Method for obtaining whole cell wall antigen from microorganism and immunoassay for detecting antibody associated with microbial infection using same

Abstract

The present invention relates to a method for obtaining whole cell wall antigens from microorganisms. The invention also relates to an immunoassay for detecting an antibody associated with a microbial infection, comprising the step of using a whole cell wall antigen. The invention also relates to the use of said immunoassay in a method for analyzing breast health, colostrum quality or mixed raw milk quality.

Description

Technical Field

[0001] This invention relates to the field of immunology. Specifically, it relates to a method for obtaining whole-cell wall antigens from microorganisms. The invention also relates to an immunoassay based on whole-cell wall antigens from microorganisms derived from animal body fluids (e.g., milk), suitable for detecting specific antibodies associated with microbial infections. Background Technology

[0002] The detection of specific antibodies against microorganisms in body fluids has significant limitations due to a lack of sensitivity and specificity, as well as the presence of interfering antibodies.

[0003] Immunoassays based on microbial expression-specific proteins as antigens rely on the expression of these proteins, and therefore lack sensitivity when a strain does not express these proteins. Immunoassays based on crude extracts as antigens often lack specificity because several microbial proteins are widely expressed, and some even bind to immunoglobulins (e.g., Staphylococcus aureus protein A).

[0004] In addition, the presence of microbial antibodies usually has low diagnostic value because these antibodies can interfere with the results, due to the presence of innate antibodies and related antibodies produced to overcome infection.

[0005] WO 97 / 26007 discloses a purified antigen characterized by a high molecular weight antigen, which is obtained from an aqueous extract of autoclaved cells of Mycobacterium spp. by digesting the extract with proteinase K and collecting the ultrafiltration residue from the digested extract by ultrafiltration through an ultrafiltration membrane, wherein the high molecular weight antigen is retained in the ultrafiltration residue.

[0006] Severin Anatoly et al.'s article, "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 for identifying surface antigens of Streptococcus pyogenes by proteolytic digestion of surface-exposed proteins.

[0007] Previous literature has not disclosed a suitable method for obtaining whole cell wall antigens from microorganisms.

[0008] Therefore, it is necessary to improve the diagnostic value of immunoassays for detecting microbial antibodies by increasing the sensitivity and specificity of immunoassays and reducing interference.

[0009] This invention overcomes the aforementioned drawbacks by providing immunoassays based on whole-cell wall antigens from microorganisms, such as immunochromatography, ELISA, and dot blot, as useful alternatives to existing immunoassay methods. Summary of the Invention

[0010] In a first aspect, the present invention relates to a method for obtaining whole cell wall antigens from microorganisms.

[0011] In a second aspect, the present invention relates to an immunoassay for detecting antibodies associated with microbial infection, the immunoassay comprising the step of using a whole-cell wall antigen obtained according to the first aspect.

[0012] In a third aspect, the present invention relates to the use of the immunoassay according to the second aspect in a method for analyzing breast health status, colostrum quality, or mixed raw milk quality. In other words, the present invention also relates to a method for analyzing breast health status, colostrum quality, or mixed raw milk quality, the method comprising the step of using the immunoassay according to the second aspect. Detailed Implementation

[0013] In a first aspect, the present invention relates to a method for obtaining whole-cell wall antigens from microorganisms, the method comprising the following steps: a) Cultivate microorganisms in a suitable culture medium; b) Washing and inactivating and / or denaturing and / or removing the external components of the cell wall of the microorganisms obtained in step a), preferably cell wall surface proteins, by subjecting the microorganisms obtained in step a) to at least autoclaving. The method described herein does not include the step of lysing microorganisms.

[0014] Therefore, in this way, useful antigens can be obtained from the whole cell wall of microorganisms, which is surprisingly achieved after the microorganisms have been autoclaved.

[0015] In a preferred embodiment, the microorganism is a bacterial microorganism.

[0016] In one preferred embodiment, after step b), the autoclaved microorganisms are centrifuged to obtain soluble and insoluble fractions. In another preferred embodiment, the whole cell wall antigen is obtained from the centrifuged precipitate or the insoluble fraction.

[0017] In a preferred embodiment, the method according to the invention further includes the step of washing the microorganisms obtained in step b).

[0018] In this disclosure and claims, terms such as “comprising,” “including,” “containing,” and “having” are open-ended terms that can mean “comprising,” “including,” etc.; while terms such as “composed of” refer to the elements mentioned after these terms and exclude other elements not mentioned.

[0019] Unless otherwise defined, 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 pertains. The singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0020] It should be noted that the terms “approximately” or “about” apply to the values ​​used in this document, both before and after the document, including a range of error of ±5%, such as ±4%, ±3%, ±2%, ±1%.

[0021] In the context of this invention, the term "antigen" refers to a molecule or group of molecules (e.g., whole microorganisms) that has a unique surface feature or epitope capable of stimulating a specific immune response. Antibodies (immunoglobulins) are produced by the immune system in response to exposure to antigens.

[0022] In the context of this invention, the term "whole cell wall antigen" means any component of a microbial cell wall that has been treated by the methods disclosed in this invention or any other equivalent method within the spirit and scope of this invention.

[0023] In the context of this invention, the term "inactivation and / or denaturation and / or removal of external components of the microbial cell wall, preferably cell wall surface proteins" means subjecting any microorganism to harsh environments known to significantly affect its composition, structure, and activity (preferably cell wall surface proteins) without disrupting its cell wall structure. This inactivation and / or denaturation and / or removal is carried out by at least autoclaving, preferably in solution, but other treatments may also be applied.

[0024] In the context of this invention, the term "soluble fraction" refers to the supernatant obtained after centrifuging microorganisms sterilized by autoclaving, while the term "insoluble fraction" is equivalent to the "precipitate" obtained after centrifuging microorganisms sterilized by autoclaving.

[0025] In step a), the microorganisms, preferably bacteria, are typically cultured overnight to several days at 30-40°C in a suitable culture medium, including but not limited to LB (lysozyme broth), BHI (brain and heart infusion), TSB (tryptone soybean broth), and thioglycolate broth. For example, the cells are washed 3 to 5 times by centrifugation and autoclaved for 5 to 60 minutes to inactivate and / or denature and / or remove external components of the microbial cell wall, preferably cell wall surface proteins, without damaging the cell wall structure. After this treatment, the cells are optionally washed 3 to 5 times, preferably by centrifugation, before being used as antigens in immunoassays.

[0026] Autoclaving can be performed using pressurized saturated steam for 5-60 minutes at 121-132℃ and 15-30 psi.

[0027] Finally, it should be noted that obtaining whole-cell wall antigens from microorganisms sterilized by autoclaving is well known in the art and can be done by several suitable methods, including but not limited to: A: Centrifugation, because centrifugation separates soluble fractions from insoluble fractions (this is the preferred method).

[0028] B: Filtration is also a valuable method because it allows for separation based on size.

[0029] C: Precipitation can also be used for separation based on the specific properties of microbial fractions that have been autoclaved.

[0030] D: Other well-known methods (e.g., chromatographic separation).

[0031] In another preferred embodiment, the microorganism in step a) is a Gram-positive bacterium or a Gram-negative bacterium.

[0032] In a more preferred embodiment, the microorganism in step a) is selected from Staphylococcus aureus and Streptococcus lactis. S. uberis ) and Streptococcus lactis ( S. dysgalactiae Gram-positive bacteria.

[0033] In another preferred embodiment, the microorganism in step a) is a Gram-negative bacterium selected from Escherichia coli and Klebsiella spp.

[0034] The procedure developed by the applicant removes most of the cell wall antigens (in fact, a significant reduction in antibody recognition patterns was observed in Tables 1-5 when whole cell wall antigens were evaluated in parallel with untreated cell walls), but unexpectedly it significantly improves the diagnostic value of the assay because the remaining whole cell wall antigens are better correlated with infection. Unexpectedly, the procedure can be applied to a wide variety of different microorganisms.

[0035] Furthermore, based on the aforementioned whole-cell wall antigen, the inventors have developed a rapid and effective immunoassay to obtain results with diagnostic value. In the context of this invention, the term "diagnostic value" refers to valuable information that can be obtained from the results of an immunoassay. This includes, but is not limited to, infection status, health condition, or sample-specific values ​​(e.g., determining the colostrum protection value of a sample).

[0036] Therefore, in a second aspect, the present invention relates to an immunoassay for detecting antibodies associated with microbial infection (preferably bacterial infection), the immunoassay comprising the step of using a whole-cell wall antigen obtained according to the first aspect of the invention (including any embodiment thereof, alone or in combination).

[0037] In a preferred embodiment, the immunoassay is performed as follows: i) Obtain aggregated and / or complexed antibodies from the sample; ii) Using the same type of antibody obtained in step i) with the whole cell wall antigen obtained according to the embodiment of the first aspect of the present invention; iii) Detect the same type of combination described in step ii).

[0038] In the context of this invention, the term "immunoassay" refers to any assay based on antigen-antibody binding. This includes, but is not limited to, ELISA, Western blotting, and immunochromatographic assays. In a preferred embodiment, the immunoassay according to the second aspect of the invention is selected from enzyme-linked immunosorbent assay (ELISA), immunochromatography, and dot blot.

[0039] In the context of this invention, the term "unprocessed milk" means raw milk or milk that has not undergone heat treatment or chemical treatment.

[0040] In the context of this invention, the term "aggregated antibody" means an antibody that binds to other molecules in a liquid solution, which can be separated from the liquid solution by, for example, centrifugation precipitation of 13,500 g.

[0041] In the context of this invention, the term "complex antibody" means an antibody that binds to other molecules in a liquid solution and cannot be precipitated by, for example, centrifugation at 13500g. They require filtration or precipitation procedures (e.g., with polyethylene glycol (PEG)) to be separated from the liquid solution.

[0042] In the context of this invention, the term "antibody isotype" means any class of antibody (e.g., IgA, IgM, IgE, IgG1, IgG2).

[0043] In the context of this invention, the term "whole cell wall immunoassay" means any immunoassay containing the whole cell wall antigen described in this invention.

[0044] In the context of this invention, the term "immunochromatography" (also known as lateral flow chromatography and flow assays) refers to any immunoassay involving a chromatographic migration step. It can detect a single parameter or multiple parameters simultaneously.

[0045] In the context of this invention, the term "whole cell wall immunochromatography" or similar terms mean any immunochromatography that contains the whole cell wall antigens described in this invention.

[0046] In the context of this invention, the term "dot blot" refers to any immunoassay involving incubating a sample with a membrane blotted with a target antigen. It can detect a single parameter or multiple parameters simultaneously.

[0047] In the context of this invention, the term "whole cell wall dot blot" or similar term means any dot blot containing the whole cell wall antigen described in this invention.

[0048] In the context of this invention, the term "ELISA" (enzyme-linked immunosorbent assay) refers to any immunoassay involving a signal recognition step related to an enzyme-catalyzed reaction. It can detect a single parameter or multiple parameters simultaneously.

[0049] In the context of this invention, the term "whole cell wall ELISA" or similar terms means any ELISA containing the whole cell wall antigen described in this invention.

[0050] In the context of this invention, the term "infection" means the invasion of a tissue by a pathogen, its reproduction, and the host tissue's response to the source of infection and the toxins it produces.

[0051] In the context of this invention, "specific antibodies related to microbial infection" means the detection of antibodies associated with or related to protecting animals from a certain microbial infection. In a preferred embodiment, the microbial infection is a bacterial infection.

[0052] In another preferred embodiment, antibodies associated with microbial infection are detected in animal milk samples, preferably mammalian milk, and even more preferably cow's milk.

[0053] In the context of this invention, the term "milk of an infected lactating cow" means milk in which a specific microbial infection has been confirmed.

[0054] In the context of this invention, the term "milk of an uninfected lactating cow" means milk that is confirmed to be free from a specific microbial infection.

[0055] In the context of this invention, the term "lactating milk" means milk obtained after the transition period and before the dry period.

[0056] In the context of this invention, the term "bovine colostrum" refers to milk obtained immediately after a calf has given birth.

[0057] In the context of this invention, the term "blended raw milk" refers to a blend of raw milk obtained after the milking process.

[0058] In a third aspect, the present invention relates to the use of an immunoassay according to the second aspect of the present invention (including any embodiment thereof, alone or in combination) for analyzing breast health status.

[0059] The present invention also relates to the use of an immunoassay according to a second aspect of the invention (including any embodiment thereof, alone or in combination) in a method for analyzing colostrum quality.

[0060] The present invention also relates to the use of an immunoassay according to a second aspect of the invention (including any embodiment thereof, alone or in combination) in a method for analyzing the quality of mixed raw milk.

[0061] In other words, the present invention also relates to a method for analyzing breast health status, colostrum quality, or mixed raw milk quality, the method comprising the step of using an immunoassay according to the second aspect of the present invention.

[0062] Several embodiments will be provided below to illustrate the invention, but not in any way to limit the scope of the invention as defined by the appended claims.

[0063] Example Example 1: Obtaining whole cell wall antigen.

[0064] Whole cell wall antigens can be obtained through the following procedure: Option A: Culture microorganisms (Staphylococcus aureus, Streptococcus lactis, Streptococcus pyogenes, Escherichia coli, or Klebsiella spp.) overnight in TSB medium at 37°C. Wash the cells three times by centrifugation and autoclave at 121°C in a solution containing 0.1% Tween for 20 minutes. Wash the autoclaved cells three to five times by centrifugation to remove debris and excess detergent, and then use them as antigens in immunoassays.

[0065] Option B: Culture microorganisms (Staphylococcus aureus, Streptococcus lactis, Streptococcus pyogenes, Escherichia coli, or Klebsiella spp.) overnight in TSB medium at 37°C. Wash cells three times by centrifugation and autoclave in PBS at 121°C for 20 minutes. Centrifuge the autoclaved cells. Mix the supernatant with ethanol to a final concentration of 70% v / v and incubate at -20°C for 1 hour. Resuspend the precipitate in PBS and use it as an antigen in immunoassays.

[0066] Staphylococcus aureus, Streptococcus lactis, Streptococcus dysgalactiae, Escherichia coli, and Klebsiella spp. have been successfully used to obtain whole cell wall antigens, but any other target microorganisms may also be used.

[0067] The procedure for obtaining whole-cell wall antigens depends on the microorganisms used as antigens and the requirements of the immunoassay. Option A is preferred for most Gram-positive microorganisms, and option B is preferred for most Gram-negative microorganisms.

[0068] Untreated whole-cell wall antigen (control antigen) can be obtained using the following procedure: Control antigen: Microorganisms (Staphylococcus aureus, Streptococcus lactis, Streptococcus pyogenes, Escherichia coli, or Klebsiella spp.) are cultured overnight in TSB medium at 37°C. Cells are washed three times by centrifugation and then used as antigens in the immunoassay.

[0069] Immunochromatography was used to evaluate the comparative performance of each antigen (see Tables 1-5, showing the performance of whole cell wall antigens compared to untreated cell wall antigens (control antigens)).

[0070] Table 1:

[0071] Table 2:

[0072] Table 3:

[0073] Table 4:

[0074] Table 5:

[0075] Example 2: Immunoassay using whole cell wall antigens.

[0076] Immunoassays are produced according to known methods, taking into account the specific characteristics of whole-cell wall antigens and sample analysis requirements: A: Option A, requirements for whole cell wall antigens: Most whole-cell wall antigens obtained according to option A should be partitioned onto immunochromatographic test lines and dot blot membranes at a relatively high concentration compared to typical protein antigens (1 μL per band, OD600 of 10), and nitrocellulose membranes with relatively large pore sizes (e.g., MDI CNPH membranes) should be used to avoid nitrocellulose being blocked by antigens.

[0077] B: Option B requires whole-cell wall antigen: Most whole-cell wall antigens obtained according to option B should be cross-linked or coupled to molecules using recognized methods to facilitate their binding to solid carriers.

[0078] C: Detection of fully specific antibodies in milk samples: Different immunoassay buffers can be used to dilute the milk from 1 / 10 to 1 / 10000. Samples can be diluted using standard immunoassay buffers (PBS containing Tween), Tween / SDS buffer, or stringent buffers (glycine, EDTA, NaCl, Tween, BSA).

[0079] D: Detection of specific antibodies aggregated in milk samples: Antibodies aggregated in milk can be separated from milk samples by centrifugation and / or filtration steps using recognized methods.

[0080] E: Detection of complex specific antibodies in milk samples: Milk complex antibodies can be separated from milk samples by incubation with 2-5% w / v polyethylene glycol (PEG) according to recognized methods, followed by centrifugation and / or filtration.

[0081] F: Antibody isotype: All isotypes (IgA, IgM, IgG1, IgG2, etc.) of specific antibodies binding to whole-cell wall antigens can be detected using known methods, or only a single isotype can be detected. Protein G is the preferred detection system for simultaneously detecting multiple isotypes of antibodies; for Protein A, it primarily detects IgG2.

[0082] Immunochromatography was used to evaluate the comparative performance of each whole-cell wall antigen immunoassay feature (see Table 6, showing the performance of whole-cell wall antigens compared using different assay versions).

[0083] Table 6

[0084] Example 3: Evaluation of milk from lactating cows by whole cell wall immunochromatography, dot blot and ELISA.

[0085] Mix 200 μL of previously filtered 0.45 μm milk with 800 μL of PEG solution to a final concentration of 3.5% w / v. Centrifuge the mixture and wash the precipitate twice with 3.5% PEG. Resuspend the precipitate in carbonate buffer at pH 10.5 and neutralize. Use the neutralized sample for assays.

[0086] In a whole-cell wall immunochromatographic assay using colloidal gold protein A conjugate, 120 μL of sample (from infected and uninfected lactating cows) was added. If specific antibodies against the whole-cell wall antigen were present, a test line would appear.

[0087] Add 1000 μL of sample (from infected and uninfected lactating cows) to a dot blot membrane coated with whole-cell wall antigens and incubate for 1 hour with stirring. After washing, add a colloidal gold protein A conjugate diluted in 1 mL of immunoassay buffer and incubate for another hour. If specific antibodies against the whole-cell wall antigens are present, a red blot will appear.

[0088] Add 100 μL of sample (from infected and uninfected lactating cows) to whole-cell wall-coated ELISA wells. After a 30-minute incubation and washing cycle, add the HRPO protein A conjugate. After another 30-minute incubation, perform a fresh wash and add TMB to the wells. A blue color will appear if a specific antibody against the whole-cell wall antigen is present.

[0089] Diagnostic results were obtained using three immunoassays (see Tables 7-11, showing the results of immunochromatography, dot blot, and ELISA for whole cell wall antigens of Staphylococcus aureus, Streptococcus lactis, Streptococcus dysgalactiae, Escherichia coli, and Klebsiella spp. in infected and uninfected lactating milk).

[0090] Table 7

[0091] Table 8

[0092] Table 9

[0093] Table 10

[0094] Table 11

[0095] Example 4: Evaluation of bovine colostrum by whole cell wall immunochromatography, dot blot and ELISA.

[0096] Whole-cell wall immunochromatography was used to determine the concentration of bovine colostrum diluted 1 / 1000 in 120 μL using colloidal gold protein G conjugate. A test line appeared if specific antibodies against the whole-cell wall antigen were present.

[0097] The bovine colostrum group was tested using a dot blot membrane coated with whole-cell wall antigen at a concentration of 1000 μL and incubated for 1 hour with stirring. After washing, a colloidal gold protein G conjugate diluted in 1 ml of immunoassay buffer was added, and the mixture was incubated for another hour. A red blot appeared if specific antibodies against the whole-cell wall antigen were present.

[0098] The bovine colostrum group was assayed using 100 μL of 1 / 1000 dilution in ELISA wells coated with whole cell wall. After a 30-minute incubation and washing procedure, HRPO protein G conjugate was added. After another 30-minute incubation, a fresh wash was performed, and TMB was added to the wells. A blue color appeared if a specific antibody against the whole cell wall antigen was present.

[0099] Diagnostic results were obtained using three immunoassays (see Tables 12-14). ， Immunochromatographic, dot blot, and ELISA results of whole cell wall antigens of Staphylococcus aureus, Streptococcus lactis, Streptococcus dysgalactiae, Escherichia coli, and Klebsiella spp. in bovine colostrum.

[0100] Table 12

[0101] Table 13

[0102] Table 14

[0103] Example 5: Evaluation of bovine mixed raw milk by whole cell wall immunochromatography, dot blot and ELISA Mix 200 μL of emulsion with 800 μL of PBS solution. Centrifuge the mixture and wash the precipitate twice with PBS. Resuspend the precipitate in carbonate buffer at pH 10.5 and neutralize. Use the neutralized sample for assays.

[0104] In a whole-cell wall immunochromatographic assay using colloidal gold protein A conjugate, 120 μL of sample (from infected and uninfected lactating cows) was added. If specific antibodies against the whole-cell wall antigen were present, a test line would appear.

[0105] Add 1000 μL of sample (from infected and uninfected lactating cows) to a dot blot membrane coated with whole-cell wall antigens and incubate for 1 hour with stirring. After washing, add a colloidal gold protein A conjugate diluted in 1 mL of immunoassay buffer and incubate for another hour. If specific antibodies against the whole-cell wall antigens are present, a red blot will appear.

[0106] Add 100 μL of sample (from infected and uninfected lactating cows) to whole-cell wall-coated ELISA wells. After a 30-minute incubation and washing cycle, add the HRPO protein A conjugate. After another 30-minute incubation, perform a fresh wash and add TMB to the wells. A blue color will appear if a specific antibody against the whole-cell wall antigen is present.

[0107] Diagnostic results were obtained using three immunoassays (see Tables 15-17, showing the results of immunochromatography, dot blot, and ELISA for whole cell wall antigens of Staphylococcus aureus, Streptococcus lactis, Streptococcus dysgalactiae, Escherichia coli, and Klebsiella spp. in infected and uninfected lactating milk).

[0108] Table 15

[0109] Table 16

[0110] Table 17

[0111] Examples and Conclusions 1. The applicant developed immunochromatography, dot blot, and ELISA according to the procedures described in the literature (References 1 and 2) but obtained unsatisfactory results. A weak correlation was obtained between milk from infected and uninfected lactating cows due to a large number of false positives and false negatives. The applicant explored alternative methods in an attempt to improve the diagnostic value of the assays.

[0112] Surprisingly, when evaluating the whole-cell wall antigen obtained according to Example 1, more specific and sensitive results were obtained.

[0113] The applicant has developed a whole-cell wall antigen production process that inactivates and / or denatures and / or removes most cell wall surface proteins and other external components of the cell wall without damaging the cell wall. This process can be achieved by incubating microbial cells in a harsh environment involving autoclaving. Soluble and insoluble fractions of this process can be used as antigens (Example 1).

[0114] The procedure developed by the applicant removes most of the cell wall antigens (in fact, a significant reduction in antigen recognition patterns was observed in Tables 1-5 when whole cell wall antigens were evaluated in parallel with untreated cell wall antigens), but unexpectedly it significantly improves the diagnostic value of the assay because the remaining whole cell wall antigens are better correlated with infection. Unexpectedly, the procedure can be applied to a wide variety of different microorganisms.

[0115] The specificity of whole-cell wall antigens was examined using milk samples, and the results were very satisfactory. Surprisingly, milk infected with *Streptococcus mammologica* contained antibodies binding to *Streptococcus mammologica* whole-cell wall antigens, but not to *Staphylococcus aureus* whole-cell wall antigens, and vice versa (Table 7-11). Furthermore, the binding profiles of different colostrums varied considerably when using different whole-cell wall antigens (Tables 12-14). These results indicate that only specific antibodies bind to each whole-cell wall antigen.

[0116] The literature describes that cell wall composition within the same microbial species can show significant differences (Reference 3). When measuring milk from infected and uninfected lactating cows from different sources, cell wall antigens obtained under different culture conditions (18 hours of culture versus 28 days of culture) provided equivalent results (Tables 1-5), thus whole cell wall antigens are antigens with surprisingly good consistency.

[0117] Staphylococcus aureus, Streptococcus lactis, Streptococcus dysgalactiae, Escherichia coli, and Klebsiella spp. have been successfully used to obtain whole-cell wall antigens. Any other target microorganisms can be used.

[0118] Most of the antigen production processes described for detecting specific microbial antibodies rely on using specific recombinant or purified proteins as antigens, while our method is based on detecting whole cell wall components.

[0119] The published whole-cell antigens used to detect microbial antibodies are usually extracts that have been sonicated, in which the cell wall is disrupted and most proteins are not significantly modified, resulting in a mixture of internal and external cell components (Reference 5). Our method is designed to detect only the whole cell wall components.

[0120] 2: Using the protocols described in the literature (References 1 and 2), whole-cell wall immunochromatography, dot blot, and whole-cell wall ELISA were developed, but unsatisfactory results were obtained (very poor signals were obtained). To overcome this problem, the applicant explored different methods.

[0121] Due to the specific characteristics of whole cell wall antigens and the requirements for sample analysis, some modifications were made to the procedure described in the literature, as shown in Example 2.

[0122] Surprisingly, when the whole-cell wall antigen obtained according to protocol A was partitioned at a relatively high concentration onto the immunochromatographic test line and dot blot membrane, and a nitrocellulose membrane with a relatively large pore size was used, a very strong positive signal was obtained.

[0123] Surprisingly, a very strong positive signal was obtained when the whole-cell wall antigen obtained according to option B was cross-linked or coupled with the molecule.

[0124] When analyzed using immunoassays containing the antigen of this invention and the buffer and sample diluent described in the literature (References 2 and 4), a significant proportion of milk samples from uninfected cows tested positive. Different tests were performed to improve the diagnostic value of the antigen of this invention.

[0125] Surprisingly, when high-concentration antigens were used in combination with significantly diluted, untreated milk samples (1 / 10–1 / 10,000) and protein G as a conjugate for assays (see Example 2), a significant improvement in diagnostic value was observed. In other words, the antigen of the present invention allows for the accurate detection of most assessed infections in milk when using, for example, high-concentration antigens and significantly diluted samples (1 / 10–1 / 10,000) and protein G as a conjugate (see Example 2).

[0126] This form of assay using untreated milk samples is particularly significant for identifying known infections or classifying colostrum. However, this method cannot identify the source of infection. To allow for the identification of the source of infection, different sample pretreatments were explored.

[0127] Surprisingly, when using only aggregated or complexed antibodies (see Example 2) and protein A as detection reagents, an immunoassay based on whole cell wall antigens can be used to identify the source of infection.

[0128] However, it should be understood that while describing preferred embodiments of the invention, detailed descriptions and specific examples are given only by way of illustration, as various variations and modifications of immunoassay buffers and detection systems, as well as immunoassay formats, will become apparent to those skilled in the art from this detailed description, within the spirit and scope of the invention. Particularly important is the consideration of the value of flow-through or vertical immunoassays (Reference 6) in assays requiring the evaluation of aggregated or complexed antibodies.

[0129] 3: Finally, to confirm the diagnostic value of the whole cell wall immunoassay, the applicant evaluated it using milk samples from different sources.

[0130] The whole-cell wall antigen developed by the applicant significantly improves the diagnostic value of immunoassays. The whole-cell wall antigen can be easily mixed (e.g., allowing simultaneous detection of different infections) and is readily available in a variety of different antigens (e.g., allowing screening for the presence of various microorganisms in lactating cow's milk, colostrum, and mixed raw milk). Furthermore, because high-dilution cow's milk can be evaluated, mixed milk samples can be easily evaluated by reducing the dilution (e.g., allowing mixed raw milk analysis at different dilutions).

[0131] Surprisingly, whole-cell wall immunoassay allowed for the accurate classification of milk from infected and uninfected lactating cows (Example 3 and Tables 7-11). Immunoassays based on whole-cell wall antigens can be used to analyze mammary gland health by detecting the presence of microbial infection (the presence of specific antibodies in the samples evaluated in Example 3 and Tables 7-11 correlated with the presence of infection).

[0132] Surprisingly, whole-cell wall immunoassay was able to differentiate bovine colostrum samples (Example 4 and Tables 12-14). Immunoassay based on whole-cell wall antigens can be used to analyze colostrum quality by detecting the presence of different antimicrobial antibodies (different passive immunizations expected to be delivered to calves in the different samples evaluated in Example 4 and Tables 12-14).

[0133] Finally, and surprisingly, whole-cell wall immunoassay allowed for the analysis of the quality of mixed raw milk samples from different sources (Example 5 and Tables 15-17). Immunoassays based on whole-cell wall antigens could be used to analyze the quality of mixed raw milk by detecting the presence of different infection-associated antimicrobial antibodies (in Example 5 and Tables 15-17, Staphylococcus aureus infection was suspected in mixed raw milk sample 2, and Streptococcus mammologicus infection was suspected in mixed raw milk sample 3).

[0134] References Reference 1: Pernthaner, A (2019) Device for detection of antibodies to a pathogen WO 2019216775A1 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 Reference 3: Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae. Beaussart A, Péchoux C, Trieu-Cuot P, Hols P, Mistou MY, Dufrêne YF. Nanoscale. 2014 Dec 21;6(24):14820-7. doi: 10.1039 / c4nr05280c. Epub 2014 Oct 31. PMID: 25358409 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 Appendix 5:Development of a Gold Nanoparticle Based Lateral Flow Assayfor Rapid Diagnosis of Contagious Agalactia in Goats TR Arun, R. Rana, P.Singh, P. Choudhuri, VP Singh, P. Thomas, V. Rekha, K. Nehra, J. Usharaniand K. Dhama. Asian Journal of Animal and Veterinary Advances Year: 2014 |Volume: 9 | Issue: 7 | Page No.: 405-413 DOI: 10.3923 / java.2014.405.413 Table 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;

Claims

1. A method for obtaining whole-cell wall antigens from microorganisms, the method comprising the following steps: a) Cultivate microorganisms in a suitable culture medium; b) Washing and inactivating and / or denaturing and / or removing the external components of the cell wall of the microorganisms obtained in step a), preferably cell wall surface proteins, by subjecting the microorganisms obtained in step a) to at least autoclaving. The method described herein does not include the step of lysing the microorganism.

2. The method according to claim 1, wherein the microorganisms sterilized by autoclaving are centrifuged after step b).

3. The method according to claim 2, wherein the whole cell wall antigen is obtained from a centrifuged precipitate or insoluble fraction.

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

5. The method according to any one of claims 1 to 4, wherein the microorganism is a Gram-positive bacterium or a Gram-negative bacterium.

6. The method according to claim 5, wherein the microorganism is a Gram-positive bacterium selected from Staphylococcus aureus, Streptococcus lactis, and Streptococcus faecium.

7. The method according to claim 5, wherein the microorganism is a Gram-negative bacterium selected from Escherichia coli and Klebsiella spp.

8. An immunoassay for detecting antibodies associated with microbial infection, comprising the step of using the whole cell wall antigen obtained according to any one of claims 1 to 7.

9. The immunoassay according to claim 8, wherein the immunoassay is performed by the following steps: i) Obtain aggregated and / or complexed antibodies from the sample; ii) Using the same type of antibody obtained in step i) of the whole cell wall antigen binding according to any one of claims 1 to 7; iii) Detect the same type of combination described in step ii).

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

11. The immunoassay according to any one of claims 8 to 10, wherein the immunoassay is selected from enzyme-linked immunosorbent assay (ELISA), immunochromatography, and dot blot.

12. The immunoassay according to any one of claims 8 to 11, wherein the antibody is detected in an animal milk sample.

13. The immunoassay according to claim 12, wherein the animal milk is mammalian milk.

14. The immunoassay according to claim 13, wherein the mammalian milk is bovine milk.

15. Use of the immunoassay according to any one of claims 12 to 14 in a method for analyzing breast health status.

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

17. Use of the immunoassay according to any one of claims 12 to 14 in a method for analyzing the quality of mixed raw milk.

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