Patent Application
20230243842
The present disclosure relates to a method for measuring the amount of a target protein in body fluid using an isotope-labelled standard protein by means of mass spectrometry. The disclosure furthermore relates to a kit comprising at least one binding agent and at least one isotope-labelled internal standard protein.
Measurement of protein levels in body fluid is an essential component of assessing the health state of an individual. A large number of proteomics technologies have successfully been established and implemented into clinical practice, and are capable of providing information describing patients at the molecular level. More than one hundred clinical protein assays have been approved by the US Food and Drug Administration (FDA) for use in serum or plasma, and an equally large number of targets have been cleared for standardized laboratory tests in the US.
The antibody-based enzyme-linked immunosorbent assay (ELISA) is considered as the gold standard for quantitation of soluble proteins. ELISA provides rapid and robust results capable of sample analysis in high-throughput. However, this and other antibody-based assays are often limited to analysis of a single target protein or a limited number of target proteins (single-plex or low-plex assays). The reason may be cross-talk between probes, especially when using colorimetric read-out.
On the other hand, mass spectrometry (MS) technologies are capable of simultaneous analysis of a plurality of target proteins (multiplex), due to the high speed of the detector and the separation by mass. This is especially true when MS is used together with liquid chromatographic separation of proteins or peptides (LC-MS). The read-out, in combination with the use of affinity capture, can efficiently compensate for any selectivity biases introduced by antibodies in e.g. ELISA when quantifying proteins from a complex matrix.
An example of a technology for quantifying proteins by MS is the SISCAPA technology (“Stable Isotope Standards and Capture by Anti-Peptide Antibodies”). The technology uses a synthetic stable isotope labelled peptide as standard. A sample comprising target protein is digested using a proteolytic enzyme, such as trypsin. An antibody is then used to capture and thus enrich peptides of the target protein and a stable isotope labeled standard of that target protein. The natural (sample derived) and internal standard (isotope labeled) peptides are quantitated by LC-MS/MS, and their measured abundance ratio is used to calculate the abundance of the target protein.
Stable Isotope Labeling with Amino acids in Cell culture (SILAC) is a technique based on MS that detects differences in protein abundance among samples using isotopic labeling. In short, cells are differentially isotopically labeled by growing them in light vs. heavy medium. A heavy medium comprises isotopic labelling of one of the amino acids therein. Samples of heavy vs. light medium grown cells are combined, and a mass spectrometer is able to distinguish between the different isotope labelled proteins. An advantage of this technology is that this methodology is very accurate and introduces very low quantitative bias due to spiking of multiple versions of the next to identical proteomes. However, the method is not feasible for plasma or serum as no equivalent standard matrix is available.
Kim et al (Clinical Chemistry 64(8):1230-1238, 2018) teaches that a target protein can be quantified via the addition of a synthetic, isotopically labelled version of that protein. This article discloses a serum sample comprising a target protein and a stable isotope-labeled internal standard protein analog thereof, which is subjected to antibody enrichment and subsequent tryptic digestion. The digested sample may be analyzed using MS.
US2011/0143379 relates to a method for detecting both full-length thioredoxin as well as a truncated form thereof (a naturally occurring cleavage product) in a sample. The protein and its cleavage product may be identified by an antibody that recognizes a 7 amino acid long sequence shared between the two forms. The sample may be digested and analyzed by for example MS.
Although several methods for determination of proteins in body fluid exist, accurate determination of protein concentration in complex mixtures often suffers from inherent bias in and/or inefficiency of the methods used. There is still a need for improved accuracy and effectiveness. In other words, there is a call for new methods for measuring the amount of a target protein in body fluid.
It is an object of the present invention to at least partly reduce or overcome challenges in the prior art, and provide means for accurate measurement of an amount of a target protein in body fluid.
It is another object of the present invention to provide means for accurate measurement of more than one target protein in the same sample, the sample being a sample from body fluid.
These and other objects, which will be apparent to a skilled person from the present disclosure, are achieved by the different aspects of the disclosure as defined in the appended claims and as generally disclosed herein.
In a first aspect of the disclosure, there is provided a method for measuring the amount of a target protein in body fluid. The method comprises the initial step of preparing a sample suspected to comprise a target protein and adding a known amount of an isotope-labelled internal standard protein. The standard protein consists of a fragment of the target protein. Furthermore, the method comprises a step of bringing the prepared sample into contact with a solid support comprising a binding agent. The solid support is then washed in order to remove unbound members of the sample. Remaining target protein and standard protein are thereafter digested, providing a digested sample. After digestion, the digested sample is subjected to mass spectrometry, whereafter the amount of target protein in the sample can be determined by comparing with the standard protein. In the method as disclosed herein, the binding agent is capable of binding an epitope present in both the target protein and the standard protein.
An embodiment of the first aspect of the disclosure is illustrated in
The method of the present disclosure is based on co-capture and digestion of a target protein together with its corresponding stable isotope-labelled standard protein. As disclosed herein, the target protein and its corresponding standard protein are captured simultaneously, i.e. co-captured, using the same binding agent. As the proteins are treated the same in all steps, there is no inherent bias of the method.
In the method disclosed herein, the capture step precedes the digestion step. An advantage of the capture occurring first is that no capture related bias can occur from incomplete digestion of the sample. Conversely, when digestion of a sample precedes the capture step, kinetic parameters of the entity digesting the sample may be of importance. For example, it may be difficult to obtain a complete digestion of the proteins in the sample when a proteolytic enzyme is used before capture. This may be due to different factors, such as limited diffusion, impaired enzyme activity, precipitation or aggregation of components in the sample, chemical modifications introduced by the enzyme (e.g. deamidation), and/or mis-cleavages (e.g. trimmed ends). Another risk is that the sample is over-digested if an enzyme with inferior specificity is used, in particular when incubation is prolonged or if digestion conditions, e.g. pH or temperature, are not optimal for that specific enzyme. That is, the proteolytic enzyme may cleave unspecifically at protein sites in the sample which do not correspond to an actual cleavage site for that enzyme.
When digesting proteins in a sample, the three dimensional folding structure of the digested proteins may disappear. An advantage of the capture step preceding the digestion step is that binding agents directed towards folded proteins may be used. For example, it is well known in the art that high quality antibodies towards peptides may be difficult to generate, as compared to generation of antibodies towards undigested proteins. The reason may be that the latter may maintain folded structures while the former may not, or may not maintain a three-dimensional structure to the same extent. The provision of antibodies towards folded structures may enable increased accuracy due to e.g. satisfactory binding kinetics.
The method of the present disclosure uses a labeled standard protein, which is added at a known amount. The standard protein is a fragment of the target protein, as discussed above. Because it is identical to the target protein over the length of the standard protein, except in a label, which label preferably is in the form of an isotope, the ratio between the standard protein and the target protein can be detected by MS. This enables the concentration of the natural target protein in the sample to be calculated.
Another advantage of the present disclosure is that the use of a binding agent for capturing target protein in the sample before digestion enables determination of the quantity of low abundant target proteins in a sample, since low abundant proteins can be enriched in the capturing step.
A related advantage is that when the sample comprises high abundant target proteins, determination of the quantity of the high abundant target proteins is enabled because the number of binding agents can be adjusted to capture a fraction of the high abundant proteins present. This concept is discussed further below.
When a sample is brought into contact with a binding agent, unbound members of the sample may be depleted by washing, resulting in a less complex sample. A further advantage of using the binding agent in a capture step before digestion in this way is that it allows higher sensitivity in the analysis, because background complexity in the sample is decreased. Another effect of having a less complex sample is that a lower amount of a digestive entity, such as a proteolytic enzyme, needs to be used.
Yet another advantage of using the binding agent in a capture step before digestion is that this may enable a faster analysis time due to an increased purity of the sample.
In some embodiments, the target protein is a soluble protein. In some embodiments, the target protein is a tissue specific biomarker. Non-limiting examples of tissue specific biomarker are prostate specific antigen (KLK3) or troponin originating from the heart. In preferred embodiments, the target protein is a water soluble protein. As used herein, water soluble proteins are defined as proteins that are at least partly soluble in water, as apparent to a person of skill in the art. In some embodiments, the target protein is a leakage product. Non-limiting examples of leakage products are highly abundant proteins such as histones; vimentin; or enzymes such as aspartate transaminase, prostate specific antigen (KLK3) or troponin originating from the heart. In some embodiments, the target protein is an actively secreted protein, i.e. a secretory protein that is secreted by a cell. In some embodiments, the secretory protein is a protein secreted into blood. Non-limiting examples of a target protein that may be secreted into blood are presented in Table 1. In some embodiments, the target protein is an FDA qualified biomarker. At the time of filing, a non-limiting list of FDA qualified biomarkers could be found at The U.S. Food and Drug Administration website, “List of Qualified Biomarkers” [retrieved on 2020-02-28]. Retrieved from <https://www.fda.gov/drugs/cder-biomarker-qualification-program/list-qualified-biomarkers>
In some embodiments, the target protein is selected from the group consisting of a cytokine, a chemokine, an interleukin, an interferon, a hormone, a neuropeptide, a growth factor, a receptor, a protein involved in transport, a protein involved in development, an enzyme, an enzyme inhibitor, a protein involved in the immune system, a protein involved in coagulation, a protein involved in the complement pathway, an acute phase protein and a cell adhesion protein.
In some embodiments, the solid support is a bead, such as a magnetic bead, or a column. Other types of solid supports are also possible, as apparent to the person of skill in the art. The binding agent may be attached to the solid support using techniques known to a person of skill in the art. According to the method disclosed herein, a sample suspected of comprising the target protein and its corresponding standard protein is brought into contact with a solid support. The contact enables binding of any target protein and its corresponding standard protein to the support via the binding agent. Due to the specificity of the binding agent, the remaining members of the sample do not bind, or bind only to a lesser extent. Any unbound members are discarded in a subsequent washing step, resulting in a higher purity of the sample.
The amount of target protein and its corresponding standard protein being captured can be adjusted by modifying the amount of binding agents used in the capturing step. For example, when capturing a high abundant target protein, all target proteins may not be bound by the binding agent. Thus, any unbound target protein and corresponding standard protein are discarded in a subsequent washing step. This results in a reduced amount of high abundant target protein and its corresponding standard protein in the captured sample. Moreover, when at least two target proteins of different abundance are to be quantified, the amount of binding agent used can be adjusted to compensate for this difference. This concept is further discussed below.
According to the method as disclosed herein, a sample comprising the at least one target protein and the at least one standard protein is digested. The digestion occurs after the washing step. In the digestion step, any captured material present on the solid support may be cleaved off.
Several different techniques for digesting proteins or peptides into smaller entities are contemplated, and would be apparent to the person of skill in the art. In some embodiments, the sample may be chemically digested. In other embodiments, the sample may be digested by means of a biological molecule, such as an enzyme. In preferred embodiments, the digestion is carried out by means of a proteolytic enzyme. The proteolytic enzyme may for example be trypsin.
In order to provide for accurate measurement of the digested sample, which comprises digested fragments of the target proteins and digested fragments of the standard protein, the digestion may be carried out such that the digested sample comprises at least one isotopically labeled standard peptide from the isotopically labeled standard protein, said peptide consisting of between 6 and 25 amino acids.
It is well known in the art how to produce a protein. A protein may for example be produced by means of recombinant DNA technology, or may be produced by means of a peptide synthesizer. In some embodiments, the standard protein is a recombinant protein. In other embodiments, the standard protein is a synthetic protein.
In some embodiments, the standard protein comprises a cleavage site for a proteolytic enzyme. In some embodiments, the standard protein comprises at least two cleavage sites for a proteolytic enzyme. In some embodiments, the standard protein comprises at least three cleavage sites for a proteolytic enzyme. An advantage of having one or more cleavage sites for a proteolytic enzyme is that multiple peptides may be used for protein quantitation, which may improve accuracy and/or precision. This can also help increase the quantitative accuracy across the protein sequence, based on sequence coverage, as it is known by persons of skill in the art that longer sequences provide higher accuracy in general.
As discussed above, the target protein and a corresponding standard protein may be distinguished from one another by at least one isotopically labelled amino acid in the standard protein. In some embodiments, the standard protein is labelled with at least one isotope selected from the group consisting of 15N, 13C and/or 18O. In another embodiment, the standard protein is labelled with deuterium. As apparent to the person of skill in the art, incorporation of other isotopes is also possible.
In some embodiments, the binding agent is an antibody or an antibody fragment. In some embodiments, the antibody or antibody fragment is a monoclonal antibody or fragment thereof. In some embodiments, the antibody or antibody fragment is a polyclonal antibody or fragment thereof. In some embodiments, the antibody fragment is an scFv. In some embodiments, the binding agent is an antibody mimetic. The binding agent may be produced and developed as disclosed in the appended examples.
The method as disclosed herein may be used to measure a plurality of target proteins in parallel (multiplex mode). An advantage of measuring several proteins together is that the result may give a more accurate basis for providing a diagnosis, when several proteins are associated with the particular diagnosis. In some embodiments, the measurement of target protein comprises measuring the amount of at least two target proteins, such as three target proteins, such as four target proteins, such as five target proteins, such as six target proteins, such as seven target proteins, such as eight target proteins, such as nine target proteins, such as ten target proteins. As apparent by the person of skill in the art, the method as disclosed herein may enable measurement of at least 20 target proteins, of at least 30 target proteins, of at least 40 target proteins, of at least 50 target proteins, of at least 60 target proteins, of at least 70 target proteins, of at least 80 target proteins, of at least 90 target proteins, or of at least 100 target proteins. In order to measure more than one target protein, more than one binding agent may be used in the step of capture. It is preferred that for each target protein, a corresponding binding agent is used. For example, when measuring 10 target proteins, 10 binding agents may be used. The corresponding binding agent may bind a target protein selectively. It is preferred that the binding agent does not bind other target proteins, or only does so with low affinity.
An advantage of capturing the sample is that this step may allow the dynamic range of highly and lowly abundant proteins to be adjusted. In some embodiments, at least two target proteins are present in the sample at a relative concentration of at least 10× in difference, such as 100× in difference, such as 1000× in difference, such as 10 000× in difference, such as 100 000× in difference, such as 1000 000× in difference, such as 10 000 000× in difference. For example, when a first target protein is present in low amounts in the sample while a second target protein is present in higher amounts in that sample, one may adjust the amount of binding agents used so that a greater number of binding agents are used for the low abundant target protein and a smaller number of binding agents are used for the high abundant target protein. This may provide a sample in which substantially all of the low abundant target protein is captured while only a certain fraction of the high abundant target protein is captured. In this way, the sample may, after the capture step, comprise amounts of the first and the second target proteins, i.e. here the low and high abundant target proteins respectively, that are within the same range, or at least closer in range, when subjected to mass spectrometry. This can be seen as the low abundant target being enriched while the amount of the high abundant target is reduced.
Another advantage of adjusting the amount of binding agent in this and related embodiments is that the sample may not be need to be diluted before mass spectrometry, which is otherwise usually the case for samples comprising high abundant proteins. As apparent to persons of skill in the art, needing to dilute a sample comprising a low abundant protein for the purpose of reducing the concentration of high abundant protein also present, may lead to such a reduction in the content of the low abundant protein that it may practically disappear.
Adjusting the amount of high and low abundant proteins in this way enables analysis of the sample with increased accuracy, because the concentrations of target proteins can be brought to be within the same range. Adjustment of the amount of binding proteins is also plausible when the at least two target proteins are three target proteins, such as four target proteins, such as five target proteins, such as six target proteins, such as seven target proteins, such as eight target proteins, such as nine target proteins, such as ten target proteins. Furthermore, one may adjust the number of different binding proteins to be added when measuring an amount of at least 20 target proteins, such as at least 30 target proteins, such as at least 40 target proteins, such as at least 50 target proteins, such as at least 60 target proteins, such as at least 70 target proteins, such as at least 80 target proteins, such as at least 90 target proteins, such as at least 100 target proteins, enabling analysis of the sample when the concentrations of target proteins are within the same range. As used herein, the phrase “within the same range” is an approximate description of an amount, allowing relative concentrations of for example 1:1, up to 5:1, up to 10:1, up to 20:1, up to 30:1, up to 40:1, up to 50:1, up to 60:1, up to 70:1, up to 80:1, up to 90:1, up to 100:1, up to 1000:1, of a first target protein and a second target protein.
In some embodiments, the at least one target protein suspected to be present in the sample, is present in a concentration of between 10−4 and 10−10 M, such as between 10−6 and 10−7 M. An advantage of using the method according to the present disclosure is that the use of a binding agent for capture before digestion enables accurate determination of a target protein which is present in very small amounts in a sample. In some embodiments, the binding agent is present in substantial excess over the sum of the target protein and the corresponding standard protein. Thus, the highest possible amount of bound target protein and bound corresponding standard protein may be achieved.
In some embodiments, the binding agent binds the target protein and the corresponding standard protein with different affinities. The difference in affinity will lead to an established ratio in which the target protein and the corresponding standard protein are present after the capture step. This ratio may be maintained throughout the remaining steps of the method. One may account for the ratio when determining the amount of target protein by comparing with the standard protein.
In some embodiments, the target protein and the standard protein bind to the binding agent with comparable affinity, at a ratio of KD values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15.
In order for the invention to work, the binding protein epitope must be shared between the target protein and its corresponding standard protein. That is, the target protein and the corresponding standard protein comprise the same epitope, and the binding agent recognizes this epitope when present on the target protein as well as when present on the corresponding standard protein. In some embodiments, the epitope comprises at least 4 amino acids, such as 5 amino acids, such as 6 amino acids, such as 7 amino acids, such as 8 amino acids, such as 9 amino acids, such as 10 amino acids, such as 11 amino acids, such as 12 amino acids, such as 13 amino acids, such as 14 amino acids, such as 15 amino acids, or more. In some embodiments, the epitope is linear. In other embodiments, the epitope is a conformational epitope.
In some embodiments, the body fluid is selected from the group consisting of plasma, serum, cerebrospinal fluid, urine, dry blood spots and saliva. In some embodiments, the body fluid is from a mammal. In some embodiments, the mammal is a human.
In some embodiments, the method is preceded by a step of approximation of the amount of target protein by establishing a standard curve, such as a forward standard curve or a reverse standard curve. An advantage of performing an approximation step is that one can estimate the amount of target protein in a sample, and adjust the addition of a standard protein to an amount within the same range as the amount of target protein. A definition of “within the same range” is given elsewhere herein. When at least two target proteins are to be measured, the amounts of the binding agents may be adjusted, as discussed above. Another advantage of performing an approximation of the amount of target protein in a sample, is that such approximation may enable further optional steps of ensuring that all the peptides are within a mass spectrometer's dynamic range and of using this dynamic range optimally. In some embodiments, the affinity of the binding agent for the target protein and its corresponding standard protein may differ. In those embodiments, the affinity of the binding agent to the full length target protein and for the full length standard protein may be measured to establish a ratio between the two affinities. The ratio may be used for determining a suitable amount of a standard protein to be added to a sample, in order to enable an outcome within the same range when carrying out a mass spectrometry analysis.
In some embodiments, the step of digesting the target protein and the standard protein is preceded by a step of eluting said target protein and said standard protein. An advantage of eluting the target proteins and their corresponding standard proteins before digestion is that the solid support may then be reused in another experiment.
In some embodiments, the step of subjecting the digested sample to mass spectrometry is preceded by a step of liquid chromatography of the digested sample. A step of liquid chromatography may aid in obtaining a purer sample or a less complex sample. Liquid chromatography may separate fractions of a sample in terms of size, level of hydrophobicity, electrostatic attraction, or by affinity for a functional group. After separation, fractions of the sample that comprise target protein are selected and fractions that do not are discarded, thereby improving sample purity.
In some embodiments, the standard protein is added to the sample in an amount approximately equal to the amount of the target protein suspected to be present in that sample. An advantage of providing a sample wherein a target protein and a corresponding standard protein are present in approximately equal amounts may be that an MS reading within the same range is enabled. As understood by the person of skill in the art, approximately equal amounts of a target protein and a corresponding standard protein is understood to mean within the same order of magnitude or within the same range. For example, the approximately equal amounts may be at a ratio of target protein:standard protein of 1:10, such as 1:9, such as 1:8, such as 1:7, such as 1:6, such as 1:5, such as 1:4, such as 1:3, such as 1:2, such as 1:1, such as 2:1, such as 3:1, such as 4:1, such as 5:1, such as 6:1, such as 7:1, such as 8:1, such as 9:1, such as 10:1.
In some embodiments, the type of mass spectrometry used is tandem mass spectrometry with data dependent acquisition mode. In other embodiments, the mass spectrometry used is tandem mass spectrometry with data independent acquisition mode. In yet other embodiments, the mass spectrometry used is tandem mass spectrometry with selective reaction monitoring mode. As apparent for a person of skill in the art, other types of mass spectrometry methods are also possible to use. The mass analyzer of the mass spectrometry instrument may be an ion trap, a triple quadrupole, an ESI-TOF, a Q-TOF type instrument, an orbitrap, or any other instrument of suitable mass resolution (>1,000) and sensitivity.
As apparent to the person of skill in the art, any target protein in a sample may be analyzed according to the first aspect of the present disclosure. The following is a non-limiting list of possible target proteins, present in human plasma and secreted into blood (Table 1).
In a second aspect of the disclosure, there is provided a kit for carrying out the method according to the first aspect, in any one of the embodiments described herein. The kit comprises at least one binding agent, at least one isotope-labelled internal standard protein, and instructions for carrying out the method. The binding agent comprised in the kit is selected such that it binds a target protein and the standard protein with comparable affinity, at a ratio of KD values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15. In one embodiment of the second aspect, the binding agent of the kit may be a monoclonal antibody. In another embodiment of the second aspect, the binding agent may be an scFv fragment. All of the alternative embodiments of the first aspect of the disclosure discussed above are equally applicable to the second aspect of the disclosure, as would be readily apparent to a person of skill in the art.
As used herein, a “fragment” of a protein means any part of a protein or polypeptide, which is not the full length of that protein or polypeptide.
As used herein, a “body fluid” may be any liquid of the mammalian body. Non-limiting examples are intravascular fluid, intracellular fluid, extracellular fluid, interstitial fluid, lymphatic fluid and transcellular fluid.
As used herein, “comparable affinity” is defined as affinity within the same range, such as within a ratio of within 1:100, preferably within a ratio of 1:10, when comparing KD-values for a target protein and a corresponding standard protein. A comparable affinity may be for example at a ratio of KD values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15. In order to enable readings within the same range for both the target protein and the added standard protein, the amount of added standard protein may be adjusted, as discussed above.
As used herein, a “binding agent” may be any molecule, such as a biological molecule, that binds to a target protein such that the target protein is captured when carrying out the method of the disclosure. The binding agent may do so in a specific manner, such that both a target protein and a corresponding standard protein are bound and captured. As non-limiting examples, a binding agent may be selected from the group consisting of proteins, polypeptides, peptides, nucleic acids (oligonucleotides and polynucleotides), antibodies, ligands, polysaccharides, microorganisms, receptors, antibiotics and synthetic organic compounds.
As used herein, an “antibody” may belong to any class of immunoglobulin molecules of any species, or be derived therefrom, or be another specific binding agent constructed by variation of a conserved molecular scaffold so as to specifically bind an analyte or target protein or fragment thereof. In general, any use made of an antibody is understood as a use that could also be made of a binding agent as defined above.
The term “bind” includes any physical attachment or close association, which may be permanent or temporary (i.e. reversible). Generally, reversible binding includes aspects of charge interactions, hydrogen bonding, hydrophobic forces, van der Waals forces etc., that facilitate physical attachment between the molecule of interest and the analyte being measured.
The terms “internal standard”, “standard protein”, standard peptide”, “isotope-labeled or internal standard protein”, each mean an altered version of a respective internal standard protein that 1) is recognized as equivalent to the target protein or a fragment thereof by the appropriate binding agent and 2) differs from the target protein or a fragment thereof in a manner that can be distinguished by a mass spectrometer. It is preferred that the target protein and its corresponding standard protein are distinguishable through direct measurement of molecular mass, where a difference lies in the use of isotopic labeling of the standard protein. The distinguishing may also be made through mass measurement of fragments (e.g., through MS/MS analysis), or by another equivalent means.
Phage display selections was used to find binding agents having affinity for secretome targets using an in-house scFv library. For the development of binding agents, the following target proteins were used during the selection process: TNF, KLK3, REN, IL6, CRP, EPO, IL8, CLU, APOA1 and CGA. All proteins were produced and purified in-house.
Each target protein was diluted to a final concentration of 0.25 μg/μl in a 1×PBS solution containing a 10 times molar excess of biotinylation reagent (EZ-Link Sulfo-NHS-LC-Biotin) and incubated for 1 h in a rotomixer at room temperature (RT). After incubation, the target protein was transferred into a pre-hydrated Slide-A-Lyzer® cassette and any remaining air in the cassette was removed. The Slide-A-Lyzer® was then incubated in dialysis buffer (1×PBS) for 2 hours on a magnetic stirrer at RT. The dialysis buffer was replaced after two hours and the Slide-A-Lyzer® cassette was subsequently incubated overnight on a magnetic stirrer at 4° C. The sample was retrieved from the Slide-A-Lyzer® and transferred to 1.5 ml tubes and the concentration of the sample was determined using absorbance measurements at 280 nm.
In order to evaluate whether or not biotinylation had been successful, the target protein was subjected to a bead binding test. 100 μl magnetic beads (Dynabeads™ M-280 Streptavidin, SA beads) were washed thoroughly in PBST (1×PBS, 0.05% Tween20) and then resuspended in 100 μl PBST. 5 μg of biotinylated protein in 200 μl PBST was then incubated with 25 μl beads for 15 min in a rotomixer. The beads were separated from the supernatant and washed 4 times in 1,000 μl PBST and stored on ice. The supernatant was transferred to a new tube with 25 μl beads and this process was repeated a total of three times. The final supernatant was precipitated by addition of 1 ml of acetone and incubated at −20° C. for 60 min. The supernatant was then removed from the precipitate. The beads from the different fractions and the precipitate were then evaluated using SDS-PAGE to ensure sufficient biotinylation and retrieval.
40 μl SA beads per target protein were washed in PBST and then transferred to a 1.5 ml tube. The SA beads were then incubated in 200 pmol biotinylated target protein diluted in PBST to a total volume of 500 μl for 1 h on a rotomixer at RT. Additionally, 40 μl of beads were washed in PBST and incubated with 800 μl of phage library for 1 h in a rotomixer. The beads, incubated in target protein, were washed in 2×1 ml PBST and the supernatant from the beads incubated in the phage solution was mixed with the washed target protein beads and transferred to 96 well plate. The beads and phages were incubated on a KingFisher™ for 3 hours with slow mixing. The beads were then washed in 800 μl PBST 5×1 min with slow mixing and eluted using 500 μl elution buffer (0.25% trypsin, 0.02% Tween20) and 30 min incubation. The eluate was transferred to 1.5 ml tubes and 250 μl aprotinin was added to each tube (0.2 mg/ml).
In order to amplify the eluted phages, 0.6 ml of the eluted solution was transferred to 50 ml of actively growing XL1-blue cells (Agilent Technologies, cat no 200249) and incubated for 30 min at 37° C. The cells were subsequently spun down, and the cell pellet was resuspended in 2 ml supernatant. The resuspended cells were spread on an agar plate supplemented with tetracycline (10 mg/ml), carbenicillin (100 mg/ml), kanamycin (25 mg/ml) and 1% glucose and incubated at 37° C. overnight (O/N).
The colonies on the agar plate were resuspended in 2×10 ml 2×YT Broth and OD600 was measured for the cell suspension. 45 ml medium (2×YT Broth, TET10/CARB100/GLU1%) was inoculated with the cell suspension to a final OD600 of 0.15-0.2. The cells were incubated at 37° C. until they reached an OD600 of 0.5. The cells were then infected with M13K07 helper phages and incubated for 1 h at 37° C. The cells were subsequently pelleted through centrifugation and the medium was discarded. The pellet was resuspended in 1 ml 2×YT Broth and transferred to baffled flask containing 45 ml 2×YT Broth (tetracycline (10 mg/ml), carbenicillin (100 mg/ml), kanamycin (25 mg/ml), and isopropyl β-d-1-thiogalactopyranoside (0.25 mg/ml)) and incubated O/N at 30° C.
The culture was transferred to a 50 ml tube and centrifuged at 10000 g and the supernatant was transferred to a 50 ml containing 10 ml PEG/NaCl (20% PEG8000, 2.5 M NaCl) and incubated on ice for 60 min before being centrifuged at 12000 g for 30 min at 4° C. The supernatant was then discarded, and the pellet was resuspended in 2 ml PBST. The resuspended pellet was centrifuged at 10000 g for ten minutes and the supernatant containing the phages was collected in 2 ml tubes.
For the second selection round, 20 μl of SA beads per target protein were washed in PBST and transferred to a 1.5 ml tube and incubated in 100 pmol target protein diluted in PBST to a final volume of 500 μl and incubated on a rotomixer at RT for 1 h. The beads were washed with 2×1 ml PBST and 800 μl of the eluted phages from round 1 were added to the beads. The beads were incubated for 1 h at RT in a rotomixer. The beads were washed in 2×1 ml PBST and transferred to a 96-well plate. The beads and phages were incubated in a KingFisher™ for 1.5 hours with slow stirring and then washed 5×1 min with slow stirring in 800 μl PBST. The phages were then eluted with 500 μl elution buffer for 30 min with slow stirring and transferred to 1.5 ml tubes containing 250 μl aprotinin solution.
The phages were amplified by adding 350 μl of the eluate to 10 ml of actively growing (OD600: 0.5-0.7) XL1-blue cells (Agilent Technologies, cat no 200249) in a 50 ml tube and incubated at 37° C. for 30 min. Subsequently 10 ml of 2×YT Broth (TET10/CARB100/GLU1%) was added to the tube and it was incubated for 30 min at 37° C. with shaking at 180 rpm. The cells were then infected with M13K07 helper phages and incubated for 1 h at 37° C. The cells were then pelleted by centrifugation and the supernatant was discarded. The pellet was resuspended in 50 ml 2×YT Broth (TET10/CARB100/KAN25/IPTG0.25) and incubated O/N at 37° C. with shaking at 140 rpm.
The phages were precipitated as described above.
For the third round, 800 μl of the precipitated phages from the second round was mixed with 50 pmol target protein and incubated in a rotomixer at RT for 1 h. Meanwhile, 20 μl SA beads per target protein were washed in 2×1 ml PBST. The target protein-phage suspension was transferred into a 96-well plate together with the beads and incubated in a KingFisher™ for 30 min with slow mixing. The beads were subsequently washed with 6×1 min with 800 μl PBST with medium mixing and then eluted over 30 min in elution buffer with slow mixing. The eluates were then transferred to 1.5 ml tubes with 250 μl aprotinin solution.
The phages were amplified as described for round 2 and then precipitated.
For the final selection round, 800 μl of the precipitated phages from round three were incubated with 10 pmol target protein and then mixed with 20 μl washed beads in a 96-well plate, as described for round three. The mixture was incubated for 30 min with slow mixing in a KingFisher™ with slow mixing and the beads were subsequently washed 9×1 min in 800 μl PBST with medium mixing. The phages were eluted in 500 μl elution buffer over 30 min with slow mixing.
The phages from rounds three and four were then amplified as described for round two. 3 ml of each culture were saved for Miniprep purification.
In order to prepare the genetic material for re-cloning into an scFv expression vector, minipreps were performed using the GeneJET Miniprep kit (Thermo Scientific, #K0503) according to the supplier's instructions. The material was eluted in a final volume of 50 μl and the concentration was determined using a NanoPhotometer NP80 (Implen GmbH; MOnchen, Germany) and stored at −20° C.
The genetic material was subsequently cloned into an expression vector for optimized production of scFv fused to a FLAG tag, and transformed for production of scFv. The cells were subsequently grown on agar plates and 47 colonies from round three and 48 colonies from round four were selected for each target protein and transferred to 96-well plates and incubated O/N.
In order to determine which binding agents (in this case scFv fragments) performed the best in terms of signal to noise, all selected binding agents were evaluated against their respective target protein and SA.
A 384-well plate was coated in SA by addition of SA diluted in PBS to a final concentration of 1 mg/ml and incubation O/N at 4° C. The solution was removed and the plate was subsequently washed in assay buffer (PBS, 0.5% BSA, 0.05% Tween20). Any remaining liquid was removed. The biotinylated target proteins were diluted ten times in assay buffer and were added to half of the wells of the plates. The plate was incubated and subsequently washed in assay buffer. Each clone of the different binding agents was subsequently added to four wells of the plates (two wells with the respective target protein and two wells with SA) and incubated before being washed. HRP-anti-FLAG antibody was added to each well and the plates were incubated before being washed. Substrate was subsequently added to all wells and after quenching, the plates were analyzed through a plate readout at 450 nm.
For each target protein, the binding agents showing the highest signal to noise were selected and the clones (a total number of 384 clones) were sent for DNA sequencing by Eurofin Genomics. All unique clones were subsequently transferred to new 96-well plates and used for further analysis.
An initial screening was performed on a BIAcore T200 system for which a CM5 chip with immobilized anti-FLAG antibody (#M8592, Sigma-Aldrich) was used for the analysis. For the analysis, the chip was flowed with target protein specific scFv from the bacterial supernatants to saturate the surface of the chip by binding to the FLAG tag present on all scFvs. The chip was thereafter flowed with 50 nM of non-biotinylated target protein. The chip was regenerated using a 10 mM glycine HCl buffer with a pH of 2.1. The binding agents which bound to the target protein with the highest affinity were selected for a more thorough kinetics analysis.
The same workflow as described above was used for determining the kinetic constants for the binding agents, but instead of flowing the chip with one concentration of target protein a four-fold dilution curve ranging from 0.3 nM to 80 nM was flowed across the chip. The kinetic constants were determined by fitting a 1:1 Langmuir binding model to the response curves.
To assess the specificity of the binding agents an ELISA was used. The plates were prepared as described above, but instead of evaluating each binder against its respective target protein and SA, the binding agents were evaluated against all target proteins used in the selection process. Only binding agents showing high specificity towards their target protein were selected.
In order to assess the ability of the binding agents to bind recombinant protein standards, the affinity of the binding agents to different protein fragments from each respective protein target was evaluated by SPR as described above. Binding agents that were able to bind both target protein and recombinant protein fragments with similar affinity were used for co-capture of target protein and protein fragments for subsequent mass spectrometry (MS) readout.
Protein A-coupled beads were coated with the obtained binding agents for the targets APOA1, IL6 and IL8. 15 μl Protein A-coupled beads (Pierce/Thermo Fisher) per target protein were washed three times in 500 μl washing buffer (0.1 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, PBS). The washing buffer was removed, and the beads were resuspended in 30 μl binding buffer (0.5 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, 1×PBS). The beads were subsequently incubated with 1.2 μg of the respective binding agent in individual tubes and diluted to a final volume of 100 μl in binding buffer. The samples were incubated for 30 min at RT in a rotomixer. The supernatant was removed, and the beads were resuspended in 30 μl binding buffer. Beads coated with binding agent, i.e. “binding agent coated beads” had thus been obtained.
For the capture of target proteins and stable isotope labeled (Protein Recombinant Isotope Standard (PRecIS)) protein fragments of APOA1, IL6 and IL8, 10 μl of each of the binding agent coated beads were transferred to tubes with 20 μl pre-aliquoted binding buffer in duplicates. The amount of added binding agents was adjusted based on the endogenous amount of target proteins in order to decrease the dynamic range of captured molecules. The relative concentration between APOA1, IL6 and IL8 binding agents was 10:1:1 Additionally, a negative sample consisting of washed bare beads was prepared.
A mixture of the target proteins and the corresponding isotope-labelled standard proteins (PRecIS) in binding buffer (Table 2) was added to each tube. The samples were diluted to a final volume of 200 μl in binding buffer and incubated for 1 h at RT in a rotomixer. The supernatant was subsequently removed. The beads were thereafter washed twice in 500 μl 1× wash buffer and the supernatant was discarded. 10 μl of 10 mM dithiothreitol in 50 mM ammonium bicarbonate was added to the beads and the samples were incubated at 56° C. for 30 min. 1 μl of 500 mM 2-chloroacetamide was subsequently added to the tubes and the samples were incubated at RT, shielded from light, for 30 min. SOLu-Trypsin (#EMS0004, Sigma Aldrich) was thereafter added to the samples to a final amount of 0.5 μg and the samples were incubated O/N at 37° C. The digestion was quenched by addition 10% formic acid (FA) to a final concentration of 0.5% FA and the supernatant was transferred to a HPLC vial for analysis by MS.
For the MS analysis of captured proteins digested into peptides, samples were analyzed using a TSQ Altis (Thermo Fisher) coupled to a Dionex Ultimate 3000 liquid chromatography (LC) system (Thermo Fisher) equipped with a cartridge type trap column (160434, Thermo Fisher) and a 15 cm analytical EASY-spray column (ES806A, Thermo Scientific) with a mobile phase consisting of solvent A (3% acetonitrile (ACN), 0.1% FA) and solvent B (95% ACN, 0.1% FA). For the analysis, 10 μl of each sample was loaded onto the column and separated over a 15 min long run with a flow rate of 3 μl/min. The gradient used for the separation of peptides was as follows: 1% solvent B for 0.75 min, 1-30% solvent B for 9.25 min, 30-95% solvent B for 0.1 min, followed by a cycling between 0% and 95% solvent B three times during 3 min, finally followed by a re-equilibration of the column at 1% solvent B for 1.5 min. The MS was operated in an unscheduled SRM mode, monitoring the 10 most intense product ions for all tryptic peptides present both in the PRecIS protein fragments and the target proteins, with a dwell time of 0.5 ms. All data analysis was performed in Skyline Targeted Mass Spec Environment (University of Washington).
A set of eight target proteins were selected for assay development based on clinical relevance and reference concentration in plasma. They are listed in Table 3, sorted by increasing plasma concentration reference range for the corresponding protein.
Phage display selections were performed to find binding agents for the target proteins. An in-house scFv library was used as described in Example 1. The biotinylated recombinant proteins of Table 3 were subjected to western blot analysis in order to verify their molecular weight prior to phage display selection.
Successfully biotinylated target proteins were bound to magnetic streptavidin beads and used for phage display selection in a four round setup. Selected binding phages (successfully enriched clones) were eluted using trypsin/aprotinin and re-cloned into a scFv expression vector.
Binding of the scFv clones from the phage selection was verified by ELISA. Positive scFv clones were selected for surface plasmon resonance (SPR) measurement towards their target protein. Furthermore, those clones were in parallel screened towards isotope-labelled internal standard proteins corresponding to their target protein. The identity of the scFv binding agents and their binding constants were determined using a kinetic SPR setup (Table 4).
In total, six scFv binding agents towards IL8, eight towards IL6, three towards KLK3 and two binding agents towards APOA0 that successfully recognized the two different forms of their target were obtained. That is, the obtained scFv binding agents recognized their target standard protein as well as its corresponding isotope-labelled internal standard protein.
The affinity of the binding agent denoted “IL8-12” towards a corresponding isotope-labelled standard protein was determined to be 78 nM, while the affinity of the same binding agent towards the IL8 target protein was more than 3 times lower, determined to be 250 nM (Table 4, entry 5).
A pool of target proteins and corresponding standard proteins was established by pooling 10 pmol secretome APOA1 (30736 Da), 1 pmol secretome IL8 (11766 Da) and 1 pmol secretome IL6 (23470 Da) into a buffer comprising 1% casein hydrolysate, 0.3% (w/v) Chaps, and 1×PBS. To this mixture, the corresponding standard proteins were pooled in equimolar amounts (10 pmol PRecIS APOA1, HPRR3450265; 1 pmol PRecIS IL6, HPRR330007; and 1 pmol PRecIS IL8, HPRR2700195; Edfors et al (2019), J Proteome Res 18(7):2706-2718).
In addition, a sample of the mixture was spiked into a complex background of bovine serum albumin (BSA). This mixture served as a baseline for the following experimental procedure.
60 μl Protein A (Thermo Scientific) magnetic beads were washed three times with 500 μl wash buffer (0.1 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, 1×PBS). Supernatant was removed after first collecting the beads using a magnetic stand and beads were re-dissolved in 120 μl binding buffer (0.5 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, 1×PBS). A total of 30 μl beads were split into four separate tubes (for the binding agents towards IL6, IL8, APOA1 and negative control, respectively). The negative control consisted of bare beads with no binding agent. A total of 1.2 μg binding agent (anti-APOA1, anti-IL6 and anti-IL8 scFv (denoted IL8-5)), respectively, was immobilized onto the Protein A magnetic beads, by incubation for 30 minutes. Excess buffer was removed after first collecting the beads using a magnetic stand.
10 μl of the prepared pool of target proteins and corresponding standard proteins in casein background were added to each respective tube of beads comprising binding agent, together with 190 μl binding buffer. Target proteins and corresponding standard proteins were captured by incubation for 1 h at RT on a rotor mixer. The supernatant was then removed, and the beads were washed once with 500 μl 1× wash buffer (0.01 μg/μl casein hydrolysate, 0.03% (w/v) Chaps, 0.1×PBS) and once with 500 μl 0.1× wash buffer.
Captured proteins were reduced on the beads following the addition of 10 μl 10 mM DTT in 50 mM ammonium bicarbonate and incubated for 30 minutes at 56° C. The proteins were alkylated following addition of 2-chloroacetamide (CAA) to a final concentration of 50 mM and incubated in the dark for 30 minutes at RT. Digestion was performed by the addition of 0.5 μg trypsin to the sample. The sample was incubated overnight at 37° C. The reaction was quenched with formic acid to a final concentration of 0.5% (v/v). The beads were removed using a magnet, and the peptide digest was analyzed using LC-MS/SRM after addition on BSA.
The mass spectrometry read-out revealed that the binding agent anti-IL8 scFv successfully co-captures the target protein together with its corresponding standard protein (PRecIS) (
In addition, the ratio between target protein and corresponding PRecIS was quantified for the pool of target proteins (APOA1, IL6 and IL8 and corresponding PRecIS) diluted in BSA, and quantified without capturing (“Mix”). The “Mix” can be considered as the baseline for this experimental procedure. The same ratio was quantified for a non-diluted pool of the target proteins APOA1, IL6 and IL8 and corresponding PRecIS without capturing (“Target”). The “Target” represents a pool of non-diluted recombinant proteins and serves as a positive control.
Each protein name on the x-axis in
This experiment shows that IL8 and PRecIS-IL8 were specifically captured in the co-capturing using an anti-IL8 scFv, compared to anti-APOA1, anti-IL6 and the negative control. Thus, binding agents generated towards full length protein sequences can co-capture proteins with identical and or similar epitopes presented on their surface. The difference in affinity of the generated scFv binding agent for the target protein and the internal standard, respectively, resulted in less effective capturing of the PRecIS relative to its full-length recombinant protein (secretome). However, it does not matter which of the target protein or the PRecIS that binds with higher affinity, as long as one knowns the difference in affinity. As discussed in the general sections of the disclosure, this effect can be accounted for if the difference in efficiency is known. To conclude, such pull-down or co-capturing experiments can therefore successfully be used for quantitative proteomics.
1. A method for measuring the amount of a target protein in body fluid, the method comprising the following consecutive steps:
said binding agent is capable of binding an epitope present in both said target protein and said standard protein.
2. The method according to item 1, wherein said target protein is a soluble protein.
3. The method according to item 2, wherein said target protein is a water soluble protein.
4. The method according to any one of the preceding items, wherein said target protein is an actively secreted protein.
5. The method according to any one of the preceding items, wherein said secretory protein is a protein secreted into blood.
6. The method according to any one of the preceding items, wherein said target protein is an FDA qualified biomarker.
7. The method according to any one of the preceding items, wherein said target protein is selected from the group consisting of a cytokine, a chemokine, an interleukin, an interferon, a hormone, a neuropeptide, a growth factor, a receptor, a protein involved in transport, a protein involved in development, an enzyme, an enzyme inhibitor, a protein involved in the immune system, a protein involved in coagulation, a protein involved in the complement pathway, an acute phase protein and a cell adhesion protein.
8. The method according to any one of the preceding items, wherein said solid support is selected from the group consisting of a bead, such as a magnetic bead, and a column.
9. The method according to any one of the preceding items, wherein said digestion is carried out by means of a proteolytic enzyme.
10. The method according to item 9, wherein said proteolytic enzyme is trypsin.
11. The method according to any one of the preceding items, wherein the digested sample comprises at least one isotopically labeled standard peptide consisting of between 6 and 25 amino acids.
12. The method according to any one of the preceding items, wherein said standard protein is a recombinant protein.
13. The method according to any one of items 1-11, wherein said standard protein is a synthetic protein.
14. The method according to any one of the preceding items, wherein said standard protein comprises at least two cleavage sites for said proteolytic enzyme.
15. The method according to any one of the preceding items, wherein said standard protein is labelled with at least one isotope selected from the group consisting of 15N, 13C and/or 18O.
16. The method according to any one of the preceding items, wherein said binding agent is an antibody or an antibody fragment.
17. The method according to item 16, wherein said antibody or antibody fragment is a monoclonal antibody or fragment thereof.
18. The method according to item 16, wherein said antibody or antibody fragment is a polyclonal antibody or fragment thereof.
19. The method according to any one of items 16-18, wherein said antibody fragment is an scFv.
20. The method according to any one of items 1-16, wherein said binding agent is an antibody mimetic.
21. The method according to any one of the preceding items, wherein said measuring comprises measuring the amount of at least two target proteins, such as three target proteins, such as four target proteins, such as five target proteins, such as six target proteins, such as seven target proteins, such as eight target proteins, such as nine target proteins, such as ten target proteins.
22. The method according to item 21, wherein said at least two target proteins are present in said sample at a relative concentration of at least 10× in difference, such as 100× in difference, such as 1000× in difference, such as 10 000× in difference, such as 100 000× in difference, such as 1000 000× in difference, such as 10 000 000× in difference.
23. The method according to any one of the preceding items, wherein said at least one target protein suspected to be present in said sample, is present in said sample in a concentration of between 10−4 and 10−10 M, such as between 10−6 and 10−7 M.
24. The method according to any one of the preceding items, wherein said target protein and said standard protein bind to the binding agent with comparable affinity, at a ratio of KD values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15.
25. The method according to any one of the preceding items, wherein said epitope comprises at least 4 amino acids, such as 5 amino acids, such as 6 amino acids, such as 7 amino acids, such as 8 amino acids, such as 9 amino acids, such as 10 amino acids, such as 11 amino acids, such as 12 amino acids, such as 13 amino acids, such as 14 amino acids, such as 15 amino acids.
26. The method according to any one of the preceding items, wherein said epitope is linear.
27. The method according to any one of the preceding items, wherein said body fluid is selected from the group consisting of plasma, serum, cerebrospinal fluid, urine, dry blood spots and saliva.
28. The method according to any one of the preceding items, wherein said body fluid is from a mammal, e.g. human.
29. The method according to any one of the preceding items, wherein said method is preceded by a step of approximation of the amount of target protein by establishing a standard curve, such as a forward standard curve or a reverse standard curve.
30. The method according to any one of the preceding items, wherein said step of digesting said target protein and said standard protein is preceded by a step of eluting said target protein and said standard protein.
31. The method according to any one of the preceding items, wherein said step of subjecting said digested sample to mass spectrometry is preceded by a step of liquid chromatography.
32. The method according to any one of the preceding items, wherein said standard protein is added to said sample in an amount approximately equal to the amount of said target protein suspected to be present in that sample.
33. The method according to any one of the preceding items, wherein said mass spectrometry is selected from the list consisting of tandem mass spectrometry with data dependent acquisition mode, tandem mass spectrometry with data independent acquisition mode and tandem mass spectrometry with selective reaction monitoring mode.
34. A kit for carrying out the method according to any one of the preceding items, comprising
at least one binding agent,
at least one isotope-labelled internal standard protein, and
instructions for carrying out the method.
35. The kit according to item 34, wherein a target protein and said standard protein bind to said binding agent with comparable affinity, such as at a ratio of KD values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15.
36. The kit according to any one of items 34 or 35, wherein said binding agent is a monoclonal antibody.
37. The kit according to any one of items 34-36, wherein said binding agent is an scFv fragment.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20182679.9 | Jun 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/067375 | 6/24/2021 | WO |
