Development of lateral flow immunochromatographic assay with Anti-Pythium insidiosum antibodies for point-of-care testing of vascular pythiosis

Abstract

The pathogenic oomycete Pythium insidiosum causes a fatal infectious illness known as pythiosis, impacting humans and certain animals in numerous countries in the tropics and subtropics. Delayed diagnosis is a primary factor contributing to the heightened morbidity and mortality associated with the disease. Several new serodiagnostic methods have been developed to improve the identification of pythiosis. However, these assays provide only indirect evidence of pythiosis and are not readily available in the commercial market. Here, we have developed an affordable point-of-care test (POCT) kit based on an immunochromatographic assay to detect P. insidiosum antigens directly. Our recent findings reveal that the lateral flow sandwich immunological testing cassette can accurately identify vascular pythiosis antigens using a small volume of patient’s plasma, accomplishing diagnostic efficiency, with a limit of detection (LOD) of 8 ng/mL. This prototype cartridge represents a significant stride toward advancing enriched POCT for pythiosis serodiagnosis.

Introduction

Pythiosis is a severe infectious illness affecting humans and animals, particularly horses, pets, and livestock. The causative agent is the pathogenic aquatic fungus-like oomycete Pythium insidiosum¹. The incidence of this infection is mainly detailed in tropical, subtropical, and temperate area^1–3. While the infected animals commonly exhibit clinical manifestations, involving cutaneous/subcutaneous lesions and disorders of the gastrointestinal tract, the symptoms in humans, especially those with hematological disorders like thalassemia hemoglobinopathy, typically present with arterial occlusions in the lower extremities (vascular pythiosis) and ocular infections^1–3. Treatment with common antifungal agent demonstrates ineffective against P. insidiosuminfections. The desired management for pythiosis occupies the surgical removal of the infected organ, for instance, the eye or leg. Unfortunately, numerous pythiosis patients accede to advanced infections or inadequate treatment⁴. The prognosis for pythiosis patients is notably unfavorable when there is a delay in diagnosis, leading to late treatment^4,5.

The gold-standard diagnostic method for pythiosis is a fungal culture designed to isolate P. insidiosumfrom infected specimens. This procedure, however laborious and time-consuming, requires knowledge for execution and outcome analysis^1,6. Additionally, it frequently evidences unsuccessful in separating the organism, particularly when sporadically present in such samples, and may yield to low temperatures during transportation and storage^1,7,8. Hence, a more sensitive method is very important for early P. insidiosum detection, supporting timely patient management. Various alternative detection approaches, utilizing sensitive molecular and immunological technologies, have been proposed to address the limitations of the microbial culture method in identifying P. insidiosum^6,9. In addition, the identification of a target pathogen’s DNA or RNA sequence through nucleic acid-based tests (NATs) is an effective method for microbial identification^10,11, facilitating the rapid detection of small quantities of the pathogen’s nucleic acid in clinical specimens, and improving diagnostic sensitivity.

Despite the appropriateness and efficiency of existing immunological methods, distinguishing between antibodies indicative of a past or recent P. insidiosum infection can be challenging. Furthermore, these methods cannot detect anti-P. insidiosum antibodies in patients with localized infections, such as in the cornea, an immunologically privileged site lacking specific types of host immune responses, such as antibody production^12,13. Notably, there is currently no commercially available immunological test for pythiosis, and its in-house development is hindered by the absence of a P. insidiosum-specific antigen. This unavailability and inaccessibility limit the incorporation of immunological tests in clinical laboratories. Unlike in-house immunological assays, all essential reagents (such as primers, probes, nucleotides, and DNA polymerases) and necessary equipment for developing NATs are readily accessible in molecular diagnostic laboratories or can be procured from commercial sources.

The lateral flow immunochromatographic assay (LFA) has grown extensively in popularity for the serodiagnosis of numerous infectious diseases due to its user-friendly format, rapid result turnaround, and high levels of detection sensitivity and specificity. Notably, this test is particularly valuable in faint or endemic areas where diagnostic facilities for pythiosis may be lacking, thus addressing critical healthcare needs in underserved regions¹⁴.

Due to the distributed evidence of published information on LFA in fungal detection strategy, we aimed to develop a prototype of the point-of-care testing (POCT) with mouse monoclonal anti-Pythium insidiosum antigen (PIA) immunoglobulin G (IgG) for P. insidiosum antigens detection (PyT-LFA) in serum samples of a patient with vascular pythiosis. It is noteworthy that recent research has identified specific monoclonal antibodies, such as anti-PIA IgG, as a potential tool for detecting P. insidiosum. However, our study exclusively focuses on the prototype test, signaling a forthcoming shift in the technology utilized for diagnosing vascular pythiosis.

Results

Principle and designing of PyT-LFA detection system

The proof-of-concept approach outlined in our recent work ulitlizes lateral flow immunochromatography assay for pythiosis diagnosis, referred to as PyT-LFA for P. insidiosum detection. The serum samples, when added to the sample pads of the PyT-LFA strips, combine with mouse anti-PIA mAb-conjugated colloidal gold (colloidal AG-anti-PIA) on the conjugate pad due to the specific binding of a PIA (no.1). As the products migrate along the strip, the complexes are captured by PIA-specific mAb at the test line (T line), resulting in a red band at the T line (no.2). The remaining colloidal gold-anti-PIA continue to move and are captured by goat anti-mouse IgG at the control line (C line), forming another red band that confirms the LFA system’s efficacy (no.3). In the absence of target PIA, no red band is observed at the T line as shown in Fig. 1A. Results can be visually assessed based on the presence of a red band at the T line. The LFA procedure can be completed within 25 min from serum preparation to result in readout as indicated in Fig. 1B. Therefore, in Fig. 1C, LFA employs a cassette strip containing five components: a sample pad, a conjugate pad, a nitrocellulose membrane, a Wick (absorbent) pad, and a plastic cushion. Serum samples are dropped onto the sample pad and can be visually identified using the PIA-specific monoclonal antibody, which is conjugated with colloidal gold.

Fig. 1.

Schematic Illustration of the PyT-LFA prototype. (A) PIA detection using a specifically designed sandwich immunoassay (corresponding to T and C lines). (B) Result interpretation: when the serum sample is added to the LFA cassette, they are driven forward by buffer action, and colloidal gold signals and color appear at the control line (C line) or both the control and test lines (T line). The results can be read visually. (C) Configuration and compartment of PyT-LFA device.

Establishment of PyT-LFA platform

The optimization of P. insidiosum-specific mAb clones was initially explored. Table 1 shown specificity of P. insidiosum-specific mAb clones that validated by immunohistochemistry (IHC) assay. The presence of a diverse set of candidate P. insidiosum-specific mAb clones, including 1.0 mg/mL of each PyT 1F5, PyT 3–24, and PyT 3–29, at the test (T) line were indicated in Table 2. Variations were observed in the outcomes of different antibody clones when different running buffers were employed. Interestingly, all strips, except Strips No. 7 (which depicted a true negative result, TN), showed the false positive (FP) results when tested with 100 µL of 1xPBS + 0.1% TX405 buffer only (data not shown). However, Strip No. 9 exhibited a positive band (+ 1), Strip No. 3 and No. 6 resulted in false negative (FN) when 95 µL CMF1 (pH 8.0) buffer with 5 µL of PIA-spiked serum (10 µg/mL; Positive test) was used. Conversely, Strip No. 1 (+ 3.5) and No. 4 (+ 1) displayed positive outcomes alongside FN results in the other strips when using the 95 µL of FL (pH 8.0) buffer with 5 µL of PIA-spiked serum (10 µg/mL; Positive test) as shown in Fig. 2A. Therefore, the PyT 1F5 clone and FL (pH 8.0) buffer were selected for the downstream process.

Table 1.

Specificity of monoclonal antibodies clones.

FFPE sample	PCR* (n = 75)		IHC** (n = 75)
	Positive	Negative	Positive	Negative
Pythium spp.
P. insidiosum	18	0	18	0
P. aphanidertum	1	0	1	0
Other fungal infection	56	0	0	56

* Reference method for pathogenic fungal infection.

** Monoclonal antibodies were analyzed using Immunohistochemistry (IHC).

Table 2.

Detail of each component in the PyT-LFA cassette.

Strip No.	Capture (Test line)	Detector (Conjugate)
Strip 1	Clone PyT 1F5	Clone PyT 1F5
Strip 2		Clone PyT 3–24
Strip 3		Clone PyT 3–29
Strip 4	Clone PyT 3–24	Clone PyT 1F5
Strip 5		Clone PyT 3–24
Strip 6		Clone PyT 3–29
Strip 7	Clone PyT 3–29	Clone PyT 1F5
Strip 8		Clone PyT 3–24
Strip 9		Clone PyT 3–29

Fig. 2.

Optimization of major compartments in PyT-LFA development. The variation of antibody clones (A), pretreated sample pad (Cytosep^® 1662) reagents (B), running buffer usage volume (C), and surfactant reagents (D) was indicated. The intensity of the band at the T and C lines was observed when testing with serum samples (pooled negative and spiked serum), Consequently, the results were analyzed for each ratio, with “+” representing the band intensity at the T line.

We then enhanced the efficiency of the sample pad (Cytosep^® 1662) using several pretreated reagents. When the pooled negative serum (20 µL) with FL buffer (pH 8.0, 80 µL) was applied to the V228 (pH 9.6)-pretreated sample pad, no band was observed (TN), whereas an inverse result (FP) was noted in the sample pad pretreated with CMF-1 or F1 supplemented with 0.5% casein solution at 15 min testing as demonstrated in Fig. 2B). Moreover, the maximum volume of the serum sample, resulting in the true negative outcome was defined as 40 µL as indicated in Fig. 2C. Thus, the pretreatment of the sample pad with V228 (pH 9.6) reagent and the maximum serum sample volume were optimized in the current study.

To improve the detection sensitivity of the apparatus, we subsequently evaluated the potential of several surfactants added to the running buffer. Similar TN results were observed when testing pooled negative serum with FL (pH 8.0) without surfactants and FL (pH 8.0) supplemented with Tween 20 or 0.1% Biot constituents. However, a band of undemanding intensity was observed when experimenting with the FL (pH 8.0) buffer supplemented with TX100 as shown in Fig. 2D; upper. Testing with PIA-spiked serum (final conc. 1,000 ng/mL) revealed that both the no surfactant buffer and all supplementation exhibited a positive band (True positive, TP) as demonstrated in Fig. 2D; lower. Additionally, 0.1% Biot (pH 8.0) enhanced the TN result when testing with pooled negative serum samples, compared to other concentrations. In conclusion, we utilized the PyT 1F5 clone as the captured antibody system, FL with 0.1% Biot (pH 8.0) solution, and V228 (pH 9.6) reagent as the running buffer surfactant and pretreated sample pad reagents, respectively, for the developed device.

Preliminary assessment of PyT-LFA efficacy

Upon establishing the PyT-LFA system, we first evaluated its the limit of detection (LOD) feasibility using an assays on serial dilutions of PIA-spiked serum, employing a signal reader to assure accuracy. For a direct and semi-quantitative determination of the LOD, we prepared serial dilutions of the samples in physiological serum and analyzed them with the PyT-LFA platform. Figure 3 illustrates the results, with serum samples containing different concentrations of PIA. The LFA efficiency (observed as band intensity) notably improved with increasing sensitivity of the running buffer surfactant. Even at a low concentration of 8 ng/mL, the test exhibited high specificity, with no FN results. Typically, the concentration of PIA in serum falls within the range of 4–8 ng/mL, indicating that the LOD of the PyT-LFA system is suitable for clinical testing. Furthermore, Fig. 3 demonstrates an imaged band for detection, exhibited as the measurable signal at the T line relative to the respective concentrations of PIA.

Fig. 3.

Assessment of PyT-LFA tool. The band image and its intensity at the T and C lines were employed to determine the detection limit (LOD) range of the tool in the testing with PIA spiked serum. The PIA concentration was indicated and results were analyzed for each ratio, with “+” representing the band intensity at the T line.

We also evaluated the stability (Reproducibility) of the PyT-LFA platform by analyzing the intensity band of the samples with a long-term storage period (30 days, 90 days, and 120 days) of the device. The triplicate of the same sample was prepared, followed by visual detection using conditioned lateral flow strips. The three spiked- and healthy serum pairs were prepared using three batches of strips under the same protocol. The results presented in Fig. 4 indicate no differences among the three pairs of strips. This study thus demonstrates the high reproducibility and reliability of our PyT-LFA platform, offering a valuable method for diagnosing pythiosis in clinical samples.

Fig. 4.

Stability of PyT-LFA device. The detected results of the cassette stored for 30 days (A), 90 days (B), and 120 days (C) are presented. Triplicate tests were conducted using the same sample types (spiked serum; PS and Negative serum; NS).

Discussion

Human and animal pythiosis is endemic in several tropical and subtropical countries³. In human pythiosis, four distinct pathological forms have been documented. While cutaneous pythiosis is characterized by granulomatous and ulcerating lesions typically affecting the face or limbs, vascular pythiosis involves arteries and can lead to arterial occlusion or aneurysm formation. The major clinical manifestation of ocular pythiosis is presented as keratitis. However, disseminated pythiosis is marked by the infection of internal organs^2,15. In the fashionable microbial infection, both immunological and nucleic acid-based methods have been utilized for pathogen identification.

Several serodiagnostic tests have been developed to aid in the early diagnosis of pythiosis⁶. In-house enzyme-linked immunosorbent assay (ELISA), Immunodiffusion (ID)^16–18, and Immunoblotting assays have demonstrated high sensitivity and specificity for diagnosing pythiosis^19–22. However, these tests require skilled personnel, stable and reproducible reagents, expensive equipment, and lengthy turnaround times.

The lateral flow immunochromatography has gained significant acceptance for the serodiagnosis of various infectious diseases owing to its user-friendly format, rapid result generation, and high levels of detection sensitivity and specificity¹⁴. Krajaejun et al.¹³ introduced an immunochromatographic test (ICT) for human pythiosis diagnosis (target to antibodies), with evaluation revealing a rapid turnaround time of less than 30 min for obtaining results. The ICT revealed 88% sensitivity and 100% specificity.

While existing immunological methods are deemed appropriate and efficient, there are still limits in their applications. The distinguishing between antibodies indicative of past or recent P. insidiosum infections can pose challenges. Moreover, these methods are difficult to detect anti-P. insidiosum antibodies in patients with localized infections, particularly in an immunologically privileged site such as the eyes, notably antibody production (actually it can detect by ELISA with titier < 1:800). Moreover, there is currently no commercially available immunological test for pythiosis, and the development of in-house assays is impeded by the absence of a P. insidiosum-specific antigen. Consequently, the unavailability and inaccessibility of such tests hinder their integration into clinical laboratories^12,13.

Due to the distributed evidence of published information on P. insidiosum-antigen (PIA) in the context of pythiosis therapy, we, therefore, aimed to develop a prototype of the point-of-care testing with mouse monoclonal anti-PIA immunoglobulin G (IgG) for P. insidiosum antigens detection in serum samples collected from the patients with vascular pythiosis (PyT-LFA).

A recent work produced and validated the anti-P. insidiosum mAb (Table 1). The IHC demonstrated that the PyT 1F5, PyT 3–24, and PyT 3–29 clones were potential monoclonal antibodies. The discrepancy between PCR and immunohistochemistry (IHC) results in fungal identifications, particularly the positive PCR outcomes for various fungi (excluding P. insidiosum) juxtaposed with negative IHC results for these fungi (Table 1), likely stems from differences in detection techniques and specificity. PCR has high sensitivity and recognizes DNA sequences, facilitating the identification of minute quantities of fungal genetic material, including fragments from non-viable organisms. In contrast, IHC relies on the binding of particular mAb to target antigens inside tissue specimens. We generated P. insidiosum-specific mAb for IHC validation. Consequently, it is shown to be completely negative against other fungi (TN). Insufficient recognition of fungal epitopes by monoclonal antibodies directed against P. insidiosum, due to antigen masking, degradation, or suboptimal production of target proteins, may lead to FN results. The structural distinctions between P. insidiosum and other fungal species may result in antigenic variations, hindering its identification using immunohistochemistry, despite the fact that its genetic material can be identified through PCR.

The limit of detection (LOD) of the PyT-LFA prototype for detecting P. insidiosum antigens in all pythiosis sera was 8 ng/mL. The inability to identify P. insidiosum antigens in patient sera has been documented, perhaps resulting from prolonged serum storage and insufficient antigen presentation in localized bloodstream infections^23–25. Therefore, the serodiagnosis of vascular infection should be approached cautiously, considering the expected rate of false-negative results. The prototype could serve as a point-of-care instrument for vascular pythiosis, while other serodiagnosis approaches require specific compartment preparation before testing. The turnaround time of the device was remarkably shorter compared to others (15–20 min. for the PyT-LFA; 1–24 h for others). Therefore, the PyT-LFA demonstrates better performance and greater convenience than previous tools, making it suitable for the serodiagnosis of vascular pythiosis. However, further evaluation is necessary to assess its performance in diagnosing ocular and cutaneous pythiosis.

Our findings show that the devices, which had been stored for 6 months, were suitable for future investigation with the vascular pythiosis sera assays. The assay’s repeatability was shown to be stable, with PIA-spiked samples yielding positive results when reanalyzed. However, this correlation might be decreased after a week of sample storage. Interestingly, the existing studies have also found a decline in BG test accuracy following both short- and long-term freezing. The BG test identifies Beta-D glucan, which can be hydrolyzed or degraded over time due to acidic conditions, even when samples are maintained at low temperatures to prevent these processe^26–31. Furthermore, subsequent PyT-LFA test findings differed from earlier tests when samples were held at −80 °C, implying that the test’s diagnostic performance may be stabilized. It has been postulated that the P. insidiosum antigen identified by the PyT-LFA test becomes stable over time due to autolytic breakdown. As a result, it is assumed that the PyT-LFA test be performed as soon as feasible to ensure a good correlation with the BG assay results. The LOD and specificity of the prototype developed in this study might be in line with previous reports. However, enhancements to the PyT-LFA test could be achieved through its combination with other tests, such as PCR or the CRISPR/Cas system. The rationed LOD observed in the LFD test in our study may be linked to patients being exposed to multiple antibiotics and/or antifungal agents before sample collection.

However, nucleic acid-based methods, particularly PCR, offer distinct advantages over immunological techniques due to their superior sensitivity, specificity, and rapidity^32,33. Moreover, advancements in biosensor technology have further enhanced the sensitivity, specificity, and simplicity of nucleic acid-based assays³⁴, paving the way for the development of convenient, cost-effective, and specific diagnostic tools for Pythium spp. pathogen monitoring applications. This can greatly aid in implementing management practices aimed at reducing the risk of epidemics.

Collectively, our prototype offers several notable advantages. This can greatly aid in implementing management practices aimed at reducing the risk of epidemics. Still further evaluation and verification, including specificity, sensitivity, accuracy of the proposed device using appropriately scaled-clinical specimens will be fulfilled at the time to its implement and adoption as a new technology for healthcare in efforts to improve public health.

Limitations of the study

Here, we have successfully developed a sensitive, cost-effective, and user-friendly PIA detection platform by lateral flow immunochromatography. This platform allows detection results to be obtained within 25 min. using clinically relevant samples (such as serum) directly without the complicated steps. We anticipate that the PyT-LFA system could be readily adapted for detecting various types of human pythiosis, particularly, vascular and systemic pythiosis. However, detecting variations in PIA number remains a challenge. We plan to refine the LOD value and read-out system further to enable the LFA system to detect the lowest PIA number variations effectively as well as an evaluation and verification, including specificity, sensitivity, and accuracy of the recent prototype.

Method details

Clinical specimens

Seventy-five formalin-fixed paraffin-embedded tissue (FFPE) from animals and patients were used for monoclonal antibodies (mAb) assessments. Whole blood samples were obtained from five healthy participants using clotted-blood tubes. Serum was separated from each sample via a standard centrifugation technique³⁵ and subsequently served up with antigen-spiked samples. Each individual provided informed consent, and the study received approval from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, a WHO-certified ethics committee (IRB No. 0427/67). All methods adhered to these approved guidelines.

Preparation of mouse monoclonal anti-P. Insidiosum antibodies

The crude (i.e., endo and exo) antigens of Pythium insidiosum were prepared from the P. insidiosum strain CBS574.85 (ATCC 58643) as previously described³⁶. The P. insidiosum strain CBS574.85was subcultured on Sabouraud dextrose agar (SDA) and incubated at 37°C for 72 h. Several pieces of agar parts containing hyphal components from the active culture were inoculated into 350 mL of 2% Sabouraud dextrose broth (SDB) and incubated with shaking (180 rpm) at 37 °C. for 5 days. Thimerosal (Merthiolate; final concentration, 0.02% [wt/vol]) was used to eliminate the cultures before filtration using a Durapore membrane filter (0.22-µm pore size; Millipore, County Cork, Ireland). The mycelial substance was homogenized using a pestle and mortar coupled with liquid nitrogen. The precipitated proteins from the mixture of disrupted mycelial masses and supernatants were lyophilized and keep at −80°C. The protein concentration was measured using BCA assay^27,29.

In this study, high-affinity monoclonal antibodies (mAb) were produced using a structured immunization protocol with female BALB/c mice. After a 7-day acclimatization, the mice received an initial immunization of 50 µg of PIA in Freund’s Complete Adjuvant, followed by three booster doses at 21, 42, and 71 days with Freund’s Incomplete Adjuvant. Blood samples were collected at key intervals to monitor antibody titers via ELISA. On Day 84, spleens were harvested for splenocyte isolation, which were then fused with Sp2/0-Ag14 myeloma cells using polyethylene glycol to generate hybridomas. Positive clones producing antigen-specific antibodies were identified through ELISA and subcloned for monoclonality. The resulting supernatants were purified by affinity chromatography utilizing TOYOPEARL AF-rProtein A HC-650 F resin (Tosoh Bioscience LLC, Grove City, OH, USA), and the mAbs were further processed through filtration, dialysis in 1X PBS using Slide-A-Lyzer (Thermo Scientific™, Waltham, MA, USA), and purity assessment via SDS-PAGE. Endotoxin levels and aggregate presence were evaluated using HPLC-SEC analysis, ensuring the quality of the produced antibodies.

Construction of a PyT-LFA cassette

• (i) Conjugation of antibody to colloidal gold.

The 40-nanometer-particle colloidal gold suspension (Arista, Allentown, PA, USA) was adjusted to pH 9.65 using 0.2 M Na₂CO₃. As optimized by a gold saturation test, subsequently 3 µg of mouse anti-P.insidiodum (PyT) mAb was added to each 500 µL of colloidal gold, followed by a 30-minute incubation at room temperature. The residual surfaces of the colloidal gold particles were then blocked by incubating with 5% (w/v) bovine serum albumin (Sigma, St. Louis, MO, USA) for 10 min. After centrifugation at 6,000 × g for 15 min, the supernatant was discarded, and the conjugate pellet was washed in 0.5% (w/v) casein. This was followed by another centrifugation at 6,000 × g for 15 min, after which the supernatant was removed. The conjugate was resuspended in a solution containing 0.5% (w/v) casein and 20% (w/v) sucrose in 0.02 M Tris-HCl (pH 8.0), with a volume 40 times smaller than the original suspension. A 2.5 by 2.5-millimeter piece of glass fiber (GF33; Whatman Schleicher & Schuell, Dassel, Germany) was impregnated with 2.5 µL of this IgG-colloidal gold conjugate and dried in a dehumidifier cabinet for an hour.

• (ii) Immobilization of antigen and antibody onto a nitrocellulose membrane.

A nitrocellulose membrane measuring 1.5 centimeters wide (CN140; Whatman Schleicher & Schuell, Dassel, Germany) was coated with PyT (1 mg/mL) to create the test line and goat anti-mouse IgG (1 mg/mL) in PBS to create the control line, each at a volume of 1 µL per centimeter using a dispenser (ZX 1000; BioDot, Irvine, CA). Subsequently, the membrane was dried, blocked with 1% (w/v) bovine serum albumin, and dried again in a dehumidifier cabinet.

• (iii) Assembly of PyT-LFA strips.

The nitrocellulose membrane, glass fiber with colloidal gold conjugate, sample pad (Cytosep 1622 / V228, pH 9.6), and wicking pad (18 mm.; 470 chromatography paper; Whatman, Maidstone, England) were carefully arranged and mounted onto a plastic backing (60 mm.). This assembly was then precisely cut into strips measuring 25 mm. in width using a strip-cutting machine (CM 4000 R; BioDot, Irvine, CA).

• (iv) Detection of human P. insidiosum antigens by the PyT-LFA.

Each serum sample (40 µL) underwent an optimized dilution in 60 µL running buffer (FL with 0.1% Biot pH 8.0). Subsequently, the lateral flow immunochromatographic test (LFA) was conducted in duplicate using 100 µL of the diluted serum in the indicated buffer. Following a 15-minute incubation period, three independent laboratory personnel evaluated the test signal of each LFA strip using visual inspection.

Quantification of PyT-LFA

To quantify the LFA band intensities, each strip was scanned using a scanner (Epson Perfection 1670 photo scanner; Seiko Epson Corp., Japan) to generate a tagged image file format picture. Test and background signal intensities were analyzed utilizing the Image J software (https://imagej.net/ij/docs/intro.html). The resulting intensity value (+ 1 to + 3.5) is obtained by subtracting the background signal from the test signal. +/- does not exclude the band intensity. Any occurrence of a “+” sign is classified as “no band observed.” The signal of each LFA strip was evaluated by three independent experiments.

Author contributions

N.W. and P.T. carried out assays and analyzed the results; P.T., A.B., NL, and A.C. supported the optimization of assays and prepared data; A.C. developed the strip; A.B., and A.C. performed analyses of assay results; N.W., A.B., and A.C. conceived and designed the experiments; P.T., A.B., and A.C. wrote the manuscript.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Materials availability

This study did not generate unique reagents.