Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

Nanobodies-based colloidal gold immunochromatographic assay for specific detection of parathion.

Parathion is one of organophosphorus pesticide, which has been prohibited in agricultural products due to its high toxicity to human beings. However, there are still abuse cases for profit in agricultural production. Hence, we established nanobodies-based colloidal gold immunochromatographic assay (GICA) in which nanobodies (Nbs) as an excellent recognition element, greatly improving the stability and sensitivity of ICA. Under the optimal conditions, the developed Nbs-based GICA showed a cut-off value of 50 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 2.39 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.15 ng/mL which was significantly 50-fold higher sensitivity than the commercial mAb-ICA. Additionally, this method exhibited good recoveries for the detection of cabbage, cucumber, and orange samples and excellent correlation with the UPLC-MS/MS method. The results showed that this method developed in this work based on nanobody can be used in practical detection of parathion in foods and nanobody is novel prospective antibody resource for immunoassays of chemical contaminants.

[Establishment of a rapid detection method of colloidal gold immunochromatographic competition assay for organophosphorus pesticides].

Objective To establish a colloidal gold immunochromatography and develop the corresponding test strip for detecting organophosphorus compounds including omethoate, phoxim, dipterex, and parathion in fruits, vegetables and drinking water. Methods Artificial antigen molecules of organophosphorus compounds were synthesized using N-hydroxysuccinimide esters. Acetylcholinesterase antigen was prepared and purified, and the serum containing the corresponding antibody was prepared, purified, and labeled. The working parameters of the test strip were optimized, and the performance evaluation of it was conducted. Results The titer of the antisera ranged from 1:32 to 1:64, with a protein content of approximately 2 mg/mL. The purified polyclonal antibodies displayed target bands at relative molecular masses (Mr) of 25 000 and 55 000, indicating satisfactory purity. The reaction time of the test strips was between 5 to 10 minutes, with a detection limit for samples at 200 ng/mL. Both specificity and accuracy were satisfactory, and the test strip remained valid for 6 months. Conclusion A simple and rapid colloidal gold immunochromatography is established successfully for detecting several organophosphorus compounds and may be useful for on-site preliminary screening of samples in large quantities.

Lateral Flow Assay Biotesting by Utilizing Plasmonic Nanoparticles Made of Inexpensive Metals─Replacing Colloidal Gold.

Nanoparticles (NPs) can be conjugated with diverse biomolecules and employed in biosensing to detect target analytes in biological samples. This proven concept was primarily used during the COVID-19 pandemic with gold-NP-based lateral flow assays (LFAs). Considering the gold price and its worldwide depletion, here we show that novel plasmonic NPs based on inexpensive metals, titanium nitride (TiN) and copper covered with a gold shell (Cu@Au), perform comparable to or even better than gold nanoparticles. After conjugation, these novel nanoparticles provided high figures of merit for LFA testing, such as high signals and specificity and robust naked-eye signal recognition. Since the main cost of Au NPs in commercial testing kits is the colloidal synthesis, our development with the Cu@Au and the laser-ablation-fabricated TiN NPs is exciting, offering potentially inexpensive plasmonic nanomaterials for various bioapplications. Moreover, our machine learning study showed that biodetection with TiN is more accurate than that with Au.

Biomarker-Driven DNA-Functionalized Colloidal Programmed Simultaneous Assembly and Disassembly in Cells.

Complex structures and devices, both natural and artificial, can often undergo assembly and disassembly. Assembly and disassembly allow multiple stimuli to initiate, for example, the assembly and disassembly of primary cilia under the control of E3 ubiquitin ligases and deubiquitinases. Although biology relies on such schemes, they are rarely available in materials science. Here, we demonstrate a DNA-functionalized colloidal Au response to endogenous biomarkers to trigger simultaneous assembly and disassembly techniques. Colloidal Au is initially inert because the starting DNA strands are paired and prehybridized. TK1 mRNA competes to bind one of the paired strands and release its complement. The released complement binds to the next colloidal Au to initiate assembly, and APE1 can shear the colloidal Au assembly binding site to initiate disassembly. Our strategy provides temporal and spatial logic control during colloidal Au assembly and disassembly, and this simultaneous assembly and disassembly process can be used for sequential detection and cellular imaging of two biomarkers, effectively reducing signal false-positive results and shortening detection time. This work highlights biomarker-controlled colloidal Au simultaneous assembly and disassembly in ways that are simple and versatile, with the potential to enrich the application scope of DNA nanotechnology and provide an idea for the application of precision medicine testing.

Development of Detection Antibody Targeting the Linear Epitope in SARS-CoV-2 Nucleocapsid Protein with Ultra-High Sensitivity.

The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens.

Quantitative measurement of acute myocardial infarction cardiac biomarkers by "All-in-One" immune microfluidic chip for early diagnosis of myocardial infarction.

Acute myocardial infarction (AMI) is a life-threatening condition with a narrow treatment window, necessitating rapid and accurate diagnostic methods. We present an "all-in-one" convenient and rapid immunoassay system that combines microfluidic technology with a colloidal gold immunoassay. A degassing-driven chip replaces a bulky external pump, resulting in a user-friendly and easy-to-operate immunoassay system. The chip comprises four units: an inlet reservoir, an immunoreaction channel, a waste pool, and an immunocomplex collection chamber, allowing single-channel flow for rapid and accurate AMI biomarker detection. In this study, we focused on cardiac troponin I (cTnI). With a minimal sample of just 4 µL and a total detection time of under 3 min, the chip enabled a quantitative visual analysis of cTnI concentration within a range of 0.5 âˆ¼ 60.0 ng mL-1. This all-in-one integrated microfluidic chip with colloidal gold immunoassay offers a promising solution for rapid AMI diagnosis. The system's portability, small sample requirement, and quantitative visual detection capabilities make it a valuable tool for AMI diagnostics.

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

BACKGROUND: Timely 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. METHODS: Colloidal 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. RESULTS: AMA1C-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. CONCLUSIONS: This 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.

Ultrasensitive detection of imatinib in human serum using a gold-based paper sensor.

Imatinib is the tyrosine kinase inhibitor of choice for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, imatinib has drawbacks such as drug resistance and significant differences in pharmacokinetics within patients. Therefore, a colloidal gold-based immunochromatographic assay (CG-IA) was developed for measuring and monitoring imatinib in human serum. An imatinib derivative containing carboxyl groups was used for the synthesis of the immunogen, and 4-(4-methyl-1-piperazinylmethyl) benzoic acid was selected as the hapten for the heterologous coating antigen. Next, a highly sensitive and specific monoclonal antibody (mAb), 2F7 was screened for the construction of a CG-IA, with an IC50 value of 0.091 ng/mL. For the qualification of imatinib in human serum, the visual limit of detection (vLOD) and cut-off values of the CG-IA were 2 and 20 ng/mL, respectively. For quantitative detection, the calculated LOD value of the CG-IA was 0.068 ng/mL, with a linearity range of 1.004 and 23.087 ng/mL. The recovery rate of spiked serum samples was between 88.24 % and 104.75 %. In addition, the concentration of imatinib in the serum samples from 10 patients was detected by CG-IA and revealed a good correlation with those from LC-MS/MS. These results indicated that the developed gold-based paper sensor could become an effective tool for the rapid monitoring of imatinib in human serum samples.

Establishment of a Colloidal Gold Immunochromatographic Method for the Detection of Cypermethrin in Tobacco.

In this study, the establishment of a colloidal gold immunochromatographic method for the detection of cypermethrin in tobacco was achieved by using colloidal gold immunochromatography: strong specificity and high sensitivity of cypermethrin semi-antigens and encapsulants were prepared during the study. The best colloidal gold solution was prepared by spectrophotometer and transmission electron microscope screening; the preparation process of gold-labeled antibodies was optimized, and finally the product of colloidal gold rapid detection test strips for cypermethrin was developed. The results of technical parameters and detection indexes showed that the detection limit of cypermethrin in tobacco was 1 mg/kg, and there was no cross-reaction with bifenthrin, cypermethrin, cyfluthrin and phenothrin, and the detection results of 30 tobacco samples were consistent with those of gas chromatography.

Development of Colloidal Gold Test Strips for the Detection of Oxamyl Residues in Tobacco.

In this study, monoclonal antibodies against oxamyl were prepared, and colloidal gold immunochromatography was used to design a rapid test strip product for the detection of oxamyl in tobacco with high specificity, accuracy and stability without cross-reactivity to commonly used tobacco fungicides based on the optimization of conditions such as pH value of diluent, diluent dosage, concentration of antibody marker, type of confining solution and complex solution. 5 The results of five samples of post-harvest ready-to-bake tobacco and first-harvest tobacco were consistent with the gas chromatographic method, which proved the reliability of the test strips. The limits of detection for the post-harvest and first-harvest tobacco samples were 0.1 mg/kg, and the test strips developed in this study are suitable for mass testing in tobacco laboratories with good application prospects because of their short detection time, simple pre-treatment and detection methods.

Development of Colloidal Gold Test Strips for the Detection of Triadimenol Residues in Tobacco.

The rapid and accurate determination of triadimenol residues is of great significance. In this study, based on the advantages of high efficiency, rapidity, reliability, simplicity and low cost of immunology, a test strip product for the rapid detection of triadimenol residues in tobacco was designed based on the optimization of conditions such as pH and dosage of diluent, concentration of antibody stock solution, type of confining solution and complex solution, with high specificity, accuracy and The results of 20 samples of fresh and first roasted tobacco were all consistent with the method of gas chromatography, which proved the reliability of the test strips. The detection limit for fresh and roasted tobacco was 5 mg/kg, and the test strips developed in this study are suitable for mass testing of tobacco samples in tobacco-related laboratories because of their short detection time, simple pre-treatment and detection methods, and good application prospects.

Rapid Colloidal Gold Immunoassay for Pharmacokinetic Evaluation of Vancomycin in the Cerebrospinal Fluid and Plasma of Beagle Dogs.

Vancomycin (VAN), a glycopeptide antibiotic, is the preferred therapeutic agent for treating Gram-positive bacteria. Rapid and precise quantification of VAN levels in cerebrospinal fluid (CSF) and plasma is crucial for optimized drug administration, particularly among elderly patients. Herein, we introduce a novel clinical test strip utilizing colloidal gold competitive immunoassay technology for the expedient detection of VAN. This test strip enables the detection of VAN concentrations in clinical samples such as plasma within 10 min and has a limit of detection of 10.3 ng/mL, with an inhibitory concentration 50% (IC50) value of 44.5 ng/mL. Furthermore, we used the test strip for pharmacokinetic analysis of VAN in the CSF and plasma of beagle dogs. Our results provide valuable insights into the fluctuations of the drug concentration in the CSF and plasma over a 24 h period after a single intravenous dose of 12 mg/kg. The test strip results were compared with the results obtained via liquid chromatography-mass spectrometry methods, and the measured VAN concentrations in the CSF and plasma via both of the methods showed excellent agreement.

Design and development of a lanthanide-labeled immunochromatographic strip for simultaneous detection of morphine, methamphetamine and ketamine in hair.

Colloidal gold immunoassay is the most widely used method in the field of drug detection. However, this method often has poor quantitative identification ability and low analytical sensitivity, which is not suitable for the analysis of hair poisoning ingredients. In order to solve these limitations, we developed an immunochromatographic test strip for simultaneously screening multiple drugs in this study. This hand-held test strip used fluorescent nanoparticles loaded with lanthanide chelates as the signal carrier of fluorescence reading, and conducted quantitative testing of various drugs based on the competitive immune reaction between the analyte and antigen. Under the optimal conditions, the competition curves of morphine (MOP), methamphetamine (MET) and ketamine (KET) were obtained on a single band. The detection limit (LOD) of this analytical method was 100-1000 times lower than that of colloidal gold test strips. The detection limits of MOP, MET and KET were 0.06 ng mL-1, 0.1 ng mL-1 and 1.0 ng mL-1, respectively. No cross-reaction was observed when morphine, methamphetamine and ketamine were tested simultaneously with this method. And 184 hair samples were tested simultaneously, and the detected amount was very close to the results of LC-MS. The immunochromatographic strip showed good stability in repeated tests, and the coefficient of variation was less than 15%. Fluorescence immunochromatography strips and handheld strip readers have the characteristics of portability, speed, ease of operation and high sensitivity, and may become powerful tools for screening drug abuse in hair in forensic medicine.

Establishment of ultrasensitive immunoassay strip based on colloidal gold-McAb probe for detecting cyproheptadine hydrochloride and six phenothiazines in feed.

An ultrasensitive and broad-specific monoclonal antibody recognising cyproheptadine hydrochloride and six phenothiazines was produced. The 50% inhibition concentration against cyproheptadine hydrochloride was 0.036 ng/mL, and the cross-reactivities for six phenothiazines were from 6.33% to 63.16%. Based on the developed monoclonal antibody, an immunochromatographic strip was established, with the visual detection limits (cut-off values) of seven drugs ranging from 5 to 100 ng/g in feedstuffs. With the strip reader, the 50% inhibition concentration of the developed immunochromatographic strip for seven drugs ranged from 0.570 to 7.750 ng/g. The intra-assay recoveries were from 79.8% to 103.4% with the highest coefficient of variation of 11.3%. The inter-assay recoveries were from 79.0% to 96.6% with the highest coefficient of variation of 12.7%. In summary, the proposed immunochromatographic strip was considered suitable for simultaneously monitoring cyproheptadine hydrochloride and phenothiazines in feedstuffs.

Development of a Lateral Flow Immunoassay Based on a Highly Specific Monoclonal Antibody To Detect 4-Methylaminoantipyrine.

To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues, especially metabolites of target compounds, the preparation of highly specific antibodies is crucial. Preserving the characteristic structure of a target compound when designing a hapten is important when preparing highly specific antibodies. Here, we designed a novel hapten, 4-(((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4yl)amino)methyl)benzoic acid, named AA-BA, to improve the specificity of antibodies for detection of 4-methylaminoantipyrine (MAA), a residual marker of dipyrone, an important antipyretic-analgesic and anti-inflammatory drug. The structural features of the hapten remained almost the same as those of MAA. After experimental validation, monoclonal antibody 6A4 (mAb 6A4) was prepared with the half maximal inhibitory concentration (IC50) value of 4.03 ng/mL and negligible cross-reactivity with dipyrone metabolites and other antibiotics. In addition, a specific lateral flow immunoassay (LFA) strip based on colloidal gold was developed for screening MAA with a cutoff value of 25 ng/mL in milk. The developed LFA is a useful tool for rapid and accurate detection of MAA.

A colloidal gold immunoassay strip assay for cadmium detection in oilfield chemicals.

Cadmium ions (Cd2+) are some of the major pollutants in oilfield chemicals. To reduce the pollution of oilfield chemicals, it is necessary to detect and control the content of Cd2+. In this study, we synthesized a highly sensitive and specific monoclonal antibody against Cd2+ with an IC50 of 1.97 ng mL-1 and no cross-reactivity. Based on this antibody, a colloidal gold immunoassay strip detection assay with an IC50 of 1 mg kg-1 and a detection range of 1.0-20 mg kg-1 in oilfield chemicals was developed. This assay could be completed in 20 min and can be used for Cd2+ on-site testing in oilfield chemicals and improve supervision efficiency in oil exploration and development.

Immunochromatographic test strip for quantitative and rapid detection of tolfenpyrad in food samples.

In the study, a hapten was designed to preserve the molecular structure of tolfenpyrad while introducing a carboxyl group and was coupled with a carrier protein to synthesize an immunogen and coating antigen. A monoclonal antibody was fabricated against tolfenpyrad and its performance was assessed by indirect competitive enzyme-linked immunosorbent assay. Finally, we developed a colloidal gold nanoparticle immunochromatographic test strip (CGN-ICTS) for the detection of tolfenpyrad in kale, Chinese cabbage, and eggplant samples. The results shows that CGN-ICTS was sensitive, with calculated detection limits of 0.49 ng/g for kale and Chinese cabbage and 0.99 ng/g for eggplant. Subsequently, CGN-ICTS and LC-MS were used to analyze the tolfenpyrad-spiked samples. The recovery rate of the CGN-ICTS for kale samples was 97.1-103.0%, for Chinese cabbage samples was 93.7-103.4%, and for eggplant samples was 92.7-105.7%. Recovery rates were similar between CGN-ICTS and LC-MS. Therefore, CGN-ICTS can be used to quickly screen tolfenpyrad residues in foods.

Colloidal gold-based immunochromatographic biosensor for quantitative detection of S100B in serum samples.

Traumatic brain injury has become a serious public health problem. Timely detection, diagnosis and treatment of brain injury are closely related to the prognosis of patients, so identification of highly sensitive and specific biochemical markers of brain injury has important clinical value. Currently, the most studied and most promising marker is the protein S100B. In this study, a rapid quantitative biosensor for S100B was established using colloidal gold labeling and double antibody (8C10-6B8) sandwich immunochromatography. The biosensor was capable of quantifying S100B within 15 min, and showed no cross-reactivity with S100A, NSE, GFAP, or PGP9.5. The detection limit was determined to be 4.6 pg mL-1 with a linear range of 0.01-2 ng mL-1. Recovery experiments also indicated that the method had an acceptable accuracy. Moreover, the quantitative colloidal gold assay correlated well with the results of a chemiluminescence immunoassay when testing 40 clinical serum samples. Our developed colloidal gold quantitative immunochromatographic biosensor is a rapid, sensitive, specific and accurate method for the detection of S100B protein in serum, which is useful in the clinic for early diagnosis, as well as assessment of disease progression and prognosis of traumatic brain injury.

Colloidal gold and fluorescent immunochromatographic test strips for canine parvovirus detection.

Canine parvovirus (CPV) is an acute and highly infectious virus causing disease in puppies and, thus, affecting the global dog industry. The current CPV detection methods are limited by their sensitivity and specificity. Hence, the current study sought to develop a rapid, sensitive, simple, and accurate immunochromatographic (ICS) test to detect and control the spread and prevalence of CPV infection. More specifically, 6A8, a monoclonal antibody (mAb) with high specificity and sensitivity, was obtained by preliminary screening. The 6A8 antibody was labelled with colloidal gold particles. Subsequently, 6A8 and goat anti-mouse antibodies were coated onto a nitrocellulose membrane (NC) as the test and control lines, respectively. Furthermore, 6A8 and rabbit IgG antibodies were labelled with fluorescent microspheres and evenly sprayed onto a glass fibre membrane. Both strips could be prepared in 15 min with no noticeable cross-reactivity with other common canine intestinal pathogens. The strips were simultaneously used to detect CPV in 60 clinical samples using real-time quantitative PCR, hemagglutination, and hemagglutination inhibition assays. The colloidal gold (fluorescent) ICS test strip was stable for 6 (7) and 4 (5) months at 4 °C and room temperature (18-25 °C). Both test strips were easy to prepare and rapidly detected CPV with high sensitivity and specificity. Moreover, the results were easily interpretable. This study establishes a simple method for two CPV diseases, colloidal gold and fluorescent immunochromatographic (ICS) test strips. KEY POINTS: • CPV test strips do not exhibit cross-reactivity with other canine intestinal pathogens. • The strips are stable for months at 4 °C and at room temperature (18-25 °C). • These strips are a promising approach for the timely diagnosis and treatment of CPV.