SE1550048A1 - Method for the formation and use of an immunolabeling complex - Google Patents

Description

15 20 25 30 For example if the primary antibody is a mouse IgG antibody, the secondary antibody is an anti-mouse IgG antibody.

The reporter molecules used in immunolabeling vary depending on the nature of the detection method. The reporter molecules are either conjugated to the primary antibody (direct method) or conjugated to the secondary antibody or streptavidin (indirect method).

Usually, several reporter molecules are attached to each antibody/streptavidin molecule. The most common reporter molecules are enzymes for chromogenic detection or ?uorochromes for ?uorescence signals. Other examples are particles (e.g. gold particles, quantum dots), phosphorescent compounds (e.g. carbocyanide dyes), radioactive compounds (e. g. 3H or 32P labeled molecules) and transition metals (for mass spectrometry). For some classes of reporter molecules, several signals can simultaneously be separated and analyzed Within one sample. This means that several different target molecules can be immunolabeled and detected simultaneously Within one sample. This multi-immunolabeling technique is for example commonly used by ?uorescence microscopy, in Which ?uorochrome-labeled antibodies are used to immunolabel a sample and a ?uorescence rnicroscope are used for detection of the ?uorochrome-labeled antibodies in the sample. By using different ?uorochromes that can be separated from each other in a microscope by different light filter set arrangements and/or different illumination light Wave lengths, a sample can be immunolabeled With a panel of different antibodies, Where each antibody (primary or secondary antibody) is labeled With a unique ?uorochrome. Standard ?uorescence microscopes today can detect up to four ?uorochromes Without bleed-through signals.

However, the technique can be improved and it has recently been showed that it is possible to detect at least seven different ?uorochromes Within one sample, Which means that at least seven different target molecules can potentially be immunolabeled Within one sample.

Although the indirect method is beneficial When it comes to signal amplification, the use of secondary antibodies for multi-immunolabeling causes problems With cross-binding of antibodies, i.e. the secondary antibodies do not only bind to the primary antibodies they are intended to bind, but in addition bind to other antibodies that is not intended. These other antibodies can be 1) endogenous antibodies Within the tissue, 2) other primary antibodies, and 3) other secondary antibodies. The problem arises from that the secondary antibodies are antibody class specific and therefore reco gnizes all antibodies that belong to that specific antibody class. Hence, each primary antibody needs to be of a different antibody class, and each secondary antibody must selectively recognize only one of those classes. In addition none of the secondary antibodies can be of the same antibody class 10 15 20 25 30 as any of the primary antibodies. This is a well-known problem in the art, and greatly limits the number of antibodies that can be combined in multi-immunolabeling.

The antibody class is determined by the animal species that antibody is made in (e. g. mouse, rat, rabbit, goat) and the antibody subclass (such as IgGl, lgG2a, IgG2b, IgM). Often the primary antibody is of an IgG class and most secondary antibodies recognize all IgG subclasses. Consequently, the animal species of the primary antibody is often the main determinant of the antibody class. In order to detect two target molecules in one sample, the primary antibodies must typically be made in two different animal species, for example mouse and rat. In addition the sample should preferably not be a mouse or rat tissue that may contain endogenous mouse or rat antibodies, and the secondary antibodies must be made in a third species other than mouse or rat, for example donkey or goat. In this example two labeled secondary antibodies can be added to simultaneously detect each primary antibody, one that recognizes mouse IgG (e.g. donkey anti-mouse IgG) and is labeled with one ?uorochrome, and one that recognizes rat IgG (e. g. donkey anti-rat IgG) and is labeled with second ?uorochrome, where the two ?uorochromes can be separately detected by a ?uorescence microscopy (or using another detection device).

Because most primary antibodies are made in a few animal species (mostly mouse, rat, rabbit and goat), it follows that combining even two antibodies can be difficult.

For example, the majority of antibodies against human proteins are made in mouse, and hence the probability is high that the two antibodies picked for immunolabeling a human sample are of the same antibody class, i.e. mouse IgG antibodies. Furthermore, the probability of ending up with two antibodies of the same antibody class 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. This makes the design of each multi-immunolabeling setup (the choice of primary and secondary antibodies) difficult, time-consuming and expensive. Often a range of different primary antibodies are needed to be tested in order to find well working primary antibodies of different lg classes.

Thus, the potential risk for antibody cross-binding in the indirect method makes it difficult to multi-immunolabel for many target molecules in one sample. Still, the amplification step of the indirect method is desirable and often crucial in order to receive sufficiently strong signals for proper signal detection. 10 15 20 25 30 To address the antibody cross-binding problem in the indirect method, several approaches have been developed. One such approach is to stain the sample sequentially with two primary antibodies of the same class, primary antibody A and B. First, primary antibody A is added to bind target molecules A in the sample. The non-bound primary antibodies are then washed away, and the secondary antibody A is added to detect primary antibody A. The secondary antibody A is labeled with a ?uorochrome A. Since antibodies (whole IgG and F(ab)2 fragments) have two antigen-binding sites, one antigen-binding site of a secondary antibody A is still exposed and reactive, and thus needed to be blocked with unlabeled IgG antibodies, or serum. Then, the primary antibody B is added. After incubation it is washed away and secondary antibody B labeled with ?uorochrome B is added. Another approach is to use monovalent secondary antibodies that only have one antigen-binding site, which avoids the IgG blocking step. The disadvantage with this sequential approach is 1) that it includes many incubation steps, 2) the primary antibody- secondary antibody complex is an equilibrium binding reaction, meaning that secondary antibody A will start to detach from primary antibody A over time. This will expose new binding sites for secondary antibody B, to bind primary antibody A. This can be improved by brief fixation with fonnaldehyde that covalently attaches the primary and secondary antibodies to each other. However, some labels do not tolerate formaldehyde and it also can cause increased background ?uorescence signals since formaldehyde ernits light when activated by light in the blue- green light spectra.

It also includes an additional step.

An alternative approach is to pre-rnix the primary antibody A brie?y with the secondary antibody A at a certain molar ratio, and then block unbound secondary antibodies with whole IgG or serum from the animal species of the primary antibody. The same is done with primary antibody B and secondary antibody B. However, premixing the primary antibodies with whole IgG or F(ab)2 secondary antibodies results in antibody aggregates, since they have two antigen-binding sites. The fonnation of aggregates decreases the quality of the sample immunolabeling. To avoid formation of antibody aggregation, monovalent secondary antibodies can be used. Furthennore, to avoid that the secondary antibodies sterically blocks the antigen-binding sites of the primary antibody, the monovalent secondary antibodies can be raised against the Fc portion of the primary antibody.

SUMMARY OF THE INVENTION According to an aspect of the invention, the above is at least partly alleviated by a method for forrning an immunolabeling complex, wherein the method comprises 10 15 20 25 30 selecting a first biotinylated primary antibody, selecting a first monovalent biotin-binding composition labeled With a first reporter element, and mixing, in a reaction vessel, the first biotinylated primary antibody With the first labeled monovalent biotin-binding composition.

As discussed above, it is possible to use biotinylated primary antibodies and regular streptavidin to achieve amplification during immunolabeling of a sample. However, such an approach must be performed sequentially in tWo steps because regular streptavidin contains four biotin-binding sites and thus can only be used on biotinylated antibodies that are bound to a solid phase (such as a fixed tissue sample). Adding regular streptavidin to biotinylated antibodies in a liquid phase has the very undesirable effect of rapid aggregation into big antibody complexes. However, in accordance to the invention, a monovalent biotin- binding composition is instead used, With only one functional binding site for biotin, Which thereby avoids antibody aggregations during the rnixing step in a liquid phase (in the reaction vessel). Thus, the monovalent biotin-binding composition is used as a second labeling reagent instead of using secondary antibodies.

In accordance to the invention, the expression “monovalent biotin-binding composition” is to be interpreted broadly. For example, the monovalent biotin-binding composition can comprise at least one of streptavidin, avidin, traptavidin, captavidin, tamavidin, bradavidin, neutravidin, and rhizavidin, etc., or any further developed or found equivalent molecule having an appropriate biotin-binding configuration.

In a possible embodiment of the invention the monovalent biotin-binding composition comprises monovalent streptavidin. There exist different methods for engineering monovalent streptavidin. The streptavidin molecule is a tetramer consisting of four equal subunits, Where each subunit has a binding site for biotin. Monovalent streptavidin can be achieved by generating monomers of streptavidin, i.e. single subunits, forming monomeric streptavidin. However, monomeric streptavidin has much lower binding affinity than tetrameric streptavidin, since part of its biotin binding site comes from a neighboring subunit. Monovalent tetrameric streptavidin With remained high binding affinity for biotin can be generated by at least two different approaches. First, by assembling streptavidin that consists of one functional subunit and three mutated subunits that lack affinity for biotin (Nature Methods; 4:267-73, (2006)). Second, by treating tetrameric streptavidin With trisbiotinylated oligonucleotide that blocks three of the four biotin-binding sites on streptavidin (AngeW Chem Int Ed Engl; 52(21):5509-12, (2013)). It should be understood that any future designed avedin derivative being a monovalent biotin-binding molecule could be possible and is to be considered equivalent to and comprised Within the generic term 10 15 20 25 30 monovalent streptavidin composition. However, When using monovalent streptavidin for the monovalent biotin-binding composition, the use of monovalent tetrameric streptavidin is currently preferred.

It should be understood that streptavidin binds extremely tight to biotin, and the present method uses a biotinylated primary antibody to form complexes with streptavidin.

Biotinylated antibodies are antibodies that are covalently conjugated with several biotin molecules, usually 3-10 biotin molecules per antibody. Because several streptavidin molecules bind to each biotinylated antibody, signal amplification may be achieved. The streptavidin-biotin binding is the strongest known non-covalent binding, and it is >l000 times stronger than the binding that can be generated from an antibody. Thus, the complex between biotinylated primary antibodies and streptavidin results in a much more stable complex than comparable methods.

The present invention is advantageous in 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.

Accordingly, this makes the monovalent biotin-binding labeling method according to the invention much more versatile, for example when used in a multi- immunolabeling setting. A further advantage relating to the multi-immunolabeling setting is that antibodies of the same class are made distinguishable from each other through pre- labeling of streptavidin, where the streptavidin used for each antibody is conjugated with unique reporter elements.

It is preferred (but optional) to further comprise adding a blocking agent to block unbound portions of the monovalent of the monovalent biotin binding composition after the first immunolabeling complex has been formed. The blocking agent is preferably free biotin. The addition of the blocking agent will make any surplus e. g. monovalent streptavidin inreactive, thereby reducing the risk of an unwanted reaction between the surplus monovalent streptavidin and a further biotinylated primary antibody. It may as an altemative be possible to reduce this unwanted reaction by the introduction of a careful titration step and allowing the mixing step (the reaction between the biotinylated primary antibody and the monovalent streptavidin composition) to be long “enough”.

The reporter element may in one embodiment be selected from a group comprising a ?uorochrome, an enzyme, a peptide, quantum dots, and a transition metal. 10 15 20 25 30 Some of the disclosed reporter elements are known to the skilled person, it should however be understood that other/future reporter elements may equally be used in relation to the invention.

For example, using a ?uorochrome as a reporter element in relation to “regular” tetravalent streptavidin is well known to the skilled person. Labeling of regular streptavidin with a range of different molecules, including ?uorochromes, proteins, peptides, oligonucleotides, is sirnilarly a well known process that in a similar manner may be used for labeling e.g. monovalent streptavidin for forming the first monovalent streptavidin composition.

In a preferred embodiment of the invention the ratio between the first monovalent biotin binding composition and the first biotinylated primary antibody is selected such that the risk of the first monovalent biotin binding composition blocking active biding sites of the first biotinylated primary antibody is reduced.

On the other hand, if too few molecules of the monovalent biotin binding composition are attached to each antibody molecule there will instead be a weak detection signal. However, the signal can be further enhanced using additional enhancer steps. For example, in regards to the use of monovalent streptavidin, the monovalent streptavidin can be labeled with antigens that are recognized by antibodies that are used to enhance the signal.

These antibodies can either be directly conjugated with a reporter molecule, such as a ?uorochrome, or conjugated with another second antigen that is recognized by a second antibody that is conjugated with a reporter molecule. This concept is further disclosed in SEl55004l-6 by the applicant, which content is fully incorporated by reference. The streptavidin may also be labeled with oligonucleotide probes that are recognized by Padlock probes for rolling-circle amplification (Nature Methods; 32725 - 727 (2006)). Thus, by adding downstream amplification steps the number of monovalent streptavidin molecules that are attached to each biotinylated antibody can be kept low, which minimizes the risk of sterical hindrance of the antibody°s antigen-binding site.

In accordance to the invention the inventive immunolabeling complex is used for detecting a target in a sample, the sample being a biological sample. Thus, in an embodiment of the invention the first immunolabeling complex as discussed above is contacted with the sample, and incubated for a time sufficient to permit the first immunolabeling complex to selectively bind to the target, and then the immunolabeling complex is detected by development of a signal from the label, for example illumination of 10 15 20 25 30 the sample under a microscope to detect a light from a ?uorochrome. In such an embodiment the reporter element is preferably a ?uorochrome.

In a preferred embodiment of the invention a second immunolabeling complex formed as discussed above is also contacted With the sample, and the sample is in a similar manner incubated for a time sufficient to perrnit the second immunolabeling complex to selectively bind to the target. In the preferred embodiment, the second immunolabeling complex comprises a second biotinylated primary antibody mixed With a second monovalent biotin binding composition, the second biotinylated primary antibody being different from the first biotinylated primary antibody, and the second monovalent biotin binding composition being labeled With a second reporter molecule being different from the first reporter molecule. The second immunolabeling complex is preferentially added to the sample together With the first immunolabeling complex.

The method can be used alone or in combination With any other immunolabeling method, for example in combination With directly conjugated primary antibodies (direct method) or in combination With primary antibody + labeled secondary antibody (two-step method), or in combination With biotinylated primary antibody + labeled regular streptavidin (two-step method). The latter requires that the present method is added after labeling With biotinylated primary antibody + labeled standard streptavidin, and that the labeled sample is blocked With free biotin before adding the immunolabeling complexes of the present method.

According to further aspect of the invention there is provided a kit of immunolabeling complexes, comprising a first and a second immunolabeling complex formed in accordance to the above discussion, Wherein the second immunolabeling complex comprising a second biotinylated primary antibody mixed With a second monovalent biotin- binding composition, the second biotinylated primary antibody being different from the first biotinylated primary antibody, and the second monovalent biotin binding composition being labeled With a second reporter molecule being different from the first reporter molecule.

As Will be understood by the skilled person, the invention Will for example allow for the possibility to have the freedom of labeling, in an embodiment, four primary antibodies, independently if they are made in mouse, rat, rabbit or goat, and for this you only need a kit of four monovalent streptavidin labeled With four different ?uorochromes. The equivalent kit With monovalent secondary antibodies requires four different secondary antibodies (anti-mouse, anti-rat, anti-rabbit and anti-goat), each labeled With the four different ?uorochromes, Which in total is 16 different monovalent secondary antibodies. 10 15 20 25 30 Furthermore, While the inventive implementation using monovalent streptavidin only needs free biotin as blocking reagent, the monovalent secondary antibodies need four different blocking reagents (one for each animal species). Thus, a kit of five components of monovalent streptavidin (four labeled monovalent streptavidin + one blocking reagent), requires a kit of 20 components for a corresponding kit of monovalent secondary antibodies (16 labeled monovalent antibodies + four blocking reagents).

Further features of, and advantages with, the present invention will become apparent when studying the appended claims and the following description. 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.

BRIEF DESCRIPTION OF THE DRAWINGS 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: Figs. la and lb shows an example of a biological sample which has been incubated with biotinylated antibodies, Figs. 2a and 2b illustrates a pre-incubation process in a liquid phase involving regular tetravalent streptavidin, and monovalent streptavidin.

Pig. 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 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 lirnited 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.

Referring now to the drawings and to Fig. la in particular, showing a standard immunolabeling procedure using regular streptavidin. There is on the left hand side depicted 10 15 20 25 30 10 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 immuno?uorescence 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. la). The labeling component provided with the streptavidin is a reporter element, such as for example a ?uorochrome 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. la, 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 Streptavidin can only be used for detection of one biotinylated antibody. 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. lb.

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 ?uorochromes.

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.

However, in accordance to the invention, a monovalent biotin-binding molecule 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 forn1ing an immunolabeling complex between the biotinylated antibody 104 and the labeled monovalent streptavidin 114 without aggregation.

This pre-incubation step will further enable formation of multiple immunolabeling complexes between biotinylated antibodies and monovalent streptavidin; such that a large plurality of 10 15 20 25 30 11 different primary antibodies 104 each conjugated With labeled monovalent streptavidin 114 could be fonned (i.e. no aggregates are formed as compared to Fig. 2a).

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 ?uorochromes 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”.

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” forrning 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 ?uorochromes, 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.

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.

In summary, the present invention relates to a method for 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.

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 10 15 20 25 12 binding sites for biotin, which causes rapid ag gregation if premixed with biotinylated antibodies in a liquid phase.

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.

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 comprise different monovalent biotin-binding molecules, such as for example avidin, traptavidin, captavidin, tamavidin, bradavidin, neutravidin, and rhizavidin.

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.

Claims (7)

PATENTKRAV 1. Metod for att detektera en malmolekyl i ett prov, varvid metoden innefattar att: 1. tillsatta ett fOrsta immunobaserat inmarkningskomplex till provet; 2. inkubera provet under en tid tillräcklig for att tillâta det forsta immunobaserade inmarkningskomplexet att selektivt binda till malet; och - detektera malet, varvid det forsta immunobaserade inmarkningskomplexet bildas genom: 3. att valja en forsta biotinylerad primarantikropp; 4. att valja en forsta monovalent biotinbindande komposition markt med ett forsta reporter element, och - blanda, i ett reaktionskarl, den forsta biotinylerade primarantikroppen med den forsta inmarkta monovalenta biotinbindande kompositionen. 2. Metoden enligt krav 1, varvid den forsta inmarkta monovalenta biotinbindande kompositionen innefattar atminstone en av streptavidin, avidin, traptavidin, captavidin, tamavidin, bradavidin, neutravidin och rhizavidin. 3. Metoden enligt nagot av kraven 1 och 2, varvid metoden vidare innefattar att tillsatta ett blockeringsreagens for att blockera obundna delar av den monovalenta biotinbindande kompositionen efter att det forsta immunobaserade inmarkningskomplexet har bildats. 4. Metoden enligt nagot av foregaende krav, varvid reporterelementet är valt ur en grupp som innefattar en fluorokrom, ett enzym, en peptid, quantum dots, och en overgangsmetall. 5. Metoden enligt krav 4, varvid den forsta monovalent biotinbindande kompositionen är konjugerad med reporterelementet. 6. Metoden enligt nagot av foregaende patentkrav, varvid forhallandet mellan 30 den forsta monovalent biotinbindande kompositionen och den forsta biotinylerade primarantikroppen valjs sâ att risken minskas for att den forsta monovalent biotinbindande 2 komposition blockerar aktiva bindningssite hos den forsta biotinylerad primarantikroppen. 7. Metoden enligt krav 1, varvid reporterelementet är en oligonukleotid. 8. Metoden enligt krav 1, varvid reporterelementet är ett antigen. 9. Metoden enligt krav 1, varvid det forsta reporterelementet är en fluorokrom och detektion av malet innefattar belysning av det immunobaserade inmarkningskomplexet. 10. Metoden enligt krav 1, vidare innefattande att: 1. tillsatta ett andra immunobaserat inmarkningskomplex med provet, och 2. inkuberinginkubera provet under en tid tillracklig for att tillata det andra immunobaserade inmarkningskomplexet att selektivt binda till malet, varvid det andra immunobaserade inmarkningskomplexet bildas av: - att valja en andra biotinylerad primarantikropp; 3. att valja en andra monovalent biotinbindande komposition markt med ett andra reporterelement, och 4. blanda, i ett reaktionskarl, den andra biotinylerade primarantikroppen med den andra inmarkta monovalent biotinbindande kompositionen, varvid den andra biotinylerade primarantikroppen skiljer sig fran den forsta biotinylerade primarantikroppen, och den andra reportermolekylen skiljer sig fran den forsta reportermolekyl en . 11. Kit av immunobaserade inmarkningskomplex, innefattande: - ett forsta immunobaserat inmarkningskomplex; och

1. en andra immunobaserat inmarkningskomplex, varvid det forsta immunobaserade inmarkningskomplexet bildas genom att:

2. valja en forsta biotinylerad primarantikropp;

3. valja en forsta monovalent biotinbindande komposition markt med ett forsta 30 reporterelement, och

4. blanda, i ett reaktionskarl, den forsta biotinylerad primarantikroppen med den forsta inmarkta monovalenta biotinbindande kompositionen, varvid det andra immunobaserade inmarkningskomplexet bildas genom att:

5. valja en andra biotinylerad primarantikropp; 3

6. valja en andra monovalent biotinbindande komposition inmarkt med ett andra reporterelement, och

7. blanda, i ett reaktionskarl, den andra biotinylerade primarantikroppen med den andra imarkta monovalent biotinbindande kompositionen, varvid den andra biotinylerade primarantikroppen skiljer sig fran den forsta biotinylerade primarantikroppen, och den andra reportermolekylen skiljer sig fran den forsta reportermolekylen. 1/3 106' 106 104 102 Fig- lb 104 104