DE29900214U1 - Western blot strips with control zones - Google Patents

Description

Western blot strips with control zones

The invention relates to Western blot strips with at least two control zones, which enable improved differentiated, additional control and thus provide increased security in the interpretation of test results.

The Western blot plays an important role in routine serology as a so-called confirmatory test, since this procedure ultimately verifies the preliminary results of other enzyme immunoassays, e.g. those of an ELISA (Enzyme-linked Immunosorbent Assay) .

Western blotting is used in routine serology to detect specific patient (especially human) antibodies, which usually belong to the immunoglobulin classes IgG, IgM and IgA and which are formed, for example, after a bacterial or viral infectious disease. The detection of these specific patient antibodies using Western blot makes it possible to indirectly conclude that a bacterial or viral infection has occurred or is currently present, and by detecting the different immunoglobulin classes, it is also possible to make statements about the temporal progression of the infection.

In Western blotting, proteins of the pathogen are separated by gel electrophoresis depending on their size, transferred to a membrane and thereby immobilized. A part of the membrane is incubated as a so-called Western blot strip with a single patient (especially human) serum, so that antibodies against the pathogen - if they are present in the serum - usually bind to these immobilized pathogen proteins. The bound specific antibody of the patient

(especially of humans) is then detected by a second antibody from another animal species (e.g. rabbit, goat), which is usually coupled or conjugated with an enzyme such as alkaline phosphatase or with peroxidase for labeling. In routine serology, this so-called conjugate is an enzyme-conjugated antibody from another animal species, which is directed against human immunoglobulin, for example (also generally referred to as anti-antibody or secondary antibody), i.e. anti-human IgG conjugate from e.g. rabbit or goat. The enzyme of the conjugate reacts with another added substance, the substrate, whereby the reaction product is deposited on the membrane as a colored "band". Individual bands or the pattern of the bands on the membrane then make it possible to make a statement about the infection with a specific pathogen.

Since the result of Western blotting only becomes apparent as a band pattern on a membrane after these successive steps, which can contain various possible sources of error, it is desirable to check individual steps of the entire procedure by means of controls, especially given the high sample throughput in routine serology.

In previous tests, only one control band appears on the Western blot strip after incubation, the presence of which determines whether the test is considered to have been carried out correctly or incorrectly. However, this one band does not allow any statement to be made about the cause of the error. A repetition of the error or a repeated finding that cannot be clearly interpreted is therefore possible.

The present invention is therefore based on the object of checking both the addition of the serum and the functionality of the conjugate used (e.g. anti-human IgG conjugate) by means of differentiated system controls in the form of at least two different control zones applied to a Western blot strip in order to identify certain errors that can be traced back to these factors.

Furthermore, according to a preferred embodiment of the invention, it is possible to determine which conjugate was added to the Western blot strip using several different control zones. This eliminates confusion.

In a further advantageous embodiment of the invention, a "cut-off" sensor zone is also provided, with the help of which the appropriate incubation time can be determined in a simple manner (previously the "cut-off" sensor zone was on a separate Western blot strip)

According to the invention, these objects are achieved with Western blot strips according to claim 1.

Further advantageous embodiments of the invention are the subject of the dependent claims.

Figures I to VI show Western blot strips with multiple control zones according to the invention. The detailed description is given below in connection with the corresponding Examples I to VI.

Western blotting, i.e. protein blotting onto a carrier for immobilization with subsequent immunodetection, is a well

introduced immunological working technique and is described, for example, by F. Lottspeich and H. Zorbas (eds.) in "Bioanalytik", Spektrum Akademischer Verlag Heidelberg 1998 (p. 94ff.).
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The electrophoretic separation of any protein mixture before Western blotting is not subject to any particular restrictions and can be carried out, for example, by SDS gel electrophoresis according to size or molecular weight or by isoelectric focusing or, to improve resolution, by a combination of both techniques, i.e. so-called 2D electrophoresis. These electrophoretic separation techniques have been well established in bioanalytics for years (see, for example, the corresponding chapters in Lottspeich/Zorbas, supra) and therefore do not require any further discussion.

The subsequent actual Western blotting, i.e. the transfer of the separated proteins to a carrier (e.g. nitrocellulose) for immobilization, is also not subject to any particular restrictions and can be carried out, for example, by capillary blotting or electroblotting. These blotting techniques have been well established in bioanalytics for years (see, for example, the relevant chapters in Lottspeich/Zorbas, supra) and therefore also require no further discussion.

Immunodetection, i.e. visualization of antibody binding, can be carried out directly using appropriately labeled primary antibodies (i.e. antibodies specifically directed against the antigen) or indirectly using appropriately labeled antibodies against the primary antibody from another animal species (i.e. so-called secondary antibodies or anti-antibodies). Since indirect immunodetection

on has a higher sensitivity, it is preferred here. Any system can be used for labeling, e.g. biotin/avidin (streptavidin), colloidal gold or an enzyme-coupled system (e.g. using peroxidase or alkaline phosphatase). These immunoassays have been well established in bioanalytics for years (see, for example, the relevant chapters in Lottspeich/Zorbas, supra) and therefore do not require any further discussion. It is also self-evident that both polyclonal and monoclonal antibodies can be used.

It should also be noted that although the term "conjugate" here generally refers to enzyme-conjugated anti-human immunoglobulin antisera or enzyme-conjugated secondary antibodies, the term should also analogously include other labeling systems for anti-human immunoglobulin antisera or generally secondary antibodies from another animal species, e.g. the biotin/avidin (streptavidin) or colloidal gold system. The conjugate in these cases would be, for example, a biotinylated secondary antibody that reacts with labeled avidin (e.g. with biotinylated alkaline phosphatase or peroxidase), or a secondary antibody to which colloidal gold particles are bound to make it visible. This also applies analogously to the terms "conjugate control zone/band".

The control zones according to the invention are applied using a conventional dot method (see, for example, the corresponding chapters in Lottspeich/Zorbas, supra), e.g. using a Hamilton microsyringe or a microspray device.

Immunodetection in Western blotting is preferably carried out as follows.

Test execution
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The following general information should be observed:

• All test reagents (ie antibody-enzyme conjugate, dilution/wash buffer, enzyme substrate, Western blot strips commonly available commercially in the form of appropriate test kits for routine serology) should be brought to room temperature (20-25 ⁰ C) before use.

• Dilutions must be mixed well before testing.

• The test is carried out at room temperature.

• Antigen strips should only be transferred to the incubation trays using plastic tweezers at the upper marked end.

• During all incubation steps, the marked side of the strip with the bound antigen must be facing upwards.

• Make sure that the antigen strips are evenly covered with liquid and that the strips are not allowed to dry during the entire test procedure.

• All incubation steps are carried out on a rocking shaker .

• Changes in the specified incubation times and dilutions may lead to incorrect results.

• Use fresh pipette tips for each reagent.

• Cross-contamination between conjugate and chromogen/substrate must be avoided at all costs.

1. For each antigen sample, 1 Western blot strip is placed into a groove of a clean incubation tray using plastic tweezers.

2. 2 ml of ready-to-use dilution/wash buffer are pipetted into the grooves of the incubation tray on the shaker to completely moisten the strips.

3. 20 μl of patient serum are added by pipetting, if possible at the upper marked end of the strip. To ensure complete mixing with the dilution/wash buffer, the serum is added while the shaker is running. Patient serum and control serum are incubated on the shaker for 30 minutes. When pipetting and then pouring off, care must be taken to avoid cross-contamination of the individual patient samples.

4. The liquid is completely sucked out of the grooves or carefully poured off. When pouring off the liquid, the Western blot strips containing the antigen remain stuck to the bottom of the grooves. Any remaining liquid is allowed to drip off on absorbent paper.

5. Washing the Western blot strips: incubate 3x5 minutes on the shaker with 2 ml of ready-to-use dilution/wash buffer each. The wash buffer is always completely aspirated or poured off.

Before the final washing step, the required amount of fresh conjugate diluent is prepared (according to the manufacturer's instructions in the test kit package insert).
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6. The liquid is completely sucked out or poured off from the gutters.

7. Pipette 2 ml of the prepared conjugate dilution into the corresponding incubation wells and incubate on the shaker for 30 minutes.

8. The liquid is completely sucked out or poured out of the gutters.

9. Washing the Western blot strips: incubate 3x5 minutes on the shaker with 2 ml of ready-to-use dilution/wash buffer each. The wash buffer is always completely aspirated or poured off. Then rinse 1x1 minute with distilled water.

10. The liquid is completely drained or poured out of the gutters (see point 4).

11. 2 ml of ready-to-use substrate solution is pipetted into the grooves of the incubation tray and then allowed to develop on the shaker for 10 minutes.

12. Stop the color development by pouring off the substrate solution. The strips are then washed 3 times with 2 ml of distilled water each, without intermediate incubation.

13. The distilled water is poured off and the Western blot strips are dried on clean absorbent paper.

14. For evaluation, the evaluation protocol and the kit-specific template of the respective manufacturer will be used.

Evaluation

For reliable evaluation, each Western blot strip is provided with a
Serum control zone and three separate conjugate control zones
fitted.
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Serum control:

Only after incubation with patient serum does the

the marking line of the Western blot strip is the serum control band. The serum control zone is an applied mixture of anti-human IgG, anti-human IgM and anti-human IgA antiserum from another animal species (e.g. rabbit, goat) that reacts with the corresponding immunoglobulins in the patient serum, whereupon a band is formed by further reaction with the enzyme-conjugated anti-human IgG/IgM/IgA antiserum used to detect the pathogen antigens.

Conjugate control zone:

The Western blot strip is also provided with at least one additional control zone, which only produces a band after incubation with a conjugate. In the simplest case, this conjugate control zone is an applied mixture of anti-animal species IgG, anti-animal species IgM and anti-animal species IgA antiserum, where the term "animal species" refers to the other animal species (e.g. rabbit, goat) whose enzyme-conjugated anti-human IgG/IgM/IgA antiserum was used to detect the pathogen antigens.

For example, if an enzyme-conjugated anti-human IgG antiserum from goat was used to detect the pathogen antigens and to form the conjugate control band, anti-goat IgG antiserum from another animal species is applied in the conjugate control zone. The same applies to the other immunoglobulin classes.

In a preferred embodiment of the invention, three conjugate control zones are provided, namely in the form of separately applied anti-animal species IgG, anti-animal species IgM and anti-animal species IgA antiserum, whereby the above statements apply in an analogous manner. If a certain conjugate is used,

, a band appears at the corresponding point on the Western blot strip.

The test has been carried out correctly if both the serum control band and the conjugate control band are clearly visible on the developed antigen strip.

In a further advantageous embodiment of the invention, a "cut-off" sensor zone is also provided, with the help of which the appropriate incubation time with the substrate can be determined in a simple manner. If the Western blot strips are incubated with the substrate for too long, non-specific bands may appear, making evaluation more difficult. The "cut-off" sensor zone produces a band whose intensity can be used to determine whether incubation with the substrate should be stopped. Bands weaker than the "cut-off" band are not taken into account.

For example, the same mixture of anti-human IgG, anti-human IgM and anti-human IgA antiserum from another animal species (e.g. rabbit, goat) as in the serum control zone can be applied in the "cut-off" sensor zone, but in a lower concentration (e.g. 1/5 of the concentration in the serum control zone). Suitable concentrations are selected by the expert according to the practical circumstances or can be determined by simple routine tests.

Alternatively, a specific pathogen antigen can be used to determine the "cut-off", whereby the above statements apply analogously.

Example I (Fig.l):

Shown are Western blot strips after incubation with patient sera that contain IgG, IgM or IgA antibodies against a bacterial pathogen, such as B. burgdorferi (positive sera) and that were developed with anti-human IgG conjugate (Fig. IA) or anti-human IgM conjugate (Fig. IB) or anti-human IgA conjugate (Fig. IC) (from goat). The strips can show different pathogen-specific bands (e) depending on the immunoglobulin class of the conjugate. A conjugate control band (b, c, d) forms when the strip was developed with a conjugate. The serum control band (a) forms when patient serum was developed together with a conjugate. The order of the control zones on the strip is: serum control zone (a), IgG conjugate control zone (b), IgM conjugate control zone (c), IgA conjugate control zone (d). However, this order does not have to be followed, but can be any order.

Example II (Fig. II)

Shown is a Western blot strip after incubation with a patient serum that does not contain IgA antibodies against a bacterial pathogen, such as B. burgdorferi (negative serum) and that was developed with an anti-human IgA conjugate. There are therefore no pathogen-specific bands visible on the strip, only the serum control band (a) and the IgA conjugate control band (d). The test has been carried out correctly as the serum control band and the conjugate control band are visible.

Example III (Fig. Ill):

Shown is a Western blot strip after incubation with an anti-human IgG conjugate. No pathogen-specific bands can be seen on the strip. Only an IgG conjugate control band (b) is visible. This finding shows that the strip was developed without the addition of serum. The test was carried out incorrectly and must therefore be repeated with the addition of serum.
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Example IV (Fig. IV):

A Western blot was performed to detect IgG antibodies. Pathogen-specific bands (e) and the IgM conjugate control band (c) are visible. This finding shows that an anti-human IgM conjugate was added instead of an anti-human IgG conjugate. The test was carried out correctly, but with the wrong conjugate. The test must therefore be repeated with the addition of the anti-human IgG conjugate.

Example V (Fig. V)

A Western blot should be performed to detect IgG antibodies. No control bands are visible on the strip and the test was therefore performed incorrectly. This finding shows that at least no conjugate was added. The test must therefore be repeated by adding an anti-human IgG conjugate.

Example VI (Fig. VI):

Western blots were performed to detect IgG anti-

bodies with strips numbered 1 to 4 from the kit with lot number (A1-A4) or for the detection of IgA antibodies with strips 1 to 4 from the kit with lot number (Bl-B4). The strips developed with the anti-human IgG conjugate or anti-human IgA conjugate were accidentally mixed up. Using the IgG or IgA conjugate control bands it is still possible to assign strips 1 to 4 to lot number A or B.

Claims (9)

Protection claims: 1. Western blot strip which has on a carrier a serum control zone, which indicates the incubation of the strip with patient serum by means of a corresponding band, and at least one conjugate control zone, which indicates the incubation of the strip with a labelled anti-patient immunoglobulin antibody from another animal species by means of a corresponding band. 2. Western blot strip according to claim 1, which has three separate conjugate control zones which indicate the incubation of the strip with labeled anti-patient immunoglobulin antibodies from another animal species by a corresponding band and whether the anti-patient immunoglobulin is directed against IgG, IgM or IgA. 3. Western blot strip according to claim 1 or 2, which further comprises a cut-off sensor zone for determining the optimal incubation time with the substrate. 4. Western blot strip according to one of the preceding claims, wherein the serum control zone comprises a mixture of immobilized anti-patient immunoglobulin antibodies of all classes. 5. Western blot strip according to one of the preceding claims, wherein in the case (a) only one conjugate control zone containing a mixture of immobilized anti-animal species immunoglobulin antibodies of all classes and (b) three separate conjugate control zones, each of which only immobilized anti-animal species immunoglobulin antibodies of one of the classes IgG, IgM or IgA, where the term "animal species" refers to the other animal species from which the labeled anti-patient immunoglobulin antibodies are derived. 6. Western blot strip according to one of the preceding claims, wherein the labeling of the anti-patient immunoglobulin antibodies is based on the biotin/avidin (streptavidin), the colloidal gold or an enzyme-conjugated system. 7. The Western blot strip of claim 6, wherein the ELISA system comprises anti-patient immunoglobulin antibodies conjugated to peroxidase or alkaline phosphatase. 8. Western blot strip according to one of the preceding claims, wherein the patient is a human. 9. A Western blot strip according to any preceding claim, wherein the other animal species is a mouse, a rabbit or a goat.