Patent Description:
In immunolabeling, antibodies are used for detection of molecules in biological and non-biological samples. Antibodies are immunoglobulin (Ig) proteins that bind with high specificity through its antigen-binding site to an antigen (target molecule). Typically the antigen is a protein, but can be any immunogenic agent such as a shorter amino acid sequence (peptide), polysacharide, lipid, toxin etc. The part of the target molecule to which the antibody binds is called epitope. Antibodies used for immunolabeling can be polyclonal or monoclonal. Polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes of one target molecule, while monoclonal antibodies show specificity for a single epitope. In general, monoclonal antibodies gender more specific immunolabeling signals than polyclonal antibodies.

The final step in immunolabeling is detection of a signal from the antibodies that has bound to the antigens in the sample. The signal is generated from some kind of reporter molecule. The reporter molecule can either be directly attached to the primary antibody, or attached to a secondary antibody that recognizes the primary antibody. Often several reporter molecules are attached to each antibody molecule. The reporter molecules used in immunolabeling vary depending on the nature of the detection method. The most common reporter molecules are enzymes for chromogenic detection or fluorochromes for fluorescence signals. Other examples are particles (e.g. gold particles, quantum dots), phosphorescent compunds (e.g. carbocyanide dyes), radioactive compounds (e.g. <NUM> or 32P labeled molecules) and transition metals (for mass spectrometry).

Immunolabeling can either be direct or indirect. The direct method is a one-step immunolabeling method and involves a primary antibody that is labeled with a reporter molecule. When the labeled primary antibody is added to a sample it binds to its corresponding target antigen in the sample and reveals the location and/or amount of the target molecule. Since the direct method utilizes only one step it is simple and rapid. However, in some applications, for example microscopy, the signal is often too weak and needs to be amplified.

The indirect method is a two-step labeling method that results in signal amplification. It involves a primary antibody (first step) that binds to the target molecule in the sample and a labeled secondary antibody (second step) that binds to the primary antibody. Since several secondary antibody molecules bind to each primary antibody molecule, the signal is amplified. The secondary antibody is usually raised against the immunoglubolin class of the animal species in which the primary antibody has been raised. For example, if the primary antibody is a mouse IgG antibody, the secondary antibody is an anti-mouse IgG antibody that recognizes all mouse antibodies of the IgG class.

Although the indirect method is beneficial when it comes to signal amplification, it gives rise to unspecific signals due to unspecific binding of the secondary antibody to endogenous antibodies present in the sample. In addition, it is also often desired to further amplify the signal, especially in fluorescence microscopy where it is crucial to override the autofluorescence of the tissue sample. One last amplification step can be introduced by using a biotinylated secondary antibody and labeled streptavidin. Streptavidin binds tightly to biotin and since several biotin molecules are conjugated to each biotinylated antibody, amplification is achieved. However, biotin is also naturally present in biological samples, which causes unspecific binding of streptavidin to the sample, unless the endogenous biotin is blocked. Hence, an alternative signal amplifying system is desired that (<NUM>) does not cause background signal from endogenous antibodies/biotin, and that (<NUM>) enables more amplification steps.

So far only single immunolabeling has been described. Additional variants of unspecific antibody cross-binding arise when using the indirect method for multi-immunolabeling. By using primary antibodies made in different species, each primary antibody can be detected with a corresponding secondary antibody that recognizes the Ig class of the animal species of the primary antibody. For example, if one primary antibody is made in rat and the other is made in rabbit, these two primary antibodies can be detected with one anti-rat and one anti-rabbit secondary antibody that are labeled with two different reporter molecules, for example two different fluorochromes. However, because most primary antibodies are made in a handful animal species (mostly mouse, rat, rabbit and goat), the probability of ending up with two primary antibodies of the same antibody class (species) greatly increases by each extra primary antibody that is included in the antibody panel for multi-immunolabeling of a sample. In addition, care must also be taken for each secondary antibody in the antibody panel, so that none of them belong to the same antibody class as any of the primary antibodies. Hence, because of this antibody cross-binding problem only a few primary antibodies can be amplified using the indirect method. Since the present invention does not cause antibody cross-binding it enables amplification of any number of primary antibodies.

Further attention is drawn to <CIT>, presenting different compositions. One embodiment of a composition comprises a first antibody having an affinity for an antigen and a second antibody having an affinity for the first antibody, wherein at least one antibody is conjugated to a marker, and wherein the antigen is not present in the composition.

<CIT> disclose methods and a reagent complex suitable for attaching detectable signals to specific-binding reagents, the complex comprising a first unlabelled antibody, wherein the first antibody has free specific--binding affinity for an analyte or an analyte-specific binding reagent; and a second antibody having specific--binding affinity for an Fc region of the first antibody, the second antibody being bound to the Fc region to form the complex and being labelled with a detectable signal.

<CIT> presents a method for detection of at least two cytokeratins selected from the group consisting of cytokeratin <NUM>, <NUM> and <NUM>, and/or soluble fragments thereof, in a sample.

<CIT> shows compositions and methods comprising proteins that bind specifically to adalimumab.

Additionally, <CIT> discloses a method and kit for quantitatively and/or qualitatively detecting one or more components in samples, including the use of metal-particle labelled reagents and an antibody conjugate.

Still further, <CIT> presents a method of detecting an antigenic substance, in animal tissue, in which a primary antibody to the substance is raised, labelled with a selected hapten such as DNP and contacted with the substance, after unwanted binding sites on the substance have been blocked by applying a wide spectrum of antibodies obtained from a different animal.

According to an aspect of the invention, the above is at least partly alleviated by a method for preparing a biological sample for use in an immunolabeling process, as defined by claim <NUM>.

In accordance to the invention, a signal enhancer system for immunolabeling enables an unlimited number of amplification steps on top of a labeling component, for example being a primary antibody, without any antibody cross-binding. The absence of antibody cross-binding also enables any number of different labeling components (e.g. the labeling component and a plurality of additional labeling components, for example being different primary antibodies) to be combined for multi-immunolabeling, regardless of what animal species the primary antibodies are made from. The invention is based on carefully chosen antigens that are used as unique tags and corresponding antibodies that are used for detection of the tags. The antigens are chosen so that the antigen is non-present in immunolabeling process, i.e. not present in the biological sample and not present in reagents that are used in sample processing or staining reagents.

As such, when introducing the labeling component provided with the first enhancer antigen, the first enhancer antigen has not been previously introduced (or comprised) in the immunolabeling process. Neither is a similar (identical, corresponding) antigen to be introduced subsequently in the immunolabeling process. In addition, the antigen should not be present in any further components used in the immunolabeling process, for example including reporter molecule used in the immunolabeling process, such as fluorochromes, chromogens, enzymes, etc. The antigen should furthermore not be present in any antibody comprised with the sample or used with the immunolabeling process, and the antigen should not be present in biotin or streptavidin.

As mentioned above, the labeling component is a primary antibody conjugated with the first enhancer antigen.

As understood from the above, the concept of the invention relies on the fact that the first enhancer antigen is not comprised with the sample or any reagents used in the immunolabeling process. The antigen may as such in accordance to some embodiments be seen as non-functional in relation to the sample, or in relation to immunolabeling process.

According to the invention, the antigens are formed from artificially formulated peptide sequences that are not present in any proteins in nature, and thereby the artificial peptides may serve as unique antigens that are not present in any biological sample. Such non-biological peptides can be designed using protein sequence databases, such as the universal proteome database. The artificially formulated peptide sequence may then be used for forming the first enhancer antigen and for subsequent generation of a corresponding first enhancer antibody for use in relation to the present invention.

The inventive concept is used for amplification, and thus the first enhancer antibody is conjugated with a second enhancer antigen, wherein the second enhancer antigen is non-present in the immunolabeling process and different from the first enhancer antigen, the method further comprising the step of providing a second enhancer antibody, the second enhancer antibody selected to solely bind to the second enhancer antigen. The unlimited amplification will, as understood based on the above, allow for the second enhancer antibody to possibly be conjugated with a third enhancer antigen, the third enhancer antigen being non-present in the immunolabeling process and different from either of the first and the second enhancer antigen. The process may of course continue with a "chain" of further antigens/antibodies.

It is preferred to allow the "last" enhancer antibody in the chain to be labeled with a reporter molecule. The reporter molecule is typically selected from a group comprising a fluorochrome, an enzyme, a peptide, quantum dots, and a transition metal. Other known or future reporter molecules are possible and within the scope of the invention, such as for example an oligonucleotide. The reporter molecule(s) are typically used in a subsequent detection/analysis process, such as for example by illumination of the biological sample under a microscope to detect a light from a fluorochrome. In such an embodiment the reporter element is preferably a fluorochrome.

The inventive concept has been described in relation to the use of a single labeling component used for labeling the biological sample. However, since the first enhancer antigen as selected in accordance to the inventive concept does not bind any labeling components, including antibodies, streptavidin or proteins used for labeling, the inventive concept may also be used in a multi-immunolabeling process, where more than one labeling component is used for labeling the biological sample. Thus, the inventive concept will essentially allow for the immunolabeling of a biological sample with an unlimited number of labeling components.

As the first enhancer antigens/first enhancer antibodies used in the multi-immunolabeling process are selected to be different from each other (as well as not previously or subsequently present in the immunolabeling process), the inventive concept allows for the use of a single first enhancer antibody for each of the different labeling components. The inventive concept also allows for the use of a chain of enhancer antibodies as discussed above. In any case, it is preferred, as above, that the last antibody in the chain is provided with a reporter molecule. In the present embodiment provided in relation to a multi color immunolabeling process, it is of course preferred that the reporter molecules are selected to generate different signals that can be separated in a subsequent analysis process.

The skilled addressee realize that different features of the present invention may be combined to create embodiments other than those described in the following, without departing from the scope of the present invention.

Referring now to the drawings and to <FIG> in particular, there is exemplified a process of preparing a biological sample <NUM> for use in an immunolabeling process. As a first step, a first enhancer antigen is selected in accordance to the criteria defined in accordance to the invention. That is, the first enhancer antigen should be previously (or subsequently) non-present in the immunolabeling process. Accordingly, the first enhancer antigen should not be present in the biological sample and not present in reagents that are used in sample processing or staining reagents. In addition, the antigen should not be present in any further components used in the immunolabeling process, for example including reporter molecule used in the immunolabeling process, such as fluorochromes, chromogens, enzymes, etc. The antigen should furthermore not be present in any antibody comprised with the sample or used with the immunolabeling process, and the antigen should not be present in biotin or streptavidin.

The selection process for the first enhancer antigen is such that it is formed, S <NUM>, from a preselected peptide sequence <NUM>, for example artificially formulated in a computerized process. The process for selecting the peptide sequence <NUM> as well as the formation of the first enhancer antigen from such a preselected peptide sequence involves numerous steps being well known to the skilled addressee and are therefore not further discussed. The antigen may also be non-peptide molecules.

Once the first enhancer antigen successfully has been formed, two separate steps are taken, including generating, S2, of a first enhancer antibody based on the first enhancer antigen, and providing, S3, a labeling component that is tagged with the first enhancer antigen. The generation process for the first enhancer antigen and first enhancer antibody also includes a plurality of steps known to the skilled addressee, including for example choice of immunogenic antigen, adjuvants, host animal, immunization, antibody selection, antibody purification, etc..

As discussed above, the labeling component is a primary antibody, where the first enhancer antigen has been conjugated with the primary antibody. The primary antibody binds directly to a target antigen comprised with the biological sample <NUM>, once being introduced with the biological sample <NUM>. Hence, the primary antibody is selected dependent on what type of target antigen comprised with the biological sample <NUM> that subsequently is to be detected/analyzed in e.g. an immunofluorescence process. As discussed, the primary antibody has been conjugated with the first enhancer antigen, and the first enhancer antibody has been generated based on the same first enhancer antigen. Thus, once the first enhancer antibody is introduced to the biological sample <NUM>, the first enhancer antibody will solely bind to the first enhancer antigen provided with the first enhancer antigen.

The first enhancer antibody may be utilized in different ways dependent on the application at hand, as will be exemplified in <FIG> and <FIG>. For example, and as is illustrated in <FIG>, it may in accordance to the invention be possible to form an enhancer chain for "amplifying" the detection of a target antigen <NUM> in the biological sample <NUM>.

As is shown in <FIG>, the enhancer chain may comprise a plurality of enhancer steps, i.e. where the first enhancer antibody has been provided (conjugated) with a second enhancer antigen selected and formed in a similar process as discussed above, as well as again taking into account the criteria set for the selection of antigen. The enhancer chain could thus be arranged to include an in essence unlimited number of enhancer steps, e.g. second, third, fourth, etc., enhancer antibody/antigen forming an expanding "three structure".

As discussed above, it is desirable to provide the last enhancer antibody in the chain (in <FIG> being the third enhancer antibody) with a reporter molecule, such as for example a fluorochrome. Other reporter molecules are possible, including for example an enzyme, a peptide, quantum dots, or a transition metal. Providing an antibody with a reporter molecule such as a fluorochrome is process known to the skilled addressee.

Turning now to <FIG>, where the biological sample <NUM> has been prepared in accordance to a multi-immunolabeling process, where a first <NUM>, a second <NUM> and a third <NUM> target antigen is to be subsequently detected/analyzed.

In a similar manner as discussed above, a primary antibody is selected for each of the target antigens <NUM>, <NUM>, <NUM>, in <FIG> denoted as primary antibodies A, B and C. A first enhancer antigen 1A is formed and provided with the primary antibody A, a first enhancer antigen 1B is formed and provided for the primary antibody B, etc. Similarly, corresponding first enhancer antibodies are generated for each of the first enhancer antigens 1A, 1B, 1C.

Each of the first enhancer antibodies are provided with a different reporter molecule, such as with different fluorochromes generating lighting within different wavelength ranges, thus making detection and analysis of each of the target antigens <NUM>, <NUM>, <NUM> possible. It would of course be possible, and within the scope of the invention, to form enhancer chains for each of the target antibodies <NUM>, <NUM>, <NUM>, in a similar manner as shown in <FIG>. Also, the concept discussed above e.g. in relation to <FIG> and <FIG> could of course be combined with known multi-immunolabeling processes, e.g. where the reporter molecules are on one or a plurality of primary antibodies, secondary antibodies or streptavidin (i.e. "prior-art" direct and indirect immunolabeling methods), and the concept involving the inventive enhancer antigens/antibodies are used for detection of one or a plurality of additional target antigens of the biological sample (still taking into consideration the antigen selection criteria as defined in accordance to the invention).

Claim 1:
A method for preparing a biological sample for use in an immunolabeling process, the method comprising:
- labeling the biological sample with a labeling component, the labeling component provided with a first enhancer antigen, and
- providing a first enhancer antibody, the first enhancer antibody selected to solely bind to the first enhancer antigen,
wherein:
- the first enhancer antigen is non-present in the immunolabeling process,
- the labeling component is a primary antibody conjugated with the first enhancer antigen, and
- the first enhancer antibody is conjugated with a second enhancer antigen, wherein the second enhancer antigen is non-present in the immunolabeling process and different from the first enhancer antigen,
- the enhancer antigens are formed from artificially formulated peptide sequences not present in any protein in nature,
wherein the method further comprises:
- providing a second enhancer antibody, the second enhancer antibody selected to solely bind to the second enhancer antigen.