DE19900103A1 - Amplification method for detection of antibodies - Google Patents

Abstract

The method comprises the steps of (1) delivering an antibody (A) directed against the target antibody (T) bound to an antigen (2) in a biological sample (1), (2) delivering another antibody (B) directed against the first antibody (A), where the A and B are from different species and mutually attractive to each other, and B and T are from different sources of the same species. Further amplification may be achieved by further separate and alternating doses of the antibodies A and B. The antibody making up the final dose is conjugated with a means for visualization such as the enzyme anti-enzyme method. An Independent claim is also included for the following: A test kit for use in amplification of positive results when determining the presence of bound target antibody.

Description

The invention relates to an improved method for Detection of antibodies attached to a solid phase, like a human tissue sample.

Immunohistochemistry is a well known technique for Evidence of the presence or absence of specific Molecules in a biological sample and their Distribution within the sample (typically one Tissue sample). Immunocytochemistry is a very similar one known area where cells are used. These techniques use a specific one Binding reaction between antibody and antigen.

In the present application, the invention with Described with reference to immunohistochemistry as an example however, the present invention is for others Areas such as ELISA (enzyme linked immunoabsorbent assay) or any method of importance the target antibody is bound to a solid phase is important.

To be able to detect a specific antigen, that an antibody is bound (i.e. the primary Antibody / Antigen Complex), it is required Detection system to use to detect the presence of the bound antibodies and their physical To be able to prove distribution. The detection system can be a direct system (where the antibody is conjugated directly to the detection system), or it can be an indirect detection system in which an easy to detect substance with a Molecule is associated, directly or indirectly binds the antibody. An example of a direct one  Detection system is a radioactive label of the primary antibody. An example of an indirect Detection system is the well-known avidin-biotin Complexation process.

Some indirect detection systems use enzymes and require the addition of a substrate of the enzyme used to make a precipitate or a to produce colored product (or to create something to be able to be colored, or to create something in a later step forms a precipitate).

From the results of immunohistochemistry tests the presence of a bound target antibody be proven, as well as its distribution, d. H. its location between the cells or within the Cells.

However, it is often necessary to see the results of the Immunohistochemistry tests to boost this for the naked eye (or under the microscope) visibly too do. Furthermore, such tests are often lacking Sensitivity in the field of Immunohistochemistry is required.

The object of the present invention is that to improve known detection methods by Sensitivity and / or gain of Immunohistochemistry tests and their results be increased.

According to a first aspect of the invention, we provide a method of reinforcing positive Results available to prove the Presence of a bound target antibody is used the method comprising the steps of: (a) Addition of a first antibody against the  bound target antibody is directed and with a first selected from a first species Source for antibody was generated, (b) adding a second antibody, against the first antibody is directed and one with a second Species selected second source for antibody was generated, which recognizes the first antibody and binds to it, the first and second Antibodies mutually mutually are specific, and being bound Target antibody with one from the second species selected further source for antibodies was, and optionally individually and alternately others Portions of the first antibody and the second Antibody can be added.

For example, the bound target antibody can also be used another individual of the same species with which the second antibody was generated. The first species is a donkey and the second species a sheep, the case is as follows: sheep Anti-X (obtained from sheep A) would be sent to that Bind target antigen X, then donkey would anti- IgG sheep (obtained from donkey A) on the bound Bind sheep antibodies and sheep anti-donkey IgG (obtained from sheep B) would be bound to the Bind donkey antibodies.

It is preferred to individually and alternately until first and second antibody added three times. In most cases, there is only one addition at a time of the first and the second antibody.

It is advantageous that by adding another Dose of the first antibody the first antibody due to the nature of the antibody source each free binding site of the second antibody binds to the second antibody. The addition cycles  for the first and second antibodies can be repeated indefinitely, creating a Reinforcement of a positive result achieved becomes.

The bound target antibody is, preferably in a biological sample bound to an antigen. Alternatively, the bound target antibody can be a be a bridging antibody to a Antibody (primary antibody) is bound to again to the antigen in the biological sample is bound.

Upon detection of the presence or absence of an antigen is usually used in a human tissue sample as an antibody bound to the antigen monoclonal mouse anti-human antibody (the Antigen is human) used. Monoclonal Antibodies are generated in vitro and are therefore easily accessible when using the inventive method, however polyclonal mouse anti "anything" antibody (e.g. Mouse anti-sheep) as the second according to the invention Antibodies needed. Polyclonal antibodies are used in manufactured in vivo, and since a mouse is a small animal is, the yield is low and the cost is high. By using a bridge-building Antibody, for example a rabbit anti-mouse Antibody, the first and second antibodies of the detection system against the bridge-building Antibodies are targeted, eliminating the need of mouse polyclonal anti-"anything" antibodies is avoided.

The biological sample is preferably a Tissue sample. Preferably the first and second Antibodies mutually sufficient to each other specifically, so they don't cross-react with  endogenous tissue immunoglobulins into one Extent show that background noise is high leads.

Preferably the first and second antibodies polyclonal antibodies.

In one embodiment, the first or second antibody, each last added with a means of making it visible conjugate (as described later). Become for example three additions of antibodies carried out (first antibody, second antibody, first antibody), the last time the first antibody added, hence the first Antibodies with this addition preferably with a Conjugation means conjugated. Become four Additions made, but with the last Adding the second antibody is added correspondingly the second antibody in the last Add the antibodies with an agent to Visualize conjugate.

In another embodiment, the last one is Add the first and / or second antibodies preferably by means of visualization conjugated. For example, four encores the last (i.e. the second) addition of the first and the second antibody with means conjugate to visualize.

In another embodiment, the first Antibody in all additions with a means of Visualize conjugate. Alternatively, the second antibody with all additions with one agent conjugate to visualize, or it can both the first and the second antibody with be conjugated to a means of visualization.  

Is the first antibody or is the last addition of the first antibody with a means for Conjugating visuals are used to prove the Presence of a bound target only three Steps required (step a, step b, step a, wherein step a the addition of the first antibody and step b the addition of the second antibody four steps are required, if the second antibody or the last addition of the second antibody with a means for Visualize is conjugated (abab - steps a and b as described above).

Instead of the first or second antibody being conjugated can be the means to Visualization can also be added separately, whereby an example of such a means of Making the enzyme-anti-enzyme process visible for which the peroxidase-anti-peroxidase is a known Example is.

The first antibody against immunoglobulins second source of antibodies.

The second antibody is against the immunoglobulins first source of antibodies.

The second antibody has the same Immunoglobulin class of like the target antibody.

The means by which the presence of the first and / or the second antibody made visible can be the same or different. The means of visualization can use of enzymes (e.g. peroxidase or alkaline phosphatase), or fluorescent compounds (e.g. fluorescein), or Particles (e.g. gold), or radiolabelled  Links. Another very well known means of The visualization of antibodies is the ABC- (Avidin Biotin complex) Process in which biotin with the Antibody is conjugated and after adding avidin a visible complex is formed. Likewise, the streptavidin-peroxidase conjugate method used. Can also be used as a means of making people visible an enzyme anti-enzyme method can be used.

If enzymes are used to make them visible, Generation of a precipitate or a colored one Product with the naked eye or the microscope a substrate for the enzyme can be detected be added.

Be the first and second sources of antibodies preferred species that are (mammals) animals (particularly preferably non-human). Alternatively species can be selected that are in vitro Generate antibodies.

The first and second sources of antibodies are differently. For example, the first source be a rabbit and the second source is a goat. In this example, the first antibody would be one "Goat anti-rabbit Imnunoglobulin" and the second antibody a "rabbit anti-goat Imnunoglobulin ", furthermore the target antibody would be in a rabbit.

The first and second antibodies can bind each other, d. H. the first antibody is both with respect to the second antibody Antibodies as well as antigen and vice versa.

This way the valence of the antibodies to each other significantly increased.  

The method according to the invention contrasts known detection methods improvements to Available, in particular on the mutual Bond and the resulting high valence of Antibodies are based and by layers of increasing size around the bound target antibody are generated that alternate from the first and the second antibodies are built up.

In the case where the first antibody with a Conjugating means binds this to the bound target antibody, and recognizes hence the bound target antibody, whereby however from a single means of making visible generated signal can be very weak. By encore the second antibody becomes a second layer that generates a larger number of antibodies comprises as the first layer since the second Bind antibodies specifically to the first antibodies and in addition every free binding site of the first Antibody to another second antibody binds.

The addition of another serving of the first Antibody, which with the means for Visualize conjugate and the specific binds the second antibody, and specifically from the free binding sites (Fab region) of the second Antibody bound leads to a third, larger layer of antibodies around the Target antibody. The accumulation of every layer increases the number of antibodies as well as the amount existing means of making visible, whereby the demonstrable result is reinforced.

This effect is achieved even if only the last one Layer of antibodies with means for Visualizing conjugate is what's additional  has a cost-saving effect, since less expensive conjugates Antibodies are needed.

The antibodies are preferably separated and added successively in aqueous solution.

The method according to the invention also improves the Sensitivity of the test as it is very possible low concentrations of the bound Target antibody (e.g. at 50-200-fold dilution) to demonstrate by stratifying the result first and second antibody is amplified.

The first and second antibodies are preferably located pure and essentially free from contamination other antibodies and physiological substances in front.

We provide a second aspect of the invention a test kit for use in amplifying positive results in the detection of the present a bound target antibody is available, which comprises a supply of a first antibody, which is directed against the target antibody and which with a first source of antibodies which was selected from a first species, and a stock of a second antibody against the first antibody is directed and with a second source of antibody was generated from a second species was selected, the Target antibody with another source for Antibody was generated from the second species was selected, the first and the second Antibodies bind each other mutually can.

The kit can also hold a supply of target antibodies contain.  

The first antibody is preferably with a Conjugation means conjugated.

The second antibody can be used with an agent for Visualize conjugate. Alternatively, you can both the first and the second antibody with be conjugated to a means of visualization.

Alternatively, the kit can be kept in stock second antibody, which with an agent conjugated to visualize.

Alternatively, the kit can keep a supply of the first Antibodies, which with means for Visualize is conjugated.

Instructions for use may be included with the kit. The kit can be a bottle with control and / or Reference material included. The antibodies are preferably contained in ampoules or bottles. All components of the kit are preferably marked.

The kit can be used in a automated test device. The first and second antibodies are preferably in of a certain concentration posed. The certain concentration can one ready-to-use solution, alternatively can dilution may also be required. The kit can with a means of adding the antibodies, such as one Pipette.

According to a third aspect of the invention, the Use of a first antibody against an bound target antibody is directed and with a first selected from a first species Source for antibody was generated, as well as one second antibody, against the first antibody is directed, and one with a second  Species selected second source for antibody was generated for the examination of tissue Provided, the first antibody to the can bind bound target antibody, and wherein the first and second antibodies mutually can bind each other, and further the bound Target antibody with one from the second species selected further source for antibodies was, and being the reinforcement and the Sensitivity of the result of the detection of the bound target antibody by the method the first aspect of the invention, or by the Means of a test kit according to the second aspect of Invention is increased.

The present invention is further discussed below Reference to the attached drawing and the Examples explained, but these only Are intended to represent examples. Show:

Fig. 1a-c schematically illustrates the general process of the invention;

Fig. 2a-c schematically illustrates the method of Figure 1, wherein the bound target antibody is a bridge-forming antibodies.

FIG. 3 shows the contents of a test kit according to the invention; and

FIGS. 4 and 5 the content of an alternative embodiment of the test kit according to the invention.

Fig. 1a shows a tissue sample 1 and 2 an antigen to which an antibody T, of the antigen 2 was added in the tissue sample 1 for the tissue sample to detect the presence, specifically bound.

The sample is used to remove unbound Antibody T rinsed and then a first Portion of antibody A added to the sample binds the antibody T. The sample becomes Removal of unbound antibody A again rinsed.

Fig. 1b shows the addition of a first portion of the antibody B to the sample, which specifically binds to the antibody A, further binds the free binding site of the antibody A to the antibody B. The sample is rinsed again to remove unbound antibody B.

Finally, FIG. 1c shows a further addition of a second portion of the antibody A, which specifically binds to the antibody B, the free binding sites of the antibody B additionally binding antibody A. The sample is rinsed again to remove unbound antibody A.

In Examples 1-3 of the invention shown in Figures 1a-1c, antibody T is a rabbit antibody raised against human antigen 2 ;
Antibody A is a goat antibody raised against rabbit immunoglobulin;
Antibody B is a rabbit antibody raised against goat immunoglobulin.

In a first example of the invention shown in FIGS. 1a-1c, both the first and the second portion of added antibody A is conjugated with a visualization agent. In the second example, only the second portion of added antibody A is conjugated with a visualizing agent, with comparable results being obtained, but reducing the amount of conjugated antibodies used.

The bound target antibody, if available, by the means associated with the antibody A for Make visible, though, if this Is an enzyme to visualize the Result another reaction with a substrate is required.

In a third example, separate means for Visualization uses how the peroxidase anti Peroxidase system (PAPS) to target the antibody make visible.

FIG. 2a shows the tissue sample 1 , the antigen 2 and the antibody T from FIG. 1a bound to the antigen. A bridge-forming antibody L was added, which specifically binds to the antibody T bound to the antigen, whereby its presence and thus the presence of the antigen 2 is detected.

As described in FIG. 1a, a portion of the antibody A is added to the sample, which binds to the bridge-forming target antibody L.

Figs. 2b and 2c show the addition of the antibody B as well as the addition of a second portion of antibody A according to the method described in Figs. 1b and 1c.

In Examples 4 to 6 of the invention shown in Figures 2a-2c, the antibody bound to the antigen is a mouse antibody raised against human antigen 2 ;
the bridging target antibody L is a rabbit antibody raised against mouse immunoglobulin; and
Antibody A and antibody B are as described in FIGS. 1a and 1c.

In Examples 4-6 the bridging is Antibody LA rabbit anti-mouse antibody, which is used in the case where the the antigen-bound antibody T a mouse anti Human is antibody. The bridging antibody L becomes the target of the detection system, causing the Use of an expensive polyclonal mouse anti "Anything" (e.g. rabbit or goat) antibody is avoided.

In Example 4 of the invention corresponding to FIGS. 2a-2c, both the first and the second portion, the added antibodies A are conjugated with a means for rendering visible.

In example 5, only the second serving of Antibody A with a visualizing agent conjugated.

In example 6, the means of making visible like the enzyme anti-enzyme system, used separately, the presence of the target antibody and thus the To make antigen visible.

Figure 3 shows a test kit comprising an ampoule 3 containing antibody A conjugated with a visualizing agent and an ampoule 4 containing antibody B. The ampoule 5 contains a control / reference material and all ampoules 3 , 4 and 5 are labeled with their contents.

In another embodiment of FIG. 3, antibodies A are not conjugated to visualization agents, while antibodies B are conjugated to visualization agents. In a further embodiment, both antibody A and antibody B are conjugated with a visualization agent.

The kit also contains a pipette 6 and instructions for use 7 .

FIG. 4 shows the test kit from FIG. 3, which further comprises an ampoule 8 which, in the event that ampoule 3 contains antibody A conjugated with visualizing agents, contains unconjugated antibody A, and in the case in which Ampoule 4 contains Antibody B conjugated with visualization agents, contains unconjugated Antibody B.

The ampoule 9 additionally shown in FIG. 5 enables the test kit to contain both unconjugated antibody A in ampoule 8 and unconjugated antibody in ampoule 9 , while ampoules 3 and 4 contain antibodies A and B, respectively, which are conjugated with visualization agents .

Claims (34)

1. A method of enhancing a positive result for use in detecting the presence of a bound target antibody, the method comprising the steps

• (a) adding a first antibody directed against the bound target antibody and generated with a first source of antibodies selected from a first species;
• (b) adding a second antibody, which is directed against the first antibody and was generated with a second source of antibodies selected from a second species, which recognizes and binds to the first antibody,
  wherein the first and second antibodies can bind to each other and the bound target antibody was generated with a further source of antibodies selected from the second species.

2. The method of claim 1 comprising the separate and alternating addition of further Servings of the first antibody and the second antibody. 3. The method according to claim 2, wherein for Amplification of the signal up to three separate and alternating additions of the first Antibody and the second antibody be carried out. 4. Method according to one of the preceding Claims wherein the bound target antibody  of an antigen in a biological sample is bound. 5. The method according to any one of claims 1 to 3, the bound target antibody is a bridging antibody to an Antibody is bound to an antigen in a biological sample. 6. The method according to claim 4 or 5, wherein the biological sample is a tissue sample. 7. The method of claim 6, wherein the first and second antibodies mutually mutually are sufficiently specific that they are not Cross reaction with endogenous Show tissue immunoglobulins to an extent which leads to a high background noise. 8. Method according to one of the preceding Claims, the first and second Antibodies are polyclonal antibodies. 9. Method according to one of the preceding Claims, wherein the first or second Antibodies that were added at the last addition of Antibodies are used with an agent are conjugated for visualization. 10. The method according to any one of claims 1 to 8, being the last addition of the first and / or second antibody with a means for Visualize is conjugated. 11. The method according to any one of claims 1 to 8, with the first antibody at all additions conjugated with a visualizing agent is.   12. The method according to any one of claims 1 to 8, the second antibody for all additions conjugated with a visualizing agent is. 13. The method according to any one of claims 1 to 8, with all additions both the first and also the second antibody with one agent are conjugated for visualization. 14. The method according to any one of claims 1 to 8, with a means of visualization separated is added instead of with the first or second antibody to be conjugated. 15. The method according to any one of claims 9 to 14, the means of making the Presence of the first and / or second Antibodies are the same or different. 16. The method of claim 15, wherein the means the use of enzymes to make them visible include. 17. The method of claim 16, wherein the enzyme Peroxidase or alkaline phosphatase. 18. The method of claim 15, wherein the means to make fluorescent compounds visible or particulate matter or radiolabelled Connections includes. 19. The method according to any one of claims 15 to 17, the means of making things visible ABC (avidin-biotin complex) process or Streptavidin is peroxidase.   20. The method of claim 15, wherein the agent an enzyme anti-enzyme to make it visible Procedure is. 21. The method according to any one of claims 15 to 20, whereby to produce a precipitate or a colored connection, the one or the visible to the naked eye or under a microscope is, a substance is added. 22. Method according to one of the preceding Claims, the first and the second Source for antibodies from one species is selected who are animals. 23. The method of claim 22, wherein the animals are not human. 24. The method according to any one of claims 1 to 21, where the first and second sources for Antibody is selected from a species which produces antibodies in vitro. 25. Test kit for use in reinforcement positive results in the detection of Presence of a bound target antibody, comprising a supply of a first Antibody directed against the target antibody is directed and one with one first species selected first source for Antibody was generated, as well as a stock of a second antibody against the first Antibody is targeted and with one out a second species selected second Source for antibody was generated, the first and second antibodies each can bind each other and the Target antibody with one from the second  Species selected another source for Antibody was generated. 26. The kit of claim 25, further comprising one Stock of target antibodies. 27. Kit according to one of claims 25 or 26, wherein the first antibody with a means of Visualize is conjugated. 28. Kit according to one of claims 25 or 26, wherein the second antibody with an agent for Visualize is conjugated. 29. Kit according to one of claims 25 or 26, wherein both the first and the second Antibodies with a means of visualization are conjugated. 30. Kit according to one of claims 25 or 26 further comprising a stock of second antibodies, with a means of making it visible are conjugated. 31. Kit according to one of claims 25 or 26, further comprising a stock of the first antibody, which with means to make visible is conjugated. 32. Kit according to one of claims 25 to 31, containing a bottle with a control and / or reference material. 33. Kit according to one of claims 25 to 32, the for use in an automated Test device is formed. 34. Use of a first antibody against is directed to a bound target antibody and with one from a first species  selected first source of antibodies was generated, as well as a second antibody, which is directed against the first antibody and with one from a second species selected second source of antibodies was created to examine tissue, the first antibody being bound to the Target antibody can bind and the first and the second antibody is mutually exclusive can bind, and being bound Target antibody with one from the second Species selected another source for Antibody was generated, and the Gain and sensitivity of the Result of the proof of the presence of the bound target antibody by a method according to claim 1, or with the aid of a test kit is increased according to claim 24.