Diagnostic kit for detecting beta-casein a1 in milk samples, method and uses.

Abstract

The invention relates to technology constituting a significant advance in the analysis of dairy products, with wide-ranging implications for industry, agriculture, nutrition and public health. The rapid diagnostic kit is designed to instantly identify cow's milk free of beta-casein A1 protein using rapid devices in the form of lateral flow immunochromatography.

Description

Diagnostic kit for the detection of beta-casein Al in milk samples, Method and Uses. Field of Application

[0001] The innovation presented in this patent represents a significant advance in the analysis of dairy products, with broad implications for industry, agriculture, nutrition, and public health. Our rapid test revolutionizes the way milk is evaluated, allowing for precise and efficient identification of samples that are free of beta-casein Al, and therefore associated with animals with the A2A2 genotype or with A2 milk purity. State of the Art

[0002] Milk proteins play a crucial role in both the nutritional composition and functional properties of dairy products. Among these proteins, caseins represent one of the main fractions, comprising approximately 80% of the total proteins in cow's milk. Caseins are known for their ability to form gelatinous structures, contributing to the texture and stability of many dairy products.

[0003] However, within the category of beta-caseins, there is an important distinction between the two main types: beta-casein variant A1 and beta-casein variant A2. This distinction lies in the structure of the beta-casein. Beta-casein variant A1 has a histidine amino acid at position 67 in its primary sequence, while in beta-casein variant A2 this amino acid is replaced by proline. This small structural difference is crucial, as it determines how these proteins are digested and what their effects are on the body.

[0004] Research suggests that partial digestion of beta-casein Al can lead to the production of a substance (peptide) called beta-casomorphin-7 (BCM-). 7), which has been associated with a number of negative health effects in some people, including gastrointestinal discomfort, inflammation, and possible links to chronic conditions such as Parkinson's disease and type 1 diabetes. On the other hand, beta-casein A2 is not known to produce BCM-7 in significant quantities during digestion.

[0005] These findings have generated increasing interest in differentiating between milks containing predominantly beta-casein A2, especially among consumers with sensitivities or intolerances to traditional milk. Dairy products derived from cows that predominantly produce beta-casein A2 have been marketed as a potentially healthier alternative for those experiencing common digestive discomforts associated with dairy consumption.

[0006] In this context, document CN108414628A describes a method for detecting the presence of A2-beta-casein in milk. The method involves analyzing high-performance liquid chromatograms of milks identified as type Al and type A2 by genotype, comparing them with the chromatograms of alpha s-casein, beta-casein, and kappa-casein standards to determine the shape and timing of the chromatographic peak of each of the Al-beta-casein and A2-beta-casein. This allows for the qualitative detection of the presence of beta-casein in unknown milk.

[0007] Document CN105925717, from 2016, entitled "Method and kit for detecting Al and A2 beta-casein in cow milk," discloses a method and kit for detecting Al and A2 beta-casein in Holstein cows, belonging to the technical area of ​​molecular biology detection. The method comprises the following steps: designing special primers using the DNA of a cow to be detected as a template, performing the amplification reaction by PCR (polymerase chain reaction), performing digestion of the EcoT22I enzyme in the amplification segment, and performing agarose gel electrophoresis. The detection of the Al beta-casein in cows occurs when the length If the length of the DNA segment subjected to gel detection is 434 bp, the cow is an A1 / A2 hybrid cow if the length of the DNA segment subjected to enzymatic digestion is 434 bp, 385 bp, and 49 bp, and the cow is an A2 cow if the length of the DNA segment subjected to enzymatic digestion is 385 bp and 49 bp. The detection method is easy to operate, has the advantages of low cost and high accuracy, and can be used for screening A2 dairy cows in the early stages, thus reducing economic losses in the livestock industry.

[0008] Document LU101241 discloses a breeding method to simplify the selection of high-yielding homozygous A2A2 dairy cows based on pedigree data. Through dairy herd improvement (DHI), pedigree and phenotype data of individual cows are collected, and genetic evaluation is performed in combination with the results of the paternal casein P genotype to obtain the genetic values ​​for milk production, milk fat production, milk protein production, and milk protein rate. Finally, the high production indices (HPIs) of the individuals are calculated to obtain the high-yielding homozygous A2A2 individuals. The present invention performs screening by applying daily DHI data, which significantly reduces the workload during the dairy farm production process itself; the present invention can track homozygous A2A2 individuals in the herd quickly, simply, and accurately.

[0009] All the methods cited in the previous patent applications differ from the present application by using methods such as chromatography and PCR (polymerase chain reaction), which require laboratory machinery that is only operated after the milk is extracted and transported to industries, while the present application uses a rapid method, through a lateral flow method. Furthermore, this method, when carried out in an organized manner, allows for the identification of the phenotype of the animal from which the milk was obtained.

[0010] Document W02024070874 describes the application of monoclonal antibodies against casein variants Al and A2, obtained through the hybridoma technique, for use in reagents for phenotypic detection. However, the methodology for producing monoclonal antibodies requires sophisticated laboratory infrastructure, such as eukaryotic cell culture, and presents significant limitations due to the high cost and low accessibility of the technology, limiting the widespread use of monoclonal antibodies in diagnostic technologies.

[0011] In contrast, the present invention proposes an innovative and more accessible technical solution: the use of affinity-purified polyclonal chicken antibodies (IgY type) for negative enrichment of beta-casein Al ligands. Laying hens are used as immunizing organisms, which represents a strategic choice since these birds do not produce milk or express beta-casein, thus avoiding the development of immunological tolerance to the protein and allowing for a more specific, robust, and efficient immune response.

[0012] Obtaining IgY anti-beta-casein A1 antibodies from birds is done by extracting them from the egg yolks of immunized animals, ensuring high yield, reduced cost, and the advantage of being a non-invasive process, without the need to sacrifice the animals. Furthermore, the hens continue to produce eggs regularly after immunization, enabling continuous collection for weeks or months, generating large volumes of antibodies from a single immunized batch. This results in greater standardization and reproducibility between batches of polyclonal antibodies, in contrast to polyclonal antibodies produced in rodents, whose production requires the immunization of new animals for each batch and their sacrifice at the end of the process, increasing inter-batch variability.

[0013] Following immunization, it was observed that IgY antibodies extracted from egg yolks showed reactivity to both the Al and A2 peptides. To overcome this challenge and obtain antibodies with high specificity for Al, an original and non-trivial negative affinity purification technique was developed. The methodology consists of removing antibodies with affinity for the A2 peptide using a chromatographic column where the A2 peptide, modified with terminal cysteine, has been previously covalently immobilized to a resin activated with thiol groups. The extract containing IgY is then applied to this column, and the antibodies that recognize A2 are retained, while those that do not bind are eluted and collected, composing a fraction enriched with antibodies with preferential affinity for beta-casein Al.

[0014] The present invention, therefore, is based on the use of IgY polyclonal antibodies (easily and cheaply produced) enriched by negative affinity chromatography, which represents a substantial difference compared to the traditional approach with monoclonal antibodies. This difference is directly reflected in the practical applicability of the invention, as the method is more accessible, less expensive, and provides a higher yield.

[0015] It is also important to highlight that the immunogenic peptides Al and A2 exhibit high structural and sequential similarity, differing only by a single amino acid: the Al peptide contains the sequence PGPIHNSLPC, while the A2 peptide has the sequence PGPIPNSLPC and is identical except for the substitution of histidine (H) for proline (P) at position 67. This subtle modification, although sufficient to distinguish the phenotypes, significantly hinders the production of specific polyclonal antibodies by conventional methods, since most residues remain unchanged. Although the production of polyclonal antibodies through the immunization of guinea pigs is a technique that generates large quantities of Given that antibodies are easy to obtain, an immunization strategy that does not include a refined purification step would inevitably result in antibodies that recognize both peptides.

[0016] The negative affinity immunopurification technique developed in the present invention is precisely the differentiating factor that allows the separation and selection of antibodies with the exclusive ability to recognize the Al peptide, by eliminating those that bind to regions common with A2. This level of specificity is achieved without resorting to hybridomas, but rather with polyclonal antibodies obtained in a scalable, ingenious, and economically viable way. This is a purification based on differential epitope subtraction, which is non-trivial and enables the production of highly specific polyclonal antibodies without the need for cloning, clonal screening, or cell manipulation.

[0017] Finally, the scientific literature already recognizes that IgY antibodies possess greater physicochemical stability compared to mammalian IgG antibodies, being more resistant to variations in pH and temperature. This characteristic makes IgY particularly suitable for field applications or "point-of-care" type tests, further consolidating the industrial and technological applicability of the present invention.

[0018] Document CN117567610 describes a rapid lateral flow immunochromatographic test for direct detection of beta-casein Al, which uses polyclonal antibodies as capture agents immobilized on the test line, combined with monoclonal anti-Al antibodies labeled with colloidal gold, located on the sample pad. The test works by forming a "sandwich" between the beta-casein Al present in the sample, the polyclonal capture antibody, and the marker antibody, generating a visual signal on the test line in the presence of beta-casein Al in the milk sample. That is, when a marked line appears in the test region, it indicates that the milk sample contains beta-casein Al.

[0019] In contrast, the present invention proposes an indirect and competitive detection methodology, which is substantially different from the approach described in CN117567610.

[0020] In this new system, the test line contains an immobilized synthetic peptide derived from beta-casein Ali, and not antibodies. The test line may contain, instead of the Ali peptide, the beta-casein of the Ali type itself. The milk sample to be analyzed is applied together with polyclonal IgY anti-beta-casein-Al antibodies previously labeled with colloidal gold. If beta-casein Ali is present in the milk sample, the labeled IgY antibodies will preferentially bind to the free Ali protein present in the milk, remaining unavailable to bind to the Ali peptide (or to the beta-casein Ali itself) immobilized on the test line. As a result, the gold-labeled anti-Ali antibody will not be captured on the test line and the line will not appear, only the control line will appear, signaling the presence of the Ali variant in the sample (one line = Ali casein).On the other hand, in the absence of beta-casein Al — as in the case of A2A2 type milk samples — IgY antibodies will remain free to bind to the Al peptide of the test line, forming two lines, one line in the test region and one line in the control region (two lines = A2 casein).

[0021] This competitive principle gives the technology significant advantages in terms of simplicity, cost, and performance. The use of synthetic peptides, or beta-casein itself, which are cheaper and easier to produce than antibodies for immobilization, makes the process more accessible. Furthermore, the competitive format reduces the likelihood of false positives and allows for a direct and intuitive interpretation to identify A2A2 milk: two lines mean a positive sample for A2 milk, one line indicates contamination with Al.

[0022] The choice of chickens as immunizing organisms also represents a relevant technical advantage of the present invention. Because they do not Since chickens do not produce milk or express beta-casein, they do not develop immunological tolerance to the protein, which ensures a more specific and robust immune response. The production of IgY antibodies in chickens is sustainable and economical, allowing for non-invasive harvesting from eggs and high yields over long periods. The possibility of obtaining large quantities of antibodies from a single immunized animal over several weeks ensures greater standardization between batches, in addition to reducing costs and process variability.

[0023] It is important to highlight that such advantages associated with the use of birds are not discussed in documents W02024070874 or CN117567610, which refer generically to the use of antibodies from mammals or any vertebrate, without addressing the specific technical, structural, and immunological benefits provided by immunization in chickens.

[0024] It is also important to highlight that documents WO2024070874 and CN1 17567610 evaluate few milk samples containing or not containing beta-casein Al for proof-of-principle of the methodology, which does not provide statistically significant data for evaluating the technology's performance. In contrast, the present invention demonstrates the technology's performance with analysis of 77 genotyped samples, conclusively demonstrating the specificity and sensitivity of the method.

[0025] Therefore, the present invention demonstrates a clear set of innovative technical elements, including the use of chickens as immunizing organisms, the purification of antibodies by negative affinity using immobilized A2 peptide, the development of a rapid test based on indirect and competitive detection with immobilized A2 peptide on the test line, and the complete absence of the use of hybridomas or monoclonal antibodies. These characteristics, considered together, demonstrate inventive activity and novelty compared to prior art documents. fully justifying the maintenance of the patent application with the proposed adjustments.

[0026] The 2023 article titled “Generation and characterization of avian IgY antibodies for detecting beta-casein Al in bovine milk” describes an ELISA test to identify A2A2 animals based on the detection of beta-casein Al-free milk. However, this test is a laboratory test, in ELISA format, requiring a laboratory to receive milk samples for processing and report issuance. The test discussed in the article above requires specialized equipment and personnel for its execution; therefore, it is not a rapid test (DE JESUS, Bruna Andersen Pereira, ECHEVERRI, Lina Maria Salazar, MAGALHÃES, Maria de Lourdes Borba, SILVA, Gustavo Felippe. Generation and characterization of avian IgY antibodies for detecting beta-casein Al in bovine milk. Analytical biochemistry, v. 678, p. 115283, 2023).The test described in the technology for which protection is sought is a quick test that the producer can perform themselves; it is a SELF-TEST and has the advantages of being done on the farm, providing results in 20 minutes, and is easy to operate.

[0027] Furthermore, the immunoglobulin purification methodology described in the article "Generation and characterization of avian IgY antibodies for detecting beta-casein Al in bovine milk" does not use negative affinity purification, as described in the present invention, resulting in low and insufficient discrimination between beta-casein Al and A2 samples. The use of polyclonal IgY not purified by the method described in the present invention is incompatible with its use in lateral flow immunochromatographic assays.

[0028] Other methods for identifying A1 and A2 caseins are cited in several articles; however, all of them utilize some kind of laboratory method, complex equipment, and specialized labor, and none of them is described in a quick, self-test format that the producer can perform themselves, without needing elaborate technical qualifications. Description of the Invention

[0029] The invention aims to instantly identify cow's milk free of the beta-casein A1 protein using rapid devices, in the form of lateral flow immunochromatography.

[0030] The lateral flow platform used resembles the structure of widely known tests, such as pregnancy tests. However, the invention stands out for being an innovative application of this rapid testing platform for the identification of specific genotypes, based on the analysis of specific phenotypes (such as the absence of beta-casein Al) in milk. To date, there are no records of its use for this purpose to our knowledge.

[0031] The invention aims to develop rapid tests designed to identify animals with the A2A2 genotype, analyzing the phenotype of absence of beta-casein Al in the evaluated milk sample. This analytical method identifies the absence of beta-casein Al in cow's milk, allowing for indirect and highly accurate correlation with specific genotypes.

[0032] This invention serves not only to identify A2A2 animals, since animals with this genotype produce milk free of beta-casein Al, but also to identify contamination in A2A2 milk with beta-casein Al, an important tool in the quality control of A2 milk.

[0033] The lack of rapid tests for the indirect identification of specific genotypes in the field makes this innovation unique and valuable for the dairy industry. Its practical application and rapid results are crucial factors that highlight it as an efficient and innovative solution in the context of quality control and genetic improvement in the sector.

[0034] For the production of the test, it was necessary to produce IgY type antibodies, anti-beta casein Al, through the inoculation of specific peptides in Chickens. The peptides used for antibody production have the following sequence: PGPIHNSLPC and are called Al peptide. The Al peptide was chemically conjugated to the KLH (Keyhole Limpet Hemocyanin) protein and inoculated into chickens to produce specific IgY antibodies against beta-casein Al. After inoculation and antibody production by the chickens, the anti-Al IgY antibodies are collected and purified from the egg yolks of the chickens and used to produce the rapid test.

[0035] The A2 peptide (sequence: PGPIPNSLPC) was immobilized on a resin activated with thiol groups (such as Sulfo-Link™) for selective purification of IgY antibodies by negative affinity.

[0036] The synthetic peptides used in the present invention have been modified by the addition of a cysteine ​​residue (C) at the C-terminal end. This modification is not for immunogenic purposes, but rather strategic, allowing the covalent coupling of the peptides to different supports:

[0037] The presence of terminal cysteine ​​allows the formation of stable thioether bonds with activated supports, without interfering with the antigenic specificity of the central epitopes of the peptides.

[0038] Anti-beta-casein Al antibodies are coupled to colloidal gold nanoparticles and used in the production of the test. As shown in Figure 1, the gold-conjugated anti-Al antibody is placed on the conjugate pad (04). The milk sample is added to the sample pad (03). If the milk contains beta-casein Al protein, it will bind to the anti-Al antibody, which will migrate through the nitrocellulose membrane bound to the milk protein and will not be captured by the Al peptide immobilized on the test line of the nitrocellulose membrane. Therefore, the milk will not generate a dark line in the test region (Figure 2).

[0039] If the milk is free of beta-casein Al, the gold-conjugated antibody will not bind to the milk protein, remaining free during migration in the nitrocellulose membrane, being captured by the Al peptide in the membrane and marking a line in the test region. Thus, there will be two lines on the membrane to identify that the milk is free of beta-casein Al (Figure 2). This is an indirect way of identifying A2A2 animals, as the test identifies milk free of beta-casein Al. Since there are predominantly these two variations in the amino acid at position 67 of beta-casein, the use of a tool that detects the absence of beta-casein Al in milk automatically identifies the A2A2 genotype.

[0040] In summary, the detection of the presence or absence of beta-casein Al in milk samples comprises the following steps: (a) a plastic cassette (01) containing a porous nitrocellulose membrane (02); (b) the membrane (02) containing a zone containing a sample pad (03) where the diluted milk is applied; (c) the membrane (02) containing a conjugate zone containing a pad (04) with anti-beta-casein Al IgY antibodies coupled to colloidal gold nanoparticles, IgY-type antibodies, anti-beta-casein Al and produced by inoculation of peptides with the sequence PGPIHNSLPC, called Al peptide, in chickens; (d) the membrane (02) containing a test zone (05) containing immobilized beta-casein Al, or Al peptide, to capture free (unbound) anti-beta-casein Al antibodies; (e) the membrane containing a control zone to verify the validity of the test (06);(f) a milk collection device (07) in the form of a plastic pipette for adding the milk sample to the tube containing diluent; (h) a dropper tube containing diluent for mixing with the milk sample (08).;

[0041] The method for detecting beta-casein in cow's milk has the following steps: (a) Collect the milk, properly cleaning the animal's teats, in a clean and dry container, avoiding any type of contamination. with other milk samples; (b) Add 10 to 30 drops of milk, using a pipette (07), to the tube containing diluent (08); Position the cassette (01) on a smooth, flat surface, drip 2 to 4 drops of the diluted milk from the bottle onto the cassette (01); (e) Wait at least 10 minutes and analyze the result; (f) If the test shows two lines, one in the test area (T) and the other in the control area (C), the milk analyzed does not contain beta-casein Al, and is therefore pure A2 milk, produced by animals with genotype A2A2; (g) If the test shows two lines, one in the test area (T) and the other in the control area (C), the milk analyzed does not contain beta-casein Al, and is therefore pure A2 milk; (h) If the test shows only one line in the control area (C), the milk analyzed is NOT A2, and is therefore produced by an animal with genotype A1A2 or Al Al; (i) If after 30 minutes, no line appears or no line appears in the control (C), the test is considered invalid.

[0042] Although rapid tests are widely used devices for identifying antibodies in biological samples, this invention is unique in that it analyzes a milk phenotype to identify the absence of a protein (specific phenotype) and thus identify a corresponding homozygous genotype. Obviously, this is possible because for this specific amino acid, there are predominantly two variants found, and therefore, the absence of Al allows for the identification of homozygosity for A2A2. Furthermore, the test is a way to identify the purity of A2 milk to ensure quality control in the dairy industry that produces A2 milk, guaranteeing that the A2 milk produced is free of beta-casein Al.

[0043] Milk should be collected manually, properly cleaning the animal's teats, in a completely clean and dry container, avoiding any type of contamination with other milk samples.

[0044] Add 10 to 30 drops of milk to the tube containing the diluent, as shown in Figure 3A. Then, attach the dropper and close the tube. as shown in Figure 3B. After shaking, as shown in Figure 3C. Position the test on a flat surface, drip 2-4 drops of the diluted milk from the bottle onto the cassette, as shown in Figure 3D. Wait at least 5 minutes and analyze the result: If the test shows two lines, one in the test area (T) and the other in the control area (C), as shown in Figure 4, the milk analyzed is A2, produced by animals of genotype A2A2, or the milk analyzed is 100% A2, therefore it does not present contamination of beta-casein Al.

[0045] If the test shows only one line in the control area (C), as shown in Figure 5, the milk analyzed is NOT A2, and is therefore produced by an animal with genotype A1A2 or Al Al, or the milk analyzed has contamination of at least 5% with beta casein Al.

[0046] If after 30 minutes, no line appears or no line appears in the control (C), the test is considered invalid as shown in Figure 6.

[0047] The present technology can be better understood through the following, but not exhaustive, examples. Example 1: AJ Ranch - animals previously genotyped by the company Zoetis.

[0048] Milk samples from 18 animals previously genotyped by Zoetis (Table 1) using traditional genotyping were analyzed. The animals came from the AJ Ranch located in Urussanga, Santa Catarina, Brazil. The milk samples were donated by the farm owner, who also provided the genotyping reports previously performed on the ranch animals. The milk was refrigerated and subjected to analysis using the rapid test methodology. The results are shown in Figure 7 and Table 1. Table 1: Genotype of each animal using the rapid test. Example 2: Milk quality control laboratory

[0049] Twenty tests were sent to the Milk Quality Control Laboratory of the State of Paraná. According to Table 2 and Figure 8, it can be observed that of the 20 samples (10 samples A2A2), all A2A2 samples were properly identified, with no false-positive results, demonstrating 100% sensitivity and specificity. Table 2: Genotype of A2A2 animals using the rapid test.

[0050] Figure 9 shows the results of 20 tests performed by the milk quality laboratory of the state of Paraná. All tests in the upper panel correspond to milk samples from A2A2 genotyped animals, and the samples in the lower panel correspond to samples from non-A2A2 genotyped animals. Example 3: AJ Ranch - animals previously genotyped by ST Genetics Brazil.

[0051] New milk samples were received from Cabanha AJ, located in Urussanga, Santa Catarina, from animals recently genotyped by the company ST Genetics Brasil. The milk samples were used in rapid tests and the results were compared to the genotyping (Table 3). Table 3: Genotype of each animal using the rapid test. Example 4: São Sebastião Community

[0052] Milk samples from 31 animals previously genotyped using traditional genotyping were analyzed. The animals came from the São Sebastião community farm, located in the municipality of Treze de Maio in Santa Catarina, Brazil. The milk samples were donated by the farm owner, who also provided the genotyping reports previously performed on the animals. The milk received was refrigerated and subjected to analysis using the rapid test methodology. The results are shown in Figure 10 and Table 4. Table 4: Names of the cows whose milk was used for the rapid test and their genotypes. >

[0053] Table 5 shows the complete compilation of results obtained through the studies presented, where milk from previously genotyped individual animals was compared to the results obtained with the technology for which protection is sought. Table 5: Technology Performance

[0054] Based on the results presented, it is safe to say that the technology has proven to be an effective and reliable tool for identifying animals with the A2A2 genotype. Internal validation, supported by results obtained from genotyped animals, confirms the consistency and accuracy of the test, providing safety to users and consolidating its applicability.

[0055] The data obtained from the analysis of the genotyped animals fully corroborated the expected results, demonstrating that... The test's ability to precisely distinguish the unique presence of the A2 allele in milk is significant. The consistent agreement between the test results and previously known genotypes reinforces the specificity of the method proposed by the technology.

[0056] It can be concluded, therefore, that the technology meets the demanding standards of reliability and precision, offering a valuable tool for the dairy industry and contributing to improving genetic management and the production of milk with specific characteristics. Example 5: Contamination Test of Raw Milk from Genotyped Cows

[0057] The contamination test performed on April 12, 2023 (Lot 230010) was conducted by mixing samples of genotyped A2A2 raw milk with samples of genotyped AlAl raw milk in different proportions. The proportions used in this test were 100:0, 95:5, 90:1, and 50:50 (A2A2:A1A1), as shown in Figure 11. The milk samples were mixed and homogenized properly, following the procedure described above. Figure 12 shows the results of the tests: a 100% A2 sample and a sample with 5% AlAl milk contamination. It is possible to observe that the control line disappears with 5% contamination.

[0058] The contamination test performed on January 24, 2024 (Lot 230011) was done by mixing a sample of genotyped A2A2 raw milk with a sample of genotyped AlAl raw milk in different proportions. The proportions used in this test were 100:0 and 95:5 (A2A2:A1A1). The milks were mixed, and 30 drops of the mixture were added to the buffer, following the procedure described previously. Figure 13 shows the test results: a 100% A2 sample and a sample with 5% AlAl milk contamination. It is possible to observe that the control line disappears with 5% contamination.

[0059] The contamination test performed on February 29, 2024 (Lot 230011) was conducted by mixing 15 samples of genotyped raw milk from A2A2 animals with one sample of genotyped raw milk from an AlAl animal in the proportions of 100:0 and 95:5 (A2A2:A1A1 mixture). The milks were mixed, and 30 drops of the mixture were added to the buffer, following the procedure described in the methodology. Figure 14 shows the test result. It is possible to observe that when several A2A2 milks are mixed and contaminated with a single AlAl milk, the control line disappears with 5% contamination. Example 6: Contamination Test using UHT Milk

[0060] The UHT milk contamination test performed on 11 / 27 / 2023 (Lot 230011) was conducted by mixing samples of A2 UHT SEMI-SKIMMED milk (PIRACANJUBA A2) with samples of regular UHT milk (PIRACANJUBA SEMI-SKIMMED - Regular milk) in proportions of 100:0, 95:5, and 90:10 (A2: regular), as shown in Table 6. Figure 15 shows the result of a 100% A2 PIRACANJUBA sample and samples contaminated with regular UHT milk from PIRACANJUBA, varying between 5% and 10% of the added volume. In this case, it is important to remember that the common UHT milks used to contaminate the samples in this test are not purely Al, therefore, they must contain an undefined mixture of beta-casein Al. To estimate the percentage of contamination, we consider that the common milks contain a 50%:50% mixture of beta-casein Al and A2 in their composition. Table 6: Percentages and proportions when mixing UHT A2 milk with a mixture of regular UHT milk.

[0061] In the contamination test of A2 UHT SEMI-SKIMMED milk, carried out on 01 / 26 / 2024 (Lot 230011), a sample of A2 UHT milk (NESTLÉ NESTLE NESTLE) was mixed with raw milk genotyped Al Al in proportions of 100:0 and 95:5 (A2A2:A1A1). The milks were mixed and homogenized properly, following the procedure. The test result is shown in Figure 16. Example 7: Contamination Test using pasteurized milk

[0062] Contamination tests on pasteurized A2 whole milk, A2 skim milk, and A2 lactose-free milk were performed on January 26, 2024 (Lot 230011). For the test, pasteurized A2 milk contaminated with fresh A1A1 genotyped milk in proportions of 100:0 and 95:5 (A2A2:A1A1) was used. Using 30 drops of each milk mixture, they were mixed and homogenized properly in the buffer, following the procedure described previously. The test results are shown in Figures 17, 18, and 19. Example 8: Contamination Test using yogurt and fermented milk

[0063] The A2A2 yogurt contamination test performed on January 26, 2024 (Lot 230011) was conducted by mixing a sample of A2A2 yogurt with non-A2A2 yogurt in proportions of 100:0 and 95:5 (A2A2: non-A2A2). The yogurt was mixed (10 drops) and properly homogenized in the buffer, following the procedure. Figure 20 shows the test result, using A2 yogurt (Trevisan) with contaminations from regular yogurt (Batavo) using the rapid test.

[0064] The contamination test using fermented milk, conducted on January 26, 2024 (Lot 230011), was performed by mixing a sample of A2 fermented milk with non-A2 fermented milk in proportions of 100:0 and 95:5 (A2: common). Ten drops of the fermented milk were used, mixed, and... The samples were properly homogenized in the buffer, following the procedure described in the methodology. Figure 21 shows the test result.

[0065] In summary, the results of this study corroborate the viability and effectiveness of the rapid testing technology as a reliable tool for evaluating the purity of bulk tank milk and A2 milk by-products to aid in dairy quality control.

[0066] The rapid test technology's ability to distinguish 100% A2 raw milk samples from samples contaminated with at least 5% Al beta-casein, with high efficiency, highlights its robustness and usefulness in industrial A2 milk processes.

[0067] Furthermore, the findings reveal that the test's sensitivity remains assured above 5% beta-casein Al contamination in processed milks, such as UHT, pasteurized, and fermented milk. In the specific context of yogurts, the test maintains its sensitivity above 10% contamination.

[0068] Therefore, based on the data presented, we conclude that the implementation of the rapid test technology represents a significant contribution to improving quality control, providing greater reliability in the detection and monitoring of the presence of beta-casein Al in different dairy products, thus consolidating excellence in the industrial processes of A2 milk. Example 9: Selective purification of IgY antibodies

[0069] The chickens were immunized with a synthetic peptide corresponding to beta-casein Al coupled to KLH (Peptide Al: Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser-Leu-Pro-Cys). Immunoglobulin Y (IgY) was extracted from the egg yolks using the caprylic alcohol precipitation method, as described by Polson et al. (1980), widely used for its effectiveness and reproducibility (Polson A, von Wechmar MB, van Regenmortel MHV. (1980). Isolation of viral IgY antibodies from yolks of immunized hens. Immunological Communications, 9(5):475-493).

[0070] Given the high homology between the Al (Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser-Leu-Pro-) and A2 (Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu-Pro-) peptides, conventional purification would result in cross-reactive antibodies. To avoid this limitation, a selective depletion differential purification step was developed: An affinity column containing the A2 peptide (with terminal cysteine) coupled to Sulfo-Link™ resin was constructed. The IgY pool was applied to the column, retaining antibodies with affinity for the A2 peptide. The collected eluate contained antibodies highly specific to the Al peptide, free of cross-reactivity.

[0071] The Al and A2 variants of bovine beta-casein differ by only one amino acid at position 67, resulting in highly similar epitopes. This small difference makes the development of highly specific polyclonal antibodies for the Al form challenging, as the immune response often generates a pool of cross-reactive antibodies, especially when using immunization with linear synthetic peptides.

[0072] To overcome this limitation, a negative depletion purification strategy was adopted, based on the principles of affinity chromatography. The central logic of the approach is: - Immobilize the A2 peptide in a solid matrix (Sulfo-Link™ resin), allowing the epitope to be exposed in its linear conformation; - Apply the pool of IgY antibodies, previously purified from the yolks of eggs immunized with Al peptide; Antibodies with cross-affinity for the A2 peptide bind to the matrix and are retained; Only antibodies with exclusive affinity for the Al peptide remain in the stream, being recovered as an eluted fraction.

[0073] This step is technically rational because it takes advantage of the similarity between epitopes to capture and remove unwanted antibodies, functioning as a "selective filter" that allows obtaining diagnostic reagents with refined specificity.

[0074] Furthermore, the use of a negative-depletion affinity column, instead of direct purification by Al, increases the functional selectivity of the antibody, eliminating any IgY that may have been generated by nonspecific structural recognition of the A2 peptide during immunization.

[0075] The practical result is the obtaining of a polyclonal antibody with preferential affinity to beta-casein Al, with an absence of reactivity towards the A2 isoform — which is crucial for the use of the test in a diagnostic context, where the presence of traces of A2 should not compromise the reading.

[0076] This strategy is not described in the prior art cited by INPI in the preliminary opinion and represents a relevant innovative differentiator, adding inventive activity and proven functional efficiency to the proposed system.

[0077] To verify the effectiveness of the purification, an ELISA assay was conducted using plates sensitized with Peptide Al or Peptide A2. Antibody samples were tested at two concentrations (0.4 pg / mL and 0.1 pg / mL), before and after the purification step with an immobilized peptide A2 column. The reading was performed at 450 nm, after development with TMB reagent. Table 7: Specificity validation by ELISA

[0078] The purified antibodies with depletion showed high reactivity exclusively with Peptide Al, and no reactivity with Peptide A2, even at low concentrations. This confirms that the purification step is effective in eliminating non-specific antibodies, resulting in a highly selective diagnostic reagent.

[0079] The modification proposed in this new application introduces an innovative step of selective purification by negative depletion using immobilized A2 peptide, allowing the production of highly specific IgY antibodies for beta-casein Al, even with a minimal difference of only one amino acid between the Al and A2 variants. This strategy ensures the practical applicability of the invention and has not been described or suggested in any of the prior art analyzed. Furthermore, it is worth noting that the documents cited in the prior art search are predominantly based on the use of monoclonal antibodies. Although these exhibit high specificity, their production depends on the hybridoma technique, which is more complex, expensive, and time-consuming, and also uses milk-producing mammalian animals, such as mice and rabbits, which express caseins and are therefore subject to immunological tolerance.

[0080] In contrast, the present invention employs polyclonal IgY antibodies obtained from chickens, offering several advantages. Since chickens do not produce beta-casein, they are able to generate a more effective immune response and are free from immunological tolerance to the protein of interest. IgY production via eggs offers high yield and allows for non-invasive collection, facilitating industrial scalability. IgY antibodies also stand out for their natural stability against pH and temperature variations, as well as exhibiting less interference with the complement system and human proteins, characteristics that make them particularly suitable for applications in diagnostic assays. The purification technique with caprylic alcohol used is simple, accessible, and highly reproducible. The selective depletion step with immobilized A2 peptide, in turn, is novel and confers... High specificity to the system, without the need to resort to advanced cloning technologies.

[0081] It is also important to emphasize that, among the patents analyzed, only one superficially mentions the possibility of using antibodies derived from chickens, along with those from mammals, without, however, presenting any technical details or experimental validation. This generic reference does not constitute a sufficient basis to question the originality of the solution presented here, which demonstrates a clearly justified technical choice supported by robust experimental data.

[0082] Thus, the proposed technology represents a concrete advance in the differential diagnosis between A1 and A2 milk, bringing together an alternative and more effective source of antibodies (IgY), a rational and innovative method of selective purification, and a practical application already validated by functional assays, with real potential for implementation in analytical routines and use on an industrial scale. These characteristics are not obvious to a person skilled in the art from the known state of the art, fully justifying the recognition of the inventive activity, descriptive sufficiency, and industrial applicability of this patent application. Designs

[0083] Figure 1 shows the items that make up the rapid test.

[0084] Figure 2 presents a schematic drawing of milk Al and A2.

[0085] Figure 3 shows an image of how to place the dropper in the bottle after adding the milk (A and B) and how to place the drops of the mixture in the cassette (C and D).

[0086] Figure 4 shows the positive result for A2A2 milk.

[0087] Figure 5 shows the negative result for A2A2 milk.

[0088] Figure 6 shows the result considered invalid.

[0089] Figure 7 shows the result of the rapid test differentiating milk with genotype A2A2 (two lines) from genotypes Al Al or Al A2 (one line).

[0090] Figure 8 shows the result of the rapid test differentiating milk with the A2A2 genotype (two lines) from non-A2A2 genotypes (one line).

[0091] Figure 9 shows the result of the rapid test differentiating milk with the A2A2 genotype (two lines) from non-A2A2 genotypes (one line).

[0092] Figure 10 shows the result of the rapid test differentiating milk with the A2A2 genotype (two lines) from non-A2A2 genotypes (one line).

[0093] Figure 11 shows the result of the test mixing genotyped milks (Al Al and A2A2) where a decrease in the intensity of the test line is observed when the percentage of beta-casein Al increases, decreasing the purity of A2 milk from 100% to 95%.

[0094] Figure 12 shows the disappearance of the test line as the percentage of A2A2 milk from genotyped cows decreases.

[0095] Figure 13 shows the test mixing milk with genotype A2A2 with AlAl milk, where it can be observed that the larger the amount of milk contaminated with AlAl, the test line disappears.

[0096] Figure 14 shows the test mixing different milks with genotype A2A2 with an AlAl milk where it can be observed that in milk contaminated with AlAl the test line disappears.

[0097] Figure 15 shows the disappearance of the test line as the percentage of A2 milk decreases.

[0098] Figure 16 shows the contamination test of Ninho brand A2 UHT semi-skimmed milk (Lot 3317A3) with raw milk genotyped Al Al (Samambaia). The image shows the disappearance of the test line.

[0099] Figure 17 shows the contamination test of Trevisan brand pasteurized A2 milk - zero lactose (Lot 12022024) with raw milk genotyped Al Al (Samambaia). The image shows the disappearance of the test line.

[0100] Figure 18 shows the contamination test of Letti brand pasteurized A2A2 skim milk (Lot 202040707) with raw milk genotyped Al Al (Samambaia). The image shows the disappearance of the test line.

[0101] Figure 19 shows the contamination test of Trevisan brand pasteurized A2A2 whole milk (Lot 120224) with raw milk genotyped Al Al (Samambaia). The image shows the disappearance of the test line.

[0102] Figure 20 shows the purity identification test for Trevisan A2 plum yogurt (Lot 160124) and Batavo plum plain yogurt.

[0103] Figure 21 shows the contamination test of Letti A2 fermented milk - zero lactose (Lot 202409) with 5% of non-A2 fermented milk (Frimesa - skimmed) (Lot 150124). The last cassette is a sample of regular fermented milk. It is important to note that although 5% of the volume of fermented milk was added in the test.

[0104] Figure 22 shows the reactivity of IgY antibodies with the Al and A2 peptides of bovine beta-casein, evaluated by indirect ELISA. The columns represent the absorbance values ​​at 450 nm obtained under different conditions: pre-immune antibodies (negative controls), total IgY before depletion purification, and IgY after purification with an affinity column containing immobilized A2 peptide. It can be observed that, after purification, reactivity with the A2 peptide is completely eliminated, while reactivity with the Al peptide is maintained, demonstrating high specificity of the purified antibody for diagnostic purposes.

Claims

CLAIMS 1. DIAGNOSTIC KIT FOR DETECTION OF BETA-CASEIN A1, characterized by being used in the detection of the presence or absence of beta-casein A1 in milk samples, and comprising the following steps: a. a plastic cassette (01) containing a porous nitrocellulose membrane (02); b. the membrane (02) containing a zone containing a sample pad (03) where diluted milk is applied; c. the membrane (02) containing a conjugate zone containing a pad (04) with anti-beta-casein A1 IgY antibodies coupled to colloidal gold nanoparticles; d. the membrane (02) containing a test zone (05) containing beta-casein A1, or peptide containing at least the SEQ ID No. PGPIHNSLPC, immobilized to capture free (unbound) anti-beta-casein A1 antibodies; e. the membrane containing a control zone to verify the validity of the test (06); f. a milk collection device (07) for adding the milk sample to the tube containing diluent; h.a dropper tube containing diluent to mix with the milk sample (08).

2. DIAGNOSTIC KIT, according to claim 1, characterized by the production of IgY type antibodies, anti-beta casein A1, by inoculation of peptides with the sequence PGPIHNSLPC, called peptide SEQ ID No. PGPIHNSLPC, in chickens.

3. DIAGNOSTIC KIT, according to claims 1 and 2, characterized by comprising an A1 peptide chemically conjugated to the KLH carrier protein, which is used for the immunization of chickens, inducing the production of polyclonal IgY antibodies specific against beta-casein A1, the IgY-anti-A1 antibodies being subsequently extracted from the yolk of the eggs of the immunized chickens and purified by affinity using a column containing resin coupled to the A2 peptide, allowing the obtaining of antibodies with high specificity.

4. DIAGNOSTIC KIT, according to claims 1 to 3, characterized by the presence of anti-beta-casein A1 antibodies coupled to colloidal gold nanoparticles.

5. METHOD FOR DETECTING BETA-CASEIN A1 in cow's milk, as defined in claims 1 to 4, characterized by comprising the steps: a. Obtaining the milk to be tested; b. Adding the milk to the tube containing diluent (08); c. Adding the diluted milk present in the bottle (8) to the cassette (1) containing the test membrane; d. Waiting at least 10 minutes and analyzing the result; e. The presence of two lines (one test line and one control line) indicates that beta-casein A1 was not detected.

6. METHOD, according to claim 5, characterized by detecting the absence of beta-casein A1 phenotype in cow's milk, indirectly identifying the A2A2 genotype of the animal.

7. METHOD, according to claim 5, characterized by the test presenting two lines, one in the test area (T) and the other in the control area (C), the milk analyzed does not contain beta casein A1, being, therefore, A2 milk, produced by animals of genotype A2A2.

8. METHOD, according to claim 5, characterized by the test presenting two lines, one in the test area (T) and the other in the control area (C), the milk analyzed does not contain beta casein A1, therefore being pure A2 milk.

9. METHOD, according to claim 5, characterized by the test showing only one line in the control area (C), the milk analyzed is not A2, therefore being produced by an animal with genotype A1 A2 or A1 A1.

10. USE OF THE DIAGNOSTIC KIT, as defined in claims 1 and 5, characterized by being used in the identification of milk free of beta-casein A1, identifying animals with the A2A2 phenotype and consequently with the A2A2 genotype.

11. USE, according to claim 12, characterized by being used in the identification of contaminations in A2A2 milk with beta-casein A1.

12. USE, according to claims 12 and 13, characterized by being used in identifying the purity of A2 milk.

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