WO2009156475A1

WO2009156475A1 - Method for the specific detection of map antibodies

Info

Publication number: WO2009156475A1
Application number: PCT/EP2009/057976
Authority: WO
Inventors: Rolf Bauerfeind; Christian Menge; Simone Schillinger; Stefanie Barth; Philip Bridger
Original assignee: Justus-Liebig-Universität Giessen
Priority date: 2008-06-25
Filing date: 2009-06-25
Publication date: 2009-12-30
Also published as: DE102008029834A1

Abstract

The present invention relates to a method and a test kit for the specific and sensitive detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in biological samples. The method and the test kit are also used to detect antibodies against Mycobacterium tuberculosis or Mycobacterium bovis.

Description

Patent application

Method for the specific detection of MAP antibodies

The present invention relates to a method for the specific and sensitive detection of antibodies against the microorganism currently designated as Mycobacterium avium subspecies (ssp.) Paratuberculosis (MAP) (without prejudice to its future name) in organ, tissue and faeces samples (biological samples). by means of flow cytometry measurement after pre-adsorption of the biological samples with intact bacteria of the species Mycobacterium avium ssp. avium (MAA) and Mycobacterium phlei (M. phlei) and a test kit for the differentiation of antibody subtypes in IgG total, IgGi and / or IgG ₂ .

State of the art

Paratuberculosis caused by the bacterium Mycobacterium avium ssp. paratuberculosis (MAP), is a chronic intestinal disease of ruminants, which is characterized mainly by a progressive, granulomatous inflammation of the small intestinal wall. The animals usually become infected within the first weeks of life, but usually show clinical symptoms only years after the infection. From an unknown point in time during the long incubation period, infected animals begin to excrete the pathogen - mostly intermittently - with the feces and thus become a source of infection for other animals. Transplacental transmission to the fetus is discussed as a further route of infection. Due to missing signs of disease during the long incubation period, infected animals remain hidden from the pet owner. Another health-related aspect is the fact that MAP is suspected to be causally involved in the disease of Crohn's disease, an incurable chronic inflammatory disease of the digestive tract, or to sustain the disease. For the rehabilitation of infected herds or for the prophylaxis of paratuberculosis diseases, it is therefore important to detect infected animals at the earliest stage of the infection (ie in the young age). Special features of the pathogen-host interaction dominate the clinically inapparent phase at the beginning of the infection by the cellular immune response, while the humoral immune response is weak. For this reason, particularly sensitive methods are required for the detection of antibodies during this period.

For the diagnosis of paratuberculosis, there are several methods known to those skilled: The so-called gold standard of Paratuberkulosediagnostik is the pathogen detection by means of cultural-bacteriological examination of animal faeces or Ileocaecallymphknoten (intestinal lymph nodes). Another method known to the person skilled in the art detects MAP-specific DNA in biological samples by means of PCR technology. These two methods are direct methods to detect MAP infections in animals. In addition, there are known to the expert indirect methods, for example by the detection of MAP-specific antibodies in the blood serum of the animals. For example, pathogen-specific IgG antibodies are detected by enzyme-linked immunosorbent assays (ELISA). For this purpose, several test kits are commercially available, such as Svanovir® paratuberculosis ELISA from Svanova, Sweden, Pourquier® ELISA from Institut Pourquier, France, HerdChek ™ Mycobacter paratuberculosis ELISA from Idexx, Germany. Paracheck® ELISA from Prionics AG, Schweitz or ID Screen® Paratuberculosis Indirect ELISA from ID VET, France. These test kits differ from each other mainly in the use and nature of the test antigen and in the preparation of bacteria for pre-adsorption.

Another indirect detection method is the antibody detection by flow cytomethe (DFZM), for example, using intact bacteria as antigen. Thus, the MAP-specific antibody detection is possible from the 170th day after an experimental infection. Disadvantages of the prior art:

In pathogen detection by means of cultural-bacteriological examination of animal excrement, the pathogen attack is very tedious (at least 8 weeks) and false negative findings are very common because pathogens in the early stages of infection are not or only intermittently excreted. A reasonably reliable statement about the MAP-free status of a single animal is therefore possible only after multiple sampling and examination with negative findings. The cultural bacteriological examination of the ileocaecally lymph node (intestinal lymph node) has, in addition to the lengthy growing period already mentioned, the further disadvantage that the intestinal lymph nodes of the animal are only accessible to abdominal cavity surgery for such an examination. Therefore, this method currently appears to be of limited practical use.

The same applies to the direct MAP detection by means of the mentioned PCR method. Although less time-consuming than culturally-bacteriological examination, it is often less sensitive.

The disadvantages of the systems for indirect detection of pathogens by means of the ELISA technique are the following: Only the MAP-specific total content of IgG is determined, but not according to IgG subclasses (IgGi, IgG ₂ ) or even the ratio of the IgG subclasses to each other (IgGi to IgG ₂ ). This may be one of the reasons why the specificity and / or sensitivity of these tests are low (around 50%), especially in the early phase of MAP infection. Another cause can be seen in the test antigens used. As a result of their production, these preparations may contain structure-conserved epitopes, which also occur in other bacterial infectious agents. In such a test, therefore, animals can react in a positive way, the corresponding antibodies have not formed in response to MAP, but on any other bacteria (false positive reaction due to serological cross-reaction). On the other hand, MAP-specific cell wall antigens may have been destroyed in the course of test antigen production, which may lead to false-negative findings. The same problems also apply to the bacterial cell preparations used in some preabsorption systems. The previously described methods for antibody detection by means of flow cytomethe aim only at the detection of MAP-specific total IgG. Furthermore, the serum sample to be tested is only combined with a single type of mycobacteria, namely MAP, and is not pre-adsorbed so that the specificity is also limited with this method.

All presently available diagnostic tests are only slightly sensitive and / or specific, especially at the early time of infection (ie up to the second year of age of the subject), so that MAP-infected and MAP-free individuals can not be identified with sufficient certainty and many animals falsely diagnosed as positive or negative.

This results in the following significant disadvantages: Although an animal may have been infected for a long time, its infection status can only be determined at a time after it itself has again born a calf or several calves and may have already infected them. Even at the inventory level, the diagnostic freedom available so far can not be used to ascertain the MAP freedom with sufficient certainty. Even if the serological and bacteriological examination of the animals leads to a negative result, it can not be ruled out that the offspring has already been infected with the pathogen. A reliable assurance of the MAP-free status of animals to be integrated into paratuberculosis-free stocks is currently impossible, but is of great importance in preventing the spread of this infectious disease. This applies in particular to the certification of breeding animals in the context of intra-community transfers and exports, which in the future will be required of more and more countries (so far, inter alia, from Austria).

Conclusion: There is currently no known method in the prior art that discloses a satisfactorily specific and sensitive detection of MAP infections using MAP-specific antibodies in biological samples, especially faeces, blood, organ and tissue samples. task

The object of the present invention is therefore to remedy the disadvantages described in the prior art by formulating a substantially improved method for the detection of MAP-specific antibodies and a test kit for differentiating the antibody subtypes in IgG total, IgGi and / or IgG ₂ provided.

The object of the present invention is therefore to remedy the described disadvantages in the prior art by formulating a substantially improved method for the detection of MAP-specific antibodies and a test kit for differentiating the immunoglobulin classes IgA, IgM and IgG-total, as well as the IgG Subclasses IgG-i and IgG _{2 is} provided.

A further object of the present invention is to remedy the described disadvantages in the prior art by formulating a substantially improved method for the detection of Mycobacterium tuberculosis or Mycobacterium bov / s-specific antibodies and a test kit for differentiating the immunoglobulin classes IgA, IgM and IgG total, and the IgG subclasses IgGi and IgG _{2 is} provided.

solution

The object is achieved by a method and a test kit according to the claims.

The method consists of a method for the detection of MAP-specific antibodies, based on the measurement of antigen-antibody compounds by flow cytomethe (DFZM).

Here are using mycobacteria, preferably Mycobacteria of intact living antigen detected as MAP-specific antibodies, specifically either for total IgG or IgG isolated by Total, IgGl and / or IgG. ₂ In addition, a further step for pre-adsorption of the non-specific antibodies is introduced by incubating the substrate to be tested with intact and standardized bacteria of the species Mycobacterium avium ssp. avium (MAA) and Mycobacterium phlei (M. phlei) is incubated. As a result, both the detection of the specific antibodies more accurately and at an earlier point in time of the infection is surprisingly possible. Also, such cross-reacting non-specific antibodies can be more effectively eliminated. The method consists of a method for the detection of MAP-specific antibodies, based on the measurement of antigen-antibody compounds by flow cytomethe (DFZM).

Here are using mycobacteria, preferably of intact mycobacteria detected as antigen MAP-specific antibodies, and that optionally for IgG whole or separated according to immunoglobulin classes IgA, IgM and IgG total, as well as the IgG subclasses IgGl and IgG. ₂ In addition, a further step for pre-adsorption of the non-specific antibodies is introduced by incubating the substrate to be tested with intact and standardized bacteria of the species Mycobacterium avium ssp. avium (MAA) and Mycobacterium phlei (M. phlei). As a result, both the detection of the specific antibodies more accurately and at an earlier point in time of the infection is surprisingly possible. Also, such cross-reacting non-specific antibodies can be more effectively eliminated.

The method consists of a method for the detection of Mycobacterium tuberculosis or Mycobacterium bows-specific antibodies, based on the measurement of antigen-antibody compounds by flow cytometry (DFZM).

Mycobacterium tuberculosis or Mycobacterium boWs-specific antibodies are detected using mycobacteria, preferably intact mycobacteria, as an antigen, optionally for IgG total or separately according to immunoglobulin classes IgA, IgM and IgG total, as well as

IgG subclasses IgG-i and IgG ₂ . In addition, a further step for pre-adsorption of the non-specific antibodies is introduced by incubating the substrate to be tested with intact and standardized bacteria of the species Mycobacterium avium ssp. avium (MAA) and Mycobacterium phlei (M. phlei). As a result, both the detection of the specific antibodies more precisely and at an earlier point in time of the infection are surprisingly possible. Also, such cross-reacting non-specific antibodies can be more effectively eliminated. The test kit consists of bacterial cells, preferably of intact living bacterial cells of the species Mycobacterium avium ssp. paratuberculosis (MAP), Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei).

The test kit for detecting MAP consists of bacterial cells, preferably intact bacterial cells of the species Mycobacterium avium ssp. paratuberculosis (MAP), Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei [M. phlei).

The test kit for the detection of Mycobacterium tuberculosis or Mycobacterium bovis consists of bacterial cells, preferably from intact bacterial cells of the species Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei).

For the first time, therefore, a laboratory diagnostic procedure is available that can detect or rule out MAP infection at a very early stage, especially in calves. The method is characterized by a high sensitivity and specificity for MAP infections, is practicable and leads to a fast and reliable diagnosis as the following examples show.

For the first time, a laboratory diagnostic procedure is now available that can detect or exclude a MAP infection at a very early stage. The method is characterized by a high sensitivity and specificity for MAP infections, is practicable and leads to a fast and safe diagnosis as the following examples show.

For the first time, therefore, a laboratory diagnostic procedure is available, with which a Mycobacterium tuberculosis or Mycobacterium bows infection can already be detected or excluded at a very early point in time. The method is characterized by a high sensitivity and specificity for Mycobacterium tuberculosis or Mycobacterium bows infections, is practicable and leads to a fast and reliable diagnosis as the following examples show. embodiments

With the method described herein according to the invention, serum samples, preferably of cattle, are tested for MAP-specific antibodies. The blood samples required for this purpose are taken from the subjects according to a method familiar to the person skilled in the art and further processed to give serum samples.

With the method described herein according to the invention, serum samples are tested for Mycobacterium tuberculosis or Mycobacterium bows-specific antibodies. The blood samples required for this purpose are taken from the subjects according to a method familiar to the person skilled in the art and further processed to give serum samples.

The cultivation of the MAP bacteria required for the process according to the invention is carried out by a method familiar to the skilled worker, such as, for example, the following: Living MAP bacterial cells, for example the MAP strain K10 (ATCC No. BAA-968), are detected Grown in Middlebrook 7H9 medium by centrifugation and resuspended with a PBS buffer containing preferably 0.5% Tween ^80® (eg Sigma-Aldrich GmbH) and a blocking buffer (eg Perbio Science, Germany). The cultivation of the Mycobacterium tuberculosis or Mycobacterium bows bacteria required for the process according to the invention is carried out by a method familiar to the person skilled in the art.

Subsequently, the flow cytometric measurement is carried out according to the method known to those skilled in the art such as described here: After re-centrifugation, the supernatant is discarded and the MAP-containing pellet with the bovine serum in question (preferably diluted to 1/50 [v / v]) for example Incubated for 1 h at 20 ° C. This is followed by three washing steps followed by incubation (eg 1 h at 20 ° C. in the absence of light) in a dilute solution of the secondary antibody which detects species-specific IgG (for example FITC-conjugated rabbit anti-bovine IgG immunoglobulin, ICN Biomedics, Costa Mesa, USA), as long as it is bound to the MAP bacterial cells. After three washes, the MAP-Baktehen cells are analyzed in the flow cytometer. Siert. The mean fluorescence intensity (MFLI) of the MAP bacterial cells of a test batch is determined. The controls or references are MAP cells incubated with standardized pools of serum samples from MAP-negative and -positive animals of the same species. Subsequently, to detect MAP, the flow cytometry measurement is carried out according to the method known to the person skilled in the art, such as described here: After renewed centrifugation, the supernatant is discarded and the MAP-containing pellet is diluted with the bovine serum in question (preferably diluted to 1/50 [v / v ]) for example for 1 h at 20 ° C incubated. This is followed by three washing steps followed by incubation (for example 1 h at 20 ° C with exclusion of light) in a dilute solution of the secondary antibody, which specifically detects the immunoglobulin classes IgA, IgM and IgG-total, as well as the IgG subclasses IgG-i and IgG ₂ if so bound to the MAP bacterial cells. After three washes, MAP bacterial cells are analyzed by flow cytometer. The mean fluorescence intensity (MFLI) of the MAP bacterial cells of a test batch is determined. The controls or references are MAP cells incubated with standardized pools of serum samples from MAP-negative and -positive animals of the same species.

Subsequently, to detect Mycobacterium tuberculosis or Mycobacterium bovis, the flow cytometre measurement is carried out according to the method familiar to the person skilled in the art. After renewed centrifugation, the supernatant is discarded and the Mycobacterium tuberculosis or Mycobacterium bows-containing pellet incubated with the serum in question. This is followed by three washing steps followed by incubation in a dilute solution of the secondary antibody, which specifically see the immunoglobulin classes IgA, IgM and IgG-total, and the IgG

Subclasses IgG-i and IgG ₂ detected, if it is bound to the Mycobacterium tuberculosis or Mycobacterium bows bacterial cells. After three washes, the Mycobacterium tuberculosis or Mycobacterium bows bacterial cells are analyzed by flow cytometer. The mean fluorescence intensity (MFLI) of the Mycobacterium tuberculosis or Mycobacterium bov / s bacterial cells of a test batch is determined. As controls or references are Mycobacterium tuberculosis or Mycobacterium bovis ZeWen, with standardized pools of serum samples from Mycobacterium tuberculosis or Mycobacterium öov / s negative and positive subjects were incubated.

The method according to the invention consists of the following additional steps:

Step 1: Pre-adsorption of live mycobacterial sera in close (MAA) and / or distant taxonomic relationship

(M. phlei) to MAP to block non-specific antibodies.

Step 2: The use of standardized bacterial cells as test antigen, according to the invention of Mycobacterium avium ssp. paratuberculosis (MAP). Step 3: Evidence of immunoglobulin subtypes IgG total and / or IgGi and / or IgG ₂ .

The method according to the invention for the detection of MAP consists of the following alternative steps:

Step 1: The pre-adsorption of the sera with intact mycobacteria in close (MAA) and / or remote taxonomic relationship

(M. phlei) to MAP to block non-specific antibodies.

Step 2: The use of standardized bacterial cells as test antigen, according to the invention of Mycobacterium avium ssp. paratuberculosis (MAP). Step 3: The detection of the immunoglobulin classes IgA, IgM and IgG total, as well as the IgG subclasses IgG-i and IgG ₂ .

The method according to the invention for the detection of Mycobacterium tuberculosis or Mycobacterium bovis consists of the following alternative steps:

Step 1: Pre-adsorption of sera with intact mycobacteria in close and / or remote taxonomic relationship to Mycobacterium tuberculosis or Mycobacterium bows bacteria and with MAP to block non-specific antibodies.

Step 2: The use of standardized bacterial cells as test antigen, according to the invention of Mycobacterium tuberculosis or Mycobacterium bows bacteria. Step 3: The detection of the immunoglobulin classes IgA, IgM and IgG total, as well as the IgG subclasses IgG and IgG ₂ . to step 1 for the detection of MAP: For pre-adsorption, for example, bacteria of the species MAA (German Collection of Micro-organisms and cell cultures DSMZ, Braunschweig, strain DSM

44156) and M. phlei (DSMZ, strain DSM 43239) are preferably grown in Middrechtrook 7H9 medium and harvested by centrifugation at the stage of exponential bacterial growth. The bacterial pellet is resuspended in a PBS buffer containing examples game, 0.5% Tween ^® 80 and 10% of a blocking buffer (such as

Superblock buffer in PBS). Subsequently, the serum sample to be tested is preferably mixed in a ratio of 1:50 [v: v] with the bacterial suspension and incubated for about one hour at room temperature. Thereafter, the bacteria are centrifuged off again and the supernatant (pre-adsorbed serum sample) as described above in reaction mixtures with MAP bacteria used.

Further exemplary embodiment of step 1 for the detection of Mycobacterium tuberculosis or Mycobacterium bovis: The pre-adsorption of the serum samples in question is carried out by a method known to a person skilled in the art. Subsequently, the test to be tested

Serum sample mixed with the bacterial suspension and incubated. Thereafter, the bacteria are again centrifuged off and the supernatant (pre-adsorbed serum sample) as described above in reaction mixtures with Mycobacterium tuberculosis or Mycobacterium bows-Baktehen used. to step 2 for the detection of MAP: It turned out, surprisingly, that the serum samples reacted differently with the replicable mycobacteria, depending on which growth phase these germs were in when they were used for the investigation. Preferably, the test bacteria are useful when harvested in the exponential growth phase. To eliminate the influence of the growth phase on the measurement results, now only bacterial cells which are grown to Middlebrook 7H9 medium to a defined phase, then harvested and stored at -70 ° C until use. This applies both to bacteria used for detection (MAP) and to those used for pre-adsorption (for example MAA and / or M. phlei).

Another exemplary embodiment of step 2 for the detection of Mycobacterium tuberculosis or Mycobacterium bovis: It turned out, surprisingly, that the serum samples reacted differently with the mycobacteria that could be propagated, depending on which growth phase these germs were in when they were used for the examination. Preferably, the test bacteria are useful when harvested in the exponential growth phase. To eliminate the influence of the growth phase on the measurement results, now only bacterial cells are used, which are grown to a defined phase, then harvested and stored at -70 ° C until use. This applies both to bacteria used for the detection of {Mycobacterium tuberculosis or Mycobacterium bovis bacteria) and to those used for pre-adsorption. to step 3 for the detection of MAP: Instead of the previous detection of MAP-specific total IgG leads according to the invention, the selective search for MAP-specific IgG total, IgGi and / or IgG ₂ (for example with the FITC conjugate sheep anti-bovine IgG-1 / IgG ₂ from ABD Serotec) to a significantly better separation of positive and negative samples. This is especially true for IgGi, as these antibodies are very antigen specific. According to the invention, the sensitivity as well as the specificity with respect to the total IgG gene can thus be determined with the sub-type-specific detection.

Detection can be significantly increased because nonspecific binding of other IgG classes can be ignored. Since it is known that in the early phase of a MAP infection IgG ₂ in relation to IgGi dominates, is the distinction of these two IgG subclasses of high diagnostic value.

Another embodiment of step 3 for the alternative detection of MAP: Instead of the previous detection of MAP-specific total IgG leads according to the invention, the selective search for the

MAP-specific immunoglobulin classes IgA, IgM and IgG-total, as well as the IgG subclasses IgG-i and IgG ₂ (for example with the FITC conjugate sheep anti-bovine IgG ₁ ZIgG ₂ , IgM or IgA of ABD Serotec) to a significantly better Separation of positive and negative samples. This is especially true for IgGi, as these antibodies are very antigen specific. Accordingly, according to the invention, the sensitivity as well as the specificity relative to the total IgG detection can be significantly increased with the subtype-specific detection, since non-specific binding of other IgG classes can be ignored. Since it is known that IgG _{2 dominates} in relation to IgGi in the early phase of MAP infection, the distinction of these two IgG subclasses is of high diagnostic value.

Further Exemplary Embodiment for Step 3 for the Detection of Mycobacterium tuberculosis or Mycobacterium bovis: Instead of detecting Mycobacterium tuberculosis or Mycobacterium bovis-specific total IgG, according to the invention the selective search for the Mycobacterium tuberculosis or Mycobacterium bows-specific immunoglobulin classes IgA leads to IgM and IgG total, as well as the IgG subclasses Igd and IgG ₂ to a much better separation of positive and negative samples. This is especially true for IgGi, as these antibodies are very antigen specific. Accordingly, according to the invention, the sensitivity as well as the specificity relative to the total IgG detection can be significantly increased with the subtype-specific detection, since non-specific binding of other IgG classes can be ignored.

Further advantageous embodiments are, for example: - Use of MAP field strains instead of the laboratory strain MAP-K10. The MAP strain K10, which has been passing through the laboratory for years, may no longer have all diagnostically important antigens. The identification and use of a field strain with better antigenic properties enhances serological differentiation between MAP-infected and MAP-negative animals.

- Use of Mycobacterium tuberculosis or Mycobacterium bows field strains. The identification and use of a field strain with better antigenic properties enhances the serological discrimination between Mycobacterium tuberculosis or Mycobacterium bows-infected and Mycobacterium tuberculosis or Mycobacterium öov / s-negative subjects.

- Use of farm-specific MAP and MAA field strains from farms, in which infected animals must be reliably distinguished from non-infected animals. It can be assumed that antigenic differences exist between different strains of the same species of mycobacteria. The use of farm-specific strains enhances serological distinctness between MAP-infected and MAP-negative animals. - Use of specific Mycobacterium tuberculosis or Mycobacterium bows strains and MAA strains. It can be assumed that antigenic differences exist between different strains of the same species of mycobacteria. The use of specific strains enhances the serological distinctness between Mycobacterium tuberculosis or Mycobacterium bovisinfected and Mycobacterium tuberculosis or Mycobacterium bovis negative subjects.

An alternative to the use of MAP bacteria as corpuscular test antigens, for example in the flow cytometer, is the use of beads or colloids which are coated with fission products or mixtures of the above-mentioned bacteria. The Analysis method is even better to standardize in this version and thus shows an increased specificity.

An alternative to the use of Mycobacterium tuberculosis or Mycobacterium öows bacteria as particulate test antigens, e.g. in the flow cytometer is the use of beads or colloids

(Colloids), which are coated with fission products or mixtures of the above-mentioned bacteria. The analysis method is even better to standardize in this version and thus shows an increased specificity. A particularly advantageous further improvement according to the invention relates to the evaluation and calculation of the measured values for a MAP antibody titer characterizing the respective serum sample. Each serum sample is pre-adsorbed with MAP-related bacteria (for example, MAA or M. phlei). Each pre-adsorbed serum sample is then analyzed once with MAP germs and once with MAA cells, initially measuring the mean fluorescence intensity (MFLI). To determine a MAP-specific antibody titer, the ratio of the two values is calculated as follows:

MFLI (MAP)

MAP - specific antibody titer of a serum sample logo CNRS logo INIST

MFLI (MAA)

With this approach, methodical and individual-related

Variations of the test results minimized.

A particularly advantageous further improvement according to the invention relates to the evaluation and calculation of the measured values for a MAP antibody titer characterizing the respective serum sample. Each serum sample is pre-adsorbed with MAP-related bacteria (for example, MAA or M. phlei). Each pre-adsorbed serum sample is then analyzed once with MAP germs and once with MAA cells, initially measuring the mean fluorescence intensity (MFLI). In order to determine a MAP-specific antibody titer, the ratio of both values is calculated as follows: MAP - spec. Antibody titer of a serum sample = MFLI (MAP) - MFLI (MAA)

This procedure minimizes methodological and individual variations of the test results. This evaluation and calculation of the measured values for a Mycobacterium tuberculosis or Mycobacterium bows antibody titer characterizing the respective serum sample is carried out in the same way.

- Use of the methodology in other infections with animal or zoonotic agents. - Application of the methodology to detect other infections with

Animal or zoonotic pathogens, especially for the detection of infections with mycobacteria, in particular for the detection of infections with Mycobacterium tuberculosis or Mycobacterium bows bacteria.

One embodiment of the invention relates to an easy-to-use test kit for the specific and sensitive detection of MAP in tissue and faeces samples. The test kit for the specific detection of Mycobacterium avium ssp. paratuberculosis (MAP) comprises live intact bacteria of the species Mycobacterium avium ssp. paratuberculosis (MAP), Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei).

In the embodiment according to the invention, the test kit furthermore comprises reagents for culturing and harvesting mycobacteria, immunodecoration for analysis by flow cytometry and labeled secondary antibodies known to the skilled person against species-specific IgG and / or IgGr and / or IgG ₂ for the differentiation of IgG total and / or or IgGr and / or IgG ₂ subtypes.

One embodiment of the invention relates to an easy-to-use test kit for the specific and sensitive detection of MAP in tissue and faeces samples. The test kit for the specific detection of Mycobacterium avium ssp. paratuberculosis (MAP) comprises intact bacteria of the species Mycobacterium avium ssp. paratuberculosis (MAP), Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei). In the embodiment of the invention, the test kit further comprises reagents for breeding and harvesting mycobacteria, for immuno-decoration for analysis by flow cytometry. Alternatively, the test kit additionally comprises labeled secondary antibodies known to the person skilled in the art against the species-specific immunoglobulin classes IgA, IgM and IgG-total, and the IgG subclasses IgG-i and IgG ₂ for differentiation into the immunoglobulin classes IgA, IgM and IgG-total, as well as into the IgG subclasses IgG-i and IgG ₂ .

One embodiment of the invention relates to an easy-to-use test kit for the specific and sensitive detection of Mycobacterium tuberculosis or Mycobacterium bows bacteria in tissue and faeces or stool samples. The test kit for the specific detection of Mycobacterium tuberculosis or Mycobacterium öows bacteria comprises intact bacteria of the species Mycobacterium tuberculosis or Mycobacterium bovis, Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei).

In the embodiment of the invention, the test kit further comprises reagents for breeding and harvesting mycobacteria, for immuno-decoration for analysis by flow cytometry. Alternatively, the test kit additionally comprises labeled secondary antibodies known to the person skilled in the art against the species-specific immunoglobulin classes IgA, IgM and IgG-total, and the IgG subclasses IgG-i and IgG ₂ for differentiation into the immunoglobulin classes IgA, IgM and IgG-total, as well as into the IgG subclasses IgG-i and IgG ₂ .

Based on the teachings of the present invention, and on the basis of general knowledge in this technical field, it is known to the manufacturer of the kit according to the invention how to use the individual components of the kit, e.g. Buffer produces, formulates and stores.

Claims

claims

1. A method for the specific detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in biological samples by means of a flow cytometric measurement characterized by the steps:

Pre-adsorption of the biological samples with mycobacteria in close (for example MAA) and remote (for example M. phlei) taxonomic relationship. Use of standardized bacterial cells (preferably MAP) as test antigen.

- Detection of immunoglobulin subtypes IgG total and / or IgGi and / or IgG2.

2. The method according to claim 1, characterized in that

- Animal-specific anti-IgG and / or anti-IgGi and / or anti-IgG ₂ can be used as a secondary antibody for the flow cytometre measurement.

The results of the flow cytometric measurement using the control results against Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei).

3. Method according to claims 1 and 2, characterized in that the biological samples are from mammals or poultry or humans.

4. Test kit for the specific detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in biological samples, characterized in that: - living mycobacteria of the species Mycobacterium avium ssp. paratuberculosis (MAP), which were preferably harvested within the exponential growth phase. - Living mycobacteria of the species Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei) are included for the pre-adsorption of the biological samples, which were preferably harvested within the exponential growth phase.

5. Testkit according to claim 4, characterized in that

The test kit contains secondary antibodies which are suitable for use in flow cytometry measurements, preferably species-specific anti-IgG and / or anti-IgGi and / or anti-IgG ₂ .

6. Method for the specific detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in biological samples by means of a flow cytometric measurement characterized by the steps:

Pre-adsorption of the biological samples with mycobacteria in close (for example MAA) and remote (for example M. phlei) taxonomic relationship.

Use of standardized bacterial cells (preferably MAP) as test antigen.

- Detection of the immunoglobulin classes IgA, IgM and IgG total, as well as the IgG subclasses IgG-i and IgG ₂ .

7. The method according to claim 6, characterized in that

- For the flow cytometre measurement as a secondary antibody, the species-specific anti-IgA, IgM and IgG total antibodies, as well as anti-IgG Ig and IgG ₂ antibodies are used.

8. A method for the specific detection of antibodies against Mycobacterium tuberculosis or Mycobacterium bovis in biological samples by means of a flow cytometric measurement characterized by the steps: - Pre-adsorption of biological samples with mycobacteria in close and distant taxonomic relationship and with MAP.

Use of standardized bacterial cells (preferably Mycobacterium tuberculosis or Mycobacterium bows bacteria) as test antigen.

9. The method according to claim 8, characterized in that - specific anti-IgA, IgM and IgG total antibodies, as well as anti-IgGi and IgG ₂ antibodies are used for the flow cytometre measurement as secondary antibodies.

10. The method according to claim 6 to 9, characterized in that the biological samples are derived from mammals or poultry or humans.

1 1.Testkit for performing a method according to claim 1 and claim 6 for the specific detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in biological samples, characterized in that

- Intact mycobacteria of the species Mycobacterium avium ssp. paratuberculosis (MAP), which were preferably harvested within the exponential growth phase.

- Intact mycobacteria of the species Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei) are included for the pre-adsorption of the biological samples, which were preferably harvested within the exponential growth phase.

12. Testkit according to claim 1 1, characterized in that

The test kit contains secondary antibodies which are suitable for use in flow cytometric measurements, preferably species-specific anti-IgA, IgM and IgG total antibodies, and anti-IgG-i and IgG ₂ antibodies.

13. Test kit for carrying out a method according to claim 1 and claim 8 for the specific detection of antibodies against Mycobacterium tuberculosis or Mycobacterium bovis in biological samples, characterized in that

- Intact mycobacteria of the species Mycobacterium tuberculosis or Mycobacterium bovis are included, which were preferably harvested within the exponential growth phase.

14. Testkit according to claim 13, characterized in that

The test kit contains secondary antibodies suitable for use in flow cytometry measurements, preferably specific anti-IgA, IgM and IgG total antibodies, as well as anti-IgG-i and IgG ₂ antibodies.