Description:FORM-2

THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patent Rules, 2003

COMPLETE SPECIFICATION
(See Section 10 and Rule 13)

Title:
POINT-OF-CARE DIAGNOSTIC KIT FOR RAPID DIFFERENTIATION OF BACTERIAL AND NON-BACTERIAL DIARRHEA

Applicant Name Nationality Address
BABYCUE PRIVATE LIMITED INDIAN C/o.: ASHA LATA ROUT, RAJENDRANAGARA, MADHUPATNA, CUTTACK, ODISHA, 753010, INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

FIELD OF INVENTION
The present invention relates to the field of healthcare diagnostics, specifically to point-of-care diagnostic devices for gastrointestinal diseases. More particularly, it pertains to a lateral flow immunoassay-based kit designed for the rapid, semi-quantitative detection of fecal calprotectin (f-CP) in stool samples to differentiate between invasive bacterial and non-bacterial causes of diarrhea, especially in children.

BACKGROUND OF THE INVENTION
Acute diarrheal diseases remain a leading cause of morbidity and mortality among children under five, particularly in low- and middle-income countries like India. According to the National Family Health Survey-4, approximately 13% of deaths in children under five are attributable to diarrheal diseases. Studies indicate that although preventive strategies such as vaccination and improvements in sanitation have reduced the disease burden, diarrhea prevalence among children in India rose from 9% in 2016 to 9.2% in 2020 (as discussed in Ghosh, K. Et al., Prevalence of Diarrhea among under Five Children in India and Its Contextual Determinants: A Geo-Spatial Analysis. Clin. Epidemiology. Glob. Health 2021).
Lakshminarayanan, S. Et al., Diarrheal Diseases among Children in India: Current Scenario and Future Perspectives. J. Nat. Sci. Biol. Med. 2015, discusses that most cases of acute diarrhea in children are viral (about 70%), while bacterial and parasitic causes constitute 10–20% and <10%, respectively. Inappropriate antibiotic prescriptions for viral infections remain a serious issue, especially in private healthcare settings where empirical antibiotic use is more frequent (discussed in Bruzzese, E. Et al., Antibiotic Treatment of Acute Gastroenteritis in Children. F1000Research 2018). Accurate and early differentiation between bacterial and non-bacterial diarrhea is thus critical to guide clinical decisions and mitigate the global concern of antibiotic overuse.
Although laboratory-based techniques like PCR, enzyme immunoassays (EIA), and stool cultures provide precise pathogen detection, they are often time-consuming, costly, and infrastructure-intensive—limiting their use in routine or point-of-care (POC) diagnostics (Pathirana WGW Et al., Faecal Calprotectin. Clin Biochem Rev. 2018 Aug). Among available biomarkers, fecal calprotectin (f-CP)—a calcium and zinc-binding protein released during neutrophilic inflammation—has emerged as a reliable, non-invasive indicator of mucosal inflammation. Elevated f-CP levels are typically observed in bacterial enteric infections, whereas viral infections generally produce minimal neutrophil response and consequently low f-CP concentrations. This makes calprotectin a promising tool for differentiating bacterial from viral causes of diarrhea.
Existing commercial tests for calprotectin, including ELISA and lateral flow assays (LFA), are largely tailored to diagnose inflammatory bowel diseases (IBD) such as Crohn’s disease or ulcerative colitis. However, these kits are not widely available in India and are often cost-prohibitive. Moreover, most available products either detect lactoferrin or calprotectin, with few addressing both. The limited diagnostic range of these tests often fails to accurately detect invasive bacterial diarrhea in pediatric populations, especially in low-resource settings.
To address this unmet need, the inventors propose a novel lateral flow immunoassay-based diagnostic kit specifically designed to detect elevated levels of fecal calprotectin in children under five, enabling rapid and accurate identification of invasive bacterial diarrhea.

OBJECT OF THE INVENTION
An object of the invention is to provide a point-of-care diagnostic kit that enables rapid, reliable, and affordable detection of fecal calprotectin levels in stool samples to differentiate between bacterial and non-bacterial causes of diarrhea.
Another object of the invention is to develop a lateral flow immunoassay (LFA)-based device that can visually distinguish calprotectin concentration through a three-line signal format, allowing for semi-quantitative interpretation without the need for specialized instrumentation.
Yet another object of the invention is to facilitate timely and evidence-based decision-making in paediatric diarrhea cases by enabling clinicians to identify bacterial infections that warrant antibiotic treatment and avoid unnecessary antibiotic use in viral cases.
A further object of the invention is to offer a diagnostic solution that is simple to use, scalable, and operable by frontline healthcare workers with minimal training, making it highly suitable for low-resource or rural healthcare settings.
Still another object of the invention is to provide a method for fabricating an optimized LFA strip that includes bio-conjugated monoclonal and polyclonal antibodies, optimized buffers, and gold nanoparticle conjugation protocols to ensure improved sensitivity, specificity, and shelf stability.
A further object of the invention is to address the limitations of existing commercial diagnostic kits, particularly those designed for IBD detection, by creating a product specifically tailored to detect elevated calprotectin levels associated with invasive bacterial diarrhea in children under five.

SUMMARY OF THE INVENTION
The invention provides a novel diagnostic solution in the form of a lateral flow immunoassay (LFA) kit, referred to as the “Diacue LFA Kit”, for the detection of fecal calprotectin, a biomarker of intestinal inflammation. The kit enables healthcare providers to rapidly and accurately distinguish between bacterial and non-bacterial diarrheal infections using stool samples at the point of care. The kit is designed to address the need for rapid, reliable, cost-effective diagnostics in low-resource settings, particularly in paediatric populations vulnerable to diarrheal diseases.
In one embodiment, the invention comprises a lateral flow device with a sample pad, conjugate pad preloaded with gold nanoparticle-conjugated monoclonal antibodies specific to calprotectin, a nitrocellulose membrane with immobilized polyclonal capture antibodies forming the test lines, and an absorbent pad to sustain capillary flow. The device uses a three-line result format to visually indicate calprotectin concentration levels: (i) three lines indicate bacterial etiology, (ii) two lines indicate viral or non-bacterial causes, and (iii) one control line indicates a negative result. The test delivers results within minutes, requires no specialized equipment, and can be operated by minimally trained personnel.
In another embodiment, the invention further provides optimized bioconjugate preparation using NXT-activated gold nanoparticles, specific centrifugal protocols, and antibody concentrations. The kit's assembly includes buffer-optimized sample and conjugate pads to enhance performance. A semi-quantitative visual intensity scale assists clinicians in decision-making regarding antibiotic therapy.
The invention also encompasses methods of using the kit for diagnostic purposes. The method involves applying a prepared stool sample to the sample pad, allowing migration through the strip, and interpreting the visual output to determine whether antibiotic intervention is warranted.
Additionally, the invention covers methods of fabricating the kit, including stepwise assembly of LFA components, antibody application, and controlled drying processes to ensure consistent quality and performance.
The kit utilizes monoclonal and polyclonal anti-calprotectin antibodies, conjugated with gold nanoparticles to ensure enhanced sensitivity and specificity. This cost-effective, point-of-care solution enables clinicians to make evidence-based decisions, promoting rational antibiotic use. The invention is particularly suited for deployment in resource-limited or rural healthcare settings where traditional diagnostic facilities are unavailable.

BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes references to the annexed drawings wherein:
Figure. 1 (a): Working principle of LFA (Lateral flow immunoassay device).
Figure. 1 (b): Internal Diagram of LFA test strip
Figure. 2: Diacue LFA test kit Package (200):
- Fig. 2(a) Lateral Flow Assay (LFA) test strip (100).
- Fig. 2(b) Pictorial image of Spatula (201).
- Fig. 2(c) Pictorial image of Dropper (202).
- Fig. 2(d) Extraction buffer vial (203).
- Fig. 2(e) Zip-lock pouch (204).
- Fig. 2(f) Information brochure (205).
- Figure 2(g) Final kit package cover (206).
Figure. 3: Flow chart for schematic representation of fabrication of Lateral Flow assay strips.
Figure. 4: Stepwise representation of the procedure for conduction of detection test.
Figure. 5: Optimization of Conjugate pad buffer concentration in the LFA strip 5(a) & 5(b).
Figure. 6: Optimization of Sample pad buffer concentration in the LFA strip 6(a) & 6(b).
Figure. 7: Optimization of monoclonal antibody concentration at the test line of the LFA strip.
Figure. 8: Test line Optimization in varying concentrations of polyclonal calprotectin antibody.
Figure. 9: Optimization of biomarker (calprotectin) concentration at the test line with synthetic stool spiked sample.
Figure. 10: Optimization Of NXT Activated Bioconjugates for Centrifugal Speed at varying rpm and time.
Figure. 11: Optimization Of optical density (OD) of gold nanoparticles (A) at OD1 (B) at OD 8 (C) at OD 16.
Figure. 12: Characteristic UV–Vis spectra of AuNPs observed at 520 nm with a redshift of 3 nm, for NXT activated‐AuNPs with antibody.
Figure. 13: Hydrodynamic diameter of the synthesized AuNPs before and after conjugation with Ab.
Figure. 14: Zeta potential shifted from -26.08 mV (AuNPs) to -15.65 mV (Bio-conjugation).

DETAILED DESCRIPTION OF THE INVENTION
The following detailed description is merely exemplary in nature and is not intended to limit the described embodiments or the application and uses of the described embodiments. This description is not intended to be a detailed catalogue of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the scope of the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.
The terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.
As used herein, the term “about” is meant to account for variations due to any experimental errors which may be commonly accepted in the field for a numeric value, for example such a variation can be considered as a ±10% of the said numeric value. All measurements reported herein are understood to be modified by the term “about,” whether or not the term is explicitly used, unless explicitly stated otherwise.
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
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 belongs. Methods and materials are described herein for use in the present disclosure; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting by any means. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict, the present specification, including definitions, will control.
Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.
The term “including” is used to mean “including but not limited to”, “including” and “including but not limited to” are used interchangeably.
As used herein the terms “device” or “kit” are used interchangeably to refer to the Diacue Lateral Flow Immunoassay (LFA) test strip (100), for the detection of fecal calprotectin (f-CP), a biomarker associated with intestinal inflammation for the simultaneous detection of mosquito-borne diseases according to the present invention. The kit according to the present invention is presented in a complete package (200) with various components for effective usage of the LFA kit (100) according to the present invention.
The term “gold nanoparticles” as used throughout the specification can also be referred to as AuNps or GNPs.
There is a critical unmet need for a paediatric-friendly, affordable, and accurate point-of-care diagnostic tool to differentiate between bacterial and Non-bacterial diarrhea, especially in countries like India where diarrhea remains a major contributor to under-five mortality. Current diagnostic methods, such as PCR, enzyme immunoassays, or culture, are time-consuming, expensive, and dependent on laboratory infrastructure—making them impractical for routine use in rural and resource-constrained settings. Existing LFA kits are primarily designed for inflammatory bowel disease (IBD) detection and lack appropriate cut-off values or sensitivity for invasive bacterial diarrhea, particularly in children.
The present invention aims to fill this gap by providing a dedicated calprotectin-based LFA kit that is optimized for detecting inflammatory responses specific to bacterial diarrhea. The kit facilitates rapid differentiation at the point of care, promoting rational antibiotic use and helping to combat the growing threat of antimicrobial resistance. Its affordability, simplicity, and scalability make it a valuable diagnostic tool for improving childhood diarrhea management and health outcomes globally.
In one key embodiment the present invention relates to a diagnostic kit, namely the Diacue Lateral Flow Immunoassay (LFA) Kit, for the detection of fecal calprotectin (f-CP), a biomarker associated with intestinal inflammation. The kit provides a rapid, non-invasive solution for identifying elevated calprotectin levels in stool samples, which are indicative of gastrointestinal inflammatory conditions.
In another embodiment, the invention is directed towards a lateral flow immunoassay (LFA)-based diagnostic system capable of distinguishing between bacterial and non-bacterial causes of diarrhea based on f-CP levels.
In one aspect the diagnostic kit according to the above embodiment is adaptable for point-of-care use, particularly in paediatric and low-resource healthcare settings, without the need for specialized instrumentation or expertise.
In yet another embodiment, the invention provides specific design features and optimized configurations—such as antibody selection, bioconjugation techniques using gold nanoparticles, and calibration of test line intensities—to improve analytical sensitivity and diagnostic accuracy. Additionally, optimized manufacturing steps ensure robust kit performance, cost-effectiveness, and scalability for widespread implementation.
In one preferred embodiment the present invention discloses a diagnostic kit which is specifically designed to detect elevated levels of fecal calprotectin (f-CP), a biomarker of intestinal inflammation indicative of invasive bacterial diarrhea. The invention comprises a general framework of the diagnostic system and that provide specific implementations to enhance sensitivity, specificity, usability, and manufacturing robustness.
In one embodiment, the invention further provides optimized bioconjugate preparation using NXT-activated gold nanoparticles, specific centrifugal protocols (3000 rpm for 5 min), and precise antibody concentrations (5 µg/mL monoclonal for conjugation, 0.5 mg/mL polyclonal at test line). The kit's assembly includes buffer-optimized sample and conjugate pads to enhance performance. A semi-quantitative visual intensity scale assists clinicians in decision-making regarding antibiotic therapy.
In one embodiment, the invention provides a lateral flow immunoassay (LFA)-based diagnostic kit for detecting calprotectin in stool samples. The kit comprises:
A sample pad for receiving the stool sample;
A conjugate pad containing gold nanoparticle (AuNP)-conjugated monoclonal antibodies specific to calprotectin;
A nitrocellulose membrane comprising test line and control line, wherein the test line contains immobilized polyclonal anti-calprotectin antibodies;
An absorbent pad to facilitate capillary flow.
In one aspect according to the above embodiment the said kit is designed to visually indicate calprotectin concentration based on the number and intensity of red lines:
Three red lines (two test, one control) indicate high calprotectin levels typical of bacterial diarrhea.
Two red lines (one test, one control) suggest moderate levels, often associated with viral or non-inflammatory diarrhea.
One control line alone indicates absence or very low calprotectin levels.
In yet another aspect according to the above embodiment the said kit utilizes monoclonal and polyclonal antibodies binding to distinct epitopes of calprotectin for a sandwich assay format, improving signal fidelity.
In some embodiments according to the present invention the disclosure provides a method for differentiating between bacterial and non-bacterial causes of diarrhea using the kit. The said method comprises:
a) Collecting a stool sample from a paediatric subject under 5 years of age;
b) Mixing the sample with the extraction buffer;
c) Applying the sample to the sample pad;
d) Allowing the sample to migrate across the conjugate and test regions;
e) Observing the number and intensity of red lines for diagnostic interpretation.
In one aspect according to the above embodiment the said method provides a visual semi-quantitative readout correlating with f-CP levels, enabling frontline healthcare providers to make decisions without laboratory infrastructure.
In another aspect according to the above embodiment the method assists in guiding antibiotic therapy by confirming bacterial etiology only in cases of elevated calprotectin.
Some embodiments according to the present invention disclose a method for fabricating a lateral flow assay kit for detecting calprotectin, wherein the kit comprises of:
- Mounting nitrocellulose membranes on a backing card;
- Dispensing test and control line antibodies using reagent dispensing systems;
- Drying, assembling and integrating sample, conjugate, and absorbent pads in a layered structure;
- Cutting the strip into individual strips and housing strips in plastic cassettes.
In one aspect according to the above method the said method comprises a step for pretreatment of the conjugate pad with a buffer comprising 5% sucrose, 1% BSA, and 0.5% Tween-20 in 1x PBS improves bioconjugate release.
In one aspect according to the above method the said method comprises a step for pretreatment of the sample pad with 1% BSA and 0.05% Tween-20 in PBS enhances sample flow, reduces background noise, and improves visibility.
In one embodiment according to the present disclosure the invention also provides a process for preparing optimized bioconjugates for the LFA comprising:
• Activating gold nanoparticles (GNPs) using NXT chemistry;
• Conjugating monoclonal anti-calprotectin antibodies with activated GNPs;
• Centrifuging at optimal conditions to isolate stable bioconjugates (3000 rpm for 5 min);
• Characterizing using UV spectroscopy, particle size analysis, and zeta potential.
In some aspect according to the above embodiment of present invention the optimal GNP optical density (OD) is determined to be 8 for ensuring both stability and intense visual signal.
In some aspect according to the above embodiment of present invention the optimal antibody concentrations include 5 µg/mL monoclonal antibodies for GNP conjugation and 0.5 mg/mL polyclonal antibodies for test line immobilization.
In one embodiment according to the present invention the disclosure provides a method for diagnosing invasive bacterial diarrhea comprising:
a) Applying a stool sample to the Diacue LFA Kit;
b) Detecting calprotectin concentration through line intensity;
c) Interpreting three lines as indicative of bacterial origin;
d) Guiding antibiotic administration accordingly.
In one aspect according to the above embodiment this method is particularly applicable in rural or resource-limited clinical settings.
In yet another aspect according to the above embodiment the method can reduce empirical antibiotic use and help mitigate antimicrobial resistance by confirming bacterial inflammation prior to prescription.
Some of the key advantages of the Present Invention can be listed as:
- Point-of-care applicability;
- No need for specialized instrumentation or trained personnel;
- High specificity and sensitivity through optimized antibody pairing;
- Rapid detection (~minutes);
- Affordable and scalable for widespread paediatric deployment.

EXAMPLES
The following examples include only exemplary embodiments to illustrate the practice of this disclosure. It will be evident to those skilled in the art that the disclosure is not limited to the details of the following illustrative examples and that the present disclosure may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive.

Example 1: Fabrication of the Lateral Flow Assay diagnostic test Strip kit:
The diagnostic kit (100) of the present invention comprises several integrated components systematically assembled to form a functional lateral flow assay (LFA) strip as in Figure 1(b). The components of the kit (100) include a sample pad (101), a sample application area (102), a conjugate pad (103), a nitrocellulose membrane (104), two test lines (T1 and T2), a control line (C), and an absorbent pad (105). Figure 3 – depicts a schematic representation of fabrication of Lateral Flow Assay (LFA) strips.
The fabrication process begins with the mounting of the nitrocellulose membrane (104) onto a backing card that serves as structural support. The membrane is activated by incubation at 37°C for 1 hour to ensure optimal surface preparation. Following activation, specific reagents including capture antibodies are applied to form the first test line (T1) and second test line (T2), as well as the control line (C) using a reagent dispensing system. This allows precise placement and ensures consistent antibody immobilization. The membrane is then dried again at 37°C to secure the applied antibodies.
On one end of the nitrocellulose membrane (104), an absorbent pad (105) is affixed. This pad is essential for maintaining capillary flow and acts as a sink for the migrated sample and reagents. Simultaneously, a conjugate pad (103) is prepared by first treating it with a conjugate pad buffer and drying it in a vacuum desiccator overnight. Monoclonal antibodies conjugated with gold nanoparticles (GNPs or AuNPs) are then sprayed onto the conjugate pad using a reagent dispenser. The coated conjugate pad is subsequently dried again in a vacuum desiccator to preserve the integrity of the bioconjugates.
The sample pad (101) is pretreated with a sample pad buffer and dried at 60°C to improve the wicking properties and sample homogenization. This sample pad (101) is affixed to one end of the strip and overlaps the conjugate pad (103), which in turn overlaps the nitrocellulose membrane (104). On the opposite end, the absorbent pad (105) is positioned to overlap the distal end of the membrane.
After the complete assembly of all functional pads — sample (101), conjugate (103), nitrocellulose membrane (104), and absorbent (105) — the entire structure is precisely cut into individual strips of approximately 4 mm width using an automated guillotine cutter. Each strip is then inserted into the plastic cassette (111) for user-friendly handling and operation.
Throughout the fabrication process, environmental moisture is controlled using a dehumidifier, and the final assembled kits (100) are stored in a vacuum desiccator to maintain stability and performance. Flow chart for schematic representation of fabrication of Lateral Flow assay strips is depicted in Figure-3.

Example 2: Optimization of Bioconjugate Parameters for Kit Performance
To ensure the sensitivity, reproducibility, and visual clarity of the lateral flow assay (LFA) strip, several experiments were conducted to optimize the properties of gold nanoparticle (GNP)–antibody bioconjugates. Below described experimental evaluations related to centrifugal processing speed, gold nanoparticle concentration (optical density), and characterization of antibody-nanoparticle conjugation.

Example 2A: Optimization of Centrifugal Speed for NXT-Activated Bioconjugate Recovery:
Experiments were done to identify the optimal centrifugation speed and duration for recovering NXT-activated gold nanoparticle (GNP)–antibody bioconjugates while preserving their stability. Bioconjugates were subjected to centrifugation under various conditions:
• 5000 rpm for 10 minutes (Figure 10A),
• 7000 rpm for 10 and 20 minutes (Figures 10B, 10C, 10D),
• 2000 rpm for 5 minutes (Figure 10F), and
• 3000 rpm for 5 minutes (Figure 10E).
Centrifugation at higher speeds and longer durations (5000–7000 rpm) led to visible loss of red color in the supernatant, indicating aggregation or breakdown of the bioconjugates. Conversely, centrifugation at 2000 rpm failed to yield a visible pellet, suggesting inadequate separation. The optimal condition was observed at 3000 rpm for 5 minutes, resulting in a distinct red-coloured pellet of gold nanoparticle–antibody bioconjugates, with minimal color remaining in the supernatant due to unbound gold nanoparticles.. This setting was found to be the most effective for preserving the integrity and yield of stable bioconjugates.

Example 2B: Optimization of Optical Density (OD) of Gold Nanoparticles for Conjugation:
Gold nanoparticles (GNPs) of varying optical densities (OD) were evaluated for their suitability in forming stable and functional antibody conjugates.
Three OD values were tested:
• OD 1 (Figure 11A),
• OD 8 (Figure 11C), and
• OD 16 (Figure 11B).
At OD 1, the concentration of gold nanoparticles was insufficient to support stable bioconjugation. Nanoparticles adhered to the tube walls, and the conjugate pad lacked visible coloration, indicating low loading efficiency. At OD 16, the high concentration caused over-deposition on the conjugate pad, leading to blockage and disrupted flow.
The optimal result was achieved at OD 8, where nanoparticles were well dispersed, the conjugate pad exhibited a bright red color, and the bioconjugate layer remained stable. This setting allowed for uniform coating and consistent visual signal development on the LFA strip.

Example 2C: Characterization of Monoclonal Antibody-GNP Bioconjugates
The successful conjugation of monoclonal antibodies with NXT-activated gold nanoparticles was confirmed through multiple characterization techniques:
1. UV-Visible Spectroscopy (Figure 12):
- Unconjugated GNPs showed a peak at 520 nm.
- After antibody conjugation, a red shift to 523 nm was observed, indicating successful surface interaction and bioconjugate formation.
2. Dynamic Light Scattering (DLS) / Particle Size Analysis (Figure 13):
- Unmodified GNPs: ~31.76 nm,
- NXT-activated GNPs: ~34.55 nm,
- Antibody-conjugated bioconjugates: ~1596 nm,
- The size increase confirmed the formation of higher-order complexes due to antibody binding.
3. Zeta Potential Analysis (Figure 14):
- GNPs: −26.08 mV,
- After NXT activation: +10.18 mV,
- After antibody conjugation: −15.65 mV,
The surface charge transitions validated successive chemical modifications and formation of a stable conjugate. The results of the study collectively confirm the successful functionalization of gold nanoparticles with detection antibodies, with retained stability and signal-generating potential suitable for use in the LFA kit (100).

Example 3: Optimization of Buffer Systems for LFA Components
To ensure optimal fluid flow, antigen-antibody interaction, and signal generation on the lateral flow assay (LFA) strip of the diagnostic kit (100), experiments were conducted to optimize the pre-treatment buffers used in both the conjugate pad (103) and sample pad (101). The aim was to enhance bioconjugate release, reduce non-specific interactions, and improve test line visibility.

Example 3A: Optimization of Conjugate Pad Buffer
Two distinct buffer formulations were evaluated for pre-treating the conjugate pad (103) to determine which condition best preserved and released the bioconjugates during testing:
• Buffer 1: 2 mM borate buffer with 10% sucrose (Figure 5A)
• Buffer 2: 5% sucrose, 1% BSA (bovine serum albumin), and 0.5% Tween-20 in 1× PBS (Figure 5B)
Following pretreatment, the conjugate pads were dried and integrated into the full LFA strip. Upon sample application and capillary migration, Buffer 2 consistently demonstrated superior release characteristics. The test lines appeared darker and more intense, indicating a higher concentration of released bioconjugates, improved antigen-antibody binding efficiency, and better signal generation. In contrast, Buffer 1 exhibited weaker signals and inconsistent release.
Therefore, Buffer 2 (5% sucrose + 1% BSA + 0.5% Tween-20 in PBS) was selected as the optimal conjugate pad pretreatment for maximizing diagnostic performance and reproducibility.

Example 3B: Optimization of Sample Pad Buffer
The sample pad (101) was evaluated for its impact on sample flow, viscosity modulation, and background signal interference. Two buffer formulations were tested as pretreatment solutions:
• Buffer A: 1 M borate buffer with 0.05% Tween-20 (Figure 6A)
• Buffer B: 1% BSA and 0.05% Tween-20 in 1× PBS (Figure 6B)
Each buffer-treated sample pad was dried and assembled onto the LFA strip, followed by testing with stool-derived samples. Results revealed that Buffer B facilitated smooth and consistent fluid migration, improved the clarity of the test lines, and reduced non-specific background staining. In contrast, Buffer A resulted in uneven flow and fainter test lines.
Consequently, Buffer B was determined to be the optimal pretreatment formulation for the sample pad (101), enhancing both signal intensity and overall assay robustness.

Example 4: Optimization of Antibody Concentrations for Enhanced Signal Performance
To achieve optimal signal intensity and specificity in the lateral flow assay (LFA) strip of the diagnostic kit (100), two critical antibody parameters were optimized:
(i) the concentration of monoclonal antibodies (mAb) used in the gold nanoparticle (GNP) conjugate formulation, and
(ii) the concentration of polyclonal antibodies (pAb) immobilized at the test line region on the nitrocellulose membrane (104).
These experiments confirmed that post optimization there is enhance antigen capture, maximize signal development, and reduce background noise.
Example 4A: Optimization of Monoclonal Antibody (mAb) Concentration for GNP Conjugation
Gold nanoparticles (GNPs) were conjugated with different concentrations of monoclonal antibodies (mAb) specific to calprotectin to determine the optimal antibody loading for effective visual detection (Refer to: Figure 7). The following concentrations were tested:
- A: 5 µg/mL
- B: 10 µg/mL
- C: 20 µg/mL
- D: Blank (no antibody control)
Each bioconjugate formulation was applied to the conjugate pad (103) and incorporated into the LFA strip. The strips were tested with 0.6 mg/mL of calprotectin spiked into 1× PBS buffer. Additionally, a polyclonal antibody (1 mg/mL) was striped at the test line on the nitrocellulose membrane (104) to serve as the capture antibody.
Results indicated that the strip containing 5 µg/mL mAb (A) generated the darkest and most distinct test line, demonstrating optimal signal intensity and antibody–antigen interaction. The strips with higher mAb concentrations (B and C) showed comparatively weaker lines, possibly due to nanoparticle aggregation or steric hindrance during conjugation. The blank strip (D) yielded no visible test line, confirming the essential role of mAb in detection.
Thus, 5 µg/mL was confirmed as the optimal monoclonal antibody concentration for GNP conjugation to ensure sensitive and reproducible detection of calprotectin in the LFA format.
Example 4B: Optimization of Polyclonal Antibody (pAb) Concentration at Test Line
To optimize the density of capture antibodies on the test lines (T1 and T2) of the nitrocellulose membrane (104), various concentrations of polyclonal antibodies (pAb) were evaluated. Each spotting position was tested using the following pAb concentrations:
- 0.3 mg/mL,
- 0.4 mg/mL,
- 0.5 mg/mL.
LFA strips were prepared using the above antibody concentrations at two test line positions (T1 and T2), and tested with 0.6 mg/mL calprotectin in 1× PBS. Three strip configurations were generated:
- Strip A: 0.3 mg/mL at both T1 and T2,
- Strip B: 0.4 mg/mL at both T1 and T2,
- Strip C: 0.5 mg/mL at both T1 and T2.
Among the tested combinations, strip C (0.5 mg/mL at both test lines) produced the most intense and clearly visible test bands, indicating superior antigen capture and signal generation. The other concentrations yielded progressively weaker signals, suggesting insufficient antibody density for effective binding.
Therefore, 0.5 mg/mL was established as the optimal polyclonal antibody concentration for spotting at test lines T1 and T2, thereby enhancing the sensitivity, clarity, and diagnostic reliability of the LFA test kit (100).

Example 5: Validation of the LFA Test Strip for Calprotectin Detection
To evaluate the functional performance and diagnostic sensitivity of the lateral flow assay (LFA) test strip incorporated in the kit (100), a validation study was conducted using synthetic stool samples spiked with varying concentrations of calprotectin (CP). The objective was to assess the test line response across a range of clinically relevant CP concentrations and determine the detection threshold of the device. (Refer to: Figure 9)
Four different CP concentrations were tested:
- 1 mg/mL,
- 0.6 mg/mL,
- 0.4 mg/mL,
- 0.2 mg/mL.
The samples were prepared in 1× PBS with 0.05% Tween 20 buffer to simulate stool matrix conditions and applied to the sample pad (101) of the assembled LFA test strips. The sample was allowed to migrate through the conjugate pad (103), where the calprotectin antigen bound to GNP-conjugated monoclonal antibodies. The resulting complex continued along the nitrocellulose membrane (104), where it was captured at the test lines (T1 and T2) by immobilized polyclonal antibodies, resulting in a visible red band. The control line (C) confirmed proper assay function.
Results showed that:
• At 1 mg/mL, the test lines appeared dark and prominent, indicating high sensitivity at elevated CP levels.
• At 0.6 mg/mL, the test lines were clearly visible, confirming the LFA strip’s ability to reliably detect CP at or above the typical clinical threshold for distinguishing bacterial from non-bacterial diarrhea.
• At 0.4 mg/mL and 0.2 mg/mL, test lines were faint, suggesting limited detection capability at lower concentrations.
These findings validate that the LFA kit (100) is suitable for identifying elevated fecal calprotectin levels ≥ 0.6 mg/mL, which are indicative of invasive bacterial diarrhea in pediatric patients. The strip thus meets the diagnostic requirement for point-of-care differentiation between bacterial and non-bacterial diarrhea and can guide appropriate antibiotic use in clinical settings.

Example 6: Clinical Validation of the LFA Test Strip Using ELISA as Reference Standard
To clinically validate the diagnostic performance of the developed LFA test strip integrated in the kit (100), a comparative study was conducted using real pediatric stool samples. The performance of the in-house LFA test strips was evaluated against a validated sandwich ELISA assay, which served as the gold-standard reference for calprotectin (CP) quantification.
Ex. 6A. ELISA Assay Validation
The sandwich ELISA protocol was first validated using known concentrations of purified calprotectin ranging from 0 to 20 ng/mL. The resulting calibration curve exhibited high linearity with a correlation coefficient (R² > 0.9) based on OD₄₅₀ readings, confirming the assay’s accuracy across the clinically relevant concentration range. Stool samples from pediatric patients (both symptomatic and asymptomatic) were processed and run in triplicate, and CP concentrations were interpolated from the standard curve.
Ex. 6B. LFA Strip Evaluation in Clinical Samples
The LFA test strips were evaluated using 25 clinically collected stool samples. Based on ELISA-derived CP levels, the following diagnostic classification thresholds were applied:
• Bacterial infection: CP > 0.60 mg/mL
• Non-bacterial infection: CP = 0.25–0.57 mg/mL
• No infection: CP < 0.25 mg/mL
Each sample was tested using the developed LFA strip, and the results (number and intensity of visible test lines) were interpreted and classified accordingly as shown in Table-1 below.
Table-1: ELISA vs LFA Comparative results (Samples 1-25):
Sample No. CP (mg/mL) ELISA ELISA Interpretation LFA Results
1. 0.161 No infection No infection
2. 0.145 No infection No infection
3. 0.201 No infection No infection
4. 0.113 No infection No infection
5. 0.089 - No infection
6. 0.207 No infection No infection
7. 0.161 No infection No infection
8. 0.145 No infection No infection
9. 0.602 Bacterial Non-bacterial infection
10. 0.521 Non-bacterial No infection
11. 0.603 Bacterial Bacterial infection
12. 0.336 Non-bacterial No infection
13. 0.459 Non-bacterial No infection
14. 0.544 Non-bacterial No infection
15. 0.521 Non-bacterial No infection
16. 0.447 Non-bacterial No infection
17. 0.514 Non-bacterial Non-bacterial infection
18. 0.719 Bacterial Bacterial infection
19. 0.591 Bacterial Bacterial infection
20. 0.616 Bacterial Bacterial infection
21. 0.567 Non-bacterial Non-bacterial infection
22. 0.666 Bacterial Bacterial infection
23. 0.567 Non-bacterial Non-bacterial infection
24. 0.567 Non-bacterial Non-bacterial infection
25. 0.626 Bacterial Bacterial infection

The LFA results were then directly compared to the ELISA-based classification (gold standard) to evaluate concordance.
Out of the 25 samples analysed following was the diagnostic performance metrics as in below Table-2:
Metrics No. of Sample Value
True Positives (TP) 6
False Negatives (FN) 1
True Negatives (TN) 18
False Positives (FP) 0
From these outcomes, standard diagnostic metrics were calculated as follows:
• Sensitivity = True Positives / (True Positives + False Negatives) = 6 / (6 + 1) = 85.7%.
• Specificity = True Negative / (True Negative + False Positives) = 18 / (18 + 0) = 100%.
These results indicate that the LFA test strip offers high sensitivity and excellent specificity for detecting elevated calprotectin levels (≥0.60 mg/mL), making it a reliable point-of-care diagnostic tool for identifying invasive bacterial diarrhea in paediatric patients. The ability to detect bacterial etiology with minimal false positives also supports appropriate antibiotic stewardship.

Example 7: Practical Use of the Diacue LFA Test Kit
The Diacue LFA test kit (200) is designed for point-of-care use and enables rapid detection of elevated fecal calprotectin levels, which are indicative of invasive bacterial diarrhea. The kit (200) includes all necessary components (As in Figure 2) for sample collection, preparation, and interpretation without the need for laboratory instrumentation.
Step 1: Sample Preparation
A stool sample is collected from the patient using the spatula (201) provided within the kit. A portion of the stool is mixed thoroughly with the extraction buffer contained in the vial (203) to form a uniform suspension. This mixture ensures that calprotectin, if present in the stool, is solubilized and available for reaction during the assay.
Step 2: Sample Application
Using the dropper (202), approximately 3–4 drops of the diluted stool extract are added to the sample application area (102) of the LFA test strip (100) housed inside the plastic cassette (As in Figure-1(b). The test strip consists of the following critical components:
• Sample pad (101) – receives and initiates capillary flow of the test solution.
• Conjugate pad (103) – contains dried monoclonal antibodies conjugated to gold nanoparticles that bind to calprotectin in the sample.
• Nitrocellulose membrane (104) – where the immune complexes flow through and are captured at:
o Test Line 1 (T1) and Test Line 2 (T2) – contain polyclonal antibodies to capture the antigen-antibody complexes, forming visible colored bands.
o Control line (C) – contains anti-IgG to capture excess conjugate, confirming successful test flow.
• Absorbent pad (105) – acts as a sink, drawing the liquid through the strip.
Step 3: Test Readout and Interpretation
As the sample migrates across the membrane via capillary action, any calprotectin present binds with the conjugated antibodies and is immobilized at the test lines. Within 10–15 minutes, the result can be visually interpreted as follows:
• Negative Result: No visible band at T1 and T2; only the control line (C) appears pink/red, confirming proper assay function.
• Positive Result: Appearance of pink to red bands at both Test Line 1 (T1) and Test Line 2 (T2), along with the control line (C), indicating elevated levels of fecal calprotectin.
• Invalid Result: Absence of the control line (C), regardless of test line results, indicates test failure and necessitates repeat testing.
Step 4: Disposal and Storage
Used test components are sealed in the zip-lock pouch (204) for safe disposal. The information brochure (205) provides clear, stepwise instructions with pictorial guides for layperson or healthcare worker use. All components are housed in the outer packaging cover (206) for easy transportation, cold chain-free storage, and distribution.
The depiction of stepwise representation of the procedure for conduction of detection test as in Figure 4 demonstrates the single-step operational simplicity, visual interpretation, and field-level applicability of the Diacue LFA test kit (200), making it ideal for clinical use in remote, pediatric, or resource-limited settings where early diagnosis and rational antibiotic prescribing are critical.

Example 8: Comparative Analysis with Existing Commercial LFA Kits for Calprotectin Detection
A comparative evaluation was conducted to assess the clinical relevance and diagnostic limitations of existing commercial lateral flow assay (LFA) kits available globally for calprotectin detection. Most of these commercial products are primarily designed and validated for the detection of Inflammatory Bowel Disease (IBD), including Crohn’s disease and ulcerative colitis, and utilize fixed calprotectin cut-off values tailored for chronic gastrointestinal inflammation.
Table-3: Comparative analysis of existing closest known solutions commercialized in the market worldwide:
Company/Product Name Approach used Cut off value
(CP-Calprotectin)
(LF-Lactoferrin) Purpose Country of origin Age group
Lactoferrin + Calprotectin Rapid Test Strip Qualitative lateral flow immunoassay CP- 13.69 nM/ml
LF- 31.99 nM/ml Diagnosis of inflammatory gastrointestinal disorders China All ages
Quantum Blue® fCAL Lateral flow test, ELISA CP- 0.0027 nM/ml Diagnosis and Monitoring of IBD Disease Switzerland All ages
CalproSmart Home
Lateral flow Assay, ELISA CP -0.0014 nM/ml Monitoring of patients with established chronic inflammatory bowel disease Norway 5 years and olde
LACTOFERRIN SCAN®
ELISA (quantitative)
LF – c Distinguish between active inflammatory bowel disease (IBD) from non-inflammatory irritable bowel syndrome (IBS) United states All ages
CerTest BIOTEC Lateral flow assay CP- 13.69 nM/ml
Chronic inflammatory bowel disease Spain All ages

As summarized in Table-3, several LFA kits employ detection limits typically ranging from 50 µg/g to 200 µg/g fecal calprotectin, which are suitable for distinguishing IBD from irritable bowel syndrome (IBS). However, these thresholds do not align with the elevated calprotectin levels consistently reported in acute bacterial diarrhea, particularly in pediatric populations. Moreover, such kits are either not commercially available in India or are cost-prohibitive, limiting their applicability in rural or low-resource settings where childhood diarrhea is most prevalent.
One such example includes the Lactoferrin + Calprotectin Rapid Test Strip, which attempts to detect both biomarkers; however, it suffers from poor clinical cut-off specificity for invasive bacterial diarrhea, and offers no classification capability between moderate viral and severe bacterial inflammation. Additionally, the reliance of existing kits on IBD-centric thresholds fails to support timely therapeutic decisions in acute infectious diarrhea cases, especially when rapid antibiotic intervention is required.
In contrast, the present invention introduces a dedicated LFA-based diagnostic kit (100) optimized specifically for differentiating invasive bacterial diarrhea from non-bacterial causes in children under five years of age. The kit incorporates anti-calprotectin monoclonal antibodies conjugated with gold nanoparticles (GNPs) and immobilized polyclonal antibodies to achieve high sensitivity and specificity. Notably, the kit operates using a semi-quantitative visual readout, allowing field-level differentiation based on calprotectin thresholds clinically relevant to bacterial diarrhea (≥0.60 mg/mL), as summarized in Table 1.
By addressing the cut-off range limitations, cost barriers, and clinical misalignment of existing IBD-focused kits, the Diacue LFA strip offers a contextualized, rapid, and reliable solution for use in pediatric diarrhea management across developing regions. This empowers frontline healthcare providers to prescribe antibiotics only when bacterial etiology is confirmed, directly contributing to antibiotic stewardship and improved health outcomes.

REFERENCES
1) Lakshminarayanan, S.; Jayalakshmy, R. Diarrheal Diseases among Children in India: Current Scenario and Future Perspectives. J. Nat. Sci. Biol. Med. 2015, 6 (1), 24.
2) Ghosh, K.; Chakraborty, A. S.; Mog, M. Prevalence of Diarrhea among under Five Children in India and Its Contextual Determinants: A Geo-Spatial Analysis. Clin. Epidemiol. Glob. Health 2021, 12, 100813.
3) Bruzzese, E.; Giannattasio, A.; Guarino, A. Antibiotic Treatment of Acute Gastroenteritis in Children. F1000Research 2018, 7, 193.
4) Saha J, Mondal S, Chouhan P, Hussain M, Yang J, Bibi A. Occurrence of Diarrheal Disease among Under-Five Children and Associated Sociodemographic and Household Environmental Factors: An Investigation Based on National Family Health Survey-4 in Rural India. Children (Basel). 2022 May 3;9(5):658.
5) Pathirana WGW, Chubb SP, Gillett MJ, Vasikaran SD. Faecal Calprotectin. Clin Biochem Rev. 2018 Aug;39(3):77-90.
, Claims:We Claim:
1. A lateral flow assay (LFA)-based diagnostic kit (100) for detecting elevated levels of calprotectin in a stool sample to differentiate between bacterial and non-bacterial causes of diarrhea, the kit comprising:
a. a sample pad (101) configured to receive the stool sample or its extract;
b. a conjugate pad (103) comprising monoclonal antibodies specific to calprotectin conjugated with gold nanoparticles (GNPs);
c. a nitrocellulose membrane (104) comprising:
(i) a first test line (T1) and a second test line (T2) containing polyclonal antibodies immobilized to bind calprotectin–antibody–GNP complexes; and
(ii) a control line (C) containing reagents to confirm valid sample migration;
d. an absorbent pad (105) disposed downstream of the nitrocellulose membrane to promote capillary flow; and
e. a sample application area (102) facilitating sample delivery to the sample pad (101);
wherein the presence of elevated fecal calprotectin is indicated by a colorimetric change at the test lines (T1, T2), and the result is visually interpretable within 20 minutes.
2. The diagnostic kit (100) of claim 1, wherein the conjugate pad (103) is pretreated with a buffer comprising 5% sucrose, 1% bovine serum albumin (BSA), and 0.5% Tween-20 in phosphate-buffered saline (PBS).
3. The diagnostic kit (100) of claim 1, wherein the sample pad (101) is pretreated with a buffer comprising 1% BSA and 0.05% Tween-20 in PBS.
4. The diagnostic kit (100) of claim 1, wherein the monoclonal antibody concentration in the conjugate pad (103) is approximately 5 µg/mL.
5. The diagnostic kit (100) of claim 1, wherein the polyclonal antibody concentration in each of the test lines (T1 and T2) is approximately 0.5 mg/mL.
6. The diagnostic kit (100) of claim 1, wherein the detection limit for calprotectin is at least 0.6 mg/mL, enabling differentiation between bacterial and non-bacterial diarrhea.
7. The diagnostic kit (100) of claim 1, wherein the appearance of three coloured lines (T1, T2, and C) indicates a positive result for invasive bacterial diarrhea, while appearance of only the control line (C) indicates a negative result.
8. The diagnostic kit package (200) comprising:
a) the diagnostic kit (100) as claimed in any of claims 1 to 7;
b) a spatula (201) for stool sample collection;
c) a dropper (202) for sample transfer;
d) an extraction buffer vial (203) for dilution of stool sample;
e) a zip-lock pouch (204) for post-use disposal;
f) an information brochure (205) containing usage instructions; and
g) a package cover (206) enclosing the complete test kit components for distribution and storage.
9. The diagnostic kit package (200) as claimed in claim 8, wherein the extraction buffer vial (203) contains a measured volume of buffer suitable for solubilizing calprotectin from stool samples and is sealed to maintain sterility and stability prior to use.
10. The diagnostic kit package (200) as claimed in claim 8, wherein the information brochure (205) comprises pictorial and textual instructions detailing sample collection, dilution, application to the LFA test strip (100), and interpretation of results based on color change at the test lines (T1, T2) and control line (C).
11. The diagnostic kit (100) or diagnostic kit package (200) as claimed in any of the preceding claims, wherein the kit is specifically configured to detect elevated fecal calprotectin levels at or above 0.6 mg/mL, and is distinguished from existing commercial calprotectin detection kits by being optimized for the diagnosis of invasive bacterial diarrhea in children under five years of age,
rather than inflammatory bowel disease (IBD), and further comprises a visual three-line result format for semi-quantitative field-level interpretation of infection severity.
Dated this 15th day of July, 2025

Biswajit Biswal
[IN/PA - 2659]
Agent for the Applicant