A process for obtaining whole cell wall antigens from a microorganism and an immunoassay for detecting antibodies related to a microbial infection using said whole cell wall antigens

Abstract

The present invention relates to a process for obtaining whole cell wall antigens from a microorganism. The present invention also relates to an immunoassay for detecting antibodies related to a microbial infection comprising the step of using the whole cell wall antigens. The present invention further relates to the use of the immunoassay in a method for analysing mammary glands health status, colostrum quality or milk tank quality.

Description

[0001] A PROCESS FOR OBTAINING WHOLE CELL WALL ANTIGENS FROM A MICROORGANISM AND AN IMMUNOASSAY FOR DETECTING ANTIBODIES RELATED TO A MICROBIAL INFECTION USING SAID WHOLE CELL WALL ANTIGENS

[0002] Field of the invention

[0003] The present invention relates to the field of immmunology. In particular the present invention relates to a process for obtaining whole cell wall antigens from a microorganism. The invention further relates to an immunoassay based on whole cell wall antigens from microorganisms in bodily fluids of an animal, for example milk, suitable to detect specific antibodies related to a microbial infections.

[0004] Background

[0005] Detection of specific antibodies against microorganisms in bodily fluids have important limitations due to the lack of sensitivity and specificity and the presence of interfering antibodies.

[0006] Immunoassays based on specific proteins expressed by microorganisms as antigen relay on the expression of these proteins so it lacks sensitivity when these proteins are not expressed by a certain strain. Immunoassays based on crude extracts as antigen often lack specificity as several microbial proteins are widely expressed and even some proteins bind immunoglobulins (for instance Staphylococcus aureus protein A).

[0007] In addition, the presence of microbial antibodies is often of poor diagnostic value due to the presence of innate antibodies and linked antibodies to overcome infections that can interfere with the results.

[0008] WO 97 / 26007 discloses a purified antigen having the characteristics of a high molecular weight antigen obtained from the aqueous extract of autoclaved cells of Mycobacterium sp. by digestion of the extract with proteinase K and collection of an ultrafiltrate residue from the digested extract by ultrafiltration through an ultrafiltration membrane, the high molecular weight antigen being retained in the ultrafiltrate residue.

[0009] Severin Anatoly et al. “Proteomic analysis and identification of Streptococcus pyogenes surface-associated proteins”, Journal of Bacteriology, American Society for Microbiology, vol. 189, no. 5, 1 March 2007, pages 1514-1522, discloses a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes.

[0010] None of the previous documents disclose a suitable method for obtaining whole cell wall antigens from a microorganism.

[0011] Accordingly, there is a need to increase diagnostic value of immunoassays that detect microbial antibodies by increasing its sensitivity and specificity as well as reducing interferences.

[0012] The present invention overcome the above mentioned drawbacks by providing an immunoassay, e.g. immunochromatography, an ELISA and a Dot Blot, based on whole cell wall antigens from microorganisms as useful alternative to existing immunoassay methods.

[0013] Summary of the invention

[0014] In a first aspect, the present invention relates to a process for obtaining whole cell wall antigens from a microorganism.

[0015] In a second aspect, the present invention relates to an immunoassay for detecting antibodies related to a microbial infection comprising the step of using the whole cell wall antigens obtained according to the first aspect.

[0016] In a third aspect, the present invention relates to the use of the immunoassay according to the second aspect in a method for analysing mammary glands health status, colostrum quality or milk tank quality. In other words, the present invention also relates to a method for analysing mammary glands health status, colostrum quality or milk tank quality comprising the step of using the immunoassay according to the second aspect.

[0017] Detailed description of the invention

[0018] In a first aspect, the present invention relates to a process for obtaining whole cell wall antigens from a microorganism comprising the steps of: a) growing a microorganism in a suitable medium; b) washing and inactivating and / or denaturalizing and / or removing the external components of the cell wall of the microorganism, preferably cell wall surface proteins, obtained in step a) by at least autoclaving said microorganism obtained in step a); wherein the process does not include a step of lysing the microorganism.

[0019] Accordingly, in this way, it is possible to obtain useful antigens from microorganism whole cell walls and surprisingly this is achieved after autoclaving microorganisms.

[0020] In a preferred embodiment, said microorganism is a bacterial microorganism.

[0021] In a preferred embodiment, the autoclaved microorganism is centrifuged after step b) obtaining thereby a soluble fraction and an insoluble fraction. In a further preferred embodiment, the whole cell wall antigens are obtained from the pellet or insoluble fraction from the centrifugation.

[0022] In a preferred embodiment, the process according to the present invention further comprises the step of washing the microorganism obtained in step b).

[0023] In this disclosure and in the claims, terms such as "comprises," "comprising," "containing" and "having" are open-ended terms and can mean "includes," "including," and the like; while terms like "consisting of" or "consists of" refer to the mentioned elements after these terms and others which are not mentioned are excluded.

[0024] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise.

[0025] It should be noted that the term "approximately" or “about” applied to the values used earlier and later in this document includes a margin of error of ± 5 %, such as, for example, ± 4 %, ± 3 %, ± 2 %, ± 1 %.

[0026] In the context of the present invention, the term "antigen" means a molecule or group of molecules (for instance a whole microorganism) having distinct surface features or epitopes capable of stimulating a specific immune response. Antibodies (immunoglobulins) are produced by the immune system in response to exposure to antigens.

[0027] In the context of the present invention, the term “whole cell wall antigen” means the components of any microorganism cell wall after being treated by a process disclosed in the present invention or any other equivalent process within the spirit and scope of the invention.

[0028] In the context of the present invention, the term “inactivating and / or denaturalizing and / or removing the external components of the cell wall of the microorganism, preferably cell wall surface proteins” means that any microorganism is subjected to a harsh environment known to significantly affect its components, structure and activity, preferably cell wall surface proteins, without breaking down cell wall structure. This inactivation and / or denaturalization and / or removing is carried out by at least autoclaving, preferably in solution, although other treatments could also be applied.

[0029] In the context of the present invention, the term “soluble fraction” means the supernatant obtained after centrifugation of the autoclaved microorganism, whereas the term “insoluble fraction” is equivalent to the “pellet” obtained after the centrifugation of the autoclaved microorganism.

[0030] In step a) microorganisms, preferably a bacterial microorganism, are usually grown from overnight to several days at 30-40 °C in a suitable culture medium including, but not limited to, LB (Lysogeny Broth), BHI (Brain Heart Infusion), TSB (Tryptic Soy Broth) and Thioglycolate broth. Cells are washed, for example, 3 to 5 times by centrifugation and are autoclaved for 5 to 60 minutes in order to inactivate and / or denaturalize and / or remove the external components of the cell wall of the microorganism, preferably cell wall surface proteins, without breaking down cell wall structure. After this treatment cells are optionally washed 3 to 5 times, preferably by centrifugation, before using them as antigen in an immunoassay.

[0031] Autoclaving can be performed to pressurized saturated steam at 121 - 132 °C for 5-60 minutes at a pressure of 15 - 30 psi.

[0032] Lastly, it is noted that obtaining the whole cell wall antigens from the autoclaved microorganism is well known in the art and can be performed by several suitable methods which include, but not limited thereto: A: Centrifugation as it separates soluble from insoluble fraction (this is the preferred method).

[0033] B: Filtration is also a valuable method as it allows to separate according to size.

[0034] C: Precipitation can be also used to separate according to specific properties of the autoclaved microorganism fractions.

[0035] D: Other well known described methods (for instance a chromatographic separation).

[0036] In another preferred embodiment, said microorganism of step a) is a gram-positive or gram-negative bacteria.

[0037] In a more preferred embodiment, said microorganism of step a) is a gram-positive bacteria selected from S. aureus, S. uberis and S. dysgalactiae.

[0038] In another more preferred embodiment, said microorganism of step a) is a gramnegative bacteria selected from Escherichia Coli and Klebsiella spp.

[0039] The procedure developed by the applicant eliminates most of the antigens from the cell wall (in fact, a significant decrease in the antibody recognition pattern is observed in Tables 1-5 when whole cell wall antigens are evaluated in parallel with non-treated cell wall) but it surprisingly significantly improves assay diagnostic value because the remaining whole cell wall antigens have a better correlation with the infection. This procedure can be surprisingly applied to a wide range of different microorganisms.

[0040] Furthermore, based on said whole cell wall antigen the inventors have developed a rapid and effective immunoassay to obtain results of diagnostic value. In the context of the present invention, the term “diagnostic value” means that valuable information can be obtained from the results of a certain immunoassay. It includes, but are not limited to, infective situation, health status or sample specific value (for instance determination the colostrum protective value of a certain sample).

[0041] Accordingly, in a second aspect, the present invention relates to immunoassay for detecting antibodies related to a microbial infection, preferably a bacterial infection, comprising the step of using the whole cell wall antigens as obtained according to the first aspect of the invention, including any of the embodiments alone or in combination.

[0042] In a preferred embodiment, said immunoassay is carried out by: i) obtaining aggregated and / or complexed antibodies from a sample; ii) using the whole cell wall antigens obtained according to the embodiments of the first aspect of the invention for binding isotypes of antibodies obtained in step i); iii) detecting said bound isotypes of step ii).

[0043] In the context of the present invention, the term “immunoassay” means any assay based on antigen-antibody binding. It includes, but are not limited to, ELISA, Western Blots and immunochromatography tests. In a preferred embodiment, said immunoassay according to the second aspect of the invention is selected from an enzyme-linked immunosorbent assay (ELISA), Immunochromatography and Dot Blot.

[0044] In the context of the present invention, the term “unprocessed milk, means raw milk o milk without thermal or chemical treatment.

[0045] In the context of the present invention, the term “aggregated antibodies” means antibodies bound to other molecules in a liquid solution that can be isolated from the liquid solution by precipitation by centrifugation at for example 13,500 g.

[0046] In the context of the present invention, the term “complexed antibodies” means antibodies bound to other molecules in a liquid solution that cannot be precipitated by centrifugation at for example 13,500 g. They require a filtration or precipitation procedure (for instance with Polyethylene glycol (PEG)) to be isolated from the liquid solution.

[0047] In the context of the present invention, the term “Antibody isotype” means any class of antibody (for instance IgA, IgM, IgE, lgG1 , lgG2).

[0048] In the context of the present invention, the term “whole cell wall immunoassay” means any immunoassay that contains the whole cell wall antigen described in the present invention.

[0049] In the context of the present invention, the term “Immunochromatography” (also named Lateral Flow assay and Flow through assay) means any immunoassay involving a chromatographic migration step. It can detect a single parameter or it can detect multiple parameters simultaneously. In the context of the present invention, the term “whole cell wall immunochromatography” or analogous terms means any immunochromatography that contains the whole cell wall antigen as described in the present invention.

[0050] In the context of the present invention, the term “Dot Blot” means any immunoassay involving incubating the sample with a membrane blotted with an antigen of interest. It can detect a single parameter or it can detect multiple parameters simultaneously.

[0051] In the context of the present invention, the term “whole cell wall Dot Blot” or analogous terms means any Dot Blot that contains the whole cell wall antigen as described in the present invention.

[0052] In the context of the present invention, the term “ELISA” (Enzyme linked immunosorbent assay) means any immunoassay involving a signal recognition step involving an enzymatic reaction. It can detect a single parameter or it can detect multiple parameters simultaneously.

[0053] In the context of the present invention, the term “whole cell wall ELISA” or analogous terms means any ELISA that contains the whole cell wall antigen as described in the present invention.

[0054] In the context of the present invention, the term “infection” means the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce.

[0055] In the context of the present invention, “specific antibodies related to a microbial infection” means the detection of antibodies which presence correlates with a microbial infection or correlates with the protection of the animal against a certain microorganism infection. In a preferred embodiment, said microbial infection is a bacterial infection.

[0056] In another preferred embodiment, the antibodies related to a microbial infection are detected in a sample of an animal milk, preferably a mammal milk, even more preferably a cow milk.

[0057] In the context of the present invention, the term “milk of infected lactating cow” means bovine milk with a confirmed presence of a specific microbial infection. In the context of the present invention, the term “milk of non-infected lactating cow” means bovine milk with a confirmed absence of a specific microbial infection.

[0058] In the context of the present invention, the term “lactating cow milk” means bovine milk obtained after the transition period and before the dry off period.

[0059] In the context of the present invention, the term “bovine colostrum” means bovine milk obtained just after calving.

[0060] In the context of the present invention, the term “cow milk tank” means the group of mixed bovine milks obtained after a milking process.

[0061] In a third aspect, the present invention relates to the use of the immunoassay according to the second aspect of the invention including any of the embodiments alone or in combination, for analysing mammary glands health status.

[0062] The present invention also relates to the use of the immunoassay according to the second aspect of the invention including any of the embodiments alone or in combination, in a method for analysing colostrum quality.

[0063] The present invention further relates to the use of the immunoassay according to the second aspect of the invention including any of the embodiments alone or in combination, in a method for analysing milk tank quality.

[0064] In other words, the present invention also relates to a method for analysing mammary glands health status, colostrum quality or milk tank quality comprising the step of using the immunoassay according to the second aspect of the invention.

[0065] A number of examples will be provided below that are intended to illustrate the invention and in no way limit the scope of the invention, which is established by the attached claims.

[0066] Examples

[0067] Example 1 : Obtaining whole cell wall antigens.

[0068] Whole cell wall antigens can be obtained by the following procedure: Option A: Microorganisms (Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli or Klebsiella spp.) are growth overnight at 37°C in TSB medium. Cells are washed 3 times by centrifugation and autoclaved at 121 °C during 20 minutes in a solution containing Tween at 0,1 %. Autoclaved cells are washed 3 to 5 times by centrifugation to remove debris and excess detergent before using them as antigen in an immunoassay.

[0069] Option B: Microorganisms (Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli or Klebsiella spp.) are growth overnight at 37°C in TSB medium. Cells are washed 3 times by centrifugation and autoclaved at 121 °C during 20 minutes in PBS. Autoclaved cells are centrifuged. Supranatant is mixed with ethanol to a final concentration of 70 % v / v and kept 1 hour at -20°C. Pellet is resuspended in PBS and used as antigen in an immunoassay.

[0070] Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli and Klebsiella spp. have been successfully used to obtain whole cell wall antigens but any other microorganism of interest can be used.

[0071] Procedure used to obtain whole cell wall antigen depends on the microorganism used as antigen and the immunoassay requirements. Option A is the preferred option for most of Gram positive microorganisms and Option B is the preferred option for most of Gram negative microorganisms.

[0072] Non treated whole cell wall antigen (control antigen) can be obtained using the following procedure:

[0073] Control antigen: Microorganisms (Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli or Klebsiella spp.) are growth overnight at 37°C in TSB medium. Cells are washed 3 times by centrifugation before using them as antigen in an immunoassay.

[0074] Comparative performance of each antigen was evaluated with immunochromatography (see tables 1-5 showing whole cell wall antigens performance compared with nontreated cell wall antigen (control antigen)). Table 1:

[0075] Table 2:

[0076] Table 3:

[0077] Table 4: Table 5:

[0078] Example 2: Immunoassay using whole cell wall antigens.

[0079] The immunoassays are produced according to well described methods taking into account the specific characteristics of the whole cell wall antigens and the sample analysis requirements:

[0080] A: Option A whole cell wall antigens requirements:

[0081] Most Whole cell wall antigens obtained according to the Option A should be dispensed in the immunochromatography test line and Dot Blot membrane at a relative high concentration compared to typical protein antigens (1 microliter at OD600 of 10 each strip) and relatively large porous size nitrocellulose membranes should be used (for instance MDI ON PH membrane) to avoid nitrocellulose clogging by antigen.

[0082] B: Option B whole cell wall antigens requirements:

[0083] Most Whole cell wall antigens obtained according to the Option B should be crosslinked or conjugated to molecules according to well established methods to facilitate its binding to a solid support.

[0084] C: Whole specific antibodies in a milk sample detection:

[0085] Milks can be diluted from 1 / 10 to 1 / 10.000 using different immunoassay buffers. A standard immunoassay buffer (PBS with Tween), a Tween I SDS buffer or an stringent buffer (Glycine, EDTA, NaCI, Tween and BSA) can be used to dilute samples.

[0086] D: Aggregated specific antibodies in a milk sample detection: Milk aggregated antibodies can be separated from milk samples by centrifugation and I or filtration steps according to well established methods.

[0087] E: Complexed specific antibodies in a milk sample detection:

[0088] Milk complexed antibodies can be separated from milk samples by Polyethylene glycol (PEG) at 2-5 % w / v incubation followed by a centrifugation and / or filtration steps according to well established methods.

[0089] F: Antibody isotypes:

[0090] It is possible to detect all isotypes of specific antibodies (IgA, IgM, I gG 1 , lgG2...) bound to whole cell wall antigens or detect only a single isotypes according to well established methods. Protein G is the preferred detection system to detect several isotypes of antibodies simultaneously and protein A to detect mainly lgG2.

[0091] Comparative performance of each whole cell wall antigen immunoassay characteristic was evaluated with immunochromatography (see table 6 showing whole cell wall antigens performance compared using different assay versions).

[0092] Table 6

[0093] Example 3: Milk from lactating cows evaluated by whole cell wall immunochromatography, Dot Blot and ELISA.

[0094] 200 microliters of milk previously filtered at 0.45 micras were mixed with 800 microliters of PEG solution at final concentration of 3.5 % w / v. Mixture was centrifuged and the pelled washed twice with PEG 3.5 %. Pelled was resuspended with Carbonate buffer pH 10.5 and neutralized. This neutralized sample was used to perform the assays.

[0095] 120 microlitres of sample (infected lactating cows and non-infected lactating cows) were added in a whole cell wall immunochromatography assay with a colloidal gold protein A conjugate. If specific antibodies against the whole cell wall antigen were present test line appeared.

[0096] 1000 microlitres of sample (infected lactating cows and non-infected lactating cows) were added in a Dot Blot membrane coated with whole cell wall antigen and incubated for 1 h in agitation. After washing, a colloidal gold protein A conjugate diluted in 1 ml of immunoassay buffer was added for 1 additional hour. If specific antibodies against the whole cell wall antigen were present a red blot appeared. 100 microlitres of sample (infected lactating cows and non-infected lactating cows) were added in a whole cell wall coated ELISA well. After 30 minutes incubation and a wash procedure a HRPO protein A conjugate was added. After an additional 30 minutes incubation a new wash was performed and TMB was added into the well. If specific antibodies against the whole cell wall antigen were present a blue colour appeared.

[0097] Results with diagnostic value were obtained using the 3 immunoassays (see tables 7- 11 showing Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli and Klebsiella spp. whole cell wall antigen immunochromatography, Dot Blot and ELISA results with infected lactating cow milk and non-infected lactating cow milk).

[0098] Table 7

[0099] Table 8

[0100] Table 9

[0101] Table 10

[0102] Table 11

[0103] Example 4: Bovine colostrum evaluated by whole cell wall immunochromatography, Dot Blot and ELISA. 120 microlitres of 1 / 1000 of a panel of diluted bovine colostrums were assayed with a whole cell wall immunochromatography assay with a colloidal gold protein G conjugate. If specific antibodies against the whole cell wall antigen were present test line appears.

[0104] 1000 microlitres of 1 / 1000 of a panel of diluted bovine colostrums were assayed with a Dot Blot membrane coated with whole cell wall antigen and incubated for 1 h under agitation. After washing, a colloidal gold protein G conjugate diluted in 1 ml of immunoassay buffer was added for 1 additional hour. If specific antibodies against the whole cell wall antigen were present a red blot appears.

[0105] 100 microlitres of 1 / 1000 of a panel of diluted bovine colostrums were assayed with a whole cell wall coated ELISA well. After 30 minutes incubation and a wash procedure a HRPO protein G conjugate was added. After an additional 30 minutes incubation a new wash was performed and TMB was added into the well. If specific antibodies against the whole cell wall antigen were present a blue colour appears. Results with diagnostic value were obtained using the 3 immunoassays (see tables 12- 14 showing Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli and Klebsiella spp. whole cell wall antigen immunochromatography, Dot Blot and ELISA results with bovine colostrum). Table 12

[0106] Table 13

[0107] Table 14

[0108] Example 5: Cow milk tank evaluated by whole cell wall immunochromatography, Dot Blot and ELISA

[0109] 200 microliters of milk were mixed with 800 microliters of PBS solution. Mixture was centrifuged and the pelled washed twice with PBS. Pelled was resuspended with Carbonate buffer pH 10.5 and neutralized. This neutralized sample was used to perform the assays.

[0110] 120 microlitres of sample (infected lactating cows and non-infected lactating cows) were added in a whole cell wall immunochromatography assay with a colloidal gold protein A conjugate. If specific antibodies against the whole cell wall antigen were present test line appeared.

[0111] 1000 microlitres of sample (infected lactating cows and non-infected lactating cows) were added in a Dot Blot membrane coated with whole cell wall antigen and incubated for 1 h in agitation. After washing, a colloidal gold protein A conjugate diluted in 1 ml of immunoassay buffer was added for 1 additional hour. If specific antibodies against the whole cell wall antigen were present a red blot appeared.

[0112] 100 microlitres of sample (infected lactating cows and non-infected lactating cows) were added in a whole cell wall coated ELISA well. After 30 minutes incubation and a wash procedure a HRPO protein A conjugate was added. After an additional 30 minutes incubation a new wash was performed and TMB was added into the well. If specific antibodies against the whole cell wall antigen were present a blue colour appeared.

[0113] Results with diagnostic value were obtained using the 3 immunoassays (see tables 15- 17 showing Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli and Klebsiella spp. whole cell wall antigen immunochromatography, Dot Blot and ELISA results with infected lactating cow milk and non-infected lactating cow milk). Table 15

[0114] Table 16 Table 17

[0115] Discussion of examples and conclusion 1 : Unsatisfactory results developing an immunochromatography, a Dot Blot and an

[0116] ELISA following procedures described in the literature (Reference 1 and Reference 2) were obtained by the applicant. A weak correlation between milk of infected and noninfected lactating cows were obtained due to the presence of large number of false positive and negative results. Alternative approaches were explored by the applicant in order to try to improve assay diagnostic value.

[0117] Surprisingly, a more specific and sensitive result was obtained when whole cell wall antigens as obtained according to Example 1 were evaluated. The whole cell wall antigen production procedures developed by the applicant inactivates and / or denaturalizes and / or removes most of the cell wall surface proteins as well as other external components of the cell wall without breaking down the cell wall. This procedure can be achieved by incubating microorganism cells in a harsh environment involving autoclaving. It is possible to use the soluble fraction and the insoluble fraction of this procedure as antigen (example 1).

[0118] The procedure developed by the applicant eliminates most of the antigens from the cell wall (in fact, a significant decrease in the antibody recognition pattern is observed in tables 1-5 when whole cell wall antigens are evaluated in parallel with non-treated cell wall antigens) but it surprisingly significantly improves assay diagnostic value because the remaining whole cell wall antigens have a better correlation with the infection. This procedure can be surprisingly applied to a wide range of different microorganisms.

[0119] Specificity of whole cell wall antigens were checked using milk samples with very satisfactory results. Surprisingly, cow milks with Streptococcus uberis infection have antibodies that bind to Streptococcus uberis whole cell wall antigen but do not have antibodies that bind to Staphylococcus aureus whole cell wall antigen and vice versa (Tables 7-11). In addition, binding profile of different colostrums is very variable using different whole cell wall antigens (Tables 12-14). These results suggest only specific antibodies are bound to each whole cell wall antigen.

[0120] It is described in the literature that cell wall composition can show notable differences within the same microbial specie (Reference 3). Cell wall antigens obtained using different culture conditions (18 h culture versus 28 days culture) provide equivalent results when milk of infected and non-infected lactating cows from different sources were assayed (Tables 1-5) so whole cell wall antigens are antigens with a surprisingly good uniformity.

[0121] Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli and Klebsiella spp. have ben successfully used to obtain whole cell wall antigens. Any other microorganism of interest can be used.

[0122] Most of the described antigen production processes used to detect specific microbial antibodies relay on the use of specific recombinant or purified proteins as antigens while our approach is based on detecting whole cell wall components. Published whole cell antigens used to detect microbial antibodies are usually sonicated extracts where cell wall is disrupted and most of the proteins are not significantly modified and a mixture of internal and external cell components is obtained (Reference 5) while our approach is intended to detect only whole cell wall components.

[0123] 2: A whole cell wall immunochromatography, a Dot Blot and whole cell wall ELISA were developed using protocols described in the literature (Reference 1 and Reference 2) but no satisfactory results were obtained (a very poor signal was obtained). In order to overcome this problem different approaches were explored by the applicant.

[0124] Due to the specific characteristics of the whole cell wall antigen and the sample analysis requirements some modifications in the procedure described in the literature were incorporated as described in Example 2.

[0125] Surprisingly a very strong positive signal was obtained with the whole cell wall antigens obtained according to the Option A when dispensed in the immunochromatography test line and Dot Blot membrane at a relative high concentration using relatively large porous size nitrocellulose membranes.

[0126] Surprisingly a very strong positive signal was obtained with the whole cell wall antigens obtained according to the Option B when cross-linked or conjugated to molecules.

[0127] A significant proportion of milk samples from uninfected cows tested positive when analyzed with an immunoassay containing the antigens of the present invention together with buffers and sample dilutions described in the literature (references 2 and 4). In order to improve the diagnostic value of the antigen of the present invention different tests were carried out.

[0128] Surprisingly when the assay was performed using a high concentration of antigen combined with a marked dilution of the untreated milk sample (1 / 10 - 1 / 10,000) and protein G as conjugate (see example 2) an improvement in the diagnostic value was significantly observed. In other words, the antigen of the present invention allows for the correct detection of most infected milks evaluated when using, for example, a high concentration of antigen and a marked sample dilution (1 / 10-1 / 10,000) and protein G as conjugate (see example 2). This assay format using untreated milk sample is of special interest to identify infections in a heard or classify colostrums. Nevertheless, using this approach it was not possible to identify the origin of the infection. In order to allow to identify the origin of the infection different sample pretreatments were explored.

[0129] Surprisingly when the assay was performed using only aggregated or complexed antibodies (see example 2) and protein A as detection reagent was it was possible to identify the origin of the infection using whole cell wall antigens based immunoassays.

[0130] It should be understood, however, that the detailed description and the specific example, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications of the immunoassay buffer and the detection system and the format of the immunoassay within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It is specially relevant to take into consideration the value of immunoassays based on flow-through or vertical format (Reference 6) to perform assays where aggregated or complexed antibodies should be evaluated.

[0131] 3: Finally, in order to confirm the diagnostic value of the whole cell wall immunoassays they were evaluated with cow milk from different sources by the applicant.

[0132] Whole cell wall antigen developed by the applicant significantly increases diagnostic value of the immunoassays. Whole cell wall antigens can be easily mixed (for instance it allows to detect different infections simultaneously) and a wide range of different antigens are readily available (for instance, it allows lactating milk, colostrum and tank milk screening for the presence of a wide range of microorganisms). In addition, as high dilutions of cow milk can be evaluated it is possible to easily evaluate pooled milk samples by decreasing the dilution (for instance it allows milk tank analysis at different dilutions).

[0133] Whole cell wall immunoassays surprisingly allowed to correctly classify milks of infected lactating cows and milks of non-infected lactating cows (Example 3 and Tables 7-11). An immunoassay based on whole cell wall antigen can be used to analyse mammary glands health status by detecting the presence of microbial infections (presence of specific antibodies correlates with the presence of an infection in samples evaluated in Example 3 and Tables 7-11). Whole cell wall immunoassays surprisingly allowed to differentiate bovine colostrum samples (Example 4 and Tables 12-14). An immunoassay based on whole cell wall antigen can be used to analyse colostrum quality by detecting the presence of different antimicrobial antibodies (different passive immunity transfer to calves is expected from the different samples evaluated in Example 4 and Tables 12-14).

[0134] Finally, whole cell wall immunoassays surprisingly allowed to analyse cow milk tank quality of samples from different sources (Example 5 and Tables 15-17). An immunoassay based on whole cell wall antigen can be used to analyse milk tank quality by detecting the presence of different antimicrobial antibodies linked to infections (Staphylococcus aureus infection is suspected to be present in milk tank sample 2 and Streptococcus uberis infection is suspected to be present in milk tank sample 3 in Example 5 and Tables 15-17).

[0135] REFERENCES

[0136] Reference 1 : Pernthaner, A (2019) Device for detection of antibodies to a pathogen WO2019216775A1

[0137] Reference 2: Systemic and local immune response of cows to intramammary infection with Staphylococcus aureus. Leitner G, Yadlin B, Glickman A, Chaffer M, Saran A. Res Vet Sci. 2000 Oct;69(2):181-4. doi: 10.1053 / rvsc.2000.0409. PMID: 11020372

[0138] Reference 3: Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae. Beaussart A, Pechoux C, Trieu-Cuot P, Hols P, Mistou MY, Dufrene YF. Nanoscale. 2014 Dec 21 ;6(24): 14820-7. doi: 10.1039 / c4nr05280c. Epub 2014 Oct 31. PMID: 25358409

[0139] Reference 4: Wondu W-M (2002) A method for simultaneous detection of multiple microbial antigens in biological specimens from mastitic animals. Patent application number WO 2002075310 A

[0140] Reference 5: Development of a Gold Nanoparticle Based Lateral Flow Assay for Rapid Diagnosis of Contagious Agalactia in Goats T.R. Arun, R. Rana, P. Singh, P. Choudhuri, V.P. Singh, P. Thomas, V. Rekha, K. Nehra, J. Usharani and K. Dhama. Asian Journal of Animal and Veterinary Advances Year: 2014 | Volume: 9 | Issue: 7 | Page No.: 405-413 DOI: 10.3923 / ajava.2014.405.413

[0141] Reference 6: Design of Gold Nanoparticle Vertical Flow Assays for Point-of-Care Testing Rongwei Lei, David Wang, Hufsa Arain and Chandra Mohan. Diagnostics 2022, 12, 1107. htps: / / doi.org / 10.3390 / diagnostics12051107.

Claims

CLAIMS1. A process for obtaining whole cell wall antigens from a microorganism comprising the steps of: a) growing a microorganism in a suitable medium; b) washing and inactivating and / or denaturalizing and / or removing the external components of the cell wall of the microorganism, preferably cell wall surface proteins, obtained in step a) by at least autoclaving said microorganism obtained in step a); wherein the process does not include a step of lysing the microorganism.

2. The process of claim 1, wherein the autoclaved microorganism is centrifuged after step b).

3. The process according to claim 2, wherein the whole cell wall antigens are obtained from the pellet or insoluble fraction from the centrifugation.

4. The process according to any of claims 1 to 3, further comprising the step of washing the microorganism obtained in step b).

5. The process according to any of claims 1 to 4, wherein said microorganism is a gram-positive or gram-negative bacteria.

6. The process according to claim 5, wherein said microorganism is a gram-positive bacteria selected from S. aureus, S. uberis and S. dysgalactiae.

7. Process according to claim 5, wherein said microorganism is a gram-negative bacteria selected from Escherichia Coli and Klebsiella spp.

8. An immunoassay for detecting antibodies related to a microbial infection comprising the step of using the whole cell wall antigens obtained according to any of claims 1 to 7.

9. The immunoassay according to claim 8, wherein said immunoassay is carried out by: i) obtaining aggregated and / or complexed antibodies from a sample; ii) using the whole cell wall antigens obtained according to any of claims 1 to 7 for binding isotypes of antibodies obtained in step i);iii) detecting said bound isotypes of step ii).

10. The immunoassay according to claim 8 or 9, wherein said microbial infection is a bacterial infection.

11. The immunoassay according to any of claims 8 to 10, wherein said immunoassay is selected from an enzyme-linked immunosorbent assay (ELISA), Immunochromatography and Dot Blot.

12. The immunoassay according to any of claims 8 to 11 , wherein the antibodies are detected in a sample of an animal milk.

13. The immunoassay according to claim 12, wherein said animal milk is a mammal milk.

14. The immunoassay according to claim 13, wherein said mammal milk is a cow milk.

15. Use of the immunoassay according to any of claims 12 to 14 in a method for analysing mammary glands health status.

16. Use of the immunoassay according to any of claims 12 to 14 in a method for analysing colostrum quality.

17. Use of the immunoassay according to any of claims 12 to 14 in a method for analysing milk tank quality.

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