METHOD FOR THE FORMATION AND USE OF AN IMMUNOLABELING COMPLEX

Abstract

The present invention generally relates to method for forming an immunolabeling complex, the immunolabeling complex comprising a labeled monovalent biotin-binding composition. The invention also related to the use of the antibody-reporter molecule complex for detecting a target in a sample.

Claims

1. A method for forming an immunolabeling complex for use in contacting with sample, wherein the method comprises: providing a biotinylated primary antibody comprising a primary antibody conjugated with a plurality of biotin molecules; providing a monovalent biotin-binding composition labeled with a reporter element, the monovalent biotin-binding composition comprising at least one of monovalent streptavidin or traptavidin; mixing, in a liquid phase in a reaction vessel, the biotinylated primary antibody with the labeled monovalent biotin-binding composition to form the immunolabeling complex; and adding a blocking agent to block unbound portions of the monovalent biotin-binding composition after the biotinylated primary antibody with the labeled monovalent biotin-binding composition are mixed, wherein the block agent comprises free biotin.

2. The method according to claim 1, wherein mixing the biotinylated primary antibody with the first labeled monovalent biotin-binding composition attaches the reporter element to the biotinylated primary antibody.

3. The method according to claim 1, wherein the sample is different from the first biotinylated primary antibody.

4. The method according to claim 1, further comprising: providing an instruction to use the immunolabeling complex for immunolabeling of a sample.

5. The method according to claim 1, wherein providing the biotinylated primary antibody and providing the monovalent biotin-binding composition are performed separately from mixing the biotinylated primary antibody with the labeled monovalent biotin-binding composition.

6. The method according to claim 1, wherein the reporter element comprises at least one of a fluorochrome, an enzyme, a peptide, quantum dots, or a transition metal.

7. The method according to claim 6, wherein the monovalent biotin-binding composition is conjugated with the reporter element.

8. The method according to claim 1, wherein the reporter element is an oligonucleotide.

9. The method according to claim 1, wherein the ratio between the monovalent biotin-binding composition and the biotinylated primary antibody is selected such that a risk of the monovalent biotin-binding composition blocking active binding sites of the biotinylated primary antibody is reduced.

10. The method according to claim 1, wherein the reporter element is an antigen.

11. The method according to claim 1, wherein the monovalent biotin-binding composition is modified to increase a number of conjugation sites to permit attachment of an increased number of reporter elements.

12. The method according to claim 1, wherein the monovalent biotin-binding composition is conjugated with macromolecules containing a plurality of molecules of a reporter element.

13. A method for immunolabeling a sample, comprising: forming an immunolabeling complex by: providing a biotinylated primary antibody selected to bind to target molecules in the sample, providing a monovalent biotin-binding composition labeled with a reporter element, the monovalent biotin-binding composition comprising at least one of monovalent streptavidin or traptavidin, mixing, in a liquid phase in a reaction vessel, the biotinylated primary antibody with the labeled monovalent biotin-binding composition, and adding a blocking agent to block unbound portions of the monovalent biotin-binding composition after the biotinylated primary antibody with the labeled monovalent biotin-binding composition are mixed, wherein the block agent comprises free biotin; and contacting the sample with the formed immunolabeling complex.

14. The method according to claim 13, wherein mixing the biotinylated primary antibody with the first labeled monovalent biotin-binding composition attaches the reporter element to the biotinylated primary antibody.

15. The method according to claim 13, wherein the sample is different from the first biotinylated primary antibody.

16. The method according to claim 13, wherein providing the biotinylated primary antibody and providing the monovalent biotin-binding composition are performed separately from mixing the biotinylated primary antibody with the labeled monovalent biotin-binding composition.

17. The method according to claim 13, wherein the reporter element comprises at least one of a fluorochrome, an enzyme, a peptide, quantum dots, or a transition metal.

18. The method according to claim 17, wherein the monovalent biotin-binding composition is conjugated with the reporter element.

19. The method according to claim 13, wherein the reporter element is an oligonucleotide.

20. The method according to claim 13, wherein the ratio between the monovalent biotin-binding composition and the biotinylated primary antibody is selected such that a risk of the monovalent biotin-binding composition blocking active binding sites of the biotinylated primary antibody is reduced.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The various aspects of the invention, including its particular features and advantages, will be readily understood from the following detailed description and the accompanying drawings, in which:

[0041] FIGS. 1a and 1b shows an example of a biological sample which has been incubated with biotinylated antibodies,

[0042] FIGS. 2a and 2b illustrates a pre-incubation process in a liquid phase involving regular tetravalent streptavidin, and monovalent streptavidin.

[0043] FIG. 3 exemplifies a multi-immunolabeling process according to an embodiment of the invention, the pre-incubation process making use of a monovalent biotin-binding molecule.

DETAILED DESCRIPTION

[0044] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which currently preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and fully convey the scope of the invention to the skilled addressee. Like reference characters refer to like elements throughout.

[0045] Referring now to the drawings and to FIG. 1a in particular, showing a standard immunolabeling procedure using regular streptavidin. Regular streptavidin can only be used in the indirect two step method, and it can only be used for one biotinylated antibody. There is on the left hand side depicted a biological sample 102 which has been incubated with a biotinylated antibody 104. The biotinylated antibody 104 binds to a first target antigen in the biological sample 102, and has been specifically selected for allowing subsequent detection/analysis in e.g. a subsequent immunofluorescence process. For allowing detection of the first target antigen, the sample 102 with the bound biotinylated antibody 104 is incubated with labeled streptavidin 106 (right hand side of FIG. 1a). The labeling component provided with the streptavidin is a reporter element, such as for example a fluorochrome conjugated with streptavidin, thereby forming the labeled streptavidin 106. Other types of reporter elements are possible. The selection of reporter element is done based on the application at hand. In FIG. 1a, a biotin-binding site of the labeled streptavidin 106 is indicated as a “black circle” denoted as 108. Similarly, a biotin molecule 110 is indicated as a “white circle” at the biotinylated antibody 104, where the biotin molecule 110 is conjugated to the antibody 104

[0046] Streptavidin can only be used for detection of one biotinylated antibody in the indirect immunolabeling method (two-step). If the sample is incubated with a first biotinylated antibody 104 recognizing a first target antigen together with a second biotinylated antibody 112 recognizing a second target antigen, followed by incubation with two (different) labeled streptavidin 106 and 106′, both antibodies 104 and 112 will be labeled by both labeled streptavidins 106 and 106′, FIG. 1b. Hence, in this two-step method each biotinylated antibody will generate a mix of two signals, for example emit light at two different colors, if the labels are fluorochromes.

[0047] One could possibly believe that this problem could be solved by pre-labeling the first biotinylated antibody 104 with the first labeled streptavidin 106 and the second biotinylated antibody 112 with the second labeled streptavidin 106′, before adding them to the sample 102. However, when using “regular” streptavidin having four biotin-binding sites (tetravalent), an aggregate will be formed because each streptavidin molecule 106 will bind to more than one biotinylated antibody molecule 104, and each biotinylated antibody 104 will bind to more than one streptavidin molecule 106 as is shown in FIG. 2a and as has been further discussed above.

[0048] However, in accordance to the invention, a monovalent biotin-binding composition is used, having only a single biotin-binding site. As is shown in FIG. 2b, using labeled monovalent streptavidin 106′ in a pre-incubation process with the first primary antibody 104 in a liquid phase will enable forming an immunolabeling complex between the biotinylated antibody 104 and the labeled monovalent streptavidin 114 without aggregation, by means of a click chemistry process. Advantages of using click chemistry over conventional conjugation chemistry includes that it is fast, no extra reactive chemicals are needed, no by-products are formed (the product is ready to use), and the amounts and concentrations of the reagents can be altered from very low to very high. These features make the inventive method suitable for small scale conjugation ‘at the lab bench’ to large scale conjugation for distribution. This pre-incubation step will further enable formation of multiple immunolabeling complexes between biotinylated antibodies and monovalent streptavidin; such that a large plurality of different primary antibodies 104 each conjugated with labeled monovalent streptavidin 114 could be formed (i.e. no aggregates are formed as compared to FIG. 2a).

[0049] As the monovalent streptavidin may be labeled with not only a single type of reporter molecule, but rather with a large plurality of different reporter molecules, e.g. different fluorochromes being active within different wavelength ranges, it may in accordance to the invention be possible to create a set comprising a plurality of different primary antibodies (e.g. the first 104 and the second 112 primary antibody as is shown in FIG. 3, each of the primary antibodies targeting a different antigen comprised with the biological sample 102), each of the primary antibody to be (separately) pre-incubated with a differently labeled monovalent streptavidin, such as labeled monovalent streptavidin 114 and 114′. This is possible because the immunolabeling complexes can be used for the direct immunolabeling method (1-step).

[0050] A first primary antibody 104 may thus be pre-incubated with a first monovalent streptavidin composition 114 forming a first immunolabeling complex in a liquid phase, and a second primary antibody 112 may be pre-incubated with a second monovalent streptavidin composition 114′ forming a second immunolabeling complex in a liquid phase. As the first 114 and the second 114′ monovalent streptavidin composition advantageously are labeled with different types of reporter elements, such as for example being labeled with different types of fluorochromes, it will in a subsequent analysis process be possible to separately detect/analyze a first and a second target antigen in the sample by incubating the sample with the first and the second immunolabeling complexes.

[0051] It should be noted that it is desirable to add a blocking agent to block unbound monovalent streptavidin after the pre-incubation process for each of the first 104 and the second 112 primary antibodies. Such a blocking agent is preferably free biotin. The addition of the blocking agent will make any surplus monovalent streptavidin inreactive, thereby reducing the risk of an unwanted reaction between the surplus monovalent streptavidin and a further biotinylated primary antibody, e.g. the first 104 vs. the second 112 primary antibody.

[0052] In summary, the present invention relates to a method for attaching reporter molecules to an antibody by using biotin click chemistry. This is alleviated by forming an immunolabeling complex, wherein the method comprises selecting a first biotinylated primary antibody, selecting a first monovalent biotin-binding composition labeled with a first reporter element, and mixing in a liquid phase in a reaction vessel, the first biotinylated primary antibody with the first labeled monovalent biotin-binding composition.

[0053] In accordance to the invention, a monovalent biotin-binding composition with only one functional binding site for biotin is used, thereby avoiding antibody aggregations during the mixing step in a liquid phase. As a comparison, e.g. regular streptavidin has four binding sites for biotin, which causes rapid aggregation if premixed with biotinylated antibodies in a liquid phase.

[0054] Advantages with the invention include that only one labeling reagent (e.g. monovalent streptavidin) and one blocking reagent (biotin) is needed. This is because the present invention is completely antibody-class independent. Other comparable methods are typically based on secondary antibodies, and hence dependent on adjusting the secondary antibodies and the blocking reagents to what classes the primary antibodies belong to. This makes the inventive labeling method much more versatile, for example when used in a multi-immunolabeling setting. Furthermore, the invention creates a much more stable immunolabeling complex than a complex between two antibodies. The high stability of the complex makes it comparable to a directly conjugated antibody using conventional conjugation chemistry.

[0055] The above description has been specifically exemplified in relation to the use of monovalent tetrameric streptavidin as a component of the monovalent biotin-binding composition being labeled with a reporter element. However, as is outlined above, it may in accordance to the invention be possible to allow the monovalent biotin-binding composition to be based on different monovalent biotin-binding molecules, such as for example monomeric streptavidin, avidin, traptavidin, captavidin, tamavidin, bradavidin, neutravidin, and rhizavidin, or any further developed or found equivalent molecule or combinations of molecules having an appropriate biotin-binding configuration.

[0056] Although the figures may show a specific order of method steps, the order of the steps may differ from what is depicted. Also two or more steps may be performed concurrently or with partial concurrence. Such variation will depend on designer choice. All such variations are within the scope of the disclosure. Additionally, even though the invention has been described with reference to specific exemplifying embodiments thereof, many different alterations, modifications and the like will become apparent for those skilled in the art. Variations to the disclosed embodiments can be understood and effected by the skilled addressee in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Furthermore, in the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality.