Immunochromatographic Strip Assay Research Articles

Immunochromatographic assay for detection (2-benzothiazolylthio) methyl thiocyanate in food packaging paper materials

The residue of fungicides in food packaging paper materials is one of the important factors leading to food safety problems, which poses a serious threat to public health. (2-benzothiazolylthio) methyl thiocyanate (TCMTB) is an important anti-mildew fungicide, which is widely used in wood and paper industry. In order to ensure the health of consumers, it is of great significance to establish an efficient and sensitive rapid detection method for TCMTB in food packaging materials. In this study, we prepared monoclonal antibody (mAb) against TCMTB with the IC50 value of 7.60 ng/mL, and subsequently developed an immunochromatographic assay (ICA) strip based on the mAb for the rapid detection of TCMTB residues. The calculated detection limit of the ICA strip in food packaging paper was 3.20 ng/g, and the recovery rate was within an acceptable range, ranging from 97.7% to 102.7%. Therefore, the developed ICA is expected to realize the rapid screening of TCMTB residues in food packaging paper.

Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/μL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

An immunochromatographic strip sensor for marbofloxacin residues.

Marbofloxacin (MBF) was once widely used as a veterinary drug to control diseases in animals. MBF residues in animal food endanger human health. In the present study, an immunochromatographic strip assay (ICSA) utilizing a competitive principle was developed to rapidly detect MBF in beef samples. The 50% inhibitory concentration (IC50) and the limit of detection (LOD) of the ICSAs were 2.5 ng/mL and 0.5 ng/mL, respectively. The cross-reactivity (CR) of the MBF ICSAs to Ofloxacin (OFL), enrofloxacin (ENR), norfloxacin (NOR), and Ciprofloxacin (CIP) were 60.98%, 32.05%, 22.94%, and 23.58%, respectively. The CR for difloxacin (DIF) and sarafloxacin (SAR) was less than 0.1%. The recovery rates of MBF in spiked beef samples ranged from 82.0% to 90.4%. The intra-assay and interassay coefficients of variation (CVs) were below 10%. In addition, when the same authentic beef samples were detected in a side-by-side comparison between the ICSAs and HPLC‒MS, no statistically significant difference was observed. Therefore, the proposed ICSAs can be a useful tool for monitoring MBF residues in beef samples in a qualitative and quantitative manner.

Quantitative immunochromatographic assay for rapid and cost-effective on-site detection of benzo[a]pyrene in oilfield chemicals

Contamination of oilfield chemicals (OFCs) by benzo[a]pyrene (B[a]P) is increasingly becoming a severe environmental security issue. There is an urgent need to develop a rapid and accurate method for B[a]P detection in OFCs. In this study, B[a]P hapten was designed using computer aided molecular design. A high-affinity, specific, and matrix-insensitive monoclonal antibody (mAb) with IC50 values of 6.77 ng/mL was obtained. Based on this mAb, we developed a rapid gold nanoparticle-based immunochromatographic strip assay (GICA) with double T-line mode for on-site detection of B[a]P in OFCs samples. The GICA exhibited excellent detection performance in OFCs samples with strong acidity, strong alkalinity, and deep color. Under optimal conditions, the proposed method detected B[a]P in OFCs at 0.42–300 mg/kg, and limit of detection was 0.23–1.07 mg/kg. The recovery rate was 88–106% with a coefficient of variation of 1.46–6.35%. Confirmed by natural positive OFCs samples and high-performance liquid chromatography, this GICA is accurate and reliable, with great potential for rapid and cost-effective on-site detection.

On-site rapid detection of perfluorooctanoic acid by visual immunochromatographic strip biosensor in domestic water and real human samples

The International Agency for Research on Cancer (IARC) classifies PFOA as a Class 1 carcinogen. Here, a new naked-eye PFOA immunochromographic strip was developed to recognize PFOA in domestic water and real human samples within 10 min based on a novel custom designed anti-PFOA monoclonal antibody (mAb) 2A3, which was firstly an immune rapid detection method for PFOA has been proposed. Using computer simulation techniques such as quantum computing to assist in designing the structural formula of PFOA semi antigen, which hapten was firstly proposed. The half maximal inhibitory concentration of PFOA monoclonal antibody (mAb) 2A3 was 2.4 μg/mL. Using mAb 2A3, we developed an immunochromatographic strip (ICS) for detecting PFOA in real samples. The developed method generated results in 10 min, with visual detection limits of 20, 20, and 200 μg/mL and limit of detection of 50, 200, and 500 μg/mL for water, blood and urine samples, respectively. The established ICS and indirect competitive enzyme-linked immunosorbent assay were used to analyze the actual samples, and the results were confirmed by LC-MS/MS. Our study findings showed that the ICS and ic-ELISA can quickly detect PFOA in actual samples.

Development and evaluation of a time-resolved fluorescence labelled immunochromatographic strip assay for rapid and quantitative detection of bovine herpesvirus 1.

Bovine herpes virus 1 (BoHV-1) causes a wide variety of diseases in wild and domestic cattle. The most widely used method for viral identification is real-time PCR, which can only be performed in laboratories using sophisticated instruments by expert personnel. Herein, we developed an ultrasensitive time-resolved fluorescence lateral flow immunochromatographic strip (ICS) assay for detecting BoHV-1 in bovine samples using a monoclonal antibody against BoHV-1 labelled with fluorescent microspheres, which can be applied in any setting. The intact process from sample collection to final result can be achieved in 15 min. The limit of detection of the assay for BoHV-1 was 102 TCID50/100 μL. The coincidence rate of the ICS method and real-time PCR recommended by the World Organization for Animal Health (WOAH) was 100% for negative, 92.30% for positive, and 95.42% for total, as evaluated by the detection of 131 clinical samples. This detection method was specifically targeted to BoHV-1, not exhibiting cross-reactivity with other bovine pathogens including BoHV-5. We developed an ICS assay equipped with a portable instrument that offers a sensitive and specific platform for the rapid and reliable detection of BoHV-1 in the field. The Point-of-Care test of BoHV-1 is suitable for the screening and surveillance of BoHV-1 in dairy herds.

Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species

BackgroundTimely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results.MethodsColloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera.ResultsAMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%–23.4% of these samples were found to be positive for T. gondii.ConclusionsThis study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.Graphical

Determination of Morphine Using Carboxylated Multiwalled Carbon Nanotubes (MWCNTs) as the Label for an Immunochromatographic Assay (ICA)

This study evaluated the application of carboxylated multi-walled carbon nanotubes (MWCNTs) as a label in immunochromatographic assay for morphine detection. MWCNTs were carboxylated using nitric acid and characterized by Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, field emission scanning microscopy (FESEM), and dynamic light scattering (DLS). The optimum concentration of polyvinyl pyrrolidone (PVP) as a surfactant for homogenous dispersion of MWCNTs was determined by ultraviolet-visible spectroscopy to be 0.5 mg/mL. The zeta potential of MWCNTs before adding PVP was −41.9 ± 1.02 mV, and after adding PVP, −54.1 ± 0.25 mV. On the surface of MWCNTs, the anti-morphine antibody (AM antibody) had a binding effectiveness ranging from 91.1% to 95.5%. The AM antibody-MWCNTs were conjugated to create immunochromatographic test strips. The visual limit of detection (vLOD) was 1.0 ng/mL and the cutoff concentration (COC) was 10.0 ng/mL. Compared to previous study, carboxylated-MWCNTs as a one-dimensional nanomaterial improved the sensitivity of immunochromatographic assay strips and may be utilized to develop the sensitive immunochromatographic assays.

Development of an ic-ELISA and immunochromatographic assay strip for the rapid detection of chloridazon in oranges and celery.

Chloridazon (CLZ) is a selective herbicide used in the control of annual broadleaf weeds. The misuse or abuse of CLZ may result in the accumulation of CLZ in crops and water, which can pose a risk to human health. In this study, a hapten of CLZ with three carbon spacer arms was designed and a highly sensitive and specific antibody against CLZ was prepared with a half-maximal inhibitory concentration of 0.630 ng mL-1 and a linear range of 0.181-2.195 ng mL-1.Based on this antibody, we developed an immunochromatographic assay (ICA) strip for the detection of CLZ in oranges and celery. Under optimized conditions, the visual limit of detection was 2 ng mL-1 and 10 ng mL-1 in oranges and celery, respectively, and the cut-off value was 50 ng mL-1. In CLZ-spiked samples and the recovery test, the results of the ICA strip were consistent with those of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Therefore, the ICA strip developed in our study represents an efficient and reliable method for the rapid screening of CLZ in oranges and celery.

An immunochromatographic strip sensor for rapid and sensitive detection of candesartan, olmesartan medoxomil, and irbesartan in herbal beverages.

Sartans, as a class of antihypertensive drugs, pose a threat to human health when illegally added to herbal beverages. It is crucial to detect sartans in herbal beverages. We have developed a highly sensitive monoclonal antibody against candesartan (CAN), olmesartan medoxomil (OLM), and irbesartan (IRB), with 50% inhibitory concentrations (IC50) that were obtained via indirect enzyme-linked immunosorbent assay (ic-ELISA) as 0.178 ng mL-1, 0.185 ng mL-1, and 0.262 ng mL-1 against CAN, OLM, and IRB, respectively. Based on this monoclonal antibody, we developed a rapid screening method for CAN, OLM, and IRB in herbal beverage samples using an immunochromatographic assay (ICA) strip. Test for 15 minutes after simple and rapid sample pre-treatment and the results of this method can be obtained through naked eye observation. The detection limits (LODs) of the ICA strip for CAN, OLM, and IRB in herbal beverage samples are lower than 0.15 ng mL-1, and the results of the ICA strip and ic-ELISA are consistent in spiked samples and recovery experiments. Therefore, this method can quickly, efficiently, and reliably achieve high-throughput on-site rapid detection of illegally added CAN, OLM, and IRB in herbal beverages.

Broad-Spectrum Antibody-Based Immunochromatographic Strip Assay for Rapid Screening of Bisphenol A Diglycidyl Ether and Its Derivatives in Canned Foods.

Bisphenol A diglycidyl ether (BADGE) is widely present in the inner coating of metal food cans, from which it can migrate into food and generate harmful derivatives during storage, such as bisphenol A (2,3-dihydroxypropyl) glycidyl ether, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, and bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) glycidyl ether. Here, a gold-nanoparticle-based immunochromatographic strip assay based on a broad-spectrum polyclonal antibody was developed for the simultaneous detection of BADGE and its derivatives, which could be accomplished within 15 min. The quantitative analysis of the visualization results was performed using Adobe Photoshop CC 2021, and the detection limit, defined as the concentration causing 15% inhibition, was 0.97 ng/mL. The recoveries of BADGE and its derivatives at various spiking levels in canned food samples ranged from 79.86% to 93.81%. The detection results of the proposed immunochromatographic strip assay were validated via high-performance liquid chromatography, showing a good correlation coefficient (R2 = 0.9580).

A fluorescence-based immunochromatographic assay using quantum dot-encapsulated nanoparticles for the rapid and sensitive detection of fetuin-B

Coronary artery disease (CAD) is the leading cause of death worldwide. Earlier detection of CAD improves treatment outcomes and secondary prevention. The circulating fetuin-B protein is considered to be a promising biomarker for the early detection of CAD. However, a facile and reliable clinical test for fetuin-B is still lacking. Herein, we describe a reliable fluorescent biosensor for detecting fetuin-B in plasma that combines quantum dots-doped polystyrene nanoparticles with an immunochromatographic assay strip (QNPs-ICAS). The QNPs served as detection signals in the QNPs-ICAS sensor system, which was based on a double-antibody sandwich structure. Under optimum experimental conditions, the biosensor exhibited a broad linear range of 1–200 ng mL−1 and a low detection limit of 0.299 ng mL−1. Furthermore, the proposed immunosensor demonstrated high sensitivity, satisfactory selectivity, good reproducibility, and excellent recovery. Finally, the performance and applicability of our QNPs-based ICAS system were validated in clinical samples using a commercial ELISA kit with excellent correlations (r = 0.98451, n = 116). To conclude, the proposed sensor served as a rapid, sensitive, and accurate method for detecting fetuin-B in actual clinical samples, thereby demonstrating its potential for preliminary CAD screening and diagnosis.

Introduction of Multilayered Dual-Signal Nanotags into a Colorimetric-Fluorescent Coenhanced Immunochromatographic Assay for Ultrasensitive and Flexible Monitoring of SARS-CoV-2.

Timely, accurate, and rapid diagnosis of SARS-CoV-2 is a key factor in controlling the spread of the epidemic and guiding treatments. Herein, a flexible and ultrasensitive immunochromatographic assay (ICA) was proposed based on a colorimetric/fluorescent dual-signal enhancement strategy. We first fabricated a highly stable dual-signal nanocomposite (SADQD) by continuously coating one layer of 20 nm AuNPs and two layers of quantum dots onto a 200 nm SiO2 nanosphere to provide strong colorimetric signals and enhanced fluorescence signals. Two kinds of SADQD with red and green fluorescence were conjugated with spike (S) antibody and nucleocapsid (N) antibody, respectively, and used as dual-fluorescence/colorimetric tags for the simultaneous detection of S and N proteins on one test line of ICA strip, which can not only greatly reduce the background interference and improve the detection accuracy but also achieve a higher colorimetric sensitivity. The detection limits of the method for target antigens via colorimetric and fluorescence modes were as low as 50 and 2.2 pg/mL, respectively, which were 5 and 113 times more sensitive than those from the standard AuNP-ICA strips, respectively. This biosensor will provide a more accurate and convenient way to diagnose COVID-19 in different application scenarios.

Development of Dot-ELISA and Colloidal Gold Immunochromatographic Strip for Rapid and Super-Sensitive Detection of Plum Pox Virus in Apricot Trees.

Plum pox virus (PPV) is a causal agent of the stone fruit tree sharka disease that often causes enormous economic losses. Due to its worldwide distribution and economic importance, rapid and reliable diagnostic technologies are becoming increasingly important for successful management of sharka disease. In this study, we have produced two super-sensitive and specific anti-PPV monoclonal antibodies (i.e., MAbs 13H4 and 4A11). Using these two MAbs, we have now developed a dot enzyme-linked immunosorbent assay (dot-ELISA) and a colloidal gold immunochromatographic strip (CGICS) assay. These two technologies can be used to quickly and reliably detect PPV. The results of these sensitivity assays confirmed that the dot-ELISA and CGICS assays could detect PPV infection in apricot tree leaf crude extracts diluted up to 1:5120 and 1:6400 (w/v), respectively. Further analyses using field-collected apricot tree leaf samples showed that the detection endpoint of the dot-ELISA was ~26 times above that obtained through RT-PCR, and the CGICS was as sensitive as RT-PCR. This present study is to broaden the knowledge about detection limits of dot-ELISA and CGICS for PPV monitoring. We consider that these newly developed dot-ELISA and CGICS are particularly useful for large scale PPV surveys in fields.

Monoclonal antibody production and development of immunochromatographic strip assays for screening of the herbicide bispyribac-sodium in rice.

Bispyribac-sodium (BIS) is a new broad-spectrum and efficient herbicide, which is widely used for the control of weeds in rice. To protect the human body from the threat of BIS exposure, it is essential to establish a sensitive and simple detection method. In this work, a high-affinity monoclonal antibody against BIS was produced for the first time, and a colloidal gold immunochromatographic strip assay (ICSA) was developed to screen for BIS in rice samples. The visual limit of detection and the calculated limit of detection of the ICSA were 0.2 μg kg-1 and 0.018 μg kg-1, respectively, which could be accurately obtained within 8 min. The average recoveries of BIS ranged from 90.0% to 109.0% in tests, with CVs ranging from 4.0% to 8.9% for rice samples. Therefore, our ICSA would be a good option for the sensitive and rapid detection of BIS in rice samples.

Hapten synthesis and a colloidal gold immunochromatographic strip assay to detect nitrofen and bifenox in fruits.

In this study, we synthesized two haptens similar in structure to nitrofen (NIT), and screened out five monoclonal antibodies with the ability to recognize NIT and bifenox (BIF) by competitive ELISA, with the lowest IC50 values of 0.87 ng mL-1 and 0.86 ng mL-1, respectively. The antibody 5G7 was selected to be combined with colloidal gold to establish a lateral flow immunochromatographic assay strip. This method was shown to qualitatively and quantitatively detect the residues of NIT and BIF in fruit samples. The visual limits of detection for qualitative detection were 5 μg kg-1 and 10 μg kg-1 for NIT and BIF, respectively. The calculated limits of detection for quantitative detection were 0.75 μg kg-1, 1.77 μg kg-1 and 2.55 μg kg-1 respectively, for nitrofen in orange, apple and grapes, and 3.54 μg kg-1, 4.96 μg kg-1 and 5.26 μg kg-1, respectively, for bifenox. Thus the strip assay could be used for rapid analysis of fruit samples.

Highly resistant and sensitive colorimetric immunochromatographic assay for sibutramine (SBT) illegally adulterated into diet food based on PDA/AuNP labelling.

A gold nanoparticle (AuNP) based immunochromatographic assay strip is a valuable tool for monitoring chemicals in foods. However, the sensitive ICA strip for SBT is rarely reported due to the fact that monoclonal antibodies (mAbs) against SBT with high affinity are commercially unavailable. Herein, a monoclonal antibody against SBT was prepared through a designed hapten with a carboxyl end-capped space arm. The obtained mAb showed high affinity for SBT and N-desmethylsibutramine, a metabolite of SBT. Furthermore, a series of core-shell NPs, polydopamine (PDA) coated AuNPs (PDA/AuNPs) with controlled shell thickness and packing density were synthesized. The obtained PDA/AuNP-mAb conjugate demonstrated high tolerance to salt and good stability in a wide pH range, which is beneficial for improving the matrix interference common in ICA. As a result, PDA/AuNP-based ICA could quantify SBT in the range of 3.39-69.60 ng mL-1 with a limit of detection (LOD) of 0.98 ng mL-1. This novel ICA improved the sensitivity of the traditional AuNP-based ICA by nearly 12 times. Method validation was conducted with spiked samples of slimming food and human serum and compared with HPLC-MS/MS. Consistent results indicated that high sensitivity, accuracy, and reliability of the PDA/AuNP-based ICA approach were achieved. To the best of our knowledge, this study reported the most sensitive immunoassay for SBT thus far.

Universal probe with oriented antibody to improve the immunochromatographic assay of lead ions inProcambarus clarkii

AbstractObjectivesBased on the information from the random inspection of foods by the China Food and Drug Administration in 2022, the contamination levels of lead ions are high in many edible products. Traditional methods of detecting lead ions cannot meet the requirements of on-site analysis of food due to the need for large equipment. The immunochromatographic assay (ICA) is an effective, rapid, on-site analytical technique for determining lead ions in foods. However, the performance of ICA based on the traditional probe (AuNP-mAb) is limited by ignoring the influence of the antibody orientation.Materials and MethodsIn this study, we developed an efficient technology for constructing a universal probe (AuNP-PrA-mAb) based on the oriented immobilization of antibody. The performance of ICA was largely improved due to specific binding of the Fc region of the antibody with recombinant protein A (PrA) on the surface of a gold nanoparticle (AuNP). The ICA based on a universal probe was applied for the qualitative and quantitative detection of lead ions in Procambarus clarkii within 30 min. Meanwhile, a simple and fast pretreatment method based on dilute acid extraction was developed for pretreating the P. clarkii containing lead ions.ResultsThe visual limit of detection and the scanning limit of quantization of the developed ICA strip for lead ions were 0.5 ng/mL and 0.28 ng/mL, respectively. The sensitivity of ICA based on universal probe was 10-fold higher than that of the ICA using traditional probe. Furthermore, the detection results had no obvious difference between the ICA and ICP-MS with t-test statistical method.ConclusionsThe developed ICA based on a universal probe presented broad application prospects in detecting contaminants in foods.

Ratiometric fluorescent immunochromatography for simultaneously detection of two nitrofuran metabolites in seafoods

Competitive immunochromatographic assay (ICA) has been widely used in food safety on-site monitoring. However, traditional competitive ICA relies on the change of single-signal intensity to discriminate the results, leading to limited sensitivity and accuracy of naked-eye visual inspection. In this study, we developed a sensitive ratiometric fluorescent immunochromatographic assay (RFICA) judging results based on the color changes of fluorescence. Two different color quantum dot nanobeads (QBs) were introduced as signal labels for rapid and accurate detection of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM) in seafoods. Under the optimal conditions, the visual detection limit for 2-NPAOZ and 2-NPSEM reached up to 1 and 0.5 ng/mL, which were 5- and 14-fold lower than that of traditional single-signal response fluorescent ICA strips. In addition, the RFICA strips were applied to real samples with acceptable recoveries of 81.33 ∼ 99.23 %. These results demonstrate that the developed RFICA platform possesses considerable potential in the on-site detection.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China

BackgroundThe existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host’s immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed.ResultsThe truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%.ConclusionsThis study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.