Immunochromatographic Assay Research Articles

A sensitive intelligent point-of-care test method for tert-butylhydroquinone in edible oil via a test strip with a smartphone

Tert-butylhydroquinone (TBHQ) is easily overused or illegally added to edible oil and attracts a growing concern because of its cytotoxic, liver-damaging, and carcinogenic effects. Thus, a sensitive and intelligent point-of-care testing (iPOCT) method is developed to fulfill the on-site monitoring. This iPOCT method depended on a fluorescent immunochromatographic assay within 15 min. Under optimization, the limit of quantification (LOQ) was calculated as 0.03 μg mL-1. The iPOCT method provided a low limit of detection (LOD) of 0.02 μg mL−1, a wide linear range of 0.03–100 μg mL−1, and great selectivity. Recoveries by the spiking experiments ranged from 97.4% to 103.5% with relative standard deviations (RSDs) of 2.4%–4.9% in soybean, peanut, rapeseed, and corn oil samples. The results showed that the iPOCT method is highly consistent with the high-performance liquid chromatography (HPLC) method.

Multi-Shell Nanourchin-Integrated Dual Mode Lateral Flow Immunoassay for Sensitive and Rapid Detection of Clinical Cardiac Myosin-Binding Protein C.

Cardiac myosin-binding protein C (cMyBP-C) is a novel cardiac marker of acute myocardial infarction (AMI) and acute cardiac injuries (ACI). Construction of point-of-care testing techniques capable of sensing cMyBP-C with high sensitivity and precision is urgently needed. Herein, we synthesized an Au@NGQDs@Au/Ag multi-shell nanoUrchins (MSNUs), and then applied it in a colorimetric/SERS dual-mode immunoassay for detection of cMyBP-C. The MSNUs displayed superior stability, colorimetric brightness, and SERS enhancement ability with an enhanced factor of 5.4 × 109, which were beneficial to improve the detection capability of test strips. The developed MSNU-based test strips can achieve an ultrasensitive immunochromatographic assay of cMyBP-C in both colorimetric and SERS modes with the limits of detection as low as 19.3 and 0.77 pg/mL, respectively. Strikingly, this strip was successfully applied to analyze actual plasma samples with significantly better sensitivity, negative predictive value, and accuracy than commercially available gold test strips. Notably, this method possessed a wide range of application scenarios via combining with a color recognizer application named Color Grab on the smartphone, which can meet various needs of different users. Overall, our MSNU-based test strip as a mobile health monitoring tool shows excellent sensitivity, reproducibility, and rapid detection of the cMyBP-C, which holds great potential for the early clinic diagnosis of AMI and ACI.

A multi-colloidal gold immunochromatography assay for rapid and simultaneous detection of 13 herbicides

A multi-colloidal gold immunochromatography assay for rapid and simultaneous detection of 13 herbicides

A sensitive immunochromatography assay based on quantum dot nanobeads for determination of acid orange II

Acid orange II (AO2) is a synthetic azo dye commonly used illegally in food production and processing. The development of a fast and effective analytical method is crucial for controlling the unlawful use of AO2 in food, especially in seasoning. Here, an antibody specific for AO2 was generated, with 1.01 ng/mL IC50 and minimal cross-reactivity with other analogs (<1 %, including sunset yellow, tartrazine, para red, chromotrope FB, Sudan Ⅰ, rhodamine B, 2-naphthol, amaranth, ponceau 4 R and allura red AC). Furthermore, a highly sensitive quantum dot beads-based fluorescence immunochromatography assay (QBs-FICA) was established for AO2 determination. The assay had a working range of 23–287 μg/kg with a 11 μg/kg limit of detection (LOD) in chili powder and hotpot seasoning samples. Using spiked samples, the results of QBs-FICA were found to be consistent with those of liquid chromatography-tandem mass spectrometry. This indicates that QBs-FICA is a reliable, rapid, and portable tool for testing of AO2.

Domestic Dog Infection with Trypanosoma cruzi from Northern and Southern Regions of Mexico.

Background: Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico, infections in domestic animals, such as dogs, may affect the risk of human disease when they serve as a source of infection to vectors that subsequently infect humans. Materials and Methods: We conducted a cross-sectional study of 296 dogs from two cities near the northern and southern borders of Mexico: Reynosa, Tamaulipas, and Tuxtla Gutierrez, Chiapas. Infection was measured based on testing of blood using T. cruzi quantitative PCR (qPCR) and up to three antibody detection assays. The StatPak immunochromatographic assay was used to screen samples and the indirect fluorescent antibody (IFA) and multiplex microsphere immunoassay (MIA) tests were used as secondary tests on all samples that screened positive and a subset of negatives. Serologic positivity was defined based on reactivity on at least two independent tests. Results: Of the 280 samples tested for parasite DNA, two (0.7%) were positive, one of which (0.4%) was confirmed as T. cruzi discrete typing unit TcIV. Overall, 72 (24.3%) samples were reactive for T. cruzi antibodies via StatPak of which 8 were also positive using MIA and 2 were also positive using IFA (including one of the PCR-positive dogs). Overall, nine dogs (3.4%) met study criteria of positivity based on either/both serology or PCR tests. Positive dogs were found in both regions of Mexico; five (2.7%) from Reynosa and four (3.6%) from Tuxtla Gutierrez. We found no association between infection status and state of origin, sex, age group, breed group, neighborhood, and whether other pets lived in the home. Conclusion: Our results re-emphasize dogs' utility as sentinels for T. cruzi in Mexico and underscore the need for improved veterinary diagnostic tests and parasite surveillance at the household level in endemic countries.

Epidemiological and clinical characteristics of patients with carbapenem-resistant Enterobacterales in a university hospital of Colombia: Enzyme coproductions in rise

The distribution of carbapenemases in Carbapenem-Resistant Enterobacterales (CRE) has recently undergone a change in our region. According to the Colombian National Institute of Health, there is an increasing prevalence of NDM and NDM-KPC co-producing strains. We carried-out an ambispective cohort study of adult inpatients from Hospital Universitario San Ignacio (2021–2023), infected or colonized with CRE, in which carbapenemases immunochromatographic assay was performed. Out of the 150 patients included in the study, 71.3 % presented with an infection, and carbapenemases were detected in 92.7 % of these cases. Among them, KPC predominated (54 %), while 16.7 % demonstrated enzyme coproductions, mainly KPC-NDM. CRE infected patients had an 18.7 % 30-days mortality, but we could not demonstrate an association between type of carbapenemase and mortality rate (p = 0.82). Logistic regression analysis suggested that ICU admission was independently correlated to fatality (OR 5.08; CI 1.68–16.01). NDM and KPC-NDM presence in CRE poses a public health threat and a therapeutic challenge, with unknown mortality differences according to the carbapenemases pattern. Nevertheless, there was not an association between enzyme type and mortality.

An ultrasensitive strip sensor for rapid detection of African swine fever virus

African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting pigs, which has caused huge economic losses in countries around the world. Currently, there is no effective vaccine, and the prevention and control of ASF is mainly through rapid detection, so it is particularly important to carry identify and develop rapid detection methods for ASFV. In this study, recombinant plasmid PET-28a(+)-p30 was constructed, and the recombinant protein was obtained by inducing expression and Ni+ resin affinity column purification. Mice were immunized with recombinant p30 protein, and after three immunizations, ten strains of hybridoma cells that stably secreted monoclonal antibodies (mAbs) against p30 protein were obtained by cell fusion and subcloning. A colloidal gold immunochromatography assay (GICA) based on double antibody sandwich technology was established to screen the paired antibodies, and the trapping and detecting antibodies were mAb-11F11 and mAb-7A8, respectively, with a detection limit of 1 ng/mL, which laid an important material foundation for the early detection of ASF in the future.

Evolución de la seroprevalencia de COVID-19 en bancos de sangre durante la pandemia en la región Lima, Perú

Introduction. Peru was one of the countries most affected by the COVID-19 pandemic, with more than 220,000 deaths recorded due to SARS-CoV-2 infection. Given the constant mutations of the virus and registered variants, it is important to carry out research that reflects the evolution of the humoral immune state. Objective. Measure the seroprevalence of COVID-19 during the pandemic in the department of Lima in the period between April 1, 2020 and June 30, 2022, in donor samples from 6 hospital blood banks public of Lima, Peru. Methods. A descriptive, transversal, observational and retrospective study was carried out evaluating the presence of IgM/IgG antibodies against SARS-CoV-2 using lateral flow chromatographic immunoassay in samples from healthy and asymptomatic donors from blood banks of 6 health facilities in the department of Lima. Results. A progressive increase in the seroprevalence of IgG and total reactivity of IgM/ IgG antibodies against SARS-CoV-2 will be observed as follows: 22,6% - 32,7% during 2020; 53,9% - 58,1% during 2021; 88,9% - 93,3% during the first half of 2022. Conclusions. The reactivity and presence of anti- SARS-CoV-2 antibodies progressively increase during the COVID-19 pandemic from 11,2% to 98,4% in the samples evaluated, possibly with the impact of the pandemic waves and the active vaccination campaign against COVID-19.

Effect of Ziziphus Spina-Christi Leaves on the Level of Testosterone Hormone and ‎Semen Parameters of Iraqi Bucks Semen

The study aimed to assess the effects of different concentrations of Ziziphus spina-Christi (ZSC) leaves on the fertility of buck goats. Twelve buck goats aged 12–15 months with an average body weight of 22±2 kg were divided randomly into three dietary treatments: T1, T2, and T3, each given a basal diet daily plus feeds of 20 g/kg (2%) of ZSC for T1 and 40 g/kg (4%) of ZSC for ‎T2; and T3 was used as the control that consumed a basal diet only. The experiment lasted for 3 months. On days 45 and 90, blood samples were collected for plasma testosterone measurement using an immunochromatographic assay. Additionally, semen samples were collected at the same time points for physical (volume, color, and pH) and microscopic (semen concentration, mass and individual motility percentage, and live-dead sperm percentages) evaluations. ‎ The results showed that ZSC at 4% significantly (P&lt;0.05) increased the testosterone hormone concentration and all physical parameters at 45 and 90 days comparing to 2% ZSC and control ‎groups. ‎Additionally, ZSC at 4% significantly improved (P&lt;0.05) the microscopic parameters at both ‎time periods compared to 2% ZSC and control groups. The present findings revealed that ZSC at 4% diet could enhance goat semen processing and ‎goat fertility enhanced significantly (P&lt;0.05) the testosterone hormone that had higher values ‎compared to ‎‎(2%) of the ZSC control group. Similarly, (4%) of ZSC affected the physical ‎assessment of ‎semen, such as the volume, color, and pH values. Thus, adding ZSC with a high ‎concentration ‎‎(4%) could enhance goat semen processing and goat fertility in general.

Phenylboronic Acid-Modified Membrane-Like Magnetic Quantum Dots Enable the Ultrasensitive and Broad-Spectrum Detection of Viruses by Lateral Flow Immunoassay.

Although lateral flow immunochromatographic assay (LFIA) is an effective point-of-care testing technology, it still cannot achieve broad-spectrum and ultrasensitive detection of viruses. Herein, we propose a multiplex LFIA platform using a two-dimensional graphene oxide (GO)-based magnetic fluorescent nanofilm (GF@DQD) as a multifunctional probe and 4-aminophenylboronic acid (APBA) as a broad-spectrum recognition molecule for viral glycoprotein detection. GF@DQD-APBA with enhanced magnetic/fluorescence properties and universal capture ability for multiple viruses was easily prepared through the electrostatic adsorption of one layer of density-controlled Fe3O4 nanoparticles (NPs) and thousands of small CdSe/ZnS-MPA quantum dots (QDs) on a monolayer GO sheet followed by chemical coupling with APBA on the QD surface. The GF@DQD-APBA probe enabled the universal capture and specific determination of different target viruses on the test strip through an arbitrary combination with the antibody-modified LFIA strip, thus greatly improving detection efficiency and reducing the cost and difficulty of multiplex LFIA for viruses. The proposed technique can simultaneously and sensitively diagnose three newly emerged viruses within 20 min with detection limits down to the pg/mL level. The excellent practicability of GF@DQD-APBA-LFIA was also demonstrated in the detection of 34 clinical specimens positive for SARS-CoV-2, revealing its potential for epidemic control and on-site viral detection.

A novel competitive color-tone change fluorescence immunochromatographic assay for the ultrasensitive detection of pesticide and veterinary drug residues

One important challenge faced by competitive immunoassay techniques is the inability to visually distinguish weak color changes in the T-line during the detection of trace analyte concentrations. In this study, we introduced multi-color fluorescence into the immunoassay system and developed a novel competitive dual-channel color-tone change fluorescent immunochromatographic assay (CFICA) for the visual and quantitative detection of kanamycin (KAN) and carbendazim (CBZ). At the same time, we have developed matching, portable fluorescence-reading equipment, which can analyse the spectral data of the RGB channel individually and can effectively solve the problems of the traditional single-fluorescence reader – large reading error and poor detection sensitivity. After optimization, our CFICA, combined with the corresponding instrument, achieved low limits of detection (LOD) for KAN and CBZ, as low as 1.98 and 12.82 pg/mL, respectively. We believe that this CFICA detection system, with its high accuracy, low error, and multi-color detection capabilities, holds tremendous potential for visual and multi-channel competitive point-of-care testing.

Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

The impact of infrared radiation, solar radiation, and burial exposure on the efficacy of forensic immunoassay testing for blood, semen, and saliva

Determining the biological source of a stain can be important information for both investigators and the judiciary in criminal cases. Immunochromatographic assays are commonly used in forensic science for the identification of human biological material. It has previously been demonstrated that various environmental, thermal and chemical insults can affect the efficacy of ABAcard® HemaTrace® in the detection of human blood. In this study, the efficacy of three tests – ABAcard® HemaTrace®, ABAcard® p30, and RSID™-Saliva – was determined for the detection of blood, semen, and saliva respectively, after the fluids had been exposed to adverse environmental conditions. Each biological fluid was deposited on cotton swatches and exposed to infrared (IR) light using a 100 W heat lamp emitting IR light between 620 and 750 nm and heat of 32° for 24, 36 and 48 h. Cotton swatches bearing biological fluids were also buried in outdoor soil for 3, 4 and 5 weeks. To test common forensic scenarios where biological material may be exposed to solar light, samples were placed on a car bonnet and left for 24, 36 and 48 h. ABAcard® HemaTrace® was able to detect haemoglobin in blood that had been exposed to IR and solar light up to 48 h. False negative ABAcard® HemaTrace® results were obtained from 60 % of blood samples buried for 3 and 4 weeks, and 80 % of blood samples buried for 5 weeks. ABAcard® p30 was able to detect p30 in semen that had been exposed to IR and solar light up to 48 h, except for one false negative after 48 h of IR exposure. False negative ABAcard® p30 results were obtained from all semen samples buried for 3, 4 and 5 weeks. RSID™-Saliva was able to detect α-amylase in saliva in all instances, with no false negative results observed. The findings from this study highlight the need to consider the context in which human blood, semen and saliva are found when reporting on negative immunoassay results.

In-house Carba NP - II test to identify and differentiate carbapenemase-producing Gram-negative bacteria among various clinical isolates in comparison with immunochromatography assay

In-house Carba NP - II test to identify and differentiate carbapenemase-producing Gram-negative bacteria among various clinical isolates in comparison with immunochromatography assay

Polydopamine-modulated anisotropic co-growth plasmonic blackbody for efficient ultra-broad-spectrum quenching to establish multicolor fluorescent immunoassay

Fluorescence quenching immunoassays have received increasing attention due to their high signal-to-noise ratio, among which multi-color fluorescence quenching methods have great advantages in detecting multiple targets. Preparing an efficient and broad-spectrum quencher is a prerequisite for establishing high-performance multicolor fluorescence quenching immunoassays. In this study, we propose a polydopamine (PDA) modulated seedless co-growth method to synthesize anisotropic plasmonic blackbody (APB) with highly branched chrysanthemum −like shape for efficient ultra-broad-spectrum quenching and utilize APB to establish a sensitive multicolor fluorescent immunoassay. The key to APB synthesis lies in the reduction of Au3+ by dopamine, which simultaneously undergoes oxidation and self-polymerization in situ to form a PDA film. This film continuously deposits on the Au crystal seeds, creating a robust end-capping effect and inducing continuous anisotropic growth of Au. The dual synergistic effects of PDA and the unique 3D-spatial structure contribute to APB’s robust ultra-broad-spectrum UV absorption (6.86 × 1011 L cm−1 mol−1) from 300 to 800 nm, along with efficient ultra-broad-spectrum fluorescence quenching ability (90.1 %). This quenching ability extends to various types of fluorescence microspheres covering the range of 300–800 nm with different excitation and emission spectra. Subsequently, we applied APB in the development of a multicolor fluorescence quenching immunochromatographic assay, enabling simultaneous detection of chloramphenicol (limit of detection, LOD = 0.0045 ng/mL) and sulfadimidine (LOD = 0.038 ng/mL) in milk. This research not only offers a novel direction for designing efficient broad-spectrum quenchers but also advances the practical application of fluorescence quenching sensors.

Etiological surveillance of viral diarrhea from 2017 to 2019 in Zhangzhou city, Fujian province, China.

Viral diarrhea is one of the major causes of morbidity and mortality in children. This study aimed to conduct etiological surveillance of viral diarrhea in Zhangzhou city, Fujian province, China, from 2017 to 2019 to identify the prevalence, distribution, and characteristics of viral pathogens causing gastrointestinal infections in the region. Stool samples were collected from patients with acute diarrhea in Zhangzhou city, Fujian province, China, from 2017 to 2019. Rotavirus, norovirus, astrovirus, and adenovirus were detected using fluorescence immunochromatography assay. Of the total 5,627 samples that were collected, at least one of the viruses (rotavirus, norovirus, astrovirus and adenovirus) was found to be positive in 1,422 samples. Rotavirus, norovirus, astrovirus, and adenovirus, were detected in 53.73, 16.68, 15.52, and 14.97%, respectively. Mixed infections were determined in 17.65% of the positive samples. The predominant mixed infections observed were a combination of norovirus and astrovirus, followed by rotavirus and norovirus, and rotavirus and astrovirus. The highest positive rate was observed in the 12-23-month group for rotavirus and adenovirus, while a significantly higher positive rate was observed for norovirus and astrovirus in the 6-11-month group. These findings from this etiological surveillance highlight the significant burden of viral diarrhea in Zhangzhou city, with rotavirus being the predominant pathogen. The identification of common mixed infections provides insights into the complex nature of viral diarrhea transmission. Target interventions and public health strategies should be implemented, particularly during the winter and spring seasons, to prevent and control the spread of viral pathogens causing gastrointestinal infections in this region.

Janus plasmonic-aggregation induced emission nanobeads as high-performance colorimetric–fluorescent probe of immunochromatographic assay for the ultrasensitive detection of staphylococcal enterotoxin B in milk

Herein, a colorimetric–fluorescent hybrid bifunctional nanobead with Janus structure (J-cf-HBN) was synthesized via one-pot microemulsification. Oleylamine-coated AuNPs and aggregation-induced emission luminogens (AIEgens) were suggested as building blocks to obtain high-performance colorimetric–fluorescent signals. The as-prepared J-cf-HBNs were used as a signal amplification probe to construct an immunochromatographic assay (J-cf-HBNs-ICA) platform for the ultrasensitive detection of staphylococcal enterotoxin B (SEB) in milk samples. Owing to the rational spatial distribution of AuNPs and AIEgens, the J-cf-HBNs present a highly retained photoluminescence and enhanced colorimetric signals. Combined with a pair of highly affinitive anti-SEB antibodies, the J-cf-HBN-ICA platform enabled the fast naked-eye visualization and fluorescent quantitative detection of SEB in various milk matrices. Given the advantages of the dual-mode high-performance J-cf-HBNs, the proposed strip achieved a high sensitivity for SEB qualitative determination with a visual limit of detection (LOD) of 1.56 ng mL−1 and exhibited ultrasensitivity for SEB quantitative detection with a LOD of 0.09 ng mL−1, which is 139-fold lower than that of ELISA using same antibodies. In conclusion, this work provides new insights into the construction of multimode immunochromatographic methods for food safety detection in the field.

Development of Monoclonal Antibody against PirB and Establishment of a Colloidal Gold Immunochromatographic Assay for the Rapid Detection of AHPND-Causing Vibrio.

Acute hepatopancreatic necrosis disease (AHPND) poses a significant threat to shrimp aquaculture worldwide, necessitating the accurate and rapid detection of the pathogens. However, the increasing number of Vibrio species that cause the disease makes diagnosis and control more difficult. This study focuses on developing a monoclonal antibody against the Photorhabdus insect-related (Pir) toxin B (PirB), a pivotal virulence factor in AHPND-causing Vibrio, and establishing a colloidal gold immunochromatographic assay for the enhanced early diagnosis and monitoring of AHPND. Monoclonal antibodies targeting PirB were developed and utilized in the preparation of colloidal-gold-labeled antibodies for the immunochromatographic assay. The specificity and sensitivity of the assay were evaluated through various tests, including antibody subclass detection, affinity detection, and optimal labeling efficiency assessment. The developed PirB immunochromatographic test strips exhibited a good specificity, as demonstrated by the positive detection of AHPND-causing Vibrio and negative results for non-AHPND-causing Vibrio. The study highlights the potential of the developed monoclonal antibody and immunochromatographic assay for the effective detection of AHPND-causing Vibrio. Further optimization is needed to enhance the sensitivity of the test strips for improved practical applications in disease prevention and control in shrimp aquaculture.

Neonatal sepsis due to NDM-1 and VIM-2 co-producing Pseudomonas aeruginosa in Morocco.

Carbapenem-resistant Pseudomonas aeruginosa are being increasingly described worldwide. Here, we investigated the molecular mechanisms underlying carbapenem resistance in an extremely drug-resistant P. aeruginosa isolate from a neonatal intensive care unit in Morocco. P. aeruginosa strain O82J1 was identified using MALDI-TOF-MS. Carba NP, immunochromatographic assay NG Carba5 and antimicrobial susceptibility testing using disc diffusion and microbroth were performed. Whole-genome sequencing using the Illumina and MinION technologies and different software packages available at the Center of Genomic Epidemiology were used to predict the resistome, sequence type and plasmid types. P. aeruginosa O82J1 co-expressed two metallo-β-lactamases, blaNDM-1 and blaVIM-2, and was susceptible to colistin and apramycin only. It belonged to ST773 that is frequently reported worldwide as a high-risk P. aeruginosa clone. The blaVIM-2 gene was integron-borne on a IncP-2 465-kb plasmid, whereas the blaNDM-1 gene was chromosomally encoded and embedded in an integrative conjugative element, probably at the origin of its acquisition. A total of 23 antimicrobial resistance genes were detected including a blaPER-1 ESBL gene, and an 16S-rRNA methyltransferase gene rmtB. The isolation of XDR P. aeruginosa isolates expressing several carbapenemases in a neonatal intensive care unit is of great concern due to the reduced treatment options, relying only on colistin, but not recommended in neonates, and apramycin, not yet approved for human therapy. Concerns were further elevated due to the resistance to cefiderocol and ATM/AVI, two novel and last-resort antibiotics recommended to treat infections caused by Gram-negative bacteria, particularly XDR P. aeruginosa in adults.

Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2–3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 μg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 μg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 μl of GNP conjugate and 50 μl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.