencing. In addition, we have also developed a qPCR assay that has the ability to discriminate and detect the bar-coding region of our probe(s). Deep sequencing analysis typically will be used primarily for high throughput screening of samples whereas qPCR is more likely to be the platform of choice for low-throughput applications of our technology. These new methodologies are outlined in
Step 2—Detection: The magnetic beads are collected directly into a 50 μl PCR mastermix that contains Ion Torrent specific primers. PCRs are performed with Pfu proofreading polymerase. The P-O specific region of the Ion Torrent primer binds the 18nt primer complementary sequences of the probes located outside the P-O barcode. This 18nt sequence on either side of the P-O barcode is typically identical for all P-O conjugates, allowing a multiplexed Ion Torrent PCR. Most preferably, each Ion Torrent primer set also contains a unique tag or label sequence, in addition to an adapter sequence. The captured oligos are amplified by PCR, and then column purified to remove PCR reagents and magnetic beads. The purified PCR product is eluted in 10 μl and the quality and quantity of the DNA is interrogated on a bioanalyzer DNA 1000 Chip. This sample is then analysed by Ion Torrent NGS. The sample may also be monitored by Taqman quantitative PCR, where the Taqman probe is specific for the P-O barcode.
In addition to the probes shown in
In each case, a specific enrichment was observed thus illustrating the functionality of the P-O probe design.
The results of quantification by deep sequencing are presented in
Whilst the above information exemplifies the technology using a protein or peptide in a sample and thus a probe with a protein or peptide binding partner specific for the protein or peptide in the sample, we have also worked the invention using a glycan/carbohydrate in a sample and thus a probe with a binding partner specific for the glycan/carbohydrate in the sample. In FIG. 10, we show data for the glycan-oligo concept using a modified, indirect conjugation method employing biotin-streptavidin binding. These data show that using this methodology, we can enrich and detect specific glycans up to 14-fold relative to the negative control.
1) Functionality of P-O probes: the trial confirmed that it is possible to conjugate a peptide or protein binding partner P with an oligonucleotide O and maintain the functionality of both entities
| Number | Date | Country | Kind |
|---|---|---|---|
| 1218909.8 | Oct 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2013/000455 | 10/22/2013 | WO | 00 |
