Instrument-free multi-analyte diagnostic strip system for rapid detection of bovine mastitis in milk

Abstract

The system comprises a laminated 5 diagnostic strip including a backing layer (105), an absorbent pad layer (106) configured for controlled capillary transport of milk, and a reagent deposit layer (107) defining multiple spatially separated dry reagent zones. The reagent zones include a pH indicator zone (101), a leukocyte esterase detection zone (102), a hemoglobin detection zone (103), and a nitrite detection zone (104), each adapted 10 for operation in the milk matrix. A calibrated sample application point (108) receives a predefined volume of untreated milk and fluidically communicates the sample to the reagent zones via the absorbent pad layer. Colorimetric reactions develop in a result window area (109), enabling simultaneous visual interpretation without analytical instruments, typically within three minutes. The invention further 15 includes a moisture-controlled storage container comprising desiccant elements (204, 205), a reference color chart (208), and a calibrated pipette (209), ensuring reagent stability and standardized use.

Description

[0001] TITLE OF THE INVENTION:

[0002] INSTRUMENT-FREE MULTI-ANALYTE DIAGNOSTIC STRIP SYSTEM FOR RAPID DETECTION OF BOVINE MASTITIS IN MILK

[0003] DESCRIPTION OF INVENTION

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the field of veterinary diagnostics and dairy health monitoring.

[0006] More particularly, the invention pertains to rapid, instrument-free diagnostic systems for detecting bovine mastitis in milk, and relates to multi -analyte diagnostic strips employing dry reagent chemistries for on-site analysis of mastitis-associated biochemical and physiological markers directly in milk samples.

[0007] BACKGROUND OF THE INVENTION

[0008] Bovine mastitis remains one of the most significant and persistent challenges within the dairy industry, affecting animal health, milk quality, and overall farm productivity. It is widely recognized as a leading cause of economic loss due to reduced milk yield, discarded milk, treatment expenses, and the premature culling of affected animals. Early identification of mastitis, especially its subclinical form — which presents without visible abnormalities — is critical for preventing progression, minimizing herd-level transmission, and ensuring judicious antimicrobial use. Despite decades of research, existing mastitis detection tools have not fully met the operational demands of dairy farms, particularly in settings where rapid, accurate, and on-farm decision-making is essential.

[0009] The California Mastitis Test (CMT) and other conventional chemical cow-side tests have historically served as inexpensive screening tools for somatic cell elevation. However, their diagnostic accuracy is limited, especially in early or mild cases of mastitis where changes in viscosity or gel formation are subtle and highly userdependent. These tests rely heavily on subjective interpretation, making results vulnerable to operator variability, lighting conditions, and inconsistencies in mixing technique. Numerous studies in veterinary science have reported significant rates of false positives and false negatives for CMT, reducing its reliability for treatment decisions or herd-level monitoring. Furthermore, CMT and similar tests do not provide insights into the underlying cause of mastitis — such as the degree of inflammation, tissue damage, or the likelihood of bacterial involvement — thereby restricting their clinical utility.

[0010] In contrast, laboratory-based somatic cell count (SCC) analysis and microbiological culture methods offer superior diagnostic precision but suffer from inherent logistical and temporal constraints. SCC measurement typically requires transport of milk samples to centralized laboratories equipped with specialized analytical instruments, leading to delays often exceeding 24 hours. Microbiological culture requires incubation periods of one to two days before pathogens can be identified. These time frames are incompatible with the immediacy required for on-farm decision-making, resulting in delayed intervention, increased risk of spread within the herd, and a tendency toward empirical antibiotic use without pathogen-specific justification. Such delays undermine antimicrobial stewardship and compromise herd health outcomes.

[0011] Some dairy practitioners have attempted to repurpose commercially available urine dipsticks for mastitis screening. However, urine test strips are fundamentally unsuitable for application in milk due to the unique physicochemical characteristics of the milk matrix. Milk contains substantial quantities of fat globules, casein micelles, endogenous enzymes, and naturally opaque components, all of which interfere with reagent dissolution, diffusion, and color development. As a result, urine-derived dipsticks commonly exhibit poor sensitivity, inconsistent reaction times, and unpredictable color changes when used with milk. These limitations render them scientifically unreliable and unsuitable for standardized mastitis detection.

[0012] Instrument-based mastitis and SCC analysers — such as optical counters, fluorometric detectors, or electronic inline monitoring systems — have also been developed to improve rapid detection. While such instruments offer improved analytical capabilities, they are associated with high acquisition costs, ongoing maintenance requirements, reliance on electricity, and the need for technical training. Their complexity and expense limit adoption among small and medium dairy operations and make them impractical for field or pen-side use. Many of these devices focus narrowly on somatic cell count alone and provide no concurrent information on other physiological or microbial indicators relevant to mastitis, limiting their diagnostic scope.

[0013] Existing diagnostic approaches that target individual biomarkers — such as nitrite testing, pH monitoring, esterase assays, or hemoglobin detection — are available in various forms. However, mastitis is a multifactorial condition, and reliance on a single biomarker often yields incomplete or misleading information. Individual parameters may be influenced by factors unrelated to mastitis, such as stage of lactation, nutrition, environmental stressors, or sampling variability. Consequently, single-analyte tests lack robustness, provide limited diagnostic confidence, and often necessitate additional confirmatory tests, thereby reducing practicality for routine on-farm surveillance.

[0014] Taken together, these limitations highlight the absence of a diagnostic tool that is simultaneously rapid, accurate, field-deployable, cost-effective, and capable of providing a comprehensive assessment of mastitis status directly from raw milk. Dairy farmers require a solution that operates without instruments, functions within minutes, and integrates multiple physiologically relevant indicators into a single, reliable test. Such a tool would significantly enhance early detection, support timely and targeted treatment decisions, reduce unnecessary antibiotic use, and improve overall herd management.

[0015] Accordingly, there exists a clear unmet need for an improved cow-side mastitis diagnostic technology capable of overcoming the inherent shortcomings of current chemical tests, laboratory-based analyses, urine-derived strips, and instrumentdependent devices. A system that addresses these deficiencies would represent a substantial advancement in the field of veterinary diagnostics and dairy herd health management. The present invention addresses the challenges posed by the existing state of the art and describes an instrument-free multi-analyte diagnostic strip system for rapid detection of bovine mastitis in milk.

[0016] OBJECTS OF THE INVENTION

[0017] The principal object of the present invention is to provide a rapid diagnostic system for detecting bovine mastitis directly in milk without requiring laboratory infrastructure or analytical instruments.

[0018] Another object of the invention is to provide a diagnostic strip system capable of simultaneous detection of multiple mastitis-associated biomarkers from a single milk sample.

[0019] Another object of the invention is to provide an instrument-free diagnostic system that produces visually interpretable results within a short time suitable for immediate decision-making.

[0020] Another object of the invention is to provide a diagnostic strip employing stable dry reagent chemistries that are compatible with the physicochemical properties of milk and maintain analytical performance in fat- and protein-rich matrices.

[0021] Another object of the invention is to provide a diagnostic system that enables comprehensive mastitis assessment by detecting inflammatory, microbial, and tissue-damage indicators and by differentiating disease severity.

[0022] Another object of the invention is to provide a compact, portable, cost-effective diagnostic solution suitable for routine use, extended shelf life under ambient conditions, and standardized sample handling and result interpretation.

[0023] SUMMARY OF THE INVENTION

[0024] Embodiments of the present disclosure present technological improvements as a solution to one or more of the above-mentioned technical problems recognized by the inventor in existing techniques.

[0025] The present invention provides an instrument-free, multi-analyte diagnostic strip system for rapid detection of bovine mastitis directly in milk. The system is designed to operate on untreated milk samples and to generate visually interpretable results within a short time period, typically within a few minutes, without requiring laboratory infrastructure, analytical instruments, or sample pretreatment.

[0026] In one aspect, the invention comprises a laminated diagnostic strip including a backing layer, an absorbent pad layer, and a reagent deposit layer defining a plurality of spatially separated dry reagent zones. The reagent zones are configured to simultaneously detect multiple mastitis-associated biomarkers, including pH variation, leukocyte esterase activity, haemoglobin, and nitrite, thereby enabling a comprehensive assessment of inflammatory status, tissue damage, and bacterial metabolic activity from a single milk sample.

[0027] In another aspect, the diagnostic strip includes a calibrated sample application point for receiving a predefined volume of milk and a result window enabling simultaneous visual observation of reaction outcomes in all reagent zones. Milk- matrix-optimized dry chemistries allow rapid reconstitution and distinct colorimetric responses upon contact with milk while minimizing interference from fats and proteins.

[0028] In a further aspect, the invention includes a moisture-controlled packaging system comprising desiccants, reference color charts, and calibrated sample delivery tools to preserve reagent stability and support standardized testing and result interpretation. The combined strip and packaging system provides a rapid, reliable, portable, and cost-effective solution for mastitis detection.

[0029] The objects and the advantages of the invention are achieved by the process elaborated in the present disclosure.

[0030] BRIEF DESCRIPTION OF DIAGRAMS

[0031] Figure 1A illustrates a top plan view of a diagnostic strip according to an embodiment of the present invention, showing a pH indicator zone (101), a leukocyte esterase detection zone (102), a hemoglobin detection zone (103), a nitrite detection zone (104), a sample application point (108), and a result window area (109). Figure IB illustrates a cross-sectional view taken along line A-A' of FIG. 1A, showing a multilayer structure comprising a backing layer (105), an absorbent pad layer (106), and a reagent deposit layer (107).

[0032] Figure 1C illustrates an enlarged view of the reagent deposit layer (107), showing spatial separation of the reagent zones (101-104) and hydrophobic barriers therebetween.

[0033] Figure ID illustrates an enlarged detail view of the calibrated sample application point (108) configured to receive a predefined volume of milk.

[0034] Figure IE illustrates an enlarged view of the result window area (109) showing printed reference markings for visual interpretation of colorimetric responses.

[0035] Figure 2A illustrates a front elevation view of a packaging container for the diagnostic strip system, showing a product label area (201) and a viewing window (202).

[0036] Figure 2B illustrates a cross-sectional view taken along line B-B' of FIG. 2A, showing internal components including a moisture-resistant lid (203), a desiccant indicator (204), a desiccant sachet (205), and a strip storage compartment (206).

[0037] Figure 2C illustrates an exploded view of the packaging assembly illustrating relative arrangement of the lid (203), desiccant elements (204, 205), foam separator (207), storage compartment (206), and integrated reference color chart (208).

[0038] Figure 2D illustrates an enlarged view of a calibrated pipette (209) used for delivering a predefined volume of milk to the diagnostic strip.

[0039] Figure 2E illustrates different visual states of the desiccant indicator (204) corresponding to dry and saturated humidity conditions.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The following detailed description illustrates embodiments of the present disclosure and ways in which the disclosed embodiments can be implemented. Although some modes of carrying out the present disclosure have been disclosed, those skilled in the art would recognize that other embodiments for carrying out or practicing the present disclosure are also possible.

[0042] The present invention is illustrated with reference to the accompanying drawings, which conceptually represent various functional components and workflows of the system. Each figure provides a high-level architectural or process-level depiction to facilitate understanding of the invention’s modular structure, data flow, and operational logic.

[0043] As used herein, the articles “a,” “an,” and “the” are intended to mean “one or more” unless explicitly stated otherwise or clearly contradicted by the context. Similarly, the term “or” as used in this disclosure should be interpreted to mean an inclusive “or” (i.e., “and / or”), unless expressly indicated as exclusive or required by the context.

[0044] The present invention provides an instrument-free, multi-analyte diagnostic strip system for the rapid detection of bovine mastitis directly in raw milk, together with an integrated packaging system designed to preserve reagent stability and to enable standardized field operation. The system is configured to function on untreated milk samples and to generate visually interpretable results within a short time period, typically within about 60-90 seconds and in any case less than three minutes, thereby enabling timely diagnostic assessment without laboratory infrastructure, analytical instrumentation, or sample pretreatment. Rapid-reacting, milk-optimized dry chemistries are arranged across spatially separated reagent zones to provide a comprehensive multi-parameter assessment of mastitis, including pH changes, leukocyte activity, haemoglobin (blood) presence, and nitrite associated with bacterial metabolism.

[0045] The diagnostic strip system comprises a multilayer construction designed to ensure controlled sample transport, chemical stability, and reliable visual signal generation. Referring to Drawing 1, the strip includes a backing layer (105), an absorbent pad layer (106) disposed on the backing layer, and a reagent deposit layer (107) disposed above the absorbent pad layer. These layers are assembled in a laminated structure with sealed edges, forming a compact and robust diagnostic device suitable for handling in dairy environments. The layers cooperate to provide unidirectional sample flow, controlled contact time with each reagent zone, and clear visual access to color changes in a result window.

[0046] The backing layer (105) is preferably formed from a rigid polymeric material such as polyethylene terephthalate (PET). The PET backing has a thickness in the range of approximately 0.5-1 mm and provides mechanical strength to the diagnostic strip, enabling one-handed handling and preventing bending or deformation during use. In addition, the backing layer functions as a fluid-impermeable barrier, preventing reverse leakage of milk and ensuring that the liquid sample flows exclusively through the absorbent pad layer (106) toward the reagent deposit layer (107). This not only protects the user from contamination but also ensures consistent, unidirectional capillary flow.

[0047] Disposed above the backing layer (105) is the absorbent pad layer (106), which is formed of a cellulose-based or equivalent fibrous material having controlled porosity and capillary characteristics, preferably aWhatman-grade paper with GSM in the range of about 150-200. The absorbent pad layer is configured to receive a milk sample from a defined application point and to distribute it both laterally and longitudinally across the reagent deposit layer (107) at a predetermined and substantially uniform flow rate. In preferred embodiments, the absorbent pad layer controls milk migration at approximately 5-10 millimeters per minute, ensuring adequate contact time between the milk sample and each reagent zone while preventing oversaturation, pooling, or uneven wetting. This controlled capillary flow is critical for reproducible reaction kinetics and for maintaining the semi- quantitative character of the multi -analyte responses.

[0048] The reagent deposit layer (107) is positioned above the absorbent pad layer and constitutes the functional analytical interface of the diagnostic strip. The reagent deposit layer defines a plurality of spatially separated reagent zones (101-104), each containing chemically distinct dry reagent formulations tailored to detect a specific mastitis-associated biomarker. Hydrophobic barriers or physical spacing between the zones prevent cross-contamination and ensure that reactions occurring within one zone do not interfere with those in adjacent zones. The reagents are preferably deposited in freeze-dried or otherwise dried form to enhance shelf stability, reduce susceptibility to hydrolysis or oxidation, and allow rapid reconstitution upon contact with milk. Upon wetting, the dry chemistries instantly rehydrate, enabling the initiation of rapid colorimetric reactions without the need for mixing steps or additional reagents.

[0049] In a preferred embodiment, the reagent zones include a pH indicator zone (101), a leukocyte esterase detection zone (102), a haemoglobin detection zone (103), and a nitrite detection zone (104). These zones are arranged such that they are simultaneously exposed to the milk sample delivered through the absorbent pad layer (106), enabling concurrent multi -parameter analysis from a single sample. In the top plan view of the strip, the pH indicator zone (101) is positioned as the first reagent zone from the left, the leukocyte esterase zone (102) as the second, the haemoglobin zone (103) as the third, and the nitrite zone (104) as the fourth or rightmost zone, thereby providing a standardized spatial layout for interpretation.

[0050] The pH indicator zone (101) comprises a pH-sensitive dye system based on phenol red in a milk-protein-compatible buffer system. The phenol red is present at a concentration of approximately 0.2-0.5%. The zone is calibrated to detect pH in a range of about 6.5-8.0, covering the physiologically relevant and mastitis- associated pH spectrum of bovine milk. Healthy milk typically exhibits a pH in the range of approximately 6.6-6.8, whereas mastitis-affected milk becomes progressively more alkaline, commonly shifting toward about 7.0-7.5. The pH indicator zone (101) is formulated to generate rapid and visually distinct color transitions within this range, providing an immediate indication of udder health status and allowing differentiation between normal and mastitis-related pH shifts.

[0051] The leukocyte esterase detection zone (102) is configured to detect esterase activity associated with leukocytes present in milk. Elevated leukocyte levels are a hallmark of mastitis and correlate directly with somatic cell count. In contrast to conventional urine-based esterase tests, which are not optimized for the fat- and protein-rich milk matrix, the leukocyte esterase zone (102) employs a modified indoxyl ester substrate system specifically adapted for use in milk. The reagent composition incorporates one or more lipase inhibitors to suppress interference from milk lipids, which would otherwise degrade ester substrates, generate non-specific background coloration, or alter reaction kinetics. Upon enzymatic cleavage of the indoxyl ester substrate by leukocyte esterase released from neutrophils, a chromogenic cascade is initiated, producing a visually detectable color change proportional to leukocyte concentration. The zone is calibrated to respond to leukocyte levels corresponding to somatic cell counts associated with subclinical and clinical mastitis. In particular, the leukocyte esterase detection zone (102) is designed to detect leukocyte concentrations of at least about 200 cells / pL and to achieve a sensitivity of at least about 93% and a specificity of at least about 95% relative to somatic cell count benchmarks.

[0052] The haemoglobin detection zone (103) is configured to detect haemoglobin present in milk as a result of vascular or tissue damage within the udder, which is frequently associated with moderate to severe mastitis, especially due to gram-positive pathogens. This zone utilizes an enhanced peroxidase-based chromogenic system in which haemoglobin from lysed red blood cells acts as a catalytic agent for color development. The peroxidase substrate is present at about 2-3 times the standard concentration used in comparable colorimetric assays, thereby providing increased sensitivity to low levels of haemoglobin while compensating for the presence of milk proteins and endogenous enzymes that might otherwise quench or interfere with the reaction. The haemoglobin detection zone (103) is capable of detecting hemoglobin concentrations of at least about 20 pg / dL in milk and produces a distinctive color change that indicates the presence of blood and, by implication, tissue damage and vascular compromise associated with mastitis.

[0053] The nitrite detection zone (104) is configured to detect nitrite arising from bacterial metabolic activity in milk, particularly the metabolism of gram-negative bacteria such as Escherichia coli. Certain mastitis-causing bacteria reduce nitrates naturally present in milk or feed to nitrites, which can serve as indicators of infection. The nitrite detection zone (104) employs a modified Griess reagent system, including diazotization and coupling components such as sulfanilamide and N-(l- naphthyl)ethylenediamine, formulated in a buffer optimized for milk’s native pH range of approximately 6.5-7.5. The chemistry is designed to detect nitrite concentrations of at least about 0.5 mg / L in milk and to develop a rapid and characteristic color, typically in the magenta or pink range, without requiring prior bacterial culture or incubation. This zone therefore functions as a gram-negative pathogen marker, with particular relevance to E. coli mastitis.

[0054] A calibrated sample application point (108) is provided on the diagnostic strip to ensure controlled introduction of the milk sample. The sample application point is defined as a circular area on the strip surface having a diameter of approximately 12-15 mm and is dimensioned and positioned to receive a predefined volume of milk, preferably about 0.5 mb ± 0.05 mb. This volumetric calibration ensures consistent test performance, prevents over-saturation or under-dosing of the reagent zones, and reduces variability arising from user technique. The application point (108) is located in an upper region of the strip above the reagent zones, such that the milk is drawn downward or laterally into the absorbent pad layer (106) and from there distributed uniformly toward the reagent deposit layer (107).

[0055] As the milk sample migrates from the sample application point (108) through the absorbent pad layer (106), it simultaneously contacts each of the reagent zones (101-104) on the reagent deposit layer (107). The dry reagents rapidly reconstitute upon contact with the liquid sample, initiating their respective chemical reactions essentially in parallel. Within less than three minutes, and typically within about 60-90 seconds, each reagent zone develops a visually distinguishable color response. The resulting multi -zone color pattern provides a composite diagnostic profile of mastitis status, integrating pH deviation, leukocyte-mediated inflammation, hemoglobin-associated tissue damage, and nitrite-linked bacterial activity.

[0056] The diagnostic strip further includes a result window area (109) positioned above or adjacent to the reagent zones (101-104) to permit simultaneous visual observation of all reaction outcomes. The result window (109) is preferably a clear viewing area of approximately 50 mm x 40 mm and may include printed reference gridlines, calibration marks, and other visual aids to standardize interpretation. These markings facilitate comparison of the color development in each reagent zone against expected standards and can be used in conjunction with an external reference color chart for semi -quantitative grading of mastitis severity and probable pathogen class.

[0057] To preserve reagent stability and extend shelf life, the strip perimeter is provided with protective edge sealing (110). The edge sealing (110) is formed by heat-sealed polyester or other suitable plastic borders, which create an airtight barrier around the laminated strip assembly. This sealing prevents or substantially reduces the ingress of moisture and oxygen, and protects the interior layers from dust and contaminants. As a result, the functional reagents in the zones (101-104) remain stable and active for prolonged periods, with shelf life preferably extended to approximately 18-24 months under typical ambient storage temperatures (about 25-30°C). The perimeter sealing also maintains the structural integrity of the strip during handling and transport.

[0058] The overall assembly of the diagnostic strip thus integrates multiple biomarker detection systems into a single, field-deployable device for rapid mastitis diagnosis. In operation, the use sequence begins when a measured 0.5 mL milk sample is applied to the sample application point (108). The absorbent pad layer (106) immediately wicks the sample and distributes it uniformly across the reagent deposit layer (107) at a controlled capillary flow rate. As the milk migrates through the strip, it sequentially and concurrently contacts the four reagent zones (101-104), each designed to detect a specific mastitis biomarker. The freeze-dried reagents reconstitute upon contact with milk, initiating colorimetric reactions that develop within 60-90 seconds and become stable within approximately three minutes. The backing layer (105) provides structural support and prevents reverse leakage, while the protective edge sealing (110) ensures that reagent activity is maintained throughout the product’s storage life. The multi -parameter approach provides differential diagnostic capabilities: the pH indicator zone (101) confirms mastitis-associated pH elevation; the leukocyte esterase zone (102) quantifies immune response via leukocyte activity; the hemoglobin zone (103) identifies tissue damage and bleeding associated particularly with gram-positive infections such as .S' aureus', and the nitrite zone (104) detects gram-negative bacterial contamination such as E. coli. This comprehensive analysis enables farmers, veterinarians, or trained operators to make informed decisions about treatment protocols, animal management, and antimicrobial usage, with greater confidence than single-parameter or purely subjective methods.

[0059] In some embodiments, the diagnostic strip system is provided as part of a diagnostic kit in combination with a dedicated packaging and storage assembly as illustrated in Drawing 2. The packaging system is designed to maintain the functional performance of the strips from manufacturing through end-use by controlling humidity, preventing contamination, and organizing accessories needed for standardized testing.

[0060] The exterior of the packaging system includes a product label or identification area (201) and a viewing window (202). The product label (201) is a moisture-resistant printed label on the container exterior, providing essential product information such as product name, batch number, lot identification, manufacturing date, expiry date, and basic usage instructions. The viewing window (202) is formed of clear plastic on the container front panel and allows visualization of the integrated reference color chart (208) and, if desired, strip color development when a used strip is presented against the window, all without opening the container. This arrangement minimizes moisture exposure to unused strips and thereby extends their shelflife.

[0061] The container is closed by a moisture-resistant lid (203), which is formed of a polymer material and includes a silicone gasket seal. The lid (203) utilizes a twistcap or snap-lock mechanism to create an airtight closure that prevents moisture ingress and maintains a low-humidity environment within the container. Attached to the interior surface of the lid (203) is a desiccant indicator pellet (204), which is typically a color-changing silica gel indicator. The indicator pellet (204) changes color from blue in the dry, active state (relative humidity below about 10%) to pink in the saturated state (relative humidity above about 40%), thereby providing realtime visual feedback on the effectiveness and remaining capacity of the desiccant system.

[0062] Within the upper interior of the container, below the lid (203), is an integrated desiccant sachet (205) containing silica gel beads. The sachet (205), typically in the range of 3-5 grams per container, actively removes moisture from the air inside the container, maintaining relative humidity below approximately 10%. This low- humidity environment is essential for preserving the freeze-dried reagents in the diagnostic strips (101-104), preventing premature reconstitution, hydrolysis, or degradation, and thereby ensuring a shelf stability of approximately 18-24 months at room temperature conditions.

[0063] The main interior volume of the container defines a strip storage compartment

[0064] (206), which may include molded vertical slots or a foam holder. The storage compartment (206) supports and organizes 25-50 diagnostic strips in individual compartments, preventing physical contact between strips that could cause mechanical damage or reagent cross-contamination. The vertical orientation facilitates easy removal of a single strip at a time without disturbing the remaining inventory, further minimizing humidity exposure to strips that remain in storage.

[0065] A protective foam separator (207) is positioned as a horizontal divider between the desiccant sachet (205) and the strip storage compartment (206). The foam separator

[0066] (207) serves as a physical barrier preventing direct contact between the desiccant sachet and the stored diagnostic strips, thereby avoiding localized over-drying or accidental absorption of desiccant particles onto the reagent zones. The foam also provides cushioning to protect strips from shock or vibration during transport.

[0067] The packaging system further includes an integrated reference color chart (208), preferably in laminated form. The color chart (208) displays standardized color blocks or gradients corresponding to expected color development patterns for various mastitis states, including negative, subclinical, and clinical mastitis, as well as paterns indicative of gram-positive and gram-negative infections. The chart (208) may be attached to the interior surface of the lid (203) and / or arranged so that it is visible through the viewing window (202). Users can hold a developed strip against the viewing window and directly compare the observed colors in the reagent zones (101-104) with the reference color chart (208), thereby enabling consistent and semi-quantitative result interpretation across different users and lighting conditions.

[0068] The kit further includes one or more calibrated pipetes (209), stored within the container alongside the diagnostic strips. Each pipete (209) is a sterile disposable plastic device with graduated volume markings, a bulb actuator, and a tapered tip. The pipetes are configured to deliver a precise volume of 0.5 m ± 0.05 m of milk, compatible with the viscosity of milk and aligned with the capacity of the sample application point (108). By using the pipete (209) supplied with the kit, users ensure consistent sample volume and reliable test performance, while the disposable nature of the pipetes prevents cross-contamination between animals.

[0069] The complete packaging assembly thus provides a field-ready, integrated diagnostic solution that maintains reagent integrity from manufacturing through end -use. The moisture control system comprising the lid (203), desiccant indicator (204), and desiccant sachet (205) creates and maintains a low-humidity internal environment essential for preserving the functional performance of the dry reagent zones (101— 104). The storage compartment (206) and foam separator (207) organize and protect the strips. The viewing window (202) and reference color chart (208) allow users to interpret test results without opening the container unnecessarily, thereby reducing humidity infiltration. The calibrated pipetes (209) supply the correct sample volume for each test, ensuring consistency and accuracy.

[0070] In practical use, the user opens the container, retrieves a single diagnostic strip from the storage compartment (206), and obtains a calibrated pipete (209). A raw milk sample is drawn into the pipete and 0.5 mb is dispensed onto the circular sample application point (108) of the strip. The strip is then placed on a flat surface or held in the hand while the absorbent pad layer (106) distributes the sample across the reagent deposit layer (107). Within about 60-90 seconds, color development occurs in the pH indicator zone (101), leukocyte esterase zone (102), hemoglobin zone (103), and nitrite zone (104). The strip is viewed through or against the result window area (109), and the observed color pattern is compared with the reference color chart (208), typically visible through the viewing window (202) of the container. This workflow allows the user to complete a comprehensive mastitis diagnostic assessment in under three minutes, without the need for additional reagents, instruments, or laboratory analysis.

[0071] The integrated design of the diagnostic strip and packaging system ensures that the strips maintain their performance specifications across their intended shelf life, while providing users with all necessary components for accurate, rapid mastitis diagnosis in a variety of dairy environments. The combination of milk-matrix- optimized chemistries, freeze-dried reagent stability, controlled capillary transport, multi -analyte detection, and robust environmental protection delivers a rapid, reliable, and comprehensive diagnostic tool for bovine mastitis. The system produces actionable diagnostic results without the need for specialized expertise, and while specific embodiments and materials have been described herein, it will be understood that variations and modifications in materials, dimensions, reagent compositions, and packaging configuration may be made by those skilled in the art without departing from the scope of the invention as defined in the appended claims .

Claims

CLAIMS:We Claim:

1. An instrument-free multi-analyte diagnostic strip system for rapid detection of bovine mastitis in milk, the said system comprising: a) a backing layer (105) configured to provide structural rigidity and to prevent reverse fluid migration; b) an absorbent pad layer (106) disposed on the backing layer and configured to receive and distribute a milk sample by capillary action at a controlled flow rate; c) a reagent deposit layer (107) disposed on the absorbent pad layer and defining a spatially separated dry reagent zone, each isolated from adjacent zones, the reagent zones comprising:- a pH indicator zone (101) configured to provide a visually detectable response to pH variations in milk;- a leukocyte esterase detection zone (102) configured to detect esterase activity associated with leukocytes present in milk;- a haemoglobin detection zone (103) configured to detect haemoglobin indicative of blood contamination and gram -positive pathogens in milk; and- a nitrite detection zone ( 104) configured to detect nitrite associated with gram-negative bacterial metabolic activity in milk; d) a calibrated sample application point (108) configured to receive a predefined volume of untreated milk and fluidically communicate the milk sample to the absorbent pad layer; and e) a result window area (109) positioned to permit simultaneous visual observation of reaction outcomes in all of the reagent zones, characterized in that the leukocyte esterase detection zone (102) consists of a modified indoxyl ester substrate system combined with one or more lipase inhibitors to suppress interference from milk lipids, the nitrite detection zone (104) consists of a Griess-based reagent chemistry buffered to operate withinthe native pH range of milk, and the reagent zones (101-104) are compositionally calibrated to generate mutually distinguishable colorimetric responses within less than three minutes upon contact with untreated milk, without use of analytical instruments.

2. The diagnostic strip system as claimed in Claim 1, wherein the leukocyte esterase detection zone (102) is responsive to leukocyte concentrations corresponding to somatic cell count ranges associated with subclinical and clinical mastitis.

3. The diagnostic strip system as claimed in Claim 1, wherein the pH indicator zone (101) is calibrated to detect pH variations in milk within a range of approximately 6.6 to 7.5.

4. The diagnostic strip system as claimed in Claim 1, wherein the haemoglobin detection zone (103) includes a peroxidase-based chromogenic system configured to detect haemoglobin concentrations associated with vascular or tissue damage in mastitis-affected udders.

5. The diagnostic strip system as claimed in Claim 1, wherein the reagent deposit layer (107) includes dry, preferably freeze-dried, reagent formulations that rapidly reconstitute upon contact with milk.

6. The diagnostic strip system as claimed in Claim 1, wherein the absorbent pad layer (106) is configured to control milk migration at a capillary flow rate of approximately 5 to 10 millimetres per minute.

7. The diagnostic strip system as claimed in claim 1, wherein protective edge sealing (110) is provided around the perimeter of the strip to form a moisture barrier and enhance shelf stability.

8. The diagnostic strip system as claimed in Claim 1, wherein the result window area (109) includes printed reference markings adapted to cooperate with a corresponding colour reference chart (208) for standardized interpretation of test results.

9. The diagnostic strip system as claimed in Claim 1, wherein the predefined volume of milk received at the sample application point (108) is approximately 0.5 millilitres.

10. The diagnostic strip system as claimed in Claim 1, wherein the reagent zones (101-104) are configured to enable differentiation of mastitis severity based on combined biochemical response patterns.

11. The diagnostic strip system as claimed in Claim 1, in combination with a storage container comprising a moisture-resistant lid (203), at least one desiccant (205), a desiccant indicator (204), a strip storage compartment (206), and a calibrated pipette (209).

Images